fluid test cassette

专利摘要:
the present invention relates to a disposable cassette for detecting nucleic acids or performing other assays. the cassette can be inserted into a base station during use. the cassette has several features to ensure correct operation of the device under gravity, such as ventilation pockets to allow sample fluid to flow from one chamber to the next when the ventilation bag is unsealed. ventilation pockets have protrusions to help prevent accidental leakage. the cassette may also have a gasket to ensure free air movement between open ventilation pockets. a flexible circuit with standardized metallic electrical components arranged in a thermostable material can be in direct contact with the fluid in the chambers and has resistant heating elements aligned with the ventilation bags and chambers. recesses in channels or cassette chambers may have structures, such as ribs or grooves to direct the flow of fluid to increase rehydration of lyophilized reagents disposed in the recess. flow diverters in the chambers can reduce the sample fluid flow rate and increase the effective length of the fluid flow path, allowing more precise control of the fluid flow in the case. the top of each chamber can have a projection that prevents the flow of capillary fluid through the top of the chamber, reducing or preventing the sequestering of resuspended reagent from the mass of the reaction solution volume.
公开号:BR112019020876A2
申请号:R112019020876
申请日:2018-04-20
公开日:2020-04-28
发明作者:J Thomas Donald；Cai Hong；B Cary Robert
申请人:Mesa Biotech Inc；
IPC主号:

专利说明:

Descriptive Report of the Invention Patent for FLUID TEST CASSETTE.
CROSS REFERENCE TO RELATED APPLICATIONS [001] This application claims priority for and the filing benefit of Provisional Patent Application 62 / 488,453, entitled “Fluidic Test Cassette”, filed on April 21, 2017, the descriptive report and claims for which are here incorporated by reference. BACKGROUND OF THE INVENTION
Invention field (Technical field):
[002] The embodiments of the present invention relate to an integrated device and related methods for detecting and identifying nucleic acids. The device may be fully disposable or may comprise a disposable portion and a reusable portion. Basics [003] Note that the following discussion may refer to various publications and references. The discussion of such publications is given for a more complete basis of scientific principles and should not be interpreted as an admission that such publications are prior art for purposes of determining patentability.
[004] As the impact on public health and awareness of infectious and emerging diseases, bireactive agents, genetic diseases and environmental reservoirs of pathogens increased, the need for more informative, sensitive and specific rapid tests at the point of use increased demand using tools based on polymerase chain reaction (PCR). Molecular testing based on nucleic acid by methods such as PCR-based amplification is extremely sensitive, specific and informative. Unfortunately, currently available nucleic acid tests are inadequate or of limited utility for use in the field because they require elaborate and expensive instrumentation, laboratory materials and
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2/103 specialized and / or multiple manipulations dependent on user intervention. Consequently, most samples for molecular testing are sent to centralized laboratories, resulting in long response times to obtain the necessary information.
[005] To address the need for rapid molecular testing at the point of use, previous efforts have focused on product designs that employ a disposable cartridge and a relatively expensive associated instrument. The use of external instrumentation to perform fluid movement, amplification temperature control and detection simplifies many of the engineering challenges inherent in integrating the multiple processes required for molecular testing. Unfortunately, the reliance on elaborate instrumentation imposes enormous economic barriers on small clinics, local and state governments and law enforcement agencies. In addition, reliance on a small number of instruments to perform tests could cause unnecessary delays during periods of greatest need, such as during a suspected release of a biological agent or an emerging epidemic. In fact, the reagent cartridge and disposable instrument model has a potentially significant bottleneck when an outbreak requires peak capacity and higher throughput. In addition, reliance on instrumentation complicates the ad hoc distribution of test devices to deployment sites where logistical constraints that prevent the transportation of bulky associated equipment or infrastructure requirements are absent (for example, reliable power sources).
[006] Gravity has been described as a means of fluid movement in existing microfluidic devices. However, the typical device does not allow programmable or electronic control of such fluid movement or the mixing of more than two fluids. In addition, some devices use a pressure drop generated by a flow
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3/103 of the inert or pre-packaged drop to induce a slight vacuum and extract reagents into processing chambers when oriented vertically, which increases the complexities of storage and transport to ensure the stability of pre-packaged fluids. Existing devices that teach the movement of a fluid in a plurality of different steps require frangible seals or valves between the chambers, which complicates operation and manufacture. These devices do not teach the use of separate and remotely located openings for each chamber.
[007] Typical microfluidic devices use lower reaction volumes than those used in standard laboratory procedures. PCR or other nucleic acid amplification reactions, such as circuit-mediated amplification (LAMP), nucleic acid-based sequence amplification (NASBA), and other thermal and isothermal cycling methods are typically conducted in test and research laboratories using volumes of reaction of 5 to 100 microliters. These reaction volumes accommodate sufficient test specimen volumes to ensure detection of scarce test targets in diluted specimens. Microfluidic systems that reduce reaction volumes compared to those used in traditional laboratory molecular tests also reduce the volume of samples that can be added to the reaction. The result of the lower reaction volume is a reduction in the ability to accommodate sufficient specimen volume to ensure the presence of detectable amounts of target in diluted specimens or where assay targets are scarce.
SUMMARY OF THE INVENTION [008] The present invention is a cassette for detecting a target nucleic acid, the cassette comprising a plurality of chambers, a plurality of ventilation pockets connected to the chambers, and a
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4/103 thermo-labile material to seal one or more of the ventilation pockets, in which at least one of the ventilation pockets comprises a protuberance. The protrusion preferably comprises a ripple or roughness and preferably sufficiently prevents the melted thermo-labile material from attaching to a thermostable material disposed adjacent to the thermo-labile material to prevent the ventilation bag from sealing after the heat-sensitive material is broken.
[009] The present invention is also a cassette for detecting a target nucleic acid, the cassette comprising a plurality of chambers, a plurality of ventilation pockets connected to the chambers, a heat-labile material to seal one or more of the ventilation pockets, a material thermostable and a joint disposed between the thermosetting material and the thermostable material, the joint comprising an opening comprising the plurality of ventilation pockets. The joint is preferably thick enough to provide a sufficient volume of air to balance the pressures and to ensure the movement of free air between the open ventilation pockets. The cassette preferably comprises a flexible circuit, the flexible circuit comprising standardized metallic electrical components, arranged on the thermostable material. The joint preferably comprises a second opening, or is limited in size, such that the flexible circuit will be in direct contact with fluid in at least one of the chambers. The electrical components preferably comprise resistive heating elements or conductive traces. The resistive heating elements are preferably aligned with the ventilation bags and chambers. The cassette preferably comprises one or more room temperature sensors to adjust a heating temperature, heating time and / or heating rate for one or more of the chambers.
[010] The present invention is also a cassette for detecting
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5/103 a target nucleic acid, the cassette comprising a vertically oriented detection chamber, a lateral flow detection strip arranged in the oriented detection chamber such that a sample receiving end of the detection strip is at the lower end of the strip detection space and a space in the detection chamber below the side flow detection strip for receiving fluid comprising amplified target nucleic acids, the space comprising sufficient capacity to accommodate an entire volume of the fluid at a height that allows the fluid to flow upward from the detection strip by capillary action without flooding or otherwise bypassing regions of the detection strip. The space preferably comprises detection particles, such as microspheres of dye polystyrene, latex, colloidal gold, colloidal cellulose, nano-gold or semiconductor nanocrystals. The detection particles preferably comprise oligonucleotides complementary to a sequence of the amplified target nucleic acids or Hgants, such as biotin, streptavidin, a hapten or an antibody, capable of binding to the amplified target nucleic acids. The detection particles were preferably dried, lyophilized or present on at least a portion of the inner surface as a dry mixture of detection particles in a carrier, such as a polysaccharide, a detergent or a protein, to facilitate the resuspension of the detection particles. A capillary assembly of the fluid is preferably formed in space, providing better mixing and dispersion of the detection particles to facilitate the irradiation of the detection particles with the amplified target nucleic acid. The cassette optionally performs a test with a volume of less than about 200 pL and preferably less than 60 pL. [011] The present invention is also a cassette for detecting a target nucleic acid, the cassette comprising one or more recesses to contain at least one lyophilized or dry reagent, at least
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6/103 in one of the recesses comprising one or more structures for directing fluids to facilitate the rehydration of at least one dry or lyophilized reagent, the recesses disposed in one or more detection chambers or one or more channels connected to the detection chambers. The structures preferably comprise ribs, grooves, undulations or combinations thereof.
[012] The present invention is also a cassette for detecting a target nucleic acid, the cassette comprising at least one chamber comprising a feature to prevent fluid entering vertically from the top of the chamber from flowing directly into an outlet from the chamber. The feature preferably diverts the fluid to the side of the chamber opposite the outlet. The resulting flow path of the fluid preferably comprises a horizontal component, thereby increasing the effective length of the flow path and sufficiently decreasing the flow rate of the fluid to restrict the amount of fluid leaving the outlet. The feature preferably creates a swirl of fluid within the chamber, thereby increasing the mixture of reagents within the fluid. The feature is preferably triangular or trapezoidal. The outlet is optionally tapered. A channel located downstream of the outlet optionally comprises loops to increase an effective channel length. The resource is preferably located near or at the bottom of the chamber or near the middle of the chamber.
[013] The present invention is also a method for controlling the vertical flow of a fluid through a chamber in a cassette to detect a target nucleic acid, the method comprising deflecting a flow of fluid entering the top of the chamber, thereby preventing the fluid to flow directly to an outlet of the chamber. The method preferably comprises reducing a fluid flow rate, thereby reducing the distance that the fluid flows through a channel
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7/103 connected to the outlet before the fluid stops. The method preferably comprises dividing a flow of fluid in the chamber into a first flow of fluid that contacts a wall of the chamber and is directed upwards and a second flow of fluid that enters the outlet. The first fluid flow preferably rotates in the chamber, thereby increasing the mixture of reagents within the fluid. The second fluid flow preferably forms a meniscus and travels through a channel connected to the outlet, the meniscus increasing the pressure in the closed air space in the channel downstream of the fluid until the pressure stops the fluid flow in the channel. The outlet is optionally conical, thus increasing the volume of compressible air at the outlet inlet. The method optionally comprises providing turns on a channel connected to the outlet, thereby increasing an effective travel length of the channel and reducing a fluid flow rate in the channel.
[014] The present invention is also a cassette for detecting nucleic acid, the cassette comprising at least one reaction chamber, in which, when the cassette is oriented vertically, a top of the reaction chamber comprises an entrance and a projection that extends downwards in the reaction chamber to minimize or prevent the flow of capillary fluid through said top of the reaction chamber. The projection is preferably generally triangular in shape. A first side of the projection preferably extends substantially vertically adjacent to the entrance. A second side of the projection preferably extends upwards towards the top of the reaction chamber at an angle less than approximately 60 degrees from the vertical, more preferably less than approximately 45 degrees from the vertical, even more preferably less than approximately 30 degrees from the vertical. vertically and optionally vertically. The cassette preferably comprises a recess to contain at least one lyophilized or dry reagent, the recess disposed in a channel
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8/103 connected to the reaction chamber input. The projection preferably reduces or prevents sequestration of the newly resuspended reagent from the mass of the reaction solution volume. The recess preferably comprises one or more structures for directing fluids to facilitate the rehydration of at least one dry or lyophilized reagent. The structures preferably comprise ribs, grooves, undulations or combinations thereof. Alternatively or additionally, the reaction chamber comprises a recess to contain at least one lyophilized or dry reagent.
[015] Objects, advantages and innovative features, and an additional scope of applicability of the present invention will be presented in part in the detailed description below, taken in conjunction with the attached drawings, and in part will become apparent to those skilled in the art upon examination of the following, or can be learned by practicing the invention. The objectives and advantages of the invention can be realized and achieved by means of the instruments and combinations particularly pointed out in the attached claims.
BRIEF DESCRIPTION OF THE DRAWINGS [016] The attached drawings, which are incorporated and form part of the specification, illustrate modalities of the present invention and, together with the description, serve to explain the principles of the invention. The drawings are only intended to illustrate certain embodiments of the invention and are not to be construed as limiting the invention. In the Figures:
[017] FIG. 1A is a drawing illustrating an embodiment of a test cassette of the present invention.
[018] FIG. 1B is an exploded view of a test cassette embodiment revealing the sliding seal, sample port, sample cup and the internal region of the expansion chamber.
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9/103 [019] FIG. 2A is a representation of the fluidic network in one embodiment of a test cassette of the invention.
[020] FIGS. 2B-2C are schematic representations before and after the vent opening, respectively, of how a heat-driven vent can be employed to vent into an expansion chamber to perform fluid flow control in the context of a hermetically sealed test cassette .
[021] FIG. 2D is a drawing of a disposable test cassette modality that shows the placement of the printed circuit assembly (PCA) comprising resistive heating elements and temperature sensors.
[022] FIG. 2E is a photograph of an injection molded plastic test cassette that includes the features described in FIG. 2A.
[023] FIG. 3A is a representation of the operating principle of an expansion chamber modality.
[024] FIG. 3B is a cross section of the piston-based expansion chamber before the gas expands into the test cassette.
[025] FIG. 3C is a cross section of the expansion chamber based on the piston after gas expansion inside the test cassette.
[026] FIG. 4A is an illustration of an approach to forming an expansion chamber, in which an expandable bladder is employed to provide an expanding internal volume.
[027] FIG. 4B is a cross section of the bladder-based expansion chamber before the gas expands into the test cassette.
[028] FIG. 4C is a cross section of the bladder-based expansion chamber after gas expansion into the test cassette.
[029] FIG. 5A is an illustration of an approach to forming an expansion chamber, in which an expandable bellows is employed
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10/103 to provide an expanding internal volume.
[030] FIG. 5B is a cross section of the bellows-based expansion chamber before the gas expands into the test cassette.
[031] FIG. 5C is a cross section of the bellows-based expansion chamber after the gas expands into the test cassette.
[032] FIG. 6A illustrates the use of a barrier, membrane or semipermeable material that allows gas to pass freely, while particles such as bacteria, viruses or large molecules, such as DNA or RNA, are retained within the device.
[033] FIG. 6B is a cross-section of the semipermeable barrier used in place of an expansion chamber to equalize internal pressures with environmental pressures or to reduce internal pressure.
[034] FIG. 7 is an exploded view of a test cassette design in which an expansion chamber is created by a spacer between a layer of biaxially oriented polystyrene film (BOPS).
[035] FIG. 8A is a design of a flexible circuit embodiment comprising resistive heating elements for two fluid chambers, a detection strip chamber and three ventilations and electrical contact pads.
[036] FIG. 8B is a flexible circuit modality comprising resistive heating elements for two fluid chambers, a detection strip chamber and three ventilations and electrical contact pads to energize the resistive heating elements.
[037] FIG. 8C is an exploded view of a test cassette modality.
[038] FIG. 8D is a view of the assembled test cassette of FIG.
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10/113
8C.
[039] FIG. 9 depicts lateral flow strips of devices with and without a capillary assembly at the receiving end of the strip. More uniform distribution of detection particles and more uniform signal across the strip is observed when a capillary assembly is present.
[040] FIG. 10 is a diagram that illustrates a hierarchical approach to splitting samples.
[041] FIG. 11 is an illustration of a multi-channel fluidic network for multiplexing and subdividing the sample showing the fluid flow path for each test. Additional fluid paths or channels can be incorporated into the network to further increase the number of parallel tests that can be performed simultaneously on a single disposable test cassette.
[042] FIG. 12 is a representation of the fluidic network in a modality of a test cassette of the invention, in which a sample is divided after the introduction of the sample cup through the sample port to allow parallel independent tests on the same inlet sample. A bifurcated fluid path from the sample cup allows the sample solution to be divided into two distinct fluid channels or test cassette paths to allow simultaneous tests to be performed in parallel on the divided sample.
[043] FIG. 13A is a drawing of an assembled sample preparation subsystem that shows the arrangement of internal components.
[044] FIG. 13B is an exploded view of the sample preparation subsystem, showing components of the nucleic acid purification apparatus configured for integration with a test cassette.
[045] FIG. 14 is a cross section through the subsystem
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12/103 sample preparation that illustrates the movements of the components that occur during the processing of a sample.
[046] FIG. 15 is an exploded view drawing showing the sample preparation subsystem with airtight sealing components, injection molded fluid subsystem, corresponding cassette holder and PCA.
[047] FIG. 16 is a photograph of a test cassette modality with an integrated sample preparation subsystem.
[048] FIG. 17A is an exploded view drawing showing a sample preparation subsystem with airtight sealing components and injection molded fluid subsystem design.
[049] FIG. 17B is a drawing of the test cassette modality with the integrated sample preparation subsystem presented in interface with the PCA.
[050] FIG. 17C is a cross-sectional drawing of the test cassette modality that represents fluid paths, electronic interface components and sample preparation.
[051] FIG. 18A is a drawing of an embodiment of a plug-in unit of the present invention shown with the lid in the open position and a test cassette inserted.
[052] FIG. 18B is a drawing of the plug-in unit shown with the lid in the closed position.
[053] FIG. 19 is a photograph of an embodiment of the plug-in unit shown with the lid in the open position and a test cassette inserted. The LCD screen indicates the detection of the insertion of an A / B influenza test cassette.
[054] FIG. 20 illustrates an embodiment of a cassette sealing mechanism of the present invention.
[055] FIG. 21A is a drawing of the placement of the cassette seal sensor inside the plug-in unit with an inserted cassette
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13/103 with the seal in the open position.
[056] FIG. 21B is a cross sectional view of the placement of the cassette seal sensor within the plug-in unit with a cassette inserted with the seal in the open position.
[057] FIG. 21C is a drawing of the placement of the cassette seal sensor inside the plug-in unit with a cassette inserted with the seal in the closed position.
[058] FIG. 21 D is a cross sectional view of the placement of the cassette seal sensor inside the plug-in unit with a cassette inserted with the seal in the closed position.
[059] FIG. 22 is a drawing of an embodiment of the cassette sealing mechanism, in which a drive gear is employed to mediate the seal closure using a rotary valve.
[060] FIG. 23A is a drawing illustrating an embodiment of the test cassette in which the lid is a hinged lid comprising an a-ring seal and an empty air volume that serves as an expansion chamber. In this drawing the cover is in the open position.
[061] FIG. 23B is a drawing showing the lid in the closed position, where the ring forms a hermetic seal with the sample door frame.
[062] FIG. 24A is an exploded view of the heating plate components and the test cassette holder of the plug-in unit that forms the test cassette receiver subset.
[063] FIG. 24B is a drawing of an embodiment of the test unit receiver subassembly of the plug-in unit.
[064] FIG. 25 is a sliding view of the test cassette holder and the heating plate mounting system in the engaged and disengaged positions.
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14/103 [065] FIG. 26 is a drawing depicting the placement of infrared temperature sensors in an embodiment of the plug-in unit to monitor the temperature of the first and second heated fluid chambers.
[066] FIG. 27A is a drawing showing the placement of the optical sensor within a modality of the plug-in unit to allow the reading of a bar code located near the bottom of the test cassette.
[067] FIG. 27B is a detail of FIG. 27A.
[068] FIGS. 28A and 28B are exploded drawings and assembled, respectively, of a dual heat plate configuration in which the test cassette is placed between two heating plate assemblies.
[069] FIGS. 29A and 29B are solid and transparent designs, respectively, of an embodiment of the plug-in unit in which a pivoting door is used to receive a test cassette. Closing the pivoting door places the back of the test cassette in contact with the heating plate mounted inside the plug-in unit.
[070] FIGS. 30A and 30B are front and side section views, respectively, of the internal components of a plug-in unit comprising servo motors to drive the sample preparation and an optical system for collecting test results.
[071] FIGS. 31A and 31B are photographs of the front and side view of an optical subsystem for a modality of the plug-in unit that incorporates a test reader.
[072] FIG. 32A and 32B are photographs of an embodiment of the plug-in unit with a hinged test cassette door in the open position and in the closed position, respectively.
[073] FIG. 33 shows a reusable subset for a unit
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15/103 snap fit of the present invention.
[074] FIG. 34 shows test results obtained in Example 1 described here.
[075] FIG. 35 shows the test results obtained in Example 2 described here.
[076] FIG. 36 shows test results obtained in Example 3 described here.
[077] FIG. 37A is a perspective view of a cassette comprising three chambers.
[078] FIG. 37B is an exploded view of the cassette of FIG. 37A.
[079] FIG. 38 is a transparent view of the cassette of FIG. 37A showing fluidic resources.
[080] FIG. 39 shows an embodiment of a chamber of the present invention comprising a triangular protruding flow feature and a conical outlet.
[081] FIG. 40 shows an embodiment of a chamber of the present invention comprising a triangular protruding flow feature and a parallel outlet.
[082] FIG. 41 shows an embodiment of a chamber of the present invention comprising a trapezoidal protruding flow feature and a parallel outlet.
[083] FIG. 42 shows an embodiment of a chamber of the present invention comprising stacked triangular flow features and a parallel outlet.
[084] FIG. 43 shows an embodiment of a chamber of the present invention comprising a flow feature protruding approximately in the middle of the chamber.
[085] FIG. 44 shows a reagent recess comprising internal features to direct fluid flow.
[086] FIG. 45 shows a ventilation bag modality
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16/103 of the present invention comprising a corrugated structure.
[087] FIG. 46A shows a drawing of a fluidic layer embodiment of a cassette of the present invention comprising recesses of Hofilized reagents disposed in the fluid flow paths. FIG. 46B is an enlarged view of a recess and reaction chamber, showing a vertical projection that extends into the chamber.
[088] FIG. 47A shows a drawing of a fluidic layer embodiment of a cassette of the present invention comprising recesses of Hofilized reagents arranged in one or more reaction chambers.
[089] FIG. 47B is an enlarged view of a recess in a reaction chamber, showing a vertical projection that extends into the chamber.
[090] FIG. 48 shows a reaction chamber without a vertical projection.
DETAILED DESCRIPTION OF THE INVENTION [091] One embodiment of the present invention is a sealable disposable platform for detecting a target nucleic acid, the disposable platform preferably comprising a sample chamber for receiving a sample comprising the target nucleic acid, an amplification chamber connected via a first channel to the sample chamber and connected through a second channel to a first ventilation bag, a marking chamber connected through a third channel to the amplification chamber and connected through a fourth channel to a second ventilation bag, a detection subsystem connected to the marking chamber via a fifth channel and connected via a sixth channel to a third ventilation bag, a plurality of resistive heating elements and one or more temperature measurement devices, in
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17/103 that the ventilation bags are each sealed from communicating with an air chamber by a thermolabile material in a suitable form, such as a membrane, a film or a plastic sheet located in the vicinity of one or more of the elements resistive heating elements. The disposable platform optionally comprises a seal to seal the platform before the detection test begins. The disposable platform preferably comprises recesses along the channels between the chambers to accommodate the incorporation of dry or lyophilized reagents on the sliding platform. These recesses may optionally comprise structures on one or more of the surfaces facing the reagent (s) to help direct fluids, preferably using capillary or surface tension effects, to the included dry reagents to facilitate rehydration of the dry reagents . Such features may comprise grooves, such as the summit 7001 of FIG. 44, grooves, corrugations or other structures to direct fluids into the internal space of the recess as the fluid passes through the recess or otherwise assist in the flow of fluid into the internal space of the recess during fluid flow. Alternatively, a recess can be located directly within one (or more) of the chambers.
[092] The disposable platform optionally further comprises a sample preparation step comprising an outlet in direct fluid connection with an inlet of the sample chamber. The dimensions of a substantially flat surface of the amplification chamber are preferably approximately the same as the dimensions of a substantially flat surface of a resistive heating element in thermal contact with the amplification chamber. The amplification chamber optionally contains an amplification solution and a recess in the channel of the sample chamber for the amplification chamber optionally comprises a mixture
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18/103 lyophilized amplification reagent and preferably there is a recess in the channel of the amplification chamber for the marking chamber comprising dry or lyophilized detection particles. The amplification and marking chambers are preferably heated using resistive heating elements. The detection subsystem preferably comprises a side flow strip comprising detection particles. The chambers, channels and ventilation bags are preferably located in a fluid mounting layer, and the electronic elements of the device are preferably located in a separate layer comprising a printed circuit board, the separate layer connected to the fluid mounting layer. or placed in contact with the fluid mounting layer by a plug-in unit. The detection subsystem is preferably located in the fluid mounting layer or, optionally, in a second fluid mounting layer. The volume of at least one of the chambers is preferably between approximately 1 microliter and approximately 150 microliters. The disposable platform preferably further comprises a connector for fitting the disposable platform with a docking unit, which preferably keeps the disposable platform in a vertical or tilted orientation and optionally provides electrical contacts, components and / or a power supply.
[093] One embodiment of the present invention is a method for detecting one or more target nucleic acids, the method preferably comprising distributing a sample comprising the target nucleic acid in a sample chamber of a disposable platform; orient the disposable platform vertically or on an incline; open a first ventilation bag connected to an amplification chamber to a closed volume of air, thus allowing the sample to flow into the amplification chamber, to react the sample with
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19/103 a mixture of previously lyophilized amplification reagent located in a recess of the channel between the sample chamber and the amplification chamber, amplifying the target nucleic acid in the amplification chamber, open a second ventilation bag connected to a marking chamber an enclosed volume of air, thus allowing the amplified target nucleic acid to flow into the marking chamber, labeling the amplified target nucleic acid using detection particles in a recess in the channel between the amplification chamber and the marking chamber, opening a third ventilation bag connected to a detection subsystem to a closed air volume, thus allowing the marked target nucleic acid to flow into the detection subsystem and detect the amplified target nucleic acid. The amplification step preferably comprises amplifying target nucleic acid using a resistive heating element located within the disposable platform in the vicinity of the amplification chamber. Preferably, the method further comprises passively cooling the amplification chamber. The method preferably further comprises heating the marking chamber during the marking step using a resistive heating element located within the disposable platform in the vicinity of the marking chamber. The method preferably also comprises controlling the operation of the disposable platform, using a plug-in unit that is not an external instrument.
[094] Modalities of the present invention comprise a disposable platform that integrates independent means of an external instrument to conduct all necessary steps of a nucleic acid molecular assay and complement rapid immunolateral current assays with a new generation of nucleic acid tests offering analyzes more informative and sensitive. The modalities of the present invention facilitate the broader use of rapid acid tests
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20/103 of the nucleic in small clinics and austere or remote environments, where infectious diseases, biotreatment agents, agriculture and environmental tests are the most likely to have the greatest impact. Certain modalities of the present invention are completely self-contained and disposable, which allows for “surge capacity” in times of increased demand, allowing parallel tests to be carried out without constraints imposed by external instruments. In addition, in areas of application where a low-cost disposable cartridge coupled to a cheap battery-powered plug-in unit or powered by an AC adapter is preferable, a modality of the invention where a simple plug-in unit is used further reduces testing costs , placing reusable components on a reusable, but inexpensive basis. The platform technology disclosed here offers sensitivity similar to methods based on laboratory nucleic acid amplification, minimum user intervention and training requirements, sequence specificity transmitted by amplification and detection, multiplex capability, stable reagents, low cost manufacturing compatibility in large scale, operation with battery or solar energy to allow use in austere configurations and a flexible platform technology that allows the incorporation of additional or alternative biomarkers without the redesign of the device.
[095] The modalities of the present invention provide systems and methods for detection and identification at the point of use, of low cost of nucleic acids suitable for carrying out analyzes in remote locations from a laboratory environment in which the test would normally be performed. Advantageously, the nucleic acid amplification reaction volumes can be in the same volume range commonly used in traditional laboratory tests (for example, 5-150 pL). The reaction conducted in the modalities of this
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21/103 The invention is thus directly comparable to acceptable laboratory tests, and allows the accommodation of the same volumes of specimens typically used in traditional molecular tests. In addition, the amplification of nucleic acids preferably takes place in a hermetically sealed test cassette which is preferably permanently sealed before the start of amplification. The retention of amplified nucleic acids within a sealed system prevents contamination of the test environment and surrounding areas with amplification products and therefore reduces the likelihood of subsequent tests generating false positive results. The integration of a sealing system in the test cassette allows the use of a corresponding sealing engagement system in the fitting unit to impose the formation of a seal at the start of the test. In one embodiment of the invention, a rack and pinion mechanism is employed to slide an integrated sealing mechanism from the test cassette to ensure the seal closes before amplification. A sensor placed on the plug unit interrogates the test cassette to confirm that the seal was formed before starting the test reaction.
[096] The modalities of the present invention can be produced using injection molding and ultrasonic welding processes to achieve high-throughput manufacturing and low-cost disposable components. In some embodiments, one or more recesses are provided in the fluid component to each accommodate a dry reagent pellet. The recesses allow the use of lyophilized or otherwise dry materials to be present in the fluid component during final assembly, when ultrasonic welding can be used without interrupting the pellet by any energy introduced into the system during welding.
[097] The modalities of the present invention can be used
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22/103 to detect the presence of a target nucleic acid sequence or sequences in a sample. Target sequences can be DNA, such as chromosomal DNA or extra-chromosomal DNA (for example, mitochondrial DNA, chloroplast DNA, plasmid DNA, etc.) or RNA (for example, rRNA, mRNA, small RNAs or viral RNA). Similarly, the modalities of the invention can be used to identify nucleic acid polymorphisms, including single nucleotide polymorphisms, deletions, insertions, inversions and sequence duplications. In addition, modalities of the invention can be used to detect gene regulatory events, such as overregulation of the gene and overregulation of the gene at the transcription level. Thus, the modalities of the invention can be used for applications such as: 1) the detection and identification of pathogenic nucleic acids in agricultural, clinical, food, environmental and veterinary samples; 2) detection of genetic biomarkers of the disease; and 3) the diagnosis of the presence of a disease or a metabolic state by detecting relevant biomarkers of the disease or metabolic state, such as gene regulation events (mRNA over-regulation or over-regulation or the induction of small RNAs or other molecules of nucleic acid generated or suppressed during a disease or metabolic state) that occur in response to the presence of a pathogen, toxin, other etiologic agent, environmental stimulus or metabolic state.
[098] The embodiments of the present invention comprise a means of preparing, amplifying and detecting a target nucleic acid sample after adding a nucleic acid sample, comprising all aspects of fluid control, temperature control and reagent mixing. In some embodiments of the invention, the device provides a means of conducting nucleic acid tests using a portable power source, such as a battery, and is
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23/103 fully disposable. In other embodiments of the invention, a disposable nucleic acid test cartridge works in conjunction with a simple reusable electronic component that can perform all laboratory instrumentation functions, such as an external instrument typically required for nucleic acid testing without requiring use such laboratory instrumentation or external instrument.
[099] The embodiments of the present invention provide a nucleic acid amplification and detection device comprising, but not limited to, a housing, a circuit board and a fluidic or microfluidic component. In certain embodiments, the circuit board may contain a variety of surface-mount components, such as resistors, thermistors, light-emitting diodes (LEDs), photodiodes and microcontrollers. In certain embodiments, the circuit board may comprise a flexible circuit board comprising a thermostable substrate, such as polyimide. Flexible circuits may, in some embodiments, comprise copper or other coatings or conductive layers deposited or bonded to the thermostable substrate. These coatings can be engraved or standardized to understand the resistive heating elements used for biochemical reaction temperature control and / or conductive traces to accommodate such heaters and / or surface mount components, such as resistors, thermistors, light emitting diodes light (LEDs), photodiodes and microcontrollers. The fluidic or microfluidic component is the portion of the device that receives, contains and moves aqueous samples and can be made from a variety of plastics and by a variety of manufacturing techniques, including ultrasonic welding, bonding, fusing or laminating, laser cutting, water jet cutting and / or injection molding. The fluidic and circuit board components are held together
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24/103 reversibly or irreversibly, and its thermal coupling can be improved by heat-conducting materials or compounds. The housing preferably serves in part as a cosmetic and protective sheath, hiding the delicate components of microfluidic layers and circuit boards, and can also serve to facilitate sample entry, buffer release, nucleic acid elution, sealing and the beginning of the processes necessary for the functionality of the device. For example, the housing may incorporate a sample inlet port, a mechanical system for forming or engaging a seal, a button or similar mechanical feature to enable user activation, release of the buffer, initiation of sample flow, elution of nucleic acids and formation of thermal or other interface between electronic components and fluidic components.
[0100] In some embodiments of the invention, the fluidic or microfluidic component comprises a series of chambers in controlled fluid communication, in which the chambers are optionally controlled by temperature, thus subjecting the fluid contained therein to programmable temperature regimes. In some embodiments of the invention, the fluidic or microfluidic component comprises five chambers, preferably including an expansion chamber, a sample inlet chamber, a reverse transcription chamber, an amplification chamber and a detection chamber. The sample inlet chamber preferably comprises a conduit for the expansion chamber, a sample inlet hole where a sample containing nucleic acid can be added, a first recess in which dry materials can be placed during manufacture to mix with the inlet sample, an outlet conduit that leads to a second recess, in which dry materials can be placed during manufacture and a conduit that leads to the
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25/103 reverse transcription chamber. In other modalities, functions of two or more chambers are consolidated into a single chamber, allowing the use of fewer chambers.
[0001] The first and second recesses may also comprise lyophilized reagents which may include, for example, suitable buffers, salt, deoxyribonucleotides, ribonucleotides, oligonucleotide primers and enzymes such as DNA polymerase and reverse transcriptase. Such lyophilized reagents are preferably solubilized after the nucleic acid sample enters the recess. In some embodiments of the invention, the first recess comprises salts, chemicals and buffers useful for the lysis of biological agents and / or the stabilization of nucleic acids present in the incoming sample. In some embodiments of the invention, the inlet sample is heated in the sample inlet chamber to lyse cells or viruses present in the sample. In some embodiments of the invention, the second recess comprises lyophilized reagents and enzymes, such as reverse transcriptase useful for the synthesis of RNA cDNA. In an embodiment of the invention, the second recess is sufficiently isolated from the sample inlet chamber to allow the materials within the second recess to maintain a temperature lower than the temperature of the sample inlet chamber during heating. In some embodiments of the invention, the reverse transcription chamber comprises a conduit comprising a third recess comprising lyophilized reagents for the amplification of nucleic acids. The sample inlet chamber, the reverse transcription chamber, the amplification chamber and the detection chamber are preferably located in the register and in sufficient proximity to the heating elements on the heater circuit board to provide thermal conduction when mounted on the heater plate. directly or by inserting the fluidic component
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26/103 or microfluidic or cassette in a plug-in unit. Likewise, the electronic components present on the heater circuit board are preferably placed in physical contact or close to ventilation pockets in the fluidic component to allow electronic control by opening the ventilation. The physical layout of the heater circuit board is designed to provide registration with elements of the fluidic or microfluidic component so that the resistive heating elements of the heater circuit board for lysis, reverse transcription, amplification, hybridization and / or flow control fluid elements are situated to form a thermal interface with elements of the fluidic component with which they interact.
[0002] In some embodiments of the invention, the fluidic or microfluidic component preferably comprises five chambers, including a sample entry chamber, a lysis chamber, a reverse transcription chamber, an amplification chamber and a detection and recess chamber for dry or lyophilized reagents located along the channels between each chamber. In this embodiment, reverse transcription of RNA to cDNA and amplification of cDNA occurs in separate chambers. In this embodiment, a first recess, located along the conduit leading from the sample inlet cup to the lysis chamber, comprises salts, chemicals (eg, dithiothreitol) and buffers (for example, to stabilize, increase or decrease the pH ) useful for the lysis of biological agents and / or the stabilization of nucleic acids present in the input sample. In some embodiments of the invention, the inlet sample is heated in the heat lysis chamber, having first fluid from the sample inlet cup through the first recess, in which the sample optionally mixed with the substances comprising the first recess. In other embodiments of the invention, lysis is carried out by means of chemical treatment resulting from mixing the sample
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27/103 with chemicals in the first recess and incubating the sample in the presence of these chemicals in the lysis chamber.
[0003] After substantial completion of treatment in the lysis chamber, the sample solution is released via electronic control of a heater that mechanically breaks through an opening to allow the sample solution to flow through a channel through a second recess and in the reverse transcription chamber. The second recess may optionally comprise lyophilized reagents which may include suitable buffers, salt, deoxyribonucleotides, ribonucleotides, oligonucleotide primers and enzymes such as DNA polymerase and / or reverse transcriptase necessary to perform the RNA reverse transcription in the cDNA sample. After substantial completion of a reverse transcription reaction, a second vent is opened to release the sample solution to flow through a third channel and recess composed of reagents necessary for nucleic acid amplification, such as lyophilized reagents that may include suitable buffers , salt, deoxyribonucleotides, ribonucleotides, oligonucleotide primers and enzymes such as DNA polymerase and in an amplification chamber.
[0004] After substantial completion of the amplification of the nucleic acid in the amplification chamber, a third vent is opened to release the sample solution into a channel leading to the detection chamber. Said channel can optionally, but preferably comprise a fourth recess comprising dry or lyophilized detection reagents, such as chemicals and / or detection particle conjugates useful for the detection of nucleic acids in the detection chamber. The detection chamber preferably comprises a set of capillaries, reagents for the detection of the amplified nucleic acid and a lateral flow detection strip. The capillary set preferably provides a space of capacity
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28/103 enough to accommodate the entire volume of fluid in the detection chamber at a height that allows fluid to flow over the detection strip by capillary action without flooding or otherwise bypassing the regions of the detection strip designed to receive the fluid for the correct capillary migration on top of the detection strip. In some embodiments of the invention, the detection reagents are lyophilized reagents. In some embodiments of the invention, detection reagents comprise microspheres of dyed polystyrene, colloidal gold, semiconductor nanocrystals or cellulose nanoparticles. The sample solution comes with the detection reagents in the detection chamber and flows by capillary action on the detection strip. Micro heaters registered with the detection chamber can optionally be used to control the temperature of the solution as it migrates upwards on the detection strip.
[0005] In some embodiments of the invention, the amplification reaction is an asymmetric amplification reaction in which one primer from each pair of primers in the reaction is present in a different concentration than the other primer from a given pair. Asymmetric reactions can be useful for the generation of single-stranded nucleic acid to facilitate detection by hybridization. Asymmetric reactions can also be useful to generate amplicons in a linear amplification reaction allowing quantitative or semi-quantitative analysis of target levels in a sample.
[0006] Other embodiments of the invention comprise a reverse nucleic acid transcription, amplification and detection device that is integrated with a sample preparation device. The modalities including the sample preparation device provides a means for the communication of fluids between the ports or outlet valves of the sample preparation subsystem and the port or ports of the fluidic or microfluidic components of the
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29/103 device.
[0007] Other embodiments of the invention comprise a means of dividing the incoming sample into two or more fluid paths in the fluidic or microfluidic component. A means of dividing the inlet sample comprises a branched conduit for transporting inlet fluids to a volume measuring chamber designed to divide the fluid through multiple fluid paths. Each measuring chamber comprises a channel conduit for a ventilation bag and a channel conduit for the next chamber in the fluid path, for example a lysis chamber or a reverse transcription chamber or an amplification chamber.
[0008] Unless otherwise defined, all terms of the technique, notations and other scientific terminologies used in this document have the meaning commonly understood by those skilled in the art to which this invention belongs. The techniques and procedures described or referenced here are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely used molecular cloning methodologies described in Sambrook et aL, Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Current Protocols in Molecular Biology (Ausbel et al, eds., John Wiley & Sons, Inc. 2001. As appropriate, procedures involving the use of kits and Commercially available reagents are generally carried out according to the protocols and / or parameters defined by the manufacturer, unless otherwise specified.
[0009] As used throughout the specification and claims, the terms' target nucleic acid 'or' model nucleic acid means a fragment or sequence of DNA or RNA single or double strand that is to be detected.
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30/103 [0010] As used throughout the specification and the claims, the terms "microparticle" or "detection particle" mean any compound used to label the nucleic acid product generated during an amplification reaction, including fluorescent dyes specific for duplex nucleic acid, fluorescence-modified oligonucleotides and quantum dots conjugated to oligonucleotide or solid phase elements such as polystyrene, latex, cellulose or paramagnetic particles or microspheres.
[0011] As used throughout the specification and the claims, the term "chamber" means a fluidic compartment where the fluid resides for some period of time. For example, a chamber can be the sample chamber, the amplification chamber, the marking chamber, or the detection chamber.
[0012] As used throughout the specification and the claims, the term "cassette" is defined as a disposable or consumable cassette, housing, component or cartridge used in the performance of a test or other chemical or biochemical analysis. A cassette can be for single or multiple use.
[0013] As used throughout the specification and the claims, the term "pouch" means a compartment that serves as a ventilation mechanism. A pouch is preferably adjacent to or superimposed on a resistor or other mechanism for opening the pouch. For example, unlike fluid chambers as described above, a pocket created in the fluid component of the cassette may have an open face that aligns with a resistance in the PCA. This open face is preferably covered with a thin membrane, film or other material to create a sealed cavity that is easily broken by energizing the underlying resistor.
[0014] As used throughout the specification and the claims, the term 'channel' means a narrow conduit within the
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31/103 fluidic set that typically connects two or more chambers and / or bags or combinations thereof, including, for example, an inlet, outlet or ventilation channel. In the case of an inlet or outlet channel, the fluid sample migrates through the channel. In the case of a ventilation channel, the duct preferably remains free of fluid and connects a fluid chamber to a ventilation bag.
[0015] As used throughout the specification and the claims, the term "external instrument" means a reusable instrument that has one or more of the following characteristics: performs a mechanical action on a disposable test or cassette other than sealing the cassette, including but not limited to piercing buffer and / or pumping packages or otherwise actively providing a transport force for fluids, comprises moving parts to control valves and other components for controlling fluid flow in the cassette or disposable assay, controls flow of fluid other than by selective heating of the assay, or requires periodic calibration.
[0016] As used throughout the specification and the claims, the term "plug-in unit" means a reusable device that controls the tests but does not have any of the characteristics listed above for external instruments.
[0017] The modalities of the present invention are devices for nucleic acid tests at the point of use and of low cost, suitable for carrying out analyzes in remote locations in a laboratory environment, where the tests would normally be performed. Certain devices comprise fluidic and electronic components or layers, optionally encapsulated by a protective housing. In embodiments of the present invention, the fluidic component is composed of plastic and comprises a series of chambers and pouches connected by narrow channels in which the chambers are oriented vertically
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32/103 in relation to each other during the operation. The fluidic component is superimposed or placed in physical contact with electronic components, preferably controlled by a microcontroller, such as a printed circuit board containing surface mount devices (SMDs) and / or a flexible circuit comprising conductive material etched to form resistive elements of heating and optionally containing SMDs. In some embodiments of the device, the entire set is disposable. In other modalities, fluid and physically connected electronic layers are disposable, while a small, low-cost control unit is reusable. In another embodiment, the fluidic component is disposable, and a small control fitting unit or fitting unit is reusable. For all embodiments, the present invention can be integrated with a nucleic acid sample preparation device, as described in International Publication WO 2009/137059 A1, entitled “Highly Simplified Lateral Flow-Based Nucleic Acid Sample Preparation and Passive Fluid Flow Control ”(incorporated herein by reference) and / or methods of use described herein.
[0018] Modalities of the present invention comprise an integrated nucleic acid testing device that can be manufactured inexpensively with established manufacturing processes. The invention provides molecular test data while maintaining simplicity from the end-user perspective of widely accepted hand-held immunoassays, overcoming the challenges of regulating fluid temperatures within the device, transporting small sample volumes in sequential steps, adding reagents, mixture of reagents, detecting nucleic acids. In some embodiments of the invention, subsystems for collecting, interpreting, reporting and / or transmitting test results are incorporated into the invention. The modalities of the present invention are adapted exclusively for
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33/103 ra use ready-to-use electronic elements that can be built using standard assembly techniques and require little or no moving parts. In addition, the fluid layer design allows the use of readily available plastics and manufacturing techniques. The result is a cheap, disposable and reliable device capable of isolating, amplifying and detecting nucleic acids without the need for a dedicated laboratory infrastructure.
[0019] Existing nucleic acid testing devices generally use sophisticated heating elements, such as deposited film heaters and Peltier devices that add significant cost and / or require specialized manufacturing methods. In embodiments of the invention, the heating of the reaction solution is preferably carried out using simple, surface-mount resistant devices that can be purchased for a few cents and are assembled and tested by common manufacturing standards. By layers of fluid chambers over these resistive elements and associated sensor elements, the temperature of the fluid of the reaction solutions can be conveniently regulated. The widespread use of SMD resistors and flexible circuits in the electronics industry ensures that the present invention is amenable to well-established quality control methods. In other embodiments of the invention, resistive heating is carried out using heating elements formed by patterns manufactured in the conductive layer of a flexible circuit substrate. Many nucleic acid amplification techniques, such as PCR, require not only rapid heating of the reaction solution, but also rapid cooling. The reaction chambers in the present invention are preferably heated on one side and the ambient temperature on the opposite side is used to help reduce the temperature of the fluid. In addition, the vertical orientation of the device modalities allows faster cooling by
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34/103 passive convection than if the device were horizontally oriented, thus reducing the thermal cycle period without using expensive devices, such as Peltier devices. In some embodiments of the invention, a fan is used to facilitate cooling.
[0020] Fluid control is another challenge associated with low cost nucleic acid testing device designs. Devices known in the art generally employ electromechanical, electrokinetic or piezoelectric pumping mechanisms to manipulate fluids during operation of the device. These pumping elements increase the complexity and cost of the device. Likewise, valves that use elaborate micromechanical designs or moving parts can increase manufacturing costs and reduce reliability due to complications, such as failure of moving parts or bio-encrustation. Unlike the nucleic acid testing devices previously described, the embodiments of the present invention use hydrostatic pressure under microcontroller control together with capillary forces and surface tension to manipulate fluid volumes. The vertical orientation of some embodiments of the present invention allows the reaction solution to cascade from chamber to chamber under control microcontroller to accommodate the necessary manipulations of the assay. The fluid can be maintained in individual reaction chambers through a balance of channel size, hydrostatic pressure and surface tension, where surface tension and hydrostatic pressure prohibit the advance of the fluid by displacing the gas. A sample advances to the lower chamber, preferably only after activating a simple ventilation mechanism under the control of the microcontroller. Once opened, ventilation allows the fluid to move from a first chamber to a second chamber, providing a path for the displaced air
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35/103 escape from the second chamber as the fluid enters. Each chamber (or each channel between the chambers) within the fluidic component preferably connects to a ventilation bag sealed through a narrow ventilation channel. The ventilation bag is preferably sealed on one side with a thin thermolabile membrane or plastic sheet, and which is easily broken by heating a small underlying surface mounting resistor, close to or adjacent to the membrane or sheet. Once the ventilation of a lower chamber is opened, the flow of the fluid continues, even under low hydrostatic pressures.
[0021] As described more specifically below, the fluidic or microfluidic ventilation mechanism used in some embodiments of the present invention preferably uses a heating element in physical (optional) and thermal contact with a thermal seal to allow electronic control of fluid movement by venting a lower elevation chamber to allow fluid from a higher elevation chamber to flow into the lower chamber. In one embodiment, a resistor is mounted on a printed circuit board, using widely used and well-established electronic manufacturing methods, and placed in physical contact with a channel seal comprising a thermolabile material. When energized, the surface mount resistor generates sufficient heat to break the seal, which results in venting the chamber to allow pressure balance in the region or chamber where the fluid is being moved with the region or chamber where the fluid resides before ventilation. The pressure balance between the chambers allows fluid to move from a higher elevation chamber to a lower elevation chamber. A direct seal between the highest and lowest lift chambers is preferably not employed. The ventilation channel and seal may be located
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36/103 of them remotely from the fluid chambers, thus facilitating the layout of the fluid device in efficient configurations for manufacturing. The sealing material can comprise any material that can seal the ventilation duct and be broken off from heating as described, for example, a thin plastic sheet. This approach to controlling the movement of fluids in the device benefits from low material costs, suitability for manufacturing using established manufacturing techniques, providing the ability to move fluids through a series of chambers under the control of electronic control circuits, such as microprocessors or microcontrollers. The use of vents, a heat-labile material to seal vents (and not to seal fluid chambers or fluid microchannels) and an electronic means of breaking the seal with heat provides a means of controlling the flow of fluid through the device to allow the movement of fluid at predetermined times or after the completion of specific events (for example, reaching a temperature, a temperature change or a series of temperature changes, or the completion of an incubation time or times or other events). In some embodiments, a blockage can be introduced in the channel between the chambers, when the water in the gas phase must be isolated from a chamber connected by said channel. The block can be a soluble material that dissolves in contact with liquid water after the ventilation opening or a readily fused material, such as paraffin, which can be removed by introducing heat at the block site.
[0022] In addition, the ventilation approach has several advantages over the sealing of the fluid chambers themselves. Ventilation bags can be located anywhere in the fluid layout and simply communicate with the chamber that they regulate through a ventilation channel. From a manufacturing point of view,
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37/103 ventilation can be located so that only a single sealing membrane for all ventilation bags (which may include a ventilation bag collector) is attached to the fluidic component, preferably by well-established methods, such as adhesives, laminating by heat, ultrasonic welding, laser welding, etc. In contrast, directly sealing a fluid chamber requires that the sealing material be placed in different locations corresponding to each chamber location, which is more difficult to manufacture. This represents a more challenging scenario during manufacturing compared to a single vent bag collector sealed by a single membrane. In addition, if the chambers are sealed directly, the molten sealing material can remain in the channels between the chambers, occluding the flow. The viscosity of the sealing material may require more pressure in the fluid column than is achieved in a miniaturized gravity driven device.
[0023] In embodiments of the present invention, mixing reagents does not require more complexity than other systems. Re-agents necessary for nucleic acid amplification, such as buffers, salts, deoxyribonucleotides, oligonucleotide primers and enzymes are preferably incorporated stably by the use of lyophilized pellets or pies. These freeze-dried reagents, sealed in a fluid chamber, a recess in a fluid chamber or a recess in a channel, can be readily solubilized by contact with aqueous solution. In the event that additional mixing is required, the vertical orientation of the modalities of the present invention offers opportunities for new methods of mixing solutions. Using heaters underlying the fluid chambers, the gas can be heated, supplying bubbles to the reaction solution in the above chamber, when the solution contains thermally sensitive components. Alternatively, heaters can be used to heat
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38/103 a solution directly to the point where boiling occurs, if the solution does not contain thermally sensitive components. The occurrence of air bubbles is often undesirable in previously disclosed fluidic and microfluidic devices, as they can accumulate in fluid chambers and channels and displace reaction solutions or prevent fluid movement within the device. The vertical design of the modalities of the invention presented here allows the bubbles to rise to the surface of the fluid, resulting in only minimal and transient displacement of the fluid, effectively improving any disadvantageous impacts of the bubbles on the fluidic or microfluidic system. Boiling mixing is also convenient with this vertical design, as the fluid displaced during the process simply returns to the original fluid chamber by gravity after the heating elements are turned off.
[0024] In embodiments of the invention, a colorimetric detection strip is used to detect amplified nucleic acids. Lateral flow assays are commonly used in immunoassay tests due to ease of use, reliability and low cost. The prior art contains descriptions of the use of side flow strips for the detection of nucleic acids using porous materials such as a sample receiving zone that is located at or near the marking zone also comprised of a porous material and placed at or near one end of the lateral flow tester. In these earlier inventions, the marking portions are in the marking zone. The use of porous materials such as the sample receiving zone and the marking zone results in the retention of some sample solutions, as well as detection particles in the porous materials. Although the marking zones comprising porous materials having reversibly immobilized portions necessary for detection can be used in
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39/103 invention, the embodiments of the present invention preferably use particles or detection portions maintained in a region of the device distinct from the sample receiving zone of the lateral flow strip and comprising non-porous materials with low fluid retention characteristics. This approach allows samples containing nucleic acid to be labeled prior to introduction to the porous components of the sample receiving end of the side flow component of the device and thus eliminates the retention and / or loss of sample material and detection particles in a marking zone. porous. This method also allows the use of several sample treatments in the presence of detection portions, such as high temperature treatment, to perform denaturation of a double-stranded target or secondary structures within a single-stranded target without concern for the temperature impacts on the porous materials from the sample receiving or marking zone or side flow detection strip materials. In addition, the use of a side flow non-contact marking zone with the sample receiving zone, but subject to the control of fluidic components, allows the target and the label to remain in contact for periods of time controlled by control systems fluid flow. Thus, modalities of the present invention may be different from traditional lateral flow test strips, in which the times and conditions of interaction with the sample and the detection of particles are determined by the capillary transport properties of the materials. By incorporating the detection particles in a temperature-regulated chamber, it is possible to denature the duplex nucleic acid, allowing efficient detection based on hybridization. In alternative modalities, fluorescence is used to detect the amplification of nucleic acids using a combination of LEDs, photodiodes and optical filters. These optical detection systems can be used to perform detection
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40/103 tion and quantification of nucleic acid in real time during amplification and detection of the end point after amplification.
[0025] The embodiments of the invention comprise a low cost point-of-use system, in which a nucleic acid sample can be selectively amplified and detected. Other modalities include integration with a nucleic acid sample preparation device, such as that described in International Publication WO 2009/137059 A1, entitled “Highly Simplified Lateral Flow-Based Nucleic Acid Sample Preparation and Passive Fluid Flow Control”. One embodiment of the device preferably comprises a plastic fluidic component and a printed circuit (PCA) and / or flexible circuit assembly, and is optionally enclosed in a housing that protects the active components. The temperature regulation, and the mixture of fluids and reagents are preferably coordinated by a microcontroller. The reaction cassette is preferably oriented and executed vertically so that gravity, hydrostatic pressure, capillary forces and surface tension, together with the openings activated by the microcontroller, control the movement of the fluid inside the device.
[0026] In embodiments of the present invention, the prepared or raw sample fluid enters a sample port and partially fills or fills a sample cup. The sample can be retained, for varying periods of time, in the sample cup where the dry or lyophilized reagents can mix with the sample. Reagents such as positive control reagents, control models or chemical reagents are beneficial for carrying out the test and can be introduced into the sample solution by inclusion in dry, liquid or lyophilized form in the sample cup. Other treatments, such as incubations at controlled temperature or heat lysis of bacterial or viral analytes, can optionally be performed in the sample cup
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41/103 tra, which means an underlying micro heater and temperature sensor system interconnected to temperature control electronics. A fluid network comprises the sample port through which the sample is inserted into the cassette manually by the user or through an automated system, for example, a subsystem integral to the plug-in unit or a sample processing subsystem; the sample cup in which the sample is held to facilitate accumulation during sample introduction and to add reagents, components to perform the necessary treatments before further movement of the sample in the downstream portions of the fluid network (eg heat treatment to perform the lysis of a bacterial cell or virus); a recirculating ventilation passageway for the balance of air, gas or solution fluidic channels and / or chambers pressure with the pressure of the cassette expansion chamber; a granule recess, in which a reagent granule (for example, a granule or pellet of material, reagents, chemicals, biological agents, proteins, enzymes or other substances or mixtures of these substances) in a dry / dried or lyophilized state or semi-dry can be rehydrated by the sample solution or a buffer solution introduced into the cassette before adding the sample to rehydrate the granule or granule contained therein and thus mix the materials into the sample solution; a set of one or more openings that can be opened to control the movement of the fluid within the cassette; a first chamber where the sample can be subjected to a temperature regime; an optional barrier within the fluidic channel that connects the first chamber to a second chamber to prevent premature invasion of liquids and / or gases in the second chamber or to temporarily control the movement of the solution or gases in the second chamber; a second chamber, in which the sample solution can be subjected to additional temperature regimes op ~
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42/103 optionally after adding reagents from an optional recess of the reagent granule optionally located between the first and the second chamber; a recess of the test strip that forms a chamber in which a test strip is mounted to detect an analyte or a reporter molecule or other substance Indicating the presence of an analyte. In some embodiments, the cassette is inserted into a plug-in unit that performs the functions of sealing the cassette, eluting, detecting and transmitting data. Preferably, no user intervention is required when the cassette is inserted into the docking unit, the sample is loaded and the lid is closed.
[0027] With reference to the representative drawings of cassette 2500 in FIGS. 1-2, a sample of nucleic acid is added to the sample cup 10 in the fluid component 5 through the sample port 20. The sliding seal 91 is moved to the closed position by closing the cover of the plug-in unit at the start of the test. Cover 25 keeps slide 91 in place to seal expansion chamber 52. The nucleic acid sample can be derived from an online process (i.e., integrated nucleic acid preparation subsystem), a nucleic acid preparation process separated (as one of the many commercially available methods, for example spin columns), followed by the addition of the purified nucleic acid to the device by pipette or a sample containing unprocessed nucleic acid. Already present in the sample cup, or preferably in the recess 13 inside or adjacent to the sample cup, is the mixture of reagents 16, which can be in liquid or dry form, containing components useful to facilitate the lysis of cells and viruses and / or stabilize the released nucleic acids. For example, dithiothreitol and / or pH buffering reagents can be used to stabilize nucleic acids and inhibit RNases. Similarly, reagents can be used to perform acid-mediated lysis or
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43/103 base. In some embodiments, the reagent mixture is lyophilized to form lyophilized reagents. In some embodiments, a positive control, such as a virus, bacteria or nucleic acid, is present in the reagent mixture. Introducing the sample into the sample cup causes reagents and samples to mix in such a way that the reagents act on the sample. An optional bubble mixing step to further mix the sample with the reagents or resuspend the reagents, can be optionally performed. The fluid is then optionally heated in the sample cup 10 to lyse virus cells and particles. The fluid is then preferably directed through channel 40 to a first chamber 30 which resides below the sample cup when the device is in vertical orientation. The reagent recess 15 is preferably located along the inlet channel, so that the fluid passes through the recess to mix with dry or lyophilized reagents contained therein before entering the first chamber 30. In the modalities in which the first chamber is a reverse transcription chamber, preferably present in the reagent recess 15 all components are required for a reverse transcription reaction, such as buffering reagents, dNTPs, oligonucleotide and / or enzyme primers (for example, reverse transcriptase) in form lyophilized. The reverse transcription chamber is preferably in contact with the heating elements to provide a means for regulating the temperature necessary to support the reverse transcription of RNA into cDNA. Channel 35 connects chamber 30 to reagent recess 37. After synthesis of cDNA in chamber 30 _s the vent 50 is opened to allow the reverse transcription reaction to flow through channel 35 into the reagent recess 37. The dry reagents or lyophilisates show the recess of reagent 37 mixed with the fluid that passes through the recess to the second chamber 90 through the inlet
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44/103 such that the reagents act on the sample in the second chamber, which is preferably an amplification chamber. Preferably present in the reagent recess 37 are all the components necessary for the amplification reaction, such as buffering agents, salts, dNTPs, rNTPs, oligonucleotide primers and / or enzymes. In some embodiments, the reagent mixture is lyophilized to form lyophilized reagents. To facilitate multiplexed tests, in which multiple amplicons are generated, multiplexed amplification can be obtained by depositing multiple sets of primers within the amplification chamber (s) or preferably within the reagent recesses upstream of the (s) said amplification chamber (s). In addition, circuit board and fluidic designs, in which multiple amplification and detection chambers are incorporated into the device, support multiple parallel amplification reactions, which can be single or multiplex plex reactions. This approach reduces or eliminates the complications known to those skilled in the art that result from multiplexed amplification using multiple pairs of primers in the same reaction. In addition, the use of multiple amplification reaction chambers allows simultaneous amplification under different temperature regimes to accommodate requirements for optimal amplification, such as different melting or annealing temperatures required for different target and / or primers. [0028] After nucleic acid amplification, the vent bag 150 is opened to allow the product of the amplification reaction to flow through channel 135 to chamber 230. Detection strip 235 located in chamber 230 allows detection of acids target nucleicles marked by detection particles located in a region of the detection strip 235 or optionally in the capillary assembly 93.
[0029] The movement of the fluid from the sample cup 10 to the first chamber 30 occurs because the chamber 30 is ventilated to the chamber
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45/103 expansion channel 52 through opening 51. The movement of fluids from the first chamber to the second chamber of the device is preferably carried out by opening a vent connected to the second chamber. When the fluid enters the first chamber 30, the ventilation bag 50, connected to the downstream chamber, is sealed and, therefore, the fluid does not pass through channel 35 that connects the two chambers. Referring now to FIG. 2A, the movement of fluid from chamber 30 to chamber 90 can be performed by allowing the air inside chamber 90 to communicate with the air in expansion chamber 52 by breaking a seal over the ventilation bag 50. Breaking the seal in the bag vent 50 allows air communication in chamber 90 through ventilation channel 60 with air in expansion chamber 52, which is connected through opening 51 to ventilation bag 54. The seal on ventilation bag 54 is preferably open, or it was previously broken, as shown in FIG. 2B. As shown in FIG. 2C, the rupture of the ventilation bag seal 50 allows the ventilation bag 50 (and therefore chamber 90) to communicate with the ventilation bag 54 (and therefore the expansion chamber 52). This method of fluid movement is preferably incorporated within a hermetically sealed space to contain biohazardous samples and amplified nucleic acids within the test cassette. To allow a hermetically sealed cassette, the selectively heat-resistant and thermolabile materials are layered in the manner shown schematically in the cross section of FIGS. 2B-2C. Referring now to Figs. 2B-2C, the heat source 70, which preferably comprises a resistor, on the printed circuit board or PCA 75 is placed in the register with ventilation pockets 50, 54 and in close proximity as the sealing material of the thermo labile ventilation bag 80. The ventilation bag seal may comprise a thermolabile material, such as polyolefin or polystyrene. a
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46/103 thermostable material (such as polyimide) 72 is preferably disposed between the heat source 70 and the thermo labile ventilation bag sealing material 80 to form an airtight barrier. In some embodiments, the sealed space 55 between or around the ventilation pockets is increased by the inclusion of an optional gasket or spacer 56 comprising an adhesive layer that joins thermostable material 72 with the thermo-labile material 80 and / or fluidic component 5 and maintains a seal tightness of the test cassette in the ventilation region after one or more of the ventilation openings are opened, while preferably also providing an air space for air communication between the open ventilation and / or the optional expansion chamber. In this embodiment, the heat is transferred from the heat source 70 through the thermally stable material 72 and the sealed space 55 to the thermally labile ventilation bag sealing material 80 _{s by} breaking and opening the ventilation bag 50. A microcontroller is preferably responsible by sending electric current to the heat source 70. The ventilation bag 50 opens preferentially to a closed space, such that the gas inside the test cassette can remain sealed in relation to the environment outside the test cassette. The enclosed space can comprise air within the test cassette, optionally including an open air chamber to allow for gas expansion, such as an expansion chamber. As shown in FIG. 2C, the opening of the ventilation bag 50 results in the communication of gases in the ventilated fluid chamber with the gas of the expansion chamber, since the ventilation bag 54 was previously broken by the heat source 71 _s and the ventilation bag 54 is in gaseous communication with the expansion chamber. The resulting reduced pressure in the ventilated fluid chamber allows the fluid to flow by gravity into the ventilated chamber from a chamber located above. Other modalities of the ventilation bag can with
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47/103 attach seals that are not a thermosensitive membrane and can use other methods to break the seals, such as drilling, tearing or dissolving. A photograph of such a cassette is shown in FIG. 2E.
[0030] The face opposite the open face of the ventilation bag can optionally comprise a ripple, protuberance, roughness or other similar structure, such as the ripple 7004 of FIG. 45, to facilitate the formation of an opening during the rupture of the ventilation sealing material. Such a structure preferably also avoids resealing the ventilation after the seal breaks. This can occur in embodiments comprising a circuit board with surface-mounted components. In such modalities, the surface mount resistors can stretch the polyimide film, pushing it towards the opening of the joint and against the thermolabile material. Once the seal breaks, the molten seal material can form a secondary seal with that polyimide, thereby closing off ventilation. In modalities with a flexible circuit comprising metallic traces that form heating elements, the heater can cause the flexible polymide circuit to deform locally, often forming a protrusion (often comprising the heating material) that extends inwards opening in the joint, possibly blocking the ventilation opening due to the molten seal material. The 7004 ripple can help prevent these occurrences.
[0031] The sealed space 55 optionally provides a conduit for other ventilation, ventilation bags or chambers (such as expansion chamber 52). After opening the ventilation, the fluid component 5 remains sealed from the external environment 59. The expansion chamber 52 preferably accommodates the expansion of the gas during heating, buffering the volume of air / water vapor, providing
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48/103 a volume large enough that the expansion of the gas due to temperature changes does not significantly affect the pressure of the system or accommodating the expansion of the gas by displacing a piston (FIG. 3), a flexible bladder (FIG. 4) , a bellows (FIG. 5) or a hydrophobic barrier that allows gas, but not macromolecules, to pass freely through the barrier (FIG. 6). In FIG. 3 the expansion chamber makes use of a piston which is displaced by the increase in pressure within the sealed fluidic system. The expansion chamber serves to reduce or eliminate pressure build-up within the sealed system. The displacement of the piston occurs in response to pressure build-up inside an airtight test cassette, reducing the internal pressure inside the test cassette resulting from processes such as gas expansion during heating. In FIG. 4, deflection of the bladder occurs in response to increased pressure within a hermetically sealed test cassette. The displacement of the bladder reduces the internal pressure inside the test cassette resulting from processes such as gas expansion during heating. In FIG. 5, the stretching of the bellows occurs in response to increased pressure within a hermetically sealed test cassette. Stretching the bellows reduces the internal pressure inside the test cassette resulting from processes such as gas expansion during heating.
[0032] The expansion chambers can be incorporated as an empty air volume, like the included volume shown in the expansion chamber 52 at the top of the test cassette illustrated in FIG.
1. As illustrated in FIG. 7, to facilitate the manufacture of a cassette of minimum thickness, the expansion chambers can also be incorporated in the air space 440 formed by a properly designed joint 420 when sealed to the thermolabile material 410 and thermostable material 430 to form the support of the fluidic component
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49/103
400. Minimizing the physical dimensions of the test cassette is desirable to reduce shipping costs, reduce thermal mass and provide an aesthetically pleasing and convenient design. In addition to forming air volume for gas expansion, the gasket 420 generates a space between the thermostable material 430 and the thermo-labile material 410 to facilitate the free circulation of air through open ventilations, maintaining a sealed system to avoid exposure to the environment . Gasket 420 is preferably thick enough to provide sufficient air clearance to balance pressures between open vents, but is also thin enough not to substantially impact the interface between the heaters and the corresponding ventilation pockets or the cassette seal by thermostable material. In the embodiments of the present invention comprising a flexible circuit, the flexible circuit may comprise a thermostable material, such as polyimide, in which case the sheet separated from the thermostable material 430 is not necessary, for example, as shown in FIG. 8C. The use of an expansion chamber to reduce or balance the pressure within the sealed test cassette ensures that pressure imbalances do not result in unfavorable or premature solution movements within the test cassette and that the accumulation of pressure does not adversely affect the movement of the desired fluid, such as movement between chambers or through channels. This pressure control, that is, the establishment of designated pressure distributions throughout the device, allows the system to function as designed, regardless of atmospheric pressure. The expansion chamber, therefore, allows controlled fluid movements that depend on the stable pressure within the system to be employed, and also allows the use of a hermetically sealed test cassette, thus avoiding the disadvantages of venting the test cassette into the atmosphere. , for example, the potential release of am
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50/103 plicon into the atmosphere. In addition, the method of allowing fluid flow by reducing pressure downstream of a fluid, such as opening a vent for the expansion chamber, eliminates the need for pumps, such as those that create positive pressure upstream of the fluid, or others devices with moving parts. Similar advantages are possible when venting an area downstream of a fluid to a relatively larger reservoir (such as the expansion chamber) at substantially the same pressure as the area downstream, thus allowing the fluid to flow under the force of gravity (provided the device is in the proper orientation). The size of the expansion chamber is preferably large enough to accommodate the reaction vapors produced during the test without increasing the system pressure to a point where it exceeds the capillary or gravitational force required for the fluid to flow.
[0033] In the modalities in which the second chamber is an amplification chamber, the chamber is preferably in contact with heating elements to provide a means for regulating the temperature necessary to support the amplification of the nucleic acid. In some embodiments of the invention, the amplification chamber may contain oligonucleotides on at least a portion of the inner surface. At the interface between the wall 95 of the chamber 30 and one or more heating elements 100, as illustrated in FIG. 2D, it may be advantageous to place a thermally conductive material, such as a thermal grease or compound. A microcontroller preferably modulates the current in the resistive heating element (s), preferably by means of metal oxide semiconductor field effect transistors (MOSFETs), based on data collected from the temperature sensor 110 in PCA 75, using simple on / off or proportional integral derivative (PID) temperature control or other algorithmic temperature control known to those see
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51/103 used in the technique.
[0034] The placement of the heating elements and, in some modalities, the corresponding temperature sensor (s) in the disposable component allows the manufacture of highly reproducible thermal couplings between the temperature control subsystem and the amplification chambers and detection with which they interact. This approach allows a highly reliable means of coupling the fluidic subsystem to the electronic thermal control subsystem, forming the thermally conductive interface during manufacture. The resulting upper thermal contact between the electronic temperature control components and the fluid subsystem results in rapid temperature balance and therefore rapid testing. The use of a flexible circuit to provide disposable resistive heating elements that are fused to the rear of the fluid component directly or with an intermediate joint, allows a low-cost means of obtaining excellent thermal contact, rapid temperature cycles and manufacturing reproducibility. Resistive heating elements for reverse transcription, amplification and control of fluid flow ventilation can be formed directly on the flexible circuit by recording the conductive layer of the flexible circuit to form geometries that exhibit the necessary resistance. This approach eliminates the need for additional electronic components and simplifies manufacturing, reducing costs.
[0035] In an embodiment of the present invention, flexible circuit 799 for resistive heating and ventilation opening is shown in FIG. 8. The use of a flexible heater as a component of the disposable cassette allows the cassette holder to be configured to allow the fluid in the heated fluid chambers to make direct contact with the material comprising the flexible heating circuit. For example, as shown in FIG. 8C. windows 806
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52/103 in thermally labile material 807 (which preferably comprises BOPS) that forms the rear of the cassette can be located on the fluid chambers to allow direct contact of fluid with the flexible circuit 799. Direct contact between the circuit layer flexible and the fluid to be controlled by temperature in the flexible circuit provides a low thermal mass system capable of rapid temperature changes. To allow the collection of temperature data for use in temperature regulation, a temperature sensor can be optionally incorporated into the flexible circuit, and / or a non-contact means of temperature monitoring, such as an infrared sensor, can be employed. Resistive heating elements, such as heating element 800, in a flexible circuit can be used to break ventilation when they are located in a register with a ventilation bag. The electrical pads §12 supply current to the heating elements 800. Likewise, the flexible circuit or circuits may comprise resistive heating elements 802 and §03 to heat the fluid chambers and optional resistive heating element 804 to regulate the temperature of the detection strip.
[0036] In this embodiment, the flexible circuit 799 also preferably serves as a thermostable seal to maintain a hermetically sealed cassette, similar to the thermostable material 72 described above. Optionally, an additional thermostable layer (for example, comprising polyimide) can be placed between flexible circuit 799 and housing or rear panel 805. A spacer or gasket 808 is preferably placed around the ventilation resistors 800 between the material thermolabile 807 and flexible circuit 799 to ensure free movement of air through open vents while maintaining a sealed cassette. The rear housing or the 80S panel preferably comprises thin plastic and is preferably
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53/103 placed on the exposed surface of the flexible circuit to protect it during handling. The rear compartment or panel 805 can comprise windows over the heating elements in the 799 circuit to facilitate cooling and temperature monitoring. Electrical contact with the plug-in unit control electronics (described below) can optionally be provided by a set of 810 electrical pads, preferably comprising an edge connector or connector pins, such as spring loaded pins.
[0037] The test cassette chamber modalities preferably comprise materials capable of withstanding repeated heating and cooling temperatures in the range of approximately 30 ° C to approximately 110 ° C. Even more preferably, the chambers comprise materials capable of withstanding repeated heating and cooling temperatures in the range of approximately 30 ° C to approximately 110 ° C at a rate of temperature change in the range of approximately 10 ° C to approximately 50 ° C per second . The chambers are preferably capable of maintaining solutions in them at temperatures suitable for heat-mediated lysis and biochemical reactions, such as reverse transcription protocols, thermal cycling or isothermal amplification, preferably controlled by microcontroller programming. In some nucleic acid amplification applications, it is desirable to provide an initial incubation at an elevated temperature, for example a temperature between approximately 37 ° C and approximately 110 ° C for a period of 1 second to 5 minutes, to denature the target nucleic acid and / or activate a hot start polymerase. Subsequently, the reaction solution is maintained at the amplification temperature in the amplification chamber for isothermal amplification or, for amplification based on thermocycling, it is varied in temperature between at least two temperatures including, but not limited to, a
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54/103 temperature that results in denaturing nucleic acid duplex and a temperature suitable for annealing the primer by hybridization to the target and extension of the primer through polymerization by nucleic acid catalyzed by the polymerase. The duration of incubations at each temperature required in a thermal cycling regime can vary with the composition of the target nucleic acid sequence and the composition of the reaction mixture, but is preferably between approximately 0.1 second and approximately 20 seconds. Repeated heating and cooling is typically performed for approximately 20 cycles to approximately 50 cycles. In modalities involving isothermal amplification methods, the temperature of the reaction solution is maintained at a constant temperature (in some cases after an initial incubation at an elevated temperature) for approximately 3 minutes and approximately 90 minutes, depending on the amplification technique used. Once the amplification reaction is complete, the amplification reaction solution is transported, opening the ventilation that is in communication with a chamber below the chamber used for amplification, to the lower chamber to perform additional manipulations of the amplified nucleic acids. In some embodiments of the invention, manipulations comprise denaturation of the amplified nucleic acids and hybridization with detection oligonucleotides conjugated to detection particles. In some embodiments of the invention, the amplified nucleic acids are hybridized to detect oligonucleotides conjugated to detection particles and to capture probes immobilized on a detection strip.
[0038] In some embodiments, additional biochemical reactions can be performed in the amplification chamber before, during or after the amplification reaction. Such processes may include, but are not limited to, reverse transcription in which the RNA is transcribed in
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55/103 cDNA, multiplexing in which multiple pairs of primers simultaneously amplify multiple target nucleic acids and real-time amplification in which amplification products are detected during the amplification reaction process. In the case of the latter, the amplification chamber may not contain a valve or outlet channel, and the amplification chamber should preferably comprise an optical window or otherwise configured to allow interrogation of amplicon concentration during the amplification reaction process. . In a real-time amplification mode, fluorescence-labeled oligonucleotides complementary to the target nucleic acid or fluorescent dyes specific to the duplex DNA are monitored for fluorescence intensity using an excitation light source, such as LEDs or diode laser ( s) and a detector, such as a photodiode, and appropriate optical components including, but not limited to, optical filters.
Detection [0039] Modalities of the detection chamber 230 preferably provide the specific labeling of amplified target nucleic acids generated in the amplification chamber. As shown in FIG. 2A, the detection chamber 230 preferably comprises a capillary assembly or space 93 and a detection strip 235. Detection particles comprising dye polystyrene microspheres, latex, colloidal gold, colloidal cellulose, nano-gold or semiconductor nanocrystals are preferably present in the capillary set 93. Said detection particles may comprise oligonucleotides complementary to the target analyte or comprise ligands capable of binding to the amplified target nucleic acid, such as biotin, streptavidin, hapten or antibody directed against a marker, such as a hapten present in acids amplified target nucleic acids. The detection chamber 230 may contain detection particles that are dried,
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56/103 lyophilized or present on at least a portion of the interior surface as a dry mixture of detection particles in a carrier, such as a polysaccharide, detergent, protein or other compound known to those skilled in the art to facilitate the resuspension of the detection particles. In some embodiments, the side flow detection strip may comprise detection particles. In other embodiments, a reagent recess channel 135 leading to the detection chamber can comprise detection particles. The detection chamber may be capable of being heated and / or cooled. Suitable detection particles include, but are not limited to, fluorescent dyes specific for duplex nucleic acid, fluorescence-modified oligonucleotides or stained microparticles conjugated to oligonucleotides or colloidal gold or colloidal cellulose. The detection of the amplicon involves a detection oligonucleotide 'or another' detection probe 'that is complementary or capable of specifically binding to the amplicon to be detected. The conjugation of a detection oligonucleotide to a microparticle can occur by the use of particles coated with streptavidin and biotinylated oligonucleotides, or by the carbodiimide chemistry, in which the carboxylated particles are activated in the presence of carbodiimide and react specifically with primary amines present in the detection oligonucleotide. The conjugation of the detection oligonucleotide with the detectable portion can occur internally or at the 5 'end or at the 3' end. The detection oligonucleotides can be attached directly to the microparticle, or more preferably through a spacer portion, such as ethylene glycol or polynucleotides. In some embodiments of the invention, the detection particles can be linked to multiple species of amplified nucleic acids resulting from processes such as multipixed amplification. In these modalities, the specific detection of each species of
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57/103 amplified nucleic acid can be performed by detection on the detection strip using a specific method for each species to be detected. In such an embodiment, a tag introduced into the target nucleic acids during amplification can be used to tag all of the amplified species present, while subsequent hybridization of the nucleic acids tagged with species-specific capture probes immobilized on the detection strip is employed to determine which specific species of amplified DNA are present.
[0041] In the case of a duplex DNA amplification product, heating the reaction solution after introduction into the detection chamber can facilitate detection. Fusion of the duplex DNA or denaturation of the secondary structure of the single-stranded DNA and cooling in the presence of the detection oligonucleotide results in the specific targeting of the amplified target nucleic acid sequence. The heating element underlying the detection chamber can be used to heat the fluid volume for approximately 1 to approximately 120 seconds to initiate the fusion or denaturation of the duplex DNA of the secondary single-stranded DNA structure. As the solution is allowed to cool to room temperature, the amplified target nucleic acid can hybridize specifically with detection microparticles. The reaction volume is then directed preferentially to a region of the detection chamber below the marking chamber opening the ventilation of the detection chamber.
[0042] For efficient marking to occur, the solubilized detection particles are preferably well mixed with the reaction solution. In the embodiments of the invention, the detection particles can be located in the capillary assembly 93 at the outlet of channel 135 to facilitate mixing with the solution as it enters the chamber 230. The detection particles in the capillary assembly 93 can op
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58/103 to be lyophilized detection particles. The capillary assembly provides improved mixing and dispersion of particles to facilitate the fusion of the detection particles with the nucleic acids to which the detection particles bind. The capillary assembly also increases the uniformity of particle migration in the detection strip, as shown in FIG. 9. A capillary set is especially advantageous for low volume tests, such as those less than 200 μΙ, or more specifically less than about 100 μί, or even more particularly less than about 60 μΙ, or even more particularly about 40 μΙ in volume.
[0043] Modalities of the detection chamber of the present invention provide specific detection of amplified target nucleic acids. In certain embodiments of the invention, the detection is carried out by capillarization of a solution containing marked amplicon through an absorbent strip composed of a porous material (such as cellulose, nitrocellulose, polyethersulfone, polyvinylidine fluoride, nylon, charge-modified nylon or polytetrafluoroethylene) standardized with lines, dots, microarrays or other visually discernible elements comprising a binding portion capable of specifically binding to the labeled amplicon, either directly or indirectly. In some embodiments, the absorbent strip component of the device comprises up to three porous substrates in physical contact: a surfactant pad comprising antipathic reagents to enhance absorption, a detection zone comprising a porous material (such as cellulose, nitrocellulose, polyethersulfone, fluoride polyvinylidine, nylon, charge-modified nylon or polytetrafluoroethylene) to which at least a binding portion capable of selectively binding the labeled amplicon is immobilized and / or an absorbent pad to provide additional absorbent capacity. Although the detection particles can optionally be incorporated into the mate
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59/103 porous lateral flow sensors in the detection chamber, unlike the previously described lateral flow detection devices, the detection particles are preferably kept upstream in a capillary assembly where they substantially intensified the formation of binding complexes between the amplicon and particle detection can be conducted before or concurrently with the introduction of the resulting labeled nucleic acids into the porous components of the device.
[0044] A 'capture oligonucleotide' or 'capture probe' is preferably immobilized on the detection strip element of the device by any of the various means known to those skilled in the art, such as UV irradiation. The capture probe is designed to capture the marked nucleic acid as a solution containing the marked nucleic acid crosses the capture zone, resulting in an increase in the concentration of the marker at the location of the capture probe immobilization, thus producing a detectable signal indicative of the presence of the target nucleic acid target amplifier (s). A single detection strip can be standardized with one or multiple capture probes to allow multiplexed detection of multiple amplicons, determination of amplicon sequence, quantification of an amplicon by the extension of the detection signal linearity and assay quality control (positive and negative controls).
Fluidic component [0045] Modalities of the fluidic component preferably comprise plastic, such as acrylic, polycarbonate, PETG, polystyrene, polyester, polypropylene and / or other similar materials. These materials are readily available and can be manufactured using standard methods. Fluidic components comprise both chambers and channels. Fluid chambers comprise walls, two faces and connect to one or more channels, such as an entrance, a
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60/103 exit, recess or ventilation. The channels can connect two fluid chambers or a fluid chamber and a recess, and comprise walls and two faces. The fluid chamber design preferably maximizes the surface area for volume ratio to facilitate heating and cooling. The internal volume of a chamber is preferably between approximately 1 pL and approximately 200 pL. The area on one side of the chamber in contact with the solution preferably corresponds to the area to which the heating elements are connected to ensure a uniform temperature of the fluid during heating. The shape of the fluid chambers can be selected to couple with heating elements and to provide favorable geometries for entering and leaving the solution. In some embodiments, the volume of the chamber may be greater than the volume of fluid, in order to provide a space for bubbles that appear during the course of operation of the device. The fluid chambers can have extended extensions that lead to ventilation channels, to ensure that the fluid does not interfere with the channel by capillary action or block the ventilation mechanism.
[0046] In some modalities, it may be desirable to reduce or eliminate the invasion of liquid or gaseous water in a chamber before the moment of the solution release. The high temperatures used in the processes of some modalities generate vapors (for example, water in gaseous phase) that can result in the premature invasion of humidity in a channel, chamber or recess. Reduction of invasion of the liquid phase or gas phase may be desirable to retain, for example, the dry state of dry reagents or lyophilized reagents present in a chamber or recess. In some embodiments, the channels can be temporarily blocked, totally or partially, with a material that can be removed by external forces, such as heat, humidity and / or pressure. Suitable materials for blocking
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61/103 temporary channels include, but are limited to, latex, cellulose, polystyrene, hot glue, paraffin, waxes and oils.
[0047] In some embodiments, the test cassette comprises a fluid component preferably injection molded comprising sample cup, chambers, channels, ventilation bags and energy directors. The fluidic component of the injection molded test cassette is preferably made of a plastic suitable for ultrasonic welding to a support plastic of similar composition. In an embodiment of the invention, the fluid component of the test cassette comprises a single injection molded part which is ultrasonically welded to a support material. Energy directors are optional features of the fluid component that direct ultrasonic energy only to areas of the thermolabile layer that are intended to join the fluid component. The injection molded fluidic component can optionally be housed in a housing. FIG. 7 illustrates a cassette preferably comprising an injection molded fluidic component 400 (preferably comprising a polymer, such as high impact polystyrene (HIPS), polyethylene, polypropylene or NAS 30, a styrene acrylic copolymer), a thermolabile material 410 (comprising, for example, BOPS, which has a relatively low melting temperature of about 239 ° C and a glass transition temperature of about 100 ° C, which is sufficient to withstand high temperatures during denaturation, or polycarbonate, melting temperature of 265 ° C and glass transition temperature of 150 ° C), adhesive spacer 420 (comprising, for example, a silicone transfer adhesive that preferably does not incorporate a carrier, an acrylic adhesive with a carrier of polyester or any adhesive that can withstand the high temperatures of the device) and the heat-resistant layer 430. The thermo-labile material 410 breaks through the
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62/103 heat which is preferably transmitted through the superimposed thermo-resistant layer 430 (comprising, for example, polyimide or other polymer with high heat resistance). The fusion of the thermo labile material over the ventilation resources of the cassette opens the ventilation channel and the ventilation for the expansion chamber, thus allowing pressure equalization within the cassette. The overlapping thermo-resistant layer 430 preferably remains intact, thus allowing the cassette to maintain an airtight seal after opening the vent.
[0048] In some embodiments, the adhesive spacer comprises a vacant 440 region that can serve as an expansion chamber to buffer the expansion of gases during heating to reduce the internal pressure of a sealed cassette. The thermolabile layer 410 is bonded to fluid component 400 by a bonding method or process, such as ultrasonic welding or the use of adhesive. The resulting part is then connected to a spacer and a heat-resistant layer. In some embodiments, the heat-resistant layer is constructed in such a way that it is not present in heated chambers. In other embodiments, the heat-resistant layer is present on the heating chambers. In still other modalities, the adhesive spacer and the thermo-resistant layers are present only in a region registered with the resources of the fluid component ventilation bag. In this modality, a thermoresistant layer can optionally be placed directly on the thermolabile material in the regions registered with the heated chambers.
[0049] In some embodiments of the invention, the thickness of the fluid chambers and the channel walls are in the range of approximately 0.025 mm to approximately 1 mm and, preferably, in the range of approximately 0.1 mm to approximately 0.5 mm. It is
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63/103 thickness preferably meets the structural integrity requirements of the fluid component and to withstand the closed chamber seal under high temperatures and associated pressures. The thickness of the channel walls, particularly the ventilation channel walls, is preferably less than that of the chambers and in the range of approximately 0.025 mm to approximately 0.25 mm. The width of the input and output channels is preferably chosen to enhance capillarity. A shallow ventilation channel provides improved rigidity to the fluid component, with no adverse effect on ventilation. The plastic forming faces of the fluidic component are preferably thinner than those forming the walls, in order to maximize heat transfer. Optional thermal breaks cut through some components of the fluidic component and involve the amplification and detection chambers, contributing to the thermal insulation of temperature-controlled chambers.
[0050] In some embodiments of the invention, before fluid component 400 is bonded to the thermolabile support material 410 additional components of the test cassette, such as lyophilized reagents 16, detection strip assembly 230, and detection particles can be incorporated. In some embodiments, components can be laminated by applying pressure to ensure good adhesion. In some embodiments, the components can be bonded by a combination of methods, such as pressure sensitive adhesives and ultrasonic welding. Known adhesives or those that negatively impact the performance of nucleic acid amplification reactions should be avoided. Acrylic or silicone based adhesives have been used successfully in the invention. A preferred adhesive film is S17876 provided by Advanced Adhesives Research. Other adhesives can be used if they are considered chemically compatible with used plugs, plastics and chemicals
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64/103 of reaction, while providing a robust seal on the temperatures encountered during the operation of the device.
[0051] Referring to FIG. 2 and 7, the ventilation bags are preferably differentiated from other chambers in their construction. After the construction of the fluidic component, as described above, the ventilation bags have an open face on the side of the fluidic component that will meet the PCA 75, directly or in some modalities, indirectly, through an intermediate air gap 420 or the ventilation bag 54 and heat-resistant material 430. To form the ventilation bag, an additional plastic component is attached to seal the chamber, preferably comprising a thin, thermolabile membrane 410 adjacent to the ventilation resistor 70 of the PCA. Film 410 comprises a material suitable for ultrasonic welding to the injection molded fluidic component, such as polystyrene, although other similar materials can be used. This film is suitable to seal the ventilation bag and allow easy drilling and thus discharge to a lower pressure chamber when the current is passed through the ventilation resistor, generating a rapid increase in temperature. Preferably, the film is sufficiently stable when heated so that the material can withstand the temperatures employed in other operations of the test cassette, such as heat lysis, reverse transcription and nucleic acid amplification. The use of a material with stability in the temperature ranges used for denaturation, labeling, reverse transcription, nucleic acid amplification and detection, but with a melting temperature readily achieved by resistor 70 allows a single material to be used to support the fluidic component injection molded 400 to serve as a face of the chambers and a face of the pouch
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65/103 ventilation. In some embodiments, additional temperature stability in the areas of temperature-controlled chambers can be achieved by an overlying film of heat-resistant material, such as polyimide. In other embodiments of the invention, a window in the heat-labile film is registered with the temperature-controlled chambers to allow direct contact between the fluids in the chamber and the substrate of a flexible circuit fused to the rear of the test cassette.
Additional Components of the Fluid Component [0052] As described above, several additional components are preferably incorporated into the fluid component of the present invention prior to final binding. Reagents including buffers, salts, dNTPs, NTPs, initiator oligonucleotides and enzymes such as DNA polymerase and reverse transcriptase can be lyophilized or lyophilized in pellets, spheres or pies before mounting the device. The freeze-drying of the reagent is well known in the art and involves the dehydration of aliquots of frozen reagents by sublimation under applied vacuum. By adding specific formulations of lyoprotectants like sugars (dl and polysaccharides) and polyalcohols to the reagents before freezing, the activity of the enzymes can be preserved and the rate of rehydration can be increased. Pellets, spheres or pies of lyophilized reagents are manufactured by standard methods and, once formed, are reasonably durable and can be easily placed in specific chambers of the fluidic component before lamination of the final face. More preferably, the recesses are incorporated into the fluidic network to allow the pellets, spheres or pies of lyophilized reagents to be placed in the fluidic component prior to the connection of the fluidic component to the support material. By selecting the geometry of the fluid network and the location and order of the recess, the sample can react with the desired lyophilized reagent in the
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66/103 desired moment to optimize performance. For example, depositing lyophilized (or dry) reverse transcription (RT) beads and amplification reagent into two separate recesses in the flow paths of the RT reaction chamber and the amplification chamber allows for an ideal reverse transcription reaction without the interference of enzymes. amplification. In addition, to minimize the interference of the RT enzymes in the subsequent amplification reaction, the RT enzymes after the RT reaction presented in the RT reaction can be inactivated further before introduction to the amplification reagents to minimize their interference in the amplification. Optionally, other salt, surfactants and other chemical intensifiers can be added to different recesses to modulate the performance of an assay. In addition, these recesses facilitate the entry of lyophilized reagents with liquids as they pass through the recess and also serve to isolate lyophilized materials from ultrasonic energy during ultrasonic welding and isolate lyophilized reagents from extreme temperatures during the heating steps of a test before its solubilization. In addition, the recesses ensure that lyophilized pellets are not compressed or crushed during manufacture, allowing them to remain porous to minimize rehydration times.
[0053] In some embodiments of the invention, the detection microparticles are another additional component of the fluidic component. In some embodiments, these microparticles can be lyophilized as described for the above reaction reagents. In other embodiments, microparticles in liquid buffer can be applied directly to an inner face of a fluid chamber and dried before the final assembly of the test cassette. The liquid buffer containing the microparticles also preferably comprises sugars or polyalcohols which assist in rehydration. The incorporation of microparticles
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67/103 in aqueous buffer directly in the fluid component before drying can simplify and reduce the final cost of manufacture and complete the agglomeration of lyophilized particles with reaction solution and the denaturation of double-stranded nucleic acids or double-stranded regions of a Nucleic acid in single-stranded nucleic acid can be facilitated by heating or nucleated boiling. In some embodiments, lyophilized detection particles are placed in recesses in the fluidic network. In other embodiments, lyophilized or dried detection particles are placed in a space 93 directly below the detection strip. In other embodiments, the detection particles are dried or lyophilized on a biblical substrate in capillary communication with the detection strip or are dried or lyophilized directly on the detection strip. Capillary communication can be direct physical direct contact of said biblical substrate with the detection strip or indirect where the capillary communication is along an intermediate distance comprised by a channel or chamber region through which capillary transport is achieved to transport fluid from the biblical substrate loaded with detection particles to the detection strip.
[0054] In some embodiments of the present invention, a side flow detection strip assembly is also incorporated into the fluidic component. The detection strip preferably comprises a membrane assembly consisting of at least one porous component and, optionally, may comprise an absorbent pad, a detection membrane, a surfactant pad and a backing film. The detection membrane is preferably made of nitrocellulose, cellulose, polyethersulfone, polyvinylidine fluoride, nylon, charge-modified nylon or polytetrafluoroethylene and can be coated with a plastic film. As described above, the capture probe can be deposited and irreversibly immobilized in the memory
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68/103 white detection line, dots, microarrays or any pattern that can be visualized with the naked eye or by an automated detection system, such as an image system. The deposited oligonucleotides can be permanently immobilized by UV irradiation of the detection membrane after deposition of the capture probe. The surfactant pad may comprise a porous substrate, preferably with minimal nucleic acid binding and fluid retention properties, which allow for unobstructed migration of the nucleic acid product and the detection of microparticles. The surfactant pad may comprise materials, such as fiberglass, cellulose or polyester. In embodiments of the invention, formulations including at least one amphipathic reagent are dried on the surfactant pad to allow uniform migration of the sample across the detection membrane. The absorbent pad can comprise any absorbent material and helps to induce sample drainage through the detection membrane assembly. Using an adhesive backing film, such as a double-sided adhesive film as a base, the detection membrane component is assembled by placing the detection membrane first, followed by the optional absorbent pad and / or the surfactant pad in physical contact with the membrane. approximately 1 mm and approximately 2 mm overlap. In some embodiments of the invention, the detection membrane may be in indirect capillary communication with the surfactant pad, in which there is a physical separation between the surfactant pad and the detection pad with the intermediate space, comprising a capillary space in which the fluids can pass through space through capillary action. In some embodiments, the surfactant pad or a region of the surfactant pad may comprise detection particles, dry detection particles or lyophilized detection particles.
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69/103
Three-chamber cassette [0055] In some embodiments of the invention, additional reaction chambers and / or additional recesses for dry or lyophilized reagents can be incorporated. In some embodiments, such a design facilitates tests in which it is desirable to provide a separate initial lysis reaction before reverse transcription and amplification. As shown in FIGS. 37A, 37B and 38, cassette 5000 comprises cover 5020 sealing expansion chamber 5021, flexible heating circuit 5022 preferably arranged in intimate contact with fluid component 5023, and rear cover 5024 which hides the circuit from the user. A sample containing nucleic acids is introduced into sample cup 5002 through sample port 5001. The sample flows freely into recess 5003, where it reconstitutes the first lyophilized granule 5004, preferably comprising lysis reagents, before flowing through channel 5005 in the first reaction chamber 5006. This free flow is facilitated by the ventilation channel 5007 that connects to the top of the sample cup 5002. The ventilation channel 5007 can additionally connect to the expansion chamber 5021 through the orifice 5008. The air space sealed below the first reaction chamber 5006 pressurizes slightly due to the fluid flow and causes the flow to stop just below the first reaction chamber 5006. The first reaction chamber 5006 is then preferably heated to a temperature to facilitate the appropriate reaction with the lysis reagents, lysing the particles and / or biological cells in the sample, exposing any nucleic acids present in it.
[0056] The opening of the ventilation valve 5009 connected to the top of the second reaction chamber 5011 facilitates the flow of the sample to a second recess, where the second lyophilized granule 5010, preferably comprising reagents for reverse transcription, is reconstituted. The fluid enters the second 5011 reaction chamber, where its
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70/103 flow to as a result of increased air pressure in the volume of air enclosed below the flow. The second reaction chamber 5011 is then preferably subsequently heated to a suitable temperature to facilitate the process of reverse transcription.
[0057] Opening the next ventilation valve 5009 connected to the top of the third reaction chamber 5013 initiates the sample flow from the second reaction chamber 5011 through a third recess where the lyophilized granule 5012, preferably comprising POR amplification reagents lyophilized, is reconstituted. The sample then flows to the third reaction chamber 5013, where it is subjected to thermal cycles to amplify targeted analytes present in the sample.
[0058] Subsequently, the opening of the final ventilation valve 5009 connected to the furthest end of the side flow strip! 5014 allows the sample that now contains amplified analytes to flow to the side flow strip 5014 for detecting the analytes as described above.
Flow control features [0059] The design of the fluidic component can optionally comprise flow control features inside, or at the outputs of the reaction chambers. These features divert the flow that enters the chamber to the side of the chamber opposite the outlet, before the flow enters the outlet. As a result, the flow enters the outlet channel at a lower speed, reducing the distance that the fluid flows through the channel before stopping. In addition, the horizontal component of the flow path adds length to the channel without adding vertical spacing between the chambers, increasing the effective length of the flow path, so that it is sufficient to stop the flow at the desired location based on the reduced flow speed. This allows for closer vertical spacing between the cassette chambers, since less vertical channel is
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71/103 required. In addition, redirecting the flow through the reaction chamber creates a swirling action on the flow within the chamber, improving the mixture of reagents with the sample fluid. The flow control feature can include any format.
[0060] In the embodiment shown in FIG. 39, the fluid enters the reaction chamber 4003 from the input channel 4002 and flows to the bottom of the reaction chamber, where it is redirected to the side of the reaction chamber 4003 opposite the opening of the input channel 4002 by the control feature triangular flow 4001. As the flow proceeds to the opposite corner 4004, the flow divides, with a little going into outlet 4005, while the rest comes into contact with the wall and is directed upwards, creating a whirlwind effect that improves the mix. The flow to the outlet preferably forms a meniscus and travels through outlet channel 4006 towards the next reaction chamber or lyophilized granule recess. As the outlet channel 4006 is sealed below the reaction chamber 4003, while the fluid moves along the outlet channel 4006 the air pressure increases in the channel below the flow until it reaches equilibrium with the fluid pressure head, interrupting the flow. In this embodiment, outlet 4005 tapers from reaction chamber 4003 to outlet channel 4006, in order to effectively form a meniscus which can subsequently increase the pressure in the closed air space downstream of the flow. This larger opening for the outlet channel preferably provides an increase in the volume of compressible air, so that a meniscus can be formed reliably in the widest opening.
[0061] In the embodiment shown in FIG. 40, the fluid enters the reaction chamber 4103 from the inlet channel 4102 and flows to the bottom of the reaction chamber, where it is redirected to the side of the reaction chamber 4103 opposite the opening of the inlet channel 4102 by the control feature triangular flow rate 4101. As the flow
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72/103 proceeds to the opposite corner 4104, the flow divides, with a little going into exit 4105, while the rest comes into contact with the wall and is directed upwards, creating a swirling effect that improves the mixture. The flow to the outlet preferably forms a meniscus and travels through outlet channel 4106 towards the next reaction chamber or lyophilized granule recess. As outlet channel 4106 is sealed below reaction chamber 4103, as the fluid moves along outlet channel 4106 the air pressure increases in the channel below the flow until it reaches equilibrium with fluid pressure, interrupting so the flow. In this embodiment, output 4105 and output channel 4106 are of uniform width. In this modality, the formation of a meniscus in the reaction chamber can be a little more reliable with a result of the narrower channel. The meniscus subsequently increases the pressure in the closed airspace downstream of the flow.
[0062] In the embodiment shown in FIG. 41, the fluid enters the reaction chamber 4103 of the input channel 4202 and flows to the bottom of the reaction chamber, where it is redirected to the side of the reaction chamber 4203 opposite the opening for the input channel 4202 by the trapezoidal flow 4201. As the flow proceeds to the opposite corner 4204, the flow divides, with a little going into exit 4205, while the rest comes into contact with the wall and is directed upwards, creating a whirling effect that improves mixing. In this embodiment, output 4205 is oriented substantially vertically. The flow to the outlet preferably forms a meniscus and travels through the outlet channel 4206 towards the next reaction chamber or lyophilized granule recess. As the outlet channel 4206 is sealed below the reaction chamber 4203, while the fluid moves along the outlet channel 4206 the air pressure increases in the channel below the flow until it reaches equilibrium with the
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73/103 fluid pressure head, interrupting the flow. In this embodiment, output 4205 and output channel 4206 are of uniform width. In this modality, the formation of a meniscus in the reaction chamber can be a little more reliable with a result of the narrower channel. The meniscus subsequently increases the pressure in the closed airspace downstream of the flow.
[0063] In the embodiment shown in FIG. 42, the fluid enters the reaction chamber 4305 from the input channel 4304 and flows to the bottom of the reaction chamber, where it is redirected to the side of the reaction chamber 4305 opposite the opening of the input channel 4304 by the control feature triangular flow 4303. As the flow proceeds to the opposite corner 4306, the flow divides, with a little going into outlet 4307, while the rest comes into contact with the wall and is directed upwards, creating a whirling effect that improves the mix. The flow to the outlet preferably forms a meniscus and travels through outlet channel 4306 towards the next reaction chamber or lyophilized granule recess. In this embodiment, the outlet channel 4306 moves through the stacked serial flow control features 4303, 4302 and 4301 that provide a tortuous route for the fluid to flow, providing an increase in the length of the outlet channel in a small vertical space.
[0064] In the embodiment shown in FIG. 43, the fluid enters the reaction chamber 4403 from the inlet channel 4402 and is redirected to the side of the reaction chamber 4403 opposite the opening to the inlet channel 4402 by the flow control feature 4401 disposed above the bottom of the reaction chamber , preferably approximately halfway along the length of the reaction chamber 4403. In contrast to the previous embodiments, the 4401 flow control feature does not form the 4403 reaction chamber outlet. The 4401 flow control feature deflects the flow from output channel 4405 to the
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74/103 opposite corner 4404, thus reducing the flow speed before leaving the chamber. Similar to the previous modalities, fluid redirection promotes turbulence and the mixing of reagents.
[0065] To facilitate the effective combination of the reaction solution with lyophilized reagents, the modalities of the test cassette portion comprising the fluid flow path may comprise dedicated 4600 reagent recesses incorporated in the fluid flow path between the chambers, such as shown in FIGS. 46A and 46B. In alternative embodiments, the recesses of the 4700 reagents are disposed within the reaction chamber itself, as shown in FIGS. 47A and 47B. Lyophilized reagent pellets are preferably disposed in the reagent recesses during manufacture. In the case of the reagent recess located within a fluid chamber, the presence of reaction solution in the fluid chamber results in resuspension of the lyophilized reagent or reagents placed within the recess during manufacture. Placing lyophilized reagent (s) into a fluid chamber recess may be preferable to placing reagent (s) into a reagent recess in the fluid channel when reagent resuspension requires longer resuspension times than provided by transitory passage of fluid through a recess located in the channel during the flow of solution from one chamber to another chamber. By housing the lyophilized reagent inside the fluid chamber, the residence time of the reaction solution with the reagent is extended, increasing the exposure of the lyophilized material to the fluid and ensuring a more complete resuspension of the lyophilized reagents and a more complete mixing of the reagents with the reaction. In addition, lyophilized reagents disposed in recesses in the flow path may be susceptible to infiltration (or capillary drip) from the above chamber before the reaction is complete. This can give a syrupy consistency to the reagents, causing
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75/103 fluid flow problems when the volume of the solution is transported through the recess of the upper chamber. This problem is preferably avoided when the recess is arranged in the lower chamber itself.
[0066] In applications where it is desirable to place the reagent recess within the fluid channel, such as that shown in the standard embodiment shown in FIG. 48, improvements in resuspension of the reagent and in the combination of the reagent and the reaction solution can be achieved by incorporating a projection into the fluid chamber, such as the 4605 projection shown in FIG. 46B, or projection 4705 shown in FIG. 47B. The sharp vertical increase on the projection side inside the chamber discourages the flow of capillary fluid through the top or roof of the fluid chamber, reducing or preventing reagent sequestration by recently resuspending most of the reaction solution volume.
Assay Multiplexing [0067] In some embodiments of the invention, several independent assays can be performed in parallel, employing a fluidic design that allows to divide a sample of inlet fluid into two or more parallel fluid paths through the device. FIG. 10 is a schematic representation of the division of one volume of fluid, for example 80 µl, in two sequential steps in the first two separate volumes of 40 µl and subsequently into four volumes of 20 µl. The illustrated scheme is useful to allow separate independent manipulations, such as biochemical reactions to be conducted on the divided volumes. Such configurations are useful to increase the number of analytes that can be detected in a single device, facilitating the multiplexed detection of multiple targets, such as nucleic acid sequences in reverse transcription reactions and / or reverse amplification of multiplexed nucleic acids. Of
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76/103 likewise, the use of multiple detection strips at the end of the independent fluid paths can provide better legibility of the strips for detecting multiple targets or to distinguish sequence differences or mutations in nucleic acid analytes. In addition, providing additional detection strips for independent interrogation of multiple amplification reaction products can increase specificity by reducing the likelihood of spurious cross-reactivity, such as cross-hybridization during the detection step of the test. FIG. 11 illustrates a test cassette comprising two fluid paths in a single test cassette. Each fluid path can be controlled independently with respect to time, type of reaction, etc. Referring now to FIG. 12, a sample introduced into the sample cup 1000 is divided into approximately equal volumes and flows into volume dividing chambers 1001 and 1002, which flow is regulated by ventilation valves 1003 and 1004. Dividing chambers 1001 and 1002 control the sample volume in each test path by passively balancing the sample amount in each chamber. After dividing the volume, the solution is allowed to flow through the reagent recesses 1007 and 1008 opening the vents 1005 and 1006. Reagents such as lyophilized reagents are disposed in recesses 1007, 1008 and coated with the sample as it flows through the recesses. and in a first set preferably temperature-controlled chambers 1009 and 1010. Reactions such as heat lysis, reverse transcription and / or nucleic acid amplification are conducted in each of the first heated chambers sets facilitated by the reagents provided in the reagent 1007 recesses , 1008. Such reagents may include, but are not limited to, lyophilized positive control agent (e.g., nucleic acid, viruses, bacterial cells, etc.), lyophilized reverse transcriptase and associated accessory reagents,
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77/103 as nucleotides, buffers, DTT, salts, etc. necessary for the reverse transcription of RNA to DNA and / or DNA amplification using lyophilized DNA polymerase or thermostable lyophilized DNA polymerase and necessary accessory reagents such as nucleotides, buffers and salts.
[0068] Upon completion of biochemical reactions, such as reverse transcription, nucleic acid amplification or concomitant reverse transcription and nucleic acid amplification (eg polymerase chain reaction with single tube reverse transcription (RT-PCR) or RT-PCR in one step or RT Oscar in one step) in the first set of chambers, seals for ventilation bags 1011 and 1012 are broken to allow fluid to flow from the first set of chambers through a second set of 1013 reagent recesses and 1014 and in a second set of chambers preferably with controlled temperature 1015 and 1016. Reagents such as lyophilized reagents can be arranged in recesses 1013 and 1014 so that they mix with the sample solution as the fluid flows from chambers 1009 and 1010 to chambers 1015 and 1016. Reagents such as lyophilized reagents for nucleic acid amplification or dry or lyophilized detection particles, such as polystyrene microspheres dyed conjugated with probe or colloidal gold conjugated with probe, can optionally be placed in reagent recesses 1013 and / or 1014. After completion of reactions or other manipulations, such as ligation or hybridization to probe conjugate detection particles in heated chambers, is The solution is allowed to flow into the detection strip chambers 1017 and 1018 by opening the vent valves 1019 and 1020. In some embodiments, a third set of reagent recesses can be placed in the fluid paths of chambers 1015 and 1016 so that additional reagents, such as detection reagents comprising particles
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78/103 of detection, salts and / or surfactants and other substances useful to facilitate hybridization or other detection modalities, can be treated with the solution flowing to the strip chambers 1017 and 1018. The detection strip chambers 1017 and 1018 they can be heated and preferably comprise detection strips, such as lateral flow strips, for detecting analytes, such as amplified nucleic acids. The detection strips may comprise a series of absorbed materials doped or standardized with dry or lyophilized detection reagents, such as detection particles (eg, dyed microsphere conjugates and / or colloidal gold conjugates), capture probes for capturing analytes, such as hybridization capture oligonucleotides for the capture of nucleic acid analytes by sequence specific hybridization, ligands such as biotin or streptavidin for the capture of suitably modified analytes and absorbent materials to provide sufficient absorbent capacity to ensure complete volume migration of the sample solution through the detection strip by means such as capillary action or absorption.
Sample preparation [0069] In some embodiments of the invention, it may be desirable to incorporate a sample preparation system into the cassette. A sample preparation system, such as a nucleic acid purification system, can comprise encapsulated solutions for carrying out sample preparation and eluting purified molecules, such as DNA, RNA or purified proteins, in the test cassette. FIG. 13 represents a 1300 nucleic acid sample preparation subsystem designed for integration with a test cassette. The sample preparation subsystem comprises a main compartment 1302 and compartment cover 1301 for housing components of the subsystem. A partitioning component
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79/103 of solution 1303 comprises crude sample reservoir 1312 preferably open on the top face, but sealed below by the bottom seal 1305. The compartment component of solution 1303 preferably also comprises reservoir 1314 containing a first wash buffer and reservoir 1315 containing a second wash buffer, both of which are preferably sealed by means of the upper seal 1304 and the lower seal 1305. A nucleic acid binding matrix 1306 is placed in the capillary flow path of the solution provided by the absorbent materials 1307 and 1308. Glass fiber or silica gel exhibiting nucleic acid binding properties and absorption properties are examples of materials suitable for use as a 1306 binding matrix. A wide range of absorbent materials can comprise absorbent materials 1307 and 1308, including polyester, fiberglass, nitrocellulose, polysulfone, cellulose, cotton or combinations of m esmos, as well as other absorption materials, as long as they offer adequate capillarity and minimal connection to the molecules to be purified by the subsystem. Any readily broken or frangible material capable of being sealed in the compartment component of solution 1303 and chemically compatible with encapsulated solutions is suitable for use as sealing material 1304 and 1305. Sealing material 1305 comes into contact with the sample or lysate of the sample and must furthermore be chemically compatible with the sample or lysate solution sample. Examples of suitable sealing material are metallic film, plastic film that can be heat sealed. The sealing material 1305 is broken at the time of use by displacing the compartment component of the solution 1303, so that the seal 1305 is perforated by the structures 1311 present in the housing 1302. Crude sample or crude sample mixed with a lysis buffer, as a compos buffer
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80/103 t of a chaotropic agent, is introduced at the time of use in sample reservoir 1312 through sample port 1309 in cap 1301. In some embodiments, the lysis buffer can optionally be encapsulated in reservoir 1312, extending seal 1304 to cover the upper orifice of reservoir 1312. In such embodiments, it may be desirable to include a flap or other means for partial removal of that region of seal 1304 covering reservoir 1312 to allow the addition of crude sample to reservoir 1312, so that the crude sample may come into contact with or mix with the lysis buffer contained therein. The sample solution or lysate containing sample material is introduced into the sample addition port 1309 and retained in the sample reservoir 1312 of the buffer reservoir 1303 until the start of the sample preparation process.
[0070] At the time of sample preparation, the partitioning component of solution 1303 is pushed into the seal drilling structures 1311, resulting in the simultaneous release of sample solution or lysate in reservoir 1312 and the first and second plugs rinses in reservoirs 1314 and 1315 respectively. The mechanical displacement of the 1303 component can be performed manually or by using an actuator or actuators present in a reusable instrument in which the disposable test cassette is placed at the time of use. The access of the actuator or the manual displacement mechanism to access the reservoir 1303 is preferably provided through access door 1310 of the compartment lid 1301. The sample solution or lysate and the first and the second wash buffers are moved by the materials 1307, 1306 and 1308 by capillary action. The physical arrangement of the reservoirs and the geometric configuration of the absorbent material 1307 guarantee the sequential flow of the raw lysate from the first wash buffer
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81/103 and the second wash buffer via the connecting matrix 1306. Additional absorbent capacity to ensure continuous capillary transport of all volumes of solution through the system are provided by absorbent pad 1313 placed in contact with absorption 1308. At the conclusion of the transport of the solution through the absorbent materials, the spent solutions remain in the absorbent pad 1313. After the capillary transport of all solutions through the system is exhausted, the purified nucleic acids are linked to the binding matrix 1306, from which the acids nucleic acids can be eluted in the sample cup 1402 of the integrated test cassette, as shown in FIG. 15.
[0071] The movement of the components of the sample preparation subsystem that occur during the sample preparation process is shown in FIG. 14, which represents the modality of the sample preparation subsystem in cross section before and after sample processing. Elution is preceded by displacement of the connecting matrix 1306 out of the capillary flow path and through the sealing component 1316 by the action of an actuator on the associated reusable test instrument. The sealing component 1316 forms a seal with a portion of the elution buffer duct 1318 to allow injection of the elution buffer through the connection matrix 1306 and into sample cup 1402 without loss of solution in the capillary flow path of the subsystem of sample preparation. The conduit 1318 is attached to or part of the elution buffer injector component composed of the elution buffer reservoir 1317 and the piston 1316. The piston 1319 can optionally comprise o-rings to facilitate the sealing of the elution buffer within the 1317 reservoir. During the elution of the purified nucleic acid, an actuator moves the component of the elution reservoir 1317, so that the connected conduit 1318 forms a seal
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82/103 with the sealing component 1316 and moves the connection matrix 1306 to the chamber 1321. Mechanical access to depress the elution reservoir 1317 is provided through the access door of the actuator 1320. After the connection matrix 1306 is moved out of the main capillary solution flow path of the sample preparation subsystem, the binding matrix 1306 resides in the elution chamber 1321. Elution of purified nucleic acid in the sample cup 1402 is performed by forcing the elution buffer from the elution buffer reservoir 1317 by an actuator acting through actuator port 1322 to move piston 1319 through reservoir 1317 in a syringe-like action. The elution buffer proceeds through conduit 1318 through the binding matrix 1306, resulting in the injection of elution buffer containing purified nucleic acids eluted into sample cup 1402.
[0072] With reference now to FIG. 15, the sample preparation subsystem 1300 is preferably connected to the fluid component
1403 of the 1500 cassette by widely used manufacturing methods, such as ultrasonic welding to form a sample test cassette for integrated single-use results. In some embodiments, it is desirable to hermetically seal the fluids from the test cassette after the introduction of eluate containing purified nucleic acid, in order to reduce the probability of leakage of amplified nucleic acid from the cassette. A slide seal 1404 can optionally, but preferably, be placed between the sample preparation subsystem and the fluid housing of test cassette 1403 to seal the cassette at the entrance of sample cup 1402. The slide seal
1404 is moved to the sealed position by the action of an actuator to form an airtight seal comprising o-ring 1405. Cassette holder 1406 is attached to the fluidic housing after introducing dry reagents and test strips. As described above for the support of
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83/103 other types of test cassette, support 1406 comprises materials for ventilation functionality, maintenance of airtight seal, thermal interface, expansion chamber (s) and can optionally comprise a printed circuit board or a flexible circuit layer that carries electronic components for temperature and fluid control. Electronic components can optionally be housed in a reusable plug-in unit. PCA 1501 comprising electronic components is preferably constructed of low thermal mass materials and surface-mounted electronic components. Surface mounted resistor arrays and temperature sensors located proximally provide a means of regulating chamber temperatures in the test cassette. The surface mount resistors and temperature sensor arrangements of the PCA 1501 are located to be registered on the test cassette when the test cassette is loaded into the docking unit. An integrated sample-to-result cassette is shown in FIG. 16. FIG. 17 illustrates the integrated cassette with an underlying electronic layer based on traditional and surface-mounted printed circuit board components. In some embodiments, a flexible circuit can be connected to the rear of the test cassette.
Electronics [0073] In some embodiments, it is desirable to place electronic components in a reusable component such that heaters, sensors and other electronic components are connected to the disposable test cassette by means capable of establishing a favorable thermal interface and accurate registration of components electronics with overlying elements of the disposable test cassette with which they must interact. In other embodiments, it is desirable to use a combination of reusable components and des
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84/103 cardable for temperature control. For example, stand-off temperature monitoring can be performed with infrared sensors placed in a reusable plug-in unit, while resistive heaters for temperature control and fluid control are placed in a flexible circuit integrated with the disposable test cassette.
[0074] In some embodiments, the printed circuit board (PCB) comprises a laminated material coated with FR4 copper of 0.062 inch of standard thickness, although other standard materials and thickness of the plate may be used. Electronic components such as resistors, thermistors, LEDs and the microcontroller, preferably, comprise ready-to-use surface mount devices (SMDs) and are placed according to industry standard methodology.
[0075] In alternative modes, the PCA can be integrated with the cassette wall and comprise a flexible plastic circuit. Flexible circuit materials, such as PET and polyimide, can be used as shown in FIG. 8. The use of flexible plastic circuits is well known in the art. In another modality, heating elements and temperature sensors can be printed on canvas on the plastic fluid component with technology developed by companies such as Soligie, Inc.
[0076] In some embodiments of the invention, the thickness of the PCB, as well as the quantity and placement of copper in the regions around the resistive heaters is tailor-made for the thermal management of the reaction solution in the fluidic component. This can be done using standard manufacturing techniques already mentioned.
[0077] In some embodiments of the invention, the resistor is a 2512 thick film package, although other resistors may be used. Heating chambers in the fluidic component are
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85/103 preferably of dimensions similar to those of the resistor to ensure uniform heating throughout the chamber. A single resistor of this size is sufficient to heat approximately 15 pL of solution, assuming a fluid component thickness of 0.5 mm. The drawing in FIG. 2D shows two resistors 100 forming a heater sufficient to heat approximately 30 pL of solution, assuming a fluid component thickness of 0.5 mm. In this case, the resistors are preferably 40 ohms each and arranged in a parallel configuration.
[0078] In some embodiments of the invention, the temperature sensor 110 preferably comprises a thermistor, such as a 0402 NTC device, or a temperature sensor such as the Atmei AT30TS750, each of which is similar in height to the 2512 resistor package. The thermistor is preferably aligned with or adjacent to, or between, the resistance resistors, in the case of a resistance of one or two sets of resistor, respectively. When closely aligning these electronic elements, only a very thin air gap results between them. In addition, the application of a thermal compound before mounting the fluid with the electronic layer ensures good thermal contact between the fluid component, the resistor and the thermistor.
[0079] In some embodiments of the invention, the ventilation resistors 70 _s 71 comprise a thick film package 0805, although similar resistors can be used. In place of a resistor, a small gauge nichrome wire heating element, such as a 40 gauge nichrome wire, can also be used.
[0080] In some embodiments of the invention, the microcontroller is a PIC16F1789 from Microchip Technologies. The microcontroller is preferably adapted to the complexity of the fluidic system. For example
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86/103 pio, with multiplexing, the number of individual fans and heaters is compatible with the number of lines of the I / O microcontroller. The memory size can be chosen to accommodate the size of the program.
[0081] In certain embodiments of the invention, the N-channel MOSFETs in the SOT-23 package operating in an ON-OFF mode are used to modulate the current load for the ventilation and heating resistors. Modulation signals are sent through the microcontroller. In alternative modalities, a pulse width modulation scheme and / or other control algorithms can be used for more advanced thermal fluid management. This would typically be handled by the microcontroller and may require additional hardware and / or software features known to those skilled in the art.
[0082] Depending on the application, some modalities comprise a device in which a small control fitting unit or fitting unit operates a smaller disposable unit comprising fluidic systems that come into contact with biological materials, referred to as a test cassette. In one of these modalities, the plug-in unit comprises the electronic components. The elimination of electrical components from the disposable test cassette reduces costs and, in some cases, the environmental impact. In another embodiment, some electronic components are included in the plug-in unit and the test cassette. In this specific modality, the test cassette preferably comprises a low-cost PCA or, preferably, a flexible circuit to provide some electrical functions, such as temperature control, fluid flow control and temperature sensor, which are energized , controlled and / or interrogated by the plug-in unit through an appropriate interface. As described above, the electronic functions of such a device
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87/103 are preferably divided into two separate subsets. The 2500 disposable cassette preferably comprises a rear surface designed to interact with resistive heating elements and sensors of the plug-in unit. The materials that comprise the rear face of the test cassette are preferably selected to provide adequate thermal conductivity and stability, while allowing control of the flow of fluid through ruptured ventilation. In some embodiments, the rear face of the test cassette or part of it comprises a flexible circuit made of a substrate, such as polyimide. Flexible circuits can be used to provide low cost resistive heating elements with low thermal mass. The flexible circuit substrates can preferably be placed in direct contact with the solutions present in the fluid network of the test cassette to allow highly efficient and rapid heating and cooling. The connector 810 as shown in FIG. 8 preferably provides current for the resistive heaters, together with a power line and signal for the optional thermistor (s).
[0083] If flexible circuit 799 is used, one or more infrared sensors located in the plug-in unit can monitor the temperature of the heated chambers (for example, amplification or detection chambers) by reading the signal through a window on the support 805 or directly at the rear of flexible circuit 799. Optionally, thermistors on the PCA or flexible circuit 799 can be used to monitor temperatures. Optionally, running a weighted average of the outputs of the IR sensors and thermistors improves the correlation between the readings and the fluid temperature in the cassette. In addition, sensors can also detect ambient temperature, allowing the system to correct it to ensure that the sample fluid quickly equilibrates to desired temperatures.
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88/103 [0084] Referring now to FIG. 33, the plug-in unit preferably comprises a subset of reusable component 3980 comprising the microcontroller, MOSFETs, switches, the power supply or a socket and / or battery, optional cooling fan 903, optional user interface, infrared temperature sensors 901 , 902 and 900 connector compatible with the 810 connector on the 2500 cassette. When the subassemblies are coupled via the §10 and 900 connectors, the docking unit preferably supports the 2500 disposable cassette in a substantially vertical or almost vertical orientation. Although a substantially vertical orientation is preferable in some of the embodiments described herein, similar results can be obtained if the device is operated at an inclination, especially if certain routes are coated to reduce the wetting angle of the solutions used.
[0085] Another modality of the device can be used to minimize operating costs, reducing the cost of the consumable part of the system, eliminating all electronic circuits located in the disposable part. The microcontroller, heaters, sensors, power supply and all other circuits are located on several PCAs and electrically connected to each other via industry standard conductor cables with high conductor count. A screen can also be added to assist the user in operating the device. An optional serial control port can also be used to allow the user to upload changes to the test parameters and monitor the progress of any test. One version of this modality comprises five different PCAs. The main board PCA contains the control circuits, the serial port, the power supply and the connectors to connect to the other system boards. The heater board PCA contains the elements
Petition 870190099370, of 10/04/2019, p. 155/173
89/103 of the heating resistor, temperature sensors and heating elements for ventilation firing. In order to facilitate the thermal interface between this heating plate and the disposable fluid cassette, this plate is mounted on a spring conveyor that is moved to the rear side of the fluid cassette by the closing action of the cover, until contact with the fluidic cassette. A thin, thermally conductive heating pad is attached to the top of the chamber heater resistors and the temperature sensor, improving heat transfer between the heater plate and the fluidic cassette. A durable ventilation firing heating element can be realized using nichrome wire wrapped around a small ceramic conveyor. The PCA of the IR sensor board is mounted a short distance from the opposite side of the cassette and is used to monitor the temperatures of the heating chamber. This allows closed-circuit temperature control of the heating and cooling process and accommodates ambient temperature variations. Also mounted on the IR sensor plate are multiple optical couplers for reflective sensing that allow the detection of the presence of the cassette, and can be used to identify the type of cassette indicated by the configurable reflective pattern located on the cassette. A screen card PCA can be located approximately behind the infrared card to allow the user to see the screen from the front of the device. A final PCA, the shutter plate is located in front of the upper edge of the cassette and contains a switch and a reflective optical coupler that is used to detect whether the cassette has been used or not, and when the lid closes, holding the cassette in place for testing.
[0086] The cooling of the system is optionally increased using a fan, such as a muffin fan, which is activated by the microcontroller only during the cooling phase of the test.
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90/103 te. A ventilation system is preferably used to direct the cooler external air against the heating chambers and to expel it from the sides of the device.
[0087] To provide a sample molecular test for complete result, any of the above embodiments of the invention can interface with a sample preparation system 1300 that provides nucleic acids as output to sample chamber 1402. This has been demonstrated using the sample preparation technology described in International Publication WO 2009/137059 A1, entitled “Highly Simplified Lateral Flow-Based Nucleic Acid Sample Preparation and Passive Fluid Flow Control”. One embodiment of the resulting integrated device is illustrated in FIG. 15 and FIG. 16.
Plug-in unit [0088] The reusable plug-in unit comprises the electronic components necessary to obtain the functionality of the test cassette. Several modalities of the plug-in unit have been invented to interact with corresponding variations in the design of the test cassette. In one embodiment, the plug-in unit, shown in FIG. 18 and FIG. 19, comprises all electronic components required to perform a test, eliminating the need for electronic components in the test cassette. Referring now to FIG. 18, before adding the sample, cassette 2500 is inserted into the plug-in unit 2501. The plug-in unit 2501 comprises a screen such as the LCD screen 2502 to communicate information such as test protocols and test status to the user. After inserting the cassette into the plug-in unit 2501, the sample is inserted into the sample port 20 of the cassette 2500 and the plug-in unit cover 2503 is closed to start the test. A plug-in unit with the test cassette inserted is shown in FIG. 19, and the plug-in unit 2501 with the lid in the closed position is illustrated in FIG. 18B.
Petition 870190099370, of 10/04/2019, p. 157/173
91/103 [0089] In some embodiments of the plug-in unit, a mechanism is incorporated in the hinge of cover 2503 that moves the slide seal 91 of the test cassette to the closed position. A sealed test cassette is useful to ensure that the amplified nucleic acids remain contained in the test cassette. Referring now to FIG. 20, a manual or automated method can be used to slide a valve over the sample port to seal the cassette. In some embodiments, the slip seals the sample port by engaging an o-ring. The expansion chamber cover secures the valve to slide in place above the sample port o-ring. The seal is moved into position by a servomotor or by a manual action, such as closing the cover of the reusable plug-in unit, which in turn activates a mechanism to close the cassette seal. In the illustrated embodiment, the rack and pinion mechanism 2504 employs the sliding seal actuator 3979 to move the sliding seal 91 to the closed position. The rack and pinion mechanism 2504 can be motorized or moved by the closing action of the cover unit 2503 by means of a mechanical coupling to the cover hinge. Optionally, a sensor such as optical sensor 2505 can be located to interrogate the position of the slide seal 91 to ensure correct placement of the seal before the test starts, as illustrated in FIG. 21. The optical sensor detects the state (that is, the position) of the cassette sample door seal. The optical sensor allows the plug-in unit to be programmed to detect accidental insertion of a previously used test cassette and to detect the successful closing of the test cassette seal. An error message indicating a seal malfunction may be displayed on screen 2502 and the test program may be interrupted if sensor 2505 fails to detect the seal closing. In other modali
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92/103 of the test cassette and plug-in unit, the sealing mechanism may comprise other means of mechanically sealing the chamber, such as a rotary valve, as illustrated in FIG. 22. In yet another embodiment, a test cassette seal can be placed on a hinged cassette cover, so that insertion into the plug-in unit is not possible without first closing the cassette cover and thus settling the seal. In this mode, the sample is added to the test cassette before insertion into the plug-in unit. A test cassette comprising a hinged, sealed lid is illustrated in FIG. 23. In general, after the cassette is inserted into the docking unit and the sample is loaded into the cassette, it is preferable that closing the docking unit cover seals the cassette automatically and starts the test, preferably without the use of servos or other mechanical devices.
[0090] In some plug-in unit types, a set of components preferably facilitates the proper insertion of the test cassette, while ensuring that the electronic components that must interface with the test cassette do not physically interfere with the cassette insertion, but form a reliable thermal interface during the test. These components form a mechanism for keeping the PCA 75 away from the cassette insertion path until the lid 2503 closes. Referring now to FIG. 24, inside the plug-in unit, the heating plate is mounted on the PCA support 2506, which preferably serves as low thermal mass scaffolding, while the test cassette is loaded on a low thermal mass cassette support 2507, where rails 2509 guide the cassette to the docking unit and hold it in the correct position, parallel to the surface of the heating plate, to interface with the PCA 75 mounted on the PCA 2506 bracket. In the open position of the cover, the prominences
Petition 870190099370, of 10/04/2019, p. 159/173
93/103
2508 on the cassette holder 2507 interferes with the PCA holder 2506 to maintain an open path along the tracks 2509 for inserting the cassette. Preferably, an inclined surface measures the distance between the surface of the prominences 2508 and the lowest elevation of the component 2507 to facilitate the smooth movement of the prominences 2508 to depressions 2511 in the PCA support 2506 during closing of the cover 2503. After closing the cover the cover of the plug-in unit, the prominences 2508 engage with the depressions 2511, thus moving the heating plate holder closer to the rear surface of the test cassette 2500. Closing the cover 2503 exerts downward force on the cassette holder 2507 , thereby moving cassette holder 2507 to a position where prominences 2508 rest on depressions 2511 resulting in movement of PCA holder 2506 such that PCA 75 is pressed against the rear of cassette 2500. Preferably, the holder of PCA 2506 is under constant force, like spring force, to allow reproducible pressure to be exerted against the rear of the cassette by PCA 75 after closing the cover. Placing the PCA 75 against the rear of the 2500 cassette forms the thermal interface that conducts the heat from the resistive elements of the heater in the PCA to the chambers and ventilation vents of the test cassette. Preferably, components 2506 and 2507 are built to contribute minimum thermal mass to the system and provide access to test cassette surfaces for refrigeration devices, such as fans, and temperature monitoring by sensors, such as infrared sensors. After closing the lid, the heater plate is therefore preferably pressed firmly against the back of the test cassette, forming a thermal interface that allows the micro heaters on the heating plate to heat solutions in the fluid chambers of the cassette.
Petition 870190099370, of 10/04/2019, p. 160/173
94/103 test and fuse the thermally labile ventilation films of the test cassette, preferably according to the control of the microcontroller or microprocessor. FIG. 25 illustrates the cassette-PCA interface mechanism in cross section in the disengaged (lid opening) and engaged (closed lid) positions.
[0091] In some embodiments, the plug-in unit comprises additional sensors for applications such as temperature detection, detection of the presence or removal of a test cassette and detection of specific test cassettes to allow automated selection of test parameters. Referring now to FIG. 26, the 2600 infrared sensors detect the temperature of the test cassette in regions overlaid with temperature controlled chambers, such as chambers 30 and 90. The sensors allow the collection of temperature data in addition to or instead of temperature data collected by the PCA 75 localized temperature sensors, such as 110 sensors. Optical sensors can optionally, but preferably, be used to detect specific test cassettes to identify cassettes for specific diseases or conditions and allow automated selection of temperature profiles suitable for a specific test . Referring now to FIGS. 27A and 27B, an optical sensor or a set of optical sensors, such as the 2601 optical sensor set, can be used in conjunction with barcode 2602-like features on the test cassette to determine the type of test cassette. test and confirm complete insertion and correct seating of the test cassette. The sensor array 2602 in conjunction with the sensor 2505 can be used to detect the insertion of a previously used test cassette, detecting a closed seal before closing the cover. The plug-in unit can include sensors to detect the type of test cassette inserted in the plug-in unit and / or to confirm
Petition 870190099370, of 10/04/2019, p. 161/173
95/103 mar the correct insertion, positioning and alignment of the cassette into the docking unit. The detection of an influenza A / B test cassette is illustrated in the plug-in unit and test cassette system shown in FIG. 19. The plug-in unit can also preferably read a bar code or other symbol on each cassette and change its programming according to the programs stored for different tests.
[0092] In some embodiments of the invention, it is desirable to heat both sides of a test cassette. A double heater PCA configuration in which the test cassette is inserted between two heater PCAs is shown in FIGS. 28A and 28B.
[0093] In another embodiment, the plug-in unit comprises servo actuators, an optical subsystem for automated reading of results, a wireless data communication subsystem, a touch screen user interface, a battery power source refillable and a test cassette receiver that accepts a test cassette comprising an integrated sample preparation subsystem. Referring now to FIGS. 29A, 29B and 30, the plug-in unit 2700 accepts the test cassette 1500 and places the test cassette in thermal contact with PCA 1501 to activate the temperature control and fluid flow control of the test cassette. The test cassette 1500 is inserted into the slot of the 3605 cassette receiver on the pivoting docking unit door 2702. After adding sample or Used Raw to the test cassette, closing the docking unit door places the rear of the cassette test record and in contact with PCA 1501 and aligned with servo actuators. The 3602 servo actuator is located to access the partitioning component of the 1303 solution through the actuator 1310 port of the 1500 cassette and provide the mechanical strength necessary to break the seal material 1305. Breaking the mechanical seal 1305 releases
Petition 870190099370, of 10/04/2019, p. 162/173
96/103
Used crude and wash buffers to flow through the sample preparation capillary materials from the sample preparation subsystem, as described above. Upon completion of capillary fluid transport, servo actuator 3601, which is located to access elution reservoir 1317 through actuator port 1320 of cassette 1500 provides mechanical force to move component 1317 so that connected conduit 1318 forms a seal with seal 1316 and move connection matrix 1306 into elution compartment 1321. Servo actuator 3604 located to access elution piston 1319 through actuator port 1322 on cassette 1500 then provides mechanical force to piston 1319 to eject the plug elution from the elution reservoir 1317 although the binding matrix 1306, resulting in the elution of nucleic acids in the sample cup 1402 of the 1500 cassette. The 3603 servo actuator seals the cassette after the elution, as described above. Control of the actuator is preferably provided by a microcontroller or microprocessor in the PCA 3606 control electronics, in accordance with the firmware or software instructions. Likewise, the temperature and fluid flow control inside the test cassette is in accordance with the instructions provided in the firmware or software routines stored in the microcontroller or in the microprocessor's memory. The optical subsystem 3607 comprising LED light source 3608 and CMOS sensor 3609, shown in FIG. 31, digitizes the signal data from the detection strip. The collected detection strip images are stored in memory inside the docking unit, where the interpretation of the result can be performed using an integrated processor and reported to the LCD 2701. A ring of LEDs provides uniform illumination during image collection with a CMOS-based digital camera. Images collected with the postage stamp-sized device can provide high-resolution data (5 megapixels, 10 bits)
Petition 870190099370, of 10/04/2019, p. 163/173
97/103 for colorimetric side flow signal analysis. A preferably low-profile (-1 cm) design, coupled with short working distance optics allows the system to be integrated into a slim device housing.
[0094] Optionally, the digitized results can be transmitted for offline analysis, storage and / or visualization through a wireless communication system incorporated in the plug-in unit, using standard WiFi networks or cellular communications networks. Photographs of this embodiment of the plug-in unit are shown in FIGS. 32A and 32B.
Examples
Example 1: Multiplexed amplification and detection method of purified viral RNA (infA / B) and an internal positive control virus [0095] An influenza A and B test cassette was placed in the docking unit. 40 pL of a sample solution was added to the sample port. Sample solutions comprised purified RNA from influenza A / Puerto Rico at a concentration equivalent to 5000 TCIDso / mL, purified influenza B / Brisbane RNA at a concentration equivalent to 500 TCIDso / mL or molecular grade water (no model control sample ). Upon entering the sample port, the 40 pL sample comes with a lyophilized granule as it flows into a first chamber of the test cassette. The lyophilized granule consisted of viral phage particles MS2 as positive internal control and DTT. In the first chamber of the cassette, the sample was heated to 90 ° C for 1 minute to promote viral Use and then cooled to 50 ° C before opening the ventilation connected to a second chamber. Opening the vent connected to the second chamber allows the sample to flow into the second chamber, allowing air to move in the second chamber to an expansion chamber. As the sample was transferred to the second chamber, it was combined with
Petition 870190099370, of 10/04/2019, p. 164/173
98/103 oligonucleotide amplification primers for influenza A, influenza B and MS2 phages, and reverse transcription amplification reagents and nucleic acid present as a lyophilized pellet in a recess in the fluid path between the first and second chambers.
[0096] The amplification chamber was heated to 47 ° C for 6 minutes, during which time the RNA model was reverse transcribed into cDNA. After the completion of reverse transcription, 40 cycles of amplification of the thermal cycle were conducted in the second chamber. After the thermal cycling was completed, a vent connected to a third chamber was opened to allow the reaction solution to flow into the third chamber. The third chamber comprised a test strip and a lyophilized granule comprising three conjugates of blue-stained polystyrene microspheres used as detection particles. The conjugates consisted of 300 nm polystyrene microspheres, covalently linked to complementary oligonucleotide probes to amplified influenza A or influenza B or MS2 phage sequences. The solution reconstituted the lyophilized detection particles as they flowed into the third chamber. Three capture lines were immobilized on the lateral flow membrane, from the bottom of the device were: A negative control oligonucleotide not complementary to any tested targets; a capture probe complementary to the influenza B amplification product; a capture probe complementary to the influenza A amplification product; and an oligonucleotide complementary to the MS2 phage amplification product. The side flow strip was left to develop for six minutes before visual interpretation of the results. After the development of the lateral flow strip, positive samples for influenza A exhibited the formation of blue test lines at the positions of the influenza A and MS2 phages, positive samples for influenza B positive samples exhibited the formation of blue lines.
Petition 870190099370, of 10/04/2019, p. 165/173
99/103 blue tests on influenza B phage positions and negative samples exhibited the formation of blue test lines only on the MS2 phage position, as shown in FIG. 34.
Example 2: Method of multiplexed amplification and detection of viral lysate in buffer and an internal positive control virus [0097] An influenza A and B test cassette was placed in the docking unit. 40 pL of a sample solution was added to the sample port. The sample solutions comprised influenza A / Puerto Rico virus at a concentration equivalent to 5000 TCIDso / mL, influenza B / Brisbane virus at a concentration equivalent to 500 TCIDso / mL or molecular grade water (no model control sample). Upon entering the sample port, the 40 pL sample comes with a lyophilized granule as it flows into the first chamber of the test cassette. The lyophilized granule consisted of viral phage particles MS2 as positive internal control and DTT. In the first chamber of the cassette, the sample was heated to 90 ° C for 1 minute to promote viral Use and then cooled to 50 ° C before opening the ventilation connected to a second chamber. Opening the vent connected to the second chamber allows the sample to flow into the second chamber, allowing air to move in the second chamber to an expansion chamber. As the sample moved to the second chamber, it mixed with oligonucleotide amplification primers for influenza A, influenza B and MS2 phages, and reverse transcription and nucleic acid amplification reagents and enzymes present as a lyophilized granule in a recess in the fluid path between the first and the second chamber.
[0098] The amplification chamber was heated to 47 ° C for 6 minutes, during which time the RNA model was transcribed back into cDNA. After the completion of reverse transcription, 40 amplification cycles of the thermal cycle were conducted in the second chamber
Petition 870190099370, of 10/04/2019, p. 166/173
100/103 ra. Upon completion of the thermal cycling, an opening connected to a third chamber was opened to allow the reaction solution to flow into the third chamber. The third chamber comprised a test strip and a lyophilized granule comprising three conjugates of polystyrene microspheres dyed blue used as detection particles. The conjugates consisted of 300 nm polystyrene microspheres, covalently linked to ollgonucleotide probes complementary to amplified influenza A or influenza B or MS2 phage sequences. The solution reconstituted the lyophilized detection particles as it flowed into the third chamber. Three capture lines were immobilized on the lateral flow membrane, from the bottom of the device were: A negative control oligonucleotide not complementary to any tested targets; a capture probe complementary to the influenza B amplification product; a capture probe complementary to the influenza A amplification product; and an oligonucleotide complementary to the MS2 phage amplification product. The side flow strip was left to develop for six minutes before visual interpretation of the results. After the development of the lateral flow strip, positive samples for influenza A showed the formation of blue test lines at the phage positions influenza A and MS2, positive samples for influenza B positive samples showed the formation of blue test lines at the phage positions influenza B and negative samples exhibited the formation of blue test lines only at the MS2 phage position, as shown in FIG. 35.
Example 3: Method of amplification and multiplexed detection of influenza virus (purified) added in negative nasal clinical samples and an internal positive control virus [0099] Nasal smear samples collected from humans were placed in 3 ml of a Triton X- 100 to 0.025%, Tris
Petition 870190099370, of 10/04/2019, p. 167/173
101/103 mM, pH 8.3 and tested for the presence of influenza A and influenza B using a real-time RT-PCR test approved by the FDA. The samples were confirmed as negative for influenza A and influenza B before use in this study. The confirmed negative influenza nasal sample was contaminated with influenza A / Puerto Rico virus at a concentration equivalent to 5000 TCIDso / mL or used without the addition of a virus as a negative control. 40 pL of the resulting control samples, either increased or negative, were added to the sample port of an influenza A and B test cassette. Upon entering the sample port, the 40 pL sample is mixed with a lyophilized grain as it flows into a first chamber of the test cassette. The lyophilized granule consisted of viral phage particles MS2 as positive internal control and DTT. In the first chamber of the cassette, the sample was heated to 90 ° C for 1 minute to promote viral lysis and then cooled to 50 ° C before opening the ventilation connected to a second chamber. Opening the ventilation connected to the second chamber allows the sample to flow into the second chamber, allowing air to move in the second chamber to an expansion chamber. As the sample moved to the second chamber, it mixed with oligonucleotide amplification primers for influenza A, influenza B and MS2 phages, and reverse transcription and nucleic acid amplification reagents and enzymes present as a lyophilized granule in a recess in the fluid path between the first and the second chamber.
[00100] The amplification chamber was heated to 47 ° C for 6 minutes, during which time the RNA model was transcribed back into cDNA. After the completion of reverse transcription, 40 cycles of amplification of the thermal cycle were conducted in the second chamber. Upon completion of the thermal cycling, an opening connected to a third chamber was opened to allow the reaction solution
Petition 870190099370, of 10/04/2019, p. 168/173
102/103 flowed into the third chamber. The third chamber comprised a test strip and a lyophilized granule comprising three conjugates of polystyrene microspheres dyed blue used as detection particles. The conjugates consisted of 300 nm polystyrene microspheres, covalently linked to complementary oligonucleotide probes to amplified influenza A or influenza B or MS2 phage sequences. The solution reconstituted the lyophilized detection particles as it flowed into the third chamber. Three capture lines were immobilized on the lateral flow membrane, from the bottom of the device were: A negative control oligonucleotide not complementary to any tested targets; a capture probe complementary to the influenza B amplification product; a capture probe complementary to the influenza A amplification product; and an oligonucleotide complementary to the MS2 phage amplification product. The side flow strip was left to develop for six minutes before visual interpretation of the results. After the development of the lateral flow strip, positive samples for influenza A exhibited the formation of blue test lines at the positions of the influenza A and MS2 phages, negative control samples exhibited the formation of blue test lines only at the position of the MS2 phage, as shown in FIG. 36.
[00101] It is noted that in the specification and in the claims, "about" or "approximately" means within twenty percent (20%) of the numerical value quoted. As used in this document, the singular forms "one", "one" and "o / a" include plural references, unless the context clearly indicates otherwise. Thus, for example, the reference to "a functional group" refers to one or more functional groups, and the reference to "the method" includes reference to equivalent steps and methods that would be understood and appreciated by those skilled in the art, and so on. onwards.
Petition 870190099370, of 10/04/2019, p. 169/173
103/103 [00102] Although the invention has been described in detail with particular reference to disclosed modalities, other modalities can achieve the same results. The variations and modifications of the present invention will be obvious to those skilled in the art and are intended to cover all such modifications and equivalents. All disclosures of all patents and publications cited above are hereby incorporated by reference.

权利要求:
Claims (12)
[1]
1. Cassette to detect a nucleic acid, the cassette characterized by the fact that it comprises at least one reaction chamber;
wherein, when said cassette is oriented vertically, a top of said reaction chamber comprises an inlet and a projection extending downwards in said reaction chamber to minimize or prevent the flow of capillary fluid through said top of said chamber reaction.
[2]
2. Cassette, according to claim 1, characterized by the fact that the projection is generally triangular in shape.
[3]
Cassette according to claim 1, characterized by the fact that a first side of said projection extends substantially vertically adjacent to said entrance.
[4]
4. Cassette according to claim 3, characterized by the fact that a second side of said projection extends upwards towards said top of said reaction chamber at an angle less than approximately 60 degrees from the vertical.
[5]
5. Cassette according to claim 4, characterized by the fact that said angle is less than approximately 45 degrees from the vertical.
[6]
6. Cassette according to claim 5, characterized by the fact that said angle is less than approximately 30 degrees from the vertical.
[7]
7. Cassette, according to claim 6, characterized by the fact that said second side of said projection extends vertically towards said top of said reaction chamber.
[8]
8. Cassette according to claim 1, characterized by the fact that it comprises a recess to contain at least one
Petition 870190099370, of 10/04/2019, p. 171/173
2/2 lyophilized or dry reagent, said recess disposed in a channel connected to said inlet of said reaction chamber.
[9]
9. Cassette, according to claim 8, characterized by the fact that said projection reduces or prevents the sequestration of reagent recently resuspended from most of the volume of the reaction solution.
[10]
10. Cassette according to claim 8, characterized in that said recess comprises one or more structures for directing fluids to facilitate the rehydration of at least one dry or lyophilized reagent.
[11]
11. Cassette according to claim 10, characterized by the fact that said structures comprise grooves, grooves, undulations or combinations thereof.
[12]
12. Cassette according to claim 1, characterized in that said reaction chamber comprises a recess to contain at least one lyophilized or dry reagent.

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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US201762488453P| true| 2017-04-21|2017-04-21|

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PCT/US2018/028668|WO2018195493A1|2017-04-21|2018-04-20|Fluidic test cassette|

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