method for detecting the presence or concentration of an analyte in a sample of fluid contained in a

专利摘要:
SYSTEMS AND METHODS FOR MAXIMIZING SAMPLE USE In some embodiments, the present invention relates to point of care and / or point-of-service devices. In some embodiments, the present invention relates to systems, devices, user interfaces, and methods for testing samples using a point of care and / or point-of-service device. In one aspect, the devices and methods described here are designed to identify the type of sample (plasma and blood, against etc.), to measure the sample volume early enough in the test process to ensure that a suitable sample is used is one that is intended for testing. In another aspect, the present invention also allows the correction of the significant volume errors that occur when carrying out a test.
公开号:BR112013018656B1
申请号:R112013018656-9
申请日:2012-01-20
公开日:2021-03-02
发明作者:Ian Gibbons；Tony Nugent；Anthony Delacruz；Daniel L. Young；Elizabeth Holmes；Andrew Drake；Timothy Michael Kemp；Sunny Balwani；Chinmay Pangarkar
申请人:Labrador Diagnostics Llc；
IPC主号:

专利说明:

Cross-reference
[001] This application claims priority to provisional Patent Application No. US 61/435, 250, filed on January 21, 2011, an application that is incorporated herein by reference. Background of the invention
[002] The discovery of a large number of disease biomarkers, new therapies and the establishment of miniaturized medical systems have opened new avenues for forecasting, diagnosing and monitoring disease treatment in a "point of care" setting or other test settings distributed. Point-of-care systems can quickly deliver test results to medical personnel, other healthcare professionals and patients. Early diagnosis of a disease or disease progression and monitoring of therapy are often essential for the treatment of deadly diseases, such as certain types of cancer and infectious diseases.
[003] Disease diagnosis and treatment can take advantage of multiplexed biomarker measurements, which provide additional knowledge of a patient's condition. For example, when monitoring the effects of a drug, three or more biomarkers can be measured in parallel. Typically, microtiter plates and other similar apparatus have been used to perform assays based on multiplexed separation. A microtiter plate (for example, a 384 microtiter plate) can perform a large number of tests in parallel.
[004] In a Point of Care (POC) device, the number of tests that can be performed in parallel, and often limited by the size of the device and the volume of the sample to be analyzed. In many POC devices, the number of tests performed is about 1 to 10. A POC device capable of performing multiplexed tests on a small sample would be desirable.
[005] A problem with many multiplexed POC test devices and the high cost of manufacturing the device components. If the device is disposable, the cost of the components can make the production of a POC device impractical. In addition, for multiplexed POC devices that incorporate all of the necessary reagents on board the device, if any of the reagents exhibits instability, an entire batch of the devices may have to be discarded, even if all other reagents are still usable. .
[006] When a customer is interested in customizing a POC device for a given set of analytes, manufacturers of multiplexed POC assay systems are often faced with the need to mix and match the device's tests and reagents. A multiplexed POC assay suitable for each customer can be very expensive and difficult to calibrate, and difficult to maintain quality control.
[007] POC methods have proven to be very valuable in disease monitoring and treatment (eg, blood glucose systems in diabetes therapy, measurement of prothrombin time in anticoagulant therapy with warfarin). When measuring multiple markers, it is believed that complex diseases (such as cancer), for which multidrug therapies are needed, can be better controlled and monitored.
[008] There is no need to use various sources of information to monitor the health status or disease condition of individuals, as well as treatments for various diseases. Especially important is the measurement of the concentrations of several selected analytes (biomarkers, antibodies, levels of gene expression, metabolites, therapeutic concentrations of the drug and the like) over time. In order to make this process convenient and maximally effective, technologies that allow the measurement of any and all necessary analytes (of any type) with a small sample of blood (drop of blood obtained by pricking the finger) or another suitable sample are particularly valuable. This technology will ideally be operated by non-technical users trained in distributed testing environments, for example, homes, clinics, doctors' offices, pharmacies and retail stores. The present invention addresses these issues and allows one to be able to make such measurements routinely in the patient's home or other non-laboratory environment.
[009] There is also no need to make the greatest use of available samples, particularly in the case where the samples (eg blood samples) are limited by the size of the sample. Blood samples are used for the vast majority of medical / clinical tests. Blood cells have to be separated from plasma (or serum) before most types of analysis, since the presence of cells will compromise the test chemicals. For example, glucose and cholesterol are often measured by color-forming chemicals that would interfere with the presence of formed elements, especially in red cells, or hemoglobin (from sanded red blood cells).
[0010] An ideally distributed test system requires a small blood sample obtained by fingerstick methods. These samples can be as small as 20 microliters (ul) (one drop) or less. Larger volume samples (say up to 200 Ul) generally cannot be taken by fingerstick methods without repeated, inconvenient ("milking") fingers. Alternatively venous samples of several milliliters (ml) can be taken, but this requires a phlebotomist with medical training.
[0011] It is generally very difficult to perform more than a single test with a small blood sample of 20 ul or less. this is especially true when the blood sample has to be filtered to remove the cells and the recovery of usable plasma from such small volumes and inefficient. Typically, only about 5 µl or less of plasma can be recovered. Samples as large as 200 µl can be effectively separated by automatic POC systems (Abaxis, Biosite etc.), but this cannot be done routinely, unless a technician is available to extract the sample. Summary of the invention
[0012] In view of the limitations of current methods, there is a great need for better methods for the automatic separation of plasma and / or other materials from blood cells. There is also a need to improve the accuracy of these measurements on the concentration of the analyte. When measuring biomarkers and other blood components for the purpose of monitoring therapy and diagnosis, it is important that the correct sample volume is used. In a laboratory environment, this is achieved through the use of complex automated instruments and qualified professional staff. In contrast to point-of-care environments, such as homes, pharmacies and stores, and the like, the methods and equipment used should allow untrained people to be technically reliable in obtaining and processing samples.
[0013] The present invention responds to the aforementioned needs and provides related advantages.
[0014] In some embodiments, the present invention relates to point of care and / or point-of-service devices. In some embodiments, the present invention relates to systems, devices, user interfaces, and methods for testing samples using a point of care and / or point-of-service device.
[0015] In one aspect, the devices and methods described here are designed to identify the type of sample (plasma and blood, counter etc.), to measure the sample volume early enough in the test process to ensure that a suitable sample used and one that is intended for testing. In another aspect, the present invention also allows the correction of the significant volume errors that occur when carrying out a test.
[0016] In yet another aspect, this invention allows the simultaneous measurement of several different types of analytes, with high precision.
[0017] One aspect of the invention can be addressed to an automated system for the separation of one or more components in a biological fluid. The automated system may comprise a closed pipette tip or tube adapted to engage with a vacuum cleaner wherein said pipette tip or tube comprises two opposite ends, at least one of which is closed or sealed, and a centrifuge configured to receive said sealed or closed tip of the tube to effect said separation of one or more components in a biological fluid. In one embodiment, the one or more components are selected from the group consisting of blood plasma, blood serum, blood cells, and the particles. In another embodiment, when the pipette tip is engaged with the aspirator to effect a coupling of the biological fluid. In another embodiment, the tip of the pipette has an open end, which forms a hermetic seal with the vacuum cleaner. In another embodiment, the system further comprises an imaging device, and at least one other tip of the pipette sized to allow the distribution of a liquid into the tip of the pipette or tube (a) or to allow the aspiration of liquid to from the tip of the pipette or tube (a). In another embodiment, the pipette tip or tube is closed and vertically oriented when the centrifuge is at rest. In another embodiment, the pipette tip or tube is closed and oriented horizontally when the centrifuge is rotating at a predetermined speed of rotation.
[0018] Another aspect of the invention may be a method for the isolation of components from a sample comprising one or more of the following steps: placing a sample on a pipette tip or a tube comprising two opposite ends, at least one of which and sealable or sealed; sealing the pipette tip on at least one end of the pipette tip; centrifuging the sealed pipette tip, thus forming an interfacial region that separates the sample in a supernatant and a sediment; Imaging centrifuged the tip of the pipette to determine the location of the interfacial region, and automatically aspirating the supernatant based on the location of the interfacial region. In one embodiment, the method further comprises determining the location of the supernatant by said step of supernatant imaging and aspiration automatically based on the location of the supernatant. In another embodiment, the determination takes place with the aid of a processor, said processor provides instructions for a suction device, which performs the automated suction step. In another embodiment, the creation of the image occurs through the use of a camera that is configured to capture the image of the side profile of the tip of the pipette or tube. In another embodiment, the supernatant includes one or more of the following: blood plasma or blood serum. In another embodiment, the pellet includes one or more of the following: the globules or particles.
[0019] A computer aided method for characterizing an object of analysis that is suspected to be present in the sample can be provided, in accordance with an additional aspect of the invention. The computer-assisted method may comprise obtaining a digital image of the sample, in which the digital image comprises at least a two-dimensional array of pixels, and in which each pixel comprises a plurality of intensity values, each of which corresponds to to a spectral detection distinct region; correlating, with the aid of a programmable device, the intensity values obtained with a predetermined set of values that define a dynamic range of each spectral region, the detection and prediction of the presence and / or quantity of said analyte in the sample based on said correlating the intensity values obtained with a set of predetermined values. In one embodiment, the plurality of intensity values comprise intensity values for detecting red, green and blue spectral regions. In another embodiment, the method further comprises the selection of an illumination wavelength, which illuminates the sample and with the illumination of the selected wavelength before and / or concurrently with obtaining the digital image. In another embodiment, the method further comprises, after obtaining the digital image, (a) selecting another illumination wavelength, (b) illuminating the sample with the other selected illumination wavelength, (c) obtaining another digital image of the sample, where the digital image comprises at least a two-dimensional array of pixels, and where each pixel comprises a plurality of intensity values, each of which corresponds to a distinct spectral region detection, and (d) predict the presence and / or quantity of said analyte in the sample based on the intensity values obtained from the digital image and said another digital image.
[0020] In addition, one aspect of the invention can be addressed to a method of measuring the concentration of analyte in a fluid sample which comprises providing the sample contained in a container sized with a plurality of different widths to allow light transmission over a plurality of different paths corresponding to a plurality of different widths, illuminating the container along at least one of a plurality of path lengths, and imaging the container to measure a first intensity of light transmitted through said, at at least, one of a plurality of path lengths, for determining the concentration of the substance to be analyzed based on the intensity of the light measured first.
[0021] According to another aspect of the invention, a method for detecting the presence or concentration of an analyte in a sample of fluid contained in a container (e.g., cuvette) can comprise the illumination of the container over a first region has a first path length to obtain a first measurement of the intensity of the light transmitted through the first path length; moving the sample fluid to another region of the container having another if the length of the first measurement path falls outside a predetermined dynamic range of the transmitted light intensity; illuminating the container along the other region to provide another measurement of the intensity of the light transmitted through the other path length, and optionally repeating, second and third steps until the measurement of the light intensity falls within the predetermined dynamic range, detecting thus the presence or concentration of the substance to be analyzed. In one embodiment, the method further comprises deconstructing a line of exploration of the image, thereby detecting the presence or concentration of a substance to be analyzed. In another embodiment, the sample is moved from a first region of the container having a first path length to a second region of the container having another path length by aspirating the sample. In another embodiment, one end of the container is connected to a pipette that is configured to aspirate the sample. In another embodiment, the sample is moved up or down the length of the container. In another embodiment, the container and a pipette tip. In another embodiment, the container is tapered. In another embodiment, the container has two open ends. In another embodiment, a first open end has a larger diameter than a second open end. In another embodiment, the container has a plurality of different widths to allow light to be transmitted over a plurality of different path lengths. In another embodiment, the volume of the container is less than 100 microliters. In another embodiment, a plurality of different path lengths are recorded simultaneously.
[0022] A method can be provided as an additional aspect of the invention. The method can be provided for the characterization of an object of analysis that is suspected to be present in a sample of biological fluid, comprising said method: providing said sample of biological fluid, allowing said analyte to react with one or more reagents that react specifically with said analyte to generate an optically detectable signal, and optically measure said detectable signal with a plurality of spectral detection regions, wherein the presence of said detectable optical signal within a dynamic range of at least one region of the spectrum. detection and indicator of the concentration of said analyte in said biological fluid sample. In one embodiment, the measurement is performed by an imaging device configured to measure a plurality of spectral detection regions. In another embodiment, the imaging device is configured to measure a plurality of spectral regions of detection simultaneously. In another embodiment, the imaging device is configured to measure a plurality of spectral regions of detection sequentially.
[0023] One aspect of the invention provides a method for increasing the accuracy of an assay comprising an image sample at a first tip to determine the volume of the first example; imaging one or more reagents on a second tip to determine the volume of the one or more reagents, mixing the sample and one or more reagents to form a reaction mixture, the image reaction mixture; correct the calibration based on the said certain sample volumes and one or more reagents, and calculating a concentration of an analyte using the corrected calibration. In one embodiment, the method further comprises mixing the image reaction to determine the volume of the reaction mixture. In another embodiment, the image of the sample at the first tip is conducted using a camera configured to capture a side profile of the first tip. In another embodiment, the imaging of one or more reagents on the second tip is conducted using a camera configured to capture a side profile of the second tip. In another embodiment, the height of the sample and the one or more reagents is calculated based on the captured profiles. In another embodiment, the determination of the volume is based on the height of the sample and the one or more reagents and the known cross-sectional areas of the sample and one or more reagents, respectively. In another embodiment, the calibration is also based on the determined volume of the reaction mixture.
[0024] Another aspect of the invention provides a configuration, comprising: a container configured to accept and confine a sample, wherein the container comprises an inner surface, an outer surface, an open end and an opposite closed end, and a tip configured for extend into the container through the open end, where the tip comprises a first open end and the second open end, where the second open end is inserted into the vessel, where the container or tip further comprises a surface feature projecting that prevents the second open end of the tip from contacting the bottom of the inner surface of the closed end of the container. In one embodiment, the surface feature is integrally formed on the inner surface of the bottom of the vessel. In another embodiment, the surface feature comprises a plurality of projections on the inner surface of the bottom of the vessel. In another embodiment, the surface feature protrudes at or near the closed end.
[0025] Another aspect of the invention provides a sample processing apparatus comprising a sample preparation station, the test station, and / or the detection station, a control unit having executable computer commands to perform a sample service. service point at the designated location with the aid of at least one of said sample preparation station, the test station and the detection station, and at least one centrifuge configured to perform the centrifugation of a sample from a finger prick. In one embodiment, the centrifuge is contained within the sample preparation station and / or the test station. In another embodiment, the executable computer commands are configured to perform the service point service at a location selected from the group consisting of a local distributor, of the subject's origin, or a local health assessment / treatment.
[0026] Another aspect of the invention provides a method for the return dynamics, comprising said method: making an initial measurement of a sample inside a container using a detection mechanism, based on said initial measurement, determining, using a processor, if the sample concentration falls within a desired range, and determining, using a transformer, (a) a degree of dilution to be performed if the sample concentration is greater than the desired range or (b) a degree of concentration to be performed if the sample concentration is less than the desired range, and adjusting the sample concentration according to the degree of dilution or the degree of concentration determined. In one embodiment, the method further comprises making a subsequent measurement of the sample inside the container. In another embodiment, the method further comprises, based on the subsequent measurement determination, using a processor, whether the sample concentration falls within a desired range. In another embodiment, the subsequent measurement is carried out by means of the detection mechanism. In another embodiment, the method further comprises determining a characteristic of the sample based on the subsequent measurement. In another embodiment, the characteristic is selected from one or more of the following: the presence or concentration of an analyte, the presence or concentration of a cell, and the morphology of the cell. In another embodiment, the subsequent measurement is carried out by means of a detection mechanism separate from the initial detection mechanism. In another embodiment, the initial measurement provides a measurement of the crude cell concentration of the sample. In another embodiment, the subsequent measurement provides a measure of the concentration of cells in the sample of higher resolution than the initial measurement. In another embodiment, the initial measurement is made using the sample image. In another embodiment, adjusting the sample concentration allows the detection of the analyte that would otherwise fall outside the desired range.
[0027] Another aspect of the invention provides a method for providing quality control, said method comprising capturing an image of conditions in which a detection mechanism measures a characteristic of a sample, and determining, using a processor, with based on the image if there are undesirable conditions under which the detection mechanism is used. In one embodiment, undesirable conditions include the presence of one or more undesirable materials. In another embodiment, undesirable materials include one or more of the following: bubbles, particles, fibers, debris and precipitates, which interfere with the measurement of the sample characteristic. In another embodiment, the detection mechanism is a different mechanism from a mechanism used to capture the image. In another embodiment, the image is captured using a camera. In another embodiment, the method further comprises providing an alert if an undesirable condition is detected. In another embodiment, the method further comprises adjusting the sample if an undesirable condition is detected. In another embodiment, the image includes an image of the sample. In another embodiment, the image includes an image of one or more of the following characteristics: the sample container, the detection mechanism.
[0028] Another aspect of the invention is an automated system for separating one or more components in a biological fluid comprising a centrifuge comprising one or more containers configured to receive a container for effecting said separation of one or more components from a sample fluid, and the container, wherein the container includes one or more features than the shape and is complementary to a container-shaped structure. In one embodiment, the container-shaped structure includes one or more shelves on which a protruding portion of the container is configured to rest. In another embodiment, the container is configured to be able to accept a plurality of containers having different configurations, and in which the container-shaped structure includes a plurality of shelves, wherein a first container having a first configuration is configured to rest on a first shelf, and a second container with a second configuration and configured to rest on a second shelf.
[0029] Another aspect of the invention provides a test unit comprising a first end and a second end, an outer surface and an inner surface comprising one or more selected patterns each of which is immobilized on it or in it with a capture reagent capable of capture an object of analysis that is suspected to be present in a biological sample, in which the first end and the second end have different dimensions.
[0030] Another aspect of the invention provides a test unit comprising an identifier that is used to determine (a) one or more capture reagents immobilized on the inner surface and (b) the source of the biological sample, if the test unit contains said sample.
[0031] Another aspect of the invention provides an assay unit comprising a plurality of selected patterns, each pattern of said plurality comprising a distinct capture agent.
[0032] Other objectives and advantages of the invention will be even more appreciated and understood when considered together with the following description and attached drawings. While the following description may contain specific details describing particular embodiments of the invention, this should not be interpreted as limiting the scope of the invention, but rather as an exemplification of preferable embodiments. For each aspect of the invention, many variations are possible, as suggested herein, which are known to those skilled in the art. A wide variety of changes and modifications can be made within the scope of the invention, without departing from the spirit of the invention. The various compounds / devices described herein can be used separately or in any combination, by any methods disclosed in the present invention individually or in any combination. Reference incorporation
[0033] All publications, patents and patent applications mentioned in this description are hereby incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. Brief description of the drawings
[0034] The new features of the invention are presented with particularity in the appended claims. A better understanding of the characteristics and advantages of the present invention will be obtained with reference to the following detailed description, which presents illustrative embodiments, in which the principles of the invention are used, and the attached drawing (s), of which:
[0035] Figure 1 shows a side view of a centrifuge.
[0036] Figure2 shows a face in the view of a centrifuge.
[0037] Figure 3 shows a perspective view of the rear of a centrifuge.
[0038] Figure 4 shows a top view of a sample tip.
[0039] Figure5 shows a side view of a sample tip.
[0040] Figure 6 shows a cross-sectional view of a sample stitch.
[0041] Figure 7 shows a diagram of a sample tip positioned in the example above a plasma / globular interface.
[0042] Figure 8 shows a graph of the spin time, such as a function of revolutions per minute.
[0043] Figure 9 shows a graph of the centrifugation time, as well as a function of the radius of the centrifuge rotor.
[0044] Figure 10 shows an empty cap of the capped sample.
[0045] Figure 11 shows a capped sample tip containing a sample of a body fluid, for example, blood.
[0046] Figure 12 shows a capped sample tip containing a sample of about 23% blood hematocrit after centrifugation.
[0047] Figure 13 shows a capped sample tip containing a sample of about 31% hematocrit in the blood, after centrifugation.
[0048] Figure 14 shows a capped sample tip containing a blood sample of about 40% hematocrit after centrifugation.
[0049] Figure 15 shows a capped sample tip containing a sample of approximately 52% hematocrit in the blood, after centrifugation.
[0050] Figure 16 shows a capped sample tip containing a blood sample of 68% hematocrit, after centrifugation.
[0051] Figure 17 shows a comparison of hematocrits measured using the digital image system of a centrifuged sample ("hematocrit, reported%") and hematocrit measured by the standard microhematocrit device ("hematocrit,% target").
[0052] Figure 18 shows a diagram of a tip used for reactions and a tip used for blood / plasma (dimensions shown in mm).
[0053] Figure 19 shows a cylindrical capillary tube containing a sample.
[0054] Figure 20 shows the angles and dimensions for calculating volumes inside a conical container, for example, a capillary.
[0055] Figure 21 shows the angles and dimensions for calculating volumes inside a conical container, for example, a capillary tube.
[0056] Figure 22 shows the angles and dimensions to calculate the volume of a spherical cap.
[0057] Figure 23 shows the dimensions for calculating the volume of a sample contained within a cylindrical tip, in which the sample has a single meniscus.
[0058] Figure 24 shows the dimensions for calculating the volume of a sample contained within a cylindrical tip, in which the sample has two menisci.
[0059] Figure 25 shows the dimensions for calculating the volume of a sample contained inside and / or in association with a cylindrical tip, in which the sample has two menisci and of which one and the cylindrical tip outside.
[0060] Figure 26 shows the dimensions for calculating the volume of a sample contained within a cylindrical tip, where there is a bubble in the sample.
[0061] Figure 27 shows the dimensions for calculating the volume of a sample contained within a cylindrical tip, where there is a bubble in the example that covers the width of the cylindrical tip.
[0062] Figure 28 shows the dimensions for calculating the volume of a sample contained within and / or in association with a cylindrical tip, in which the sample includes a pendent drop of sample outside the cylindrical tip.
[0063] Figure 29 shows the dimensions for calculating the volume of a residual sample contained within a cylindrical tip.
[0064] Figure 30 shows a blood sample inside a tip before being mixed with a magnetic reagent.
[0065] Figure 31 shows a blood sample to be mixed with a magnetic reagent.
[0066] Figure 32 shows a blood sample, mixed with a reagent.
[0067] Figure 33 shows a blood sample, mixed with a magnetic reagent contained inside a tip.
[0068] Figure 34 shows a blood sample, mixed with a magnetic reagent moved to a selected position inside a tip.
[0069] Figure 35 shows a magnetic force applied by an ima (M) to a blood sample, mixed with a magnetic reagent.
[0070] Figure 36 shows a blood sample that has been separated into a component of red blood cells and a component of plasma using magnetic force.
[0071] Figure 37 shows a well positioned underneath a tip that contains a blood sample that has been separated into a component of red blood cells and a component of plasma.
[0072] Figure 38 shows a representation of blood plasma to be transferred from one end of a well.
[0073] Figure 39 shows a tip after dispensing blood plasma to a well.
[0074] Figure 40 shows a high contrast image of a cylindrical tip containing a liquid with low absorption.
[0075] Figure 41 shows an image of a conical tip, containing a liquid with high absorbency.
[0076] Figure 42 shows a tip with a high absorption liquid showing two meniscus on the tip.
[0077] Figure 43 shows a tip with a liquid sample and large bubbles that cover the tip diameter.
[0078] Figure 44 shows a tip containing water showing a clear upper meniscus in a transparent or capillary tip.
[0079] Figure 45 shows a graph of the C-protein concentration computed as a function of the sample volume.
[0080] Figure 46 shows an image of a sample transfer device with a capillary, housing, plunger, groove, and elevated feature. The raised feature can help to locate the plunger in the housing.
[0081] Figure 47 shows an example contained with the capillary of a sample transfer device.
[0082] Figure 48 shows a sample transfer device after a sample has been injected by a plunger.
[0083] Figure 49 shows a sample transfer device after an incomplete sample has been injected.
[0084] Figure 50 shows a conical tip containing a sample, with the position l3 indicated by the arrow shown.
[0085] Figure 51 shows a graph of the relationship between the distance between ll and l2 and the distance between l3 and lG as a function of the sample volume.
[0086] Figure 52 shows a scheme of a chemical reaction that produces a colored product.
[0087] Figure 53 shows a scheme of a chemical reaction that produces a color product from cholesterol.
[0088] Figure 54 shows a scheme of a chemical reaction that uses reducing equivalents for the production of a colored product.
[0089] Figure 55 shows an example of a compound that changes color by being complexed with a metal fon.
[0090] Figure 56 shows a series of hint images with double decreasing concentration of albumin from right to left, except for the leftmost tip, which has no albumin.
[0091] Figure 57 shows a series of hint images with double cholesterol-lowering concentration, from right to left, except for the leftmost tip, which has no cholesterol.
[0092] Figure 58 shows a series of hemispherical cavities machined from a block of white opaque plastic, which each well will have twice the analyte concentration from the decrease from right to left, except for the better left. , which has no analyte. In some embodiments, the analyte can be calcium.
[0093] Figure 59 shows a series of hemispherical cavities machined from a block of white opaque plastic, which each well will have twice the analyte concentration from the decrease from right to left, except for the better left. , which has no analyte. In some embodiments, the analyte may be magnesium.
[0094] Figure 60 shows a series of hemispherical cavities machined from a block of white opaque plastic, which each well will have twice the analyte concentration from the decrease from right to left, except for the better left , which has no analyte. In some embodiments, the analyte may be urea.
[0095] Figure 61 shows a series of tips for solutions containing bromophenol blue.
[0096] Figure 62 and an illustration of tips with a plurality of different optical path lengths.
[0097] Figure 63 shows a light path through a rectangular tub.
[0098] Figure 64 shows a light path through a microtiter well.
[0099] Figure 65 shows a light path through a conical tub.
[00100] Figure 66 shows a graph of light intensity as a function of location, as measured at the tips containing samples with different concentrations of solutions from bromophenol blue to red, green, and blue color channels.
[00101] Figure 67 shows an image of the tips that were analyzed in Figure 66.
[00102] Figure 68 shows a signal graph as a function of the bromophenol blue concentration as measured by the red, green and blue color channels. The optical density can be measured at 589 nm.
[00103] Figure 69 shows a logarithmic scale of signal response as a function of the concentration of bromophenol blue as measured by the blue (diamonds) and red (squares) color channels.
[00104] Figure 70 shows a graph of the concentration measured by color analysis of digital images as a function of actual concentration.
[00105] Figure 71 shows a signal response graph, measured by red (squares), green (diamonds), and blue (triangles) color channels as a function of albumin concentration.
[00106] Figure 72 shows three graphs of signal response measured for green, red, and blue color channels for polystyrene latex particles.
[00107] Figure 73 shows tips that each contain separately NADH, WST-1, PMS reagents, and two tips that contain a mixture of the reagents.
[00108] Figure 74 shows a digital image of tips containing double decreasing concentration of lactate dehydrogenase (lDH) from left to right.
[00109] Figure 75 shows a graph of the optical density measured at 450 nm as a function of lDH.
[00110] Figure 76 shows the potassium chloride solutions added to potassium dosing strips.
[00111] Figure 77 shows tips containing blood samples mixed with blood typing reagents for Anti-A, Anti-B, Anti-D, and control (from left to right).
[00112] Figure 78 shows signals measured for the signal as a function of position for red (left column), green (middle column) and blue (right column) for samples mixed with Anti-A, Anti-B, Anti-D, and control reagents.
[00113] Figure 79 shows the normalized signal as a function of the relative concentration measured for narrow and wide path lengths using red, green, and blue color channels.
[00114] Figure 80 shows a graph of the measured concentration log as a function of the actual concentration, which illustrates the accuracy of the measurement algorithm.
[00115] Figure 81 shows a fluorescence image of products in test tubes.
[00116] Figure 82 shows an image of the reaction products on tips. Figure 83 shows an image of the spiked reaction products. Figure 84 shows an image of the spiked reaction products. Figure 85 shows an image of the spiked reaction products. Figure 86 shows an image of the spiked reaction products. Figure 87 shows an image of the spiked reaction products.
[00117] Figure 88 shows a background color image obtained for the calibration.
[00118] Figure 89 shows a fluorescence image of the reaction products in tips.
[00119] Figure 90 shows the red and blue color channel response and fluorescence response as a function of the number of DNA copies.
[00120] Figure 91 shows the 3-color signal transform graph as a function of the fluorescence signal.
[00121] Figure 92 shows a graph of the signal response of the green channel, as a function of the pixel position.
[00122] Figure 93 shows an image of tips containing solutions of bromophenol blue and water.
[00123] Figure 94 shows an image of additional tips that may contain solutions of bromophenol blue and water.
[00124] Figure 95 shows a schematic diagram of a tip containing reaction mixtures to perform multiple tests.
[00125] Figure 96 shows an image of tips containing solutions of bromophenol blue and water.
[00126] Figure 97 shows a graph of the signal response for the sample, water, and control between multiple patterns.
[00127] The samples can be aqueous calibrators containing known concentrations of analyte.
[00128] Figure 98 shows tests of tips containing both Ca 2 + (upper tip region) and Mg 2 + (lower tip region).
[00129] Figure 99 shows four tips with various types of serum samples: hemolysis (reddish), lipemic (gray), jaundice (yellow), and normal (from left to right).
[00130] Figure 100 shows a schematic of a camera and optical components.
[00131] Figure 101 shows a cross-sectional view of a camera and optical components, including a white light source, an aperture, and a sensor.
[00132] Figure 102 shows a schematic diagram of an optical signal installation for measuring light, using (a) a sensor that is positioned to detect light at an angle perpendicular to an excitation beam, and (B) a sensor which is positioned in line with an excitation beam.
[00133] Figure 103 shows images obtained using (a) an excitation beam perpendicular to a sensor and (B) an excitation beam that is in line with a sensor.
[00134] Figure 104 shows a matrix of printed inks that can be used to calibrate the optical configuration.
[00135] Figure 105 shows a signal graph as a function of the sample volume.
[00136] Series 1-5 can correspond to different concentrations of analytes, such as 0, 20, 40, 60 and 80, respectively.
[00137] Figure 106 shows a signal graph as a function of the sample volume. Series 1-5 can correspond to different concentrations of analytes, such as 0, 20, 40, 60 and, respectively.
[00138] Figure 107 shows a signal graph as a function of the sample volume. Series 1-5 can correspond to different concentrations of analytes, such as 0, 20, 40, 60 and 80, respectively.
[00139] Figure 108 shows a graph of the measured analyte concentration as a function of the actual analyte concentration.
[00140] Figure 109 shows a graph of the measured analyte concentration as a function of the actual analyte concentration.
[00141] Figure 110 schematically illustrates an exemplary method for an EllSA assay. vertical.
[00142] Figure 111 shows an example of a rotor at rest with containers
[00143] Figure 112 shows an example of a rotor at a container speed of a small angle in relation to the horizontal.
[00144] Figure 113 shows an example of a container configuration.
[00145] Figure 114 shows an example of a centrifuge container mated to the container.
[00146] Figure 115 shows an example of another centrifuge container that can be coupled with the container.
[00147] Figure 116 shows an example of a centrifuge container. Figure 117 shows an example of an extraction tip.
[00148] Figure 118 provides an example of how the centrifuge and tip extraction vessel can mate.
[00149] Figure 119 and an image that was taken from the original reaction mixture before centrifugation.
[00150] Figure 120 and another image, which was made of the initial reaction mixture before centrifugation.
[00151] Figure 121 is an additional image that was taken of the initial reaction mixture before centrifugation
[00152] Figure 122, Figure 123, Figure 124, Figure 125, Figure 126, Figure 127, Figure 128, Figure 129, Figure 130 relating to accuracy, precision, predicted for the concentration.
[00153] Figure 131 shows images collected by a digital camera.
[00154] Figure 132 illustrates examples of images taken from the reaction product.
[00155] Figure 133 presents examples of images that were analyzed before rotating in the centrifuge and, after rotating in the centrifuge.
[00156] Figure 135 illustrates the spectra of several serum samples.
[00157] Figure 136 illustrates an example detection process of the invention using a matrix.
[00158] Figure 137 illustrates an example detection process of the invention using pearls.
[00159] Figure 138 illustrates an example detection process of the invention using labeled aptamers.
[00160] Figure 139 illustrates the detection of aptamer connection to a complementary probe.
[00161] Figure 140 illustrates the lack of connection between aptamer and a non-complementary probe.
[00162] Figure 141 illustrates the specificity of linking aptamers in an arrangement.
[00163] Figure 142 shows a more detailed view of the detection of the analyte in a matrix.
[00164] Figure 143 shows an example matrix.
[00165] Figure 144 shows a graph of chemiluminescence against concentration for a vitamin D assay.
[00166] Figure 145 shows a graph of chemiluminescence against concentration for an estradiol assay.
[00167] Figure 146 shows a spectrophotometric measurement of the WBC concentration. Figure 147 shows turbidity plots as a function of time.
[00168] Figure 148 and a plot of inflection points for the three experiments in 800 copies / ul and 80 copies / ul.
[00169] Figure 149 is a graphical representation of an example in which the magnetic spheres are used for the analysis of proteins and small molecules through EllSA assays.
[00170] Figure 150 is a graphical representation of an example in which magnetic spheres are used for the analysis of proteins and small molecules through EllSA assays. Detailed description of the invention
[00171] As long as preferred embodiments of the invention have been shown and described, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes and substitutions will now occur to those skilled in the art without departing from the invention. It is to be understood that several alternatives to the embodiments of the invention described herein can be employed in the practice of the invention.
[00172] The invention provides mobile applications for the system and methods for using the maximization sample. Various aspects of the invention described herein can be applied to any of the particular applications presented below, or by any other types of diagnostic or therapeutic applications. The invention can be applied as an independent system or method, or as part of an integrated pre-clinical, clinical, laboratory or medical application. It should be understood that the different aspects of the present invention can be appreciated individually, together, or in combination with each other.
[00173] The devices and systems described herein can provide an effective means for the real-time detection of analytes in a subject's body fluid. Detection methods can be used in a wide variety of circumstances, including the identification and quantification of analytes that are associated with specific biological processes, physiological conditions, diseases or stages of disorders. As such, the systems have a wide spectrum of utility in, for example, drug screening, disease diagnosis, phylogenetic classification, forensic and parental identification, disease onset and recurrence, from individual response to treatment against population of bases, and / or monitoring of therapy. Subject devices and systems are also particularly useful for advancing the pre-clinical and clinical phase of development of therapies, improving patient compliance, monitoring ADRs associated with a prescribed drug, developing individualized medicine, outsourcing blood testing central laboratory for the home or on a prescription basis and / or monitoring of therapeutic agents after regulatory approval. The present devices and system can be used by outsourcing blood tests from a central laboratory. The devices and systems can provide a flexible system for personalized medicine. Using the same system, a device can be changed or exchanged, along with a protocol or instructions for a programmable processor of the systems to perform a wide variety of tests, as described. The systems and devices described here, while being much smaller and / or portable, incorporate new features and offer many functions of a laboratory instrument.
[00174] In one aspect, a system of the invention comprises a device comprising test units and reagent units, which include reagents, for example, liquids and / or solid phase reagents. In some embodiments, at least one of the entire device, a dosing unit, a reagent unit, or a combination thereof is disposable. In a system of the invention, the detection of a substance to be analyzed with the object device is typically automated. Such automation can be carried out using an embedded protocol or a protocol supplied to the system by the manufacturer.
[00175] The devices and systems as described here can offer many features that are not available in the POC systems or analysis of existing integrated systems. For example, many POC cartridges rely on a closed circuit or fluid system to handle small volumes of liquid efficiently. Fluidic devices such as cartridges described herein can have fluid movement open between the units within a given cartridge. For example, a reagent can be stored in a unit, a sample stored in a sample collection unit, a diluent stored in a diluent unit, and the capture surface can be a dosing unit, where in a cartridge state, none of the units is in fluid communication with any of the other units. The units can be mobile relative to each other in order to bring some units in fluid communication with a system fluid transfer device. For example, a fluid transfer device may comprise a head that engages a dosing unit and the dosing unit brings fluid communication with a reagent unit. In some cases, the head and a pipette head that moves the test unit (eg, top) of fluid in communication with a reagent unit.
[00176] Thus, in one embodiment, the present invention provides a method for detecting and / or measuring the concentration of an analyte in a body fluid or tissue sample, the method typically comprises the steps of providing a sample (e.g. example, blood, urine, saliva, tissue), for a device or system of the invention, allowing the sample to react at least within a dosing unit of the device, and to detect the detectable signal generated from the analyte in the blood sample.
[00177] One aspect of the invention provides for the analysis of samples using a point of care device that is configured to maximize sample utilization. For example, more than about 15, 25, 50, 75, or 100 tests can be performed on a sample with a volume of less than about 1, 20, 50, 100, or 500 μf. The sample may be a blood sample taken from a finger prick. The sample can be collected in a capillary tip or sealed. The sample can be prepared by one or more tests, subjecting the sample to separation (eg, centrifugation) and / or the dilution process. The one or more tests can be prepared by combining the sample, which may have been separated and diluted with one or more reagents in a reaction chamber. The reaction chamber can be a pipette tip, test tube, a sample transfer device, and / or a cuvette. The one or more tests can be configured in such a way that if an optical signal can be measured, it is indicative of the concentration of one or more analytes in the sample. The reaction chamber can contain a plurality of tests, which can be spatially separated. A plurality of optical signals can be generated within a single reaction chamber of an assay or a plurality of spatially separate assays. The one or more optical signals can be measured by a digital imaging camera that can measure a plurality of regions or spectral detection detection bands, for example, red, green and blue. The optical signal can be measured with the reaction product of the assay in the reaction chamber, which may be a pipette tip or other sample containers. Systems, devices and methods can be fully automated or semi-automated by programmable logic.
[00178] Another aspect of the invention provides systems, devices and methods for preparing samples for analysis. Samples can be prepared for analysis by one or more separation devices. For example, a sample can be prepared for analysis by centrifugation inside a centrifuge. Other separations based on load, size, hydrophobicity / hydrophilicity and / or volatility can also be implemented.
[00179] One aspect of the invention provides for the sample and analysis of the reaction product through image-based analysis. The system can include a camera that can measure an optical signal with one or more regions of the detection spectrum. For example, a camera can measure an optical signal using regions of the red, green, blue detection spectrum.
[00180] The measurement signal can include the three measured values, which can be interpreted using one or more algorithms described here. Using more than one region of the detection spectrum can increase the dynamic range of an assay and can increase the accuracy of a measurement, compared to measurements using a single region of the detection spectrum.
[00181] The invention also provides systems, devices and methods for performing optical measurements on test samples and the reaction products that are contained within reaction chambers, each with a plurality of different path lengths. The reaction chambers can have a plurality of different path lengths in such a way that a greater or lesser amount of light absorption is observed. The plurality of different path lengths (such as, for example, through the sample and / or the reaction chamber), allows an increase in the dynamic range of a chosen test protocol. The image from the reaction chamber can be analyzed as described here to obtain information about the sample, or the test reaction products. The combination of using a plurality of path lengths available within a single reaction chamber and using three regions of the detection channel spectrum significantly increases the dynamic range of each assay.
[00182] A system for performing sample preparation and analysis can include instrumentation, disposable components, and reagents. The system can accept samples and automatically performs a plurality of tests without user intervention. When desired, the instrumentation may include a graphical user interface, a cartridge insertion mechanism, which may be disposable, a motorized stage, which may have mobility in three dimensions, one or more single-head liquid handling devices, one or more more multi-head liquid handling devices, one or more devices for carrying out sample preparation, optical sensors, which may include a PMT and / or an imaging device, temperature controllers, and communication devices. The disposable component may include a disposable cartridge that contains sample tips, tip seals, and reagents. In some embodiments, the disposable cartridge may also contain neutralizing assemblies configured to absorb and neutralize the liquid test products.
[00183] Instrumentation, disposable components, and reagents can be housed within an environment that can be closed, such as a box or cabinet. In some embodiments, the process has a cross-sectional area of less than about 4 m2, 2 m2, 1 m2 of 0.5 m2, 0.1 m2, 0.05 m2, or less. The invention provides a distributed test system, such as a point-of-treatment device, which may include one or more of the following: Efficient (centrifugal) blood separation and recovery of the separated plasma The dilution of the plasma sample from one or more levels (for example, 1: 10 ,: 100, 1: 1000) so that each assay can be performed at an optimal dilution Optimized example distribution for several different assays that may involve several different methodologies Optimal testing protocols
[00184] The use of open-ended cuvettes of circular section for sample analysis, mixing with reagents, incubation and presentation of optical systems
[00185] Analysis of the tests using image technology (scanning and / or photography, and / or microscopy)
[00186] In one embodiment, the device of the invention is self-contained and comprises all reagents, solid-liquid and reactants, necessary to perform a plurality of tests in parallel. When desired, the device is configured to perform at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 500, 1000 or more tests. One or more control tests can also be incorporated into the device to be carried out in parallel if desired.
[00187] Calibrators can also be provided for the calibration of the test system. Some examples of dry controls and calibrators useful for calibrating the assay system may include aqueous solutions of analytes, serum, plasma or samples with known levels of analytes, such known amounts of calibrators and controls may also be dried by lyophilization, drying under vacuum , and fabrication of other processes (and dissolved during the test).
[00188] When incorporating these components within a point-of-treatment system, the patient or the user can have a plurality of analytes, for example, more than about 10, 20, 30, 50, 75, 100, 150, or 200 analytes quantified in less than about, 5, 1, 2, 3, 4, 5, 10, 20, 30, 60, 120, 180, 240, 480 or 600 minutes.
[00189] The devices and systems of subjects can be used for conducting immune quantitative assays, which can be completed in a short period of time. Another type of assay can be performed with a device of the invention, including, but not limited to, nucleic acid sequence measurements and metabolite measurements, such as cholesterol or electrolytes, such as magnesium and chloride ions. In some embodiments, the test is completed in no more than an hour, preferably less than 120, 60, 30, 15, 10, 5, 4, 3, 2 or 1 minute. In other embodiments, the test is performed in less than 5 minutes. The duration of the detection test can be adjusted according to the type of test that is to be performed with a device of the invention. For example, if necessary for the highest sensitivity of an assay, it can be incubated for more than an hour or even more than a day. In some examples, trials that require a long period may be more practical in other applications, such as household POC, than in a POC clinical setting.
[00190] In other embodiments of the invention, the reagent units of a subject device are configured to be a set of mixing and phosphorous components.
[00191] A reagent unit typically stores liquid or solid reagents needed to perform an assay that detects a particular analyte. The test units can sometimes (or, optionally not always) comprise at least one capture surface capable of reacting with an analyte from the body fluid sample. The dosing unit can be a tubular tip with a capture surface inside the tip.
[00192] Examples of hints of the invention are described herein. Each individual dosing unit and reagents can be configured to function independently in the assay. For the assembly of a device, the units can be assembled in a just-in-time manner for use in an integrated device, which can take the form of a cartridge.
[00193] A housing for a device of the invention can be made of a polymeric material, a metallic material or a composite material, such as, for example, aluminum, polystyrene or other moldable or machinable plastic, and may have set to place test sites units and reagent units. The housing can include a metal or any other material. The housing may include, partially or totally, the test units and / or reagent units. The housing can support the weight of the test units and / or reagent units. In one embodiment, the housing has means for drying tips or test units for removing excess liquid. The blotting media may be a porous membrane, such as cellulose acetate, or a piece of absorbent material, such as filter paper.
[00194] In some embodiments, at least one of the components of the device can be manufactured with polymeric materials. Non-limiting examples of polymeric materials include polystyrene, polycarbonate, polypropylene, polydimethylsiloxanes (DMS), polyurethane, polyvinyl chloride (PVC), polysulfone, polymethylmethacrylate (PMMA), acrylonitrile-butadiene-styrene (ABS), and glass.
[00195] The device or subcomponents of the device can be manufactured by various methods, including, without limitation, stamping, injection molding, embossing, molding, blow molding, machining, welding, ultrasonic welding, and bonding thermal. In one embodiment, a device manufactured by injection molding, thermal bonding, and ultrasonic welding. The subcomponents of the device can be fixed to each other by a thermal connection, ultrasonic welding, friction fitting (pressure fitting), adhesives or, in the case of certain substrates, such as glass, or semi-rigid and non-rigid polymeric substrates, one natural adhesion between the two components.
[00196] A system like the one described can be performed from a variety of tests, regardless of the analyte to be detected from a sample of body fluid. A protocol depending on the identity of the device can be transferred from an external device, where it can be stored in a reader, to allow the assembly of the reader set to carry out the specific protocol in the device. In some embodiments, the device has an identifier (lD), which is read or detected by an identifier detector described herein. The identifier can allow bidirectional communication between the device and a sensor or receiving system. The identifier detector can communicate with a communication module through a controller that transmits the identifier to an external device. When desired, the external device sends a protocol stored on the external device to the communication module based on the identifier. The protocol to be performed on the system may comprise instructions for the system controller to execute the protocol, including, but not limited to, a particular test to be performed and a detection method to be performed. Once the test is performed by the system, a signal indicating a substance to be analyzed in the body fluid sample is generated and detected by a system detection set. The detected signal can then be communicated to the communications set, where it can be transmitted to the external processing device, including, without limitation, the calculation of the analyte concentration in the sample.
[00197] The systems, devices and methods for performing sample analysis using point-of-care devices and tips that can function as reaction chambers are described in US Patent Publication No. 2009/0088336 and US Provisional Application n ° 60/997, 460, each of which is incorporated herein by reference in its entirety for all purposes. Handling of sampling and reaction chambers
[00198] The samples, reagents, and assays collected here described can be handled and contained by a variety of types of reaction chambers. An example of a reaction chamber and delivery device can be a well, a tube or an open end tip, which can also be a cuvette. As used herein, a tip can also be referred to as a sampling tip, a cuvette tip, a reaction chamber, a cuvette, a capillary sample delivery device or a sample transfer device. Samples can be collected from a source at a tip or a tube. The tips can be sealed. Such seals can be permanent or reversible. The diluted samples can be combined with one or more reagents and mixed (as described in the previous orders) into "test elements", such as tips (open tubs) or open or closed wells. Once the assay is prepared for reading, the dosing element can be presented to the optical system for image analysis or other types of reading. Alternatively, the reaction mixtures in the assay can be transferred from one type of element to another. For example, assays incubated in tips can be transferred to an assay medium or absorbent or absorbent in wells can be aspirated into tips. Many tests can be run in parallel. Assay reading can be serial or simultaneous, depending on the assay protocol and / or the incubation time. For tests involving the measurement of a rate of change, the dosing element may be presented to the optical system more than once on different occasions. Fluid and material transfer devices
[00199] A fluid transfer device can be part of a system. The liquid transfer device can comprise a plurality of heads. Any number of heads as needed to detect a plurality of analytes in a sample is designed for a fluid transfer device of the invention. In one example, a fluid transfer device has about eight heads mounted in line, separated by a distance. In one embodiment, the heads have a conical nozzle that engages by pressure fitting with a variety of tips, such as the unit test or sample collection units, as described herein. The tips may have a feature that allows them to be removed automatically by the instrument and placed in a box inside a device as described, after use. In one embodiment, the test leads are clear and transparent, and can be similar to a crucible in which a test is performed that can be detected by an optical detector such as a photomultiplier tube or sensor.
[00200] In an example, a programmable processor (for example, a central processing unit CPU) of a system can include, or be configured to accept (such as, for example, from a memory location) instructions or commands and can operate a fluid transfer device according to the instructions for transferring liquid samples by either withdrawing (for aspirating liquid a) or extending (to expel liquid) from a piston to an enclosed air space. The processor can be configured to facilitate aspiration and / or distribution. Both the volume of air displaced and the speed of movement can be precisely controlled, for example, by the programmable processor.
[00201] Mixing samples (or reagents) with diluents (or other reagents) can be achieved by aspirating components to be mixed into a common suction tube and then several times a significant fraction of the volume of liquid gathered up and down to one end. Dissolving dry reagents in a tube can be done in a similar way e. The incubation of samples and liquid reagents, with a capture surface to which a capture reagent (for example, an antibody) is attached can be accomplished by pulling the appropriate liquid into the tip and holding it for a predetermined time. The removal of samples and reagents can be accomplished by expelling the liquid into a reservoir or an absorbent pad within a device as described. Another reagent can then be placed on the tip according to the protocol or programmable processor instructions.
[00202] A system can include a support or hitch to move the units of analysis or tips. A hitch can include an empty set or a set designed to comfortably fit a head of a test tip unit. For example, a means for moving the tips can be moved in a similar way to the head of the fluid transfer device. The device can also be moved on a stage according to the position of a holder or hitch.
[00203] In one embodiment, an instrument for moving the tips and the same as an instrument for moving a sample volume, such as a device for transferring fluids, as described herein. For example, a sample collection tip can be fit in a pipette head according to the head on the collection tip. The collection tip can then be used to distribute the liquid throughout the device and the system. After the liquid has been dispensed, the collection tip can be disposed of, and the pipette head can be fitted in a dosing unit according to the projection on the dosing unit.
[00204] The tip of the tester can then be moved from the reagent unit to the reagent unit, and reagents can be distributed to the dosing unit according to the action or aspiration of the pipette type provided by the head of the pipette. The pipette head can also mix within a collection tip, the analysis unit, or reagent unit by type of aspiration or action syringe.
[00205] In another embodiment, tips containing liquids, including assay reaction mixtures can be disconnected from the pipetting device and "stationed" at specific locations within the instrument, or within a disposable unit. If necessary, tips can be covered using a sealant (as used in the centrifuge) to prevent draining liquids out. In some embodiments, the seal may be a vinyl seal. Sample tip specimens
[00206] A variety of container shapes can be used as sample tips, reaction chambers, and vats. For example, a tub can be circular, cylindrical, square, rectangular, cubic, conical, pyramidal or any other form capable of containing a sample of liquid. Rectangular cuvettes, where a beam of light hits the cuvette surfaces perpendicularly as shown in plan and in section in Figure 63 can be used. In these rectangular cuvettes, the liquid sample which is also illuminated is rectangular and is defined by a cuvette. Cuvettes with circular cross sections can also be used. For example, some types of micro-titration plates in which the sample volume is illuminated in the part defined by the meniscus sample, as shown below in plan and in section in Figure 64.
[00207] Variable path length cuvettes can be used to optimize and prolong the assay response and minimize the sample volume needed to measure the assay. Cuvettes can be larger, in relation to their cross-section in at least one region. In some cases, the length of the trajectory of a cuvette can be selected based on the geometry of the cuvette and / or material. Different cuvettes can be selected for different tests.
[00208] In the present invention, a preferred version of the test cuvette has a circular cross-section in the direction of the light beam, as shown in Figure 65. The use of a cuvette with a circular cross-section has several advantages, including, but not limited to, not limited to the following: The optical path length can be precisely defined. Dimensional accuracy of injection molded parts were found to be better than 1 -2% CV.
[00209] In conventional polystyrene plates the unrestricted liquid meniscus may introduce inaccuracy in the path length.
[00210] The open character and circular section of the tips gives excellent fluid handling characteristics, making the liquid aspiration very precise.
[00211] The optical image of the tips provides the ability to identify the location of the tip and the limits of the liquid column and much to accurately locate the center of the tip, where the signal is maximum.
[00212] More than one liquid sample can be incubated and analyzed from the same tip. This is because in the narrowest part of the tip, very little material transfer occurs (in the axial direction) between adjacent "slugs" of liquid.
[00213] An exemplary tip may have the following general characteristics: Tip length: 0.5 - 4 cm OD: 0.2 - 1.0 cm ID tip: 0.1 - 0.5 cm Capacity for tip liquids: 5 - 50 ul Dimensional accuracy of tip: generally better than 2% or +/- 0.001 cm.
[00214] Configuration Tip: The tip will generally have a characteristic that engages with a pipette (cylindrical), in order to form a fluid-tight seal. There is a generally cylindrical or conical region, which is used for imaging.
[00215] In general, the optical part of the tip will have at least two different sections, with different path lengths. The lower end of the tip will typically be narrow, to assist in the retention of vertical liquid columns by gravity
[00216] Tip material: clean or evenly speculate plastic (polystyrene, polypropylene, etc.) (light transmission in the visible> 80%)
[00217] For imaging purposes, the tip can usually be transparent or translucent, but the tips do not have to be clear to work well as test tubes when three-color analysis is used. Tip tubs that appear "cloudy" can work in a similar way when cleaning tips. The cloudy tips are made in injection molds with unpolished surfaces
[00218] or textured or by adding some light scattering material to the plastic used to manufacture the tips. The intensity of light scattering from such cloudy tips can be chosen to not be as large as obscuring the color liquid to be measured. In general, the effects of light scattering for transmitted light can be selected to be less than 10, (20 and 30%) in relation to the impact of the colored material. The light scattering effect can be selected in such a way that the light scattering of the tips is cloudy and uniform.
[00219] The ends and reaction chambers described here may consist of a cylindrical (or conical) shaft, about 2 cm long and with an inside diameter of about 1-5 mm that corresponds to a capacity of about 10 - 50 ul.
[00220] In one example, at the top end of the cylinder and a truncated cylindrical "boss" fluidly attached to the cylinder and adapted so as to be able to engage with the conical feature of a pipettor. The lower end of the tip can be reduced to provide functionality that allows the tip to hold its liquid contents when oriented vertically and not attached to the pipette. The tip can be a tip. The external shape of the lower end of the tip is typically also somewhat pointed with the diameter, having been reduced from the main part of the cylindrical shaft to the end in order to be able to be fluidly sealed with a flexible cover (vinyl) in which the end of the tip and adapted by pressure. Tips are usually made of molded plastic (polystyrene, polypropylene and the like). The tips can be transparent or translucent in such a way that information about the sample can be acquired by image.
[00221] Figure 4, Figure 5, and Figure 6 shows an example of a tip. The tip is configured with (1) a superior feature that can engage to form an airtight seal with a pipette head, (2) a basically cylindrical shaft (actually tapered with a very small angle design) and a narrow, pointed tip bottom. This tip can form a liquid-tight seal with a cap. The pointed shape helps to achieve good compliance with the cap with moderate force. The material used is injection molded polystyrene. The dimensions are: 32 mm long, about 7.6 mm larger outside diameter, the useful capacity of about 20 ul. The tip dimensions can be scaled to a larger volume. For example, for a 50 μl sample, the IDs can be increased by about 1.6 times.
[00222] Sealing can be achieved using a cover made of vinyl or other material that can be easily adjusted by pressure to the narrowest part of the sample containing means using the force generated by the movement of the instrument phase in the z direction. An air bubble can get stuck inside the tip when the tip is limited. A centrifugation step can be used to direct the bubble to the top of the blood column in order to eliminate the effects of the bubble. The dimensions of the tip and / or the dimensions of the tip holder in a centrifuge can be compensated in such a way that a tip can be secured by centrifugation. Sample Preparation
[00223] The invention provides systems, methods and apparatus for the treatment and analysis of samples that can be collected from a variety of sources. For example, the sample can be collected from patients, animals or the environment. The sample can be a body fluid. Any body fluids suspected to contain an analyte of interest can be used in conjunction with the system or devices of the invention. Commonly used body fluids include, but are not limited to, blood, serum, saliva, urine, gastric and digestive fluid, tears, feces, semen, vaginal secretions, interstitial fluid derived from tumor tissue, and cerebrospinal fluid.
[00224] In some embodiments, the body fluid and a blood sample from a human patient. The blood source can be collected from a finger prick and has a volume of less than about 0.5, 1, 5, 10, 20, 50, 100, 200, 300, 400, 500, or 1000 Ul.
[00225] A body fluid can be withdrawn from a patient and delivered to a device in a variety of ways, including, but not limited to, puncture, injection, or pipetting.
[00226] As used herein, the terms "subject" and "patient" are used interchangeably herein, and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, apes, humans, farm animals, animals and pets.
[00227] In one embodiment, a lancet pierces the skin and takes a sample, using, for example, gravity, capillarity or vacuum force aspiration. The lancet can be an integral part of the device, or part of a system or an independent component. Whenever necessary, the lancet can be activated by a variety of known mechanical, electrical, electromechanical activation mechanisms, or any other or a combination of these methods. In another embodiment in which no active mechanism is needed, a patient can simply provide a body fluid for the device, as for example, they could occur with a saliva sample. The collected fluid can be placed in the sample collection unit inside the device. In yet another embodiment, the device comprises at least one micro needle that pierces the skin.
[00228] The volume of body fluid to be used with a device can be less than about 500 microliters, typically between about 1 to 100 microliters. When desired, a sample of 1 to 50 microliters, 1 to 40 microliters, 1 to 30 microliters, 1 to 10 microliters, or even 1 to 3 microliters can be used for the detection of a substance using the device. In one embodiment, the volume of body fluid used to detect an analyte using the subject devices or systems and a drop of fluid. For example, a drop of blood from a pricked finger can provide the sample of body fluid to be analyzed, using a device, system or method described here.
[00229] A sample of body fluid can be collected from a subject directly to a tip of the one described here, or can be later transferred to a tip. Sample Dilution
[00230] In some cases, the direct effects processor configuration of fluid transfer of a degree of dilution of the body fluid sample in the range of test units to bring signals indicative of the plurality of analytes to be detected within a range detectable, such that said plurality of analytes are detectable with said system. In one example, the body fluid sample comprises at least two analytes that are present in concentrations that differ by at least 1, 2, 5, 10, 15, 50, 100, 500, 1000, 10000, 105, 106, 107, 108, 10, 9, 10 or 10 times. In one example, the sample of body fluid and a single drop of blood. In one embodiment, the concentrations of at least two analytes in a sample differ by up to 10 orders of magnitude (for example, a first analyte is present at 0.1 pg / ml and a second analyte is present at 500 ug / ml). In another example, some protein analytes are found in concentrations greater than 100 mg / ml, which can extend the range of interest to about twelve orders of magnitude. In the case of the measurement of exponential nucleic acid analytes such as DNA and NA using amplification methods such as the polymerase reaction, the number of copies of the analyte can be increased by one thousand times before the measurement.
[00231] If desired, a degree of dilution of the body fluid sample can bring the indicative signals of at least two analytes within the detectable range.
[00232] As described, the systems and devices here can activate many characteristics of flexibility of the laboratory environment in a POC environment. For example, samples can be collected and handled automatically on a table top or smaller size of the device or system. A common problem in POC devices is reaching different dilution ranges when carrying out a series of tests, where the tests may have significantly different sensitivity or specificity. For example, there may be two analytes in a sample, one analyte, but it has a high concentration in the sample and the other analyte
[00233] has a very low concentration. As predicted, the systems and devices described herein can dilute the sample to significantly different levels in order to detect both analytes. Alternatively, a sample can be divided into two or more samples, which can allow individual analytes to be detected at various levels of dilution.
[00234] For example, if the analyte is in a high concentration, a sample can be diluted in series for the appropriate detection interval and supplied to a capture surface for detection. In the same system or device, having an analyte sample in a low concentration may not need to be diluted. In this way, the dosing range of the POC devices and systems provided herein can be expanded from many of the current POC devices.
[00235] In POC test systems using disposable cartridges that contain the diluent, a practical limit to the extent of the dilution is often established. For example, if a small blood sample is obtained by pricking the finger (for example, about 20 microliters) and to be diluted and the maximum volume of diluent that can be placed in a tube and
[00236] 250 microliters, the practical limit of dilution of the entire sample and about 10 times. In an example here, a system can aspirate a smaller volume of the sample (for example, about 2 microliters) causing the maximum dilution factor to be about 100 times. For many assays, such dilution factors are acceptable, but for an assay similar to that of CP (as described in the examples), there is a need to dilute the sample much more. Separation-based ELISA assays may have an intrinsic limitation on the ability of the capture surface to bind to the analyte (for example, about a few hundred ng / ml for a typical protein analyte). Some analytes are present in the blood at hundreds of micrograms / ml. Even when diluted 100 times, the analyte concentration may be outside the calibration range. In an exemplary embodiment of a fluid transfer system, device and device, multiple dilutions can be achieved by performing multiple fluid transfers from the diluent to an individual test unit or sample collection unit. For example, if the concentration of an analyte is very high in a sample, as described above, the sample can be diluted several times, until the concentration of the analyte is within an acceptable detection range. The systems and methods of this invention can provide accurate measurements or estimates of dilutions in order to calculate the original concentration of the analyte. Sample separation
[00237] In some embodiments of the invention, a sample can be prepared, for analysis by an initial separation step. For example, if the assay consists of analyzing DNA, a DNA separation step can be used to eliminate or reduce contaminants or materials of unwanted origin. The separation step can use chromatography, centrifugation, liquid-liquid extraction, solid-liquid extraction, binding affinity, or any other mechanisms known to a person skilled in the art.
[00238] In some embodiments, a blood sample to be analyzed and processed first, separating the plasma component from the blood sample. This step can be performed using a variety of techniques, such as filtration, centrifugation, and binding affinity. Centrifugation can be an efficient method for separating components from the blood sample, and can be employed in the present invention. The separation of the plasma.
[00239] Blood can be introduced at a nearby end or sealed sealable in a variety of ways, for example, samples can be supplied in a tube and a sealable tip can receive the sample from the tube through a capillary action or by pneumatic force. A preferred means of introduction and the use of capillary action.
[00240] Alternatively, the container used to store the sample for centrifugal separation can be configured with just one opening, as in a conventional centrifuge.
[00241] The tip, once filled with blood, can be automatically transferred to a location on a disposable cartridge, where there is a sealing element. The sealing element can be a small "bowl", made of a deformable (flexible) material (vinyl, silicone or similar) shaped to fit the lower end of the tip and seal it. The tip is pressed into the seal by the instrument, thus forming a liquid-tight joint. The sealed end is then moved to a small centrifuge (typically located on and forming part of the instrument) and fitted to a centrifuge rotor positioning element such that the lower (sealed) end of the tip ends up to a rigid shelf that supports the tip during the centrifugation step.
[00242] The centrifugal rotor can be circular with about 10 cm in diameter. The mass of the tip containing blood is either (1) small in relation to that of the rotor, or (2), where desired, in relation to the weight of a counter located on the opposite side of the rotor in such a way that any vibration during the step of centrifugation and minimized. An example of the orientation of the centrifuge and vertical rotor (horizontal axis of rotation). The rotor is mounted on the drive shaft with one and driven by an electric motor.
[00243] Centrifugation can be achieved by rotating the rotor at about 15,000 rpm for 5 minutes. During this process, the particular elements in the blood (red blood cells and white blood cells) pellet to the sealed end of the tip, and form a compact column with separate free cell plasma at the distal end part of the seal.
[00244] The tip containing the separate sample can then be placed vertically in an accessible location for a fluid handling device composed of a narrow tip ("sample acquisition tip") mounted on a pipetting device, which in turn are mounted on an xyz phase.
[00245] The plasma can now be efficiently recovered from the centrifuged sample. This is accomplished by moving the sample acquisition tip vertically along the axis of the centrifuge tip so that it comes into contact with the fluid with the plasma and can draw upwards using the plasma, for example, pneumatic means.
[00246] Optionally, this operation can be controlled by means of a camera or other imaging device that can be used both to measure the hematocrit of the sample and to provide information on the location of the plasma / red cell boundary for the blood pressure controller. phase / pipette. With the aid of the separate images of the blood, a narrow pipette tip mounted on a pipette and slowly moved vertically downwards, in such a way that the tip is directed axially downwards, the containment of the sample means even touching the plasma. The tip is then moved further until it is close (within less than about 3, 2, 1, 0.5, or 0.1 mm) to the hematocrit interface. At the same time, the plasma is aspirated to the narrow pipette tip under the control of the computer. The plasma can be aspirated simultaneously, moving the tip of the narrow pipette in a plasma column so that the plasma does not move to the upper part of the sample confinement means. Aspiration can be controlled to prevent air being sucked in during the plasma removal step.
[00247] In general, the tip of the pipette with a very narrow end, such as those used to apply a sample system for electrophoresis, can be used to aspirate plasma from the tip of the centrifuged sample. The narrow tip is typically tapered or tapered and has dimensions 1-3 x 0.1-0.5 cm (length x diameter) and made of any of a variety of materials (polypropylene, polystyrene, etc.). The material can be transparent or translucent, not visible. One end of the tip of the fitting with a pipetting device. The other is very narrow (0.05-0.5 mm outside diameter), so that it can move to the tip of the sample, without touching the inner surface of the sample tip. Even if there is contact between the plasma aspiration tip and the sampling tip, the plasma aspiration is not impaired.
[00248] A schematic of the plasma aspiration process at the stage where the plasma aspiration tip is located above the interface of plasma-packed cells during the aspiration step and shown in Figure 7.
[00249] In this way, it was found that almost all of the plasma can be removed, leaving, for example, as little as 1 ul at the tip of the centrifuged sample. This corresponds to about 11 ul of plasma (90% recovery) of 20 ul of blood with a hematocrit of 40%. In addition, the quality of the plasma sample (with respect to hemolysis, lipemia and jaundice) can be determined from an image of the centrifuged sample.
[00250] The aspirated plasma can be transferred to other sites for dilution and mixing with the assay reagents so that assays for the analyte industry but not limited to electrolytes, metabolites, biomarkers of proteins, drugs and nucleic acids can be fulfilled. The separation of white blood cells
[00251] Another use of the invention is to isolate and concentrate white blood cells. In one aspect of the invention, the blood sample is first subjected to a process that smooths red blood cells (and optionally fixes white cells) by adding a reagent (for example, BD Pharmlyse M 555.899 or BD FACS M Lysis solution 349202) _to the blood and mixing. After a brief incubation, the sample is subjected to centrifugation lysate, as described above such that the white cells are concentrated at the sealed end of the blood tip. The lysed red blood cell solution can then be removed by aspiration.
[00252] Recovery of the white cells and achieved by either (1) adding a small amount of a buffer solution and aspiration was repeated and down to resuspend the cells, followed by displacement to a receptacle or ( 2) removal of the seal and downward displacement of erythrocytes in the receptacle using air pressure.
[00253] An alternative scheme allows the recovery of white cells without lysis of red cells. After centrifuging the blood (as is well known), the white blood cells form a layer on top of the red blood cells known as Buffy Coat. After removing most of the plasma (as above), white blood cells can be effectively recovered by (1), optionally, adding a small volume (for example, about 5 μl) of isotonic buffer solution, or ( 2) using a residual plasma and resuspending the white cells by repeated aspiration and / or mechanical agitation using the sample acquisition tip. Once suspended, the resulting white blood cell mixture, along with a small proportion of red cells also resuspended, can be acquired by aspiration for analysis of white blood cells. In this way most white blood cells (typically all) can be recovered with only a small amount of red blood cells (usually less than 5% of the initial) (contaminant). Centrifuges
[00254] Figure 1, Figure 2 and Figure 3 shows perspective of a centrifuge (Figure 1 - side view, Figure 2 - Front face view, Figure 3 - rear view) that can be integrated into the system. The centrifuge can contain an electric motor capable of rotating the rotor at 15,000 rpm. A type of centrifuge rotor and similar to a fan blade and mounted on the motor shaft, in a vertical plane. Attached to the rotor and an element containing the sample that holds the middle (top) and provides a ledge or shelf in which the end of the distal end of the motor shaft supports and which provides support during centrifugation, so that the sample does not can escape. The tip can also be supported at its proximal end by a mechanical stop on the rotor. This can be provided so that the force generated during centrifugation does not cause the tip to cut through the soft vinyl cover. The tip can be inserted and removed using standard "pick and place" mechanisms, but preferably by a pipette. The rotor is made of a single piece of acrylic (or other material) in order to minimize vibration and noise during the operation of the centrifuge. The rotor is (optionally) shaped so that, when it is oriented at specific angles, to the other vertical movable components in which the instrument can move after centrifugation. The sample that holds the means is by centrifugation in relation to the mass counter on the opposite side of the rotor in such a way that the center of rotation of the inertia is relative axial to the motor. The centrifuge motor can provide positioning data to a computer, which can then control the rotor's resting position (typically vertical before and after centrifugation).
[00255] As can be seen from the two graphs in Figure 8 and Figure 9, to minimize the centrifugation time (without generating a lot of mechanical stress during the centrifugation) according to the published standards (DlN 58933-1; for the USA the standard ClSl H07- A3 "Process for the determination of hematocrit by the microhematocrit method"; Approved Standard - Third Edition) dimensions suitable for the rotor are in the range of about 5 - 10 cm, which rotate at approximately 0 - 20,000 rpm, giving the red cells time to tidy for about 5 min.
[00256] An example of an equation for calculating the spin force is shown below:

[00257] In some embodiments, a centrifuge can be a horizontally oriented centrifuge with an oscillating container design. In some preferred embodiments, the axis of rotation of the centrifuge is vertical. In alternative embodiments, the axis of rotation may be horizontal or at any angle. The centrifuge may be able to simultaneously rotate two or more vessels and can be designed to be fully integrated into an automated system using computer-controlled pipettes.
[00258] In some embodiments, the vessels may be close to the bottom. The tilting design can allow the centrifuge containers to be passively oriented in a vertical position when stopped, and to rotate out of a fixed angle during centrifugation. In some embodiments, shuttle containers can allow the centrifuge containers to rotate outward to a horizontal orientation. Alternatively, they can rotate to any angle between a vertical and horizontal position (for example, about 15, 30, 45, 60 or 75 degrees from the vertical. Swinging the centrifuge with container card can meet the positional accuracy and repeatability requirements of a robotic system a number of positioning systems are employed.
[00259] A computer-based control system can use position information from an optical encoder in order to rotate the rotor at controlled slow speeds. Because an appropriate engine could be designed for high speed performance, precise static positions do not need to be performed using position feedback alone. In some embodiments, a chamber, in combination with a solenoid cornered lever, can be employed to achieve very accurate and stable stopping at a certain number of positions. Using a separate control system and return from Hall effect sensors, mounted on the motor, the rotor speed can be controlled very precisely at high speed.
[00260] Since a number of sensitive instruments must operate simultaneously within the test instrument system, the design of the centrifuge, preferably minimizes or reduces vibration. The rotor can be aerodynamically with a smooth exterior - completely enclosing the containers when they are in their horizontal position. In addition, vibration dampening can be employed at various locations in the design of the process. Rotor
[00261] A centrifugal rotor can be a component of the system, which can hold and rotate the centrifuge container (s). The axis of rotation can be vertical and, therefore, the rotor itself can be positioned horizontally. However, in alternative embodiments, different rotor axes of rotation and positions can be employed. There are two components known as containers positioned symmetrically on both sides of the rotor, which hold the centrifuge containers. Alternative configurations are possible in which containers are oriented with radial symmetry, for example, three segments oriented at 120 degrees. Any number of containers can be provided, including, but not limited to 1, 2, 3, 4, 5, 6, 7, 8 or more containers. The containers can be spaced from each other. For example, if n containers are provided where n is an integer, then the containers can be spaced about 360 / degrees apart from each other. In other embodiments, the containers do not need to be spaced evenly around one another or with radial symmetry.
[00262] When the rotor is stopped, these containers, influenced by the force of gravity, can fall passively, as position the vessels vertically and make them accessible to the pipette. Figure 111 shows an example of a rotor at rest with vertical containers. In some embodiments, the containers may passively fall to a predetermined angle, which may or may not be vertical. When the rotor rotates, the containers are forced to an almost horizontal position, or at an angle predetermined by centrifugal forces. Fig. 112 shows an example of a rotor at a container speed of a small angle to the horizontal. There can be no physical rigid stops for both vertical and horizontal positions that act to enforce your position accuracy and repeatability.
[00263] The rotor can be aerodynamically with a disk shape and as few physical characteristics as possible in order to minimize the vibration caused by the turbulence of the air. To achieve this, the outer geometry of the container can correspond exactly to that of the rotor such that when the rotor is rotating and the container can be forced horizontally from the container and rotor they can be perfectly aligned.
[00264] To facilitate the extraction of the plasma, the rotor can be tilted down towards the ground in relation to the horizon. Because the blade angle can be combined with that of the rotor, it can impose a fixed angle of rotation for the container. The sediment resulting from such a configuration could be tilted in relation to the container, when placed in the vertical position. A narrow extraction tip can be used to aspirate plasma from the top of the centrifuge container. By placing the extraction tip close to the bottom of the ramp created by the sediment angle, the final volume of plasma can be extracted more efficiently without disturbing the sensitive buff coat.
[00265] A variety of tube models can be accommodated in the device's containers. In some embodiments, the various models of tubes can be closed finished. Some are in the form of conventional centrifuge tubes with a tapered bottom. Other tube designs can be cylindrical. Tubes with a low relationship between height and cross-sectional area can be favored for cell transformation. Tubes with a large ratio (> 10: 1) may be suitable for accurate measurement of hematocrit and other imaging needs. However, any time the cross-sectional area ratio can be used. The containers can be made of any of several plastics (polystyrene, polypropylene), or any other material discussed elsewhere in this document. Containers have capacities ranging from a few microliters to about a milliliter. The tubes can be inserted and removed from the centrifuge, using a "pick and place" mechanism. Control system
[00266] Due to the spinning and positioning requirements of the centrifuge device, a dual control system approach can be used. For the rotor rotation index, specific guidelines, a position-based control system can be implemented. In some embodiments, the control system may employ a PlD (Proportional Integral Derivative) control system. Other feedback control systems known in the art can be employed. Positional feedback for the position controller can be provided by a high resolution optical encoder. To operate the centrifuge from low to high speeds, a speed controller can be implemented, by employing a tuned PlD control system for speed control. Rotation rate feedback for the speed controller can be provided by a set of simple Hall effect sensors placed on the motor shaft. Each sensor can generate a square wave in one cycle per rotation of the motor shaft. Stop mechanism
[00267] In order to consistently and firmly position the rotor in a particular position, a physical interruption mechanism can be employed. In one embodiment, the stop mechanism can use an eccentric, coupled to the rotor, together with a solenoid operated lever. The meat may be in the form of a circular disc with a series of "C" shaped notches machined around the perimeter. To position the centrifuge rotor, its rotation speed can be reduced to a maximum of 30 rpm. In other embodiments, the rotation speed can be reduced to any other amount, including, but not limited to, about 5 rpm, 10 rpm, 15 rpm, 20 rpm, 25 rpm, 35 rpm, at 40 rpm or 50 rpm when the speed is sufficiently slow, the lever can be operated. At the end of the lever and a cam follower that can slide along the perimeter of the chamber, with minimal friction. Once the cam follower reaches the center of a particular notch in the chamber, the force of the actuated solenoid lever can exceed that of the motor and the rotor can be brought to a stop. At that point, the motor can be braked electronically, and in combination with the stop mechanism of a rotating position it can be very precisely and firmly maintained indefinitely. Centrifuge Containers
[00268] Centrifuge containers can be configured to accommodate different types of centrifuge tubes. In preferred embodiments, the various types of tubes may have a collar or flange on their final (open) upper part. This feature can stick or flange rests on the top end of the container and supports the tube during centrifugation. As shown in Figures 113, 114, and 115, conical and cylindrical tubes of different lengths and volumes can be accommodated. Figures 113, 114, and 115 provide examples of containers and other models of container can be employed. For example, Figure 113, shows an example of a container configuration. The container can have side portions that mate with the centrifuge and allow the container to move freely. The container can have a closed bottom and an opening in the top. Fig. 114 shows an example of a centrifuge container coupled with the container. As mentioned earlier, the container can be shaped to accept various configurations of centrifuge containers. The centrifuge vessel may have one or more protruding members that can rest on the vessel. The centrifuge container can be molded with one or more characteristics that can be coupled with the centrifuge container. The feature may be a vessel-shaped feature or one or more protrusions. Fig. 115 shows an example of another centrifuge container that can be coupled with the container. As previously described, the container may have one or more characteristics that the shape may allow different configurations of centrifuge containers to mate with the container.
[00269] Centrifuge tubes and proof extraction means: The centrifuge tube and extraction tip can be supplied individually and can be coupled together for the extraction of material after centrifugation. The tip of the centrifuge and extraction tube can be designed to handle complex processes in an automated system. Fig. 116 shows an example of a centrifuge container. Figure 117 shows an example of an extraction tip. Figure 118 provides an example of how the centrifuge and tip extraction vessel can mate. The dimensions are provided as an example only, and other dimensions of the same or different proportions can be used.
[00270] The system can allow one or more of the following: Rapid transformation of small blood samples (typically 5 - 50 ul) from the blood) Accurate hematocrit measurement Efficient plasma removal
[00271] Efficient resuspension of formed elements (red and white cells The concentration of white blood cells (hereinafter marking with fluorescent antibodies and fixation in addition to analysis of red blood cells) Optical confirmation of red blood cell lysis and recovery of white blood cells . Centrifugal return and extraction tip view
[00272] A personalized tip container and can be used for the operation of the centrifuge, in order to satisfy the various restrictions imposed on the system. The centrifuge container can be a closed bottom tube designed to be rotated in the centrifuge. In some embodiments, the centrifuge container can be the container shown in Figure 116, or it can have one or more features illustrated in Figure 116. It can have a number of unique features that allow for the wide range of necessary features, including the hematocrit measurement, BC lysis, pellet resuspension and efficient plasma extraction. The extraction tip can be designed to be inserted into the centrifuge container for precise fluid extraction and resuspending of the pellet. In some embodiments, the extraction tip may be the tip shown in Figure 117, or it may have one or more features illustrated in Figure 117.
[00273] Exemplary specifications for each tip are discussed here. Centrifuge vessel
[00274] The centrifuge vessel can be designed to handle two separate usage scenarios, each associated with a volume of blood and anticoagulant and totally different.
[00275] A first use scenario may require that 40ul of whole blood with heparin be pelleted, the maximum plasma volume be recovered, and hematocrits measured using computer vision. In the case of 60% hematocrit, or below the volume of plasma needed or preferable, it can be about 40ul * 40% = 16ul.
[00276] In some embodiments, it will not be possible to recover 100% of the plasma because the yellowish coverage must not be disturbed, thus a minimum distance must be maintained between the base of the tip and the top of the pellet. This distance can be determined experimentally, but the volume (V), sacrificed as a function of the required safety distance (d) can be estimated using: V (d) = d * 7il 0.25 mm 2. For example, for a distance of 0.25 mm the required security, the volume can be sacrificed 1.23ul for the case of 60% hematocrit. This volume can be reduced by decreasing the internal radius of the hematocrit part of the centrifuge return.
[00277] However, because in some embodiments, the narrow portion must fully accommodate the outer radius of the extraction tip which may be less than 1.5 mm, the actual dimensions of the centrifuge container may be close to the minimum.
[00278] Along with the extraction of plasma, in some modalities, it may also be necessary that the hematocrit be measured using computer vision. To facilitate this process, the total height for a given volume of hematocrit can be maximized by minimizing the inner diameter of the narrow portion of the container. By maximizing height, the relationship between changes in hematocrit volume and physical change in column height can be optimized, thereby increasing the number of pixels that can be used for measurement. The height of the narrow part of the vessel may also be sufficient to accommodate the worst case scenario of 80% hematocrit, while leaving a small portion of plasma at the top of the column to allow efficient extraction. Thus, 40ul * 80% = 32ul may be the volume capacity required for accurate hematocrit measurement. The volume of the narrow portion of the tip can be projected as about 35.3ul which can allow some plasma volume to remain, even in the worst case.
[00279] A second usage scenario and much more involved, and may require one, more or all of the following: pelleted whole blood extracted plasma pellet resuspended in lysis buffer and stain remaining white blood cells (leukocytes) pelleted supernatant removed white blood cells re-suspended WBC suspension fully extracted.
[00280] In order to fully resuspend a packaged pellet, experiments have shown one can physically disturb the sediment with a tip capable of completely reaching the bottom of the container containing the sediment. A preferred geometry of the bottom of the container used for resuspension appears to be a hemispherical shape, similar to commercial standard PC tubes. In other embodiments, other bottom shapes of the vessel can be used.
[00281] The centrifuge container, together with the extraction tip, can be designed to facilitate the resuspension process, adhering to these geometric requirements at the same time, allowing the extraction tip to physically contact the bottom.
[00282] During the manual resuspension experiments it was noted that physical contact between the bottom of the container and the lower part of the tip can create a seal that prevents the movement of the fluid. A delicate spacing can be used in order to both disturb the sediment completely, while allowing the flow of the fluid. To facilitate this process in a robotic system, a physical feature can be added to the bottom of the centrifuge return. In some embodiments, this feature may comprise four small hemispherical projections placed around the perimeter of the vessel's bottom portion. When the extraction tip is fully inserted into the container and physical contact is allowed, the tip can rest on the protrusions, and the fluid is allowed to flow freely between the protrusions. This can result in a small amount of volume (0.25 ~ Ul) lost in the gaps.
[00283] During the lysis process, in some implementations, the maximum fluid volume is expected to be 60ul, which, along with 25ul displaced by the extraction tip, may require a total volume capacity of 85ul. A project with a current maximum volume of 1000 ul may exceed this requirement. Other aspects of the second usage scenario require similar cutting edge characteristics or have already been discussed.
[00284] The upper geometry of the centrifuge container can be designed to be coupled with a pipette tip. Any pipette nozzle described or known in the art can be used. The outer geometry of the upper portion of the vessel can exactly match that of a reaction tip that both the current injector and the cartridge can be designed around. In some embodiments, a small ridge can circumscribe the inner surface of the upper portion. This summit can be a visual marker of the maximum height of liquids, intended to facilitate the automatic detection of errors using the computer vision system.
[00285] In some embodiments, the distance between the bottom of the nozzle completely engaged to the beginning of the maximum fluid line is 2.5 mm. This distance is less than 1.5mm to 4mm recommended distance adhered by the extraction tip. This decrease in distance can be motivated by the need to minimize the length of the extraction tip while respecting the minimum volume requirements. The justification for this decrease in distance results from the particular use of the vessel. Because, in some implementations, the fluid can be exchanged with the vessel from just the top, the maximum liquid you will ever have while coupled with the mouthpiece and the maximum amount of whole blood expected at any time (40ul). The height of this fluid can be well below the bottom of the nozzle. Another concern is that at other times the volume of liquid in the container may be much greater than this and wet the walls up to the height of the spout. In some embodiments, it will be up to those who use the vessel to ensure that the meniscus of any fluids contained within the container does not exceed the maximum height of the fluid, even if the total volume is less than the specified maximum. In other embodiments, other resources can be provided to maintain the fluid contained within the vessel.
[00286] The dimensions, sizes, volumes, or distances provided in this document are provided by way of a single example. Any other dimension, size, volume or distance can be used which may or may not be proportional to the mentioned values.
[00287] The centrifuge container can be subjected to a number of forces during the process of fluid exchange and rapid insertion and removal of tips. If the container is not limited, it is possible that these forces will be strong enough to lift or otherwise move the container from the centrifuge. In order to prevent movement, the vessel must be protected in some way. To accomplish this, a small ring that circumscribes the bottom outer part of the container has been added. This ring can be easily coupled with a compatible mechanical feature on the container. While the retaining force of the protrusion is greater than the forces experienced during fluid manipulations, but less than the frictional force, when coupled with the nozzle, the problem is resolved. Extraction Tip
[00288] The extraction of the tip can be designed to interface with the centrifuge container, efficient extraction of plasma, and resuspend the pelleted cells. When desired, its total length (for example, 34.5 mm) can correspond exactly to that of the other end of the blood including, but not limited to those described in the USA. Serial No. 12/244, 723 (incorporated herein by reference), but may be long enough to physically touch the bottom of the centrifuge container. The ability to touch the bottom of the vessel may be required in some embodiments, both for the re-suspension process, and for the complete recovery of the white blood cell suspension.
[00289] The required volume of the extraction tip can be determined by the maximum volume that is expected to aspirate from the return of centrifugation at any given time. In some embodiments, this volume may be approximately 60ul, which may be less than the maximum tip capacity which is 85ul. In some embodiments, a tip of greater volume than the required volume may be provided. As with the centrifuge container, an internal feature that circumscribes the inside of the upper portion of the tip can be used to mark the height of the maximum volume. The distance between the maximum volume line and the top of the coupled nozzle can be 4.5 millimeters, which can be considered a safe distance, to avoid contamination of the nozzle. Any distance sufficient to prevent contamination of the nozzle can be used.
[00290] The centrifuge can be used for precipitated sediment of lDl-cholesterol. Image can be used to check whether the supernatant is clear, indicating complete removal of the precipitate.
[00291] In one example, the plasma can be diluted (for example, 1: 10) to a mixture of dextran sulfate (25mg / dl) and magnesium sulfate (100mM), and can then be incubated for 1 minute, to precipitate LDL-cholesterol. The reaction product can be aspirated into the centrifuge tube, then capped and centrifuged at 3000 rpm for three minutes. Figures 119, 120 and 121 are images that were taken from the initial reaction mixture before centrifugation (showing the white precipitate), after centrifugation (showing a clear supernatant), and the LDL-cholesterol pellet (after removing the cap), respectively.
[00292] Other examples of centrifuges that can be employed in the present invention are described in U.S. Patent Nos. 5,693,233, 5,578,269, 6,599,476 and US Patent Publication Nos. 2004/0230400, 2009/0305392, and 2010/0047790, which are incorporated by reference in their entirety for all purposes. Examples of protocols
[00293] Many protocol variations can be used for processing and centrifugation. For example, a typical protocol for using the centrifuge to process and concentrate white cytometry cells may include one or more of the following steps. The steps can be provided in different orders or other steps can be replaced by any of the steps below: Receive 10 µl of anti-clotted blood with EDTA (pipette injects blood into the bottom of the centrifuge container) Sediment the red and white cells by centrifugation (<5 min x 10,000 g). Measure hematocrit by image. Slowly remove plasma by aspiration into the pipette (4 ul corresponding to the worst case scenario [60% hematocrit]), without disturbing the cell pellet.
[00294] Resuspend the pellet after adding 20 μl of an appropriate cocktail of up to five fluorescence-labeled antibodies 1 dissolved in BSA buffered saline solution (1 mg / ml) (total reaction volume of about 26 μl of 2).
Incubate for 15 minutes at 37 ° C.
[00296] 7. Prepare the lysis reagent / fixative by mixing red cell lysis solution (ammonium chloride / potassium bicarbonate) with the white cell fixing reagent (formaldehyde).
[00297] Add 30 ul of lysis / fixative reagent (total reaction volume of about ul
[00298] incubate 15 minutes at 37 ° C.
[00299] Sediment the leukocytes by centrifugation (5 min, 10,000 g).
[00300] Remove the supernatant hemolysate (about 57 Ul).
[00301] Resuspend the white cells, adding 8 ul buffer (saline isotonic)
[00302] Measure the volume accurately.
[00303] Deliver sample (c 10 ul) for cytometry.
[00304] The steps may include receiving a sample. The sample can be a body fluid, such as blood, or any other sample described elsewhere. The sample can be a small volume, just like any of the volume measurements described elsewhere in this document. In some cases, the sample may have an anticoagulant.
[00305] Concentration will be adjusted to properly handle the different volume ratio compared to the standard laboratory method (specifically about 5 x lower)
[00306] If necessary, this volume can be larger to have the ideal color, but not more than 50 ul.
[00307] A separation step can occur. For example, density-based separation may occur. This separation can occur through centrifugation, magnetic separation, lysis, or any other separation technique known in the art. In some embodiments, the sample can be blood, and the red and white blood cells can be separated.
[00308] The measurement can be done. In some cases, the measurement can be made by means of an image, or by any other detection mechanism described elsewhere in this document. For example, the hematocrit of a separate blood sample can be imaged. Filming can take place using a digital camera or any other image capture device described here.
[00309] One or more components of a sample can be removed. For example, if the sample is separated into solid and liquid components, the liquid component can be moved. Plasma from a blood sample can be removed. In some cases, the liquid component, such as plasma, can be removed using a pipette. The liquid component can be removed without disturbing the solid component. The image can assist in removing the liquid component, or any other selected component from the sample. For example, the image can be used to determine where the plasma is located and can assist in placing the pipette to remove the plasma.
[00310] In some embodiments, a reagent or other materials can be added to the sample. For example, the solid portion of the sample can be resuspended. A material can be added with a label. One or more incubation steps may occur. In some cases, lysis and / or a fixing reagent may be added. Additional separation and / or resuspension steps may occur. As necessary, dilution and / or concentration of steps may occur.
[00311] The sample volume can be measured. In some cases, the sample volume can be measured accurately and / or precisely. The sample volume can be measured in a system with a low coefficient of variation, such as the coefficient of variation of the values described elsewhere in this document. In some cases, the sample volume can be measured using images. An image of the sample can be captured and the volume of the sample can be calculated from the image.
[00312] The sample can be delivered to a desired process. For example, the sample can be delivered by cytometry.
[00313] In another example, a typical protocol that may or may not make use of centrifugation for nucleic acid purification may include one or more of the following steps. The system can allow the DNA / RNA extraction to deliver template nucleic acid for exponential amplification reactions for detection. The process can be designed to extract nucleic acids from a variety of samples, including, but not limited to, blood, serum, viral transfer media, human and animal tissue samples, food samples, and cultures bacterial. The process can be fully automated and can extract DNA / RNA in a consistent and quantitative way. The steps can be provided in different orders or other steps can be replaced by any of the steps below: Lysis sample. The cells in the sample can be lysed using a chaotropic de-salt buffer. The chaotropic salt buffer can include one or more of the following: ground tropic salt, such as, but not limited to, guanidine hydrochloride 3-6 M guanidine or thiocyanate, sodium dodecyl sulfate (SDS) at a concentration typical of 0.1-5% v / v, ethylenediaminetetraacetic acid (EDTA) for a typical concentration of 1-5 mM, lysozyme at a typical concentration of 1 mg / ml, of proteinase-K, at a typical concentration of 1 mg / nil, and the pH can be adjusted to 7 - 7.5 using a buffer, such as HEPES. In some embodiments, the sample can be incubated in the buffer at a typical temperature of 20-95 ° C for 0-30 minutes. Isopropanol (50% -100% v / v) can be added to the mixture after lysis.
[00314] Loading surface. Lysate sample can be exposed to a functionalized surface (often in the form of a packed ball bed), such as, but not limited to, a resin holder packed in a style chromatography column, magnetic beads mixed with the sample in a one-way batch model, pumped through a resin sample suspended in a fluidized bed mode, and sample pumped through a closed channel tangentially along the surface. The surface can be functionalized in order to bind nucleic acids (for example, DNA, RNA, hybrid DNA / NA) in the presence of the lysis buffer. Surface types can include silica and functional ion exchange groups, such as diethylaminoethanol (DEAE). The lysed mixture can be exposed to the surface and the nucleic acids bind.
[00315] The solid surface is washed with a salt solution such as sodium chloride M and 0-2 ethanol (20-80% v / v), at pH 7.0-7.5. Washing can be done in the same way as loading.
[00316] Elution. Nucleic acids can be eluted from the surface, exposing the surface to water or buffer with pH 7-9. Elution can be carried out in the same way as loading.
[00317] Many variations of these protocols or other protocols can be used by the system. Such protocols can be used in combination or in the vicinity of any protocols or methods described herein.
[00318] In some embodiments, it is important to be able to recover red blood cells and concentrates by centrifugation for cytometry. In some embodiments, this can be achieved through the use of the pipetting device. liquids (typically isotonic buffered saline, a lysis agent, a mixture of a lysis agent and a fixative or labeled antibody fixer, in buffer) can be dispensed into the centrifuge container and repeatedly aspirated and re-dispensed. The tip of the pipette can be forced into the packed cells in order to facilitate the process. Image analysis helps in the objective verification process that all cells have been suspended again.
[00319] The use of the pipette and centrifuge to process samples before analysis: According to an embodiment of the invention, the system can have pipetting, take and position and centrifugal capacities. These capabilities can allow almost any type of sample pretreatment and complex test procedures to be performed efficiently with very small sample volumes.
[00320] In particular, the system can allow the separation of the figured elements (red and white blood cells) from the plasma. The system can also allow the resuspension of figurative elements. In some embodiments, the system may allow the concentration of lysed white blood cells to be fixed and haem. The system can also allow cell lysis to release nucleic acids. In some embodiments, purification and concentration of nucleic acids, by filtration through tips (typically crimped) reactants in solid phase (for example, silica) can be activated by the system. The system can also allow elution of the purified nucleic acids after solid phase extraction. The removal and collection of the precipitate (e.g., LDL-cholesterol precipitate using polyethylene glycol) can also be activated by the system.
[00321] In some embodiments, the system may allow for affinity purification. Small molecules such as vitamin D and serotonin can be adsorbed onto beads (particles) on hydrophobic substrates, and then eluted using organic solvents. The antigens can be supplied on substrates coated with antibody and eluted with an acid. The same methods can be used to concentrate the analytes were found in low concentrations, such as 6-keto-prostaglandin Fla Thromboxane B2 antigens and can be provided for antibodies or aptamero coated substrates and then eluted.
[00322] In some embodiments, the system may allow chemical modification of the analytes prior to testing. For testing serotonin (5-hydroxytryptamine), for example, it may be necessary to convert the analyte to a derivative (such as an acetylated form) using a reagent (such as acetic anhydride). This can be done to produce a form of the analyte that can be recognized by an antibody.
[00323] Liquids can be moved using the pipette (aspiration and pumping). The pipette can be limited to relatively low positive and negative pressures (about 0.1-2.0 atmospheres). A centrifuge can be used to generate much higher pressures when needed to force liquids through beaded solid phase media. For example, using a rotor with a radius of 5 cm at a speed of 10,000 rpm, forces of about 5000 xg (about 7 atmospheres) can be generated, enough to force the liquid through resistive means, such as packed beds. Any of the centrifuge models and configurations discussed elsewhere in this document or known in the art can be used.
[00324] Measurement of hematocrit, with very small volumes of blood can occur. For example, cheap digital cameras are capable of making good images of small objects, even when the contrast is poor. Using this capability, the system of the present invention can allow the measurement of the automated hematocrit with a small volume of blood.
[00325] For example, 1 ul of blood can be used in a capillary microcap glass. The capillary can then be sealed with a curable adhesive, and then subjected to centrifugation at 10,000 xg for 5 minutes. The hematocrit can be easily measured and the plasma meniscus (indicated by an arrow) can also be visible to the hematocrit can be measured accurately. This can allow the system not to lose a relatively large volume of blood to make this measurement. In some embodiments, the camera can be used "as and" without operating under a microscope to make a larger image. In other embodiments, an optical microscope or other techniques can be used to enlarge the image. In one implementation, the hematocrit was determined using the digital camera without additional optical interference and the hematocrit measured was identical to that determined using a conventional microhematocrit laboratory method requiring many microliters of sample. In some embodiments, the length of the column and the sample of the erythrocyte column can be measured with great precision (+/- <0.05 mm). Since the blood sample column can be about 10 - 20 mm, the hematocrit standard deviation can be much better than 1%, corresponding to that obtained by conventional laboratory methods.
[00326] The system can allow the measurement of the erythrocyte sedimentation rate (ES).
[00327] The ability of digital cameras to measure very small distances and the rates of variation of distances can be exploited to measure the ESR. In one example, three blood samples (15 ul) were aspirated in "Reaction tips". The images were captured over an hour at two-minute intervals. Image analysis was used to measure the movement of the interface between red cells and plasma. Figure 122 shows the results as the distance from the plasma meniscus interface.
[00328] The measurement accuracy can be estimated by adjusting the data to a polynomial function and calculating the standard deviation of the difference between the data and the adjusted curve (for all samples). In the example, this was determined to be 0.038 millimeter or <2% CV when related to the distance covered for one hour. Therefore, ESR can be measured precisely by this method. Another method for determining ESR and measuring the maximum slope of the distance ratio as a function of time. Assay Preparation
[00329] In some embodiments, tips can be designed to accommodate a plurality of reactions or tests. The simultaneous measurement of several different test mixes and one or more controls or one or more calibrators that can be made within a tip of the present invention. In doing so, explore the ability to experiment with various sources of liquids by sequential aspiration of liquids at the same end. Effective segmentation and separation of liquids and greatly improved by aspiration following a small volume of air and a small amount of a washing solution that cleans the surface of the tips before aspiration of the liquid of interest.
[00330] As described above, tips can have conical shapes. In some embodiments, an assay may require oxygen as a reagent. In such reactions, increasing the availability of oxygen within a reaction can be achieved by moving the sample and / or the test mixture over a large part of the tip by increasing the surface area in relation to the volume.
[00331] In Figure 93 and Figure 94, the bromophenol blue solutions were aspirated into tips. The upper segments (aspirated first) 6 ul are from a two-fold dilution series (the maximum concentration (0.0781 mg / ml) to the right of the image, with the exception of the extreme left tip, which is a raw piece of water). Then, the air (2 µl), a washing solution (2 µl), respectively, were aspirated followed by a volume of 6 µl of a fixed concentration control solution (0.625 mg / ml).
[00332] With this approach several alternative assay configurations can be achieved, for example: Simultaneous measurement of reagent and / or blank sample and assay Simultaneous measurement of sample, blank and control In-tip calibration of the Case 2 assay and shown in Figure 95.
[00333] Serial measurements of the blank solution, sample, controls and calibrators can also be done with simple tips. In this scenario, the tip is filled with the first solution, then read emptied. The tip can be refilled and read with other optical samples, in sequence. The impact of "transferring" color product from one sample to the next and minimized by one or both: read the column of liquid in the middle portion well away from the part that first comes into contact with the previous sample and rinse the tip between the samples.
[00334] In order to measure the degree of 'carry-over' from one liquid segment to the other, if the following procedure is carried out. A small amount (for example, 6 ul) of a high concentration of bromophenol blue ( for example, 0.625 mg / ml) was aspirated for tips, followed by 2 μl of air and 2 μl of the washing solution.Finally 6 μl of two-fold serial dilutions of bromophenol blue and aspirated with the following results (highest concentration (0.0781 mg / ml) to the right, leftmost tip and a white of water).
[00335] As can be seen from the images shown in Figure 96 and in the 3-color analysis shown in Figure 97, measurable amounts of the high concentration solution and transferred to the washing solution.
[00336] Average carry-over (of high concentration control for water washing) and calculated at 1.4%. Since, in effect, the front zone (proximal to the slug previously) of a bullet after the liquid acts as a second washing step and the color reading is done at a remote location from this attack zone (typically a zone of the slug), the effective transition from one spacer to the other is usually much less than 1% and therefore generally insignificant. When the dilution series is measured using single dilution series samples to fill the tips, the results are identical to those obtained previously. The above description represents a "stress test" designed to assess the extent of the carry-over.
[00337] Figure 98 shows a tip containing reaction products from two commercially available tests for ionized calcium, Ca2 + (upper segment) and magnesium Mg2 + (lower segment), which were aspirated into measurement tips. The Ca2 + concentrations used in this experiment are 0, 2.5, 5, 10, 20, and 40 mg / dl, of Mg2 +, are 0, 1.25, 2.5, 5, 10, 20 mg / dl . The test reaction mixtures (6 μl of [Ca2 +] and 4 μl [Mg 2 +]) are well separated using 2 μl of air, 3 μl of washing and an additional 4 μl of air. The results of each assay read in this way are essentially identical to those measured having only one assay reaction mixture per tip.
[00338] As indicated above, the present invention allows the simultaneous evaluation of a plurality of assays. Images can be taken from many test tubes in the same field of view. Specifically, the simultaneous evaluation of the tests and controls in the same test cuvette can be performed. Simultaneous evaluation of several tests in the same test cuvette can also be performed. Reaction in the Environment
[00339] A system may comprise a heating block for heating the test or test unit and / or for controlling the test temperature. Heat can be used in the incubation step of an assay reaction to promote the reaction and shorten the time needed for the incubation step. A system can comprise a heating block configured to receive a test unit of the invention. The heating block can be configured to receive a plurality of dosing units from a device of the invention. For example, if tests of 8 are desired to be performed on a device, the heating block can be configured to receive eight test units. In some embodiments, the test units can be moved in thermal contact with the heating block using the means to move the test units. Heating can be carried out by a heating medium known in the art. Optimization Protocol
[00340] Test protocols for sample analysis can be optimized in several ways. When multiple assays are to be performed on a sample, all protocols can be optimized for the most stringent reaction conditions, or each of the assay protocols can be optimized based on the desired performance of a particular assay.
[00341] In some modalities, a single protocol that can be designed to meet the test requirements in all possible use cases. For example, in a multiplex cartridge, a single protocol can be specified based on the case where all the tests on the cartridge are to be performed (ie the limit case). This protocol can be designed to meet the minimum test requirements, such as the accuracy and dynamic range of each test on the cartridge. However, this approach can be sub-optimal for cases of alternate use, for example, when only a subset of tests with the cartridge is being performed. In these cases, for example the use of more, some tests can achieve a better performance in terms of sensitivity and precision. There may be a trade-off between the amount of sample assigned to a test and the sensitivity of the test. For example, a test that has a sensitivity of 1 unit / ml when the sample is diluted 1: 100 may be able to detect to 0.1 units / ml, if the dilution factor is increased to 1: 10. A disadvantage of The use of a smaller dilution factor in a test system multiplexed with the sample volume can be restricted than the fraction of the sample required for this test and increased by 10 times, even when using the minimum volume to perform the test. Likewise, the accuracy of the assay can be improved by using a higher sample concentration. For example, an assay that results in an (say) 0.1 +/- 0.02 absorbance signal (20% signal inaccuracy) at its detection limit can be improved by using 10 times the concentration of the sample such that the signal produced is 10 times greater than giving a 0.1 + / 1 0.02 OD signal at a ten-fold lower concentration of the analyte and at the 1.0 +/- 0.02 signal inaccuracy is now only 2%. The reason why this is the case, and typically the test signal (in the lower range of analyte concentrations), is directly proportional to the analyte concentration (and therefore the sample concentration), while the signal inaccuracy can generally be related to the square root of the signal and then increases as the square root of the analyte concentration (and sample concentration). Thus, the coefficient of variation (CV) of the signal can be inversely proportional to the square root of the signal, such that a 10-fold increase in the signal corresponds to approximately three times the reduction of the CV signal. Since CV concentration and typically directly related to the CV signal, the CV concentration will decrease with increasing sample concentration (decrease in dilution).
[00342] Protocols can be optimized for specific use cases instead of the typical one size fits all approach described above. For example, the protocol can be optimized to improve the accuracy of each test to be performed on the multiplex device. In addition, some tests can be prioritized over other tests for optimization. Optimization protocol can be pre-computed for use cases that are known a priori. Optimization protocol can also be performed in real time for new use cases are not known a priori. Validation system can be performed to measure the set of use cases.
[00343] An example of an optimization protocol is described below comparing two use cases. For both use cases, 8 ul of undiluted sample is available to perform the necessary tests. In this example, the multiplex cartridge has 20 plate assays, where 5 of the tests require 1: 5 dilution and 15 of the tests require 1: 25 dilution.
[00344] In the first use case, all tests are necessary to be performed on the sample. The protocol in this use case (if using B) is as follows: Prepare 1: 5 dilution (8 ul + 32 ul diluent sample) Prepare 1: 25 dilution (15ul 1: 5 + 60 ul sample diluent) for each assay (n = 20), mix with 5 μl of sample suitably diluted with 10 μl of the reagent This results in an imprecise concentration of 10% CV for all 20 protocol tests and which meets the minimum requirements. The use of sample and 1 ul of each: dilution test 5 and 0.2 ul for each: 25 dilution test (out of a total of 5 * 1 * 15 + 0.2 = 8 ul, using all the sample available).
[00345] In the second use case (of use case "B") with the same type of cartridge, only 10 tests are needed to be performed for the sample, not all 20. In addition, all of these 10 tests would be the ratio of 1: 25 dilution level was used in case of use A. The protocol was optimized for this use case to maximize the accuracy for all tests using a smaller dilution (1: 5). The protocol optimized for the specific use in this case, and the following: Prepare 1: 5 dilution (8 ul + 32 ul of diluent sample) For each assay (n = 10), mix with 4 ul of diluted sample with 11 ul of reagent Example of use and 0.8 ul undiluted sample per assay for a total of 8 ul. Since the concentration of the sample in the test is increased by 5 times in relation to the use case for A, the sensitivity of the test is improved by a factor of 5 and the test inaccuracy is reduced by about 2.4 (5 / 0.5) times to about 4.5%.
[00346] By re-optimizing the protocol, in case of use B uses five times the maximum of the original sample for each test, thus improving the overall performance. Note that the above discussion does not take into account any inaccuracy due to errors in the measurement of volumes, but only addresses errors due to inaccuracy in the measurement of the optical signal. Use case B would have less inaccuracy due to inaccuracy in volumes, since it uses fewer pipetting steps. For example, if the volume of inaccuracy introduces 5% inaccuracy in the concentration of the analyte evaluated in both use cases, there would be a total analyte inaccuracy of 11.2% (10 / 2 + 5 / 2) / 0, 5 in case of use A compared to 6.5% (4.5 / 2 +5 / 2) / 0.5 in case of use B (assuming, as is and generally true, that the factors that cause inaccuracy in aggregates as the square root of the sum of the squares of each source of inaccuracy).
[00347] The effects illustrated above can be more easily seen, in the case of tests based on luminescence in which the test signal is expressed as a number of photons emitted per unit of time. As is the case for counting radioactive emissions, for example, radioimmunoassay, the imprecision of the signal is equal to the square root of the signal and therefore the signal is 100 CV / (square root of the signal). For example, a 10,000 count sign will have a CV of 1%. In many photon assays (which produce, for example, chemiluminescence immunoassays, the signal is almost exactly proportional to the concentration of the analyte, at least in the lowest concentration range). Thus, the analyte inaccuracy scales measured with / 1 (square root of the signal) for concentrations significantly above the detection limit. In tests using the sample dilution, the measured analyte inaccuracy will therefore intensify to 1 / (the sample dilution). For example, a test using a 1: 100 dilution of sample dilution will have a CV signal and concentration about 3.2 times (10 / 0.5) greater than a test using a 1: 10 dilution (and also has a sensitivity about 10 times higher).
[00348] A variety of assays can be performed on a fluid device according to the present invention to detect an analyte of interest in a sample. Whenever a label is used in the essay, one can choose from a wide variety of labels is available in the art that can be used to perform subject tests. In some embodiments, labels are detectable by spectroscopic, photochemical, biochemical, electrochemical, immunochemical or other chemical means. For example, useful labels include nucleic acids, fluorescent dyes, electron-dense reagents, and enzymes. A wide variety of labels suitable for labeling biological components are known and are extensively reported in both the scientific and patent literature, and are generally applicable to the present invention, for labeling biological components. Suitable labels include, enzymes, fluorescent units, chemiluminescent units, bioluminescent labels or colored labels. Reagents that define the specificity of the assay optionally include, for example, monoclonal antibodies, polyclonal antibodies, aptamers, proteins, nucleic acid probes or other polymers, such as affinity matrices, carbohydrates or lipids. Detection can proceed by any of a variety of known methods, including spectrophotometric or optical control of fluorescent or luminescent markers, or other methods that follow a molecule based on size, charge or affinity. The detectable portion can be of any material having a detectable physical or chemical property. Such detectable markers have been well developed in the field of gel electrophoresis, column chromatography, solid substrates, spectroscopic techniques, and the like, and in general, labels useful in such methods can be applied to the present invention. Thus, a label includes, without limitation, any composition detectable by photochemical, biochemical, immunochemical, probe-based spectroscopic nucleic acid, electrical, optical thermal, or by other chemical means.
[00349] In some embodiments of the label (such as a color compound, fluorine or enzyme) and coupled directly or indirectly with a molecule to be detected, according to methods well known in the art. In other embodiments, the tag is attached to a receptor for the analyte (for example, an antibody, nucleic acid probe, aptamer etc.) As indicated above, a wide variety of markers are used, with the choice of the tag depending on the necessary sensitivity, ease of conjugation of the compound, requirements for stability and available instrumentation, and flow arrangements. Non-radioactive labels are often attached by indirect means. Generally, a specific receptor for the analyte is attached to a signal generation portion. Sometimes the analyte receptor is attached to an adapter molecule (such as biotin or avidin) and the assay reagent kit includes a binding portion (such as a biotin or avidin reagent) that binds to the adapter and stops the analyte. The analyte binds to a specific receptor at the reaction site. A labeled reagent can form a sandwich complex in which the analyte is at the center. The reagent can also compete with the analyte for receptors at the reaction site, or bind to vacant receptors at the reaction site not occupied by the analyte. The tag is either inherently detectable or linked to a signal system, such as a detectable enzyme, a fluorescent compound, a chemiluminescent compound, or a chemiluminogenic entity such as an enzyme with a luminogenic substrate. A number of ligands and anti-ligands can be used. Whenever a ligand has a natural anti-ligand, for example, biotin, thyroxine, digoxigenin, and cortisol, it can be used in conjunction with labeled, anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
[00350] The enzymes of interest as labels will be mainly hydrolases, mainly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl groups, umbeliferone and. Chemiluminescent compounds include acridinium esters, dioxetanes, luciferin and 2,3-dihydrophthalazinadiones, such as luminol.
[00351] The methods of detecting labels are well known to those skilled in the art. So, for example, where the label is fluorescent, it can be detected by exciting the fluorochrome with light of an appropriate wavelength and the detection of the resulting fluorescence, for example, microscopy, visual inspection, by means of a photographic film, by the use of electronic detectors such as digital cameras, charge-coupled devices (CCDs) or photomultipliers and phototubes or other detection devices. Likewise, enzymatic markers are detected, providing appropriate substrates for the enzyme and detecting the product resulting from the reaction spectroscopically or through the digital image (the object of the present invention). Finally, simple colorimetric labels are often detected simply by looking at the color associated with the label. For example, colloidal gold suns often appear pink, while several spheres doped with dyes are heavily colored.
[00352] In some embodiments, the detectable signal can be provided by luminescence sources.
[00353] Luminescence and the term generally used to refer to the emission of light from a substance for any reason other than an increase in its temperature. In general, atoms or molecules emit photons of electromagnetic energy (for example, light) when transitioning from an animated state to a lower energy state (usually the state of the ground). If the agent is a photon exciting, the luminescence process is referred to as photo luminescence or fluorescence. If the cause is exciting and an electron, the luminescence process can be referred to as electroluminescence. More specifically, the electroluminescence results from direct injection and removal of electrons to form an electron-hole pair, and subsequent recombination of the electron-hole pair to emit a photon. luminescence, which results from a chemical reaction and commonly referred to as chemiluminescence. The luminescence produced by a living organism, and is commonly referred to as bioluminescence. If photoluminescence is the result of an allowed transition rotation (for example, a transition, single singlet triplet-triplet transition), the photoluminescence process is commonly referred to as fluorescence. Typically, fluorescence emissions do not persist after the exciting cause and are removed as a result of short-lived excited states that can quickly relax through such allowed spin transitions. If photoluminescence is the result of a prohibited rotation transition (for example, a triplet-singlet transition), the photoluminescence process is commonly referred to as phosphorescence. Typically, long phosphorescence emissions persist after the exciting cause and are removed as a result of long-term excited states which can relax only through such spin-banned transitions. A luminescent label can have any of the properties described above.
[00354] Suitable chemiluminescent sources include a compound that becomes electronically excited by a chemical reaction and can then emit light that serves as a detectable signal or donates energy to a fluorescent acceptor. A diverse number of families of compounds have been found to provide chemiluminescence under a variety of conditions. A family of compounds and 2,3-dihydro-1,4-phthalazinedione. One compound used is luminol, which is the 5-amino compound. Other members of the family include 5-amino-6, 7, 8-trimethoxy and dimethylamino [ca] benz analog. These compounds can be made of luminescence with hydrogen peroxide in an alkaline medium or calcium hypochlorite and base. Another family of compounds is 2,4,5-triphenylimidazoles, with lophine as the common name for the related product.
[00355] Chemiluminescent analogs include para-dimethylamino and methoxy substituents. Chemiluminescence can also be obtained with oxalates, active esters, usually oxalyl, for example, p-nitrophenyl and a peroxide such as hydrogen peroxide, under basic conditions. Other useful chemiluminescent compounds which are also known include -N-alkyl esters and dioxidanes acridinum. Alternatively, luciferins can be used in conjunction with luciferase to provide lucigenins or bioluminescence. Especially preferred chemiluminescent sources are "luminogenic" enzyme substrates, such as phosphate-dioxetane esters. These are not luminescent, but produce luminescent products, when taken into account by phosphatases such as alkaline phosphatase. The use of substrates for luminogenic enzymes is particularly preferred because the enzyme acts as an amplifier capable of converting thousands of substrate molecules per second to product. Luminescence methods are also preferred because the signal (the light) can be detected at the same time very sensitive and over a huge dynamic range using PMT.
[00356] The duration analytes, as used herein, include, without limitation, prodrug drugs, pharmaceutical agents, drug metabolites, such as biomarkers of expressed proteins and cell markers, antibodies, whey proteins, cholesterol and other metabolites , electrolytes, metal fons, polysaccharides, nucleic acids, biological analytes, biomarkers, genes, proteins, hormones or any combination of these. Analytes can be combinations of polypeptides, glycoproteins, polysaccharides, lipids and nucleic acids.
[00357] The system can be used to detect and / or quantify a wide variety of analytes. For example, analytes that can be detected and / or quantified include albumin, alkaline phosphatase, AlT, AST, bilirubin (direct), bilirubin (total), blood urea nitrogen (BUN), calcium, chloride, cholesterol, dioxide carbon (CO 2), creatinine, gamma-glutamyl-transpeptidase (GGT), globulin, Glucose, HDl-cholesterol, hemoglobin, homocysteine, Iron, lactate dehydrogenase, magnesium, phosphorus, potassium, sodium, total protein, triglycerides and uric acid. The detection and / or quantification of these analytes can be performed using optical, electrical, or any other type of measurements.
[00358] Of particular interest are biomarkers, which are associated with a particular disease or with a specific disease stage. Such analytes include, but are not limited to, those associated with autoimmune diseases, obesity, hypertension, diabetes, neuronal and / or muscle degenerative diseases, heart disease, endocrine disorders, metabolic disorders, inflammation, cardiovascular disease, sepsis, angiogenesis , cancers, Alzheimer's disease, athletic complications, and any combinations of these.
[00359] Of interest are also the biomarkers that are present in abundance varying in one or more of the tissues of the body, including heart, liver, prostate, lung, kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, and selected tissues that are affected by different diseases, such as different types of cancer (malignant or non-metastatic), autoimmune diseases, inflammatory or degenerative diseases.
[00360] Also of interest are the analytes that are indicative of a microorganism, virus or Chlamydiaceae. Examples of microorganisms include, but are not limited to, bacteria, viruses, fungi and protozoa. Analytes that can be detected by the present method include blood borne pathogens selected from a non-limiting group, consisting of Staphylococcus epidermidis, Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Staphylococcus hominisis, Enterococcus fainisis , Pseudomonas aeruginosa, Staphylococcus capitis, Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus influenzae, Simulans Staphylococcus, Streptococcus pneumoniae and Candida albicans.
[00361] The analytes that can be detected by this method also encompass a variety of sexually transmitted diseases, selected from the following: gonorrhea (Neisseria gonorrhoeae), syphilis (Treponena pallidum), chlamydia (Clamyda tracomitis), non-gonococcal urethritis (Ureaplasm urealyticum) yeast infection (Candida albicans), soft cancer (Haemophilus ducreyi), trichomoniasis (Trichomonas vaginalis), genital herpes (HSV type le ll), HlV l, HlV ll and hepatitis A, B, C, G, as well as hepatitis caused by TTV.
[00362] Analytes that can be detected by subject methods encompass a variety of respiratory pathogens, including, but not limited to, methicillin-resistant Pseudomonas aeruginosa Staphlococccus aureus (MRSA), Klebsiella pneumoniae, Haemophilis influenzae Staphlococcus aureus, Ha Escherichia coli, Enterococcus faecalis, Serratia marcescens, parahaemolyticus Haemophilis, Enterococcus cloacae, Candida albicans, Moraxiella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii, Enterococella faecium, Klebsella oxitoca, Klebsella oxitoca, Pseudomisisis, fluse , Mycoplasma pneumoniae, and Mycobacterium tuberculosis. listed below are additional exemplary markers according to the present invention: Theophylline, PCR, CK-MB, PSA, myoglobin, CA125, Progesterone, TxB2, 6-keto-PGF-1-alpha, and theophylline, estradiol hormone, lutenizing , triglycerides, Tryptase, lDl-cholesterol, HDl cholesterol, cholesterol, lGFR.
Examples of liver markers include, without limitation, lDH, (lD5), alanine aminotransferase (AlT), Arginase 1 (liver type), alpha-fetoprotein (AFP), alkaline phosphatase, lactate dehydrogenase and bilirubin.
[00364] Examples of renal markers include, without limitation, TNFα receptor, cystatin C, urinary prostaglandin D lipocalin, synthatase (lPGDS), hepatocyte growth factor receptor, polycystin 2, polycystin 1, Fibrocystin, Uromodulin, alanine, aminopeptidase, N-acetyl-protein BD-glucosaminidase, albumin and Retinol (RBP).
[00365] Examples of cardiac markers include, without limitation, troponin 1 (TNl), troponin T (TnT), creatine dinase (CK), CK-MB, myoglobin, fatty acid binding protein (FABP), protein C- reactive (PCR), fibrinogen D-dimer, protein S-100, brain natriuretic peptide (BNP), NT-proBNP, PAPP-A, myeloperoxidase (MPO), glycogen phosphorylase isoenzyme BB (GPBB), thrombin inhibitor Activatable fibrinolysis (TAFl ), fibrinogen, ischemia-modified albumin (lMA), cardiotrophin-1, and MlC-l (myosin-l light chain).
[00366] Examples of pancrease markers include, without limitation, amylase, Pancreatitis-Associated protein (PAP-1), and Regeneratein proteins (REG).
[00367] Examples of muscle tissue markers include, without limitation, myostatin. (EPO).
[00368] Examples of blood markers include, without limitation Erythopoeitin
[00369] Examples of bone markers include, without limitation, L-type bone collagen N-telopeptides (NTx), bone collagen cross-linking carboxy terminal peptide, lysyl-pyridinoline (Deoxypyridinoline), Pyridinoline, acid phosphatase tartrate resistant,
[00370] type Procollagen lC propeptide, Type of procolagen lN propeptide, osteocalcin (bone glaprotein), alkaline phosphatase, cathepsin K, COMP (Oligimeric Matrix Protein Cartilage), Osteocrin, osteoprotegerin (OPG), RANKl, sRANK, TRAIN, 5 TRACP 5), Osteoblast specific factor 1 (OSF-1, Pleiotrophin), soluble cell adhesion molecules, sTtR, sCD4, sCD8, sCD44, and osteoblasts specific factor 2 (OSF-2, periostin).
[00371] In some embodiments, markers according to the present invention are disease specific. Examples of cancer markers include, without limitation, PSA (total prostate specific antigen), creatinine, prostatic acid phosphatase, PSA complexes, specific for Prostrate-1 gene, CA 12-5, carcino-embryonic antigen (CEA) , alpha fetus protein (AFP), hCG (human chorionic gonadotropin), lnibine, ovary CAA C1824, CA 27.29, CA 15-3, breast CAA Cl 924, Her-2, pancreas, CA 19-9, pancreas CAA, neuron-specific enolase, Angiostatin DcR3 (soluble decoy receptor 3), Endostatin, Ep-CAM (MK-1), kappa free chain immunoglobulin, lambda light chain free immunoglobulin, Herstatin, chromogranin A, Adrenomedulin, integrin, the factor receptor epidermal growth factor, receptor tyrosine kinase epidermal growth factor, N-terminal Pro-adrenomedullin peptide 20, vascular endothelial growth factor, vascular endothelial growth factor receptor, stem cell receptor factor, c-kit / KDR, KDR, and Midkine.
[00372] Examples of infectious disease conditions include, without limitation: viremia, bacteremia, sepsis and markers: PMN elastase, PMN elastase / al-Pl complex, protein D surfactant (SP-D), HBVc antigen, HBV antigen, Anti -HBVc, Anti-HlV, t-suppressor of cell antigens, the ratio of T-cell antigen, the antigen of T-helper cells, anti-HCV, pyrogens, p24-Muramyl dipeptide antigen.
[00373] Examples of diabetes markers include, without limitation of C-peptide, Ale hemoglobin, glycated albumin, advanced glycosylation end products (AGEs), 1,5-anhydroglucitol, gastric inhibitory polypeptide, glucose, hemoglobin Ale, ANGPTl3 and 4.
[00374] Examples of inflammation markers include, without limitation, rheumatoid factor (RF), Antinuclear Antibody (ANA), C-reactive protein (CRP), Cellular Protein (Uteroglobin).
[00375] Examples of exemplary allergy markers include, without limitation, total lgE and specific lgE.
[00376] Examples of exemplary autism markers include, without limitation Ceruloplasmin, Metalothioneine, Zinc, Copper, BÓ, B12, glutathione, alkaline phosphatase, and activation of alkaline apo-phosphatase.
[00377] Examples of markers of coagulation disorders include, without limitation, b-thromboglobulin, platelet factor 4, von Willebrand factor.
[00378] In some embodiments it may be a specific marker of therapy. Markers indicative of the action of COX inhibitors include, without limitation, TxB2 (COX-1), 6-keto-PGF 1 alpha (COX-2), 11-dehydro-TxB-la (COX-1).
[00379] Other markers of the present invention include, without limitation, leptin, leptin receptor, and Procalcitonin, Brain protein S100, Substance P, 8-iso-PGF-2a.
[00380] Examples of geriatric markers include, without limitation, neuron specific enolase, GFAP and S100B.
[00381] Examples of nutritional status markers include, without limitation, pre-albumin, albumin, Retinol protein (RBP), transferrin, acylating stimulating protein (ASP), adiponectin, Agouti-related protein (AgRP), Angiopoietin-like protein 4 (ANGPTl4, FlAF), C-peptide, AFABP (adipocyte Fatty Acid Binding Protein, FABP4), acylation stimulating protein (ASP), EFABP (Epidermic Fatty Acid Binding Protein, FABP5), glycentin, glucagon, glucagon-glucagon-like peptide- 1, the glucagon-like Peptide-2, ghrelin, insulin, leptin, leptin receptor, PYY, RElMs, resistin, amd sTfR (soluble transferrin receptor).
[00382] Examples of lipid metabolism markers include, without limitation, Apo-lipoproteins (various), Apo-Al, Apo-B, Apo-C-Cll, -Apo D, Apo-E.
[00383] Examples of clotting state markers include, without limitation, Factor l: fibrinogen, Factor ll: Prothrombin, Factor lll: The tissue factor, Factor lV: Calcium, Factor V: Proaccelerin, Factor Vl, Factor Vll: Proconvertin, Vlll Factor, Anti-hemolitic Factor, Factor lX: Christmas factor, Factor X: Stuart-Prower factor,
[00384] Factor Xl: Plasma thromboplastin antecedent, Factor Xll: Hageman factor, Factor Xlll: fibrin stabilizing factor, Precalicrefna, high molecular weight quininogen, protein C, protein S, D-dimer, tissue plasminogen activator, Plasminogen, a2-antiplasmin, plasminogen activator 1 (PAH).
[00385] Examples of monoclonal antibodies include those for EGF, ErbB2, and lGF1R.
Exemplary tyrosine kinase Abl inhibitors include, without limitation, Kit, PDGFR, Src, ErbB2, ErbB 4 EGFR, EphB, VEGFR1- 4, PDGFRB, FlT3, FGFR, PKC, Met, Tie2, RAF, and TrkA.
[00387] Examples of serine / threonine kinase inhibitors include, without limitation, AKT, Aurora A / B / B, CDK, CDK (pan), CDKl-2, VEGFR2, PDGFRB, CDK4 / 6, MEK1-2, mTOR, and PKC-beta.
[00388] GPCR targets include, without limitation, histamine receptors, serotonin, Angiotensin receptors, adrenergic receptors, muscarinic acetylcholine receptors, GnRH receptors, dedopamine receptors, Prostaglandin receptors, and ADP receptors. Cholesterol
[00389] The measurement of metabolites can be carried out by producing a colored product using oxidases (for example, cholesterol oxidase) (to make H202) and raban peroxidase, in addition to a chromogen (such as N-ethyl- N- ( 2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, sodium salt ["DAOS" plus anti-pyrene-amino] to form a colored product such as a Trinder dye). An example of such chemistry is shown in Figure 52 and Figure 53. NADH or NADPH
[00390] Production or consumption of NADH or NADPH are often used in clinical trials. This is because these coenzymes are common substrates for enzymes. For example, the measurement of enzymes of clinical interest, such as lactate dehydrogenase (lDH), can be measured by the rate of NADH production. Since NADH absorbs light at a maximum of 340 nm and (1) polystyrene and other plastics transmit light poorly in the near UV, (2) White light sources produce little light in the near UV and (3) camera and scanner sensors have low sensitivity to near UV light, it is not practical to measure NADH by three color image analysis. To deal with this problem NADH can be converted to a stained product using tetrazolium salts, such as tetrazole water soluble tetrazolium (egWST- 1 (Dojindo molecular technologies), plus an "electron mediator" such as 1-methoxyphenazine methosulfate (PMS ).
[00391] In some embodiments, the tests that produce or consume NADH or NADPH, can be combined with other reactions that allow colorimetric measurement. For example, NADH or NADPH is used to reduce compounds such as 2 - (4-lodophenyl) -3 - (4-nitrophenyl) -5 - (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt (WST- 1) to a colored dye formezan as shown below with the use of phenazine methosulfate as an electron mediator, as shown in Figure 54.
[00392] As shown in Figure 73, when NADH, WST-1 and PMS are combined in millimolar concentrations, a yellow product (shown at the tips indicated as a mixture) and formed.
[00393] Using this chemistry, an assay for lHD was created. lactate (mM), NAD (mM) and lDH were combined and incubated at 37 ° C for 10 minutes before the addition of WST-1 and PMS. A good dose-response for lDH was obtained as shown in Figure 74, for two serial dilutions of lDH (1000 IU / L) (from left to right), corresponding to the OD values at 450 nm shown in the graph in Figure 75. Alkaline phosphatase
[00394] In other embodiments, assays using enzymes such as alkaline phosphatase can be measured using a chromogenic substrate such as p-nitrophenyl phosphate. The enzymatic reaction can make p-nitrophenol which is yellow, in alkaline conditions. Metal ions
[00395] Measurements can also be performed in tests that form a colored complex, such as between a metallic phon of a chelating dye that changes color at the bond. For example, o-cresolftalefna Complexona (shown in Figure 55) which forms a complex with calcium, which has a different color than the reagent. The general regime of such tests is: chelating dye (color 1) + M + N <-> dye Chelating: M + N: (Color 2)
[00396] Optical signals can also be measured by testing metal ions using metal-dependent enzymes. For example, sodium ions can be determined enzymatically through sodium-galactosidase-dependent activity, with o-nitro-phenyl galactoside (ONPG) as the substrate. The absorbance at 405 nm of the o-nitrophenol product is proportional to the sodium concentration. ELISA
[00397] The assays can be performed for analytes by color-forming EllSAs.
Many ELISA methods are known to generate color using enzymes such as raban peroxidase, alkaline phosphatase and galactosidase with chromogenic substrates, such as o-phenylenediamine, p-nitrophenyl phosphate, and galactoside-nitrophenyl, respectively. Such assays can be easily performed and read by the subject invention. l Luminogenic immunoassays
[00399] Luminogenic immunoassays can also be performed. The assays can use chemiluminogenic entities, such as an enzyme with a luminogenic substrate. For example, chemiluminescent compounds include acridinium esters, dioxetanes, luciferin and 2,3-dihydrophthalazinediones, such as luminol.
[00400] In addition, suitable chemiluminescent sources include a compound that becomes electronically excited by a chemical reaction and can then emit light that serves as a detectable signal or donates energy to a fluorescent acceptor. A diverse number of families of compounds have been found to provide chemiluminescence under a variety of conditions. A family of compounds and 2,3-dihydro-1,4-phthalazinedione. One compound used is luminol, which is the 5-amino compound. Other members of the family include 5-amino-6, 7, 8-trimethoxy and analogue dimethylamino [ca] benz. These compounds can be made of luminescence with hydrogen peroxide in an alkaline medium or calcium hypochlorite and base. Another family of compounds is 2,4,5-triphenylimidazoles, with lophine as the common name for the related product.
[00401] Chemiluminescent analogs include para-dimethylamino and methoxy substituents. Chemiluminescence can also be obtained with oxalates, active esters, usually oxalyl, for example, p-nitrophenyl and a peroxide such as hydrogen peroxide, under basic conditions. Other useful chemiluminescent compounds that are also known and include N-alkyl acridinum dioxetane esters. Alternatively, luciferins can be used in conjunction with luciferase to provide lucigenins or bioluminescence. Amplification of nucleic acid
[00402] The assays that can be performed include amplification of the nucleic acid. Among these assays, isothermal amplification and Loop-mediated isothermal amplification assays (lAMP) are examples. Nucleic acid amplification can be used to produce visibly cloudy, fluorescent or stained test reaction products for analytes as nucleic acid targets (genes, etc.). Nucleic acid amplification technology can be used for isothermal amplification of DNA targets and specific RNA. Additional information on isothermal amplification of nucleic acids is described in Goto et al. ", Colorimetric detection of the loop-mediated isothermal amplification reaction using blue hydroxy naphthol", BioTechniques, vol. 46, No. 3, March 2009, 167-172.
[00403] Nucleic acid amplification can be used to measure DNA, and, together with the use of reverse transcriptase, RNA. Once the reaction has occurred, the amplified product can be detected optically using intercalating dyes or chromogenic reagents that react with released pyrophosphate generated as a secondary product of the amplification.
[00404] The reaction can be visualized by changes (increases) in color, fluorescence or turbidity. Very small number of copies of DNA can be detected in less than an hour. This technology can advantageously be read in the present invention using three-color image analysis. As shown below, images of the products of the nucleic acid isothermal amplification reaction test can be measured by (1) back-lit illumination (transmission optics) measuring the absorbance of light, (2) the images captured by a camera digital light transmitted through a reaction product or (3) the fluorescent images generated by lighting the reaction products with a UV source (or any other suitable light source) captured by a digital camera.
[00405] The nucleic acid amplification assay is generally carried out in a "one-pot" format where the sample and reagents are combined in a sealed tube, and incubated at an elevated temperature. In some formats, the reaction can be monitored in real time by changes in optical properties. In other test formats, the reaction is stopped and the reaction products are visualized after adding a chromogenic or fluorogenic reagent. The present invention allows the reading of the test nucleic acid amplification products directly into the reaction vessel or after aspiration for the tips described here. Turbidity
[00406] The invention also provides for optical turbidimetric tests. For example, immunoassays can be configured by measuring the agglutination of small latex particles (50-300 nm). In these assays the particles can be coated with an antigen and / or the antibody and agglutination occurs when a binding homologue in the sample such as the antibody or antigen is added. Assays can be configured as direct (eg, antibody on the particle reacts with a multi-epitope protein or biomarkers) in competitive mode (eg hapten drug on the particle reacts with the anti-drug antibody in competition with the free drug in the sample ) or. The dispersion of latex becomes more cloudy and turbidity can be measured as the decrease in light transmission using 3-color optics.
[00407] Likewise, tests based on the agglutination of large latex particles (diameter of about 1 µm) or red blood cells can be measured. Configuration test is similar to turbidimetric tests as revealed above, but the measurement can be done by image analysis (scanner or camera measurement) using the software to interpret the number and size of the agglutinates.
[00408] Reagents for carrying out chemical reactions can be included in the cartridges described here, such as in the tip. The reagents can be stored in the form of liquids or in dry, lyophilized or glassy forms. Localized reagents
[00409] In some embodiments, the location and configuration of a reaction site is an important element for a dosing device. Most, if not all, disposable immunoassay devices have been configured with their capture surface as an integral part of the device.
[00410] In one embodiment, a molded plastic testing unit is either commercially available or can be made by injection molding, with precise shapes and sizes. For example, the characteristic dimension can be a diameter of 0.05-3 mm, or it can be a length of 3 to 30 mm. The units can be coated with capture reagents and using the method similar to those used for coating microtiter plates, but with the advantage that they can be processed in large quantities by placing them in a large container, adding the coating reagents and treatment using sieves, supports, and the like to recover the pieces and wash them as needed.
[00411] The test unit (for example, involving the tip disclosed here, tips, vessels, or any other containers) can offer a rigid support on which a reagent can be immobilized. The test unit is also chosen to provide the appropriate characteristics with regard to interaction with light. For example, the test unit can be made of a material, such as functionalized glass, Si, Ge, GaAs, GAP, Si02, SiN4, modified from silicone, or any of a wide variety of gels or polymers, such as (poly) tetrafluoroethylene, (poly) vinylidenedifluoride, polystyrene, polycarbonate, polypropylene, polymethylmethacrylate (PMMA), acrylonitrile-butadiene-styrene (ABS), or combinations thereof. In one embodiment, it comprises a polystyrene test unit. In some embodiments, the dosing unit can be formed from a homogeneous material, heterogeneous material, coated material, coated with the material, the impregnated material, and / or incorporated material. Other suitable materials can be used in accordance with the
[00412] the present invention. A transparent reaction site can be advantageous. In addition, in the case where there is a window allowing optically transmissive light to reach an optical detector, the surface can advantageously be opaque and / or scattered, preferably of light. In some embodiments, the dosing unit can be formed from a transparent material. Alternatively, a portion of the dosing unit can be formed from a transparent material.
[00413] The test unit may have a reagent coated and / or impregnated therein. In some embodiments, the reagent can be a capture reagent capable of immobilizing a reagent on a capture surface. The reagent can be a cell and / or the analyte, or any other reagent described herein elsewhere. In some embodiments, the reagent can be a molecule that can be a cell capture agent. A cell capture agent can anchor the surface of desired cells during fluid transport. In some embodiments, the capture reagents may be an antibody, peptide, organic molecule (eg, which may have a chain, lipophilic lipid molecule), polymer matrix, protein, composed of protein, glycoprotein, which can interact with the cell membrane. Capture reagents can be molecules, cross-linked molecules, nanoparticles, nanostructures, and / or scaffolding. In some embodiments, microstructures can be provided that can become a vessel analysis mechanism. Capture reagents (which may include capture structures formed by the test unit material) can allow cells to be attached, bound, and / or trapped.
[00414] Capture reagents can immobilize a reagent, such as a cell, during processing. Capture techniques can be chemical, physical, electrical, magnetic, mechanical, related size, related to density, or any combination of these. In some embodiments, capture reagents can be used to concentrate reagents, such as cells, in a desired location. For example, a test unit can be coated with the capture reagents, which can cause the cells to be captured on the surface of the test unit, thus the concentration of the cells on the captured surface. Capture reagents can keep the captured reagent immobilized on the cell surface. This can help to keep the reagents (eg, cells, analytes) stationary during the examination.
[00415] Immobilizing reagents can be useful for applications where there may be occasions in long acquisition for reactions and / or detection. For example, a number of imaging applications may require prolonged exposure times (~ 1 min) or small object imaging (<lum) that can have significant Brownian motion.
[00416] In some embodiments, capture reagents can be formed from materials that can provide little or no imaging. In some cases, the dosing unit material may provide little or no background for the imaging. Capture reagents can be chosen so that they do not interfere with, or have only slight interference with, imaging and / or detection.
[00417] A reagent immobilized on the capture surface can be useful for the detection of an analyte of interest in a sample of body fluid. For example, such reagents include, without limitation, nucleic acid probes, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with aptamers a specific analyte. Various commercially available reagents, such as a set of polyclonal and monoclonal antibodies specifically developed for specific analytes, can be used.
[00418] An expert in the art will appreciate that there are many ways of immobilizing various reagents on a support where reaction can take place. The immobilization can be covalent or non-covalent, through a binding portion, or tie them to an immobilized portion. Non-limiting exemplary binding portions for attachment of either nucleic acids or protein molecules, such as antibodies to a solid support include streptavidin or avidin / biotin bonds, carbamate bonds, ester bonds, amide, thiolester, thiourea (N) - functionalized , maleimide, functionalized amino, disulfide, amide, hydrazone bonds, among others. In addition, a silyl moiety can be attached to a nucleic acid directly to a substrate such as glass using methods known in the art. Immobilization surface can also be achieved by means of a poly-l lysine rod, which provides a load coupling load to the surface.
[00419] The test units can be dried after the last step of incorporating a capture surface. For example, drying can be carried out by passive exposure to a dry atmosphere, or by using a vacuum distributor and / or applying dry, clean air through a collector or by lyophilization.
[00420] A capture surface can be applied to a test unit, using any technique. For example, the capture surface can be painted, printed, electroplated, embedded in the material, impregnated with the material, or any other technique. The capture reagents can be coated with the dosing unit material, incorporated into the material, to co-penetrate the material, or it can be formed from the material. For example, a reagent, such as a capture reagent, can be incorporated into a polymer matrix that can be used as a sensor. In some embodiments, one or more small particles, such as one of the nanoparticles, a microparticle, and / or a strand, can be coated and / or impregnated with the reagents. In some embodiments, the capture reagents may be part of the test unit material itself, or it may be something that is added to the material.
[00421] In many embodiments, a test unit has been designed to allow the unit to be manufactured in a high volume, fast manufacturing process. For example, tips can be assembled in large-scale sets to coat the capture surface in batch or on the tip. In another example, tips can be placed on a rotating belt or table for series processing. In yet another example, a wide variety of suggestions can be connected to vacuum and / or pressure collectors for simple processing.
[00422] A capture reagent can be applied to a test unit during any point in the process. For example, the capture reagent can be applied to the test unit during manufacture. The capture reagent can be applied to the test unit before sending the test unit to a destination. Alternatively, the capture reagent can be applied to the test unit after the test unit has been shipped. In some cases, the capture reagent can be applied to the test unit at a point of use, such as a service location point.
[00423] In some embodiments, the capture reagent may cover the entire surface or an area of the test unit. The capture reagent can be provided on an interior surface of the test unit. In some embodiments, the capture reagent may cover portions or sections of a test surface unit. The capture reagent can be supplied on a surface in a pattern. The unit may have portions of the surface that have a reagent capture applied to it, and portions of the surface that do not have a capture reagent applied to it. For example, there may be coated and uncoated regions. A capture reagent can be applied to a surface according to a geometric choice of how the capture reagent is to be applied. For example, the capture reagent can be applied to points, lines, columns, matrices, regions, circles, rings, or any other shape or pattern. Capture reagents can be applied at desired positions on the surface.
[00424] A plurality of capture reagents can optionally be applied to a test unit. In some embodiments, the plurality of capture reagents can be applied so that the different capture reagents do not overlap (for example, the different capture reagents are not applied to the same region or zone). Alternatively, they can overlap (for example, different capture reagents can be applied to the same region or region). Space without any capture reagents may or may not be provided between regions with different capture reagents. The various capture reagents can be used to immobilize different reagents. For example, different capture reagents can be used to immobilize different cells and / or analytes on the capture surface. By using a plurality of capture reagents stamped in selected regions, a plurality of reagents can be detected from the same test unit. In some forms of achievement, two or more, or more than three, four or more, five or more, seven or more, ten or more, or more than fifteen, twenty or more, or more than thirty, forty or more, or more than fifty, seventy or more, 100 or more, 150 or more, 200 or more, or 300 or more different capture reagents can be applied to a surface of a dosing unit. The various capture reagents can be applied in any pattern or form. For example, the different capture reagents can be applied as a matrix or a series of rings on an internal surface of a dosing unit. For example, the different capture reagents can be applied on an internal surface of the tip of one, container, container, cuvettes or other container described elsewhere herein.
[00425] The location of the different capture reagents on the dosing unit can be known before the detection of the captured reagents. In some embodiments, the dosing unit may have an identifier that can indicate the type of dosing unit and / or the pattern of capture agents therefor. Alternatively, the location of the different capture reagents in the assay unit cannot be known before the detection of the captured reagents. The location of the different capture reagents can be determined based on detected captured reagent patterns.
[00426] Capture reagents can be applied using any technique, such as those described in this document. In some cases, lithographic masking techniques or can be used to apply different capture reagents.
[00427] Any description here of a capture reagent and / or the coating applied to a test unit can be applied to any other units or containers described elsewhere in this document, including, but not limited to, tips, vessels, vats, or reagent units. Reagent Sets
[00428] In many embodiments of the invention, the reagent units are modular. The reagent unit can be designed to allow the unit to be manufactured in a high volume, rapid manufacturing process. For example, many units of reagents can be filled and sealed in a large-scale process simultaneously. The reagent units can be filled according to the type of assay or assays to be performed by the device. For example, if a user wants different tests from another user, the reagent units can be manufactured according to the preferences of each user, without the need to manufacture an entire device. In another example, the reagent units can be placed on a rotating moving belt or table for series processing.
[00429] In another embodiment, the reagent units are housed directly into the cavities of the housing of a device. In this embodiment, a seal can be made in housing areas around the units.
[00430] Reagents according to the present invention include, without limitation, wash buffers, enzyme substrates, dilution buffers, and conjugates, enzyme-labeled conjugates, DNA sample amplifiers, diluents, wash solutions, reagents sample pre-treatment, including additives such as detergents, polymer chelating agents, albumin binding reagents, enzyme inhibitors, enzymes, anticoagulants, red cell binding agents, antibodies, or other materials necessary to perform a test on a device. An enzyme-labeled conjugate can be a polyclonal antibody or an enzyme-labeled monoclonal antibody that can produce a detectable signal after reaction with an appropriate substrate. Non-limiting examples of such enzymes are alkaline phosphatase and raban peroxidase. In some embodiments, the reagents comprise immunoassay reagents. In general, reagents, especially those that are relatively unstable when mixed with the liquid, are confined separately to a defined region (for example, a reagent unit) inside the device.
[00431] In some embodiments, a reagent unit contains about 5 microliters and about 1 milliliter of liquid. In some embodiments, the unit can contain about 20-200 microliters of liquid. In another embodiment, the reagent unit contains 100 microliters of fluid. In one embodiment, a reagent unit contains about 40 microliters of fluid. The volume of liquid in a reagent unit may vary, depending on the type of assay to be performed, or the sample of body fluid provided. In one embodiment, the volumes of the reagents are not necessarily predetermined, but must be more than a known minimum. In some embodiments, the reagents are initially stored dry and dissolved after the start of the test to be performed on the device.
[00432] In one embodiment, the reagent units can be filled with a siphon, funnel, pipette, syringe and needle, or a combination of these. The reagent units can be filled with liquid, using a filling channel and a vacuum channel. The reagent units can be filled individually or as part of a mass manufacturing process.
[00433] In one embodiment, an individual reagent unit comprises a different reagent, as a means of isolating the reagents from each other. The reagent units can also be used to contain a washing solution or a substrate. In addition, the reagent units can be used to contain a luminogenic substrate. In another embodiment, a plurality of reagents is contained within a reagent unit.
[00434] In some cases, the installation of the device allows the pre-calibration capacity of the test units and the reagent units before the assembly of disposable articles of the subject device. Aptamer connection tests
[00435] The present invention allows a variety of test methods based on the use of linkers that specifically bind to one or more analytes in a sample. In general, a bonding member and a member of a bonding pair capable of selectively and specifically bonding to the other member of the bonding pair in the presence of a plurality of different molecules. Examples of linkers include, but are not limited to, antibodies, antigens, metal-binding ligands, nucleic acid probes and primers, receptors and reagents as described herein and aptamers. In some embodiments, a linker used to detect an analyte is an aptamer. The term "aptamer" is used to refer to a peptide, nucleic acid, or combination thereof, which is selected for the ability to specifically bind one or more target analytes. Aptamer peptides are affinity agents that generally comprise one or more variable loop domains displayed on the surface of a scaffold protein. A nucleic acid aptamer and a specific binding oligonucleotide, which is an oligonucleotide that is capable of selectively forming a complex with a desired target analyte. Complexation is specific to the target, in the sense that other materials, such as other analytes that can accompany the target analyte, do not stop the complex aptamero with such great affinity. It is recognized that the affinity complexation and are a matter of degree, however, in this context, it means "specific" target that aptamero binds to the target to a much higher degree than the affinity that binds to the contaminating material. The meaning of specificity in this context is therefore similar to the meaning of specificity when applied to antibodies, for example. Aptamero can be prepared by any known method, including methods of synthesis, recombinant and purification. In addition, the term "aptamer" also includes "secondary aptamer" that contains a consensus sequence derived from comparing two or more aptamer known for a given target.
[00436] In general, nucleic acid aptamers are about 9 to about 35 nucleotides in length. In some embodiments, an aptamer of nucleic acid is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 , 55, 60, 65, 70, 80, 90, 100 or more residues in length. Although the oligonucleotides of aptamers are generally single-stranded or double-stranded, it is contemplated that aptamers can sometimes take on single-stranded triple or quadruple-stranded structures. In some embodiments, a nucleic acid aptamer is circular, as in the patent US20050176940. Aptamer-specific binding oligonucleotides must contain what gives sequence specificity, but can be extended with regions flanking and derivatized or otherwise modified. The aptamers found to bind to a target analyte can be isolated, sequenced, and then re-synthesized as conventional DNA or RNA fragments, or oligomers can be modified. These modifications include, but are not limited to, the incorporation of: modified or analogous forms of sugar (for example, ribose and deoxyribose), (2) alternative linking groups, or (3) analogous forms of purine and pyrimidine bases.
[00437] Nucleic acid aptamers may comprise DNA, RNA, functionalized or modified nucleic acid bases, nucleic acid analogs, modified or alternative backbone chemistries, or combinations thereof. The oligonucleotides of aptamers can contain the conventional bases for adenine, guanine, cytosine and thymine or uridine. included within the term aptameros are synthetic aptameros that incorporate analogous forms of purines and pyrimidines. "Analogous" forms of purines and pyrimidines are those generally known in the art, many of which are used as chemotherapeutic agents. Non-limiting examples of forms of purine and pyrimidine analogues (i.e., base analogs) include aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromo uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, NÓ-isopenteniladenina, 1 - methyladenina, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-methylethine , 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, NÓ-methyladenine, 7-methylguanine, 5-methylaminomethyl-uracil, 5-Methoxyminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5-methoxyuracyl, 2-methoxyuracil, 2-methoxyuracil - methyl-thio- NÓ- isopentenyladenine, uracil-5-oxyacetic acid methyl ester, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, 5-uracil acid oxyacetic, 5-pentynyl-uracil, and 2,6-diaminopurine. action of uracil as a substitute for a thymine base in deoxyribonucleic acid (hereinafter referred to as "dU") and considered as a form of pyrimidine "analog" with the present invention.
[00438] Aptamer oligonucleotides may contain analogous forms of ribose or deoxyribose sugars that are known in the art, including, but not limited to 2 'substituted sugars such as 2'-0-methyl-, 2'-0-allyl, 2' - fluoro-ou 2'-azido-ribose, carbocyclic sugar analogues, alpha-anomeric sugars, epimeric sugars, such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, closed nucleic acids (lNA), nucleic acid peptide (PNA), acyclic analogs and abasic nucleoside analogs, such as methyl riboside.
[00439] Aptameros can also include intermediaries in its synthesis. For example, any of the Xyl hydrocarbon groups normally present can be replaced by phosphonate groups, phosphate groups, protected by a standard protecting group, or activated to prepare additional bonds to additional nucleotides or substrates. The 5 'of the OH terminus is conventionally free, but can be phosphorylated; OH substituents at the 3 'terminus can also be phosphorylated. Hydroxyls can also be derivatized for standard protection groups.
[00440] One or more phosphodiester bonds can be replaced by alternative bonding groups. These alternative linking groups include, but are not limited to embodiments in which P (0) 0 is replaced by P (0) S ("thioate"), P (S) S ("Dithioate"), P (0 ) N 2 ("amidate"), P (0) R, P (0) OR ', CO or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted, alkyl (1 -20C). optionally containing a bonding ether (- O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl.
[00441] A particular embodiment of aptamers that are useful in the present invention is based on RNA aptamers as described in the U.S. Patent. Nos. 5,270,163 and 5,475,096, which are incorporated herein by reference. The aforementioned patents describe the SElEX method, which involves selecting a mixture of candidate oligonucleotides and stepped binding, partition and amplification iterations, using the same general selection scheme, to achieve virtually any desired binding affinity and selectivity criteria . From a mixture of nucleic acids, which preferably comprises a segment of random sequence, the SElEX method includes steps of contacting the mixture with a target, such as a target analyte, under favorable conditions for binding, partition of the nucleic acids unbound nucleic acids that specifically bound target molecules, dissociation of target nucleic acid complexes, amplification of nucleic acids dissociated from target nucleic acid complexes to obtain a mixture enriched with nucleic acid ligand and then resuming the steps of ligation, partitioning, dissociation and amplification through many cycles, as desired, to obtain highly specific acidic ligands of high nucleic affinity with the target molecule. In some embodiments, negative screening is employed in which a plurality of aptamers are exposed to analytes or other materials likely to be found, along with the target analytes in a sample to be analyzed, and only aptamers that do not bind are retained .
[00442] The SElEX method encompasses the identification of high-affinity ligands of nucleic acids containing modified nucleotides that confer improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics.
[00443] Examples of such modifications include chemical substitutions in ribose and / or phosphate and / or base positions. In some embodiments, two or more aptamers are joined to form a single, multivalent aptamer molecule. Multipurpose aptamer molecules can contain several copies of an aptamer, each copy targeting the same analyte, two or more different aptamers for different analytes, or combinations of these.
[00444] Aptameros can be used as diagnostic and prognostic reagents, as reagents for the discovery of new therapies, as control reagents in response to particular drugs, and as reagents for the discovery of new therapeutic targets. Aptameros can be used to detect, modify the function of, or inhibit or interfere with the function of, one or more target analytes. The term "analyte", as used herein, includes, without limitation, prodrug drugs, pharmaceutical agents, drug metabolites, such as expressed protein biomarkers and cell markers, antibodies, whey proteins, cholesterol and other metabolites, electrolytes , metal ions, polysaccharides, nucleic acids, biological analytes, biomarkers, genes, proteins, hormones or any combination of these. Analytes can be combinations of polypeptides, glycoproteins, polysaccharides, lipids and nucleic acids.
[00445] Aptameros can inhibit the function of gene products, by any of, but not limited to, the following mechanisms: (i) modulation of the protein-protein interaction affinity, (ii) modulate the expression of a protein a a level of transcription; (iii) modulating the expression of a protein at a post-transcriptional level, (iv) modulating the activity of a protein, and (v) modulating the location of a protein. The precise mechanism of action of peptide aptamers can be determined by biochemical and genetic means to determine their specific function in the context of their interaction with other genes and gene products.
[00446] Aptameros can be used to detect an analyte, from any of the detection schemes described here. In one embodiment, aptamers are covalently or non-covalently coupled to a substrate. Non-limiting examples of substrates to which aptamers can be attached include micro arrays, microspheres, pipette tips, sample transfer devices, vats, capillary tubes or other, reaction chambers, or any other suitable format compatible with the detection system object. Production Biochip microarray can employ several semiconductor manufacturing techniques, such as solid phase chemistry, combinatorial chemistry, molecular biology, and robotics. A process used and typically a manufacturing process for the production of micro photolithographic arrays with millions of probes on a single chip. Alternatively, if the probes are pre-synthesized, they can be attached to a matrix surface, using techniques such as micro-channel pumping, "inkjet" spotting, pattern-stamping, or photoreticulation. An exemplary photolithographic process begins by coating a quartz wafer with a light-sensitive chemical compound to prevent the coupling between the quartz wafer and the first nucleotide of the DNA probe to be created. A lithographic mask is used to inhibit or allow the transmission of light at specific locations on the surface of the wafer. The surface is then put in contact with a solution that may contain adenine, thymine, cytosine or guanine, and the coupling occurs only in the regions on the glass that have been unprotected by lighting. The coupled nucleotide supports a light-sensitive protective group, allowing the cycle to be repeated. In this way, the microarray is created as the probes are synthesized through repeated cycles of deprotection and coupling. The process can be repeated until the probes reach their full length.
[00447] Commercially available matrices are typically manufactured at a density of more than 1.3 million unique characteristics per matrix.
[00448] Depending on the requirements of the experiment and the number of tests required per matrix, each insert can be cut into dozens or hundreds of individual matrices.
[00449] Other methods can be used to produce the biochip. The biochip can be a langmuir-Bodgett film, functionalized glass, germanium, silicone, PTFE, polystyrene, gallium arsenide, gold, silver, membrane, nylon, PVP, or any other material known in the art that is capable of having functional groups such as amino, carboxyl, Diels-Alder reagents, thiol or hydroxyl incorporated on its surface. These groups can then be covalently linked to crosslinking agents, so that subsequent binding of the nucleic acid ligands and their interaction with the target molecules will occur in solution, without hindrance from the biochip. Typical cross-linking groups include ethylene glycol oligomer, diamines and amino acids. Alternatively, aptamers can be coupled to a matrix using enzymatic procedures, such as those described in US20100240544.
[00450] In some embodiments, aptameros are attached to the surface of a microperola. Microbeads useful in oligonucleotide coupling are known in the art, and include magnetic, magnetizable, and non-magnetic spheres. Microbeads can be marked with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dyes to facilitate the encoding of the granules and the identification of an aptamero attached to it. Microsphere coding can be used to distinguish at least 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 5000, or more different microspheres in a single assay, each Microbead corresponding to a different aptamero with specificity for a different analyte.
[00451] In some embodiments, the reagents are coupled to the surface of a reaction chamber, such as a tip. For example, the inner surface of a tip can be coated with a specific aptamer for a single analyte. Alternatively, the inner surface of a tip can be coated with two or more different aptamers specific for different analytes. When two or more different aptamers are coupled to the same surface of the inner tip, each of the different aptamers can be coupled at different known locations, such as the formation of distinct rings or bands arranged in different positions along the axis of a tip. In this case, multiple different analytes can be analyzed in the same sample by extracting a sample from one tip and allowing the analytes contained in the sample to bond with the coated aptamer in successive positions along the tip. Binding events can then be visualized as described here, with the location of each band in a pattern of bands corresponding to a specific known analyte.
[00452] In some embodiments, the connection of one or more aptamers to one or more target analytes is detected through an optical element. In some embodiments, the optical element and fluorescence. In some embodiments, a sample containing the analytes to be analyzed and treated with a labeling compound to combine the analytes with a fluorescent label. The binding can then be evaluated by fluorescence to detect the presence and optionally the amount of one or more analytes, such as that illustrated in Figure 136, in combination with aptamers coupled to a matrix, and in Figure 137, in combination with aptamers coupled to coded spheres. In some embodiments, the sample is treated with a labeling compound to couple the analytes with a binder. After ligation the ligand is functionalized with a fluorescent marker and the positive event is measured by fluorescence. In some embodiments, the binding domain of an analyte aptamer is partially hybridized with a complentary probe that is fluorescently labeled. After binding to the analyte, the complementary probe is released, which results in a measurable decrease in the optically fluorescent signal. In some embodiments, an aptamer is fluorescently labeled and partially hybridized with a complementary probe labeled with a suppressor that is in proximity to the fluorescent label. After binding to the analyte, the complementary probe is released, resulting in a measurable increase in the fluorescence of the marker conjugated to aptamero. In some embodiments, the aptamer is partially hybridized with a complementary probe, which causes hybridization occlusion containing a secondary structure domain. After attaching to the analyte, the complementary probe is released, and the secondary structure is made available to an intercalation dye used to produce a measurable signal. Labels useful in detecting the binding between an apatamer and an analyte in a binding pair may include, for example, fluorescein, tetramethylrodamine, Texas red, or any other fluorescent molecule known in the art. The level of marker detected at each address in the biochip will vary according to the amount of target analyte in the mixture to be tested.
[00453] In some embodiments, the complementary probe is displaced and conjugated to a member of an affinity pair, such as bio tin. A detectable molecule and then conjugated to the other member of the affinity pair, for example, avidin. After the test mixture is applied to the biochip, the detectable molecule conjugate is added. The amount of detectable molecule at each location on the biochip will vary inversely with the amount of target molecule present in the test mixture. In another embodiment, the displaced complementary probe will be labeled with biotin, and can be detected by the addition of fluorescently labeled avidin, the avidin itself will then be linked to another fluorescently labeled, biotin conjugated compound. The biotin group in the displaced oligonucleotide can also be used to bind an avidin-linked reporter enzyme, the enzyme will catalyze a reaction that leads to the deposition of a detectable compound. Alternatively, the reporter enzyme will catalyze the production of an insoluble product that will extinguish locally the fluorescence of an intrinsically fluorescent biochip. In another embodiment of the displacement assay, the displaced complementary probe will be labeled with an immunologically detectable probe, such as digoxigenin. The displaced complementary probe will then be linked by a first set of antibodies that specifically recognize the probe. These first antibodies will then be recognized and linked by a second set of antibodies that are labeled with fluorescence, or conjugated to a reporter enzyme. Many variations of these examples are now known or will occur to those skilled in the art. Assays analogous to the "double sandwich" EllSA tests can also be configured using combinations of antibodies and aptamers as receptors. For example, a capture surface can be functionalized with an aptamer and the detection reagent can be an enzyme-labeled antibody. On the other hand, the antibody can be on the capture surface and the detection reagent labeling an aptamer.
[00454] In some embodiments, a sample containing an analyte to be analyzed and dispersed in a three-dimensional hydrogel matrix. The hydrogel matrix can be activated to trap covalenly proteins and small molecules. After washing the excess and unbound sample, fluorescence-labeled aptamers can be introduced for the detection of the specific analytes present, as shown in Figure 138. In some embodiments, the three-dimensional hydrogel matrix is divided into small subsets or microcavities in which a single aptamero can be added to undergo a specific analysis of the present analyte. In some embodiments, aptameros are labeled with a set of encoded quantum dots or fluorescent markings corresponding to a single signature. In some embodiments, marked aptamers are added to the three-dimensional matrix, simultaneously with the sample.
[00455] In some embodiments, an aptamer is used instead of an antibody from an EllSA assay. In general, a sample is exposed to a surface and, specifically, or not specifically, coupled to it. In an EllSA sandwich, an analyte is specifically bound to a first antibody surface ligand, which is coupled to the surface. In a typical EllSA, the analyte, whether it bound specifically or non-specifically, and then detected by binding to a second antibody carrying a marker. In an EllSA aptamero, the first antibody, the second antibody, or both are replaced with aptamers specific for an analyte. Image analysis of samples, product reaction test
[00456] In some embodiments of the invention, analysis of samples and test reaction products can be performed using the digital image. The test cuvettes can be aligned for measurement and digitized or worked in a single operation. In the instrumented system of the invention this is achieved automatically by the mechanical components. Test tubes are located at defined locations on a cartridge and moved to the scanner while maintaining the same orientation and spacing. The graph shown in Figure 92 corresponds to the response of the green channel along the width of the tub. As shown, the edges of the cuvettes are well defined, as is the location corresponding to the middle of the cuvette.
[00457] The images obtained by scanning or the image can be a two-dimensional array of pixels, in which each pixel comprises a plurality of intensity values corresponding to a distinct spectral detection region (for example, red, blue, green). The images can be interpreted by the line-scans, which can correspond to a horizontal portion of a tip. If the tip is circular in shape, then an effective absorbance can be determined by deconvolution of the scanning line using an appropriate function. Example functions include parabolic functions and functions for circles. In some embodiments, images can be averaged over several images taken from a tip or a sample over a range of physical locations.
[00458] In one embodiment, a sensor is provided to locate a test unit relative to a detector when it is detected in a test.
[00459] As shown in Figure 61 and Figure 62, the bromophenol blue solutions were aspirated to a set of conical tips and photographed with frontal face illumination (light source and the detector on the same side of the object). Small volumes (5 µl) of serial dilutions of a 0.78 mg / ml solution were used with the highest concentration at the top of the image. In Figure 61, the left tip has the sample located at the widest position at the conical tip while the right tip has the sample the narrowest part. The image in Figure 61 was made using an optical scanning system.
[00460] Figure 62 shows tips that were photographed using a backlight setting (light source and the detector on opposite sides of the digitized object). The backlight setting may be preferred because of the higher image quality.
[00461] As shown in Figure 61 and Figure 62, the length of the effective optical path of a color solution can be varied by changing the projecting tip. In particular, the path length can be varied within a single tip to increase the sensitivity of light absorbance measurement (long path length) or to increase the dynamic measurement range. The path length can be changed, for example, by changing the tip diameter.
[00462] An additional feature of the tip design may be that it allows assays to be read with a very small volume of product from the test reaction requires a very small volume of the sample. Typically, the test reaction mixtures are incubated in a narrow part of a tip that provides a high proportion of liquid / air surface area to the volume, thus minimizing evaporation. The small volume can then be transferred to a large part of the tip for measuring the colored product, thus maximizing the length of the available optical path (and thereby increasing the absorption of light) for a given volume of reaction mixture.
[00463] For example, in the table below, which compares the reading of a 10 ul test reaction mixture in which a 1 ul sample is diluted 1: 10. At the ends of the current invention, the incubation of a test mixture can be reached in a mm length of the tip region with a diameter of one millimeter, then be transferred to a region 3 mm in diameter for color measurement 13. Compared to the use of a micro titration plate of dimensions normalized (typical of 384-well plates) to incubate and read the same test, the surface area of the liquid exposed to air (allowing evaporation) is about 5 times smaller and the length of the optical path is about twice as long. Optimizing the optical path length
[00464] Spectroscopic measurements of colored solutes are traditionally measured by recording the fraction of light transmitted through a cuvette at the maximum absorbance wavelength. The data is then transformed to give absorbance (A) or optical density (OD) values. According to Beer's law, A (max) = EM * l * Concentration where EM is the molar extinction product (l / Mole.cm), 1 and the length of the optical path (cm) and the concentration and in units molars. OD = A by 1 = 1. This is done to provide a measure, A, which is directly proportional to the concentration of the solute.
[00465] There are two significant limitations to absorbance measurements for solute dosages. At low concentrations, the change in transmission is small and therefore inaccurate, due to variations in the transmission (or white) background. At high concentration and very low transmission (for example, at A = 3, the transmitted light is 1/1000 o of the incoming light. Any light "stray" or other forms of noise signal has a significant effect on the measurement and the response to concentration becomes non-linear and inaccurate. Typically, absorbance measurements are considered to be accurate and precise over a range of about 0.1 to about 2.0 (on a 20-fold scale) .
[00466] The method of the present invention overcomes these problems, to a significant degree, allowing easy measurements of color over a wide dynamic range (up to 1000 times): 1. At different path lengths: low concentrations can be measured in long path lengths and high concentrations in short path lengths.
[00467]. In different color channels: low concentrations can be measured in the color channel best matched and high concentrations, in color channels not matched to color.
[00468] This situation is illustrated by the data presented in Figure 79. Blue solutions of bromophenol diluted in series from one mg / ml stock 5 were analyzed using the three-color method in tips in two locations, one with an extension of maximum path (also "path length" here) about 5 mm ("large"), the other about 1 mm ("narrow"). The signals from the three color channels have been normalized to the highest and lowest levels as shown in the following graph. An algorithm to optimally extract the concentration of the analyte (bromophenol blue) was established as follows:
[00469] For normalized signals in the maximum 10% range <maximum <90% signal, calculate a concentration value = a + b * log (signal) + c * (log (signal)) A 2, where B and C are arbitrary constants. This operation was performed for each color in both path lengths.
[00470] Using a well-known routine optimization (for example, "Solver" in Microsoft Excel), calculate the best fit values for a, b and c for all colors and path length.
[00471] The average concentration values calculated for all colors and the path length.
[00472] As shown in Figure 80, the method produced accurate results over a concentration range of 1000 times. When the algorithm was used to calculate the concentration values for repeated measurements (n = 3), the average CV was 3.5%.
[00473] Measurements can be made in various lengths of paths. In some cases, path lengths are at least partially dependent on the container (eg, cuvette, tip, flask) geometry. Container geometry and / or container features, such as dispersion characteristics, can affect the optical path and path length in the container.
[00474] The multicolor analysis Scanners and cameras have detectors that can measure a plurality of different regions of the color channel detection spectrum (for example, red, green and blue). Because the spectral width of each of these channels and the wide and chemical color to produce colored products with broadband widths, colored reaction products can be detected using a plurality of channel detection spectra. For example, Figure 71 shows the red (square), green (rhombus), and blue (triangle) response of the spectrum detection channel as a function of the analyte concentration. The signals produced by each detector correspond to the intensity of the light within each spectrum and the detection is typically expressed as a number from 0 to 255. When white light is transmitted through a tub of circular section, containing a colored solute, as shown above, the light is absorbed and the light intensity reduced so that the detector responses change.
[00475] For example, when bromophenol blue, dissolved in alkaline buffer, in concentrations ranging from 0 to 5 mg / nil and scanned at the location indicated "C3" in Figure 62, the signs shown in Figure 66, which are the detector responses measured over a zone corresponding to seven pixels along the length of the cuvette. The signals were recorded on a backlit Epson scanner. Figure 66 shows the three-color responses from a set of 11 cuvettes containing two series of dilutions of a "blank" solution (arranged from left to right in the image) to 5 mg / ml of bromophenol blue e solution. The image of the scanned tips is shown in Figure 67. The signal in each channel that corresponds to the solution is reduced to a point related to the optical path. Therefore, the maximum variation of the signal is considered in the center of the tank. When the signals in the central region of the vat were average (relative to the area indicated by the small rectangles for the fourth vat from the left) and plotted against the concentration of bromophenol blue, the dose-response curve shown in Figure 68 was observed. In each "channel" color of the signal fell well with the concentration. The green light changed more and the blue light less. Corresponding optical densities measured on an M5 spectrometer (Molecular Devices) at the maximum absorbance wavelength (for example, 589 nm) are also shown. At higher concentrations, the spectrophotometer response becomes non-linear and changes very little with concentration. A similar effect was seen in the scanner's green and red channel responses. The response of the blue channel in contrast, is very small until the highest concentrations.
[00476] According to Beer's law, absorption of a solution is equal to eM * Concentration * optical path. Absorbency is defined as log10 (Transmission / Transmission blank), where transmission is blank and that corresponding to the solvent. Strictly Beer's law applies to a parallel beam of monochromatic light (in practice, a bandwidth of a few nm) normally passes through a rectangular tub. Spectrophotometers respond linearly to concentration up to absorption values of about 1.5. At higher absorbency, the device's response becomes non-linear due to "diffuse light" and other effects. Optical density is defined as the absorbance for an optical path length of one centimeter.
[00477] When the color signal data from the previous experiment was transformed according to an expression that linearizes optical transmission in order to obtain an absorbance value proportional to the concentration in conventional spectrophotometry (-log (signal / signal plate)) , the graph shown in Figure 69 was obtained for the green (squares) and red (diamonds) channels.
[00478] The data of the green channel followed Beer's law, but the data of the red channel did not reach a stable level of a sample with about 2 mg / nil in a similar way to the DO response of the spectrophotometer.
[00479] Better use of the test by means of three-color analysis and optimization of the optical path length.
[00480] Results of testing reaction configurations that otherwise provide uninterpretable data can be recovered using the present invention. The present invention allows for a greater dynamic range and sensitivity of the tests by combining optimization of the optical path length and three-color analysis. The inability to recover data plagued by the reduced dynamic range and a major problem in trial management, especially in the context of samples to be evaluated for diagnostic or therapy management purposes and that the trials have a limited dynamic range or a limited range of values analytes that can be reported with good confidence. There are two main reasons why a test result may not be available from laboratory test systems or distributed test situations. That is, the value of the analyte is too high or too low to be reported. This can, under certain circumstances, be rectified in clinical laboratories, by reanalyzing a portion of a sample maintained using a different dilution. In normally distributed tests, there is no recourse, but to remind the patient, obtain a new sample and use a different method (laboratory). This is because the test systems use fixed protocols and fixed levels of sample dilution. In any situation, it is very inconvenient and expensive to correct the problem. In addition, information pertinent to valuable correct diagnosis and / or management of therapy can be lost with resulting damage to the patient.
[00481] In the system of the present invention, these problems are eliminated by monitoring the tests during their execution, recognizing any problem and altering either the extension of the optical path used to measure the test product, or making use of different levels of sensitivity of the tests. three color channels for the color assay and in turn with the sensitivity of the analyte.
[00482] In particular, when the test reaction product is measured, if the signal measured is too high or too low, the system can account for: 1. making the measurement with a different path length (moving the cuvet) optics relative to the optical system so that the length of the trajectory is greater or less). This can be accomplished by (a) taking a measurement at a first, standard location,
[00483] (b) present the result to the test management software (on the device and / or on the remote server), (c), which recognizes a problem condition and (d) which modifies the reading position and a second take measurement and / or; emphasizing a channel more or less sensitive to the color of the signal analysis. This can be performed automatically by suitable test analysis algorithms. Color calibration
[00484] The signal responses can be calibrated to allow the calculation of the concentration of the colored species from image data. To obtain a set of predictive data for transforming the concentration of the color solute, the following procedure can be used. In other embodiments, other methods can also be used.
[00485] For each channel for all concentrations, the transform-log (signal / white signal) was calculated and designated "A".
[00486] For all concentrations, another transformation ("C") was calculated as a * b * A + A A 2 + C * A A 3 (initially values for a, b and c were set at arbitrary values).
[00487] For all concentrations, the C values for the three color channels were added and designated Cestimate.
[00488] The sum of the square differences between the target (known) concentration and Cestimate was calculated over all concentrations.
[00489] The values of a, b and c of parameters for all channels were obtained by a known algorithm that minimizes the sum of the squares of the differences.
[00490] The results shown in Figure 70 demonstrate an accurate calibration of the digitizer response over the entire concentration range.
[00491] Other automated calibration algorithms have been developed and found to be equally effective. For example, the following is an example of a calibration for a cholesterol assay performed on a reaction tip.
[00492] The signal measured and decomposed into Red (R), Green (G) and blue (B), the color channels.
[00493] Calibration equations are calculated to optimize the accuracy, precision and dynamic range according to the design requirements of the assay.
[00494] In this test example, only the red and green channels are used to calculate the concentration. These two signals are transformed to calculate an intermediate variable (F), as follows: F = pl + p2 ^ G + p3 ^ G2 + p4 ^ R + p5 ^ R2, where p t are calibration parameters.
[00495] It is used to calculate the concentration of (C), through a linear transformation: where C and the calculated concentration, and pe and pj are the calibration parameters, in this case, representing the parameters of a linear intercept and slope, respectively.
[00496] When the same approach was followed by a large set of assays for a variety of analytes that produced colored products, spanning the entire visible spectrum, from -700 to 400 nm), comparable results were obtained.
[00497] In conventional transmission spectrophotometric measurements, an "empty" value is used to normalize measurements. Blank method (1) are typically constructed by measuring a sample that is equivalent to the sample, but has no component to be measured. The measurement is typically done in the same vat as the one that will be used for the sample, or an optically equivalent cuvette. Thus, in a spectrophotometric assay, it would be to combine all reagents at the same concentrations using the same protocol, replacing a zero analyte solution for the sample. Method (2) uses a two-step process making measurements against an absolute reference, such as air (which will never vary in absorbency) and measuring both blank and sample against the absolute reference. The absorbance of the sample is then calculated by subtracting the blank value from the sample. Method (3), to collect the spectra of the sample or product of the test reaction and reference the measured absorbance (or transmission) at an ideal wavelength (which is generally the maximum absorbance for the species measured) against the absorbance. at a wavelength, where the species is measured and known to have zero absorbance. The absorbance and the difference between those recorded at the two wavelengths.
[00498] The digital image and analysis of three colors can be used, but in some embodiments it can be modified according to the digital character (grainy) of the test signal. Namely: For each pixel of the image, for each color and a white and worked pattern and the signal intensities adjusted to a value corresponding to the absence of absorbance. This can be done using the following example procedure: a. adjusting the intensity of the light source adjust the sensitivity of the detector (preferred), or adjustment software (not preferred by itself).
[00499] A preferred approach is that of a combination of (b) and (c) above. First, adjust the detector in the analog range, and then fine-tune the result in the digital realm.
[00500] For the analog adjustment, and the gain of the displacement amplifiers between the light sensors and the analog to digital section are adjusted to ensure a maximum resolution of the scan. The lower end of the light range of interest will be set to zero and the upper end of the range will be adjusted to below sensor saturation.
[00501] Subsequently, the images can be adjusted in the digital domain. The preferred approach, specifically, would be to use what is called "two image calibration" for an mxn image. The mechanism is the first to collect a black image, blocking all light from the detector. We will call this image BLACK [m, n]. The second calibration and recorded image consists of a light at the maximum end of the sensitivity range.
[00502] We will call this image WHITE [m, n]. Thus, an image corrected at [m, n] can be constructed, pixel-wise, as: ric m, n] - BlA CK m, n] a [m, n = l 'J 1 l-ATJ- WHlTE [m, n] - BlA CK [m, n]
[00503] Note that this digital correction does not improve the dynamic range of the digitized data, but adjusts the values so that the total references of whites and blacks are consistent.
[00504] An image of a physical structure of a tip can be used as one pixel per pixel and color to white color. The blank can be: Air; Water; Reaction product of the blank test (without analyte); Blank sample (no test reagents), or a combination of the above; The signal coming from a color channel, where there is a null or weak response, can be used to normalize the signals coming from other channels.
[00505] Another method of controlling and normalizing the optics and image of a set of (stable) physical patterns, before or during a test. For example, a matrix of printed inks (shown in Figure 104) can be made for a corresponding set of standard colors, with normalized intensities (similar to the standard color "wheels" used to calibrate cameras and scanners).
[00506] Such standards can be measured using reflectance from an opaque surface, or (preferably) by transmission through a transparent film.
[00507] According to the stability of the optical system, calibration and normalization of the optical can be (1) an exercise at once, (2), performed at regular intervals, or (3) performed for each test. Calibration of a Digital lmager range
[00508] In some embodiments, methods may be provided to calibrate a digital camera used for optical image densities.
[00509] In optical density tests of a substance to be analyzed, it may be desirable to make use of as much of the dynamic range of the image generator as possible. Under normal conditions of use, the installation may comprise a relatively homogeneous background with white lighting, the image generator and the analyte to be tested in a transparent cuvette between them. Operationally, the test can include placing the vat between the image generator and the white background light source and measuring the amount of light absorbed by the analyte in the cell. To maximize the dynamic range of the sensor, the background can be detected as the maximum measurable intensity. It may be desirable to take care not to saturate the sensor, because then the information can be lost since when the sensor is saturated, and attenuation cannot be measured correctly. The system can be configured to maximize the efficiency of the measured values of the backlight, minimizing the number of saturated pixels.
[00510] The illuminated background can emit white light of equal intensity over its entire surface. The light emission can vary slightly, producing a normal distribution of pixel intensities, as detected by the image sensor. This is illustrated by the curves shown in Figure 128. For this example, the sensor can return a value from 0 to 256 for each pixel as an indicator of the amount of light it receives. Each pixel can saturate to a value of
[00511] That is, regardless of increasing the light intensity or the sensitivity of the sensor even more, only a value of 256 can be recorded. Serie 1 in Figure 128, the dashed line, indicates where the light is very intense, cutting the normal curve. Serie 3, the dashed line, indicates that all pixels are of correct reading intensity, but the sensitivity of the image generator is lower than it could be in the maximum dynamic range. Most pixels that are less than 200. Serie 2 represents the desired settings, in which the average of the distribution is as high as possible, but small enough that a number of pixels are saturated.
[00512] In one embodiment, the intensity of the lighting can be kept constant while the settings of the image player can be adjusted. For the purposes of sensor sensitivity, two controls can be used: exposure time and gain. Exposure time can be the amount of time that the sensor's pixels are allowed to collect the value before photons and read. For a given amount of light, the reading value may be greater than the exposure time and taken more. This control can be the "thick" control for the application. Gain can be control by adjusting the amount of amplification applied to the sensor signal. Increasing the gain can increase the value of the sensor signal. Gain can be "fine" control.
[00513] An exemplary procedure for setting the image generator's sensitivity parameters may include one or more of the following steps:
[00514] Set the exposure time to the known value to be below saturation. Set to gain higher usable value.
[00515] Binary search adjust upwards from the exposure time when finding the setting in which not all pixels in the region of interest of the image are saturated. This can be detected by observing the point at which the average pixel value becomes less than 256.
[00516] Back to win until incrementally until there are not enough pixels that are at the limit of saturation. The number of pixels with an acceptable level will be determined by the shape of the distribution. Standard deviation Product will increase the number of saturated pixels to be tolerated.
[00517] Then, the white balance can be corrected. There are three groups of sensors in a digital camera. The members of each group collect light of a different wavelength, red, green or blue. When detecting white light, the sensors prefer to see the same values or red, green and blue. The white balance control adjusts the relative gains of the red and blue channel. Since the light that comes from the backlight is set to white, the procedure would be to simply adjust the white balance up to the channels to read the same values. In practice, the green channel is typically left uncorrected, and the red and blue channels are changed in opposite directions to each other, as the control is changed. However, in other embodiments, another channel, such as the red or blue channel can be left uncorrected, while the other two channels can be changed.
[00518] Finally, the images can be adjusted in the digital domain. A preferred approach, specifically, would be to use what is called "two image calibration" for an X X image, as previously described.
[00519] Assays of making a variety of colored products were analyzed in the present invention. Colors from those with low maximum absorption wavelength (yellow) to high maximum wavelength (blue) have been successfully measured. Maximum wavelength of some representative tests was: 405, 450, 500, 510, 540, 570, 612 and 620 nm, demonstrating the ability to read color throughout the spectrovisible.
[00520] Colors can be quantified using average data for many pixels (typically around 1000). A parameter (f), which produces a good fit (for example, greater than R 2) with dose-response data can be selected. The parameter can be mounted with the first form of bl ai + * R * R + cl 2 + b2 * G + c2 * G 2 + b3 * B * B + C2 2, where a, b, c are constants and R, G and B are color intensity values for red, green and blue channels, respectively. The parameter f can then be derived, forcing it to have a maximum value of 1, and a minimum value of 0. Parameter f is related to the transmission of light through the colored reaction product. As might be expected, f may be closely related to the optical density (OD) parameter used in spectrophotometry to quantify an absorption species. When 1 - f measured by 3-color image and represented in function OD measured at the maximum absorption for the same test reaction products in a micro titerplate in a spectrophotometer, it can be observed that 1 - fe essentially linearly related to the outside diameter . In Figure 129, these data are presented for five trials. OD can be normalized by "relative OD" = (OD - OD min) / (ODmax - OD min). In some cases, there is a slightly curved relationship but the correlation coefficient (R) is generally> 0.99.
[00521] The parameter f can be used to calibrate the tests measured by 3-color image analysis. When plotted against the concentration of the analyte, a smooth calibration ratio can be shown in Figure 130, for a representative cholesterol test. A concentration equation form = a + b + c * f * f 2 (where a, b and c are constant), relative to the concentration of derivative f and, as shown in Figure 130, the calculated concentration and essentially identical to that of (expected, "nominal" value (slope regression line close to 1.0, intercept close to 0.0 and R 2 = 0.998. Also shown in Figure 130 are graphs of test precision and precision. Precision and almost 100% (average 100.2%) and imprecision (represented by% CV) and low (less than 10%, average CV 3.9%). Simultaneous imaging of assays
[00522] As shown in Figure 56, Figure 57, Figure 58, Figure 59 and Figure 60, several test elements (tips, wells, stains) can be worked in parallel. In general, the elements can be placed in known locations on a cartridge or mounted on a subsystem of the instrument, so that a particular element can be associated with a particular test. Even if the elements are not perfectly oriented or localized, image analysis can be used to correct such a failed positioning by locating the characteristics of the test elements.
[00523] The commercially available assays for albumin (Figure 56) and cholesterol (Figure 57) were used according to the manufacturer's instructions. A series of analyte concentrations in the range of clinical interest was measured using a set of calibrators in which the analyte concentration was reduced twice from the highest concentration. In Figure 56 and Figure 57, the concentration of the analyte was higher on the right side and on the left side more distant from the tip corresponded to zero analyte. The volume of the test reaction mixture aspirated to the tips was 20 µl.
[00524] Figure 58, Figure 59 and Figure 60 show that the wells can be viewed in parallel. A set of shallow hemispheric wells was made by machining a block of white opaque plastic. Three commercially available tests, which form color, were carried out in these wells and the reaction products worked. As stated above, the wells to the far right have the highest concentration of analyte and each well next to it has the lowest concentration of twice, except the most to the left as soon as it has zero analyte. Seven ul of the test reaction product was introduced into each well.
[00525] The reaction products can also be worked on after drying them onto porous membranes or paper and imaging, once the liquid has soaked in the medium. It is also possible to use any of a variety of test chemicals impregnated with paper or membranes and to image the products resulting from the reaction after adding the sample. Analyzing Turbidity
[00526] Turbidimetry is performed by measuring the reduction in the intensity of the incident light after passing through the sample being measured. This technique is used in which the test result is a dispersed precipitate which increases the opacity of the liquid.
[00527] Turbidimetry can be measured in latex agglutination tests. As a model of responses to the latex agglutination assay, polystyrene latex particles (1 µm in diameter) were dispersed in said buffer (w / v) and subjected to concentrations of three image analysis colors. As can be seen in Figure 72, a good response was observed in all three channels and can be used to measure the concentration of latex particles and latex agglutination. Analyzing Agglutination
[00528] Like the turbidity analysis, the system can be used to measure agglutination, hemagglutination and its inhibition.
[00529] The system can be used to perform blood typing by agglutination of red blood cells. The blood was diluted and mixed with the blood typing reagents (anti-A and anti-B, anti-D) from a commercial typing kit. As shown below for a B + blood, the appropriate agglutination responses can easily be seen, when mixtures are recorded. In addition, when the images shown in Figure 77 were checked along the vertical axis of the tips, a quantitative measure of agglutination can be obtained by measuring the variation of the three-color signals, as shown in Figure 78. More variance indicated agglutination and can be detected in each color channel. It is evident that the method can be used to measure the extent of such agglutination reactions. Recognition Form
[00530] The images can be analyzed as to the form of recognition. Shape recognition can be performed with normal magnification and very high magnification. In high magnification image analysis it can be used to recognize the shape and size of the cells. These techniques are commonly used in cell counting to determine the relative concentrations of red blood cells, white blood cells and platelets. Below normal size, the shape recognition is used to observe the state of the sample. Bubble defect recognition and other methods are used to ensure that the measured liquid amounts are aspirated and dispensed correctly. Analyzing samples of solid phase substrates
[00531] The digital image with front face illumination can also be used to read the test responses on solid phase supports, as shown in Figure 76. Potassium chloride solutions (0, 2, 4 and 8 mm) were added to eflotronTM potassium dosing strips (Boehringer-Mannheim / Roche) designed for use in a reflectance analysis system. Analyzing the quality of the sample
[00532] Certain characteristics of the sample may render the test results invalid. For example, hemolysis causes potassium fons to leak red blood cells into the plasma causing the measured plasma or serum potassium fon concentrations to be falsely high. Likewise, jaundice and lipemia can interfere with various color-forming chemicals, changing the measured absorbances. In the present invention, it is possible to detect and quantify such interfering substances, using image analysis. Tests that give false results can then either be either (1) eliminated from the list of results provided by the analytical system or (2) optical signals can be corrected to take into account the measured level of interferer. An image of different types of serum samples is shown in Figure 99 (from left to right: hemolysates, icemic lipemics (yellow) and "normal"). Digital data analysis
[00533] Conventional methods for generating calibration data and test methods that generate and / or change color typically measure an analog signal representing the change in absorbance characteristics of a test mixture generated by mixing a sample with the reagents. A portion of the reaction mixture is illuminated and the light transmitted through or reflected from it strikes a portion of the detector and evaluated as an analog signal. The quality of the test as determined by the volume and quality of the sample, treatment of the sample, the test set for the test mixture and the material element used to present the mixture to the optical system to count on an assumed quality of the physical system used.
[00534] In the present invention, we can image (1) the sample, (2) the sample processing processes, and (3) the test mixture and collect the data as a set of one or more digital images. Each pixel of the test mix image represents a very small fraction of the total, but the average signal of 3 color pixels of many, if it collects a test signal, at least as good as that obtained by conventional analog methods. Where, however, conventional methods lose information through the mean, the present invention both aggregates the information and maintains the detail lost by conventional methods. In this context, color based assays include assays for: metabolites, electrolytes, enzymes, Biomarkers (using immunoassay), drugs (by immunoassay), and nucleic acid targets (using "lAMP" technology). The same principles can be applied to tests using fluorescence and / or luminescence. Volume confirmation and correction
[00535] The volume of a sample, or any other material, such as a liquid or a solid, can be determined optically. This can be accomplished by imaging a container whose internal dimensions are known and mathematically determining the sample volume from the observed segment of the occupied container. Solid measurements are mainly used to measure solids that are centrifuged. The most common case is the reading of the volume of red blood cells centrifuged to determine the level of hematocrit. Examples 6-11 and 16 describe the use of image analysis to calculate the sample volume and other measurements. This can allow for an improvement in the test results. For example, if the target volume to be used is 10 μl and the technology of the invention determines that the actual volume is 8 μl, the test system can correct the results for the volume (in this example, the concentration of analytes in the calculated presumption of a 10 ul sample would be multiplied by 10/8).
[00536] The knowledge of the real sample and the reagent volumes can be carried out by the image of the sample and the reagents and can be used to correct the calculations used to detect and / or quantify the analytes in the sample.
[00537] As demonstrated in many examples above, the use of images allows samples and test mixes to be evaluated for the quality and response of the test. In addition, the image of "tips" used as reaction vessels and sample acquisition methods allows (1) the accurate and precise measurement of sample volume and reagents and (2) the use of such data to correct any inaccuracies and or inaccuracies in the test results due to volume errors. To achieve this, they can have tips with precise and precisely known geometry (as is the case for tips made by injection molding). replicating tip measurements using imaging has shown that their dimensions are accurate to better than about 1%. It is thus possible to measure the volume of liquid samples and reagents in these tips with corresponding precision. If pipetting of samples and reagents is less accurate and precise, correction of the results knowing the actual volumes (by image measurement) is possible.
[00538] For example, consider an assay in which the response is directly proportional to the analyte concentration (as is true for many of the assays discussed here). An error sample volume of 10% does not induce an error of 10% of the value reported by the analytical system. If, however, the sample volume is inaccurately dispensed and accurately measured (for example to within 2% of the actual value), the system response can be corrected to reduce the error by 10% to 2%. Corresponding corrections can be made to the volume errors of the reagent volumes. The correction algorithm may depend on the test system's response to the volume or knowledge of each test component (sample, reagents), but this information can be easily determined during the development and validation of the test.
[00539] In this way, the invention provides a variety of advantages over conventional techniques. In generating the "test signal", the present invention can detect physical defects in the test cuvette, defects in the test mixture (bubbles and the like). Once these defects are identified (image analysis), the test result can be rejected so that false results do not occur or (preferably) the effect of the defect can be eliminated and an exact test signal calculated.
[00540] In the assay mix as a whole, any and all defects can be detected including: incorrect sample type (eg blood versus plasma), incorrect sample volume, for a blood sample, the inability to separate plasma from figured elements (red and white cells), example factors that may compromise the quality of the test result (eg, lipemia lcteria, hemolysis, the presence of precipitates, or other unidentified homogeneities), defects in the assembly of the test mixture (for example, the presence of bubbles, insufficiency to mix properly (non-uniformity of color)), the mechanisms for evaluating the retrospective quality and preservation of detailed file information, the mechanisms for measuring sample and reagent volumes (and for correcting errors and / or inaccuracies in such volumes). Evaluate Therapeutic Agents
[00541] In a separate embodiment, devices and methods for controlling more than one pharmacological parameter useful for assessing the efficacy and / or toxicity of a therapeutic and delivered agent. For example, a therapeutic agent can include any substances that are of therapeutic and / or potential utility. These substances include, but are not limited to, chemical or biological compounds, such as simple molecules or organic or inorganic complexes, peptides, proteins (for example, antibodies) or a polynucleotide (for example, antisense). A wide range of compounds can be synthesized, for example, polymers, such as polypeptides and polynucleotides, and synthetic organics
[00542] Compounds based on various structures of the nucleus, and these can also be included as therapeutic agents. In addition, the various natural sources can provide compounds for screening, such as plant or animal extracts, and the like.
[00543] It should be understood, although not always stated explicitly, that the agent is used alone or in combination with another agent, with the same or different biological activity as agents identified by the inventive screen. The agents and methods are also intended to be combined with other therapies. For example, small molecule drugs are often measured by mass spectrometry that can be inaccurate. EllSA (based on antibodies), assays can be much more accurate and precise.
[00544] The physiological parameters according to the present invention include, without limitation of parameters, such as the temperature rate, heart rate / pulse, blood pressure, and airway. Pharmacodynamic parameters include concentrations of biomarkers, such as proteins, nucleic acids, cells, and cell markers. Biomarkers can be indicative of disease or it can be a result of the action of a drug. The pharmacokinetic parameters (PK) according to the present invention include, without limitation drugs and drug metabolite concentration. l Identify and quantify pharmacokinetic parameters in real time from a sample volume and extremely desirable for the adequate safety and efficacy of drugs. If drug and metabolite concentrations are outside a desired range and / or unexpected metabolites are generated due to an unexpected reaction with the drug, immediate action may be necessary to ensure patient safety. Likewise, if none of the pharmacodynamic parameters (PD) falls outside the desired range during a treatment regimen, immediate action may have to be taken as well.
[00545] The ability to control the rate of change in the concentration of an analyte or the parameters of PD or PK over a period of time in a subject, or to perform trend analysis for the concentration, PD, or pharmacokinetic parameters, if they are concentrations of drugs or their metabolites, can help you avoid potentially dangerous situations. For example, if glucose were the analyte of interest, the concentration of glucose in a sample at a given time, as well as the rate of change in glucose concentration over a given period of time, can be very useful in predicting and avoiding , for example, hyperglycemic events. Such trend analysis has widespread beneficial implications in drug dosing regimens. When various drugs and their metabolites are involved, the ability to detect a trend and take proactive and often desirable measures.
[00546] In some embodiments, the present invention provides a commercial method of assisting the clinician in providing individualized medical treatment. A business method may include post-prescription monitoring of drug therapy, monitoring trends in biomarkers over time. The commercial method may comprise the collection of at least one pharmacological parameter, from an individual receiving a medication, collection of this step and carried out by treating a sample of body fluid for the reagents contained in a fluid device, which is provided to said individual to produce a detectable signal indicative of said at least one pharmacological parameter, and cross references with the aid of a computer medical records of said individual with at least one parameter of said pharmacological individual, thus assisting in the clinical supply said individualized medical treatment.
[00547] The devices, systems and methods here allow automatic quantification of a patient's pharmacological parameter, as well as automatic comparison of the parameter with, for example, the patient's medical records, which may include a history of the monitored parameter, or medical records doctors from another group of subjects. Coupling real-time monitoring of the analyte with an external device, which can store data, as well as perform any type of data processing algorithm or, for example, provides a device that can assist in the treatment of the typical patient, which may include, for example, comparing current patient data with previous patient data. Therefore, also provided here is a business method that effectively performs at least part of the monitoring of a patient that is being performed by the medical team. Optical configuration for sample and reaction imaging products
[00548] The sample and analysis of the reaction product can be performed using an optical configuration. The optical configuration can include a light source, an aperture, and a sensor or detector. A schematic diagram of an optical configuration is shown in Figure 100 and Figure 101. In some embodiments, the camera can be a logitech CÓ00 Webcamera, the camera sensor can be 1/3 "2.0 MP (1600x1200) CMOS: glass (lM-2010-S), the lens can be with an object at a distance from the standard webcam lens (lens-to-Object distance: 35mm). The light source can be a Moritex white edge lluminator MEBl- Cw25 (white) operating at 9.4 volts Camera images can be taken in a sequence in which 1, 2, 3 4 or more. Tips are moved by an xyz phase in the optical path.
[00549] In one embodiment, the detector and a housing assembly of a detector reader assembly for detecting a signal produced by at least one test on the device. The detection set can be above the device or in a different orientation with respect to the base device, for example, the type of test to be performed and the detection mechanism to be employed. The detection set can be moved to communicate with the tester or the dosing unit can be moved to communicate with the detection set.
[00550] The sensors can be PMT, wide range of photo diodes, avalanche photodiodes, single frequency photo diodes, image sensors, CMOS chips, and CCDs. The light sources can be lasers, single color LEDs, wide frequency of light from fluorescent lamps or LEDs, LED arrays, mixtures of red, green, and blue light sources, phosphors activated by an LED, fluorescent lamps, incandescent lamps , and arc sources, such as a flash tube.
[00551] In many cases, an optical detector is provided and used as the detection device. Non-limiting examples include a photodiode, photomultiplier tube (PMT), photon count detector, avalanche photodiode, or charge-coupled device (CCD). In some embodiments, a diode pin may be used. In some embodiments a diode pin can be coupled to an amplifier to create a detection device with a sensitivity comparable to that of a PMT. Some tests can generate luminescence, as described here. In some embodiments it is detected by chemiluminescence. In some embodiments, a detection array may include a plurality of fiber optic cables connected as a bundle to a CCD detector or a PMT array. The optical fiber bundle can be constructed of discrete fibers or of many small fibers fused together to form a solid package. Such packages of solids are commercially available and easily interconnected for CCD detectors.
[00552] A detector can also comprise a light source, such as a lamp or light emitting diode (LED). The light source can illuminate a test to detect the results. For example, the assay can be a fluorescence assay or an absorption assay, as they are commonly used with nucleic acid assays. The detector may also comprise optics to provide the light source for the test, such as a lens or fiber optic.
[00553] In some embodiments, the detection system may comprise non-optical detectors or sensors for detecting a specific parameter of a subject. Such sensors can include temperature, conductivity, potentiometers, amperometric signals and signals, for compounds that are oxidized or reduced, for example, 02, H2 02, and l2, or oxidizable / reducible organic compounds.
[00554] The lighting can be backlit, front lit, and oblique (side) lit. Back lighting can be used in general chemistry for the purpose of detecting either light absorption (colorimetric) or scattering (turbidity). The arrangement takes two forms, a wide and uniformly illuminated rear field, and a specifically shaped beam that is interrupted by the subject. lit front lighting can be used for fluorescence and reflectance excitation. In reflection, a subject and illuminated from the front by a light source are measured by observing the reflected light from the object. The absorbed colors produce the same information as a liquid illuminated by a back light. In reflection, a subject can also be illuminated with oblique light. The use of oblique (side) lighting gives the appearance of a 3-dimensional image and features that can otherwise stand out invisible. A more recent technique based on this method is the Hoffman modulation contrast, a system found in inverted microscopes for use in cell culture. Oblique illumination suffers from the same limitations as bright field microscopy (low contrast of many biological samples; low apparent resolution due to out-of-focus objects), but the otherwise invisible structures can be highlighted.
[00555] In fluorescence excitation, individuals can be illuminated from the front for purposes of fluorescence lighting. These are usually single color lights, most commonly lasers. The Confocal laser Scanning Microscope is a common realization of this. oblique lighting can also be used in fluorescence excitation. In fluorescence cytometry, individuals are often excited at an angle, usually at 90 degrees, from which photons deteriorate. This form of lighting allows the detection of dispersion directly behind the subject (backlit), as well as the fluorescence emissions that come out from the side.
[00556] In some embodiments, the fluorescent light is photographed at 90 degrees in relation to the excitation beam. In Figure 102a, a photon source (S), typically a high intensity light-emitting diode, passes through a diffuser beam (D) and a molding lens (ll), producing an excited or slowly diverging collimated beam. . The excitation beam passes through a bandpass filter (Fl) and illuminates the sample, which consists of a container (tube, cuvette, or pipette tip) containing a solution with a sample marked with fluorescence. Isotropic-emitted fluorescence and separated from the excitation light spectrum with a long filter or band-pass (F2) suitable for passing shifted Stokes fluorescence. The light is then photographed through a lens (l2) to a digital camera (C) or another detector. The fluorescence intensity is extracted from the resulting images through image analysis.
[00557] images taken with the optical configuration shown in Figure 102A produces images from a single tube (as shown in Figure 103 A. successive experiments show the difference in fluorescence intensity from negative and positive lAMP experiments using dye interleaving.
[00558] In other embodiments, the light transmitted and processed after optical filtering to remove the light at the exciting wavelength. In Figure 102B, a photon source (S), typically a high intensity light emitting diode, passes through a diffuser beam (D) and a molding lens (ll), producing a slowly divergent, excitation beam. elliptic. The excitation beam passes through a bandpass filter (Fl) and illuminates the samples, presented as a series of sample vessels (tube, cuvette, or pipette tip), each containing a solution with a sample marked with fluorescence . Isotropic-emitted fluorescence and separated from the excitation light spectrum with a long filter or band-pass (F2) suitable for passing shifted Stokes fluorescence. The light is then photographed through a camera lens (l2) to a digital camera (C). The fluorescence intensity is extracted from the resulting images through image analysis. The optical configuration shown in Figure 103 can be used to produce matrix images of multiple tubes simultaneously (as shown in Figure 103B).
[00559] For colorimetry, the preferred mode for detecting and backlighting the subject with white light with the result perceived by an image sensor. In this case, color absorption and transmissive measurement.
[00560] For Turbidimetry, the preferred mode for detecting and backlighting the subject with white light with the result perceived by an image sensor. For turbidimetry, the reduction in the intensity of the emitted light is measured.
[00561] Luminometry uses no method of illumination as the subject emits its own photons. The light emitted can be weak and can be detected using an extremely sensitive sensor, such as a photomultiplier tube (PMT).
[00562] In some forms of realized, the image can occur through fluorescence, darkiield lighting, bright field lighting or. This image can be used for other applications or cytometry. epi-fluorescence lighting can be achieved through the use of three lighting sources, of different wavelengths. In addition, two different sources can be used simultaneously, if necessary. Therefore, the imaging platform can be used to image a wide variety of fluorescent dyes. The combination of light sources, emission optics can be configured to achieve a plurality of independent image spectrum channels.
[00563] Dark field lighting can be achieved by using a ringlight (located above or below the sample), a dark field Abbe capacitor, a dark field capacitor, with a circular shaped mirror, an epi-darkfield capacitor built in a sleeve around the objective lens, or a combination of ringlight with a condenser equipped with a dark stop phase. Fundamentally, these optical components create a cone of light from the numerical aperture (NA) larger than the AN of the objective to be used. The choice of lighting system depends on a number of considerations, such as the required magnification, mechanical design considerations, the size of the image sensor, etc. A ringlight based lighting system generally provides uniform dark field illumination over an area wider and, at the same time, providing sufficient flexibility in the mechanical design of the overall system.
[00564] Brightfield lighting can be achieved by using a white light source, along with a condenser stage to create Koehler lighting.
[00565] In some embodiments, an automatic filter wheel may be employed. The automatic filter wheel allows the control of the optical image path to allow images of several fluorophores in the same field of view.
[00566] In some modalities, the image based on auto-focus can take place. An image-based algorithm can be used to control the Z position (for example, the vertical position) of an objective (ie, its distance from the sample) to achieve autofocus. Briefly, a small image (for example, 128x128 pixels) and captured at a fast pace using darkfield lighting. This image can be analyzed to obtain the auto-focus function, which is the measure of image sharpness. Based on a quick search algorithm and calculated the following z location of the objective. The objective can be transferred to the new z location and another small image can be captured. This closed-loop system does not require the use of any other equipment for focusing. The microscope stage can be connected to a computer controlled by stepper motors to allow translation in X and Y directions (for example, the horizontal directions). At each location, the desired number of images is captured and the phase is moved to the next XY position.
[00567] Image or other detection can be performed with the aid of a detector. A detector can include a camera or other detection device configured to convert electromagnetic radiation into an electronic signal. In one example, a camera can be a charge coupled (CCD) or electron-multiplication CCD camera (EMCCD). The detector can be a sensor, such as an active pixel sensor or the CMOS sensor. A detector can include a photomultiplier tube for detecting a signal.
[00568] The detector may be in optical communication with a sample container (for example, cuvette, tip, flask). In some cases, the detector is in direct line of sight of the sample container. In other cases, the detector is in optical communication with the sample container, with the aid of one or more optics, such as lenses, mirrors, collimators, or combinations thereof.
[00569] Cell count can be performed using imaging and cytometry. In situations where the patient can be illuminated in a bright field, the preferred embodiment is to illuminate the materials from the front with white light and to detect the cells with an image sensor. Subsequent digital processing will count the cells. When cells are rare or small, the preferred method is to attach a fluorescent marker and illuminate the field of the subject with a laser. Scanning confocal image is preferred. For flow cytometry, the subjects were marked with fluorescent markers and flowed past the detection device. There are two types of sensors, a position such that the subject is backlit, the beam scatter to determine the presence of a cell. The other sensor, aligned so that the illumination and from the side, measures the fluorescent light emitted from the marked subjects. More detailed description and provided below, related to the image cytometry methodology. End user systems
[00570] A device can be supplied after the manufacture and the system, for the end user, together or individually the device or system of the invention can be packaged with a manual or instructions for use. In one embodiment, the system of the invention is generic to the type of tests performed on different devices. Because the device components can be modular, a user may only need one system and a variety of devices or assay units or reagent units to perform a series of assays in a point of care or other distributed test environment. In this context, a system can be used repeatedly with several devices, and it may be necessary to have sensors on both the device and the system to detect such changes during transport, for example. During the change of transport, pressure or temperature it can affect the performance of a number of components of the present system, and as such a sensor located on the device or system can relay these changes, for example, the external device so that the Adjustments can be made during calibration or during processing on the external data device. For example, if the temperature of a fluid device is changed to a certain level during transport, a sensor located on the device can detect this change and transmit this information to the system when the device is inserted into the system by the user. There may be an additional detection device in the system to perform these tasks, or such a device may be incorporated into another component of the system. In some embodiments, information can be transmitted wirelessly to the system or external device, such as a personal computer or a television set. Likewise, a sensor in which the system can detect similar changes. In some embodiments, it may be desirable to have a sensor in the transport package, as well as, or instead of, or in addition to the system components. For example, adverse conditions that would render a test cartridge or system invalid that can be detected may include exposure to a temperature higher than that in which the maximum tolerable penetration or break in the integrity of the moisture cartridge.
[00571] In one embodiment, the system comprises a communication set capable of transmitting and receiving information wirelessly from an external device. Such wireless communication can be Bluetooth or RTM technology. Various communication methods can be used, such as a wired dial-up connection with a modem, a direct connection, such as a Tl, lsDN, or cable line. In some modalities, the wireless connection is established through exemplary wireless networks such as cellular, satellite or pager networks, GPRS, or a local data transport system, such as Ethernet or Token Ring on a local area network. In some embodiments, the information is encrypted before being transmitted over a wireless network. In some embodiments, the communication module may contain a wireless infrared communication component for sending and receiving information. The system can include integrated graphics cards to facilitate the display of information.
[00572] In some embodiments, the communication module may have a memory or other storage device, for example, located RAM, in which the collected information can be saved. A storage device may be necessary if the information cannot be transmitted at a given time, due, for example, to a temporary inability to connect wirelessly to a network. The information can be associated with the device identifier on the storage device. In some embodiments, the communication module may repeat the sending of the stored information after a certain period of time.
[00573] In some configurations an external device communicates with the communication module within the reader set. An external device can wirelessly or physically communicate with a system, but it can also communicate with third parties, including, without limitation, the patient, medical staff, doctors, laboratory personnel, or others in the healthcare industry.
[00574] In some embodiments, the system may comprise an external device, such as a computer system, server or other electronic device capable of storing information or processing information. In some configurations the external device includes one or more computer systems, servers or other electronic devices capable of storing information or processing information. In some embodiments, an external device may include a database of patient information, for example, but not limited to medical records or patient history, clinical trial records, or pre-clinical trial records. An external device can store protocols to be executed in a system that can be transmitted for the assembly of a communication system in which it received an identifier that indicates which device was inserted in the system. In some embodiments, a protocol may be dependent on a device identifier. In some embodiments, the external device stores more than one protocol for each device. In other embodiments of patient data on the external device it includes more than one protocol. In some cases, the server stores external mathematical algorithms to process a photon count sent from a communication module and, in some embodiments, to calculate the analyte concentration in a body fluid sample.
[00575] In some embodiments, the external device may include one or more servers, as they are known in the art and are commercially available. These servers can provide load balancing, management tasks, and support capability in the event of failure of one or more of the servers or other components of the external device, to improve server availability. A server can also be implemented in a distribution network of storage units and a processor, as known in the art, in which the data processing according to the present invention resides on workstations, such as computers, thus eliminating the need for of a server.
[00576] A server can include a database and process system. A database can reside on the server, or it can reside on another server system that is accessible to the server. Since the information in a database can contain sensitive information, a security system can be implemented that prevents unauthorized users from gaining access to the database.
[00577] An advantage of some of the features described here and that the information can be transmitted from an external device to the rear, not only to the reader assembly, but to other parts or other external devices, for example, without limitation, a PDA or cell phone. Such communication can be carried out over a wireless network, as disclosed herein. In some embodiments, a calculated patient analyte concentration or other information can be sent to, for example, but not limited to, medical personnel or the patient.
[00578] In this way, the data generated with the use of devices and systems of subjects can be used to carry out a trend analysis of the concentration of a substance to be analyzed in a subject that changes over time.
[00579] Another advantage as described here is that the test results can be substantially immediately communicated to third parties who may benefit from obtaining the results. For example, since the concentration of the analyte is determined in the external device, it can be transmitted to a staff of patients or doctors who may need to take further measures. The step of communicating to third parties can be performed wirelessly as described here, and to transmit the data by hand to a third performed device, the third party can be notified of the test results almost anytime and anywhere. Thus, in a time-sensitive setting, the patient can be contacted immediately anywhere if urgent medical intervention is needed.
[00580] As described here, the image can be used for detection. lmagiology can be used to detect one or more characteristics of a sample. For example, the image can be used to detect the presence or absence of a sample. The image can be used to detect the location, placement, volume or concentration of a sample. The image can be used to detect the presence, absence, and / or concentration of one or more analytes in the sample.
[00581] In some embodiments, a single measurement can be used to capture various information about a sample and / or analytes. For example, a single measurement can be used to capture information about the volume of a sample and the concentration of an analyte in the sample. A single measurement can be used to capture information about the presence and / or concentration of a multiplicity of analytes and / or types of analytes within the sample. A single image can be used to capture information relating to one, two, or more of the information or types of information described here.
[00582] This imaging and detection tests can provide more precise and accurate, which can be advantageous in situations with small sample volumes, such as those described in this document. Other examples of sample volumes may include μf 500 or less, 250 μf or less, 200 μf or less, 175 μf or less, 150 μf or less, with 100 μf or less, 80 or less μf, μf 70 or less, 60 μf or less, 50 or less μf, μf 30 or less, 20 or less μf, μf 15 or less, 10 or less μf, 8 μf or less, 5 or less μf, 1 μf or less, 500 nl or less, 300 nl or less, with 100 nl or less, 50 nl or less, 10 nl or less, 1 nl or less, 500 pl or less, 250 pl or less, with 100 pl or less, 50 pl or less, 10 pl or less , 5 pl or less, or 1 pl or less. In some embodiments, the sample volume may include less than or equal to about 3 drops of a finger prick, less than or equal to about 2 drops from a finger prick, or less than or equal to about 1 drop of a finger prick. These small volumes may be useful in service point applications.
[00583] This image and / or detection can produce tests with a low coefficient of variation. A coefficient of variation can be the relationship between the standard deviation and an absolute value of the mean. In one embodiment, a reaction and / or test may have a coefficient of variation (CV) (also "relative standard deviation" here), less than or equal to about 20%, 15%, 12%, 10%, 9 %, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, or 0.1%. A single reaction and / or test or a procedure with a plurality of reactions and / or the tests can have a coefficient of variation of less than or equal to about 20%, 15%, 12%, 10%, 9% , 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, or 0.1%. In some embodiments, an image and / or detection step, or a procedure with a plurality of images and / or step detection may have a coefficient of variation of less than or equal to about 20%, 15% , 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, or 0.1%.
[00584] In some embodiments, the use of the image with a device that can be placed at a service location point can improve the overall performance of the device. Accuracy and / or precision can be improved and / or the coefficient of variation can be reduced. The performance of the device can be improved when handling small samples such as those described here. The image can be used in combination with other detection systems, in combination with other processes, or as an independent system. Performance improvement may include a reduction in the coefficient of variation of about 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, or 0.1%.
[00585] Image can be useful for various types of detection of one or more types of tests or sample handling procedures. Examples of such assays or sample handling procedures may include centrifugation, separation, immunoassay cytometry, EllSA, nucleic acid assay, enzyme assay, colorimetry, or any other type of assay or reaction described elsewhere in this document.
[00586] Imaging systems can provide several advantages over other methods of data collection, data processing and interpretation of results. Imaging systems can maximize or increase the efficiency of, the use of small samples and improve performance at the system level. Imaging systems can be used for detection as stand-alone systems or can be used in combination with other detection systems or mechanisms.
[00587] In some systems, sensors and systems can be used (such as photodiodes and photomultiplier tubes and optics / associated devices), which normally do not provide any spatial information about the sample being interrogated. Instead, these systems can collect information about the sample after the information has been spatially integrated, usually the loss of spatial information related to the sample. While integrating the space signal from the sample can increase the signal levels to be detected by the sensor, advances in the sensitivity of optical sensors and others can negate the need for such integration. Imaging for detection can be used in place of such sensors, or can be used in conjunction with such sensors.
[00588] Imaging systems can be used that can advantageously have one or more of the following characteristics. Image sensors can have sensitivity and dynamic range that are and / or higher than that of non-conventional image sensors. Imaging devices can maintain the spatial aspects of the sample being interrogated, providing significant capacity for post-processing. Post-processing may include QA / QC (eg, quality control, such as automated error detection and / or review by a pathologist), and / or image analysis to extract characteristics from specific samples. The imaging device can use 3D, 2D, lD (line sensors), and / or point sensors with a means to convert the sample with respect to the collection / sensor optics, to allow spatial reconstruction of the sample. The data obtained from the imaging device can be processed to extract very specific information, such as the morphological characteristics of the sample (such as cell count), data selected from regions of the image (peak fluorescence through a sample or in a cell within the image). The data obtained from the imaging device can be processed to improve the sensitivity and resolution of the measurement. The data collected from the imaging device can allow the evaluation of the signal variation in the entire sample to be worked on. The data can be post-processed to calculate mean, standard deviation, maximum, minimum and / or other statistics applicable across the sample, or within any regions of interest identified in the sample images. Imaging devices allow the exploration of variations in the sample over time by collecting multiple images and comparing the variation of images over time and space, as would be evident in an aggregation process (for example, for a time trial) prothrombin) or others (eg, electrical, morphological changes) of the sample over time and in the chemical, physical, biological space. Imaging devices can allow you to acquire matrices, tissue sections, and other test / sample configurations faster. Cytometry application
[00589] In some embodiments, any of the embodiments described here can be adapted to allow the system to perform cytometry. Cytometry (for example, counting and analyzing cell function) in the system can be performed using image analysis. The blood can be processed using the pipette and centrifuged as previously described in this document. Typically, a known volume of blood measured (1-50 µl) can first be centrifuged and the plasma fraction removed. The cell fraction can THEN be resuspended in the pipette using buffer repeatedly to distribute and aspirate. A cocktail of fluorescent antibodies can be directed to selected cell markers (such as CD45, CD4, etc.). After a brief incubation, a reagent that can act as a fixative for white cells and a red blood cell lysis agent can be added. . After another incubation of white cells, they can be collected by centrifugation and the supernatant has been removed hemolysate by aspiration. Stained white blood cells can be resuspended in a measured volume of buffer solution (usually less than the original blood volume (eg 1 - 20. Ul) and distributed in transparent capillary channels for image analysis, typically up to three or even five or more cell types can be visualized using antibodies with different fluorescent tags or and / or antibodies labeled with different fluorine / protein ratios.When more cell types have to be counted or analyzed, more than one reaction mixture can be used. In some embodiments, a reaction mixture can be used to count or analyze various numbers of cell types.
[00590] In some embodiments, the capillary channels are typically about 10 - 100 µm deep, 0.5 - 2 mm wide and 0.5 - 5 cm long. Capillary channels can have other dimensions, including but not limited to other dimensions described here elsewhere. The dispersion of stained cells can fill the channel normally by capillary action and the cells can be allowed to settle on the lower surface of the channel. The channels can be illuminated with one or more lasers or other light sources (for example, LEDs). The optical train may have one or more optical elements, such as dichroic lenses or mirrors, and may or may not increase the field of view. In some embodiments, the field of view can be enlarged 2-100 times. A series of images can be collected normally representing a field of view of about 1 mm x 0.5 mm, and comprising 1 - 10,000 cells (preferably 300 cells of interest) photographed for a sensor having an area of about 1000 x 1000 pixels (1 million in total).
[00591] A series of images representing adjacent sections of the channel can be collected. A mechanical phase can be used to move the channels in relation to the light source. In some cases, a servomechanism can move the phase in a vertical direction, in order to focus the image. In some embodiments, the light source or one or more optical elements can move in relation to the phase of focusing the image. Images are usually made using one or more combinations of light sources and optical filters. The light sources can be turned on and off and the filters moved to the light path when needed. Preferably, up to 1000 cells of a given type can be counted. In other embodiments, various numbers of cells of any type can be counted, including, but not limited to, more than, less than, or equal to about 1 cell, 5 cells, 10 cells, 30 cells, 50 cells , cells 100, 150 cells, 200 cells, 300 cells, 500 cells, 700 cells, cells 1000, 1500, cells 2000, 3000, 5000 cells. The cells can be counted using available counting algorithms. The cells can be recognized by their characteristic fluorescence, size and shape. Pattern recognition algorithms can be employed to exclude stained cell debris and in most cases where there are cells that are aggregated or they can be excluded from analysis or interpreted as aggregates.
[00592] The cytometry platform can be an integrated automated microscopy device capable of the following tasks in a fully automated and controlled environment. One or more of the following tasks can occur in cytometry applications. The following tasks can occur in the order in which they appear or in alternate orders, or other tasks can be substituted, as appropriate. l Insulation of blood cells of the desired type
[00593] Marking cells with dyes and / or fluorescent and / or colored spheres Confining cell suspension in an optically compatible cuvette cell filming using fluorescence microscopy, dark field illumination and / or bright field illumination Automatic analysis images to extract desired cellular attributes Automated analysis of information extracted using advanced statistical and classification methods to obtain clinically reportable information.
[00594] In the following sections, each of these tasks and discussed in greater detail, images and sketches are provided whenever necessary.
[00595] Isolation of blood cells of the desired type. Blood cells of a desired type can be isolated according to one or more of the embodiments described elsewhere elsewhere. For example, such isolation can occur as referred to in the previous descriptions related to cytometry or the centrifuge.
[00596] The labeling of cells with dyes and / or fluorescent and / or colored spheres.
[00597] Specific fluorescent dyes can be employed. Cells of interest can be incubated with pre-aliquot solutions of fluorescently labeled ligands (for example, antibodies, aptamers, etc.), which are specific for markers on those cells. An important consideration may be the "brilliant" pairing or higher extinction coefficient and high quantum yield Fluor with cell markers for which it has a lower binding capacity, and vice versa, for example, the CD22 marker can be expressed in lymphocytes B at about a tenth the CD45 level. Given this relative expression, CD22 can be labeled with a "bright" dye and CD45 can be labeled with a "dimmer" dye. The markers to be labeled with this technique can be either intracellular or cell surface markers. The detection and quantification sensitivity can be improved through a secondary expression labeling system for low markers. Briefly, a primary ligand can be conjugated to another molecule that can be recognized specifically by a secondary ligand. A secondary ligand labeled with a greater number of fluorophores can then bind the primary ligand in situ and improve the fluorescence signal. One scheme for carrying out this may be the use of conjugated anti-CD22 antibody biotin which in turn can be recognized by an antibiotic antibody, which is labeled with fluorescein isothiocyanate (lTCF). Use can dramatically improve the fluorescence signal. Fig. 123 is an example of a fluorescence micrograph showing labeled leukocytes. The example illustrates an Alexa Fluor-647-anti-CD45 fluorescence micrograph of labeled human leukocytes in a fixed lysed blood sample. The pseudo scheme is used to increase the perception of the difference between 'shiny' cells (with high CD45 expression) and 'dim' cells (with low CD45 expression).
[00598] Color smears of cell smears can also be employed within the system. For example, the procedure indicated in the Sto 'Do M Wright-Giemsa manual (Polysciences lnc.) Can be automated and read on the devices of the present invention.
[00599] In some embodiments, non-specific fluorescent dyes can be used. For the purposes of differentiating leukocyte subpopulations, the platform may also use fluorescent dyes, which can bind to nucleic acids (eg, SYTO, Hoechst) or lipid membranes (eg, Dil, DiD, FM-4-64) .
[00600] Confinement of cell suspension in an optically compatible cuvette.
[00601] In some embodiments, cytometric cuvettes can be designed to confine a fixed volume of pre-labeled cell suspension to a «channel» manufactured to provide an optically transparent material for imaging above and below the cells. Sample can be introduced into the channel through a sample inlet port. At some distance from the sample inlet port, an air purge can allow the release of air pressure and sample flow within the channel.
[00602] The channel dimensions can be designed to contain a previously defined known volume of fluid, regardless of the volume dispensed at the sample inlet port. Each cuvette can have multiple channels of the same and / or different volumes, each with at least one sample inlet port and at least one air vent.
[00603] The concentration of cells of interest in the sample can be adjusted during sample preparation in such a way that after delivery, in the cuvette, a desired number of cells per field of view of the imaging system can be achieved. One method of doing this can be to image a container with cell dispersion and measure turbidity. Using a pre-established relationship between turbidity and cell count, cell density can be calculated. Normally, cell dispersion will be done in a volume of buffer such that, with the least number of susceptible cells, and the concentration of cells will be greater than ideal for image-based cell counting. More buffer can then be added to bring the dispersion to the optimum level.
[00604] The image area and the cuvette can be designed so as to provide a sufficient number of cells for the application of interest. For example, counting abundant red blood cells may require only a cell count of 1000-2000 and, consequently, a diluted sample and only a small area of the cuvette image. However, counting rare myeloblasts may require, in some cases, the ability to image more than 100,000 (total) cells. In such a situation, the system can concentrate the cell suspension so that 100,000 cells can be worked with a reasonable number of fields of view. Therefore, the dedicated channel of the cuvette with the RBC image will be smaller than the one dedicated to the image myeloblasts.
[00605] The cuvette can be designed to be removed using a standard pipetting mechanism in an automated way to allow the transfer of the tub to the imaging platform. Ejecting tip of the pipetting mechanism can eject the crucible from the pipetting mechanism onto the imaging platform. Vessel registration for the imaging platform can take place in two stages. After transferring the tub to the imaging platform, the static recording characteristics on the cuvette interface can, with mating characteristics on the imaging platform to align the cuvette parallel to the optical axis of the imaging platform (X, Y registration). The registration can then be completed by a mechanism located on the imaging platform. This mechanism can influence the cell against a flat surface perpendicular to the optical axis of the imaging platform (Z register), thereby restricting the sample within the focal range of the imaging platform.
[00606] image of cells using fluorescence, darkfield lighting, bright field lighting. The cell imaging method can also be applied to other applications of the invention described elsewhere herein. Imaging techniques, as described earlier, can be used for other imaging uses.
[00607] Lighting capabilities: The cytometry platform can be designed to have three types of lighting systems: epi-fluorescence, dark field and bright field. The modular nature of the configuration also allows for the integration of phase contrast and differential interference contrast (DlC).
[00608] The epi-fluorescence of illumination can be achieved through the use of three laser lines (for example, 488 nm, 532nm and 640nm), but the modular nature of the system also allows the integration of other light sources, such as other laser sources, the LEDs and standard arc lamps (for example, Xenon, Mercury and halogen). In addition, two different sources can be used simultaneously, if necessary. Therefore, the cytometry platform can be used to image a wide variety of fluorescent dyes. The combination of light sources, emission optics can be configured to achieve various numbers (eg 3-5) spectrally independent image channels.
[00609] Dark field lighting can be achieved by using a ringlight (located above or below the sample), a dark field Abbe capacitor, a dark field capacitor, with a circular shaped mirror, an epi- darkfield capacitor built in a sleeve around the objective lens, or a combination of ringlight with a condenser equipped with a dark stop phase. Fundamentally, these optical components can create a cone of light of the numerical aperture (NA) larger than the AN of the objective to be used. The choice of lighting regime depends on a number of considerations, such as the required magnification, mechanical design considerations, or the size of the image sensor. A base ringlight lighting scheme generally provides uniform dark-field lighting over a larger area, while at the same time providing sufficient flexibility in the mechanical design of the overall system. Figure 124 provides an example of intracellular patterns using dark field images. The example shows different intracellular patterns in dark field images of human leukocytes, (a) a strong scattering pattern due to the presence of eosinophil granules, (b) polymorphonuclear neutrophils with the characteristic nucleus lobes and (c) the cells that do not scatter light to a significant degree (lymphocytes or basophils) Brightfield lighting can be achieved by using a white light source, along with a condenser stage to create Koehler lighting. Figure 126 is an example of brightfield images of human whole blood. The example shows that the bright field images of a human whole blood smear stained with the Wright-Giemsa staining method. Characteristic patterns of staining of human leukocytes are apparent. The red blood cells, characteristically, in shape can also be identified in these images.
[00610] Automatic filter wheel: An automatic filter wheel can allow the control of the optical image path to allow images of several fluorophores in the same field of view.
[00611] The image based autofocus: The cytometry platform can use an image based algorithm to control the Z position (for example, the vertical position) of the objective (ie, its distance from the sample) to achieve auto focus. Briefly, a small image (for example, 128x128 pixels) can be captured at a fast rate using dark field lighting. This image can be analyzed to obtain the auto-focus function, which can be used to measure image sharpness. Based on a fast search algorithm, the next z location of the objective can be calculated. The sample can be transferred to the new z location and another small image can be captured. In some embodiments, this closed-loop system does not require the use of any other equipment for focusing.
[00612] The translation of the step: The microscope stage can be connected to a computer controlled by stepper motors to allow translation in X and Y directions (for example, the horizontal directions). At each location, the desired number of images can be captured and the stage can be moved to the next XY position.
[00613] Image sensor: a camera with a CCD, EMCCD, CMOS or, in some cases, a photomultiplier tube can be used to detect the signal.
[00614] Image analysis to extract desired cellular attributes.
[00615] The cytometry platform can use different lighting techniques to acquire images that reveal different properties and characteristics of the cells. Marking with specific cell-linker markers may reveal the degree of expression than the particular marker on the cell surface or in the cell. Dark field images can reveal the light scattering properties of cells. The internal and external characteristics of the cell that scatter the light appear brighter and the characteristics of dispersion, that smaller amounts of light appear darker than a dark field image. Cells such as granulocytes have internal granules in the size range (100-500nm), which can spread significant amounts of light and generally appear in bright dark field images. In addition, the outer boundary of any cell can scatter light and can appear as a ring of bright light. The diameter of the ring can directly give the cell size. Brightfield images of cells can reveal the cell size, the dense phase material inside the cells and the color characteristics of the cell, if the cells have been previously stained.
[00616] An image processing library can extract one or more of the following information elements for each cell (but are not limited to the following): Cell size
[00617] Quantitative measure of cell granularity (also popularly called lateral dispersion, based on language flow cytometry) Quantitative measure of fluorescence in each spectral channel of the image, after compensating for cross-talk between spectral channels Cell format, such as quantified by standard and custom shape attributes such as aspect ratio, Feret diameter, kurtosis, moment of inertia, circularity, solidity, etc. Color, color distribution and shape of the cell, in cases where the cells were stained with the dyes ( not bound to antibodies or other types of receptor).
[00618] Patterns of intracellular staining or dispersion or colors that are defined as quantitative measures of a biological characteristic, for example, the density of granules within cells in a dark field image, or the number and size of nucleolus lobes of an image stained with polymorphonuclear neutrophil polymorphonuclear Giemsa, etc. Co-location of cell characteristics revealed in separate images
[00619] The image processing algorithms used in this step can use combinations of image filtering, edge detection, combining model, automatic thresholding, morphological operations and object shape analysis.
[00620]. Analysis of information extracted using advanced statistical and classification methods to obtain clinically reportable information.
[00621] Any assigned measurement number can be extracted from cell images. For example, the attributes of each measurement cell extracted from the images can vary from 7 to 15, thus creating a 7 to 15 three-dimensional space in which each cell is a point. If n measured attributes are extracted from the images, a non-dimensional space can be provided, in which each cell is a point.
[00622] Based on data obtained by a large number of cells (for example, 100-100,000 cells) a complex n-dimensional scattered data set can be generated.
[00623] Statistical methods can be used for grouping cells separated into individual populations in this n-dimensional space. These methods can also use knowledge of the state of the art of cell biology and hematology to assist in the grouping and identification of the cell population.
[00624] Figure 125 is an example of acquiring multiple parameters of data from samples of labeled cells. Human leukocytes were marked with the anti-CD45-Alexa Fluor 700 panleukocyte marker (shown in green) and the anti-CD22-APC B cell marker (shown in red). The individual channels show different patterns of CD45, CD22 expression and lateral dispersion. Cells that are positive for CD22 and CD45 (B lymphocytes) show the characteristic of low lateral dispersion. On other hand cells, such as neutrophils and eosinophils, which have high lateral dispersion, they did not show CD22 marking.
[00625] Figure 127 is an example of multi-quantitative data acquisition and parametric analysis. For example, a histogram can be provided which can show the distribution of CD45 intensity in human leukocytes. Any other techniques for distributing graph data can be employed to show the distribution. In some embodiments, a scatter diagram, from the side scatter, may be provided. The lateral dispersion can be determined by analyzing the dark field image against CD45 fluorescence intensity of a human leukocyte sample. The side scatter plot can show two main populations of granulocytes (upper left) and lymphocytes (lower right).
[00626] In the previous sections, they describe the main components and capabilities of the cytometry platform and applications. Based on these resources, a wide range of cell-based assays can be designed to work on this platform. For example, an assay for performing a differential 5-part leukocyte may be provided. The reportable in this case can be the number of cells.

权利要求:
Claims (14)
[0001]
1. Method for detecting the presence or concentration of an analyte in a sample of fluid contained in a container, characterized by comprising: illuminating the container along a first region having a first path length to obtain a first measurement of the intensity of the transmitted light through the first travel length; moving the sample fluid to another region of the container having another travel length if the first measurement falls outside a predetermined dynamic range of the transmitted light intensity; illuminate the container along the other region, to obtain another measurement of the intensity of the light transmitted through the other path length, and optionally repeat steps (b) and (c) until a measurement of the intensity of the light falls within the range predetermined dynamic, thus detecting the presence or concentration of the analyte.
[0002]
Method according to claim 1, characterized in that it comprises the deconvolution of an examined line of the image, thus detecting the presence or concentration of analyte.
[0003]
Method according to claim 1, characterized in that the sample is moved from a first region of the container that has a first path length to a second region of the container having another path length by aspirating the sample.
[0004]
Method according to claim 3, characterized in that one end of the container is connected to a pipette that is configured to aspirate the sample.
[0005]
Method according to claim 3, characterized in that the sample is moved up or down the length of the container.
[0006]
Method according to claim 3, characterized in that the container is a pipette tip.
[0007]
7. Method according to claim 1, characterized by the fact that the container has a conical shape.
[0008]
Method according to claim 1, characterized in that the container has a plurality of different widths to allow the transmission of light along a plurality of different path lengths.
[0009]
Method according to claim 1, characterized in that the volume of the container is less than 100 microliters.
[0010]
Method according to claim 1, characterized in that a plurality of different path lengths are reflected simultaneously.
[0011]
11. Method of measuring the concentration of analyte in a fluid sample characterized by comprising: providing the sample contained in a container sized with a plurality of different widths to allow the transmission of light along a plurality of different path lengths corresponding to the plurality of different widths; illuminating the container along at least one of the plurality of path lengths, reflecting the container to measure a first intensity of light transmitted through said at least one of the plurality of path lengths, for determining the concentration of the analyte based on at the first measured light intensity, reflect the container to measure a second intensity of light transmitted through another path length corresponding to a different width than the container; compare the first light intensity and the second light intensity; and determining an analyte concentration based on the comparison step.
[0012]
Method according to claim 11, characterized in that it comprises selecting a desired detection path length by one or more of the following: (a) moving a light source in relation to the sample, (b) moving a detector in relation to sample, or (c) move the sample inside the container in relation to the light source.
[0013]
Method according to claim 11, characterized in that the illumination is provided by a light source and the capture and provided by a detector, where the light source and the detector are on opposite sides of the container.
[0014]
Method according to claim 11, characterized in that the illumination is provided by a light source and the capture and provided by a detector, where the light source and the detector are on the same side of the container.

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法律状态:
2018-05-29| B25A| Requested transfer of rights approved|Owner name: THERANOS IP COMPANY, LLC (US) |

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2018-12-18| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-10-08| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-07-14| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-09-24| B25D| Requested change of name of applicant approved|Owner name: LABRADOR DIAGNOSTICS LLC (US) |

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2020-12-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-02| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 20/01/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161435250P| true| 2011-01-21|2011-01-21|

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US61/435,250|2011-01-21|

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PCT/US2012/022130|WO2012100235A2|2011-01-21|2012-01-20|Systems and methods for sample use maximization|

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