FLUIDIC CENTRIPT DEVICE FOR TESTING COMPONENTS OF A BIOLOGICAL MATERIAL IN A FLUID, TEST APPARATUS A

专利摘要:
Fluid centripetal dlsposltlvo A fluid centripetal apparatus for testing components of a biological material in a fluid is presented. the fluidic centripetal device is adapted to be received within a rotating support. the apparatus comprises a fluidic component layer having fluidic characteristics on at least one front face and a lower component layer connected to a rear part of the fluidic component layer thereby creating a fluidic network through which the fluid flows under centripetal force . in one embodiment, the fluidic feature may be a lower filling chamber coupled to an inlet channel to receive the fluid, the chamber inlet being provided on the outside of the lower filling chamber. in another embodiment, the fluidic characteristic can be a holding chamber coupled to an inlet channel to receive the fluid, a container integrally supplied in the holding chamber and containing a liquid diluent, the container keeping the liquid diluent in the container until it releases the liquid. even in the holding chamber by applying a force external to the container, thus restoring the fluid connection between the liquid diluent and the fluid in the holding chamber. in addition, the holding chamber may have a flow dissociating receptacle for receiving fluid located on the outside of the holding chamber and interrupting a fluid connection between the holding chamber inlet and outlet. a test apparatus and a test method using a fluid centripetal device to test components of a biological material in a fluid are also provided.
公开号:BR112013022889B1
申请号:R112013022889-0
申请日:2012-03-07
公开日:2020-10-06
发明作者:Régis Peytavi；Sébastien Chapdelaine
申请人:UNIVERSITé LAVAL；Genepoc Inc；
IPC主号:

专利说明:

REMISSIVE REFERENCE TO RELATED ORDERS
[0001] This application claims priority for Provisional Application US 61 / 450,373 filed on March 8, 2011, the description of which is incorporated herein by reference. TECHNICAL FIELD
[0002] The invention relates to fluidic centripetal devices. BACKGROUND OF THE TECHNIQUE
[0003] Molecular diagnoses include the detection of molecular compounds useful for the identification of diseases, species, individuals, etc. These molecular compounds can be, for example, ions, metabolites, sugars, fatty acids, amino acids, nucleic acids, proteins or lipids. The nucleic acid test (NAT) comprises the identification of pathogenic agents specific nucleic acids, or the identification of specific nucleic acid sequences related to diseases such as cancer, genetic diseases, the genetic signature of species or individuals or markers for personalized medicine . NAT protocols often begin with a sample preparation step in which cells are lysed to release their nucleic acids. The nucleic acids are then prepared specifically to be prepared for a target amplification process, such as, for example, polymerase chain reaction (PCR) or Recombinase Polymerase Amplification (RPA) for isothermal amplification or other methods nucleic acid amplification. The target amplification produces amplicons that can be analyzed in real time, that is, during the amplification, or at the end of the amplification in agarose gel or in a microarray, for example. Amplification processes also exist for the amplification of a signal generated by the detection of the analyte and these approaches to signal amplification may also be associated with target amplification processes. These technologies require complex protocols performed by highly qualified personnel in dedicated facilities. For these reasons, not all laboratories, hospitals or health centers can perform molecular diagnostics.
[0004] There is a need to automate complex molecular diagnostic protocols. Some approaches have high-performance robotic units, which are often very expensive and may require a lot of space. There is an increasing need to develop more compact instruments and mobile instruments, such as Point of Care (POC) diagnostics, and to miniaturize and integrate the steps of an assay - from sample preparation to response - in a single disposable device (for example). example, lab devices on chip or micro Total Analysis Systems: pTAS).
[0005] One of the main difficult steps to integrate into a disposable microfluidic system is sample preparation. Sample preparation usually begins with a cell lysis step, which can be chemical and / or mechanical. Then, to remove or at least control potential inhibitors from the testing process, the nucleic acids can be purified. The most common techniques used to purify nucleic acids are based on solid phase adsorption of nucleic acids, under specific pH and salt conditions. Enzymatic reaction inhibitors, such as proteins, metals and other molecules are purified from nucleic acids adsorbed on the solid phase. The nucleic acids are then recovered from the solid phase using an appropriate elution solution. The whole process requires different solutions, which need to be stored and released, a solid phase matrix and different reaction chambers. This complicates the process for integration into a compact disposable microfluidic cartridge.
[0006] In the development of fluidic devices, there is a need to move fluids inside and outside the different processing zones in a controlled manner. Pumping and valve components are commonly used.
[0007] Some have developed fluidic units allowing the automation of molecular diagnostics. For example, there is a sample preparation cartridge with a rotary valve and a piston pump to move fluids in different reservoirs. There is also mechanical lysis using ultrasound and hard particles. Other devices use a flexible plastic assembly to smooth the cells and transfer fluids between sectors of the container by compressing the flexible material to a specific location. These fluidic units require multiple actuators to be able to perform the tasks.
[0008] The use of centripetal platforms provides a simple and effective format for the application of pumping and valve options. When rotating, centrifugally induced fluid pressure causes fluid flow within the fluidic device.
[0009] Centripetal pumping provides many advantages over other alternative pumping methods, such as a syringe, piston, peristaltic, or electro-osmotic pumping. Centripetal pumping has lower electrical energy requirements (the only active actuation being necessary for rotation), it is independent of the fluid pH or ionic strength, and does not need any interconnections or external fluidic pipes. Consequently, different test steps requiring different sample and buffer properties (for example, surface energy, pH) can be combined into a single fluidic centripetal device.
[0010] Another advantage of centripetal pumping is that a valve can be implemented by the geometric design of the fluidic microchannels in such a way that the capillary forces balance the centrifugal force due to the rotation of the disk. Through the design of microfluidic structures with capillary valves of different shapes and in different positions in relation to the center of rotation of the fluid centripetal device, the flow of liquid can be stopped and resumed, controlling the speed of rotation.
[0011] Since most analytical processes for biological material require several steps, passive valves can be difficult to apply robustly. For greater robustness, there is a need to implement active valves in a centripetal device. For example, it is possible to block a microfluidic channel using a phase-changing material such as a paraffin wax plug. This type of valve is independent of the rotation speed and can be activated by heat. For example, a plug of heat-generating particles and phase-changing materials can also be used. The particles absorb electromagnetic waves from an external device (for example, laser, IR lamp) and the phase change material melts with the heat generated by the particles. Valves of phase change material have been described to block a fluidic channel (US Patent 7,837,948) and used in a centripetal nucleic acid test device (EP Publication 2375256).
[0012] Some active valve approaches for centripetal devices are based on actuation by an electromagnetic wave. For example, a valve closure at a desired location can be opened without contact by means of laser ablation and without perforating the outer layer of the microfluidic device (see, for example, Publication EP 1930635, PCT Patent Application Publication W02004 / 050242 , US Patent Application Publication 2009/0189089, US Patent 7,709,249, US Patent 7,323,660).
[0013] The actuation of a valve of phase change material can be done by means of electrodes that form a resistive heater for the substrate itself. The electrodes generate heat to a specific region of interest in the microfluidic network to fuse the phase change material.
[0014] There is still a need for an improved fluidic centripetal device with sample flow control. SUMMARY
[0015] The fluid centripetal device described here can allow the combination of simplified structures and actuators that guarantee sample preparation, volume measurement, controlled displacement of the volume in a minimum of chambers and channels, while allowing the storage of both dry reagents necessary for the amplification and multiplex detection of nucleic acids.
[0016] The fluidic centripetal device described is well suited to be implemented in point of care or bench top systems to simultaneously process multiple samples and yield quick results.
[0017] According to a first aspect of the invention, a fluid centripetal device is provided in which the combined macrostructures and microstructures ensure a simplified sample preparation method. Fluids can be moved with the centripetal force applied to a rotor, which provides a centripetal force. The process is simplified in order to minimize the use of liquids and robustly use simple valves.
[0018] In accordance with a second aspect of the invention, a method for extracting and preparing nucleic acids is provided in order to control potential inhibitors present in a sample that can interfere with amplification and / or detection. In addition, the fluid circuit can provide a pre- and post-lysis measurement of the sample volume. This allows you to set the volume. The volume definition can be achieved by subtracting a liquid volume defined by the difference between the 2 menisci. This allows the use of a simple collection device, instead of the usual high-precision micropipettes needed to accurately measure small volumes introduced into the fluidic centripetal device, which greatly facilitates handling by the operator.
[0019] According to a third aspect of the invention, a fluid centripetal device combining sample preparation and detection of nucleic acid amplification in real time multiplex is provided. The fluidic centripetal device includes an input receptacle in fluidic communication with a bottom filling chamber (which can be used for homogenization, cell lysis, control of inhibitors and concentration of microbes) in fluidic communication with a holding chamber, in communication fluidic with a detection area that can use a distribution channel, to divide the sample into two or more detection chambers, if necessary, for amplification and detection. The channels and chambers of the fluidic centripetal device can be self-ventilated by a closed-loop system providing air displacement, while keeping the system closed thus helping to prevent contamination.
[0020] In accordance with a fourth aspect of the invention, an instrument is provided to control the functions of the fluidic centripetal device. The system comprises mechanical components, such as a motor to rotate the fluid centripetal device, magnets to move the translocable member in the fluid centripetal device, thermal elements to control the temperature of the fluidic device, optical components to measure fluorescence signals and an interface of electronic and human machine, for example, with a touch screen device.
[0021] In one embodiment, the instrument provides air temperature control in multiple zones of the fluidic centripetal device.
[0022] In one embodiment, the instrument provides temperature control in multiple zones in a centripetal device by placing thermal elements in contact with the fluidic centripetal device of rotation.
[0023] According to a broad aspect, a fluid centripetal device is provided for testing components of a biological material in a fluid sample, the apparatus comprising a fluid component layer with a substantially flat back side, the fluid component layer having a shape adapted to be received inside a rotating support, the rotating support having a center of rotation and with an outer edge, the fluid component layer extending radially between the center of rotation and the outer edge, an inner side of the fluidic component being located towards the center of rotation and an outer side of the fluidic component layer being located towards the outer edge, the fluidic component layer being formed to include: a sample inlet receptacle for receiving the sample, the receptacle sample inlet extending outward from the fluid component layer and being located close to the inner side, the sample inlet receptacle terminating at a sample outlet; an inlet channel for circulating the fluid sample, the inlet channel being coupled to the sample outlet on one end and to a chamber inlet on the other end; a bottom fillable chamber coupled to the inlet channel at the chamber inlet to receive the fluid sample, the chamber inlet being provided to an external side of the bottom fillable chamber.
[0024] In one embodiment, the apparatus further comprises a cover for the sample inlet receptacle to close access to the sample inlet receptacle.
[0025] In one embodiment, the bottom-filling chamber is oblong in shape and extends radially between the inside and the outside.
[0026] In one embodiment, the bottom fillable chamber includes at least one translocatable member that translocates inside the bottom fillable chamber in response to an external floating magnetic field.
[0027] In one embodiment, the translocatable member that translocates in response to a floating magnetic field is formed of paramagnetic material.
[0028] In one embodiment, the translocable member that translocates in response to a floating magnetic field is a disk or a sphere.
[0029] In one embodiment, the translocable member is ferromagnetic.
[0030] In one embodiment, the bottom-filling chamber also comprises at least one object that does not react in response to a floating magnetic field.
[0031] In one embodiment, the object is at least one of a sphere, a glass sphere, a zirconium sphere, a resin, and a sphere and resin paste.
[0032] In one embodiment, the object is coated with a chelating material adapted to interact with the sample components.
[0033] In one embodiment, each of the object and the translocatable member is larger in size than the size of the chamber entrance.
[0034] In one embodiment, the bottom fillable chamber is a homogenization chamber.
[0035] In one embodiment, the bottom fillable chamber is a lysis chamber.
[0036] In one embodiment, the background fillable chamber is a clarification chamber.
[0037] In one embodiment, the bottom fillable chamber is a target concentration chamber.
[0038] In one embodiment, the apparatus also comprises an overflow chamber coupled to an excess outlet for the bottom fillable chamber, the excess outlet allowing a part of the fluid sample to exit from the bottom fillable chamber to the overflow chamber.
[0039] In one mode, the excess outlet is provided close to the inside of the bottom fillable chamber.
[0040] In one embodiment, the excess outlet is provided on a longitudinal side of the bottom fillable chamber.
[0041] In one embodiment, the device also comprises an outlet discharge to the bottom fillable chamber, the outlet discharge allowing the sample to exit from the bottom fillable chamber.
[0042] In one embodiment, the outlet discharge is located on a longitudinal side of the bottom fillable chamber.
[0043] In one embodiment, each object and the translocatable member are larger in size than the size of the outlet discharge.
[0044] In one mode, the excess outlet is located closer to the inside than the outlet discharge.
[0045] In one embodiment, the apparatus further comprises a holding chamber, the holding chamber being coupled to the outlet discharge on the inside of the holding chamber, the holding chamber being located closer to the outside of the component layer fluidic than the bottom fillable chamber.
[0046] In one embodiment, the holding chamber is coupled to the outlet outlet via a transfer channel, the transfer channel for circulating at least a portion of the fluid sample from the bottom fillable chamber to the holding chamber .
[0047] In one embodiment, the apparatus further comprises a container integrally supplied in the holding chamber and containing a liquid reagent, the container being adapted to hold the liquid reagent in the container and to release the liquid reagent into the holding chamber by applying a external force in the holding chamber.
[0048] According to a broad aspect, a fluid centripetal device for mixing a liquid reagent with a fluid sample is provided, the apparatus comprising a fluid component layer with a substantially flat back side, the fluid component layer having a shape adapted to be received within a rotating support, the rotating support having a center of rotation and an outer edge, the fluid component layer extending radially between the center of rotation and the outer edge, an inner side of the fluidic component being located towards the center of rotation and an outer side of the fluidic component layer being located towards the outer edge, the fluidic component layer being formed to include: a sample inlet receptacle for receiving the sample, the receptacle sample inlet extending outward from the fluidic component layer and being located close to the inner side, the sample inlet receptacle terminating at a sample outlet, a holding chamber coupled to the sample inlet receptacle for receiving the fluid sample within the holding chamber, a container integrally supplied in the holding chamber and containing a liquid reagent, the container being adapted to hold the liquid reagent in the container and to release the liquid reagent in the holding chamber, after applying an external force to the holding chamber.
[0049] In one embodiment, the apparatus further comprises an inlet channel for circulating the fluid sample from the sample outlet to a holding chamber inlet of the holding chamber.
[0050] In one embodiment, the holding chamber has a receptacle for receiving the fluid sample.
[0051] In one embodiment, the receptacle is located on the outside of the holding chamber.
[0052] In one embodiment, a volume of capacity of the receptacle is at least equal to a volume of capacity of the sample transferred to the holding chamber.
[0053] In one embodiment, the container includes a dry reagent.
[0054] In one embodiment, the dry reagent is an inhibitor control reagent.
[0055] In one embodiment, the holding chamber is a dilution chamber.
[0056] In one embodiment, the holding chamber receptacle is emptied after the diluent is released.
[0057] In one embodiment, the container is made of glass, capillary glass, polymeric thermoplastic and / or heat sensitive material.
[0058] In one embodiment, the liquid reagent is a diluting agent.
[0059] In one embodiment, the liquid reagent is one of water, buffer, ion, polymer, protein, sugar, nucleic acid and / or a dryable part of a solution.
[0060] In one embodiment, the container has a lid made of a heat sensitive material, adapted to be melted at a melting temperature, allowing the liquid reagent to travel from inside the container to outside the container in the holding chamber.
[0061] In one embodiment, the container is made of heat-sensitive material.
[0062] In one embodiment, the external force is one of mechanical, electrical, electromagnetic force, heat, shock and acoustic.
[0063] In one embodiment, the container has a release port.
[0064] In one embodiment, the holding chamber has a distribution outlet for the holding chamber, the distribution outlet being located on the outside of the holding chamber, the distribution outlet being coupled to a transverse distribution channel in an internal side of the transverse distribution channel at a first transverse end of the distribution channel, the transverse distribution channel having a series of at least one bucket provided to an external side of the transverse distribution channel.
[0065] In one embodiment, the distribution outlet is coupled to the distribution channel through a transfer channel.
[0066] In one embodiment, the cuvettes include a dry reagent.
[0067] In one embodiment, the dry reagent is for amplification and may include an enzyme.
[0068] In one embodiment, the cuvettes include a set of initiators.
[0069] In one embodiment, the dry reagent in the cuvette is covered with a film of material sensitive to heat or phase change, with a density lower than that of water.
[0070] In one embodiment, the material sensitive to heat is a wax.
[0071] In one mode, the cuvette is adapted to be optically interrogated by at least one parameter.
[0072] In one embodiment, the cuvette has a cuvette body with at least one optically transparent window in the cuvette body, the optically transparent windows being aligned with a light path from a light source adapted to project the light of a length predetermined waveform along the light path.
[0073] In one mode, the parameter is one of fluorescence, absorbance, and colorimetry.
[0074] In one embodiment, the parameter is fluorescence and in which the cuvette includes one of fluorescence solution in the cuvette, particles covered with fluorophore in a solution in the cuvette, particles of fluorophore on the inner wall of the cuvette.
[0075] In one embodiment, the cuvette is a detection chamber.
[0076] In one embodiment, the cuvette is an amplification chamber.
[0077] In one embodiment, the cuvette is a nucleic acid amplification chamber.
[0078] In one embodiment, the transverse distribution channel includes a waste chamber at a second transverse end of the distribution channel.
[0079] In one embodiment, the waste chamber includes a heat activated seal, adapted to seal the entrance to the bucket, coupled to the distribution channel.
[0080] In one embodiment, the heat-activated seal is a wax.
[0081] In one embodiment, at least one of the chamber inlet, overflow outlet, outlet outlet, distribution outlet including an anti-reflux valve.
[0082] In one embodiment, at least one of the chamber inlet, the excess outlet, the outlet outlet, the distribution outlet including a rupture valve, the rupture valve opening at a predetermined applied centripetal force in the apparatus.
[0083] In one embodiment, the anti-reflux valve and the rupture valve being provided in a single anti-reflux rupture valve.
[0084] In one embodiment, the fluidic component layer is made of a plastic material.
[0085] In one embodiment, the plastic material is one made of polycarbonate, polypropylene, PDMS, COC, SU-8.
[0086] In one embodiment, the fluidic component layer is sealed at the substantially flat back, with a sheet of plastic material.
[0087] In one embodiment, the plastic material sheet one is made of polycarbonate, polypropylene, PDMS, COC, SU-8 material.
[0088] In one embodiment, the fluidic component layer is sealed with the plastic material sheet using bonding methods such as adhesive, pressure sensitive adhesive material, heat transfer, solvent bonding, UV curable adhesive, bonding by ultrasound, laser welding, RF connection.
[0089] In one embodiment, the rupture characteristic of the rupture valves is a combination of their distance from the center of rotation, the plastic material that constitutes the support plate, the material that constitutes the seal and the geometry of the valve itself molded in plastic material.
[0090] In one embodiment, the distribution channel, the cuvettes and the waste chamber are provided on a portion of the support member plate that extends beyond the outer edge of the rotating support.
[0091] In one embodiment, the fluidic component layer is rectangular.
[0092] In one mode, the support is a disk.
[0093] In one embodiment, the fluidic component layer shape is a conical section of a ring.
[0094] In one embodiment, the conical section of the ring is a fraction of a ring.
[0095] In one embodiment, the conical section of a ring is one-eighth of a ring.
[0096] In one embodiment, the device also comprises ventilation outlets from at least one of the overflow chamber, the holding chamber and the distribution channel, the ventilation outlets being connected to a self-ventilation channel.
[0097] In one embodiment, the self-ventilation channel is coupled to the sample inlet in the container on the inner side of the sample inlet receptacle.
[0098] In one embodiment, the fluidic component layer is adapted to be at least partially heated.
[0099] In one embodiment, the fluidic component layer is adapted to be temperature controlled.
[0100] In one embodiment, the fluidic component layer is adapted to be divided into at least two distinct temperature-controllable sections.
[0101] In one embodiment, a first of the two distinct temperature-controllable sections includes the bottom filling chamber and the holding chamber.
[0102] In one embodiment, a first of the two distinct temperature-controllable sections includes at least the holding chamber.
[0103] In one embodiment, the first section includes the sample input receptacle, the input channel, the overflow chamber and the measurement channel.
[0104] In one embodiment, a second of the two distinct temperature-controllable sections includes at least the distribution channel and cuvettes.
[0105] In one embodiment, the second of the two sections includes the overflow chamber and a portion of the transfer channel.
[0106] In one embodiment, the fluid sample is at least one of blood, nasal pharynx aspiration, oral fluid, resuspended oral smear liquid, resuspended nasal smear liquid, resuspended anal smear liquid, resuspended smear liquid vaginal, urine saliva (pure or diluted).
[0107] According to a broad aspect, a test apparatus is provided using a fluid centripetal device to test components of a biological material in a fluid sample, the device comprising at least one of the fluid centripetal device, a rotor assembly, a support for receiving at least one of the fluidic centripetal device using the fluidic component layer, the support being coupled to the rotor, a motor to rotate the rotor assembly, a speed controller for the motor to control at least one of a duration, acceleration and a rotational speed of the rotor assembly; a temperature conditioning subsystem for controlling a temperature of at least a portion of the microfluidic centripetal device; an excitation subsystem for exciting the sample from the fluid centripetal device and obtaining a test result; a user interface for receiving a user command and for sending a command to at least one of the speed controller, temperature conditioning subsystem and excitation subsystem.
[0108] In one embodiment, the support is a rotor assembly comprising a lower part of a rotor that receives the fluidic centripetal device and a pressure ring for fixing the fluidic centripetal device.
[0109] In one embodiment, the test apparatus further comprises a housing for the testing apparatus having a base, walls and hinged cover, the housing surrounding the rotor assembly, the support, the motor, the temperature conditioning subsystem and the excitation subsystem.
[0110] In one embodiment, the tester also comprises permanent magnets supplied under the rotor.
[0111] In one embodiment, the temperature conditioning subsystem controls the temperature of two zones of the fluidic centripetal device.
[0112] In one embodiment, the tester also comprises the compartments created by at least one of the enclosure, enclosure separation wall, rotor assembly, rotor insulation wall, support, cover insulation and the insulation wall of the cover.
[0113] In one embodiment, the tester also comprises the insulating materials that can be used to control the transfer of heat between the compartments.
[0114] In one embodiment, the temperature conditioning subsystem comprises a thermal element located in one of the above and below a heating zone.
[0115] In one embodiment, the thermal element is a resistive heating coil.
[0116] In one embodiment, the tester also comprises a thermocouple inside each heating zone to measure the individual temperature in each zone.
[0117] In one embodiment, the tester also includes a fan that forces air into the room at room temperature.
[0118] In one embodiment, the tester also includes an outlet port to eject hot air out of the heating zone.
[0119] In one embodiment, the excitation subsystem includes a light source, and optical elements to form an excitation beam.
[0120] In one embodiment, the excitation subsystem includes a detection module to collect the light emitted by species of interest within the fluidic centripetal device.
[0121] In a broad sense, a test method is provided using a fluidic centripetal device for testing components of a biological material in a fluid sample, the method comprising providing at least one of the fluidic centripetal device; provide a test device; provide a fluid sample with the biological material; loading the fluid sample into the sample inlet receptacle of the fluid centripetal device; placing the fluidic centripetal device in the tester holder, providing a user command to initiate a test sequence; rotate the rotor assembly at a first speed to transfer the fluid sample from the sample inlet receptacle in the bottom fillable chamber.
[0122] In one embodiment, the rotation also includes emptying part of the sample in the overflow chamber.
[0123] In one embodiment, the test method also comprises rotating the rotor assembly at a second speed to activate the movement of the translocation member within the bottom fillable chamber.
[0124] In one embodiment, the test method also comprises rotating the rotor assembly at a third speed to clarify the sample and break the measurement output, in which a measured volume of the sample is transferred to the holding chamber.
[0125] In one embodiment, the measured volume is transferred to the receptacle of the holding chamber.
[0126] In one embodiment, the test method also comprises rotating the rotor assembly at a fourth speed.
[0127] In one embodiment, the test method further comprises heating the holding chamber, thus releasing the liquid reagent from the container.
[0128] In one mode, the test method also comprises rotating the rotor assembly at a fifth speed to break the outlet of the holding chamber.
[0129] In one embodiment, the test method also includes keeping the cuvettes below 65 ° C.
[0130] In one embodiment, the test method also includes keeping the cuvettes below 35 ° C.
[0131] In one embodiment, the test method also includes heating the cuvettes to a first temperature.
[0132] In one embodiment, the test method also includes heating the cuvettes to a second temperature.
[0133] In one embodiment, the test method also includes cycling the temperature of the cuvettes between a high, low and medium test temperature.
[0134] In one embodiment, the test method further comprises taking the fluorescence measurement having at least one excitation wavelength at the end of each temperature cycle.
[0135] In one embodiment, the test method also comprises recording fluorescence measurements.
[0136] According to a broad aspect, a test method is provided using a fluidic centripetal device for testing components of a biological material in a fluid sample, the method comprising providing at least one of the fluidic centripetal device; provide a test device; provide a fluid sample with the biological material; loading the fluid sample into the sample inlet receptacle of the fluid centripetal device; place the fluid centripetal device in the tester holder, provide a user command to initiate a test sequence, rotate the rotor assembly at a first speed to transfer the fluid sample from the sample inlet chamber in the retention; heat the holding chamber, thus releasing the liquid reagent from the container.
[0137] In one embodiment, the method comprises rotating the rotor assembly at a fifth speed to break the outlet of the holding chamber.
[0138] In one embodiment, the sample is transferred to the receptacle of the holding chamber.
[0139] According to another broad aspect of the present invention, a fluid centripetal apparatus is provided for testing components of a biological material in a fluid. The fluidic centripetal device is adapted to be received inside a rotating support. The apparatus comprises a fluidic component layer having fluidic characteristics on at least one front face and a bottom component layer connected to a rear part of the fluidic component layer, thereby creating a fluidic network through which the fluid flows through the centripetal force. A test apparatus and a test method using a fluid centripetal device to test components of a biological material in a fluid are also provided.
[0140] In one embodiment, the fluidic feature may be a bottom-filling chamber coupled to an input channel to receive the fluid, the chamber-in being supplied to an external side of the bottom-filling chamber.
[0141] In another embodiment, the fluidic characteristic can be a holding chamber coupled to an inlet channel to receive the fluid, a container integrally supplied in the holding chamber and containing a liquid diluent, the container keeping the liquid diluent in the container until that it is released in the holding chamber after applying an external force to the container, thus restoring the fluid connection between the liquid diluent and the fluid in the holding chamber.
[0142] In addition, the holding chamber may have a flow dissociation receptacle to receive the fluid, located on the outside of the holding chamber and interrupting a fluid connection between the holding chamber inlet and outlet.
[0143] In accordance with another broad aspect of the present invention, a fluid centripetal apparatus is provided to test components of a biological material in a fluid, the fluid centripetal device having a shape adapted to be received within a rotating support, the rotating support having a center of rotation and an outer edge, the fluidic centripetal device extending radially between the center of rotation and the outer edge, an inner side of the fluidic centripetal device being located towards the center of rotation and an outer side of the fluidic centripetal device being located towards the outer edge, the apparatus comprising: a layer of fluid component having fluid characteristics on at least one front face, the fluid characteristics including an inlet channel for circulating the fluid, the inlet channel being coupled to a camera input, a bottom fillable camera coupled to the input channel at the camera input to receive the fluid, the chamber inlet being provided to an external side of the bottom fillable chamber, and a bottom component layer connected to a rear part of the fluid component layer, thus creating a fluid network through which the fluid flows by centripetal force.
[0144] In one embodiment, the fluidic centripetal apparatus further comprises an inlet receptacle for receiving fluid, the inlet receptacle extending outwardly from the fluidic component layer on a front face of the fluidic component layer and being located close to the inner side, the inlet receptacle terminating at an outlet of the inlet receptacle, the inlet channel being coupled to the outlet of the inlet receptacle at an end opposite the chamber inlet.
[0145] In one embodiment, the bottom fillable chamber includes at least one translocatable member that translocates inside the bottom fillable chamber in response to an external floating magnetic field.
[0146] In one embodiment, the bottom fillable chamber comprises at least one object unresponsive to a floating magnetic field and in which the object is at least one of a sphere, a zeolite, a particle, a particle of filtration, a glass sphere, a zirconium sphere, a resin, a sphere and resin paste.
[0147] In one embodiment, at least one of the object and the translocatable member is coated with at least one of a chelator and a binder material adapted to interact with the fluid components.
[0148] In one embodiment, the fluidic centripetal apparatus further comprises an overflow chamber coupled to an excess outlet to the bottom fillable chamber, the excess outlet allowing part of the fluid to exit from the bottom fillable chamber to the overflow chamber, in which the excess outlet is provided close to the inside of the bottom fillable chamber on a longitudinal side of the bottom fillable chamber.
[0149] In one embodiment, the fluidic centripetal device further comprises an outlet discharge to the bottom fillable chamber, the outlet discharge allowing the fluid to exit from the bottom fillable chamber, where the outlet discharge is located in the longitudinal side of the bottom fillable chamber, the outlet discharge being located closer to the outside of the bottom fillable chamber than the excess outlet, a measurement volume of the bottom fillable chamber being defined between the outlet discharge and the outlet excess.
[0150] In one embodiment, the fluid centripetal device also comprises outlet discharge to the bottom fillable chamber, the outlet discharge allowing the fluid to exit from the bottom fillable chamber, in which the outlet discharge is located on a longitudinal side of the bottom fillable chamber.
[0151] In one embodiment, the fluidic centripetal apparatus further comprises a rupture valve at the outlet discharge, the rupture valve opening at a predetermined centripetal force applied on the apparatus, the rupture valve preventing the fluid from leaving the fillable chamber of bottom to the opening.
[0152] In one embodiment, the fluidic centripetal apparatus further comprises a holding chamber, the holding chamber being coupled to the outlet discharge on the inner side of the holding chamber, the holding chamber being located closest to the outside of the layer fluid component than the bottom filling chamber, where the holding chamber is coupled to the outlet discharge through a measuring channel, the measuring channel to circulate at least a portion of the fluid from the bottom filling chamber to the holding chamber.
[0153] In one embodiment, the fluid centripetal apparatus further comprises a container integrally supplied in the holding chamber and containing a liquid diluent, the container being adapted to keep the liquid diluent inside the container and to release the liquid diluent in the holding chamber by means of application of an external force to the container, where the external force is one of mechanical, electrical, electromagnetic force, heat, and acoustic shock, thus restoring the fluid connection between the liquid diluent and the fluid in the holding chamber .
[0154] A fluidic centripetal device for testing components of a biological material in a fluid, the fluidic centripetal device having a shape adapted to be received inside a rotating support, the rotating support having a center of rotation and with an outer edge, the fluidic centripetal device extending radially between the center of rotation and the outer edge, an inner side of the fluidic centripetal device being located towards the center of rotation and an external side of the fluidic centripetal device being located towards the outer edge, the apparatus comprising : a fluidic component layer having fluidic characteristics on at least one front face, fluidic characteristics including an inlet channel to circulate the fluid, the inlet channel being coupled to an inlet receptacle outlet, a holding chamber, the chamber retainer being coupled to the input channel via the input receptacle output for rec eber fluid inside the holding chamber; a container integrally supplied in the holding chamber and containing a liquid diluent, the container being adapted to hold the liquid diluent in the container and to release the liquid diluent in the holding chamber by applying an external force to the container, where the external force it is one of mechanical, electrical, electromagnetic, heat, shock and acoustic strength, thus restoring the fluid connection between the liquid diluent and the fluid in the holding chamber; and a bottom component layer connected to a rear part of the fluid component layer, thus creating a fluid network through which the fluid flows by centripetal force.
[0155] In one embodiment, the retention chamber has a fluid dissociation receptacle for receiving fluid, where the fluid dissociation receptacle is located on the outside of the retention chamber, the fluid dissociation receptacle interrupting a connection fluidic between the outlet and inlet of the receptacle and a distribution outlet of the holding chamber.
[0156] In one embodiment, the fluid dissociation receptacle includes a dry reagent.
[0157] In one embodiment, the holding chamber has a distribution outlet for the holding chamber, the distribution outlet being located on the outside of the holding chamber, the distribution outlet being coupled to a distribution channel transverse to an internal side of the transverse distribution channel to a first transverse end of the distribution channel, the transverse distribution channel having a series of at least one bucket supplied to an external side of the transverse distribution channel.
[0158] In one embodiment, at least one of the cuvettes includes at least one of a dehydrated reagent and a phase change material.
[0159] In one mode, the cuvette is adapted to be optically interrogated for at least one parameter, the parameter is one of fluorescence, absorbance, and colorimetry.
[0160] In one embodiment, the transverse distribution channel includes a waste chamber at a second transverse end of the distribution channel.
[0161] In one embodiment, the waste chamber includes a phase change material.
[0162] In one embodiment, the distribution channel, the cuvettes and the waste chamber are supplied to a portion of the fluidic layer component that extends beyond the outer edge of the rotating support.
[0163] In one embodiment, the fluid component layer is adapted to be divided into at least two distinct temperature-controllable sections, wherein a first of the two distinct temperature-controllable sections includes at least the holding chamber and a second of the two distinct temperature-controllable sections includes at least the distribution channel and cuvettes.
[0164] A fluid centripetal apparatus for testing components of a biological material in a fluid, the fluid centripetal device having a shape adapted to be received within a rotating support, the rotating support having a center of rotation and an outer edge, the device fluidic centripetal extending radially between the center of rotation and the outer edge, an inner side of the fluidic centripetal device being located towards the center of rotation and an external side of the fluidic centripetal device being located towards the outer edge, the apparatus comprising: a fluidic component layer having fluidic characteristics on at least one front face, fluidic characteristics including an inlet receptacle for receiving fluid, the inlet receptacle extending outwardly from the fluidic component layer on a front face of the layer fluidic component and being located close to the internal side, the receptacle d and input terminating at an input receptacle outlet; an inlet channel for circulating the fluid, the inlet channel being coupled to the outlet of the inlet receptacle at one end and a chamber inlet at the other end; a bottom fillable chamber coupled to the inlet channel at the chamber inlet to receive the fluid, the chamber inlet being provided to an external side of the bottom fillable chamber, and a holding chamber, the holding chamber being attached to the fillable chamber bottom to receive the fluid inside the holding chamber; a dispensing outlet for the holding chamber, the dispensing outlet being located on the outside of the holding chamber; a transverse distribution channel having a series of at least one cuvette provided on an external side of the transverse distribution channel, the distribution outlet being coupled to the transverse distribution channel to an internal side of the transverse distribution channel on a first transverse end of the distribution channel, a waste chamber at a second transverse end of the distribution channel, and a bottom component layer attached to a rear part of the fluid component layer, thus creating a fluid network through which the fluid flows by force centripetal.
[0165] A test apparatus using a fluidic centripetal device to test components of a biological material in a fluid, the apparatus comprising: at least one of the fluidic centripetal device, a rotor assembly, a holder for receiving at least one of the centripetal device fluidic using the fluidic component layer, the support being coupled to the rotor, a motor to rotate the rotor assembly; a speed controller for the motor to control at least one of a duration and a rotational speed of the rotor assembly; a temperature conditioning subsystem for controlling a temperature of at least a portion of the microfluidic centripetal device; a detection subsystem for detecting a fluid characteristic; a user interface for receiving a user command and for sending a command to at least one speed controller, the temperature conditioning subsystem, the excitation subsystem and the detection subsystem.
[0166] In one embodiment, the temperature conditioning subsystem controls at a temperature of at least two zones of the fluidic centripetal device.
[0167] A test method using a fluidic centripetal device to test components of a biological material in a fluid, the method comprising: providing at least one of the fluidic centripetal device; providing a test apparatus, supplying a fluid with the biological material, loading the fluid into the inlet receptacle of the fluid centripetal device; placing the fluidic centripetal device in the tester holder; provide a user command to start a test sequence; rotate the rotor assembly at a first speed to transfer fluid from the inlet receptacle to the bottom fillable chamber. Definitions
[0168] In this report, the term “fluid centripetal device” is intended to mean a fluid network with the fluid motivated by the action of rotation.
[0169] In this report, the term "Macro" in the expressions "Macro Structure" and "Macro Geometry" is intended to mean a characteristic of the fluidic centripetal device greater than 1 mm. In particular, the dimensions of "Macro Structure" are, for example, from about 1 mm to about 10 mm.
[0170] In this report, the term “Micro” in the expressions “Micro Structure” and “Micro Geometry” is intended to mean a characteristic of the fluidic centripetal device less than 1 mm. In particular, “Micro Structure” dimensions about 1 pm to about 1 mm.
[0171] In this report, the term “Sample” is intended to mean any suspension of fluid, solution or mixture to be analyzed. In particular "sample" can be a "biological" sample or "raw biological sample" and is intended to mean any biological species of interest from the blood, blood, nasal and / or pharynx and / or body fluid components oral, nasal and / or oral smear liquid and / or pharyngeal resuspended, resuspended liquid from anal / vaginal smear, saliva, wound exudate, feces and urine.
[0172] In this report, the term “Diluent” is intended to mean a certain amount of liquid that can be used to dilute a sample.
[0173] In this report, the term “Receptacle” is intended to mean an element of fluidic centripetal device designed to receive a certain amount of fluid.
[0174] In this report, the term “Channel” is intended to mean a microstructure or macrostructure path of a fluidic centripetal device allowing fluid flow between the chambers of fluidic centripetal devices, receptacles, and sample receptacles.
[0175] In this report, the term “Inlet” is intended to mean an opening for a fluid centripetal device chamber allowing the fluid to enter.
[0176] In this report, the term "Exit" is intended to mean an opening for a fluid centripetal device chamber allowing the fluid to escape.
[0177] In this report, the term "rupture valve" or "fluidic valve" is used interchangeably and is intended to mean a microstructure in a fluidic centripetal device having the primary function of helping to prevent the liquid from flowing below a certain amount of pressure applied to the liquid, typically by the centripetal force created by the rotation of the fluidic centripetal device. The flow flows through a “rupture valve” when the pressure exceeds the force produced by the surface tension of the liquid. BRIEF DESCRIPTION OF THE DRAWINGS
[0178] Having thus generally described the nature of the invention, reference will be made to the attached drawings, showing by way of illustration exemplary modalities thereof in which:
[0179] FIG. 1A is a perspective view of a rotor assembly holding a fluidic centripetal device; FIG. 1B represents an oblique, exploded view of a fluidic centripetal device in a lower part of a rotor; FIG. 1C illustrates an oblique view of a fluidic centripetal device; FIG. 1D is a cross-sectional view of a fluidic centripetal device.
[0180] FIG. 2A illustrates a fluidic fan connected to the inlet receptacle; FIG. 2B illustrates a cover for the input receptacle described in FIG. 2A.
[0181] FIG. 3A illustrates the fluid construction of a bottom filling chamber; FIG. 3B illustrates an alternative construction of the bottom filling chamber with a port connection.
[0182] FIG. 4A illustrates an alternative construction of the bottom filling chamber including a translocatable member; FIG. 4B illustrates an alternative construction of the bottom filling chamber including a translocatable member and dry reagents.
[0183] FIG. 5 illustrates an alternative construction of the bottom filling chamber, including an overflow chamber.
[0184] FIG. 6 illustrates an alternative construction of the bottom filling chamber including a measurement outlet.
[0185] FIG. 7A illustrates an alternative construction of the bottom filling chamber, including a translocatable member, dry reagents, overflow chamber and a measurement outlet; FIG. 7B illustrates an alternative construction of the bottom filling chamber including a translocatable member, dry reagents, filter, overflow chamber and a measurement outlet, FIG. 70 illustrates the alternative construction of FIG. 7B after dissociation of the filter by a translocatable member.
[0186] FIG. 8A illustrates the filling of the bottom filling chamber shown in FIG. 7A, FIG. 8B illustrates the translocation of the liquid overflow to the element 309, FIG. 80 illustrates the volume setting step; FIG. 8D illustrates the translocation of the translocatable limb; FIG. 8E illustrates the pelletizing at the bottom of the bottom filling chamber of elements 308 and 307, FIG. 8F illustrates the translocation of the measured volume from the lower chamber to the chamber 313.
[0187] FIG. 9A illustrates a cross-sectional view of the inlet and outlet geometry to help prevent the translocable object and / or sphere located in the solid phase material from leaving the bottom filling chamber, FIG. 9B illustrates a top view of the inlet and outlet geometry to help prevent the spheres and / or translocatable object located in the solid phase material from leaving the bottom filling chamber.
[0188] FIG. 10A illustrates a fluid structure for mixing or diluting the sample; FIG. 10B illustrates the fluid contained in a holding chamber receptacle, FIG. 10C illustrates dry reagents in the holding chamber receptacle, FIG. 10 D illustrates a container of liquid within a holding chamber.
[0189] FIG. 11A illustrates the liquid container in a holding chamber receptacle prior to heating; FIG. 11B illustrates the fluid contained in the holding chamber receptacle during the start of the heating process, FIG. 110 illustrates the release of liquid from the fluid container into the holding chamber; FIG. 11D illustrates mixing the lysate with the fluid released from the liquid container; FIG.11E illustrates the translocation of the diluted lysate from the holding chamber to chamber 513.
[0190] FIG. 12 illustrates an alternative construction of the liquid container.
[0191] FIG. 13 represents a fluidic construction including a bottom filling chamber with overflow chamber and a measurement outlet fluidly connected to a holding chamber.
[0192] FIG. 14A describes the fluidic construction of detection cells for a fluidic centripetal device; FIG. 14B illustrates an alternative construction of the detection cuvette with pre-stored dry reagents; FIG. 140 illustrates an alternative construction of the detection cuvette with dry reagents pre-stored in the cuvettes and pre-stored in the cuvette wax in the cuvettes themselves, FIG. 14D illustrates an alternative construction of the detection cuvette with dry reagents pre-stored in cuvettes and pre-stored in cuvette wax in a waste chamber.
[0193] FIG. 15 illustrates the fluidic construction described in FIG. 14D, when the cuvettes are heated and filled with a sample.
[0194] FIG. 16 depicts a fluidic construction including the holding chamber and detection cuvettes.
[0195] FIG. 17 represents a fluid construction for sample preparation and detection;
[0196] FIG. 18 represents a perspective view of an instrument that can be used to perform a series of simultaneous fluid centripetal devices.
[0197] FIG. 19 represents an oblique view of the interior architecture of the apparatus illustrated in FIG. 18.
[0198] FIG. 20 shows a diagram of the various modules of an instrument.
[0199] FIG. 21 illustrates multiple regions of zone temperature control in a fluidic centripetal device.
[0200] FIG. 22 illustrates an alternative modality of the multiple zone temperature control regions on a fluidic centripetal device.
[0201] FIG. 23 is a cross-sectional view of the dual zone air temperature control system with the instrument shown in FIG. 18.
[0202] FIG. 24 shows a schematic cross-sectional view of a multi-wavelength excitation module.
[0203] FIG. 25 illustrates spectral profiles of LEDs, excitation filter and dichroic beam dividers adapted to excite fluorescent dyes FAM and Texas Red.
[0204] FIG. 26 illustrates a schematic section view of a detection module.
[0205] FIG. 27 illustrates spectral profiles of a double-band pass-through interferential filter adapted for the detection of FAM and Texas Red fluorescent dyes.
[0206] FIG. 28 illustrates spectral profiles of the penta-band pass-through filter adapted for the detection of common fluorescent dyes.
[0207] FIG. 29 is a flow chart of the steps involved in processing a PCR assay using the instrument illustrated in FIG. 21.
[0208] FIGS. 30A, 30B, 30C illustrate the rotor speed and temperatures of the fluidic centripetal device over time to process the PCR, using the instrument illustrated in FIG. 18.
[0209] It should be noted that throughout the attached drawings, similar characteristics are identified by reference numerals. DETAILED DESCRIPTION
[0210] Fluid centripetal device structure assembly
[0211] FIG. 1A and FIG. 1B show an example of rotor assembly 1003. An exemplary lower part of rotor 2 formed to receive up to eight fluid centripetal devices 1. The rotor assembly includes a bottom rotor 2 and pressure ring 7 to retain the fluid centripetal device 1 inserted between them. The upper pressure ring rotor assembly body part has been removed in FIG. 1B.
[0212] The fluid centripetal device 1 is composed of at least two layers of components. As shown in FIGS. 1C and 1 D, the fluidic layer has characteristics on the lower and / or upper face of the fluidic centripetal device 1. The fluidic layer 3 is composed of the inlet receptacle 5, chambers 6a, 6b, 6c, channels and fluidic valves. It should be understood that the fluidic layer 3 can be made by means of several layers connected together. The thin lower layer 4 is connected to the fluidic layer 3. The lower surface of the fluidic layer 3, when combined with the thin lower layer 4, forms a fluidic network of closed reservoirs, channels and valves through which the fluid flows by means of the centripetal force.
[0213] Fluid layer 3 and thin bottom layer 4 can be made of thermoplastic material. The thermoplastic material can be at least one of the cyclic olefin copolymers (COC), polycarbonate (PC), polystyrene (PS), polyoxymethylene (POM), perfluoralkoxy (PFA), polyvinyl chloride (PVC), polypropylene (PP) , polymethylmethacrylate (PMMA), cyclic olefin copolymer (COC), polyamide (PA), polysulfone (PSU), polyvinylidene (PVDF) as well as other materials known to those skilled in the art. They can be used with surface modifications or without surface modifications. The surface modification can be applied to one or both faces or over a specific region of interest on one or both faces.
[0214] Various combination techniques for assembling the fluid layer of the fluid centripetal device 3 with the flat bottom layer 4 are available, such as thermal bonding, radiofrequency bonding, laser welding, ultrasonic bonding, pressure sensitive adhesion or adhesion, and other techniques known to those skilled in the art.
[0215] In an exemplary modality, the combination technique allows incorporating the dry or liquid reagent inside the fluidic centripetal device prior to assembly.
[0216] In another exemplary embodiment, the combination technique is at a temperature of about 4 ° C to about 80 ° C.
[0217] In an exemplary mode, the rotation of the fluid centripetal device is created by placing the fluid centripetal device on a dedicated rotor 2, which is rotated around a center of rotation. Rotor 2 has a center of rotation and an outer edge, in this case, a circumference. The fluidic centripetal device 1 extends radially between the center of rotation and the outer edge. It extends beyond the outer edge in the example shown. An internal side of the fluidic centripetal device 1 is located towards the center of rotation and an external side of the fluidic centripetal device 1 is located towards the outer edge.
[0218] The fluidic centripetal device may be a portion of a disk having an inner diameter of about 5 mm and an outer diameter of about 20 mm to about 50 mm. The portion of a disc can be 1/8 of a disc. There are no limitations on the shape of the fluid centripetal device and the number of fluid centripetal devices that a rotor can receive.
[0219] In an alternative embodiment, the fluidic centripetal device has a disk shape and the rotor is adapted to receive a single fluidic centripetal device.
[0220] In another alternative embodiment, the shape of the fluidic centripetal device corresponds to a standard 25 mm x 75 mm microscope slide. The rotor can be adapted to receive between 2 to 12 microscope slides. Fluid layer
[0221] FIG. 1C illustrates the structure of the upper face of the fluid layer 3 including the inlet receptacle 5 for receiving a sample and several reservoirs 6a, 6b, 6c. The shape of each reservoir is adapted to the requirements and functions implemented in the fluidic centripetal device 1.
[0222] FIG. 1D illustrates a sectional view of the fluidic layer 3 with the thin lower layer 4. In an exemplary embodiment, the fluidic layer design 3 can be adapted for the injection molding process. It may be advantageous, for some applications, to respect a uniform wall thickness. For example, a wall thickness can be about 0.7 to 1.2 mm. It can be advantageous, for some applications, to ensure a constant tilt angle. Vertical faces can have an inclination angle of about 0.5 ° to 5 °.
[0223] Inlet receptacle, ventilated channels and sample inlet cover of the fluid centripetal device
[0224] FIGS. 2A and 2B illustrate an exemplary embodiment of an input receptacle. Inlet receptacle 5 is fluidly connected to chamber 901. Ventilated outlet 816 is connected to outlet channel and ventilated chamber 905.
[0225] In one embodiment, the ventilated chamber 905 is connected to the inlet receptacle 5, with the inlet vent connection 906 on the upper face of the fluidic centripetal device near the inner portion of the inlet receptacle 5.
[0226] In an exemplary embodiment, a cover 907 includes the base piece 908 in direct contact with the input receptacle 5, a flexible connection arm 909, and a cover 910 connected to the base piece 908 through the connection arm 909 The cover 907 can be placed in a closed configuration with the cover 910 attached to the base piece 908 or it can be placed in an open configuration, as shown in FIG. 2B. In this particular aspect, the base piece 908 is designed to allow communication between the ventilated chamber 905 and chamber 901 via the inlet vent connection 906 even when the cover 907 is in the closed configuration.
[0227] In an alternative mode (not shown), ventilated chamber 905 is disconnected from chamber 901 (inlet ventilation connection 906 is missing). Air ventilation is provided through a hole provided in the base piece 908 of the cover 907 that allows air to be communicated between the chamber 901 and the ventilated chamber 905 through the free cavity formed between the base piece 908 and the cover 910 when the cover 907 is in the closed configuration. Bottom fillable chamber
[0228] FIGS. 3A and 3B illustrate the bottom fillable chamber of the fluidic network. In this example, the input receptacle 5 is fluidly connected to the bottom fillable chamber 315 with the input channel 302. The connection between the input receptacle and the input channel can be optionally made via a port connection 303 or the input receptacle output can be directly connected to the input channel.
[0229] In one embodiment, specific solid phase chromatography material (such as ion exchange material) can be placed inside receptacle 5. During centrifugation, to fill the bottom fillable chamber, the solid phase chromatography material it will fill channel 302 allowing the formation of an exclusion column capable of adsorbing a nucleic acid amplification inhibitor from the crude sample.
[0230] The bottom entry 304 of the bottom fillable chamber 315 is located on the outside of the bottom fillable chamber 315. Since the sample flow will be from the inlet receptacle 5 to the outside of the bottom fillable chamber, the outer side of the bottom fillable chamber is referred to as the bottom of the bottom fillable chamber. A ventilation channel 305a is connected to chamber outlet 306 on the inside of the bottom fillable chamber.
[0231] The size of the chamber is between several centimeters wide, several centimeters high and several millimeters deep. In an exemplary embodiment, the dimension of the chamber 315 is comprised between 1 cm wide, 2 cm high and 2 mm deep. In another exemplary embodiment, the dimensions are 0.5 cm wide, 1.5 cm high and 1.3 mm deep. Reagents and translocatable members
[0232] Referring now to FIGS. 4A and 4B, the bottom fillable chamber may optionally contain a translocatable member 307. The translocatable member may be ferromagnetic and may move in the chamber in response to a floating magnetic field. In an exemplary embodiment, the magnetic field of fluctuation is generated by the rotation of the fluidic centripetal device fixed above to magnets placed alternatively in a radial position corresponding to the inside and outside edges of the bottom fillable chamber. In another embodiment, the fluctuation magnetic field is generated by the rotation of magnets above a fixed fluidic centripetal device.
[0233] In an exemplary mode, fixed magnets are permanent magnets made of rare earth magnetic material. In another modality they are electromagnets.
[0234] The chamber can also optionally contain solid material 308 that does not respond to a magnetic field. The solid material can be used to provide a chemical or biochemical reaction and can include the salt, buffer or enzyme. The solid material can be used to purify the sample by adsorption of enzyme inhibitors and may include a chromatography matrix, a solid support for binding affinity, a solid phase extraction, a chelating material, anionic and cationic resins and different types of zeolites. The solid material can be used for cell disruption and can include a hard matrix. The solid material can be used to control the process and can include bacterial cells or spores. The solid material can be used to concentrate the lysate using a hygrometric matrix for absorbing liquids. The solid material can be functionalized with binders, such as specific antibodies, and can be used to capture targets within the bottom fillable chamber. The solid material can be a filter capable of interrupting or trapping target microbes within the bottom fillable chamber. The solid material can be functionalized with ion exchange portions capable of adsorbing target microbes on its surface, immobilizing them into the bottom fillable chamber. These different solid materials can be used alone or in combination.
[0235] When the solid materials are hard matrix for the cell wall and membrane rupture, the material can be made of silica or zirconium spheres with diameters from about 50 pm to about 200 pm. The spheres can optionally be coated with a chelating agent for the absorption of enzyme inhibitors.
[0236] In an exemplary modality, the translocable object is a metallic disk and the solid material is composed of hard spheres mixed with spores and anionic and cationic resins. Overflow
[0237] FIG. 5 illustrates another fluid interconnection of the bottom fillable chamber that includes an overflow chamber 309 fluidly connected to the overflow outlet 310 of the bottom filler through the overflow channel 311. The overflow channel is placed near the inside of the chamber on one side longitudinal sections of the chamber. The overflow chamber is located towards the outer edge of the fluidic centripetal device with respect to overflow outlet 310 and the overflow chamber is ventilated through ventilation channel 305b. This configuration allows to define the volume in the bottom filling chamber while simultaneously ventilating the bottom filling chamber and the overflow chamber 309. The volume of the overflow chamber is comprised between 100 pl and several milliliters. In an exemplary embodiment, the volume of the overflow chamber is between 150 to 200 pl. Measurement
[0238] FIG. 6 illustrates an optional outlet discharge 312 for the bottom fillable chamber to fluidly connect the bottom fillable chamber to a rear chamber 313 with transfer channel 314. The outlet discharge is located on one of the longitudinal sides of the bottom fillable chamber . The outlet outlet can be a stop valve having a micrometric dimension. The size of the micrometer valve can be from 1 to 100 pm in depth, 10 pm to 1 mm in width and a few microns to a few millimeters in length. In an exemplary embodiment, the dimension of the micrometric valve is between 30 and 75 pm in depth, from 70 to 120 pm in width and 0.5 to 1.5 mm in length. The outlet outlet can be placed at any distance between the inner and outer edges of the bottom fillable chamber, while the outlet outlet is placed in an external position in relation to the overflow outlet. The distance between the discharge outlet and the overflow outlet will define the volume to be measured and sent to the next chamber.
[0239] The volume of fluid measured by the outlet discharge can be between 10 to 50 pl. In an exemplary mode, the volume set is 20 pl.
[0240] FIG. 7A illustrates a bottom fillable chamber having some of the optional configurations described above. Inlet receptacle 5 is fluidly connected to inlet channel 302, bottom fillable chamber 315 and bottom inlet 304. An overflow chamber 309 is fluidly connected to the bottom fillable chamber through overflow outlet 310 and overflow channel 311. Outlet discharge 312 allows the transfer of liquids located between the overflow outlet and the outlet discharge of a rear chamber 313 through outlet channel 312. The chamber contains translocatable member 307 and solid material 308.
[0241] FIG. 7B illustrates a bottom fillable chamber with a target stopper 316. The stopper is placed so as to force the sample through it. Water and small molecule will pass, but the target will be maintained. Since most of the liquid charged into the inlet receptacle 5 will flow through overflow 309 through target stop 316, the target will be concentrated on the small percentage of liquid present in the bottom fillable chamber.
[0242] FIG. 7C shows the release of trapped bacteria after the pathogen stopper is dissociated by the translocable movement of the translocable member 307. The target can be at least one of cells, bacteria, fungi, viruses, etc. In one embodiment, target stopper 316 is a size exclusion filter. In another embodiment, the target stopper 316 is an ion exchange resin. In another embodiment, the pathogen stopper 316 includes beads functionalized with specific antibodies.
[0243] FIG. 8 illustrates fluid progression in the bottom fillable chamber described in FIG. 7. FIGS. 8A to FIG. 8F describe the sequential fluid movement in the bottom fillable chamber. The filling of the chamber occurs in FIG. 8A, the overflow of liquid into the overflow chamber occurs in FIG. 8B and FIG. 8C, sample homogenization and lysis triggered by the translocable movement occurs in FIG. 8D, sedimentation clarification of insoluble materials occurs in FIG. 8E and the transfer of the measured liquids to the next chamber occurs in FIG. 8F.
[0244] Referring now to FIG. 9A and FIG. 9B, the geometry of the bottom inlet 304 and the optional overflow outlet 310 and the optional outlet outlet 312 are adapted to help prevent the translocatable object and / or spheres from leaving the bottom fillable chamber. In one embodiment, the smallest dimension of the translocable object and the spheres contained in the solid material must be greater than width 317a or depth 318a and greater than width 317b or depth 318b. Retention Chamber
[0245] An exemplary embodiment of a fluidic structure for retaining and / or diluting a sample is illustrated in FIGS. 10A to 10D. In this embodiment, a fluid inlet channel 401 is fluidly connected to the inlet 402, located on the inner side of the chamber 403. The ventilation outlet 404 is located on the inner side of the chamber, to allow air to flow into the chamber. The reservoir has a volume of about 1 pl to about 2 ml. The outlet 405 of the reservoir is located on the outside of the chamber, and is generally a stop valve.
[0246] In the embodiment example of FIG. 10A, the holding chamber has an optional receptacle 406 located on the outside of the chamber. The container is generally adapted to contain liquid 407 from the inlet channel, to help prevent the liquid from being in contact with the chamber outlet 405 from the initial entrance in the holding chamber, as shown in FIG. 10B.
[0247] Optionally, the receptacle may contain dry reagents 408 as shown in FIG. 10C. Dry reagents 408 can be, but are not restricted to, enzymes, buffer and / or chemicals.
[0248] In the exemplary embodiment illustrated in FIG. 10D, the holding chamber may optionally include a container of liquid 409 placed within the holding chamber and containing a diluent 410. The diluent may be, but is not limited to, water, buffer or a portion of buffer that cannot be dry. The liquid container 409 is generally, but not necessarily, made of a heat tolerant material and / or a sensitive phase change material. The heat-tolerant material can have a melting point above 100 ° C and can be a glass, thermoplastic polymer, as well as other materials known to those skilled in the art. The phase change material can melt and solidify at a certain temperature. The solid phase can be less than about 45 ° C and the melting phase temperature can be between about 45 ° C and 85 ° C. The phase change material may be wax, paraffin wax, microcrystalline wax, synthetic wax, natural wax, glue or other sealing materials known to those skilled in the art.
[0249] The structures described above can be used as a new type of valve that we call the Flow Dissociation Valve. The Flow Dissociation Valve contains two elements, a flow dissociation receptacle to interrupt the fluid connection between the inlet and outlet of a holding chamber, and a container of liquid that includes a diluent that can be released by applying a external force. The release of the diluent restores the fluid connection within the circuit.
[0250] In an exemplary embodiment, one end of the phase change material 411 of the container 410 releases the liquid when the holding chamber is heated above a certain temperature. The liquid container can help prevent the included liquid from evaporating for a period of about 1 to 3 years, and has a capacity of one microliter to two milliliters.
[0251] FIG. 11 illustrates the fluid progression of the sequential fluid movement in the holding chamber mode. Shown in FIG. 11A is the liquid 407 from the inlet, which is in the receptacle, in FIG. 11B, the holding chamber is heated 416 and the end of the phase change material is melting 411a, 411b, in FIG. 11C, diluent 410 is released, in FIG. 11 D, the liquid 407 from the inlet is mixed with the diluent 410, and in FIG. 11E, the dilute 415 is evacuated by the rupture outlet valve 405.
[0252] An exemplary alternative embodiment is illustrated in FIG. 12 to dilute a fluid in a holding chamber. A diluent from chamber 412 can be located above or on the inside of the holding chamber 403. Liquid is released into the holding chamber by activating a phase change material valve 413 placed at the diluent outlet 414. In this exemplary embodiment, the phase change material valve is a heat activated wax valve above about 50 ° C. In another embodiment, the heat is generated by an electromagnetic wave such as infrared radiation, laser, microwave and any other materials known to those skilled in the art. In an alternative embodiment (not shown), the liquid from the liquid container can be introduced mechanically. For example, a drilling mechanism can be activated by a plunger. In another alternative embodiment (not shown), the liquid from the diluent container can be released by an electromagnetic actuator.
[0253] An exemplary embodiment of a fluid measurement system connected to the holding chamber is shown in FIG. 13. In this example, sample outlet 303 is fluidly connected to bottom fillable chamber 315, via bottom inlet 304. An overflow chamber 309 is connected to bottom fillable chamber 315 with overflow outlet 310 and overflow channel 311 The measurement outlet 312 allows the liquid contained to be transferred between the overflow outlet 310 and the measurement outlet 312 of the chamber 315 to the receptacle of the holding chamber 406 through the measuring channel 314 and holding chamber entrance 402.
[0254] After heating, the diluent 410 contained in the dilution vessel 409 is released into the holding chamber 403. The released liquid 410 mixes with the measured volume contained in the receptacle in the holding chamber 406. Once the diluent and measured volume are mixed together, the total volume is large enough to bring the dilution into contact with outlet 405 acting as a burst valve. Thus, the release of liquid from container 409 takes a liquid to the right place at the right time and also reactivates the fluidic circuit. In fact, before the liquid container is heated, the liquid from chamber 315 is retained in receptacle 406. In this particular embodiment, the receptacle in the holding chamber allows a high burst rate RPM for the 312 metering outlet burst valve and helps prevent liquid from leaving the check chamber by controlling the location of the fluid to help prevent contact with the outlet valve of the 405 check chamber. The mechanism of this new Flow Dissociation Valve dissociates from the rupture valve measurement output 312 of the 405 passive output valve, allowing robust fluid control without the need for complex active valves.
[0255] In another exemplary embodiment, the phase change material 411 of the liquid container 409 has a higher density than the measured liquid 407 retained in receptacle 406 and the diluent 410. When heated, the liquid 410 contained in the liquid container 409 is released into chamber 403. Phase change material 411 will move below the mixture of diluent 410 and fluid 407 and displace the latter so that it can be in contact with outlet 405 which can act as a valve rupture. In this particular embodiment, the holding chamber can be emptied, once the liquid of greater density is released.
[0256] In some embodiments, the dry reagents 408 can be stored in the holding chamber 403. Bucket, detection chamber and distribution chamber
[0257] FIG. 14 illustrates a modality of an arrangement of fluidic detection cuvettes. In the embodiment example of FIG. 14A, a sample from reservoir 601 is fluidly connected to one or more detection cells 602a, 602b and 602c via input channel 603, distribution channel 604 and cell inputs. In the example shown, the number of buckets is three. The end of the distribution channel 604 is fluidly connected to the waste chamber 605 and a ventilation outlet 606 is located close to the distribution end of the channel 604.
[0258] In an exemplary embodiment of FIG. 14B, dry reagents 607a, 607b and 607c were stored in respective cuvettes 602a, 602b, 602c. The dry reagents can be sets of primers, mixtures of enzymes, fluorescence probes and salts to perform the detection and / or enzymatic amplification process. The fluid transferred in the cuvettes will resuspend the dry reagents.
[0259] In another embodiment shown in FIG. 14C, a heat sensitive phase change material 608 can be placed directly inside each cuvette, for example, on top of dry reagents 607. Heat sensitive phase change material 608 can have a lower specific gravity than the specific gravity of the inlet fluid. For example, the melting point of material 608 is greater than 50 ° C and has a specific gravity of less than one. In an exemplary embodiment, the phase-changing material is wax. In this embodiment, the volume of the cuvette minus the volume of the phase change material defines the volume of the amplification reaction, which is generally between 5 and 100 pl. After heating, the phase change material will melt and through the centripetal force it will pass to the 610 cuvette inlet. In this mode, a well-designed cuvette inlet, when filled with the phase-changing material will help prevent evaporation. and cross-contamination between each bucket.
[0260] In another embodiment illustrated in FIG. 14D, the waste chamber may contain a heat sensitive phase change material 612 with a specific gravity less than the specific gravity of the sample. For example, the melting point of the material is greater than 50 ° C and has a specific gravity of less than 1. In an exemplary embodiment, the phase-changing material is wax. As illustrated in FIG. 15, when the waste chamber 605, the distribution channel 604 and the cuvettes 602 are heated 611, and, when an excess of fluid 609 enters the waste chamber 605, the molten wax 612b moves within the distribution channel 604 in the top of the cuvette inlets.
[0261] In another embodiment, a phase change material 612 is placed in the waste. The liquid from the holding chamber is brought to the distribution channel at a temperature below the melting point of the phase change material present in the waste.
[0262] In one embodiment, a phase change material is placed in both the cuvettes and the waste. In this particular embodiment, the melting point of the phase change material 612 placed in the residue is equal to or less than the melting point of the phase change material 608 placed in cuvettes.
[0263] FIG. 16 shows another exemplary embodiment, in which a holding chamber 403, as described above, is fluidly connected to distribution channel 604. In one embodiment, ventilation outlet 606 of the distribution channel and ventilation outlet 404 of the holding chamber they can optionally be merged into a single ventilation channel 704, close to the ventilation outlet 404 of the holding chamber 403.
[0264] FIG. 17 shows another exemplary embodiment in which a sample port 303 is fluidly connected to the bottom fillable chamber 315, in which the chamber includes a translocatable member 307 and hard spheres 308. The bottom fillable chamber is connected to an overflow chamber 309 and a rupture valve metering output 312 is fluidly connected to a holding chamber inlet 402. Holding chamber 403 includes a receptacle 406 and a liquid container 409, as described above, to allow dilution of the fluid. The diluted fluid reaches outlet 405 and is transferred to cuvettes 602, via the outlet of the rupture valve 405 of the retention chamber 403 and residues 605 contain wax 612. In this exemplary embodiment, dry reagents 607 and wax (not shown) are stored in buckets. The ventilated outlets 606, 404 and 815 of the cuvettes, the holding chamber and the bottom filling chamber are fused together to allow ventilation of the complete fluidic circuits through a single ventilation port 816. Exemplary Instrument Configuration
[0265] FIG. 18 illustrates an exemplary instrument 1000 for processing a fluidic centripetal device, as shown above. The apparatus 1000 is, in this exemplary modality, 30 cm wide x 30 cm deep x 20 cm high. It includes a base 1001, a hinge cover 1002 and a rotor assembly 1003 placed inside the centrifuge enclosure 1004. The rotor assembly 1003 placed inside the centrifuge enclosure 1004 rotates in a plane parallel to the base of the instrument.
[0266] FIG. 19 shows the instrument 1000, in more detail, in particular the components located inside the base 1001. The rotational movement of the rotor assembly 1003 is produced by the motor 1005, located below the centrifuge enclosure 1004. The controller 1008 provides a microprocessor, a memory, electronics and software for controlling the 1000 instrument. In this example, the controller provides a hardwire communication protocol interface, such as Ethernet, serial, digital I / O and analog I / O. In this exemplary mode, the 1006 touch screen LCD provides a graphical user interface (GUI) used to operate the instrument software built into the 1008 controller. The LCD communicates with the controller using a serial communication protocol. The controller communicates with 1010 motor controllers and the 1011 optical signal acquisition board, using serial connections. The temperature conditioning board 1012 is connected to analog inputs and excitation source control boards 1013 is connected to the digital output of controller 1008.
[0267] This exemplary instrument 1000 provides multiple temperature zone controls to control the temperature in the predetermined regions of interest of a fluidic centripetal device. In this exemplary embodiment, the centrifuge enclosure 1004, rotor assembly 1003 and cover 1001 are designed to ensure double-zone air temperature control.
[0268] The excitation module 1007 provides at least one excitation wavelength. The path of the excitation beam goes upwards to excite the fluorescent species inside the buckets of fluidic centripetal devices from the bottom face.
[0269] The 1009 detection module is located at the rear of the centrifuge enclosure. The detection module 1009 houses the optical elements that collect the light emitted by fluorescent species in the fluidic centripetal device in at least one wavelength. In this exemplary embodiment, the detector is a PMT. Overview of Instrument Functions
[0270] The instrument includes integrated modules: motor 1005, centrifuge enclosure 1004, multiple zone temperature controller 2000, optics 1014, controller 1008 and a machine-human interface 1006. It must be understood that the arrangement of the various components or modules shown in FIG. 20 is exemplary and is not intended to be limiting. Centrifuge enclosure
[0271] The rotor assembly 1003 is placed inside a centrifuge enclosure 1004 that rotates to control the movement of fluid into the fluidic centripetal device 1. The rotational movement of the rotor assembly is produced by the 1005 motor. rotor can be permanently attached inside the centrifugal enclosure or can be removed from the centrifugal enclosure to allow the placement of the fluidic centripetal device (s) in which the rotor prior to placing the interior of the enclosure of the centrifugal rotor. The rotor assembly can rotate in a plane parallel to the instrument base or, alternatively, in a plane perpendicular to the instrument base. The revolution speeds of the rotor assembly can vary between 0 and 10000 RPM clockwise and / or counterclockwise with an acceleration rate between 0 and 20000 RPM / s. For example, the rotation sequence is performed automatically by controller 1008.
[0272] A permanent magnet (not shown) can be placed inside the centrifuge enclosure to magnetically activate translocatable member 307 located at the bottom of the bottom filling chamber of some types of fluidic centripetal devices. An example of magnetic action for a fluid centrifugal disk was described by Kido et al., In “A novel, compact disk-like centrifugal microfluidics system for cell lysis and sample homogenization”, Colloids Surfaces B: Biointerfaces, 58 (2007) 44- 51. Multiple zone temperature control
[0273] Instrument 1000 also allows the multi-zone temperature controller 2000 to modulate the temperature of predetermined regions of interest (ROI), 1300, 1302 from a fluidic centripetal device. Heating / cooling can be achieved with resistive techniques (nichrome wire, ceramic heater), with or without a fan, thermal techniques (Peltier), halogen heating lamp, as well as other heating / cooling systems known to those skilled in the art. technical.
[0274] Referring now to FIG. 21, a simplified top view of a fluidic centripetal device schematically illustrates two areas of ROI 1300, 1302. In this exemplary embodiment, the two areas of ROI are non-overlapping and ring-shaped. Heating / cooling of different ROI can be achieved independently at specific time points.
[0275] Returning to FIG. 20, heaters 2001 a and 2001 b can heat the air and the hot air forces the selected ROI of the fluid centripetal device to be heated and, consequently, the fluid to be heated. The centrifuge enclosure 1004 may comprise an insulating structure to confine the heated air to the respective compartments of the centrifugal enclosure to ensure ROI temperature control of the centripetal fluid device. The temperature of the heated air in each compartment can be measured by a temperature sensor. The temperature sensor can be thermocouples, thermistors, resistance temperature detectors (RTD), as well as other temperature sensors known to those skilled in the art. A temperature control feedback loop can be applied over the 1008 controller to precisely control the air temperature.
[0276] In some embodiments, a fan can be used to recirculate hot air around an ROI. Alternatively, or in addition, a fan can force fresh air to be heated by a heater before contacting the ROI of interest.
[0277] In some embodiments, at least one ventilation (not shown) allows warm air to escape from the centrifugal enclosure compartment. Ventilation can be opened momentarily or permanently.
[0278] In some modalities, the 2002 fan can be used to cool a specific ROI of the fluid centripetal device. The fan can be used to force cold air (room temperature) into a particular compartment of the centrifugal enclosure to cool a specific ROI of the fluidic centripetal device.
[0279] In some embodiments, at least one ROI of the fluid centripetal device can be kept below 35 ° C when heating another ROI between 25 ° C and 99 ° C.
[0280] Preferably, a temperature feedback circuit algorithm can be applied to controller 1008 to do an isothermal incubation of at least one of the ROI of the fluid centripetal device. Alternatively or in addition, temperature feedback circuit algorithms can be implemented to perform the thermal cycle on at least one ROI of the fluidic centripetal device.
[0281] In one embodiment, the isothermal incubation of an ROI can be used to control inhibition of nucleic acid amplification, more specifically, to control inhibition of amplification by PCR. Alternatively or in addition, isothermal incubation can be used to heat the phase change material. In a more specific embodiment, the ROI of interest within the fluid centripetal device includes at least the fluid retention chamber of the fluid centripetal device described above.
[0282] In one embodiment, isothermal incubation of at least one ROI from a fluid centripetal device can be used to perform an isothermal nucleic acid amplification. In a more specific embodiment, the ROI comprises the cuvettes of a modality of the fluidic centripetal device described above.
[0283] In another embodiment, the thermal cycle of at least one ROI of a fluidic centripetal device can be used for the performed PCR amplification. In a more specific embodiment, the ROI comprises the cuvettes of a modality of the fluidic centripetal device described above.
[0284] Referring now to FIG. 22, the fluid temperature can be controlled in more specific ROI 2201, 2202a, 2202b, 2202c from a fluid centripetal device. It may be appropriate to prevent heating of unnecessary areas of the fluid centripetal device to minimize thermal mass and increase the rate of heating / cooling. The temperature in each specific ROI can be controlled by placing heating / cooling elements and temperature sensors in contact with the lower face and / or the upper face of the fluidic centripetal device on the rotor. The energy can be transmitted to the heating elements by means of slip rings (not shown) placed between the motor and the rotor assembly. Temperature sensor data can also be transmitted via the slip ring and / or wireless set.
[0285] In another alternative modality, a subcontroller can be integrated into the rotary rotor assembly to implement the temperature control feedback cycle of one or more heating elements directly to the rotor. Electric power can be supplied to the rotating electronic plate by one of the batteries placed on the rotating electronic plate, transfer of induction energy between the non-rotating part and the electronic plate placed on the rotor or with a slip ring interface between the motor and the rotor. The communication interface between this subcontroller and controller 1008 can be implemented through serial communication via a slip ring, RF communication or any other wireless transmission mode. In some embodiments, the temperature can be measured at different ROIs in a fluid centripetal device. The conditioning of the detection element and the conversion from analog to digital can be applied directly on the speed controller, thus preventing transmission of the sensor's analog signal through a slip ring and reducing noise. This modality is suitable for calibrating the enzymatic amplification reaction, such as PCR amplification. In an alternative embodiment, the rotation controller can be used to measure the electrical signal from the coated electrode over one of the layers of the fluidic centripetal device. The electrode can be used to detect the presence of liquid in various ROIs of the fluid centripetal device.
[0286] FIG. 23 illustrates dual zone air temperature control. In this example, there are two compartments: compartment # 1 1301 to heat the holding chamber 403 and compartment # 2 1303 to heat and cool the cuvette area 602 of the fluidic centripetal device 1. Air confinement in each area is achieved through a combination of compartments bounded by the centrifuge enclosure 1004, separation wall of the centrifuge enclosure 1304, bottom of the rotor 2, rotor insulation wall 1305, pressure ring 7, cover insulation 1307 and the cover insulation wall 1308. Insulating materials can be used to control heat transfer between adjacent and / or combined components and also to prevent uncontrolled heat flow outside the 1004 centrifuge enclosure. To generate heat inside each compartment, a thermal element 1309a is placed under the fluidic centripetal device in compartment # 2 1303, and a heating element 1309b is placed above the central device fluidic type in compartment # 1 1301. Thermocouples 1310a and 1310b are placed inside each compartment to measure the individual temperature of the compartment. In the 1000 instrument, the heating elements in both compartments are resistive heating coils. To control the cooling rate of compartment # 2 1303, blower 3111 forces air at room temperature to enter compartment # 2 1303. When blower 3111 is blowing air in, outlet port 3112 is opened to eject air hot outside of compartment # 2 1303. A temperature feedback circuit algorithm is implemented in the electronic controller 1008 to precisely control the temperature in each compartment. This configuration allows an air heating rate for both compartments to be, for example, between 1 to 20 ° C / s. The air cooling rate of compartment # 2 1303 is, for example, between 0.1 to 20 ° C / s. Control supply circuit algorithms can be implemented to perform the isothermal incubation of each region of interest of the fluid centripetal device and thermal cycle programs, such as PCR amplification for compartment # 2 1303. Optics
[0287] Returning to FIG. 20, the optics 1014 of the exemplary instrument 1000 includes two modules: the excitation module 1007 and the detection module 1009. These two modules are optically configured to interrogate a liquid 1608 in the fluidic centripetal device. It is suitable for the measurement of fluorescent species in the cuvettes of the fluid centripetal device 1. In some embodiments, fluorescence optics can be used to perform isothermal detection in real time or PCR in real time.
[0288] In another embodiment, optics 1014 only includes a detection module to interrogate the liquid in the fluidic centripetal device.
[0289] The excitation module 1007 includes light source (s) and the mechanical and optical elements for both the spectral and spatial forms of an excitation beam. Several light sources can be housed in an excitation module and their outputs can be coupled to a single beam path. Alternatively, an actuator can allow switching between light sources to excite fluorescent species at different wavelengths. In one mode, the selection of wavelength and output energy adjustment is performed automatically by the instrument's controller 1008.
[0290] In one mode, the light sources are light emitting diode (LED). In another modality, laser, halogen or mercury lamps can be used.
[0291] In some embodiments, excitation module 1007 contains 1 to 6 LEDs to excite fluorescent species in 1 to 6 different wavelengths. Each LED can be spectrally filtered by a single bandpass interferential filter, before being coupled to a single beam path. Alternatively, a multi-band interferential filter can be used for the filter LEDs after they are coupled to a single beam path.
[0292] The detection module 1009 comprises optical elements to collect the light emitted by species of interest within the fluidic centripetal device. The optical elements can be a lens, to shape the spatially collected light, a photodetector interferential filter to select a wavelength band corresponding to the emission spectrum of the fluorescent species. In one embodiment, the detector is a PMT. in another mode, the detectors can be photodiodes.
[0293] In some modalities, the detection module can detect 1 to 6 different wavelengths for a single detector. Each wavelength can be filtered through a single pass-band interferential filter and an actuator can allow switching between the filter to sequentially detect fluorescent species. Alternatively, a multi-band interferential filter can be used to prevent the need for an actuator to change between wavelengths. In this case, all wavelengths will be detected simultaneously by the detector. It may be necessary to excite the fluorescent species sequentially with the excitation module to distinguish each species. For example, this task is performed automatically by controller 1008.
[0294] FIG. 24 illustrates a schematic cross-sectional view showing an excitation module 1007 according to an embodiment of the invention. In this modality, the beam combiner 1607 consists of two LEDs 1601 a and 1601 b, two source lenses 1602a and 1602b, two excitation filters 1603a and 1603b, a dichroic mirror 1604, an aperture 1605 and projection lenses 1606a and 1606b. After being focused through a lens 1602a, LED light 1601a is spectrally filtered through filter 1603a. Then, light passes through the dichroic beam splitter 1604 and lens focusing 1602a is at aperture 1605. The light emitted by LED 1601b is shaped and the lens using filter 1602b and filter 1603b is also focused on aperture 1605, reflecting over beam splitter 1604. Aperture 1605 spatially filters the light emitted from the two LEDs 1601a and 1601b. The light is then projected onto the sample 1608 in the fluidic centripetal device 1 through a pair of lenses 1606a and 1606b to excite the fluorescent species.
[0295] FIG. 25 illustrates the spectral characteristics of the exemplary LEDs 1601a and 1601b, filters 1602a and 1602b and beam splitter 1604. The spectral characteristics of the beam splitter allow combining blue LED and amber LED with peak power at, respectively, 471 nm and 590 nm. This spectral arrangement is well suited to excite both carboxyfluorescein (5-FAM) and / or Texas Red® used in real-time PCR amplification.
[0296] Referring now to FIG. 26, schematic side section views illustrate an exemplary detection module to collect the light emitted by the fluorescent species of interest at two wavelengths from sample 1608 located in the fluidic centripetal device 1. The emitted fluorescence is collected and collimated by the lens objective 1801. Then, after having been spectrally filtered through the interferential filter 1802 having two transmission bands corresponding to the emission spectrum of the fluorescent species, the fluorescence beam path 1807 is shaped separately in two planes by two cylindrical lenses 1803 and 1804. The beam is then filtered spatially by the 1805 rectangular opening field stop and the PMT 1806 rectangular photocathode.
[0297] FIG. 27 illustrates the spectral characteristic of the dual band filter. This spectral configuration allows transmission centered at 524 nm and 628 nm. This configuration is suitable for the detection of 5-carboxyfluorescein (5-FAM) and Texas Red® used in real time PCR amplification. In addition, this module can have several configurations, depending on the needs of the intended application.
[0298] FIG. 28 shows more complex spectral characteristics, with 5 transmission bands: [420-460 nm], [510-531 nm], [589-623 nm], [677-711 nm] and [769-849 nm]. This multi-band pass filter is well adapted for the sequential detection of the following five dyes: AlexaFluor350, 5-carboxyfluorescein (5- FAM), Texas Red®, Cy5, and Alexa 750. Test method for thermocycling amplification
[0299] FIG. 29 illustrates an example workflow using instrument 1000 and an example of a fluid centripetal device of FIG. 17 to perform the preparation of the sample of biological material, control potential inhibitors and detect with a real-time PCR. This flowchart lists examples of temperatures, durations, speeds and steps.
[0300] The first step 1201 consists of placing a biological sample in the input receptacle 5. Then, place the fluid centripetal device in the instrument and press the start button 1202. From this point, the instrument will take care of the entire process. The rotation will start at speed # 1 1203 to transfer the liquid from the inlet receptacle 5 to the lysis chamber 315 and evacuate part of the sample to the overflow chamber 309. The rotation speed will change to speed # 2 1204 to activate the movement of the translocation member 307 within the lower fillable lysis chamber. permanent magnets placed under rotor 2 create a floating magnetic field when fluid centripetal devices rotate on it. After a predetermined period of time, the rotation is changed back to speed # 3 1205 to clarify the lysate and disrupt the 312 measurement output.
[0301] The measured volume is transferred to the holding chamber receptacle 406. In step 1206, the rotation is changed back to speed # 4. Compartment # 1 is heated so that the ROI # 1 of the fluidic centripetal device reaches 95 ° C for 3 minutes, for example, to control inhibitors potentially present in the biological sample. This heating will also melt the wax cap of the liquid container 411 to release diluent 410 into the holding chamber 403. It should be noted that compartment # 2 of the instrument and ROI # 2 of the fluid centripetal device are kept at a lower temperature at 35 ° C, for example, by activating the fan, if necessary.
[0302] At the end of step 1206, the lysate is generally well mixed with the diluent and is ready to be transferred into the distribution channel and cuvettes 602. The transfer is made by heating compartment # 2 to a temperature such that ROI # 2 reaches a temperature above 50 ° C, for example, to melt wax 608 in residue chamber 605 and change the speed to speed # 3, step 1207. The outlet of the dilution reservoir 405 breaks and the liquid is transferred to cuvettes 602 to resuspend the pre-stored PCR dry reagents 607. In step 1208, the rotation speed is changed to speed # 4 and the hot starting enzyme contained in reagents 607 is activated by heating the compartment # 2, so that the ROI # 2 of the fluid centripetal device reaches 94 ° C, for example, for a period between 3 to 10 minutes, depending on the specific reagents used.
[0303] During this time, the heating zone as # 1 cools down naturally to a temperature of around 45 ° C. Continuing at rotation speed # 4, the 1209 real-time PCR cycling protocol is started. The temperature in compartment # 2 is cycled so that the temperature in ROI # 2 is cycled between about 95 ° C, 56 ° C and 72 ° C during periods that varied, respectively, from 1 to 15 s, from 0 to 15 s from 1 to 20 s. At the end of each 72 ° C cycle, the fluorescence measurement is taken at 1 to 6 different excitation / detection wavelengths simultaneously or sequentially. Cycling is done 35 to 45 times. The real-time PCR fluorescence curve is then analyzed and interpreted by a computer-based algorithm. The results are recorded in a database and, optionally, transmitted to the test operator or to a doctor.
[0304] FIG. 30A illustrates the rotation speed profile, the thermal temperature profile of ROI # 1 and ROI # 2, for the exemplary embodiment described in relation to FIG. 29. FIG. 30B illustrates the period before the real-time PCR and FIG. 30C illustrates three cycles of real-time PCR detection.
[0305] In another mode, the instrument can alternatively process sample preparation and isothermal detection in real time. The isothermal amplification used can be, but is not limited to, RMA (ribonuclease-mediated amplification), HDA (Helicase-dependent Amplification), RPA (Recombinase Polymerase Amplification) and SPIA (Single Primer Isothermal Amplification), LAMP (Isothermal Amplification) Circuit-mediated), SDA (Ribbon Displacement Amplification), NASBA (Nucleic Acid Sequence-based Amplification), wGA (Whole Genome Amplification), pWGA (Primate-Based Whole Genome Amplification), ICAN (Initiator Initiated Amplification) Isothermal and Chimeric Nucleic Acids), EXPAR (Exponential Amplification Reaction), NEAR (Enzyme Notch Amplification Reaction), RCA (Rolling Circle Amplification), TMA (Transcription Mediated Amplification).
[0306] It will be recognized by those skilled in the art that a plurality of fluid centripetal devices can be manufactured with the specific applications in mind, which allows the definition of the sample volume, sample homogenization, sample lysis, sample measurement, dilution sample, sample mixing and sample detection. Test method for isothermal amplification
[0307] In an alternative embodiment of the flow diagram illustrated in FIG. 29, steps 1207, 1208, 1209 and 1210 are modified to perform the preparation of the sample of biological material, control potential inhibitors and detect with real-time isothermal amplification. In a more particular modality, real-time isothermal amplification is Real-time Recombinase Polymerase Amplification (real-time RPA).
[0308] The steps are as follows: load a biological sample into the input receptacle 5. Then, place the fluid centripetal device on the instrument and press the start button. From this point on, the instrument will take care of the entire process. The rotation will start at speed # 1 to transfer the liquid from the inlet receptacle 5 to the lysis chamber 315 and evacuate part of the sample to the overflow chamber 309. The rotation speed will be changed to speed # 2 to activate the movement of the translocation member 307 within the lower fillable lysis chamber. Permanent magnets placed under rotor 2 create a floating magnetic field when fluid centripetal devices rotate over it. After a predetermined period of time, the rotation is changed back to speed # 3 to clarify the lysate and disrupt the 312 measurement output.
[0309] The measured volume is transferred to the holding chamber receptacle 406. The rotation is changed again to speed # 4. Compartment # 1 is heated so that in ROI # 1 of the fluid centripetal device the temperature is 95 ° C for 3 minutes, for example, to control inhibitors potentially present in the biological sample. This heating will also melt the wax cap of the liquid container 411 to release the diluent 410 into the holding chamber 403. It should be noted that compartment # 2 is kept at a lower temperature so that ROI # 2 is maintained at a temperature below 35 ° C, for example, by activating the blower, if necessary.
[0310] The lysate is generally well mixed with the diluent and is cooled to a temperature of 42 ° C or less and ready to be transferred into the distribution channel and cuvettes 602. In this modality, the diluent is water and magnesium . The transfer is made by keeping compartment # 2 at a temperature so that ROI # 2 is maintained at 37-42 ° C, and changing the rotation to speed # 3. The outlet of the dilution reservoir 405 breaks and the liquid is transferred to the wells 602 to resuspend the pre-stored dry PCR reagents 607. In this embodiment, the dry reagent 607 comprises RPA fluorescent probe, primers, recombinase, polymerase, exonuclease, agglomeration agent, GP32, uvsY and uvsX. The rotation speed is changed to speed # 4 and compartment # 2 is heated so that ROI # 2 reaches 37-42 ° C.
[0311] During this time, heating zone # 1 cools down naturally to a temperature below 45 ° C. Fluorescence measurement is taken at 1 to 6 different excitation / detection wavelengths simultaneously or sequentially every few minutes. The amplification step is interrupted after 20 minutes. The RPA fluorescence signal in real time is then analyzed and interpreted by a computer-based algorithm. The results are recorded in a database and, optionally, transmitted to the test operator or to a doctor.
[0312] In another mode, the instrument can alternatively process sample preparation and isothermal detection in real time. The isothermal amplification used can be, but is not limited to, RMA (ribonuclease-mediated amplification), HDA (Helicase-dependent Amplification), RPA (Recombinase Polymerase Amplification) and SPIA (Single Primer Isothermal Amplification), LAMP (Isothermal Amplification) Circuit-mediated), SDA (Ribbon Shift Amplification), NASBA (Nucleic Acid Sequence Based Amplification), wGA (Whole Genome Amplification), pWGA (Primate Based Whole Genome Amplification), ICAN (Primer Initiated Amplification) Isothermal and Chimeric Nucleic Acids), EXPAR (Exponential Amplification Reaction), NEAR (Enzyme Notch Amplification Reaction), RCA (Rolling Circle Amplification), TMA (Transcription Mediated Amplification).
[0313] It will be recognized by those skilled in the art that a plurality of fluid centripetal devices can be manufactured with the specific applications in mind, which allows the definition of the sample volume, sample homogenization, sample lysis, sample measurement, dilution sample, sample mixing and sample detection. Example 1
[0314] The following example is illustrative and is not intended to be limiting.
[0315] The present example concerns the detection of the presence of Group B streptococci from a vaginal - anal smear of a pregnant woman.
[0316] The fluid centripetal device used for the purposes of the present example has the external shape described in FIG. 1C and is composed of the following fluid elements shown and described in FIG. 17. Table 1. Summary of details of fluidic centripetal device structures


[0317] The liquid container was manufactured using the following protocol: close one end of the plastic straw with hot glue; load 140 pl of PCR treated water; seal the container with molten paraffin wax.
[0318] The fluidic centripetal device was assembled using the following protocol: place the paramagnetic disk in the lysis chamber; loading 60 pl of glass ball paste; load 2 pl of primers in each cuvette; load 0.5 pl of TaqMan probe; dry the paste and primers under vacuum overnight, place the liquid container in the holding chamber; dispense low melting paraffin wax in the waste chamber.
[0319] The following steps are carried out in a glove box under an Argon atmosphere: place an OmniMix HS sphere per detection cuvette; glue the thin bottom layer to the fluid layer using the pressure sensitive adhesive, place the fluid centripetal device mounted in an aluminum bag with desiccant and seal the bag. Experiment
[0320] During a clinical study, vaginal / anal smears were collected from pregnant women using Clinical Packaging pressure valve technology filled with 600 pL of 10 mM Tris EDTA (TE).
[0321] After resuspension of the smear with 600 pl of TE, an amount of 170 pl of the smear dilution is placed directly into the inlet receptacle of the fluid centripetal device described above.
[0322] The fluidic centripetal device is attached to the instrument and the following protocol is performed on the dual zone temperature control instrument for sample preparation.
[0323] The parameters for the measurement, lysis and control of PCR inhibitors used in this example are as follows: Table 2. Loading the bottom fillable chamber
Table 3. Lysis step
Table 4. Clarification step and transfer to the holding chamber receptacle
Tabel 5. PCR Inhibitor Control
Table 6. Filling the cuvette by PCR

[0324] The fluidic centripetal device is then transferred to an adapted rotor specifically designed to work in a RotorGene to process the PCR in real time using the following conditions. Table 7. Process conditions
Results:
[0325] Smears were found positive for the presence of GBS detection at 28.30 CT. Example 2
[0326] The following example is illustrative and is not intended to be limiting
[0327] The present example refers to the use of an exemplary modality of the fluid centripetal device to detect the presence of Group B streptococci in vaginal-anal smears of pregnant women.
[0328] The fluid centripetal device used for the purposes of the present example has the external shape described in FIG. 1C and is composed of the following fluid elements shown and described in FIG. 17. Table 8. Summary of the details of the structures of fluidic centripetal devices


[0329] The liquid container was manufactured using the following protocol: load 120 pl of the liquid PCR diluent; seal the polyalomer tube with a hot glue stick BAP 5-4.
[0330] The fluid centripetal device was assembled using the following protocol: place the paramagnetic disk in the lysis chamber; load 60 pl of glass sphere paste; load 4.6 pl of PCR reagents in each well; drying the paste and PCR reagents under heat and vacuum; place the liquid container in the holding chamber; dispense the low melting paraffin wax in the waste chamber.
[0331] Connect the pre-assembled 9795R / 467 MP / polycarbonate layers to the fluidic layer and apply pressure using a press with a torque of 90 in.lbs. Place the fluidized centripetal device mounted in an aluminum bag with desiccant and seal the bag. Experiment
[0332] During a clinical study, vaginal / anal smears were collected from pregnant women using Medical Packaging pressure valve technology filled with 600 pL of 10 mM Tris EDTA (TE).
[0333] After resuspension, smears with 600 pl of TE, 170 pl of the smear dilution are placed directly into the sample inlet receptacle of the fluid centripetal device described above.
[0334] The fluid centripetal device is placed on the instrument and the following protocol is performed on the dual zone temperature control instrument for sample preparation.
[0335] The parameters for the measurement, lysis and control of PCR inhibitors used in this example are as follows: Table 9. Loading the bottom fillable chamber
Table 10. Lysis step
Table 11. Clarification step and transfer to the holding chamber receptacle
Table 12. Control of PCR inhibitors by heating and diluting the fluid
Table 13. Filling the PCR cuvette

[0336] The fluidic centripetal device is then transferred to an adapted rotor specifically designed to work in a RotorGene to process PCR in real time using the following conditions. Table 14. Thermocycling conditions
Results:
[0337] Smears were found positive for the presence of GBS detection at CT between 27 and 32. Example 3
[0338] The following example is illustrative and is not intended to be limiting.
[0339] The present example relates to the use of an exemplary modality of the fluid centripetal device to detect the presence of the human beta-globin gene from a human cheek smear sample, Escherichia coli from human urine samples , and methicillin-resistant Staphylococcus aureus (MRSA) from samples of human nose smears. Table 15. List of selected amplification initiators and detection probes for the different assays




a The small letter on the Taqman-LNA probe indicates Blocked Nucleic Acids (LNA ™). Table 16. Summary of details of the structures of the fluidic centripetal device


[0340] The fluid centripetal devices used for the purposes of the present example have the external shape described in FIG. 1C and are composed of fluid elements shown and described in FIG. 17.
[0341] The fluid centripetal devices used for the purpose of this example contained the components listed in Table 16.
[0342] The liquid container was manufactured using the following protocol: load 120 pl of the PCR liquid diluent; seal the polyalomer tube with hot glue stick BAP 5-4.
[0343] The fluid centripetal device was assembled using the following protocol: place the paramagnetic disk in the lysis chamber; load 60 pl of glass ball suspension; load 4.6 pl of PCR reagents in each well; dry paste and PCR reagents under heat and vacuum; place the liquid container in the holding chamber; dispense low melting paraffin wax in the waste chamber.
[0344] Connect the pre-assembled 9795R / 467 MP / polycarbonate layers to the fluidic layer and apply pressure using a press with a torque of 90 in.lbs. Experiment
[0345] A face brush smear was collected from a human volunteer using a medical packaging smear with pressure valve technology filled with 600 pL of 10 mM Tris EDTA (TE). The smear was placed in contact with the inner cheek surface and rotated for 30 s. The smear was placed back on his sleeve and the pressure valve was broken to release 600 pl TE. After 5 minutes of waiting time, the smear is vortexed for 1 minute. This suspended diluted sample served for testing.
[0346] Urine samples collected from patients were diluted 1/56 in TE. This diluted sample served for testing.
[0347] During a clinical study, nasal smears were collected from volunteers and resuspended in 600 pL TE. This suspended diluted sample served for testing.
[0348] A volume of 140 pl of the diluted samples is placed directly in the sample entry receptacle of the fluid centripetal device described above.
[0349] Fluid centripetal devices are placed on the instrument and the protocols were performed in accordance with Tables 9, 10, 11, 12, and 13.
[0350] In some tests, the protocol was interrupted and restarted in step 8 to examine the fluid's position in the receptacle holding chamber.
[0351] Thermocycling was carried out under the conditions listed in Table 17 or Table 18, depending on the assay. Table 17. Thermocycling conditions for the beta-globin assay
Table 18. Thermal cycling conditions for ICU and MRSA tests
Results:
[0352] The internal controls revealed no inhibition or only minimal inhibition of samples. All samples already known to contain the target DNA by another test method were actually found positive with the fluidic centripetal device and similarly, samples already known to be negative for the target DNA did in fact have negative results with the fluidic centripetal device.
[0353] This example illustrates the versatility of the fluid centripetal technology of the present invention for the detection of nucleic acids from a variety of samples and biological cells. Diluted faecal samples were also successfully tested for the detection of bacterial pathogens responsible for diarrhea. Example 4
[0354] The following example is illustrative and is not intended to be limiting.
[0355] The present example relates to the use of an exemplary embodiment of the fluidized centripetal device and, more specifically, the bottom fillable chamber and other elements of the present invention to concentrate cells and microbes. Table 19. Summary of the details of the structures of fluidic centripetal devices.


[0356] The fluid centripetal devices used for the purposes of the present example have the external shape described in FIG. 1C and are composed of fluid elements shown and described in FIG. 17.
[0357] The fluid centripetal devices used for the purpose of this example contained the components listed in Table 19.
[0358] The fluid centripetal device was assembled using the following protocol: place the paramagnetic disk in the lysis chamber; load 60 pl of glass sphere paste; drying the paste overnight under vacuum; connect the pre-assembled 9795R / 467 MP / polycarbonate layers to the fluidic layer and apply pressure using a press with a torque of 90 in.lbs. Experiment
[0359] 10 pl (105 Colony Forming Units; CFU) of a diluted culture of the bacteria Enterococcus faecalis were mixed with 190 pl of TE.
[0360] The 200 pl mixture was placed directly in the sample entry receptacle of the fluid centripetal device described above.
[0361] Fluid centripetal devices were placed on the instrument and the following protocols were performed according to Tables 9 and 10.
[0362] The fluidic centripetal devices were disassembled, removing pressure-sensitive layers, so that the liquid in the bottom fillable chamber and the overflow chamber could be collected and diluted to perform bacterial cell counts present in each chamber. Results:
[0363] Counts revealed that the number of bacterial cells was higher in the bottom fillable chamber compared to the number of bacterial cells present in the overflow chamber by a factor of 1.5 to 3 times.
[0364] The modalities described above are intended to be an example only. The scope of the invention is therefore intended to be limited only by the appended claims.

权利要求:
Claims (49)
[0001]
1. Fluidic centripetal device for testing components of a biological material in a fluid, said fluidic centripetal device having a shape adapted to be received within a rotating support, said rotating support having a center of rotation and an outer edge, said fluidic centripetal device extending radially between said center of rotation and said outer edge, an inner side of said fluidic centripetal device being located towards said center of rotation and an external side of said fluidic centripetal device being located towards said outer edge, the device characterized by the fact that it comprises: a layer of fluid component having fluid characteristics on at least one front face, said fluid characteristics including: an inlet channel for circulation of said fluid, said inlet channel being coupled a camera entrance; a bottom fillable chamber coupled to said inlet channel in said chamber inlet to receive said fluid, said chamber inlet being provided on an external side of said bottom fillable chamber; wherein said bottom fillable chamber comprises at least one translocable member other than spheres that translocates within said bottom fillable chamber in response to an external floating magnetic field, the bottom fillable chamber and translocatable member being configured and sized to allow translocating the translocatable member into the bottom fillable chamber, while preventing the translocable member from leaving the bottom fillable chamber; an outlet discharge to said bottom fillable chamber, said outlet discharge allowing said fluid to exit from said bottom fillable chamber, wherein the outlet discharge is located on a longitudinal side of said bottom fillable chamber; an overflow chamber coupled to an overflow outlet for said bottom-fillable chamber, said overflow outlet allowing part of said fluid to exit from said bottom-fillable chamber to said overflow chamber, wherein said overflow chamber excess outlet is provided close to said inner side of said bottom fillable chamber on a longitudinal side of said bottom fillable chamber; a ventilation outlet connected to a ventilation channel, said ventilation outlet being coupled to said overflow chamber to simultaneously ventilate said bottom fillable chamber and said overflow chamber; and a bottom component layer connected to a rear part of said fluidic component layer, thereby creating a fluidic network through which said fluid flows under centripetal force.
[0002]
2. Device according to claim 1, characterized in that it further comprises an inlet receptacle for receiving said fluid, said inlet receptacle extending outwardly from said fluidic component layer on a front face of the said fluidic component layer and being located close to said inner side, said inlet receptacle terminating at an inlet receptacle outlet, said inlet channel being coupled to said inlet receptacle outlet at an end opposite to said inlet receptacle chamber. bottom fillable chamber, bottom fillable chamber
[0003]
3. Device according to claim 1, characterized by the fact that the bottom-filling chamber comprises at least one object unresponsive to a floating magnetic field and in which said object is at least one of a sphere, a zeolite, a particle, a filtration particle, a glass sphere, a zirconium sphere, a resin, a sphere and resin paste.
[0004]
Device according to claim 1, characterized by the fact that at least one of said object and said translocatable member is coated with at least one of a chelator and a binder material adapted to interact with components of said fluid.
[0005]
5. Device according to claim 1, characterized by the fact that it also comprises an outlet discharge for said bottom fillable chamber, said outlet discharge allowing said fluid to exit from said bottom fillable chamber, in that said outlet discharge is located on said longitudinal side of said bottom fillable chamber, said outlet discharge being located closer to said external side of said bottom fillable chamber than said excess outlet, a volume of measurement of said bottom fillable chamber being defined between said outlet discharge and said excess outlet.
[0006]
6. Device according to claim 5, characterized by the fact that it further comprises a rupture valve in said outlet discharge, said rupture valve opening at a predetermined centripetal force applied to said apparatus, said rupture valve preventing that said fluid leaves the bottom filling chamber until said opening.
[0007]
Device according to claim 5, characterized by the fact that it further comprises a holding chamber, said holding chamber being coupled to said outlet discharge on an internal side of said holding chamber, said holding chamber being located closer to said external side of said fluidic component layer than said bottom fillable chamber, wherein said holding chamber is coupled to said outlet discharge through a measuring channel, said measuring channel for circulating at least a portion of said fluid from said bottom fillable chamber to said holding chamber.
[0008]
Device according to claim 7, characterized in that it further comprises a container integrally supplied in said holding chamber and containing a liquid diluent, said container being adapted to hold said liquid diluent in said container and to release the said liquid diluent in said holding chamber by applying a force external to said container, in which said external force is one of mechanical, electrical, electromagnetic, heat, shock and acoustic force, thus restoring the fluid connection between said liquid diluent and said fluid in said holding chamber.
[0009]
9. Device according to claim 7, characterized by the fact that said holding chamber has a distribution outlet for said holding chamber, said distribution outlet being located on an external side of said holding chamber, the said distribution outlet being coupled to a transverse distribution channel on an internal side of said transverse distribution channel at a first transverse end of said distribution channel, said transverse distribution channel having a series of at least one bucket provided in a external side of said transverse distribution channel.
[0010]
10. Device according to claim 9, characterized by the fact that said at least one cuvette includes at least one of a dry reagent and a phase change material.
[0011]
11. Device, according to claim 9, characterized by the fact that said at least one cuvette is adapted to be optically investigated for at least one parameter, said parameter is one among fluorescence, absorbance and colorimetry.
[0012]
Device according to claim 9, characterized by the fact that said transverse distribution channel includes a waste chamber in a second transverse end of said distribution channel.
[0013]
13. Device according to claim 12, characterized by the fact that said waste chamber includes a phase change material.
[0014]
14. Device according to claim 12, characterized by the fact that said distribution channel, said at least one bucket and said waste chamber are provided in a portion of said fluidic layer component which extends beyond the said outer edge of said rotating support.
[0015]
15. Device according to claim 14, characterized by the fact that said fluid component layer is adapted to be divided into at least two distinct temperature controllable sections, wherein a first of said two distinct temperature controllable sections includes at least minus said holding chamber and a second of said two distinct temperature-controllable sections includes at least said distribution channel and said cuvettes.
[0016]
16. Test apparatus using a fluidic centripetal device for testing components of a biological material in a fluid, the tester characterized by the fact that it comprises: at least one of said fluidic centripetal device as defined in claim 1; a rotor assembly; a support for receiving said at least one of said fluidic centripetal device using said fluidic component layer, said support being coupled to said rotor; a motor for rotating said rotor assembly; a speed controller for said motor to control at least one of a duration and a rotation speed of said rotor assembly; a temperature conditioning subsystem for controlling a temperature of at least a portion of said microfluidic centripetal device; a detection subsystem for detecting a characteristic of said fluid; a user interface for receiving a user command and for sending a command to at least one of said speed controller, said temperature conditioning subsystem, said excitation subsystem and said detection subsystem.
[0017]
Test apparatus according to claim 16, characterized in that said temperature conditioning subsystem controls a temperature of at least two zones of said fluidic centripetal device.
[0018]
18. Test method using a fluidic centripetal device to test components of a biological material in a fluid, the method characterized by the fact that it comprises: providing at least one of said fluidic centripetal device as defined in claim 1; providing a test apparatus as defined in claim 16; supply a fluid with biological material; loading said fluid into said inlet receptacle of said fluidic centripetal device; placing said fluidic centripetal device on said support of said tester; provide a user command to initiate a test sequence; rotating said rotor assembly at a first speed to transfer said fluid from said inlet receptacle to said bottom fillable chamber.
[0019]
19. Test method according to claim 18, characterized by the fact that the fluid is selected from the group consisting of blood, nasal pharynx aspiration, oral fluid, liquid from resuspended oral smear, liquid from resuspended nasal smear, resuspended liquid from anal smear, resuspended liquid from vaginal smear, saliva and urine.
[0020]
20. Test method, according to claim 18, characterized by the fact that it tests at least one component selected from the group consisting of ions, sugars, metabolites, fatty acids, amino acids, nucleic acids, proteins and lipids.
[0021]
21. Fluidic centripetal device for testing components of a biological material in a fluid, said fluidic centripetal device having a shape adapted to be received within a rotating support, said rotating support having a center of rotation and an outer edge, said fluidic centripetal device extending radially between said center of rotation and said outer edge, an inner side of said fluidic centripetal device being located towards said center of rotation and an external side of said fluidic centripetal device being located towards said outer edge, the device characterized by the fact that it comprises: a fluidic component layer having fluidic characteristics on at least one front face, said fluidic characteristics including: a holding chamber for receiving said fluid, said holding chamber being coupled to a fluid inlet channel to receive said fluid within said holding chamber; and a container integrally supplied in said holding chamber, said container containing a liquid diluent, said container additionally comprising a first phase-changing material for releasing said diluent into the holding chamber; and a bottom component layer connected to said fluid component layer, the fluid component layer and the bottom component layer creating a fluid network, said fluid flowing through said fluid network under centripetal force.
[0022]
22. Device according to claim 21, characterized in that said holding chamber comprises a fluid dissociation receptacle, wherein said fluid dissociating receptacle is located on an external side of said holding chamber between the fluid inlet channel and an interior of the holding chamber, said fluid dissociation receptacle interrupting a fluid connection between the fluid inlet channel and the holding chamber.
[0023]
23. Device according to claim 22, characterized in that said fluid dissociation receptacle includes a dry reagent.
[0024]
24. Device according to claim 21, characterized in that said holding chamber has a dispensing outlet for said holding chamber, said dispensing outlet being located on an external side of said holding chamber, said distribution outlet being coupled to a distribution channel on an internal side of said distribution channel on a first transverse end of said distribution channel, said distribution channel having at least one bucket provided on an external side of said channel of distribution.
[0025]
25. Device according to claim 24, characterized by the fact that said at least one cuvette includes at least one of a dry reagent and a second phase change material.
[0026]
26. Device, according to claim 24, characterized by the fact that said cuvette is adapted to be optically investigated for at least one parameter, in which said parameter is one among fluorescence, absorbance and colorimetry.
[0027]
27. Device according to claim 24, characterized by the fact that said distribution channel includes a waste chamber at a second end of said distribution channel.
[0028]
28. Device according to claim 27, characterized by the fact that said waste chamber includes a third phase-changing material.
[0029]
29. Device according to claim 28, characterized by the fact that said distribution channel, said at least one bucket and said waste chamber are supplied in a portion of said fluidic component layer that extends beyond said outer edge of said rotating support.
[0030]
30. Device according to claim 29, characterized by the fact that said fluidic component layer comprises at least two distinct temperature controllable sections.
[0031]
31. The device according to claim 21, characterized by the fact that said container is adapted to hold said liquid diluent in said container and to release said liquid diluent in said holding chamber by applying a force external to said container, wherein said external force is one of mechanical, electrical, electromagnetic, heat, shock and acoustic force, thus allowing the fluid connection between said liquid diluent and said fluid in said holding chamber.
[0032]
32. The device according to claim 21, characterized by the fact that it additionally comprises a bottom-filling chamber, wherein said bottom-filling chamber is coupled to said inlet channel to supply fluid to the holding chamber.
[0033]
33. Device according to claim 32, characterized by the fact that said bottom-fillable chamber includes at least one translocatable member that translocates within said bottom-fillable chamber in response to an external floating magnetic field.
[0034]
34. Device according to claim 32, characterized by the fact that the bottom-filling chamber comprises at least one object unresponsive to a floating magnetic field and in which said object is at least one of a sphere, a zeolite, a particle, a filtration particle, a glass sphere, a zirconium sphere, a resin, a sphere and resin paste.
[0035]
35. Device according to claim 34, characterized by the fact that at least one of said object and said translocatable member is coated with at least one of a chelator and a binder material adapted to interact with components of said fluid.
[0036]
36. Device according to claim 32, characterized by the fact that it additionally comprises an overflow chamber coupled to an overflow outlet for said bottom fillable chamber, said overflow outlet allowing part of said fluid to flow out of said chamber bottom filler for said overflow chamber, wherein said overflow outlet is provided close to said inner side of said bottom fillable chamber on a longitudinal side of said bottom fillable chamber.
[0037]
37. Device according to claim 36, characterized in that it further comprises an outlet discharge for said bottom-filling chamber, said outlet discharge allowing said fluid to exit from said bottom-filling chamber, in that said outlet discharge is located on said longitudinal side of said bottom fillable chamber, said outlet discharge being located closer to said external side of said bottom fillable chamber than said excess outlet, a volume of measurement of said bottom fillable chamber being defined between said outlet discharge and said excess outlet.
[0038]
38. Device according to claim 37, characterized by the fact that it additionally comprises a rupture valve at said outlet discharge, said rupture valve opening at a predetermined centripetal force applied to said apparatus, said rupture valve preventing that said fluid leaves the bottom filling chamber until said opening.
[0039]
39. Device according to claim 37, characterized in that said holding chamber is coupled to said outlet discharge on an internal side of said holding chamber, said holding chamber being located closer to said side external of said fluidic component layer than said bottom fillable chamber, wherein said holding chamber is coupled to said outlet discharge through a measuring channel, said measuring channel to circulate at least a portion of said fluid from said bottom fillable chamber to said holding chamber.
[0040]
40. Device according to claim 36, characterized in that it additionally comprises an outlet discharge for said bottom fillable chamber, said outlet discharge allowing said fluid to exit from said bottom fillable chamber, wherein said outlet discharge is located on a longitudinal side of said bottom fillable chamber.
[0041]
41. Device according to claim 21, characterized in that it additionally comprises an inlet receptacle for loading the fluid comprising said biological material.
[0042]
42. Device according to claim 21, characterized by the fact that the phase-changing material is a heat-tolerant material releasing said diluent at a certain temperature.
[0043]
43. The device of claim 21, characterized in that said holding chamber comprises a flow dissociation receptacle located on an external side of said holding chamber, the flow dissociation receptacle comprising a reagent.
[0044]
44. Device according to claim 43, characterized by the fact that the reagent is isolated from the inside of the holding chamber by a fourth phase change material which is a heat-tolerant material releasing said reagent in a certain temperature.
[0045]
45. Test apparatus using a fluidic centripetal device for testing components of a biological material in a fluid, the tester characterized by the fact that it comprises: at least one of said fluidic centripetal device as defined in claim 21. a rotor assembly ; a support for receiving said at least one of said fluidic centripetal device using said fluidic component layer, said support being coupled to said rotor; a motor for rotating said rotor assembly; a speed controller for said motor to control at least one of a duration and a rotation speed of said rotor assembly; a temperature conditioning subsystem for controlling a temperature of at least a portion of said fluidic centripetal device; a detection subsystem for detecting a characteristic of said fluid; a user interface for receiving a user command and for sending a command to at least one of said speed controller, said temperature conditioning subsystem, said excitation subsystem and said detection subsystem.
[0046]
46. Test apparatus according to claim 45, characterized in that said temperature conditioning subsystem controls a temperature of at least two zones of said fluidic centripetal device.
[0047]
47. Test method using a fluidic centripetal device to test components of a biological material in a fluid, the method characterized by the fact that it comprises: providing a tester with at least one of the fluidic centripetal device as defined in claim 45; supply a fluid with biological material; loading said fluid into said inlet receptacle of said fluidic centripetal device; placing said fluidic centripetal device on said support of said tester; provide a user command to initiate a test sequence; rotating said rotor assembly to flow said fluid through said fluid network.
[0048]
48. Test method according to claim 47, characterized by the fact that the fluid is selected from the group consisting of blood, nasal pharynx aspiration, oral fluid, liquid from resuspended oral smear, liquid from resuspended nasal smear, resuspended liquid from anal smear, resuspended liquid from vaginal smear, saliva and urine.
[0049]
49. Test method, according to claim 47, characterized by the fact that it tests at least one component selected from the group consisting of ions, sugars, metabolites, fatty acids, amino acids, nucleic acids, proteins and lipids.

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

---

2019-09-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-05-19| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-10-06| 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 07/03/2012, 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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US201161450373P| true| 2011-03-08|2011-03-08|

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US61/450,373|2011-03-08|

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PCT/IB2012/051076|WO2012120463A1|2011-03-08|2012-03-07|Fluidic centripetal device|

---