system for detecting the presence of an infectious agent in a biological sample, test device for det

专利摘要:
SYSTEM FOR DETECTING THE PRESENCE OF AN INFECTIOUS AGENT IN A BIOLOGICAL SAMPLE, TEST DEVICE FOR DETECTING AN INFECTIOUS AGENT IN A BIOLOGICAL SAMPLE, SYSTEM FOR USE IN THE SAMPLING ANALYSIS, SYSTEM FOR DETECTING THE PRESENCE OF A HAZARDOUS PRESENTATION TESTING TO DETECT AN INFECTIOUS AGENT IN A BIOLOGICAL SAMPLE The present invention relates to a portable system for detecting in real time the presence of an infectious agent in a biological sample (414) that uses a reagent that detects the presence of an agent specific infectious agent in the sample (414) and emits a detectable signal when the reagent reacts with the sample (414) and detects the presence of the infectious agent. A test kit (300) has a reaction chamber (404) for receiving the sample (414) and the reagent. The reaction chamber (404) has a predetermined internal geometry and at least one internal surface. Insert the sample (414) and the reagent into the test kit (300) mix the sample (414) and the reagent. A test unit receives the test case (300) and includes a sensor (206) to detect an emitted detectable signal. The detection of the detectable signal emitted is indicative of the presence of the infectious agent in the sample (414)
公开号:BR112014014049B1
申请号:R112014014049-9
申请日:2012-12-12
公开日:2020-11-17
发明作者:Marvin R. Williams；Charles MCBRAIRTY；Daniel W. Pfautz；Thomas J. Zupancic；Lingchun ZENG；Andrew Weiman；Richard S. Brody；Joseph KITTLE；Anthony Truscott；Robert Baranowski
申请人:Fundamental Solutions Corporation；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[0002]. This patent application claims the benefit of US Provisional Patent Application serial number 61 / 570,016 filed on December 13, 2011 and entitled "Portable Detection Device," whose content is hereby incorporated by reference in its entirety and made an integral part. of the current US utility patent application for all purposes. HISTORY OF THE INVENTION
[0003]. The described invention generally relates to a system for detecting contaminants in biological samples. More specifically, the present invention relates to a system for detecting infectious agents or pathogens in food samples, in real time, using a reagent, such as a biosensor.
[0004]. Previously, testing samples for infectious agents was a time-consuming and expensive process, largely separate from the manufacturing process. To test for the presence of an infectious agent, a sample was typically enriched or cultured. This process requires the presence of a laboratory and, generally, the involvement of scientists with knowledge of carrying out the necessary test. Due to the need for additional time for cultivation or enrichment and specialized tools and techniques, the test could not be easily carried out on site during the manufacturing process. As a consequence, the manufacturing process was generally separated from the testing process, resulting in the need for expensive recalls when the testing process subsequently found the presence of infectious agents and the like. In other establishments, such as hospitals, delays in receiving testing for infectious agents can allow the spread of these infectious agents.
[0005]. Several proposals have been made to improve the speed of testing for infectious agents using biosensors to detect. For example, the application of the aequorin-Ca2 + indicator to detect contamination of food products by E. coli was reported by Todd H. Rider et a /, AB Cell-Based Sensor for Rapid Identification of Pathogens, SCIENCE, July 11, 2003, pages 213- 215, the full disclosure of which is hereby incorporated by reference. However, the Rider process had several disadvantages, such as a low signal-to-noise ratio, which made the process unreliable for use in large-scale tests.
[0006]. In general terms, a biosensor is a system or device for detecting an analyte that combines a sensitive biological component with a physicochemical detector component. The components of a typical biosensor system include a biological element, a transducer or detector element and associated electronics or signal processors that display test results in a meaningful and useful way. The biological element includes biological material, such as, for example, tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids and the like, which can be created by known biological engineering processes. The transducer or detector element works in a physicochemical way (for example, optical, piezoelectric and / or electrochemical), which transforms a signal resulting from the interaction of the analyte with the biological element into another signal that can be more easily measured and quantified. Biosensors originating from the integration of molecular biology and information technology (for example, microcircuits, optical fibers, etc.) to qualify or quantify biomolecule-analyte interactions, such as antibody-antigen interactions.
[0007]. There is a demand for fast, sensitive, easy to handle and economical detection tools to detect infectious agents, pathogens and / or toxins in food (see, for example, Mead et a /, Food Related Illness and Death in the United States, Emerging Infectious Diseases; Vol. 5, No. 5, September-October 1999 (607-625), which is incorporated herein by reference).
[0008]. Consequently, it is desirable to provide a portable and autonomous system, capable of quickly testing samples for infectious agents, in real time or in near real time. It is also desirable to improve the technique of using biosensors to test samples for infectious agents, improving the signal-to-noise ratio. It is also desirable to provide a test device capable of being used by general personnel to test foodstuffs during the manufacturing process. BRIEF DESCRIPTION OF THE INVENTION
[0009]. In one embodiment, a system for quickly detecting the presence of an infectious agent in a biological sample is described. A first reagent is effective for detecting the presence of a specific infectious agent in a sample to be tested and to emit a detectable signal when the first reagent reacts with the sample and detects the presence of the infectious agent in the sample. A test kit has a reaction chamber for receiving the sample and the first reagent. The reaction chamber has a predetermined internal geometry and at least one internal surface. Inserting the sample and the first reagent into the test kit mixes the sample and the first reagent. A test unit receives the test kit and includes a sensor to detect an emitted detectable signal. The detection of the detectable signal emitted is indicative of the presence of the infectious agent in the sample. The detection of the specific infectious agent in the sample occurs in real time.
[0010]. In another embodiment, a set of test kits is described to facilitate the real-time detection of an infectious agent in a biological sample. The test kit set includes a reservoir card and a test kit base. The reservoir card initially stores at least one reagent to test a sample for an infectious agent. The reservoir card is configured to interface with a base of the test kit through at least one fluid port. The base of the test kit includes a reaction chamber and a fluid displacement mechanism. The reaction chamber receives the sample and at least one reagent and has a predetermined internal geometry and at least one internal surface. The fluid displacement mechanism includes a plunger to move at least one reagent from the reservoir card into the reaction chamber through at least one fluid port. When at least one reagent is mixed with the sample in the reaction chamber, a detectable signal is emitted if the infectious agent is present in the sample.
[0011]. In yet another embodiment, a test device for detecting in real time an infectious agent in a biological sample is described. A test device housing includes a cover and an input / output device. Part of the analysis includes a recess in the housing to receive a test kit containing a sample to be tested. An actuator interacts with the test case when the cover is closed. The actuator causes at least one reagent in the test case to be moved to react with the sample during a test run. A sensor is associated with the recess in the housing to detect a signal emitted after at least one reagent is moved by the actuator to react with the sample and generate an output signal. A control unit is configured to receive input from a user via the input / output device to initiate a test. In response to receiving user input, the control unit activates the actuator to move at least one reagent in the test kit to react with the sample. The control unit receives an output signal from the sensor and outputs a test result to the user on the input / output device. BRIEF DESCRIPTION OF THE DRAWINGS
[0012]. The above summary, as well as the detailed description below of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustrating the invention, the drawings show the embodiments that are currently preferred. It should be understood, however, that the invention is not limited to the precise provisions and instrumentalities shown. In the drawings:
[0013]. Figure 1A is a rear perspective view of a test device with a closed hinged lid for detecting infectious agents in accordance with a preferred embodiment of the present invention;
[0014], Figure 1B is a front perspective view of the test device of Figure IA with the hinged lid open to show the recess of the case;
[0015]. Figure 1C is a front perspective view of the test device of figures IA and IB showing a test case inserted into the recess of the case;
[0016]. Figure 2A is an exploded front perspective view of the components of an analysis part of the test device of Figure 1, according to the preferred embodiment of the present invention;
[0017]. Figure 2B is a front perspective view of the analysis part of the test device of Figure 2A;
[0018]. Figure 3A is a front perspective view of a test kit assembly comprising a 6/6 reservoir card} inserted into a test case base with the base cover closed for use with the test device of figure 1, according to the preferred embodiment of the present invention;
[0019]. Figure 3B is a front perspective view of the test kit assembly of Figure 3 A with the base cover of the test kit open;
[0020]. Figure 3C is a bottom perspective view of the test kit assembly of Figures 3A and 3B;
[0021]. Figure 4A is a front perspective view of the base of the test kit with the lid of the base open from the test kit assembly of Figure 3B, according to the preferred embodiment of the present invention;
[0022], Figure 4B is an exploded front perspective view of the base components of the test case of figure 4A;
[0023]. Figure 5A is a front perspective view of the reservoir card in an initial arrangement for use in the test kit set of Figure 3A, according to the preferred embodiment of the present invention;
[0024], Figure 5B is an exploded front perspective view of the components of the reservoir card of figure 5 A;
[0025]. Figure 5C is a front perspective view of the reservoir card of Figure 5A in an arrangement inserted to reveal the fluid ports;
[0026]. Figure 6A is an enlarged side elevated view of a portion of the reservoir card in the initial arrangement of Figure 5A with a folded film covering the fluid ports;
[0027]. Figure 6B is an enlarged side elevated view of the part of the reservoir card in the inserted arrangement of Figure 5C with the folded film retracted;
[0028]. Figure 7 is a side elevational cross-sectional view αmpliαdα of a part of the test kit set of Figure 3 A;
[0029]. Figure 8 is a side elevational view in cross-section of the test kit assembly of Figure 3A;
[0030]. Figure 9 is a side elevational view in cross-section of the test kit assembly of Figure 3 A inserted in the analysis part of the test device of Figure 2B;
[0031]. Figure 10A is a side elevational cross-sectional view of the test kit assembly of Figure 3A with a plunger in the starting position;
[0032]. Figure 10B is a side elevational cross-sectional view of the test kit assembly of Figure 3A with a plunger in a second position;
[0033]. Figure 10C is a side elevational cross-sectional view of the test kit assembly of Figure 3A with a plunger in final position;
[0034]. Figure 11 is a schematic block diagram of the electrical components of the test device of Figure 1, according to the preferred embodiment of the present invention;
[0035]. Figure 12 is a schematic block diagram of the light detection circuit of the test device of Figure 1, according to the preferred embodiment of the present invention;
[0036]. Figures 13A and 13B are a flow chart of the steps of a control application of the test device of Figure 1, according to the preferred embodiment of the present invention;
[0037]. Figure 14 is an example of a graphical user interface for a home page provided by the control application of Figures 13A and 13B;
[0038]. Figure 15 is an example of a graphical user interface showing the result of a test provided by the control application of figures 13A and 13B;
[0039]. Figure 16 is a flow chart of the steps in which test kit set 300 is used in conjunction with test device 100 to perform a test; and
[0040]. Figure 17 is a graph illustrating the efficiency of the system of the present invention with respect to detecting the presence of one or more infectious agents in a biological sample. DETAILED DESCRIPTION OF THE INVENTION
[0041]. Certain terminology is used in the following description for the purpose of convenience only and not limitation. The words "left", "right", "lower" and "upper" designate the directions in the drawings to which reference is made. The words "inward" and "outward" refer, respectively, to the meanings to and from the geometric center of the named component and designated parts thereof. In addition, the words "one" and "one" used in the claims and corresponding parts of the report mean “at least one or one.” The terminology includes the words mentioned above specifically, derived from them and words of a similar meaning.
[0042], The present invention provides a portable, autonomous system for quickly detecting (ie, within one to five minutes or more) infectious agents, especially pathogens, in biological samples, particularly samples of beef, pork or other types of meat, poultry, fish or vegetables, although other biological materials, such as health instruments and hospital surfaces, can be analyzed using the present invention. This system has very high sensitivity (for example, even a single cell of a particular infectious agent) without the need for culturing infectious agents, such as bacteria, obtained from samples before testing. In an example of an embodiment, the specific infectious agent is Escherichiaco / i, although other infectious agents (such as Salmonella, Listeria and Campylobacter), toxins and various contaminants can be detected with the present invention. Escherichia coli 0157 H7, 026, 045, 0103, Olli, 0121 and 0145, in separate assays or multiplexed assays can all be detected using the present invention.
[0043]. With reference to the drawings in detail, in which the same reference numbers refer to the same elements throughout all the various figures, a portable, standalone test device 100 is illustrated to perform a variety of qualitative tests in real time (or almost time) real) to quickly detect the presence of infectious agents in biological samples, such as food and other substances. With reference to figures 1A-1C, the test device 100 is illustrated for performing a quick analysis (in real time or in near real time) of a sample 414 (Fig. 4B) to identify infectious agents according to an embodiment preferred part of the present invention. In a preferred embodiment, test device 100 uses a disposable test kit set 300 to qualitatively test specific analytes. Test device 100 is a portable analyzer that interacts with test kit set 300 and provides simple messages for the user to obtain specific test results that are designed to find aggressive analytes from a variety of sources. The test kit set 300, which interacts with the device, contains a live biosensor that is produced to detect and report an offending analyte in a 414 sample. The 414 samples to be tested include materials such as food, liquids, surfaces and that can be sources of infectious agents. Infectious agents include foodborne diseases, pathogens, viruses, bacteria and the like. The testing device 100 allows you to perform a quick analysis of the sample 414, without the need for the lengthy enrichment process or culture of the materials being tested to facilitate the test.
[0044]. Figure 1A is a front perspective view of a test device 100 with a closed hinged lid 104 according to a preferred embodiment of the present invention. The test device 100 includes an outer housing 102 which is preferably constructed of a generally rigid, preferably polymeric material, such as, for example, acrylonitrile butadiene styrene. Other materials, or combinations of materials, may be used in the construction of outer housing 102 without departing from the scope of this disclosure. Such materials are well known to those skilled in the art.
[0045]. Test device 100 includes an electrical ON / OFF disruptor 108 and a touch sensitive liquid crystal display ("LCD") 110 to allow a user to interact with test device 100 when electrical disruptor 108 is in the ON position. The touch sensitive LCD screen 110 allows the user to give commands to the test device 100 and instructions to the user displaying menus to facilitate the operation of the test device 100, as illustrated in figures 14 and 15. As will also be discussed, the menus include, among others, graphical user interfaces to provide information and / or data to the user regarding the status or results of a specific test or operation being performed by the test device 100.
[0046]. In a preferred embodiment, the touch-sensitive LCD screen 110 comprises an LCD unit and a superimposed touch screen capable of receiving input from the user through a latex glove, or the like. In the present embodiment, the LCD 110 comprises an IPS, QVGA, TFT LCD module, VL-PS-CQG-T500F2080-X1 5-inch diagonal from VARITRONIX and a glass-film resistive touchscreen. glass model AD-5.0-4RU-02-200 from AD METRO. Other models and manufacturers of the touch sensitive LCD screen 110 can be used without departing from the scope of the present invention. In addition, other sizes and types of input / output devices, such as buttons, keyboards, trackpads and the like can be used in test device 100 without departing from the scope of the present invention.
[0047]. Test device 100 includes a plurality of interface ports 112, such as Ethernet port 112a and a micro USB port 112b. Interface ports 112 allow test device 100 to interface, download and upload data (for example, test data) to or from a local or remotely located computing device, mobile device, server or similar (not shown) ). The structure and operation of the typical 112 interface ports are well known to those skilled in the art and are not described in detail here for the sake of brevity. Although particular interface ports 112 have been described in this document, other ports and methods of communication with and / or wireless, such as 802.11 Wi-Fi, can be integrated and used in test device 100 without departing from the scope of the present invention.
[0048]. Referring to figure 11, the external housing 102 of the test device 100 also contains an electrical supply system 1126 and other electrical and electronic components, circuits and software necessary to allow the test device 100 to perform the test with a kit kit installed tester 300. Preferably, the power supply system 1126 comprises one or more batteries 116 to facilitate the autonomous operation of tester 100. A battery charger connector 114 (Fig. 1 A) is also provided for charging a or more 116 batteries, which are preferably rechargeable.
[0049]. Preferably, one or more 116 batteries comprise a double cell lithium ion battery, model 503759AY from AUTEC BATTERY, with a capacity of 2200mAh at nominal 3.7 volts. The 1126 power supply system also includes a smart charging circuit for fast battery charging 1114 that works to recharge the batteries 116 and monitors the battery temperature using a temperature sensor built into the batteries 116. In the present embodiment, the circuit battery charging system 1114 is a TEXAS INSTRUMENTS model BQ240032ARHLR. If the temperature of batteries 116 is not within a safe operating range, battery charging circuit 1114 stops charging batteries 116 until a safe temperature is reached. The battery charger is activated whenever an attached DC adapter (not shown) is connected to test device 100 via battery charger connector 114 to supply power to test device 100 and allow normal use of test device 100 during battery recharge 116.
[0050]. With reference to figure 1B, the testing device 100 of figure IA is shown with its hinged lid 104 in an open position to reveal a recess in case 152. The recess in case 152 is preferably accessible to the user only when the hinged lid 104 is in the open position. As shown in figure 1A, the recess of the case 152 is covered by the hinged lid 104 when the hinged lid 104 is in its closed position. The hinged lid 104 is released by a mechanical actuator 106, preferably located in the outer housing 102, next to the hinged lid 104. The mechanical actuator 106, which is preferably a button, disruptor or similar, releases the hinged lid 104 to rotate from the closed position, which is substantially integrated with the outer housing 102, as shown in figure IA, to an open position, which is spaced from the outer housing 102, as shown in figure IB. Referring to Figure 1C, when the hinged lid 104 is in the open position, a set of test kit 300 can be inserted into the recess of kit 152.
[0051]. As shown in figures IB and 1C, the hinged lid 104 contains two projections 104a on the lid that are arranged to hold the hinged lid 104 in the closed position, while a test is being performed by the testing device 100. In the closed position, the protrusions of the cover 104a are engaged by a pair of locking latches 104b contained within the outer housing 102. Locking latches 104b are disengaged from the protrusions of cover 104a when the user presses mechanical actuator 106. Hinged cover 104 preferably includes a groove light seal 118 provided with a light seal gasket (not shown) that engages a light seal rib 120 in the structure of the analysis part 202 (Fig. 2B) that surrounds the recess of the case 152, when the hinged lid 104 is in the closed position to prevent the entry of ambient light into the recess of case 152. A generally square design 122 with tapered side walls on the inner surface of the hinged cover Lada 104 engages the conical side walls 204A of housing 204 of an analysis part 200 when the hinged lid 104 is in the closed position.
[0052]. Referring now to Figures 2A and 2B, an analysis part 200 of test device 100 according to the preferred embodiment of this invention is shown. The analysis part 200 includes a structure of the analysis part 202 which is contained within the outer housing 102. The structure of the analysis part 202 is preferably arranged in a predetermined structure and orientation, having a first end 202a and a second end 202b to facilitate acceptance of a test kit set 300 (Fig. 1C), or other compatible test container. A housing of the analysis part 204, defining the recess of the case 152, is positioned at a first end 202a of the structure of the analysis part 202. As shown in figure 1C, the cartridge case 152 allows a user to insert a case assembly test 300 in the analysis part 200 of the test device 100, when the hinge cover 104 is in the open position. The analysis part 204 housing serves as the interface between the test device 100 and the test kit set 300. As will become apparent from now on, the disposable test kit set 300 is used to collect and introduce a sample test 414 (Fig. 4B) on test device 100 in order to perform one or more tests with test sample 414.
[0053]. The housing of analysis part 204 of analysis part 200 will now be described in more detail. The housing of the analysis part 204 is preferably made of a polymeric material, generally rigid, such as acrylonitrile butadiene styrene, or some other polymeric material well known to those skilled in the art and is located within the structure of the analysis part 202. A structure of the analysis part 202 provides structural support for the housing of the analysis part 204 and is the main component in a light sealing scheme that greatly minimizes or prevents the entry of ambient light into the recess of the case 152, through the rectangular walls that surround the structure of the analysis part 202, thereby preventing ambient light emissions from reaching sensor 206. In a preferred embodiment, sensor 206 is a light sensor.
[0054]. Test device 100 performs a desired test for a sample 414 retrieved from a variety of sources, analyzing the electrical output of sensor 206. When sensor 206 is a light sensor, the output varies with the amount of light that strikes the detection surface 206a of light sensor 206, originating within test kit set 300. Based on the type of test being performed, the output of light sensor 206 determines whether the analyzed sample 414 is positive or negative for the presence of the material (infectious agent) that is being sought in a qualitative analysis. That is, there is no need to determine by the test device 100 the actual amount of material present in test sample 414. The test device 100 is able to change the parameters for the test based on the test performed and the kit sets 300 test units used.
[0055]. Whereas in the preferred embodiment, the evaluation of the material within the test kit 300 by test device 100 requires the detection of the presence of light that can be emitted by test sample 414 introduced by test kit 300, it is preferable to minimize or eliminate the amount of external or ambient light that is being introduced into the recess of the case 152 of the test device 100 during the test. To achieve this objective, analysis part 200 preferably prevents most or all ambient light emissions from reaching sensor 206. Sensor 206 is arranged on a printed circuit board ("PCB") 208, which is positioned under the analysis part 204 housing. Minimizing those external light emissions that reach sensor 206 prevents erroneous output from sensor 206.
[0056]. The structure of the analysis part 202 and the hinged lid 104 are preferably made of a solid, opaque, generally rigid material, such as aluminum, to reflect or absorb all measurable light on the material, or some other solid material. , opaque well known to those with general knowledge of the subject. The base 204B of the housing of the analysis part 204 contains a rectangular cutout 214 on a lower surface. A viewing window 216 is mounted on the rectangular cutout 214. The viewing window 216 is preferably made of a solid, transparent optical grade material, such as, for example, quartz, glass or other transparent solid material, as is known to those skilled in the art. The sensor 206 is positioned under the viewing window 216, allowing light to pass from the test case set 300 through the viewing window 216 to the sensor 206 with a minimum amount of absorption or light reflection. Therefore, sensor 206 receives the maximum possible signal through the viewing window 216.
[0057]. In the preferred embodiment, sensor 206 is a light sensor and even more preferably sensor 206 is a photomultiplier tube (PMT), as will be described further with reference to figure 12. PCB 208 further includes an RFID communication circuit (radio frequency identification) 210, a high voltage power supply 218 for use with sensor 206 and another light detection electrical circuit system 1200, as will be described further below with reference to figure 12. Preferably, the RFID communication 210 is positioned under an area of the recess of the case 152 which aligns with the RFID tags 508 (Fig. 5B) within the test case set 300 when the test case set 300 is inserted into the case recess 152.
[0058]. A protective cover for sensor 220 is positioned to substantially surround sensor 206. The protective cover for sensor 220 isolates sensor 206 from electromagnetic and magnetic interference. The protective cover of the sensor 220 is preferably made of a conductive, solid material, generally rigid with high magnetic permeability, such as, for example, mu-metal or other solid conductive material, as is well known to those skilled in the art. One of the housing walls of the analysis part 204 contains a hollow protrusion 222 that extends into the recess of the case 152, which combines with a recess in the test case set 300. The hollow projection 222 allows a piston 224 and piston rod 224A, which engages a fluid displacement mechanism 900 (Fig. 9) in the test kit 300, pass through it and come into contact with a plunger 424 (Fig. 4B) of the test kit 300 .
[0059]. The piston 224 is preferably made of a polymeric material, generally rigid, such as, for example, polystyrene or other similar polymeric material, as is well known to those skilled in the art. Piston 224 is driven by a motor 226. In the preferred embodiment, motor 226 is a linear stepper motor. However, other actuators, such as pistons, servos or the like, can be used without departing from the scope of this invention. Piston 224 is coupled to motor 226 via threaded shaft 226A on motor 226 coupled to an integral threaded hole (not shown) inside piston 224. In the currently preferred embodiment, motor 226 is a HAYDON-KERK stepper motor model 19542 -05-905. To minimize the introduction of noise from motor 226 to analysis part 200, motor 226 is located outside the housing of analysis part 204, not being in close proximity to sensor 206. This arrangement of motor 226 in relation to sensor 206 decreases the possibility of motor 226 interfering electrically or electromagnetically with sensor 206.
[0060]. A projection 228 protrudes from piston 224 and aligns with a position detector 230, which is positioned outside analysis part 200. At a certain stage in the stroke of piston 224 (described below), projection 228 fires position detector 230 to generate a position signal. In one embodiment, the trigger position of the position detector 230 corresponds to the second position of the plunger 424 shown in figure 10B. However, the trigger position may alternatively correspond to the final position of plunger 424, shown in figure 10C, or any other position in the path of plunger 424. In a preferred embodiment, position detector 230 is a photodisrupter and the position 230 is triggered by projection 228, blocking the path of the light inside the position detector 230, at which point a signal is sent to microprocessor 1102 to indicate the position of piston 224. Thus, the accurate detection of the position of the piston 224 can occur to ensure that there are no errors in the performance of the test kit 300. In the preferred embodiment, the position detector 230 is an OMRON photodisrupter model EE-SX4134. However, those skilled in the art will be able to assess what other types of devices can be used for the position detector 230 without departing from the scope of the present invention.
[0061]. Piston 224 includes a piston rod 224A extending from it, which contains spaced pairs of annular flanges extending outwardly 232A-C at spaced locations along its length. Compressible sliding seals 234A and 234B are mounted radially between ring flanges 232A and 232C, respectively. Preferably, the sliding seals 234 are made of an elastomeric material, such as silicone, or some other elastomeric material, as is well known to those skilled in the art. When the piston 224 is installed, the first sliding seal 234A, mounted between the ring flanges 232A, engages the inner surface of the hollow protrusion 222 of the housing of the analysis part 204 to create an airtight seal that prevents liquids from entering the analysis part bottom 200, from the recess of the case 152 and the arrival of the electronic components on the PCB 208 under the housing of the analysis part 204. The second sliding seal 234B, mounted between the ring flanges 232C engages the inner surface of a hollow channel through the which the piston rod 224A passes, entering the structure of the analysis part 202 to create an airtight seal that prevents the entry of environmental (ambient) light emissions into the light-sealed area of the analysis part 204 housing along the path of the piston 224.
[0062], Piston rod 224A also contains a third pair of ring flanges 232B that engage a sliding plug 236. Sliding plug 236 is preferably constructed of thin, opaque and rigid material, such as a plate of molded stainless steel, in order to keep the low profile of the analysis part 200 and sized to be portable. Alternatively, sliding plug 236 can be constructed of conductive material with high magnetic permeability, such as mu-metal, in order to provide additional protective cover to sensor 206. When initially engaged by piston rod 224A, sliding plug 236 passes between the sensor 206 and the viewing window 216. In that position, the sliding shutter 236 reflects or absorbs virtually all ambient light emissions that would otherwise reach sensor 206, when the hinged lid 104 is open and the analysis part 200 is exposed to ambient light. If sensor 206 is a PMT, sliding shutter 236 protects sensor 206, which is vulnerable to saturation and damage when fully exposed to ambient light levels. The sliding shutter 236 contains an opening 238 that fits the sensor 206 at the start of the test. Preferably, before starting a test, the slide plug 236 covers the sensor 206. It is desirable that the slide plug 236 engages the piston rod 224A and makes use of the movement of the motor 226 to slide to the position in which the opening 238 is over sensor 206 at the start of the test. The arrangement minimizes the costs of additional components and further reduces the risk of electrical or electromagnetic interference.
[0063]. With reference now to Figures 1 and 2, the analysis part 200 is located within the housing 102 of the test device 100. At least a part of the analysis part 200 is at least partially covered by the hinge cover 104 when the hinge cover 104 is in the closed position, shown in figure 1 A. The analysis part 200 preferably includes the PCB 208 with an integral RFID communication circuit 210, which is configured to communicate via radio frequency with a Radio Identification tag Single frequency ("RFID") 508 from a test kit set 300 (Fig. 3). In the preferred embodiment, the RFID communication circuit 210 is a TEXAS INSTRUMENTS RFID communication IC model TRF7961. It is evident to those skilled in the art that other types of scanners or scanning devices and other data transmission schemes can alternatively be used to provide information to test device 100 and / or write in formations on RFID tag 508 of test kit set 300.
[0064]. It should be assessed by those skilled in the art that the precise structure of the analysis part 200 and / or its components are merely those corresponding to the currently preferred embodiment and that variations can be made in the structure of the analysis part 200 and / or in its components. components, without departing from the scope and spirit of the invention. Therefore, the present invention is not limited to the precise structure of the analysis part 200 described here, but is intended to cover structural and / or operational variations, as well as other structures and arrangements that can perform the same, or substantially the same functions that those of the current analysis part 200.
[0065]. Variations can include these structural changes, such as the omission of an electromechanical engine, relying instead on the user's input force to drive the case, to activate the test case directly without the use of a piston, using various engines to different actions, place the engine within the sealed area in the light of analysis part 200, or control the engine without detecting the precise position. In addition, the shape, arrangement and size of the recess of the test case 152 in the housing of the analysis part 204, the projections of the lid 104a and locking latches 104b may vary from what is illustrated and described here, without departing from the scope of the present invention. All that is needed is that the recess of case 152 must complement and conform to the size and shape of the test case set 300, so that the recess of case 152 can receive an inserted test case set 300.
[0066]. Similarly, light detection by sensor 206 can be replaced by a different signal detection scheme, as is well known to those skilled in the art, without departing from the scope of the present invention. For example, the detection of electrical signals can be used to evaluate the test result. In this case, it may be preferable to minimize or eliminate external sources of noise, in addition to light. Structural changes to the analysis part 200 that facilitate the reduction or elimination of these external noise sources, in addition to light, are within the scope of the present invention.
[0067]. Referring now to Figures 3A and 3B, test kit set 300 for use with test device 100 in accordance with the preferred embodiment of the present invention is shown. Preferably, the test kit set 300 is a disposable kit. pαrα single use, which is used to receive a small amount of a sample 414 (Fig. 4B) collected from food products or other sources for a test to be performed by test device 100. Therefore, test kit set 300 is preferably configured to be fixedly inserted into test device 100 during the entire run of a selected test. Even more preferably, each set of test kit 300 contains all necessary reagents 504, 506 (Fig. 5B) and the like for carrying out a single test, as will be further described in this document.
[0068]. As shown in figure 3A, the test kit set 300 preferably comprises two separate parts, a test kit base 400, further described with reference to figure 4 and a reservoir card 500, further described with reference to figure 5 The base of the test kit 400 and the reservoir card 500 are configured to interact with each other to perform a test using the test device 100. The reservoir card 500 is designed as a separate part of the test kit base test 400 in order to occupy a minimum volume and achieve high packing density. The packing density is a critical consideration for the occasions when the necessary reagents 504, 506 need to be stored at low temperatures or below freezing temperature. However, a single, integrated unit test kit set 300 can likewise be produced and is within the scope of this disclosure.
[0069]. The base of the test case 400 is configured to receive the separate reservoir card 500 in a slot 402 (Fig. 4A) at a first end 400a of the base of the test case 400. The reservoir card 500 is specifically designed to provide a storage and administration vehicle, convenient and small in size, for one or more biosensors (reagents 504, 506). As illustrated in figures 3A and 3B, a user mounts reservoir card 500 on the base of test case 400 by sliding reservoir card 500 into slot 402. As soon as reservoir card 500 is inserted into the base of test case 400, the reservoir card 500 is fixedly attached to the base of the test case 400. Permanent fixing features 502 prevent the improper use of the base of the test case 400, such as reusing the base of the test case 400 with several reservoir cards 500. This prevents contamination of the base of test kit 400 and / or reservoir card 500. Reservoir card 500 can be attached to the base of test kit 400 using any device or element known suitable mechanical fastening elements, such as one-way fastening elements 502 (Fig. 5B).
[0070]. Preferably, the base of test kit 400 does not contain any specialized test components and can therefore be common for various types of tests. As such, the base of test kit 400 must be compatible with various types of reservoir cards 500. As shown in figures 4B and 9, the base of test kit 400 contains a 404 reaction chamber and a fluid displacement mechanism. 900, which together occupy a relatively large volume compared to the volume of the reservoir card 500. With reference to figures 5A and 5B, the reservoir card 500 contains all the necessary reagents 504, 506 and the like, to perform a single test by test device 100. Consequently, several different types of reservoir cards 500 may be provided, each with one or more different reagents 504, 506 to perform a particular type of test. Preferably, reaction chamber 404 promotes an adequate mixing of sample 414 and reagents 504, 506, while reducing damage to the living cells that make up reagents 504, 506. Reaction chamber 404 also maximizes the accumulation of light that reagents 504, 506 emit to sensor 206 in the presence of an aggressive analyte or to confirm the proper functioning of the first test phase.
[0071]. As best illustrated in Figure 4B, the base of test kit 400 consists of a generally rectangular housing 401, with an integral hinged lid 408. The rectangular housing 401 is preferably made of preferably polymeric material, generally rigid, such as polypropylene or other polymeric material well known to those skilled in the art. An adhesive film 410 is used to close the fluid channels 406 formed on the planar surface 400b of housing 401 for the sealed passage of reagents 504, 506 and / or air between the reservoir card 500 and the base of the test kit 400. The housing 401 of the base of test kit 400 also includes an integral reaction chamber 404 for deposition of sample 414 and eventual mixing of sample 414 with reagents 504, 506 to carry out the desired test.
[0072]. When the test kit set 300 is placed in the recess of the case 152, the lower surface 404a of the reaction chamber 404 within the housing 401 of the base of the test case 400 aligns with the light sensor 206. Referring to figure 3C, reaction chamber 404 is sealed on the bottom surface with a lens 412. Lens 412 is preferably made of rigid material, preferably polymeric, such as polycarbonate or some other polymeric material well known to those skilled in the art. Preferably, the lens material 412 is a transparent optical grade material in order to prevent unwanted light absorption or reflection between reagents 504, 506 and light sensor 206. In the preferred embodiment, lens 412 is thermally welded in housing 401 of the base of test kit 400 to provide an impermeable seal and minimize the introduction of contaminants into reaction chamber 404. With reference to figures 4A and 4B, reaction chamber 404 is opened on the upper surface 400b of the housing 401 from the base of test kit 400 to allow a user to directly deposit sample 414 (preferably in liquid form) in reaction chamber 404.
[0073]. Adhesive film 410 is placed on the upper surface 400b of housing 401 of the base of test kit 400. Preferably, film 410 is pre-scratched or perforated 416 above reaction chamber 404 to allow the user to pierce the film 410 using the tip of a deposition tool (not shown), such as a pipette for depositing sample 414 in reaction chamber 404. Pre-scratching or perforating 416 of film 410 is desirable to give a visual cue to the user that the sample deposition step in the testing process has been completed, or that the test kit set 300 has been used previously and should be discarded. A compressible gasket 418 with adhesive reinforcement 418b is placed around the perimeter of the opening on the upper surface 400b in the reaction chamber 404 (surrounding the perforated or pre-scratched area 416 of the adhesive reinforced film 410, see figure 4A), with the purpose of creating a fluid impermeable seal when the hinged lid 408 of the base of the integral test case 400 is closed. The hinged lid 408 of the base of the integral test kit 400 contains inserts 408a, 408b to hold the hinged lid 408 of the base of the test kit 400 in a closed position, interacting with the slot slots 420a, 420b after the sample 414 be deposited in reaction chamber 404.
[0074]. Still referring to figures 4B and 3C, a central hole 422 is located within housing 401 of the base of test case 400 which contains a plunger 424 as part of the fluid displacement mechanism of test case 900, which will be described below in more detail. Preferably, the plunger 424 is made of an elastomeric material, such as silicone rubber or some other elastomeric material, as is well known to those skilled in the art and is dimensioned to seal the inner wall of the central hole 422 in a sealing manner. upper 400b of the housing 401 of the base of the test kit 400 contains a relatively large ventilation overflow chamber 426 which communicates with the reaction chamber 404 through a ventilation channel 426A. Ventilation overflow chamber 426 is present to allow air to be displaced from reaction chamber 404 during the introduction of reagents 504, 506 and contains features to contain any lost amount of liquid that may enter the ventilation channel 426A. Preferably, the ventilation overflow chamber 426 contains an absorbent material 428 which uses an antimicrobial coating through which the ventilated air must pass when leaving housing 401 of the base of test case 400. This ensures that any lost amount of liquid is absorbed and contained within the ventilation overflow chamber 426 and any destroyed biological components.
[0075]. As illustrated in Figure 5B, reservoir card 500 includes a plurality of fluid ports 516, which, when reservoir card 500 is inserted into housing 401 at the base of test case 400, interfaces with a number of elements seal 702 (Fig.7), thereby making connections with the reservoir card 500 when assembled. The sealing elements 702 are embedded under a wall 401A (Fig. 7) in the housing 401 of the base of the test case 400 in order to prevent damage to the sealing elements 702 and eliminating potential locations for the introduction of contaminants of easy contact in the reagents 504, 506.
[0076]. Those skilled in the art should note that the precise structure of the test case base 400 and / or its components are merely those corresponding to a preferred embodiment, and variations in the structure of the test case base 400 and / or its components may be introduced. components, without departing from the scope and spirit of the invention. Other structural and functional variations, such as depositing sample 414 in another location except in reaction chamber 404, to be moved to reaction chamber 404 at a later time, use several parts to reach housing 401 of the base of the test kit 400 and reaction chamber elements 404, use a separate cover or closure scheme for reaction chamber 404 after depositing the sample, or alternatively locate the plunger 424 and / or other components of the fluid displacement mechanism 900 in the reservoir card 500 are all within the scope of the present invention.
[0077]. Reaction chamber 404 and fluid channels 406 leading to reaction chamber 404 within housing 401 of the base of test kit 400 are preferably designed to achieve various objectives. An inlet channel 802 (Fig. 8) for the fluid entering the reaction chamber 404 is preferably tubular in shape with a diameter that is preferably small and tapers until it becomes smaller at the entrance to the reaction chamber 404. Preferably , this structure increases the speed of the fluids that enter the reaction chamber 404, promoting a vigorous and, therefore, homogeneous mixture, due to the mass movement of reagents 504, 506 inside the reaction chamber 404.
[0078]. Referring to figure 8, a side elevational cross-sectional view of test kit 300 is shown. It is desirable to mix reagents 504, 506 and sample 414 in order to promote mixing beyond molecular diffusion, in order to minimize the duration of the test, ensuring that any infectious agent present in sample 414 quickly finds reagents 504 and 506. In the preferred embodiment, the minimum diameter of the inlet channel 802 is 0.75 mm. Preferably, the input channel 802 is further displaced from the central axis of the reaction chamber 404 to promote a clockwise or counterclockwise movement of the reagents 504 and 506 around the central axis of the reaction chamber 404, when the fluids are mixed to increase the homogeneity of the mixture.
[0079]. In the currently preferred embodiment, the inlet channel 802 is approximately tangent to the inner surface of reaction chamber 404. This is desirable to allow the inlet fluid to move from the inlet channel 802 to the fluid level inside the reaction chamber 404, while remaining in contact with the side surface of the reaction chamber 404, which allows a minimally turbulent flow and minimal introduction of air bubbles in the mixed fluids. Bubbles are undesirable due to the unpredictable refraction of light that they cause when the light emitted by reagents 504, 506 that interact with sample 414 moves through bubbles within mixed reagents 504, 506, or on the surface of mixed reagents 504, 506.
[0080]. In some embodiments of the invention, a stabilizer is included in reaction chamber 404. The stabilizer can be, for example, Pluronic F68, which is used in cell cultures as a cell membrane stabilizer protecting against membrane shear and, in addition to addition, as a defoaming agent. Certain embodiments of the present invention also include at least one additive, such as Pluronic F68, polyethylene glycol, metocel or the like, located in reaction chamber 404 to minimize bubble formation in reaction chamber 404 during mixing of sample 414 and reagents 504, 506. This additive may also include a surfactant, such as Pluronic F68, Polyvinylpyrrolidone, Polyethylene glycol, polyvinyl alcohol, Metocel (methylcellulose), or the like. Some embodiments of the present invention also include a device for breaking up individual cells in sample 414 and, in particular, the infectious agent within sample 414 before mixing sample 414 with reagents 504, 506, in order to amplify the light signal generated by reagents 504, 506 that react with an infectious agent within the sample. An example of such a device is an ultrasound device (not shown).
[0081]. The axis of the inlet channel 802 is preferably angled above the horizontal to provide a partially downward direction for the flow of the inlet fluid to ensure that reagents 504, 506 are mixed with the fluid that resides in the bottom of the reaction 404. In the currently preferred embodiment, the input channel 802 is angled above the horizontal at an angle of approximately 30 (thirty) degrees, and in addition, the optimal functional range occurs between 15 (fifteen) degrees and 60 ( sixty) degrees above the horizontal. Those skilled in the art will appreciate that the arrangement, position and structure of the 802 input channel can be varied without departing from the scope of the present invention.
[0082]. Alternatively, if desired, reagents 504, 506 can be introduced into reaction chamber 404 using alternative fluid discharge techniques, such as a vertical channel (not shown) that discharges reagents 504, 506 into reaction chamber 404, or by discharging fluid reagents 504, 506 directly on the central axis of reaction chamber 404 to create a column of reagent flowing into reaction chamber 404, promoting mixing by suspending droplets. In addition, a user can discharge one or more reagents 504, 506 manually in the same manner and, for example, at the same time, in which sample 414 is deposited in reaction chamber 404.
[0083]. Preferably, the reaction chamber 404 has a shape that maximizes the amount of photons reflected towards the base of the reaction chamber 404, to allow the photons to be read by the sensor 206 positioned under the reaction chamber 404 in the analysis part 200 In the preferred embodiment, the shape of the reaction chamber 404 is a rotated section to facilitate the clockwise or counterclockwise movement of the mixing fluids 414, 504, 506 around the central axis of the reaction chamber 404. Alternatively, if If desired, another format for the reaction chamber 404 may be used, in addition to a rotated section, for example, a rectangular or irregular shape. In the preferred embodiment, the rotated section used to form reaction chamber 404 is a part of an ellipse. The elliptical shape is desirable to assist in collecting the diffuse light emitted by reagents 504, 506 that react with sample 414 and to reflect that light towards the surface of the light sensor 206. Preferably, the shape of reaction chamber 404 is generally parabolic . Reaction chamber 404 can be a rotated half of an ellipse with an opening at the top of approximately 2.5 mm and the lower diameter located on the major or minor axis of the ellipse and equal to approximately 8 mm.
[0084]. The surface of the 404 reaction chamber is preferably reflective, in order to further improve the light-collecting properties of the elliptical shape. In the preferred embodiment, the maximum diameter of the detection surface 206a of the sensor 206 is limited so as to achieve the maximum signal-to-noise ratio of the output of the light detection circuit 1200 (Fig. 12). The diameter of at least the bottom of the reaction chamber 404 is designed to correspond approximately to the diameter of the sensor 206, which influences the elliptical shape that can be obtained in a reaction chamber 404 designed to contain a specific volume of fluids for a given type of test. In the preferred embodiment, the surface color of the preferred reaction chamber 404 is a partially diffused white, due to the additional light collection that occurs when light that would not otherwise be reflected directly on the surface 206a of the sensor 206 is partially diffused across the white surface and a fraction of the light is directed to the sensor surface 206a. Alternatively, other finishes, colors and surface materials, such as an almost mirror finish, aluminum, or transparent material can be used.
[0085]. It is desirable that the material of the reaction chamber 404 be minimally phosphorescent to prevent the light emitted by the reaction chamber 404 itself from overloading any light emitted by reagents 504, 506 that react with sample 414, thereby avoiding, or otherwise affecting detection. Although it has been found that white polymeric materials, such as acrylonitrile butadiene styrene, or other polymeric materials demonstrate a low level of phosphorescence, the collection of additional light provided by the combination of reflection and light diffusion proved to be a benefit for the signal-to-noise ratio of light detection circuit 1200 output.
[0086]. As shown in figures 5A-5C, the reservoir card 500 consists of a generally rectangular housing 501. Preferably, the housing 501 of the reservoir card 500 is made of preferably polymeric material, generally rigid, such as polypropylene or other polymeric material well known to those skilled in the art. With reference to figure 5B, fluid channels 510, 512 are formed on the upper surface 501a of the housing 501 of the reservoir card 500 to provide storage for all necessary reagents 504, 506 to perform a specific type of test.
[0087]. In the preferred embodiment, the first reagent 504 is a biosensor reagent capable of emitting light when a specific pathogen or set of pathogens is detected and the second reagent 506 is a positive control sample, such as anti-immunoglobulin M (anti-IgM) or digitonin. The second reagent 506 is used for the purpose of rapid activation of the first biosensor reagent 504 after the duration of the initial test, as confirmation of the viability of the biosensor reagent 504. The second reagent 506 functions as a negative result control test and is therefore optional. In other words, the test can be performed without the presence and / or use of the second reagent 506, but in its absence, the accuracy of the test result may be difficult to prove.
[0088]. Fluid storage channels 510, 512 for storing reagents 504, 506 are designed to provide a small cross-sectional area, preferably approximately 1 mm wide and 1 mm high. The small cross-sectional area allows stored reagents 504, 506 to be easily displaced from fluid storage channels 510, 512, using one or more additional fluids, such as air. A smaller cross-sectional area is also desirable due to the resulting decrease in thawing time on occasions when the necessary reagents 504, 506 need to be stored frozen and thawed immediately before testing. A thin layer 514, preferably of polymeric or similar material, is glued to the housing 501 of the reservoir card 500 to close the fluid storage channels 510, 512 and offer a fluid impermeable seal on the upper surface 501a of the housing 501 of the card - reservoir 500.
[0089]. With reference to figure 5B, the housing 501 of the reservoir card 500 also contains a recessed area (not shown) on the bottom surface 501b to contain the RFID tag 508. The recessed area serves to prevent damage resulting from accidental contact with sensitive components of the RFID tag 508. The RFID tag 508 is located inside or attached to the reservoir card 500 in order to minimize errors made by the user when associating the test case 300 data stored in the RFID tag 508 with the necessary reagents 504, 506 for specific test types. It is preferable to use RFID technology to automate the transfer of data between test kit set 300 and test device 100, thereby reducing the sources of possible user error.
[0090]. A terminal face 501c of reservoir card 501 housing 500 contains several fluid ports 516a-516d, which make the fluid connections to the housing 401 of the test case base 400, when mounted on the test case set 300. Each of the fluid ports 516 is attached to a compressible gasket 518 with an adhesive or similar reinforcement around the perimeter of each fluid port (516) 516. The compressible gaskets 518 create a fluid-tight seal with the housing 401 of the base of test kit 400, when reservoir card 500 is properly installed in the base of test kit 400, as illustrated.
[0091]. To prevent contaminants from coming in contact with fluid ports 516 and to avoid damage to compressible gaskets 518, the end face 501c of housing 501 of reservoir card 501 is initially covered by a film 520 (see figure 5A). In the preferred embodiment, film 520 is a polyethylene terephthalate film, or some flexible polymeric film capable of creating an impermeable seal with the housing 501 of reservoir card 500. Film 520 has a selectively applied adhesive reinforcement and is selectively glued , using the adhesive on a single side of the film 520, to the housing 501 of the reservoir card 500, so that each fluid port (516) 516 is individually sealed around its perimeter and one end 520a of the film 520 permanently glued to the upper face 501a of reservoir card 500.
[0092]. Figure 6A is an enlarged side elevated view of part of the reservoir card 500 in the initial arrangement of Figure 5A with a folded film 520 covering the fluid ports 516. As shown in Figures 5B and 6A, the film 520 is placed on top of it same at point 520b so that the remaining tip of film 520 is directed back to the upper face 501a of reservoir card 500. The remaining tip 520c of film 520 is permanently glued using the adhesive selectively applied to a part of carrier 522. Preferably , the conveyor part 522 is made of a polymeric material, generally rigid, such as polypropylene, or other polymeric material known to those skilled in the art.
[0093]. Figure 6B is an elevated side view of the enlarged side of the reservoir card part 500 in the inserted arrangement of Fig. 5C with the folded film 520 retracted to reveal the fluid ports 516. As shown in figure 6B, any movement of the conveyor part 522 away from the fluid ports 516 results in a peeling motion of the glued film 520 from the tip of the fluid port (516) 501c of the housing 501 of the reservoir card 500, removing the seal and exposing the fluid ports 516 and their gaskets 518 .
[0094]. Several actions occur when the reservoir card 500 is mounted on the base of the test case 400. When the reservoir card 500 is slid into the receiving slot 402 in the housing 401 of the base of the test case 400, the conveyor part 522 on the card reservoir 500 mechanically interferes with the top wall of the receiving slot 402 in the housing 401 of the base of the test case 400. The reservoir card 500 is shaped so that it cannot be fully inserted into the receiving slot 402 of the base of the test case 400 with reverse or inverted orientation. When the reservoir card 500 is in the correct direction as the user continues to insert the reservoir card 500, the mechanical interference between the part of the conveyor 522 and the wall of the housing 401 of the base of the test case 400 causes cause the conveyor part 522 to move relative to the reservoir card 500 away from the fluid ports 516 (see figure 5C).
[0095]. As described above, moving the conveyor part 522 away from the fluid ports 516 of the reservoir card 500 causes a peeling motion of the film 520 placed on the fluid ports 516 of the reservoir card 500. Peeling of the film 520 exposes the fluid 516 and gaskets 518 in reservoir card 500 (see figure 5C). Preferably, full exposure of the fluid ports 516 occurs after the reservoir card 500 fully engages the receiving slot 402 in the housing 401 of the base of the test case 400 so that the fluid ports 516 are protected by the upper wall of the receiving slot and never are openly exposed to the external environment. This conduct is desirable to prevent contaminants from coming into contact with fluid ports 516 and being introduced into reagents 504, 506.
[0096]. As the reservoir card 500 fully enters the receiving slot 402 in the housing 401 of the base of the test case 400, with reference to figure 7, the sealing elements 702 present in the housing 401 of the base of the test case 400 enter in contact with the gaskets 518 of the fluid ports 516 on the reservoir card 500, forming fluid-tight seals. In the currently preferred embodiment, the seal between the gaskets 518 and the sealing elements 702 is a face seal. However, other types of seals or sealing elements (such as luer seals) can alternatively be used to provide the fluid impermeable seal between the reservoir card 500 and the housing 401 of the test case base 400. Alternative sealing elements can include a radially compressible gasket (not shown) forming an annular seal. When the reservoir card 500 is fully inserted into the receiving slot 402 and the fluid impermeable seals are formed, the fastening elements of a track 502 (Fig. 5B) in the housing 501 of the reservoir card 500 engage the complementary retaining elements ( not shown) in the housing 401 of the base of the test kit 400, in a manner well known in the art, to permanently retain the reservoir card 500 in the state assembled with the base of the test kit 400, thereby creating the test kit set 300.
[0097]. Those skilled in the art will appreciate that although an arrangement of components of a particular reservoir card 500 has been described, the present invention is not limited to that particular arrangement. Possible alternative arrangements include the use of a single reagent, storage of 504, 506 reagents in a larger cylindrical volume, or alternative fluid port protection elements, such as perforated films or aluminum foils and / or covers removed by the user.
[0098]. Referring to figures 9, 10A, 10B and 10C, a fluid displacement mechanism 900 is shown within the base of test kit 400. Figure 9 is a side elevational cross-sectional view of the test kit assembly of figure 3 A inserted in the analysis part 200. The plunger 424, which is located in the housing 401 of the base of the test case 400 is preferably designed to move the air that passes through the air channels 902A-902D in the housing 401 of the base of the test test kit 400, through the sealing elements 702 constituted between the mounted reservoir card 500 and the housing 401 of the base of test kit 400. When actuated by piston rod 224A, piston 424 causes reagents 504, 506 stored in the reservoir card 500 are moved to the base of the test kit 400. As described above, the plunger 424 is preferably actuated by the piston rod 224A in the analysis part 200.
[0099]. When displaced, reagents 504, 506 are forced into housing 401 of the base of test kit 400 and eventually into reaction chamber 404. The model that uses air to move reagents 504, 506 from reservoir card 500 allows that the components of the fluid displacement mechanism 900 are located in the housing 401 of the base of the test case 400, allowing the reservoir card 500 to reach a minimum volume to facilitate the storage and transport of the reservoir card 500. In the embodiment Preferably, air channels 902A-D from central hole 422 and plunger 424 are designed to produce a stepwise discharge of reagents 504, 506 from reservoir card 500 into reaction chamber 404. Referring to Figures 10A-10C, the discharge of reagents 504, 506 occurs as air is displaced from central hole 422 through a series of air channel ports 902 that are alternately sealed and then opened when plunger 424 moves to along the central hole 422.
[0100]. Referring to figure 10A, at the beginning or the first stage, the plunger 424 is positioned at the starting point 906A of the central hole 422. The flanges 908 of the plunger 424 initially seal a first port of the air channel 902A, which is connected by means of a fluid port (516) 516C to storage area 512 for second reagent 506 on reservoir card 500 and isolate the first port of channel 902A from the other ports of channel 902B-D and the first reagent 504.
[0101]. A second 902B air channel door is opened and connects to a fourth 902D air channel door. The third port of the air channel 902C is opened and connected to the storage area 510 of the first reagent 504. In the preferred embodiment, the first reagent 504 includes the biosensor used to perform the test on sample 414. When the plunger 424 is actuated through piston rod 224A, plunger 424 advances further through central hole 422 and air displaced from central hole 422 passes through the third port of air channel 902C, displacing the first reagent 504 from reservoir card 500. The first reagent 504 flows into inside the housing 401 of the base of the test kit 400, and, eventually, to the reaction chamber 404 to mix with the sample 414 in the manner described above.
[0102], When the plunger 424 passes through a second stage towards the second end 906B of the central hole 422, with reference to figure 10B, the second port of the air channel 902B is sealed by the flanges 908. However, the seal of the second air channel port 902B has no effect due to the direct connection of the second air channel port 902B with the fourth air channel port 902D. When the plunger 424 reaches the second stage of figure 10B, the entire volume of the first reagent 504 will have been moved from the reservoir card 500 to the housing 401 of the base of the test kit 400. At that moment, the movement of the plunger 424 is paused with the second air channel port 902B sealed for the duration required for the test device 100 to complete the first test phase. In one embodiment, the movement of the plunger 424 is paused for approximately 60 (sixty) to 120 (one hundred and twenty) 37/6} seconds or more. The amount of time that plunger 424 is paused depends, preferably, on the type of test being performed by test device 100 and is determined based on the information provided by an RFID tag 508 from test case 300 read by the test device test 100 after insertion into the recess of case 152.
[0103]. After the end of the first test phase, if a second test has to be carried out, the plunger 424 is moved again by the piston rod 224A, causing the flanges 908 of the plunger 424 to seal the third port of the air channel 902C and open the second port of the air channel 902B. As the plunger 424 continues to move through the central hole 422 towards the second end 906B, the air displaced from the central hole 422 is forced through the fourth port of the air channel 902D, to the second port of the air channel. air 902B, passing through the central hole 422 in the region of the gap between the plunger 424 and the surface of the central hole 422 to the first door of the air channel 902A. The displaced air passing through the first port of the air channel 902A displaces the second reagent 506, which flows to the housing 401 of the base of the test kit 400 and eventually to the reaction chamber 404, in order to carry out the second test phase. result verification or negative phase.
[0104]. The plunger 424 continues to pass through the central hole 422, until it contacts the second end 906B of the central hole 422, as shown in figure 10C. At that time, most of the second reagent 506 will have been displaced and flowed through the reaction chamber 404. After the movement of the plunger 424 from the first end 906A to the second end 906B of the central hole 422 is complete, it is preferable that the plunger 424 does not can be retracted towards the first end 906A. This one-way movement of the plunger 424 helps to prevent the test kit assembly 300 from being reused in a subsequent test.
[0105]. The use of a single piston rod 224A and a single plunger 424 is desirable to limit the use of additional parts in the test case set 300 and the test device 100 for reasons of cost, manufacturing complexity and reduced sources of possible interference with light sensor 206. However, it should be noted by those skilled in the art that the precise structure of the fluid displacement mechanism 900 described above is merely that of a currently preferred embodiment, with variations in the structure of the displacement mechanism being possible. of fluid 900 without departing from the scope and spirit of the present invention. Possible alternative arrangements for the fluid displacement mechanism 900 include the use of multiple motors to control a specific actuation or more per motor, using multiple plungers to move one or more reagents 504, 506 per plunger, using plungers to directly move reagents 504, 506, such as a compressible membrane or blister pack.
[0106]. Referring to Figure 11, a functional schematic block diagram of hardware 1100 of electrical / electronic components and other related components of the preferred embodiment of test device 100 is shown. The operation of test device 100 is controlled by a microprocessor 1102 In the preferred embodiment, microprocessor 1102 is an application processor, such as the FREESCALE SEMICONDUCTOR processor, model number MCIMX255AJM4A, which implements the ARM926EJ-S core with the processor speed up to 400 MHz. Even more preferably , microprocessor 1102 includes an integrated 10/100 Ethernet controller and a 1108B Universal Serial Bus (USB) physical layer (PHY). Microprocessor 1102 includes universal input / output (I / O) pins or ports for connecting additional peripheral devices (not shown), as described below. The core of microprocessor 1102 operates between 1.34V - 1.45V of average voltage of electrical power supply through the 1126 power system. It is evident to those skilled in the art that microprocessor 1102 can be replaced by one or more microprocessors or other devices control systems, such as FPGAs or ASICs, with different and / or additional elements and functionalities without departing from the scope of the present invention.
[0107]. The built-in USB 112b port and USB PHY 1108B integrated in microprocessor 1102 are used to provide a USB communication port 112b that allows test device 100 to communicate with or receive communications from other USB devices (not shown). Test device 100 uses a USB client protocol that allows USB port 112b to serve as a client for other USB devices (not shown). The external connection can be used for retrieving and installing updated software, transmitting test records to remote devices (not shown), downloading test information and uploading test results to a main computer, or similar. Likewise, other driver circuit systems can be used, if desired.
[0108]. The test device 100 also includes a read-only flash memory (ROM) 1104, a random access memory (RAM) 1106 and an Ethernet interface PHY 1108A, each accessing or being accessed by microprocessor 1102 via individual parallel buses 1110 in a manner well known in the art. In the preferred embodiment, there are at least 64MB (sixty-four megabytes) of ROM 1104 and at least 16MB (sixteen megabytes) of SDRAM 1106. RAM 1106 is an integrated circuit model MICRON MT48LC8M16A2P-7E: G organized by 2Mb x 16 l / Os x 4 benches. RAM 1106 supports software that runs inside microprocessor 1102. Preferably, ROM 1104 is an integrated circuit in the SAMSUNG flash memory model K9F1208U0C-PIB00 NAND. ROM 1104 is a persistent memory responsible for storing all system software and all test files run by test device 100. Consequently, ROM 1104 keeps data stored even when test device 100 does not receive power from electricity. The ROM 1104 memory can be rewritten by a procedure well known to those skilled in the art, thereby facilitating the updating of the test device 100 system software executed by the 1102 microprocessor, without having to add or replace any of the 1104, 1106 memory components of test device 100. Different models from the same or different manufacturers can be used alternatively in ROM 1104 and / or RAM 1106, if desired.
[0109]. The microprocessor 1102 also has an integrated interface for a Secure Digital expansion port on the memory card and 1112 card reader. The SD 1112 card expansion port is located inside the test device 100 to promote additional functionality in future iterations of test device 100 by inserting an SD memory card (not shown) with additional functionality recorded on it.
[0110]. The PHY 1108A Ethernet interface is an integrated circuit model DP83640TVV from NATIONAL SEMICONDUCTOR and provides a 100MB per second connection to a local area network (LAN), computer (not shown) or other external device (not shown). The PHY 1108A Ethernet interface negotiates between a connected external device (not shown) and microprocessor 1102 via its individual parallel bus 1110C.
[0111]. The tester 100 requires the supply of several regulated voltages to function properly. The various voltages are powered by a multichannel power management integrated circuit (PMIC) 1116. The power management of PMIC 1116 addresses requires up to 8 (eight) independent output voltages with a single input power source. In the present embodiment, the PMIC 1116 is a FREESCALE MC34704 IC, but other power management circuits can be used alternatively. The PMIC 1116 includes backup outputs that are always actively feeding power to the real-time clock on microprocessor 1102 and the battery monitor circuit (not shown).
[0112]. Microprocessor 1102 controls its power system 1126 and enters hibernation mode whenever test device 100 is inactive for a predetermined period of time (for example, 10 minutes). On that occasion, most of the internal functions of microprocessor 1102 are paralyzed, preserving, to preserve batteries 116. However, a real time clock (not shown) is kept running to maintain the correct date and time of day on the test device. 100. In addition, one or more sensors, such as the touch screen portion of the LCD 110, are preferably kept in an active state so that it is possible to exit hibernation mode, for example, after detecting pressure by user from any part of the touch screen, or if the hinge cover 104 is opened by pressing the actuator 106.
[0113]. If all electrical power supplied to the power system 1126 of test device 100 is withdrawn, for example, when batteries 116 are replaced, a backup battery (not shown) installed in microprocessor 1102 maintains the minimum power required to power the watch in real time so that tester 100 can maintain the correct date and time. The ability of microprocessor 1102 to write to flash memory ROM 1104 is inhibited whenever power is turned off or restored to test device 100 until power system 1126 and microprocessor 1102 stabilize to prevent accidental alteration of the contents of flash memory ROM 1102 for the duration of the power interruption.
[0114]. A first port on microprocessor 1102 is used to connect microprocessor 1102 to RFID communication circuit 210 via sensor interface / RFID board 1118 and light detection circuit 1200 (Fig. 12) to receive data from it. A second port on microprocessor 1102 is used to connect microprocessor 1102 to peripherals (not shown) via Peripheral Experiment Support Interface 1120. Light detection circuit 1200 will be described in more detail hereinafter with reference to figure 12 .
[0115]. The light detection circuit 1200 is capable of detecting the various ranges and types of readings necessary to perform the various types of tests performed by the test device 100. The light detection circuit 1200 includes a secondary microprocessor 1202, a fast pulse counter 1204, one or more analog amplifiers and filters 1206, a PMT 206 and a PMT 218 high voltage power supply. The PMT 206 detects light signals from test kit 300 on an active surface and emits current pulses into the circuit light detection 1200. In the preferred embodiment, as soon as reagent 504 mixes with sample 414, PMT 206 starts analyzing the light signature to locate photons that are not associated with normal radiation, emitting photons from housing 401 from the base of test kit 400 and other mechanical noise from test device 100. Output current pulses are converted by light detection circuit 1200 and retransmitted by micr o secondary processor 1202 in digital format, which is sent to primary microprocessor 1102 for analysis.
[0116]. The spectral response range of the PMT 206 varies from the ultraviolet range to the visible light range (230nm - 700nm) with a peak response at 350nm and a photosensitivity response time of 0.57ns. In the present embodiment, the PMT 206 is a model R9880U-110 and the high voltage power supply 218 is a model Cl0940-53, both manufactured by HAMAMATSU PHOTONICS. Preferably, the secondary microprocessor 1202 is a TEXAS INSTRUMENTS processor model MSP430F2013IPW.
[01171. Secondary microprocessor 1202 offers a consistent interface for transmitting data to primary microprocessor 1102. Consequently, although it is desirable to include secondary microprocessor 1202 in test device 100 within light detection circuit 1200 in order to provide future flexibility and ease of implementation of additional or alternate sensors 206, or to expand light detection circuit 1200 to include multiple detectors, secondary microprocessor 1202 is optional. In other words, the functionality of secondary microprocessor 1202 can alternatively be realized by microprocessor 1102. In this case, sensor 206 can be connected directly to a serial port on microprocessor 1102.
[0118]. 206 PMTs are sensitive to sources of interference, such as temperature changes, electric fields, magnetic fields and electromagnetic fields. Therefore, the PMT 206's detection surface area is susceptible to the output of unwanted signals or background noise, due to these and other sources of interference. In the preferred embodiment, the diameter of the detection surface of the PMT 206 is limited to 8 mm in order to limit the generation of background noise signals and increase the signal-to-noise (SNR) ratio of the output of the detection circuit 1200 light. It will be apparent to those skilled in the art that other PMTs 206 and 218 high voltage power supplies can be used alternatively.
[0119]. Going back to figure 11, LCD 110 is activated by an LCD controller integrated with microprocessor 1102, which generates the signaling format required by LCD 110. Consequently, LCD 110 is connected to the general universal input / output ports of microprocessor 1102 via display / touch panel interface 1122. LCD 110 preferably includes a built-in drive circuit (not shown) that interfaces microprocessor 1102 input / output ports via standard control and data signals. The LCD 110 touch screen uses a four-wire connection to communicate with microprocessor 1102. A speaker 1124 can be connected to microprocessor 1102 to emit audible output sounds, such as alert and error messages and the like to the user .
[0120]. It should be appreciated by those skilled in the art that the various electrical / electronic components shown in figures 11 and 12 are merely an illustration of the electrical / electronic components of the preferred embodiment of the present invention. Other components can be replaced or added to any of the components shown without departing from the scope of the present invention. In other words, the present invention is not limited to the precise structure and operation of the electrical / electronic components and related components shown in figures 11 and 12.
[0121]. Referring to figure 16, a flow chart of the steps in which the test kit set 300 is used in conjunction with the test device 100 to perform a test according to the preferred embodiment of the present invention is shown. Before starting a test, a reservoir card 500 is preferably manufactured outside the test establishment. B cells produced by genetic engineering are grown in step 1610. The cultured cells are loaded with coelenterazine in step 1612 and excess coelenterazine is removed in step 1614. A cell stabilizer, such as Pluronic F68, is added in step 1616 and a Cryopreservative, such as dimethyl sulfoxide (DMSO), is added in step 1618 to complete the creation of the biosensor (ie reagent 504). The cells are loaded onto reservoir cards 500 in step 1620. In step 1622, the positive control sample (ie reagent 506), such as anti-IgM or digitonin, is loaded onto reservoir cards 500. Reservoir cards 500 are then frozen, stored and / or distributed to the test sites in step 1624. Preferably, the cards are frozen and stored at a temperature below approximately -40 ° C (minus forty degrees Celsius).
[0122]. As soon as the cards are distributed, before starting the test, in step 1626, the user may have to prepare a reservoir card 500 of a selected test type, thawing the reservoir card 500 and the reagents 504, 506 contained in the card using a specified defrost procedure. Preferably, a defrosting procedure is specified when necessary for a specific type of test with a 500 reservoir card. In step 1628, a user selects the (prepared) reservoir card 500 of the desired test type and assembles the reservoir card 500 on the base of the test case 400 until the permanent fixing elements 502 in the housing 501 of the reservoir card 500 engage the retaining elements in the housing 401 of the base of the test case 400. In the currently preferred embodiment, audible (ie a click) and / or tactile feedback is evident to the user, resulting from the engagement of the fasteners permanent 502 on the reservoir card 500 to the retaining elements in the housing 401 of the base of the test case 400.
[0123]. In step 1630, the user optionally prepares a sample 414, for example, fragmenting any infectious agent present in sample 414 using ultrasound, pressure gradient and / or enzymatic treatment or the like. Several techniques can be used, including: (i) an enzyme, such as lipase, to release O antigens from the cell surface (part of LPS); (ii) ultrasound to fragment cells; (iii) a French press or equivalent to fragment cells; or (iv) a chemical treatment to release LPS from cells. In step 1632, the user uses a sample deposition tool to pierce perforated film 410 at the base of test kit 400 above reaction chamber 404 and deposit a very small amount (for example, thirty microliters) of a sample 414 of a suspected infectious agent directly in reaction chamber 404 within the base of test kit 400. The user then removes the sample depositing tool and closes the integral hinged lid 408 from the base of test kit 400, ensuring that the retaining elements 408a and 408b engage slots 420a and 420b in housing 401 of test case base 400. Hinged lid 408 of test case base 400 is retained in the closed position and compressible gasket 418 on the upper surface of the test case base 400 is engaged by the cap 408 to form a fluid impermeable seal. At that time, reagents 504, 506 stored inside reservoir card 500 must be fully thawed in order to proceed with the rest of the test. Alternatively, the user can mount reservoir card 500 on the base of test kit 400 after depositing sample 414 in reaction chamber 404, or before reagents 504, 506 thaw. In addition, sample 414 can be deposited in reaction chamber 404 after test kit 300 is inserted into test device 100.
[0124]. Referring to Figures 1C and 3C, the bottom of the test kit set 300 is designed to be placed in the recess of the case 152 so that the lens 412 of the reaction chamber 404 aligns with the sensor 206. The test kit set 300 and the recess of the case 152 are preferably configured so that the test case set 300 cannot be fully inserted in the wrong direction and / or the hinged lid 104 of the test device 100 cannot be closed if the set test kit 300 is inserted in the analysis part 200 in the wrong direction.
[0125]. When the user inserts test kit set 300 into the recess of case 152 correctly, a physical process initiates a chain reaction of physical and electronic processes within test device 100 to perform the desired test on sample 414 in step 1634 and, if necessary, a positive control test in step 1636. The user closes the hinged cover 104 of the test device 100, mechanically locking it in the closed position. The testing device 100 is able to detect when the hinged lid 104 is closed and sends a signal to the microprocessor 1102, which activates the RFID communication circuit 210 for transmitting data to and from the RFID tag 508 via communication circuit RFID 210.
[0126]. At that time, the RFID tag 508 located inside the test reservoir card 500 is placed in the path of the RFID communication circuit 210 within the analysis part 200. In the present embodiment, the RFID tag 508 is an RI- 116-114A-S1 from Texas Instruments, which operates at 13.56 MHz and contains 256 bits of user memory for read / write functionality. The test device 100 reads detailed information for the test to be performed from the RFID tag 508 of the test kit set 300 via RFID. Information that can be communicated to and from RFID 508 tags includes the test batch or sample source, the specific test to be performed, information regarding the identity of a particular test case, as well as other information. The test device 100 also writes a value to the test kit's RFID tag 508, which indicates that the test kit set 300 was used to perform a test. Writing the RFID tag 508 prevents the test kit set 300 from being reused on the same or another compatible test device 100 in the future. With reference to figures 13A and 13B, test device 100 asks the user to confirm the type of test and start the test via user interface 1400 (Fig. 14) shown on LCD screen 110, which will be described in more detail below .
[0127]. With reference to figures 2 and 9, when the user chooses to start the test, the microprocessor 1102 sends a signal to drive the motor 226, which drives the piston 224 and the piston rod 224A forward to engage the fluid displacement mechanism 900 and to complete the introduction of the first reagent 504 into reaction chamber 404 in the manner described above. The piston rod 224A also works. preferably as a locking of the hinged cover 104. Thus, as piston 224 begins to move under the force of motor 226 at the start of the test, piston rod 224A moves under actuator 106. When the piston rod piston 224A is under actuator 106, mechanical interference between the two prevents the user from pressing actuator 106 and opening cover 104, as a precaution against a user error during the course of the test. The piston rod 224A remains under the actuator until the test is complete and piston 224 is fully retracted. Simultaneously at the end of the first stage of the fluid displacement mechanism 900, piston rod 224A moves the slide plug 236 to its second position, which exposes the surface of sensor 206 to light emitted by reaction chamber 404 of the test kit assembly 300 via sliding shutter opening 238.
[0128]. Considering that the reaction process preferably begins as soon as the fluid displacement mechanism 900 within the test kit set 300 completes the introduction of the first reagent 504 into reaction chamber 404, the light detection circuit 1200 is also activated at that moment to detect any light emissions that may occur even before the user registers the correct data, as will be described in more detail below. If light detection circuit 1200 detects an appropriate light signal, microprocessor 1102 stores and reports a positive result, light detection circuit 1200 is turned off and motor 226 moves to retract piston 224 to its starting position.
[0129]. The plunger 424 of the tested test kit assembly 300 remains in its final position even after piston 224 is retracted. The user can then open the hinged lid 104 by pressing on the actuator 106 and remove the test kit set 300 used for proper disposal. Test sample 414 and reagents 504, 506 are all contained in a sealed manner within test kit set 300. The user can also confirm a test result within the user interface (Fig. 15) displayed on the LCD screen of the test device 100. Alternatively, if a predetermined period of time (for example, 60-120 seconds) elapses during the initial test and the light detection circuit 1200 does not detect an appropriate light signal, the motor 226 moves, preferably , to drive the piston 224, still in the fluid displacement mechanism 900, until the end of the introduction of the second reagent 506 in the reaction chamber 404 for the second test, as described above.
[0130]. If the light detection circuit 1200 does not detect an appropriate light signal as a result of the second test, microprocessor 1102 stores and reports an error message. However, if as a result of the second test, the appropriate light signal is detected by the light detection circuit 1200, the microprocessor 1102 stores and reports a negative result. At that time, the light detection circuit 1200 is turned off and the motor 226 moves to retract piston 224 to its starting position. The user can then remove the test kit 300 used for proper disposal. At that time, test device 100 is restarted and is ready to receive another set of test kit 300. Subsequent testing can be performed in the same way (using a new set of test kit 300) as described above.
[0131]. As previously discussed, test device 100 has the ability to perform a variety of different tests in real time (or near real time) using a single set of disposable test kits 300 containing a reservoir card 500 that has been specifically designed for run a particular test. Each reservoir card 500 contains a predetermined reagent mixture 504, 506 to perform a particular test. The RFID tag 508 inside the reservoir card 500, as well as the labeling of the reservoir card (not shown) identifies the particular test that the reservoir card 500 must perform, as well as the relevant control parameters for the particular test . In this way, the test device 100 is adapted for automatic customization, using software, to carry out various tests.
[0132]. An example of first reagent 504 is a biosensor reagent that includes a human B lymphocyte produced by genetic engineering to express a bioluminescent protein and at least one membrane-bound antibody, specific for a predetermined infectious agent. With regard to biosensors, cell-based biosensor systems (CBB) that incorporate whole cells or cellular components respond in a way that can offer a perception of the physiological effect of an analyte. As technicians in the subject can observe, cell-based assays (CBA) are emerging as reliable and promising methods to detect the presence of pathogens in clinical, environmental or food samples, because living cells are known to be extremely sensitive to modulations or disturbances of "normal" physiological microenvironments. Therefore, CBB systems have been used to classify and monitor "external" or environmental agents capable of causing disturbances to living cells (see, for example, Banerjee et al, Mammalian cell-based sensor system. Adv. Biochem Eng. Biotechnology, 117: 21-55 (2010), which is incorporated herein by reference.)
[0133]. Compared to traditional detection methods (for example, immunoassays and molecular assays, such as PCR), a biosensor has several advantages, including (i) speed, that is, detection and analysis occur in several seconds up to less than 10 minutes; (ii) greater functionality, which is extremely important to inform active components, such as live pathogens or active toxins, and (iii) ease of expansion to perform high productivity screening.
[0134]. An aequorin-based biosensor system is used with certain embodiments of the present invention. Aequorin is a 21-kDa calcium-binding photoprotein isolated from the luminescent jellyfish Aequorea victoria. Aequorin has a covalent bond with a hydrophobic prosthetic group (coelenterazine). After binding calcium (Ca2 +) and coelenterazine, aequorin undergoes an irreversible reaction and emits a blue light (preferably 469 nm). The fractional rate of consumption of aequorin is proportional, in the physiological pCa range, to [Ca2 +]. The application of the aequorin-Ca2 + indicator to detect E. coli contamination in food products was reported in 2003 (see Rider et al, AB cell-based sensor for rapid identification of pathogens, Science, 301 (5630): 213-5 (2003 ), which is incorporated by reference). In Rider, B lymphocytes produced by genetic engineering were used to express antibodies that recognize specific bacteria and viruses. B lymphocytes have also been used to express aequorin, which emits light in response to the calcium flow activated by the binding of a cognate target to the surface antibody receptor. The resulting biosensor cell emitted light in minutes in the presence of the target microorganisms. To create these biosensor cells, heavy and light antibody chains with variable regions were cloned and expressed in the B lymphocyte cell line. The resulting immunoglobulins become part of a surface R cell receptor complex, which includes reduced accessory molecule immunoglobulin ( Iga or CD79a) and β immunoglobulin (Igβ or CD79b). When the complex is cross-linked and grouped by polyvalent antigens, such as microorganisms, a set of signaling events quickly leads to changes in the intracellular calcium-ion concentration, which then causes the aequorin to emit light. This mechanism essentially hijacks the intrinsic ability of the B cell to specifically recognize the antigen presented in E. coli by the IgG antibody of the B cell membrane and this link triggers a temporary influx of Cα2 + to the cytosol, which binds the aequorin proteins produced in that B cell. and subsequently emits a blue light. See, Reiman, Shedding light on microbial detection, N England J Med, 349 (22): 2162-3 (2003), which is incorporated herein by reference, in its entirety.
[0135]. The selection of an appropriate B cell is important for the test described. Therefore, any proposed cell line should be tested to confirm that the signaling pathway of the B cell receptor is fully functional. Individual B cell clones having the aequorin gene should be tested to identify a particular clone with high aequorin activity, as significant variation from one clone to another is possible (see, generally, Calpe et al., ZAP-70 enhances migration of malignant B lymphocytes toward CCL21 by inducing CCR7 expression via lgM-ERKI / 2 activation, Blood, 118 (16): 4401-10 (2011) and Cragg ef al, Analysis of the interaction of monoclonal antibodies with surface IgM on neoplastic B -cells, Br J Cancer, 79 (5/6): 850-857 (1999), both of which are incorporated herein by reference in their entirety).
[0136]. A high-expression aequorin B cell is important to achieve high levels of sensitivity when using this detection system. In one example of an embodiment, the receptor response to the biosensor was verified using the Ramos human B cell line. Ramos cells are first transfected with the aequorin gene and the transfected cells were then selected for aequorin expression for two weeks. Then, the mixed Ramos cells are loaded with coelenterazine (CTZ) and stimulated with anti-IgM Ab. The light signal caused is captured by a luminometer.
[0137]. As shown in figure 17, anti-IgM stimulation causes a good-sized and prolonged (45 to 65 seconds) expected flash or flicker. In figure 17, the Y axis represents the amount of sparkling light and the X axis represents the reaction time in seconds. At 30 (thirty) seconds, the anti-IgM solution is injected into the Rαmos-αequorinα cell solution. The first peak (30-37 seconds) is a noise signal and the second largest and longest peak is the biological response to anti-IgM stimulation. To improve the overall signal-to-noise ratio, CTZ is removed from the CTZ-loaded Ramos-aequorin cell solution. The removal of CTZ from the cell solution decreases the noise signal from about 150 (one hundred and fifty) to about 50 (fifty), without significantly compromising the amount of the true peak signal.
[0138]. In accordance with the present invention, an example of a protocol for cell manipulation and brightness testing includes: (i) culturing Ramos-aequorin cells with a regular culture medium and maintaining these cells healthy (ie viability> 98%); (ii) loading Ramos-aequorina cells with CTZ in a final concentration of 2 pM, the cell density being 1-2 million per milliliter; (iii) loading the cells at 370 ° C with 5% CO2 in an incubator for at least 3 hours; (iv) removing the loading medium containing CTZ; (v) glow test taking 200pl of cell solution plus 30pL stimulants (anti-IgM) and reading with a luminometer; and (vi) confirm the functionality of CTZ and aequorin by adding 30-40 µL of digitonin (770 pM).
[0139]. The test device 100 is preferably controlled by an operating system run by microprocessor 1102. In the present embodiment, the operating system is preferably an application with a custom design and programmed to run in a Linux environment. The operating system offers input / output functionality and power management functions, as described. The custom application includes a simple, menu-based user interface, as illustrated in figures 14 and 15; parameter driven functions to control and analyze the tests performed by the test device 100; and a file system for storing protocols and test results. The stored test results can be retrieved, displayed or printed. The software allows adding protocols for new tests by downloading files, or the like.
[0140]. Preferably, the 1400 user interface in Figure 14 is menu controlled, with a series of items selectable by a user using menus provided on the LCD 110 touch screen. Preferably, the 1400 user interface presents a series of choices that allow you to navigate through each specific test until its completion. In the present embodiment, the 1400 user interface allows the user to return to the previous screen using a back key provided on the LCD and on the touch screen. However, the use of the back key during the test is not allowed unless the test is canceled or aborted. A selected action can continue through a series of steps, each of which is indicated by a new alert to the user.
[0141]. Figures 13A and 13B are a flow chart showing the basic flow of a preferred embodiment of the computer program used by test device 100. Those skilled in the art will find that the software may function slightly or completely differently from that shown in figures 13A and 13B, which is included merely to illustrate a currently preferred mode for the operation of the software.
[0142]. The process starts at step 1300, where a start screen is presented to the user on display 110 while the application completes the loading of test device 100. At steps 1302 and 1304, the user must enter the username and password. The test device 100 verifies that the user name and password entered are valid and goes to an initial screen 1400 (Fig. 14) in step 1306. A user identifier (for example, a five-digit code) that uniquely identifies the The user performing the test is preferably stored by the test device 100 as part of a test record.
[0143]. The user selects one or more actions and / or functions of the test device 100 to be performed from the initial screen 1400, including α running a test (step 1308) by introducing a set of test kit 300, analyzing the recorded results ( step 1310) by pressing the Test Protocol button 1402 or configuring settings (step 1312), such as Time Zone (Step 1330) or Language (Step 1332) by pressing the 1404 button. Preferably, the user selects the desired action using the LCD screen touchscreen 110 of test device 100.
[0144]. If the user inserts a set of test kit 300 into test device 100 and closes hinge cover 104, RFID communication circuit 210 is activated after hinge cover 104 is closed and RFID tag 508 or other identification in the set installed test kit 300 identify the type of test that test device 100 should perform.
[0145]. Preferably, each RFID 508 tag stores a sequence of characters, which encodes the particular test type for the test kit set 300, an expiration date for each test kit set 300, a serial number of the test set test kit 300, which may include a test solution lot number, if test kit set 300 has previously been tested within a test device 100, as well as other information pertinent to a test kit set in particular 300. Taken together, the information presented in the RFID string uniquely identifies each test kit set 300. Information from test kit 300 is entered into test device 100 when the test kit set test 300 is inserted into the recess of the case 152 in the analysis part 200 and the hinged lid 104 is closed. The process checks the RFID 508 tag data from the read test kit to confirm that the test kit has not been used before. The RFID tag 508 data from the test kit is accepted as valid if the RFID communication circuit 210 does not detect RFID transmission error during the reading process and if the format of the RFID tag 508 data is valid. After determining if the hinged door 104 is closed and the data read on the RFID tag 508 is valid, the Run Test option of step 1308 is selected automatically and the user must confirm that the test device 100 must perform the test.
[0146]. As the test begins, the user begins to provide the necessary data. The user must provide the specific numeric code of sample 414 using the touch sensitive LCD screen 110 in step 1314, where the user must enter a type of “Sample / Location”. In the preferred embodiment, the specific numeric code of sample 414 comprises a five-digit number that refers to the batch or environment of sample 414. If the user selects “Sample” in step 1316, the user must enter the batch number using the touch sensitive LCD screen 110. If the user selects "Location" in step 1318, the user must enter a location using the touch sensitive LCD screen 110.
[0147]. After receiving the sample specific code, the information received from the RFID tag 508 of the test kit set 300 and the user input received are compared to all stored test files, as well as the data received from the RFID tag 508 indicating that test kit 300 has been previously tested, refusing test kit 300 if this test kit 300 has been tested before.
[0148]. The information read on the RFID 508 tag is also used to identify the particular test to be performed by the test device 100 and to select the appropriate test protocols. The protocols to be selected include test synchronization, light reading requirements of the 1200 light detection circuit and the like for the particular test to be performed. The parameters of a test control table recorded in ROM 1104 specify how each step of the acquisition and analysis of the test data should be performed, including alternative software routines, when necessary. In this way, new or modified test parameters can be installed by downloading new test control tables and, if necessary, supporting software modules, without modifying the operating software or the basic application. The information in the test control tables is recorded in ROM 1104 for each diagnostic test that can potentially be performed using test device 100. In alternative embodiments, additional information regarding test samples 414 can also be included in the process test device 100 initiation test. This additional information may include handling requirements, quarantine requirements, and other anomalous characteristics of the 414 test samples.
[0149]. The testing device 100 performs the test of sample 414 while the user enters the specific numeric code of sample 414 and continues to perform the test after the user finishes providing the necessary data. Preferably, the test is completed only after the user has completed the necessary data information. Applying force to open the hinged lid 104, or letting the data information complete results in failure or aborted test. Preferably, users of test device 100 understand that test device 100 needs all data to be filled in and hinge cover 104 must remain closed to minimize failures or aborted tests.
[0150], In step 1320, the test status is shown to the user. The test device 100 displays status information to the user to confirm that the test is in progress until it is complete. Test information, whether prospective, in progress or completed, is displayed on the LCD screen 110 in fixed text format, which includes identification by the test kit set 300 of the information described above. Elements of the test file not yet completed are left blank or displayed as “in progress” until the test is complete. Preferably, the user cannot perform other functions on test device 100 while a test is being performed. However, in other embodiments, the software can be changed to allow the user to perform other tasks on the test device 100, such as reviewing a test record, while performing a test.
[0151]. If during the test, it is determined that an appropriate light signal has been detected by sensor 206, the process continues at step 1322, when a positive result is reported and the user is asked to confirm. As soon as the user confirms, he must retype the Lot / Location number in step 1324. If the Lot / Location number is compatible, the test data will be recorded and the process will return to the initial screen in step 1306. If in step 1320 , sensor 206 does not detect an appropriate light signal, the process checks whether the appropriate light signal is detected for the negative control test. If detected, the negative result is reported, as shown in the Negative Result 1500 screen in figure 15 and the user must remove the case in step 1326. At this point, no RFID signal is detected and the test data is recorded and the process returns to the initial screen of step 1306.
[0152]. In addition, a test can be aborted by the software at any stage if, for example, sensor 206, engine 226, or any other hardware failure is detected or if the hinge cover 104 is open. If this problem is detected in step 1320, a test error is reported in step 1328 and the user must remove the used test kit set 300. As soon as the test kit set 300 is removed, no RFID by RFID 210 communication circuit, the error data is recorded in ROM 1104 and the process returns to the initial screen 1400 of step 1306. Similarly, the test can also be canceled by the user at any stage until the results the test are informed and stored. Aborted and canceled tests are recorded in the test results file and stored in flash memory ROM 1104 to prevent reuse of a previously used 300 test kit set.
[0153]. The test results are stored in flash memory ROM 1104 in text form, preferably as displayed on the 110 touch-sensitive LCD screen. Each test record preferably includes all the test information identified above, including sample identification of test 414, the particular test performed, the test date and time, user ID and a standard result or identification that the test failed due to an error or was aborted.
[0154]. All test results completed successfully or failed tests are stored in flash memory ROM 1104. The user can retrieve test results in flash memory ROM 1104 for display on the LCD touchscreen 110. Preferably, flash memory ROM 1104 it is large enough to store a substantial number of test records (for example, five thousand records), preferably corresponding to the number of tests that must be performed in at least one week of tests by test device 100. It is contemplated that the user cannot delete records recorded in flash memory ROM 1104 to prevent unauthorized tampering with test results. However, if flash memory ROM 1104 is completely full, test device 100 can automatically exit test mode and alert the user to start uploading data to a computer located remotely (not shown) via interface ports 112. As soon as the upload is complete and the test records are deleted from flash memory ROM 1104, the user can perform tests again using the test device.
[0155]. Conserving battery power is an important issue addressed by two-level operating software. First, the current battery charge level is provided to the user periodically or continuously. The software also provides user-specific warnings to initiate a recharge of the 116 batteries when the battery monitor circuit indicates that the charge level of the 116 batteries has fallen below a predetermined safety limit. In addition, the software prevents the start of a new test when the battery charge level in the 116 batteries is too low for the safe completion of a test, without risking the failure of sensor 206, or other software function or hardware associated with the test function of test device 100.
[0156]. The power supplied to the various peripheral devices, including the RFID communication circuit 210, the light detection circuit 1200, the touch sensitive LCD screen 110 and the microprocessor 1102, is controlled by the operating system. Thus, the power supply can be selectively switched off when the functions of the various devices are not required for the current operation of test device 100. The entire test device 100 can also be placed in a “deactivated” state after receiving a command from the user, or after a predetermined period of inactivity of test device 100. The deactivation state differs from the complete absence of power in which the clock / date continues to function and RAM 1106 is kept activated by batteries 116 instead of the backup battery , which is activated after a complete absence of power supplied by the battery.
[0157]. However, when deactivation occurs, virtually all software activity ceases, except for the processes necessary to monitor the status of the 110 touch-sensitive LCD screen. The user can “activate” the unit by touching the 110 touch-sensitive LCD screen. As mentioned earlier, after detecting the restoration of battery power 116 after a complete power interruption, the software does not need to be informed of the date and time because the backup battery maintains this minimum function. the time period set for test device 100 for automatic deactivation based on a period of inactivity depends on which menu is displayed, preferably the delay periods are adjustable using the settings menu in step 1312.
[0158]. Those skilled in the art will be able to observe that changes and modifications can be made to the embodiments described above without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited to the embodiments described above, but is intended to cover all such modifications within the scope and spirit of the invention. Those skilled in the art can observe that alternative arrangements of test kit set 300, including the combination of reservoir card 500 and test kit base 400 in a single subset, storing some of the necessary reagents 504, 506 both on the base of the kit Test 400 as on reservoir card 500, or direct deposition of the sample on the necessary reagents 504, 506 to perform the desired test, are within the scope of the present disclosure.

权利要求:
Claims (44)
[0001]
1. SYSTEM TO DETECT THE PRESENCE OF AN INFECTIOUS AGENT IN A BIOLOGICAL SAMPLE quickly, characterized by the fact that it comprises: a. a single biosensor reagent (504, 506) that includes at least one antibody specific for a predetermined infectious agent and a bioluminescent agent, where at least one antibody is expressed on the surface of living B lymphocytes produced by genetic engineering and, where the agent bioluminescent is expressed in an equine manner by living B lymphocytes produced by genetic engineering, the biosensor reagent (504, 506) being effective to: (i) detect the presence of a specific infectious agent in a biological sample (414) to be tested; and (ii) emit a detectable light signal when the biosensor reagent (504, 506) reacts with the biological sample (414) and detects the presence of the specific infectious agent in the biological sample (414); B. a disposable test kit (300) for single use, where the disposable test kit (300) for single use includes: (i) a reservoir card (500), where the reservoir card (500) is still includes the biosensor reagent (504, 506); and (ii) a test case base (400), where the test case base (400) is configured to accept the reservoir card (500) and, where the test case base (400) ) further includes: (a) a single reaction chamber (404) comprising a central axis, wherein the single reaction chamber (404) is shaped like a rotated half of an ellipse; (b) an input channel (802) connected with the reaction chamber (404), where the input channel (802) is positioned above the reaction chamber (404) at an angle of 15 to 60 degrees above the horizontal and, wherein, the input channel (802) is displaced from the central axis of the reaction chamber (404); and (c) in which when introducing biological sample (414) at the base of the test kit (400) through the input channel (802), the biological sample (414) is mixed homogeneously with the biosensor reagent (504, 506) while minimizing damage to live B lymphocytes produced by genetic engineering and minimizing any bubbling of the mixed biosensor reagent (504, 506) and biological sample (414) in the reaction chamber (404); and c. a test unit adapted to receive the disposable test kit (300), for single use, the test unit including a sensor (206) to detect a detectable light signal emitted by the biosensor reagent (504, 506) upon reaction with the biological sample (414), the detection of the detectable light signal emitted being indicative of the presence of the infectious agent in the biological sample (414) and being the only analysis performed of the detectable light signal emitted, in which the detection of the specific infectious agent in the biological sample ( 414) occurs in real time.
[0002]
2. SYSTEM according to claim 1, characterized by the fact that the sensor (206) is a photomultiplier tube (PMT) with an active surface and, in which the size of the active surface is optimized to minimize background noise and increase the signal-to-noise ratio of the detectable signal emitted.
[0003]
3. SYSTEM according to claim 2, characterized by the fact that the reservoir card (500) is configured to be inserted in the base of the test case (400) and to be permanently retained in it by one or more retaining elements (502).
[0004]
4. SYSTEM according to claim 3, characterized by the fact that the base of the test case (400) still comprises a fluid displacement mechanism (900) including a piston (424) and, in which the actuation of the piston ( 424) causes the biosensor reagent (504, 506) stored in the reservoir card (500) to move into the reaction chamber (404).
[0005]
5. SYSTEM according to claim 4, characterized by the fact that the test unit still comprises a motor (226) and piston (224) set configured to drive the piston (424).
[0006]
6. SYSTEM according to claim 1, characterized by the fact that the test unit is a portable test unit.
[0007]
7. SYSTEM according to claim 1, characterized by the fact that it still comprises at least one additive located in the test chamber, at least one additive being effective in minimizing the formation of bubbles in the test chamber during the mixing of the biological sample ( 414) and the biosensor reagent (504, 506).
[0008]
8. SYSTEM according to claim 7, characterized by the fact that at least one additive includes a surfactant.
[0009]
9. SYSTEM according to claim 1, characterized by the fact that it still comprises a disruptor for breaking the individual cells of an infectious agent in the biological sample (414) before mixing the biological sample (414) with the biosensor reagent (504, 506).
[0010]
10. SYSTEM according to claim 9, characterized by the fact that the disruptor is at least an effective enzyme for releasing O antigens from the cell surface, an ultrasound effective for fragmenting cells, an effective French press for fragmenting cells and a treatment effective chemical to release LPS from the cells of the infectious agent.
[0011]
11. SYSTEM according to claim 1, characterized by the fact that the biosensor reagent (504, 506) is preloaded with coelenterazine and, in which any excess coelenterazine is removed from the biosensor reagent (504, 506) before the reaction of the biosensor reagent (504, 506) with the biological sample (414) to be tested.
[0012]
12. SYSTEM according to claim 1, characterized by the fact that the biological sample (414) to be tested is extracted from food including at least one material between beef, poultry, pork and other meats, fish and vegetables.
[0013]
13. SYSTEM according to claim 1, characterized by the fact that the specific infectious agent is Escherichia coli.
[0014]
14. SYSTEM according to claim 1, characterized by the fact that the real time is within the range of approximately five minutes from the combination of the biological sample (414) with the biosensor reagent (504.506).
[0015]
15. SYSTEM according to claim 1, characterized by the fact that it still comprises a second biosensor reagent (504, 506) effective to react with the biosensor reagent (504, 506) to determine the proper functioning of the test unit, in which the biosensor reagent (504, 506) reacts with the biological sample (414) to be tested before the second biosensor reagent (504, 506) reacts with the biosensor reagent (504, 506).
[0016]
16. TEST DEVICE TO DETECT AN INFECTIOUS AGENT IN A BIOLOGICAL SAMPLE in real time, the test device (100) characterized by the fact that it comprises: a. an external housing (102) comprising a hinged lid (104) and an input / output device; B. an analysis part comprising: (1) a recess (152) in the external housing (102) for receiving the disposable test kit (300), for single use containing a biological sample (414) to be tested, in which the test kit disposable test (300) for single use includes: (a) a reservoir card (500), in which the reservoir card (500) still includes a single biosensor reagent (504, 506); (b) a test case base (300), where that of the test case base (400) is configured to receive the reservoir card (500) and, where that of the test case base (400) further includes: i) a single reaction chamber (404) comprising a central axis, wherein the single reaction chamber (404) is shaped like a rotated half of an ellipse; ii) an input channel (802) connected with the reaction chamber (404), wherein the input channel (802) is positioned above the reaction chamber (404) at an angle of 15 to 60 degrees above the horizontal and , wherein, the input channel (802) is displaced from the central axis of the reaction chamber (404); and (ii) a single-use interaction actuator, disposable test kit (300) when the hinged lid (104) is closed, the actuator causing the single-use biosensor reagent (504, 506), test kit (300 ) disposable to be moved to react with the biological sample (414) during the performance of the test, the biosensor reagent (504, 506) including at least one at least one antibody specific to a predetermined infectious agent and a bioluminescent agent, at which at least one antibody is expressed on the surface of living B lymphocytes produced by genetic engineering and, where the bioluminescent agent is expressed in an equorine form by living B lymphocytes produced by genetic engineering, and when introducing the biological sample (414) into the test kit base (400) through the input channel (802), the biological sample (414) is mixed homogeneously with the biosensor reagent (504, 506) while minimizing damage to live B lymphocytes pr induced by genetic engineering and minimizes any bubbling of the mixed biosensor reagent (504, 506) and biological sample (414) in the reaction chamber (404); (iii) a sensor (206) associated with the recess (152) in the external housing (102) to detect the light signal emitted after at least one biosensor reagent (504, 506) has been moved by the actuator to react with the biological sample (414 ) and to generate an output signal; and c. a control unit configured to: (i) receive input from a user through an input / output device to start the test; (ii) in response to receiving user input and after the biological sample (414) has been deposited in the recess (152) in the analysis part, it activates the actuator to move at least one biosensor reagent (504, 506) in the case test (300) to react with the biological sample (414); (iii) receiving an output signal from the sensor (206); and (iv) show the test result to the user on the input / output device.
[0017]
17. DEVICE according to claim 16, characterized by the fact that the sensor (206) is a photomultiplier tube (PMT) with an αtivα surface and, in which the αtivα surface size is optimized to minimize background noise and increase the signal-to-noise ratio of the detectable signal emitted.
[0018]
18. DEVICE according to claim 17, characterized by the fact that it still comprises an RFID communication circuit to receive one or more signals from the test case (300), in which one or more signals identified at least one of the test type for be performed when the test kit (300) has been previously used.
[0019]
19. DEVICE according to claim 16, characterized in that the biosensor reagent (504, 506) is preloaded with coelenterazine and, in which any excess coelenterazine is removed from the biosensor reagent (504, 506) before the reaction of the biosensor reagent (504, 506) with the sample (414) to be tested.
[0020]
20. DEVICE according to claim 16, characterized by the fact that it still comprises a disruptor for breaking the individual cells of an infectious agent in the sample (414) before mixing the sample (414) with the biosensor reagent (504, 506)
[0021]
21. DEVICE according to claim 20, characterized by the fact that the disruptor is at least an effective enzyme for releasing O antigens from the cell surface, an effective ultrasound to fragment cells, an effective French press to fragment cells and a treatment effective chemical to release LPS from the cells of the infectious agent.
[0022]
22. DEVICE according to claim 16, characterized in that the test device (100) is a portable test device (100).
[0023]
23. SYSTEM TO DETECT THE PRESENCE OF AN ANALYTES IN A BIOLOGICAL SAMPLE, characterized by the fact that it comprises (a) a biosensor reagent (504, 506) that includes at least one specific antibody for a predetermined analyte and a bioluminescent agent, in that at least one antibody is expressed on the surface of living lymphocytes produced by genetic engineering and, where the bioluminescent agent is expressed by living lymphocytes produced by genetic engineering the biosensor reagent (504, 506) being effective to: (i) detect the presence a specific analyte in a sample (414) to be tested; and (ii) emitting a detectable light signal when the biosensor reagent (504, 506) reacts with the sample (414) and detects the presence of the specific analyte in the sample (414); (b) a test kit (300), where the test kit (300) still includes: (i) a reservoir card (500), where the reservoir card (500) still includes the biosensor reagent (504 , 506); and (ii) a test case base (400), where the test case base (400) is configured to accept the reservoir card (500) and, where the test case base (400) ) further includes: a) a reaction chamber (404) comprising a central axis, in which the single reaction chamber (404) is shaped like a rotated half of an ellipse; b) an input channel (802) connected with the reaction chamber (404), in which the input channel (802) is positioned above the reaction chamber (404) at an angle of 15 to 60 degrees above the horizontal and , wherein, the input channel (802) is displaced from the central axis of the reaction chamber (404); and c) where by introducing the sample (414) into the base of the test kit (400) through the input channel (802), the sample (414) is mixed homogeneously with the biosensor reagent (504, 506) while minimizing damage the live lymphocytes produced by genetic engineering and minimizes any bubbling of the mixed biosensor reagent (504, 506) and sample (414) in the reaction chamber (404); and (c) a test unit adapted to receive the test kit (300), the test unit including a sensor (206) to detect a detectable light signal emitted by the biosensor reagent (504, 506) upon reaction with the sample (414), the detection of the detectable light signal emitted being indicative of the presence of the analyte in the sample (414) and being the only analysis performed of the detectable light signal emitted, in which the detection of the specific analyte in the sample (414) occurs in real time .
[0024]
24. SYSTEM according to claim 23, characterized by the fact that the sensor (206) is a photomultiplier tube (PMT) with an active surface and, in which the size of the active surface is optimized to minimize background noise and increase the signal-to-noise ratio of the detectable signal emitted.
[0025]
25. SYSTEM according to claim 23, characterized in that the reservoir card (500) is configured to be inserted in the base of the test case (400) and to be permanently retained in it by one or more retaining elements (502).
[0026]
26. SYSTEM according to claim 23, characterized by the fact that the base of the test case (400) still comprises a fluid displacement mechanism (900) and, in which the actuation of the fluid displacement mechanism (900) causes the biosensor reagent (504, 506) stored in the reservoir card (500) to move into the reaction chamber (404).
[0027]
27. SYSTEM according to claim 23, characterized by the fact that the test unit still comprises a motor (226) and piston (224) set configured to drive the fluid displacement mechanism (900).
[0028]
28. SYSTEM according to claim 23, characterized in that the test unit is a portable test unit.
[0029]
29. SYSTEM according to claim 23, characterized by the fact that it still comprises at least one additive located in the test chamber, at least one additive being effective in minimizing the formation of bubbles in the test chamber during the mixing of the biological sample ( 414) and the biosensor reagent (504, 506).
[0030]
30. SYSTEM according to claim 29, characterized by the fact that at least one additive includes a surfactant.
[0031]
31. SYSTEM according to claim 23, characterized in that it further comprises a disruptor for breaking up individual cells of an infectious agent in the biological sample (414) before mixing the biological sample (414) with the biosensor reagent (504, 506)
[0032]
32. SYSTEM according to claim 31, characterized by the fact that the disruptor is at least an effective enzyme to release O antigens from the cell surface, an effective ultrasound to fragment cells, an effective French press to fragment cells and a treatment effective chemical to release LPS from the cells of the infectious agent.
[0033]
33. SYSTEM according to claim 23, characterized in that the biosensor reagent (504, 506) is preloaded with coelenterazine and in which any excess coelenterazine is removed from the biosensor reagent (504, 506) before the reaction of the biosensor reagent (504, 506) with the biological sample (414) to be tested.
[0034]
34. SYSTEM according to claim 23, characterized by the fact that the biological sample (414) to be tested is extracted from food including at least one material between beef, poultry, pork and other meats, fish and vegetables.
[0035]
35. SYSTEM according to claim 23, characterized by the fact that the specific analyte is Escherichia coli.
[0036]
36. SYSTEM according to claim 23, characterized by the fact that the real time is within the range of approximately five minutes from the combination of the biological sample (414) with the biosensor reagent (504.506).
[0037]
37. SYSTEM according to claim 23, characterized in that it still comprises a second biosensor reagent (504, 506) to determine the proper operation of the test unit, in which the biosensor reagent (504, 506) reacts with the sample (414) to be tested before the second biosensor reagent (504, 506).
[0038]
38. TEST DEVICE FOR DETECTING AN INFECTIOUS AGENT IN A BIOLOGICAL SAMPLE IN REAL TIME, the test device (100) characterized by the fact that it comprises: (a) a housing (102) comprising a cover (104) and a entrance exit; (b) an analysis part comprising: (1) a recess (152) in the housing (102) for receiving a test kit (300) containing a biological sample (414) to be tested, in which the test kit (300 ) includes: a) a reservoir card (500), in which the reservoir card (500) still includes the biosensor reagent (504, 506); and b) a test kit base (400), where the test kit base (400) is configured to accept the reservoir card (500) and, where the test kit base (400) is still includes: i) a single reaction chamber (404) comprising a central axis, wherein the single reaction chamber (404) is shaped like a rotated half of an ellipse; ii) an input channel (802) connected with the reaction chamber (404), wherein the input channel (802) is positioned above the reaction chamber (404) at an angle of 15 to 60 degrees above the horizontal and , wherein, the input channel (802) is displaced from the central axis of the reaction chamber (404); and (ii) an actuator to interact with the test case (300) when the lid (104) is closed, the actuator causing at least one biosensor reagent (504, 506) in the test case (300) to move to react with the biological sample (414) while performing a test, biosensor reagent (504, 506) that includes at least one antibody specific for a predetermined analyte and a bioluminescent agent, in which at least one antibody is expressed on the surface of the lymphocytes produced by genetic engineering and, in which the bioluminescent agent is expressed by the living lymphocytes produced by genetic engineering, and, when introducing the biological sample (414) at the base of the test kit (400) through the input channel (802) , the biological sample (414) is mixed homogeneously with the biosensor reagent (504, 506) while minimizing damage to live lymphocytes produced by genetic engineering and minimizing any bubbling of the mixed biosensor reagent (504, 506) and the biological sample (414) in the reaction chamber (404); and (iii) a sensor (206) associated with the recess (152) in the housing (102) to detect a signal emitted after at least one biosensor reagent (504, 506) is moved by the actuator to react with the biological sample (414) and generate an output signal; and c. a control unit configured to: (i) receive input from a user via an input / output device to initiate a test; (ii) in response to receiving input from the user and after biological sample (414) has been deposited in the recess (152), activate the actuator to move at least one biosensor reagent (504, 506) in the test kit (300) to react with the biological sample (414); (iii) receiving the output signal from the sensor (206); and (iv) issue a test result to the user on the input / output device.
[0039]
39. TEST DEVICE according to claim 38, characterized by the fact that the sensor (206) is a photomultiplier tube (PMT) with an active surface and, where the size of the active surface has been optimized to minimize noise and increase the signal-to-noise ratio of the emitted signal.
[0040]
40. TEST DEVICE according to claim 38, characterized by the fact that it still comprises an RFID communication circuit for receiving one or more signals from the test case (300), in which the one or more signals identify at least one type of test to be performed and whether the test kit (300) was previously used.
[0041]
41. TEST DEVICE according to claim 38, characterized in that the biosensor reagent (504, 506) is preloaded with coelenterazine and, in which any excess coelenterazine is removed from the biosensor reagent (504, 506) before of the reaction of the biosensor reagent (504, 506) with the sample (414) to be tested.
[0042]
42. TEST DEVICE according to claim 38, characterized in that it further comprises a disruptor for breaking up the individual cells of an infectious agent in the sample (414) before mixing the sample (414) with the biosensor reagent (504, 506).
[0043]
43. TEST DEVICE according to claim 42, characterized by the fact that the disruptor is at least an effective enzyme for releasing O antigens from the cell surface, an ultrasound effective for fragmenting cells, a French Press effective for fragmenting cells and an effective chemical treatment to release LPS from the cells of the infectious agent.
[0044]
44. TEST DEVICE according to claim 38, characterized in that the test device (100) is a portable test device (100).

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法律状态:
2019-08-13| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-07-28| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-11-17| 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 12/12/2012, 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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US201161570016P| true| 2011-12-13|2011-12-13|

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US61/570,016|2011-12-13|

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PCT/US2012/069192|WO2013090394A1|2011-12-13|2012-12-12|Device for rapid detection of infectious agents|

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