NZ758378B2 - Fluidic test cassette - Google Patents

Abstract

disposable cassette for detecting nucleic acids or performing other assays which provides a more informative, sensitive and specific point-of-use rapid polymerase chain reaction (PCR)-based tool that doesn't rely on costly elaborate instrumentation, specialized lab materials and/or multiple manipulations dependent on user intervention. The cassette can be inserted into a base station during use. The cassette has numerous features to ensure correct operation of the device under gravity, such as vent pockets for enabling the flow of sample fluid from one chamber to the next when the vent pocket is unsealed. The vent pockets have protrusions to help prevent accidental resealing. The cassette also can have a gasket to ensure free air movement between open vent pockets. A flexible circuit with patterned metallic electrical components disposed on a heat stable material can be in direct contact with fluid in the chambers and has resistive heating elements aligned with the vent pockets and the chambers. Recesses in the cassette channels or chambers can have structures such as ridges or grooves to direct fluid flow to enhance rehydration of lyophilized reagents disposed in the recess. Flow diverters in the chambers can reduce the flow velocity of the sample fluid and increase the effective fluid flow path length, enabling more accurate control of fluid flow in the cassette. The roof of each chamber can have a projection that prevents capillary fluid flow across the top of the chamber, thus reducing or preventing sequestration of newly resuspended reagent from the bulk of the reaction solution volume. lations dependent on user intervention. The cassette can be inserted into a base station during use. The cassette has numerous features to ensure correct operation of the device under gravity, such as vent pockets for enabling the flow of sample fluid from one chamber to the next when the vent pocket is unsealed. The vent pockets have protrusions to help prevent accidental resealing. The cassette also can have a gasket to ensure free air movement between open vent pockets. A flexible circuit with patterned metallic electrical components disposed on a heat stable material can be in direct contact with fluid in the chambers and has resistive heating elements aligned with the vent pockets and the chambers. Recesses in the cassette channels or chambers can have structures such as ridges or grooves to direct fluid flow to enhance rehydration of lyophilized reagents disposed in the recess. Flow diverters in the chambers can reduce the flow velocity of the sample fluid and increase the effective fluid flow path length, enabling more accurate control of fluid flow in the cassette. The roof of each chamber can have a projection that prevents capillary fluid flow across the top of the chamber, thus reducing or preventing sequestration of newly resuspended reagent from the bulk of the reaction solution volume.

Description

A disposable cassette for detecting nucleic acids or performing other assays which provides a more informative, sensitive and specific point-of-use rapid polymerase chain reaction (PCR)- based tool that doesn't rely on costly elaborate instrumentation, specialized lab materials and/ or multiple manipulations dependent on user intervention. The cassette can be inserted into a base station during use. The cassette has numerous features to ensure correct operation of the device under gravity, such as vent pockets for enabling the flow of sample fluid from one chamber to the next when the vent pocket is unsealed. The vent pockets have protrusions to help prevent accidental resealing. The cassette also can have a gasket to ensure free air movement between open vent pockets. A flexible circuit with patterned ic electrical components disposed on a heat stable material can be in direct contact with fluid in the chambers and has resistive heating elements d with the vent pockets and the rs. Recesses in the cassette channels or chambers can have structures such as ridges or grooves to direct fluid flow to enhance rehydration of lyophilized reagents disposed in the recess. Flow diverters in the chambers can reduce the flow velocity of the sample fluid and increase the effective fluid flow path length, enabling more accurate control of fluid flow in the cassette. The roof of each chamber can have a tion that ts capillary fluid flow across the top of the chamber, thus ng or preventing sequestration of newly resuspended t from the bulk of the on solution volume. 758378 B2 FLUIDIC TEST CASSETTE CROSS-REFERENCE TO D APPLICATIONS This application claims priority to and the benefit of filing of US. Provisional Patent Application Serial No. ,453, entitled "Fluidic Test Cassette", filed on April 21, 2017, the ication and claims of which are incorporated herein by reference.

OUND OF THE INVENTION Field of the Invention (Technical Field): 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 le portion.

Background Art: Note that the following discussion may refer to a number of publications and references.

Discussion of such publications herein is given for more complete background of the scientific principles and is not to be construed as an admission that such publications are prior art for patentability determination purposes.

As the public health impact and awareness of ious and emerging diseases, biothreat agents, genetic diseases and environmental reservoirs of pathogens has increased, the need for more informative, ive and specific point-of—use rapid assays has increased the demand for rase chain reaction (PCR)—based tools. Nucleic acid—based molecular testing by such methods as PCR- based amplification is extremely sensitive, specific and informative. Unfortunately, currently available nucleic acid tests are unsuitable or of limited utility forfield use e they require elaborate and costly instrumentation, specialized laboratory materials and/or multiple manipulations dependent on user intervention. Consequently, most samples for molecular testing are shipped to centralized laboratories, resulting in y turn-around-times to obtain the required information.

To address the need for rapid point-of-use molecular g, prior efforts have focused on product designs employing a disposable cartridge and a relatively expensive associated ment.

The use of external instrumentation to accomplish fluid movement, amplification temperature control and detection simplifies many of the engineering challenges inherent to integrating the le processes required for molecular testing. Unfortunately, dependence upon elaborate instrumentation imposes tremendous economic barriers for small clinics, local and state government and law enforcement agencies. Further, dependence upon a small number of instruments to run tests could cause unnecessary delays during periods of increased need, as occurs during a suspected fare agent release or an emerging epidemic. Indeed, the instrument and disposable reagent cartridge model presents a potentially significant bottleneck when an outbreak demands surge capacity and increased throughput. Additionally, instrumentation dependence complicates ad hoc distribution of test devices to deployment sites where logistic aints preclude transportation of bulky associated equipment or infrastructure ements are absent (e.g. le power sources).

Gravity has been described as a means of fluid nt in existing microfluidic devices.

However, the l device does not allow for programmable or onic control of such fluid nt, or the mixing of more than two fluids. Also, some devices utilize a pressure drop generated by a falling inert or pre-packaged fluid to induce a slight vacuum and draw reactants into processing chambers when oriented vertically, which increases e and transport complexities to ensure ity of the pre-packaged fluids. Existing devices which teach moving a fluid in a plurality of discrete steps require frangible seals or valves between chambers, which complicates operation and manufacture. These devices do not teach the use of separate, remotely located vents for each chamber.

Typical microfluidic devices make use of smaller reaction volumes than are employed in standard laboratory ures. PCR or other nucleic acid amplification reactions such as loop mediated amplification (LAMP), nucleic acid based sequence amplification (NASBA) and other isothermal and thermal cycling methods are typically conducted in testing and research laboratories using reaction volumes of 5 to 100 microliters. These reaction volumes odate test specimen volumes sufficient to ensure the detection of scarce assay targets in dilute specimens. Microfluidic systems that reduce on s relative to those employed in traditional laboratory molecular testing necessarily also reduce the volume of en that can be added to the reaction. The result of the smaller reaction volume is a reduction in capacity to accommodate sufficient specimen volume to ensure the presence of detectable amounts of target in dilute specimens or where assay targets are scarce.

SUMMARY OF THE INVENTION The present invention is a cassette for detecting a target nucleic acid, the cassette comprising a plurality of chambers, a plurality of vent pockets connected to the chambers, and a heat labile material for sealing one or more of the vent pockets, wherein at least one the vent pockets comprises a protrusion. The protrusion preferably comprises a dimple or an asperity and ably sufficiently prevents molten heat labile material from attaching to a heat stable material ed adjacent to the heat labile material to prevent resealing of the vent pocket after the heat labile material is ruptured.

The present invention is also a cassette for detecting a target nucleic acid, the cassette comprising a ity of chambers, a plurality of vent pockets connected to the chambers, a heat labile material for sealing one or more of the vent pockets, a heat stable material, and a gasket disposed between the heat labile material and the heat stable al, the gasket comprising an g encompassing the plurality of vent pockets. The gasket is preferably sufficiently thick to provide a sufficient air volume to equilibrate pressures and ensure free air movement between open vent pockets. The cassette preferably comprises a flexible circuit, the flexible circuit comprising patterned metallic electrical ents disposed on the heat stable al. The gasket preferably comprises a second g, or is limited in dimension, 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 ive heating elements are preferably d with the vent pockets and the chambers. The cassette preferably comprises one or more ambient temperature sensors for adjusting a g temperature, heating time, and/or heating rate of one or more of the chambers.

The present invention is also a cassette for detecting a target nucleic acid, the cassette comprising a vertically oriented detection chamber, a lateral flow detection strip ed in the detection chamber oriented such that a sample receiving end of the detection strip is at the bottom end of the detection strip, and a space in the detection chamber below the lateral flow detection strip for receiving fluid comprising amplified target nucleic acids, the space comprising sufficient capacity to odate an entire volume of the fluid at a height that s the fluid to flow up the detection strip by ary action without flooding or othen/vise bypassing s of the detection strip. The space preferably comprises detection particles such as dye polystyrene microspheres, latex, colloidal gold, colloidal cellulose, nanogold, or semiconductor nanocrystals. The detection particles preferably comprise oligonucleotides complementary to a sequence of the amplified target nucleic acids or ligands, such as biotin, streptavidin, a hapten or an antibody, capable of binding to the amplified target nucleic acids. The detection particles have preferably been dried, lyophilized, or t on at least a portion of the interior surface as a dried mixture of detection particles in a carrier, such as a polysaccharide, a detergent, or a protein, to facilitate resuspension of the detection particles. A capillary pool of the fluid ably forms in the space, providing improved mixing and dispersion of the ion particles to facilitate comingling of the detection particles with the amplified target nucleic acid. The cassette optionally performs an assay having a volume less than about 200 uL, and preferably less than about 60 pL.

The t ion is also a cassette for detecting a target nucleic acid, the cassette comprising one or more recesses for containing at least one lized or dried reagent, at least one of the recesses comprising one or more structures for directing fluids to facilitate rehydration of the at least one dried 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 se , s, dimples, or combinations thereof.

The present ion is also a cassette for detecting a target nucleic acid, the cassette comprising at least one chamber comprising a feature to prevent fluid vertically entering a top of the chamberfrom flowing directly into an outlet of the chamber. The feature preferably deflects the fluid to the side of the chamber opposite from the outlet. The resulting flow path of the fluid preferably comprises a horizontal component, thereby sufficiently sing the effective length of the flow path and sufficiently decreasing the flow velocity of the fluid to restrict the amount of fluid exiting the .

The feature preferably creates a swirling of fluid within the chamber, y increasing mixing of reagents within the fluid. The feature is preferably triangular or trapezoidal in shape. The outlet is optionally tapered. A channel located downstream of the outlet optionally comprises turns for increasing an effective length of the channel. The feature is preferably located near or at a bottom of the chamber or near a middle of the chamber.

The present invention is also a method of controlling vertical flow of a fluid through a chamber in a cassette for detecting a target c acid, the method comprising deflecting a flow of fluid entering a top of the chamber, thereby preventing the fluid from flowing directly into an outlet of the chamber. The method ably comprises reducing a flow velocity of the fluid, y reducing a distance the fluid flows down a channel ted to the outlet before the fluid stops. The method preferably comprises dividing a flow of the fluid into the chamber into a first fluid flow that contacts a wall of the chamber and is directed upward, and a second fluid flow that enters the . The first fluid flow preferably swirls in the chamber, thereby increasing mixing of reagents within the fluid. The second fluid flow preferably forms a meniscus and s through a l connected to the outlet, the meniscus sing pressure in closed air space in the channel downstream of the fluid until the pressure stops the flow of fluid in the l. The outlet is optionally tapered, thereby increasing compressible air volume at the entrance to the outlet. The method ally comprises providing turns in a channel connected to the outlet, thereby sing an effective path length of the channel and reducing a flow velocity of fluid in the channel.

The present invention is also a cassette for detecting a c acid, the te comprising at least one reaction chamber, wherein, when the cassette is oriented vertically, a top of the reaction chamber comprises an inlet and a projection extending downward into the reaction chamber to minimize or prevent capillary fluid flow across 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 inlet. A second side of the projection preferably extends upward toward the top of the reaction r at an angle less than approximately 60 degrees from al, more preferably less than approximately 45 degrees from vertical, even more preferably less than approximately 30 degrees from vertical, and optionally vertically. The cassette preferably comprises a recess for containing at least one lyophilized or dried reagent, the recess disposed in a channel connected to the inlet of the reaction chamber. The projection preferably reduces or ts sequestration of newly resuspended reagent from the bulk of the reaction solution volume. The recess preferably comprises one or more structures for directing fluids to tate rehydration of the at least one dried or lyophilized reagent. The structures ably comprise , grooves, dimples, or ations thereof. atively or in on, the reaction chamber comprises a recess for containing at least one lyophilized or dried reagent.

Objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated into and form a part of the specification, illustrate embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The drawings are only for the purpose of illustrating certain embodiments of the invention and are not to be construed as limiting the invention. In the drawings: is a drawing illustrating an embodiment of a test cassette of the present invention. is an exploded view of one ment of the test te revealing the sliding seal, sample port, sample cup and internal region of the expansion r. is a representation of the fluidic network in one embodiment of a test cassette of the invenflon.

FIGS. 28-20 are schematic representations prior to and after vent opening, respectively, of how a heat triggered vent can be ed to vent to an expansion chamber to lish fluid flow control in the context of a hermetically sealed test cassette. is a drawing of one embodiment of a disposable test cassette showing the placement of the printed circuit assembly (PCA) comprising resistive heating elements and temperature sensors. is a photograph of an injection molded c test cassette that includes the features described in . is a representation of the operating principle of an embodiment of the expansion chamber. is a cross-section of the piston-based expansion chamber prior to gas expansion within the test cassette.

FIG. SC is a cross-section of the piston-based expansion chamber after gas expansion within the test cassette. is an ration of an approach to g an expansion chamber wherein an expandable bladder is employed to provide an expanding internal volume. is a section of the bladder-based expansion chamber prior to gas ion within the test cassette. is a cross-section of the bladder-based expansion chamber after gas expansion within the test cassette. is an illustration of an approach to forming an expansion chamber wherein an expandable bellows is employed to provide an ing internal volume. is a cross-section of the bellows-based ion chamber prior to gas expansion within the test cassette. is a cross-section of the bellows-based expansion r after gas expansion within the test cassette. illustrates the use of a semi-permeable barrier, membrane or 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. is a cross-section of the semi-permeable barrier employed in lieu of an expansion chamber to equalize internal pressures with ambient pressures or to reduce internal pressure. is an exploded view of a test cassette design wherein an ion chamber is created by a spacer between a layer of biaxially oriented polystyrene (BOPS) film. is a drawing of an embodiment of a flexible circuit comprising resistive heating elements for two fluid chambers, a detection strip chamber and three vents and electrical contact pads. is an ment of a flexible circuit comprising resistive heating elements for two fluid chambers, a detection strip chamber and three vents and electrical contact pads for zing the resistive heating elements. is an exploded view of an ment of a test cassette. is a view of the led test cassette of . depicts lateral flow strips from s with and without a capillary pool at the sample receiving end of the strip. More uniform distribution of detection particles and more uniform signal across the strip is observed when a capillary pool is t. is a m illustrating a hierarchical approach to sample splitting. is an illustration of a multiple l fluidic network for multiplexing and sample subdivision showing the fluid flow path for each test. Additional c paths or channels can be incorporated into the network to further se the number of parallel tests that can be performed simultaneously in a single disposable test cassette. is a representation of the fluidic network in one embodiment of a test cassette of the invention wherein a sample is split following introduction to the sample cup via the sample port to enable parallel independent tests on the same input sample. A ating fluid path from the sample cup allows sample solution to be split into two distinct fluidic channels or paths of the test cassette to allow simultaneous tests to run in parallel on the split sample.

A is a drawing of an assembled sample preparation subsystem showing the internal component arrangement. 8 is an exploded view of the sample preparation subsystem showing components of the nucleic acid purification apparatus configured for integration with a test cassette. is a cross-section through the sample preparation subsystem illustrating the movements of components occurring in the course of sing a sample. is an ed view drawing showing the sample preparation tem with hermetic seal components, injection molded c subsystem, corresponding cassette backing and is a photograph of a test cassette embodiment with an integrated sample preparation subsystem.

A is an exploded view drawing showing a sample ation subsystem with hermetic seal components and injection molded fluidic tem design.

B is a drawing of the test cassette embodiment with integrated sample preparation subsystem shown interfaced to the PCA. 0 is a cutaway drawing of the test cassette embodiment depicting the fluidic paths, interfaced electronics and sample preparation ents.

A is a drawing of an embodiment of a docking unit of the present invention shown with the lid in the open position and a test cassette inserted.

B is a drawing of the docking unit shown with the lid in the closed position. is a photograph of one embodiment of the docking unit shown with the lid in the open position and a test cassette inserted. The LCD display indicates detection of the insertion of an influenza A/B test cassette. illustrates an embodiment of a cassette sealing mechanism of the present invention.

A is a g of the cassette seal sensor placement within the docking unit with an inserted cassette with the seal in the open position.

B is a cutaway drawing of the cassette seal sensor ent within the g unit with an inserted cassette with the seal in the open position. 0 is a drawing of the cassette seal sensor placement within the docking unit with an inserted cassette with the seal in the closed position.

D is a cutaway drawing of the te seal sensor ent within the docking unit with an inserted cassette with the seal in the closed position. is a drawing of an embodiment of the cassette sealing mechanism wherein a drive gear is employed to mediate seal closure using a rotating valve.

A is a drawing rating an embodiment of the test te wherein the lid is a hinged lid comprising an o-ring seal and a vacant air volume that serves as an expansion chamber. In this drawing the lid is in the open position.

B is a drawing g the lid in the closed position, where the o-ring forms a hermetic seal with the rim of the sample port.

A is an exploded view of the heater board and test cassette holder components of the docking unit forming the test cassette receiving subassembly.

B is a drawing of an embodiment of the test cassette receiving subassembly of the docking unit. is a slide view of the test cassette holder and heater board mounting system in the engaged and disengaged positions. is a drawing depicting the placement of infrared ature sensors in one embodiment of the docking unit to monitor the temperature of first and second heated fluidic chambers.

A is a drawing showing l sensor placement within an embodiment of the docking unit to allow reading of a barcode located near the bottom of the test cassette.

B is a detail of A.

FIGS. 28A and 288 are exploded and assembled drawings, respectively, of a double heat board configuration wherein the test cassette is sandwiched between two heater board assemblies.

FIGS. 29A and 298 are solid and transparent drawings, respectively, of a docking unit ment wherein a pivoting door is used to receive a test cassette. Closure of the pivoting door brings the rear of the test cassette into t with the heater board mounted within the docking unit.

FIGS. 30A and 308 are front and side cutaway views respectively of the internal components of a docking unit comprising servo motors for actuating sample preparation and an optical system for test results collection.

FIGS. 31A and 31B are front and side view photographs of an optical subsystem for an embodiment of the docking unit that incorporates a test reader.

A and 32B are photographs of a docking unit embodiment with a pivoting test cassette receiving door in the open position and closed position, respectively. shows a reusable embly for a docking unit of the present invention. shows test s obtained in Example 1 described herein. shows test results obtained in Example 2 described herein. shows test results obtained in Example 3 described herein.

A is a perspective view of a cassette comprising three rs.

B is an exploded view of the cassette of A. is a transparent view of the cassette of A g fluidic features. shows an embodiment of a r of the present invention comprising a ular protruding flow feature and a tapered . shows an embodiment of a chamber of the present invention comprising a triangular protruding flow feature and a parallel . shows an embodiment of a r of the t invention comprising a trapezoidal protruding flow feature and a parallel outlet. shows an embodiment of a chamber of the present invention comprising stacked triangularflow features and a parallel outlet. shows an embodiment of a chamber of the present invention comprising a protruding flow feature in approximately the middle of the chamber. shows a reagent recess comprising internal features for directing fluid flow. shows an embodiment of a vent pocket of the present invention comprising a dimple structure.

A shows a drawing of an embodiment of a fluidic layer of a te of the t invention comprising lized reagent recesses disposed in the fluid flow paths. B is a magnified view of a recess and reaction r, showing a vertical projection extending into the chamber.

A shows a drawing of an embodiment of a fluidic layer of a cassette of the present invention comprising lyophilized reagent recesses disposed in one or more reaction chambers. B is a magnified view of a recess in a reaction chamber, showing a vertical projection extending into the chamber. shows a reaction chamber without a vertical tion.

DETAILED PTION OF THE INVENTION An embodiment of the present invention is a sealable disposable platform for detecting a target nucleic acid, the disposable platform preferably sing 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 ted via a second channel to a first vent pocket, a labeling chamber connected via a third l to the amplification chamber and connected via a fourth channel to a second vent pocket, a detection subsystem connected to the labeling chamber via a fifth channel and connected via a sixth channel to a third vent pocket, a plurality of resistive heating elements, and one or more ature measuring devices, wherein the vent pockets are each sealed from communication with an air chamber by a heat labile material in a suitable form, such as a membrane, a film, or a plastic sheet located in a vicinity of one or more of the resistive heating elements. The disposable platform optionally comprises a seal to seal the platform prior to the initiation of the detection assay. The disposable platform preferably comprises recesses along channels between chambers to accommodate the incorporation of dried or lyophilized reagents into the disposable platform. These es may optionally comprise structures on one or more of the surfaces facing the reagent(s) to assist with directing fluids, preferably using capillarity or surface n effects, to the enclosed dried reagents to facilitate rehydration of the dried reagents. Such features may comprise ridges, such as ridge 7001 of , grooves, dimples or other structures to direct fluids to the internal space of the recess as the fluid passes through the recess, or otherwise assist in fluid flow to the internal space of the recess during fluid flow. atively, a recess may be directly located within one (or more) of the chambers.

The able platform optionally further comprises a sample preparation stage comprising an output in direct fluid connection with an input of the sample r. Dimensions of a substantially flat surface of the amplification r are preferably approximately the same as dimensions of a substantially flat surface of a resistive heating element in thermal contact with the amplification r. The amplification chamber optionally contains an amplification solution and a recess in the channel from the sample chamber to the amplification chamber optionally comprises a Iyophilized amplification reagent mix, and there is preferably a recess in the channel from the amplification r to the labeling chamber comprising dried or Iyophilized ion particles. The amplification and labeling chambers are preferably heatable using resistive heating elements. The detection subsystem preferably comprises a lateral flow strip that comprises detection particles. The chambers, the ls, and the vent pockets are preferably d on a fluid assembly layer, and the electronic elements of the device are preferably d on a separate layer comprising a printed circuit board, the separate layer bonded to the fluid assembly layer or placed in contact with the fluid assembly layer by a docking unit. The detection tem is preferably located on the fluid ly layer or optionally on a second fluid assembly 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 docking the disposable platform with a docking unit, which preferably ins the disposable platform in a vertical or tilted orientation and optionally provides electrical contacts, components and/or a power supply.

An embodiment of the present invention is a method for detecting one or more target nucleic acids, the method preferably comprising dispensing a sample comprising the target nucleic acid in a sample chamber of a able platform; orienting the disposable platform vertically or at a tilt; opening a first vent pocket connected to an amplification chamber to an enclosed air , thereby enabling the sample to flow into the ication chamber, reacting the sample with a previously Iyophilized amplification reagent mix located in a recess of the channel between sample chamber and amplification chamber, amplifying the target nucleic acid in the amplification chamber, opening a second vent pocket ted to a labeling chamber to an enclosed air volume, y enabling the amplified target nucleic acid to flow into the labeling chamber, labeling the amplified target nucleic acid using detection particles in a recess in the channel between the amplification chamber and the labeling chamber, opening a third vent pocket connected to a detection subsystem to an enclosed air volume, thereby enabling the labeled target nucleic acid to flow into the ion subsystem, and detecting the amplified target nucleic acid. The amplifying step preferably comprises amplifying the target nucleic acid using a resistive heating element located within the disposable rm in a vicinity of the ication chamber. The method preferably further comprises passively cooling the amplification r. The method preferably further comprises heating the ng chamber during the ng step using a resistive heating element located within the disposable platform in a vicinity of the labeling chamber. The method preferably further comprises controlling operation of the disposable rm by using a docking unit which is not an external instrument.

Embodiments of the present invention comprise a disposable platform which integrates external instrument-independent means of conducting all requisite steps of a nucleic acid molecular assay and complements current immuno-Iateral flow rapid assays with a new generation of nucleic acid tests offering more informative and sensitive analyses. Embodiments of the present invention facilitate the broader use of rapid nucleic acid testing in small clinics and austere or remote settings where infectious disease, biothreat agents, agriculture and environmental testing are the most likely to have the greatest impact. Certain embodiments of the present invention are completely self-contained and disposable which s "surge capacity" in times of sed demand by allowing el tests to be run without external instrument-imposed bottlenecks. Additionally, in those application areas where a low cost disposable cartridge d with an inexpensive battery-powered or AC adapter energized docking unit is able, an embodiment of the invention where a simple docking unit is employed r reduces test costs by placing reusable ents in a reusable yet inexpensive base. The platform technology disclosed herein offers sensitivity similar to laboratory nucleic acid amplification-based methods, minimal user intervention and training requirements, ce specificity imparted by both amplification and detection, multiplex capacity, stable reagents, compatibility with low-cost large-scale manufacturing, battery or solar powered ion to allow use in e settings, and a le platform technology allowing the incorporation of additional or alternative biomarkers without device redesign.

Embodiments of the present invention provide systems and methods for low-cost, point-of-use nucleic acid detection and identification suitable to perform analyses in locations remote from a laboratory environment where testing would ordinarily be performed. Advantageously, nucleic acid amplification reaction volumes can be in the same volume range ly used in traditional laboratory testing (e.g. 5-150 uL). The reaction ted in embodiments of the present invention is thus directly comparable to ed laboratory assays, and allows the accommodation of the same en volumes typically employed in traditional molecular g. Furthermore, the amplification of nucleic acids preferably takes place in a hermetically sealed test cassette that is preferably permanently sealed prior to the initiation of amplification. Retaining amplified nucleic acids within a sealed system ts contamination of the testing environment and surrounding areas with amplification products and ore reduces the likelihood subsequent tests will generate false positive s. The integration of a sealing system into the test cassette enables the use of a corresponding seal engagement system in the docking unit to enforce the formation of a seal at the time of assay initiation. In an embodiment of the ion, a rack and pinion mechanism is employed to slide a test cassette integrated sealing mechanism into place to ensure seal closure prior to amplification. A sensor placed in the docking unit interrogates the test cassette to confirm the seal has been formed prior to initiating the test reaction.

Embodiments of the present invention may be produced using ion molding processes and ultrasonic welding to achieve high-throughput cture and low cost disposable components.

In some embodiments one or more recesses are provided in the fluidic component to each accommodate a dried reagent pellet. The recesses enable the use of lyophilized or ise dried materials to be present in the fluidic component during final assembly when ultrasonic welding may be used t disruption of the pellet by any energy introduced to the system during the welding.

Embodiments of the present invention may be used to detect the presence of a target nucleic acid sequence or sequences in a sample. Target sequences may be DNA such as chromosomal DNA or extra-chromosomal DNA (e.g. mitochondrial DNA, chloroplast DNA, plasmid DNA, etc.) or RNA (e.g. rRNA, mRNA, small RNAs, or viral RNA). Similarly, embodiments of the invention may be used to identify nucleic acid polymorphisms ing single nucleotide polymorphisms, deletions, insertions, inversions and sequence duplications. Further, embodiments of the ion may be used to detect gene regulation events such as gene up- and down-regulation at the level of ription. Thus, embodiments of the invention may be employed for such applications as: 1) the detection and identification of pathogen nucleic acids in agricultural, clinical, food, environmental and veterinary samples; 2) detection of genetic biomarkers of disease; and 3) the diagnosis of the presence of a disease or a metabolic state through the detection of relevant biomarkers of the disease or metabolic state, such as gene regulation events (mRNA up- or down regulation or the induction of small RNAs or other nucleic acid les generated or repressed during a e or metabolic state) that occur in response to the presence of a pathogen, toxin, other etiologic agent, environmental us or lic state.

Embodiments of the present invention comprise a means of target nucleic acid sample preparation, amplification, and ion upon on of 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 ming c acid testing using a portable power supply such as a battery, and is fully disposable. In other embodiments of the invention, a disposable nucleic acid test cartridge works in conjunction with a simple reusable electronic component which can perform all of the functions of laboratory instrumentation such as an external instrument lly required for nucleic acid testing without ing the use of such laboratory mentation or external instrument.

Embodiments of the present invention provide for a nucleic acid amplification and detection device comprising, but not limited to, a housing, a circuit board, and a fluidic or luidic component. In certain embodiments, the circuit board may contain a variety of surface-mount components such as resistors, stors, light-emitting diodes (LEDs), photo-diodes, and microcontrollers. In certain embodiments the circuit board may comprise a flexible circuit board comprising a heat stable substrate such as polyimide. Flexible circuits may, in some embodiments, comprise copper or other conductive coatings or layers deposited onto or bonded to the heat stable substrate. These coatings can be etched or otherwise patterned to so as to comprise 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- ng diodes (LEDs), photo-diodes, and microcontrollers. The fluidic or luidic component is the device portion which receives, contains, and moves aqueous samples and may be made from a variety of cs and by a variety of manufacturing techniques, including ultrasonic welding, bonding, fusing or lamination, laser cutting, water-jet cutting, and/or ion g. The fluidics and t board components are held together either reversibly or irreversibly, and their thermal coupling may be enhanced by heat conducting als or compounds. The housing preferably serves in part as a cosmetic and protective sheath, hiding the delicate components of the microfluidic and circuit board , and may also serve to facilitate sample input, buffer release, nucleic acid elution, seal formation and the tion of processes required for device functionality. For example, the housing may incorporate a sample input port, a mechanical system for the formation or engagement of a seal, a button or similar mechanical feature to allow user activation, buffer release, sample flow initiation, c acid elution, and thermal or other physical interface formation between electronic components and fluidic components.

In some embodiments of the invention, the fluidic or microfluidic component comprises a series of chambers in controlled fluid communication where the rs are optionally temperature- lled, thereby subjecting the fluid contained therein to programmable temperature regimens. In some embodiments of the invention, the c or microfluidic component comprises five chambers, preferably including an expansion chamber, a sample input chamber, a reverse transcription chamber, an amplification r, and a detection chamber. The sample input chamber preferably comprises a conduit to the expansion r, a sample input orifice where a nucleic acid containing sample may be added, a first recess wherein dried materials may be placed during cture for mixing with the input sample, an egress conduit leading to a second recess wherein dried materials may be placed during manufacture and a conduit leading therefrom to the e transcription chamber. In other embodiments functions of two or more of the chambers are consolidated into a single chamber, enabling the use of fewer chambers.

The first and second recesses may also comprise lyophilized reagents that may include, for example, suitable buffers, salt, deoxyribonucleotides, ribonucleotides, oligonucleotide primers, and enzymes such as DNA rase and reverse transcriptase. Such lyophilized reagents are ably solubilized upon entrance of the nucleic acid sample into 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 c acids present in the input . In some embodiments of the invention the input sample is heated in the sample input chamber to accomplish the lysis of 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 cDNA from RNA. In an ment of the invention the second recess is sufficiently isolated from the sample input chamber to allow materials within the second recess to maintain a lower temperature than the temperature of the sample input r 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 c acids. The sample input chamber, the reverse transcription chamber, the amplification chamber and the detection chamber are preferably situated in register with and in ient ity to the heater elements on the heater circuit board to provide thermal conduction when mounted to the heater board either directly or through insertion of the fluidic or microfluidic ent or te into a docking unit. Similarly, electronic components present on the heater circuit board are preferably placed in physical contact or proximity to vent pockets in the fluidic ent to enable electronic control by opening of the vent. The heater circuit board physical layout is designed to e registration with elements of the fluidic or microfluidic component such that ive heating elements of the heater circuit board for lysis, reverse transcription, amplification, hybridization, andfor fluid flow control are situated to form a thermal interface with ts of the fluidic component with which they interact.

In some embodiments of the invention the fluidic or microfluidic component preferably comprises five chambers, including a sample input chamber, a lysis r, a e transcription chamber, an amplification chamber, and a detection chamber and recesses for dried or lyophilized reagents located along the channels between each chamber. In this embodiment reverse transcription of RNA to cDNA and the amplification of cDNA occur in separate chambers. In this embodiment, a first recess, located along the conduit leading from the sample input cup to the lysis chamber, comprises salts, chemicals (e.g. dithiothreitol) and buffers (e.g. to stabilize, increase, or decrease 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 input sample is heated in the heat lysis chamber having first flowed from the sample input cup through the first recess wherein the sample has optionally comingled with the substances that se the first recess. In other embodiments of the ion, lysis is accomplished by means of chemical treatment resulting from the ling of the sample with als in the first recess and the incubation of the sample in the presence of these chemicals in the lysis chamber.

After substantial completion of treatment in the lysis chamber, the sample solution is released by means of onic l of a heater that chanically ruptures a vent to allow the sample solution to flow via a channel through a second recess and into the reverse transcription chamber.

Said second recess may optionally comprise lyophilized reagents that may include suitable buffers, salt, deoxyribonucleotides, ribonucleotides, ucleotide s, and enzymes such as DNA polymerase and/or reverse transcriptase ed to accomplish the reverse transcription of RNA in the sample into cDNA. Following the substantial completion of a reverse transcription on, a second vent is opened to release the sample solution to flow h a channel and third recess comprised of reagents required for nucleic acid amplification such as lyophilized reagents that may include suitable s, salt, deoxyribonucleotides, ribonucleotides, oligonucleotide primers, and enzymes such as DNA polymerase and into an ication chamber.

Following the substantial completion of nucleic acid amplification in the amplification chamber a third vent is opened to release the sample solution to a channel leading to the detection chamber.

Said channel may optionally but preferably comprise a fourth recess comprising dried or lyophilized detection reagents such as chemicals and/or detection particle conjugates useful for the ion of nucleic acids in the detection chamber. The detection chamber preferably comprises a capillary pool, reagents for the detection of the amplified nucleic acid and a lateral flow detection strip. The capillary pool preferably provides a space of sufficient capacity to accommodate the entire volume of fluid in the ion chamber at a height that enables the fluid to flow up the detection strip by capillary action without flooding or otherwise bypassing the regions of the detection strip designed to receive the fluid for t capillary migration up the detection strip. In some embodiments of the invention the detection reagents are lyophilized ts. In some embodiments of the invention the detection reagents comprise dyed polystyrene microspheres, colloidal gold, semiconductor nanocrystals, or cellulose nanoparticles. The sample solution comingles with the detection reagents in the detection chamber and flows by capillary action up the detection strip. Microheaters in register with the detection chamber may optionally be ed to control the temperature of the solution as it migrates up the detection strip.

In some embodiments of the invention the amplification reaction is an asymmetric amplification reaction wherein one primer of each primer pair in the reaction is present at a concentration different from the other primer of a given pair. Asymmetric reactions can be useful for the generation of single-stranded nucleic acid for the facilitation ofdetection by hybridization.

Asymmetric reactions can also be useful for generating amplicons in a linear amplification reaction allowing quantitative or semi-quantitative analysis of target levels in a sample.

Other embodiments of the invention comprise a nucleic acid reverse transcription, amplification and detection device that is integrated with a sample preparation . Embodiments including the sample ation device provide a means for the communication of fluids between sample preparation subsystem output ports or valves and the input port or ports of the fluidic or microfluidic components of the device.

Other embodiments of the invention comprise a means of splitting the input sample into two or more fluid paths in the fluidic or microfluidic component. A means of splitting the input sample comprises a branched conduit to carry input fluids to a metering r of a volume designed to divide the fluid across multiple fluid paths. Each ng r comprises a l conduit to a vent pocket and a channel conduit to the next r in the fluid path, for example a lysis chamber or a reverse transcription chamber or an amplification chamber.

Unless othenNise d, all terms of art, notations and other scientific terminology used herein are ed to have the meanings ly understood by those of skill in the art to which this invention ns. The techniques and procedures bed or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., lar Cloning: A Laboratory Manual 3rd. n (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 ing the use of commercially available kits and reagents are lly carried out in accordance with manufacturer defined protocols and/or parameters unless othenNise noted.

As used throughout the specification and claims, the terms ‘target nucleic acid’ or ‘template nucleic acid’ mean a —stranded or double-stranded DNA or RNA fragment or sequence that is intended to be detected.

As used throughout the ication and claims, the terms ‘microparticle’ or ‘detection particle’ mean any compound used to label nucleic acid product generated during an amplification reaction, including scent dyes specific for duplex nucleic acid, fluorescently modified oligonucleotides, and oligonucleotide-conjugated quantum dots or solid-phase elements such as a polystyrene, latex, cellulose or paramagnetic particles or microspheres.

As used throughout the specification and claims, the term ‘chamber’ means a fluidic compartment where fluid resides for some period of time. For example, a chamber may be the sample chamber, amplification chamber, labeling chamber, or the detection chamber.

As used hout the specification and claims, the term "cassette" is defined as a disposable or consumable cassette, housing, component, or cartridge used in performing an assay or other al or mical analysis. A cassette may be single use or multiple use.

As used throughout the specification and , the term t’ means a compartment that serves as a venting mechanism. A pocket is preferably adjacent or overlaid to a resistor or other mechanism to open the pocket. For example, unlike fluidic chambers as described above, a pocket created in the fluidic component of the cassette may have one open face that aligns with a resistor on the PCA. This open face is preferably covered by a thin membrane, film, or other material to create a sealed cavity that is easily ruptured by energizing the underlying resistor.

As used throughout the specification and claims, the term el’ means a narrow conduit within the fluidic assembly which typically connects two or more chambers and/or pockets or combinations thereof, including, for example, an inlet, outlet, or a vent channel. In the case of an inlet or outlet channel, fluid sample migrates through the channel. In the case of a vent channel, the t ably remains clear of fluid and connects a fluidic chamber to a vent pocket.

As used throughout the specification and 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 assay or cassette other than sealing the cassette, including but not limited to ng buffer packets and/or pumping or othen/vise actively providing a transport force for fluids, comprises moving parts to control valves and other components for fluid flow control in the cassette or disposable assay, controls fluid flow other than by selective heating of the assay, or requires periodic calibration.

As used hout the specification and claims, the term ng unit" means a le device that controls assays but does not have any of the characteristics listed above for external instruments.

Embodiments of the present invention are devices for low-cost, point-of-use c acid testing le to perform analyses in locations remote from a tory environment where testing would ordinarily be performed. Certain devices comprise fluidic and electronic components or layers, optionally d by a protective housing. In embodiments of the present invention, the fluidic component is composed of plastic and comprises a series of chambers and pockets connected by narrow ls in which chambers are ed vertically with t to one another during operation. The fluidic component is overlaid or othenNise placed in physical contact with electronic components, preferably controlled via a microcontroller, such as a printed circuit board containing off- the-shelf surface mount devices (SMDs), and/or a le circuit comprising etched conductive material to form resistive heating ts and optionally containing SMDs. In some embodiments of the device, the entire assembly is disposable. In other embodiments, the fluidic and physically bonded electronic layers are disposable, while a small inexpensive controlling unit is reusable. In another embodiment, the fluidic component is disposable, and a small controlling docking unit or docking unit is le. For all embodiments, the present invention may be integrated with a nucleic acid sample preparation device such as that described in International Publication No. ed "Highly Simplified Lateral Flow-Based Nucleic Acid Sample Preparation and Passive Fluid Flow Control" (incorporated herein by reference), and/or use methods described therein.

Embodiments of the present invention comprise an integrated c acid testing device that can be manufactured inexpensively with established manufacturing ses. The invention provides molecular test data while retaining the simplicity from the end-user perspective of widely accepted hand-held assays, overcoming the challenges of ting fluid temperatures within the device, transporting small sample volumes in sequential steps, t addition, reagent mixing, detecting nucleic acids. In some embodiments of the invention subsystems for collecting, interpreting, ing and/or transmitting assay results are incorporated into the invention. ments of the t invention are uniquely adapted to utilize off-the-shelf electronic elements that may be constructed by standard assembly techniques, and requires no or few moving parts. Furthermore, the fluid layer design enables the use of readily available plastics and manufacturing techniques. The result is an inexpensive, disposable, and reliable device e of nucleic acid isolation, amplification, and detection without the need for a dedicated laboratory infrastructure.

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, heating of the on solution is preferably accomplished by use of simple resistive surface-mount devices that may be purchased for pennies or less and are assembled and tested by common manufacturing standards. By layering fluidic chambers over these resistive elements and associated sensor ts, the fluid ature of the reaction solutions may be conveniently ted. The broad 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 realized using heating elements formed by patterns fabricated 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 rapid g as well. on chambers in the present invention are preferably heated on one side and the ambient temperature across the te face is used to help reduce fluid temperature. In addition, vertical orientation of embodiments of the device allows for more rapid cooling by passive convection than if the device was oriented horizontally, thus, reducing the thermal cycle period without the use of costly devices such as Peltier devices. In some embodiments of the invention a fan is used to facilitate cooling.

Fluid control is another challenge associated with st nucleic acid test device s.

Devices known in the art generally employ electromechanical, electrokinetic, or piezoelectric g isms to manipulate fluids during device ion. These pumping elements increase both device complexity and cost. Similarly, valves making use of elaborate micromechanical designs or moving parts can increase fabrication costs and reduce reliability due to complications such as moving part failure or bio-fouling. Unlike previously described nucleic acid testing devices, embodiments of the present invention e tatic 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 for the reaction solution to cascade from chamber to chamber under ontroller control to accommodate ed manipulations of the assay. Fluid may be held in individual reaction chambers through a balance of channel size, hydrostatic pressure and surface tension, where surface tension and hydrostatic pressure prohibits fluid advancement by gas cement. A sample advances to the lower chamber preferably only after tion of a simple venting mechanism under microcontroller control. Once open, the vent allows fluid to move from a first chamber to a second chamber by means of providing a path for displaced air to escape from the second chamber as fluid . Each chamber (or each channel between chambers) within the c component preferably connects to a sealed vent pocket through a narrow vent channel. The vent pocket is preferably sealed on one face with a thin, heat labile plastic membrane or sheet that is easily ruptured by heating a small surface mount resistor underlying, near, or adjacent to the membrane or sheet. Once the vent of a lower chamber is opened, fluid advancement proceeds, even under low hydrostatic res.

As more specifically described below, the fluidic or microfluidic vent mechanism used in some embodiments of the present invention preferably employs a heating t in thermal and (optional) physical contact with a heat labile seal to enable electronic l of fluid movement by means of venting a chamber of lower elevation to allow a fluid from a chamber of higher elevation to flow into the lower chamber. In one embodiment, a resistor is mounted on a printed circuit board, using widely used and stablished electronics cturing methods, and placed in physical contact with a channel seal comprising a heat labile material. When energized the surface mount resistor generates sufficient heat to rupture the seal, which results in the venting of the chamber to allow equilibration of pressure in the region or chamber where fluid is being moved with the region or chamber where fluid is resident prior to venting. The equilibration of the pressure between the chambers allows the movement of fluid from a r of higher elevation to a chamber of lower ion. A direct seal between higher and lower elevation chambers is preferably not employed. The channel and vent seal may be located ly from the fluid chambers, thus facilitating fluidic device layout in configurations efficient for manufacture. The seal material may comprise any material that can seal the vent channel and be ruptured from heating as described, for example a thin c sheet. This ch to fluid movement control in the apparatus benefits from low materials costs, suitability for manufacture using established manufacturing techniques while providing the capacity 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 the vents (and not to seal the fluid rs or fluid hannels themselves) and an electronic means of breaking said seal with heat provides a means of controlling fluid flow through the device to enable movement of fluid at predetermined times or following the completion of specific events (for example, attaining a temperature, a temperature change or a series of temperature changes, or the tion of an incubation time or times or other events). In some embodiments, a blockage may be introduced to the channel between chambers when gas phase water must be isolated from a chamber connected by said l. The blockage may be a soluble material that dissolves upon contact with liquid water following vent opening or a readily melted material such as paraffin that can be removed by the introduction of heat to the site of blockage.

In on, the vent approach has a number of ages over sealing the fluid chambers themselves. Vent pockets can be located anywhere on the fluidics layout and simply communicate with the chamber they regulate via a vent channel. From a manufacturing oint, vent pockets can be localized so that only a single sealing membrane for all vent pockets (which may comprise a vent pocket manifold) is affixed to the fluidic component, preferably by well established methods such as adhesives, heat lamination, ultrasonic welding, laser welding etc. In contrast, directly sealing a fluid chamber requires that the seal material be placed at different locations corresponding to each chamber location, which is more difficult to manufacture. This presents a more challenging scenario during manufacture compared to a single vent pocket manifold sealed by a single membrane.

Additionally, if chambers are directly sealed, melted sealing material can remain in the channels between chambers, occluding flow. The viscosity of the sealing material may require more pressure in the fluid column than is obtained in a miniaturized gravity driven apparatus.

In embodiments of the present invention, reagent mixing requires no more complexity than other systems. ts necessary for nucleic acid amplification such as s, salts, ibonucleotides, oligonucleotide primers, and enzymes are preferably stably incorporated by use of lyophilized pellets or cakes. These lyophilized reagents, sealed in a fluidic chamber, a recess in a fluidic chamber or a recess in a channel, may be readily solubilized upon t with aqueous solution. In the case that additional mixing is ed, the vertical orientation of embodiments of the present invention offers opportunities for novel methods of mixing solutions. By utilizing heaters underlying fluidic chambers, gas may be heated, ring bubbles to the reaction solution in the chamber above when the on contains thermally-sensitive components. Alternatively, heaters may be used to directly heat a solution to the point that boiling occurs, in the case that the solution contains no thermally-sensitive components. The occurrence of air bubbles is often undesirable in previously disclosed c and microfluidic s, as they may accumulate in c chambers and channels and displace reaction ons or impede fluid movement within the device. The vertical design of embodiments of the invention presented herein allows s to rise to the fluid surface, ing in only minimal and transient fluid displacement, effectively ameliorating any disadvantageous impacts of bubbles on the fluidic or microfluidic system. Mixing by boiling is also convenient with this vertical design as fluid displaced during the process simply returns to the al c chamber by gravity after the heating elements are turned off.

In embodiments of the invention, a colorimetric detection strip is used to detect amplified nucleic acids. Lateral flow assays are commonly used in —assay tests due to their ease of use, reliability, and low cost. The prior art contains descriptions of the use of lateral flow strips for the detection of nucleic acids using porous materials as a sample receiving zone which is at or near a labeling zone also comprised of a porous material and placed at or near one end of the lateral flow assay device. In these prior inventions labeling moieties are in the labeling zone. The use of porous materials as the sample receiving zone and the labeling zone results in the retention of some sample solution as well as ion particles in the porous materials. Although labeling zones comprising porous materials having reversibly immobilized es required for detection may be used in embodiments of the present invention, embodiments of the present invention preferably utilize detection particles or moieties held in a region of the device ct from the sample receiving zone of the lateral flow strip and comprising nonporous materials with low fluid retention characteristics. This ch allows nucleic acid target containing samples to be d prior to introduction to the porous components of the sample receiving end of the lateral flow component of the device and thereby eliminates the retention and/or loss of sample material and detection particles in a porous labeling zone. This method r enables the use of various treatments of the sample in the presence of detection moieties, such as treatment with high temperatures, to lish denaturation of a double-stranded target or ary structures within a single-stranded target without concern for the impacts of temperature on porous sample receiving or labeling zone materials or the lateral flow detection strip materials. Additionally, the use of a labeling zone not in lateral flow contact with the sample receiving zone but subject to the control of fluidic components such as vents allows target and label to remain in contact for periods of time controlled by fluid flow control systems. Thus embodiments of the present invention can be different than traditional lateral flow test strips wherein sample and detection particle interaction times and conditions are determined by the capillary transport properties of the als. By incorporating the detection les in a temperature- ted r, ration of duplex nucleic acid is possible allowing for efficient hybridization- based detection. In alternative embodiments, fluorescence is used to detect nucleic acid amplification using a combination of LEDs, photodiodes, and optical filters. These optical detection systems can be used to perform real-time nucleic acid detection and quantification during amplification and end-point detection after amplification.

Embodiments of the invention comprise a low cost, point-of-use system is provided wherein a c acid sample may be ively amplified and detected. Further embodiments include ation with a nucleic acid sample preparation device such as that described in International Publication No.

Sample Preparation and Passive Fluid Flow Control". An embodiment of the device preferably comprises both a plastic fluidic component and a printed circuit assembly (PCA) and/or flexible circuit, and is optionally encased in a housing that protects the active components. Temperature regulation, fluid and reagent mixing are preferably coordinated by a ontroller. The reaction cassette is ably oriented and run vertically so that gravity, hydrostatic pressure, capillary forces and surface tension, in conjunction with microcontroller red vents, control fluid movement within the device.

In embodiments of the present ion, prepared or crude sample fluid enters a sample port and fills or partially fills a sample cup. Sample may be retained, for g periods of time, in the sample cup where dried or lyophilized reagents can mix with the sample. Such reagents as ve control reagents, control templates, or chemical reagents beneficial to the performance of the test may be uced to the sample solution by inclusion in dry, liquid or lyophilized form in the sample cup.

Other treatments such as controlled temperature incubations or heat lysis of bacterial or viral analytes may optionally be accomplished in the sample cup my means of an ying eater and temperature sensor system interfaced to temperature control onics. A fluid k comprises the sample port through which sample is introduced to the cassette either manually by the user or via an automated system, e.g. a subsystem integral to the docking unit or a sample processing subsystem; the sample cup n sample is held to facilitate accumulation during sample introduction and to add reagents, components to perform treatments required prior to further movement of the sample into the downstream portions of the fluid network (e.g. heat treatment to perform lysis of a bacterial cell or virus); a recirculation vent passage for the equilibration of air, gas or solution pressures of the fluidic channels and/or chambers with the pressure of the expansion chamber of the cassette; a bead recess wherein a t bead (e.g. a bead or pellet of material, reagent, chemicals, biological agents, proteins, enzymes or other substances or mixes of these substances) in a dried/desiccated or lyophilized or semidry state may be rehydrated by the sample solution or a buffer solution introduced to the te prior to the addition of sample to rehydrate the bead or pellet contained n and thus comingle the materials n to the sample solution; a set of one or more vents that can be opened to control fluid movement within the cassette; a first chamber where the sample can be subjected to a regimen of temperatures; an optional barrier within the c channel connecting the first chamber with a second chamber to preclude premature invasion of liquids and/or gases into the second chamber or to temporally control the movement of solution or gases into the second chamber; a second chamber wherein the sample solution may be subjected to further temperature regimens optionally following on of reagents from an optional reagent bead recess optionally located between first and second chambers; a test strip recess forming a chamber wherein a test strip is mounted to detect an analyte or a reporter le or other substance indicative of the presence of an analyte. In some embodiments the cassette is ed into a docking unit which performs the functions of sealing the cassette, n, detection, and data transmission. Preferably no user intervention is required once the cassette is inserted into the docking unit, the sample is loaded, and the lid is closed.

Referring to the representative gs of cassette 2500 in FIGS. 1-2, a c acid sample is added to the sample cup 10 in fluidic component 5 through the sample port 20. Sliding seal 91 is moved to the closed position by closure of the docking unit lid at the time of assay initiation. Cover 25 holds slide 91 in place in order to seal expansion chamber 52. The nucleic acid sample may derive from an online (i.e. integrated nucleic acid preparation sub-system), a separate nucleic acid preparation process (such as one of many commercially available methods, e.g. spin-columns) followed by addition of the purified nucleic acid to the device by pipette, or an unprocessed nucleic acid containing sample. Already present in the sample cup, or preferably in recess 13 within or adjacent to the sample cup, is reagent mix 16, which may be in liquid or dry form, containing components useful for facilitating cell and virus lysis and/or stabilize ted nucleic acids. For example, dithiothreitol and/or pH buffering reagents may be employed to stabilize nucleic acids and inhibit . Similarly, reagents to accomplish acid or base mediated lysis may be used. In some embodiments the t mix is lyophilized to form lyophilized reagents. In some embodiments a positive control such as a virus, bacteria or nucleic acid is present in the t mix. Introduction of the sample to the sample cup causes reagents and samples to commingle such that the ts act upon the sample. An optional bubble-mixing step to further mix the sample with the reagents or re- suspend the reagents may optionally be performed. Fluid is then optionally heated in the sample cup to lyse cells and virus particles. Fluid is then preferably directed h channel 40 to a first chamber 30 that resides below the sample cup when the device is in the vertical orientation. Reagent recess 15 is preferably situated along the inlet channel such that fluid passes through the recess to commingle with dried or lyophilized reagents contained therein prior to entering the first chamber 30.

In embodiments wherein the first r is a reverse transcription chamber, preferably present in reagent recess 15 are all components necessary of a reverse ription reaction such as buffering reagents, dNTPs, oligonucleotide primers, and/or enzymes (e.g. reverse transcriptase) in dried or lyophilized form. The reverse transcription chamber is preferably in t with heater elements to provide a means for the temperature regulation necessary to support the reverse transcription of RNA into cDNA. Channel 35 connects chamber 30 to reagent recess 37. Following cDNA synthesis in chamber 30, vent 50 is opened to allow the reverse transcription reaction to flow via channel 35 into reagent recess 37. Dried or lyophilized reagents present reagent recess 37 commingle with fluid as it passes through the recess to second r 90 via inlet 39 such that the reagents act upon the sample in the second chamber, which is preferably an amplification chamber. Preferably t in reagent recess 37 are all components necessary for the amplification reaction, such as ing agents, salts, dNTPs, rNTPs, oligonucleotide primers, and/or enzymes. In some embodiments the t mix is lyophilized to form lyophilized reagents. To facilitate multiplexed tests, wherein multiple amplicons are generated, multiplexed ication can be accomplished by deposition of multiple primer sets within the amplification chamber(s) or preferably within reagent recesses upstream of said amplification chamber(s). Additionally, circuit board and fluidic designs in which multiple amplification and detection chambers are incorporated into the device support multiple parallel amplification reactions that may be single-plex or multiplex reactions. This ch reduces or eliminates the complications known to those skilled in the art that result from multiplexed ication using multiple pairs of primers in the same reaction. er, the use of le amplification reaction chambers allows simultaneous amplification under different temperature regimens to accommodate requirements for l amplification, such as different melting or annealing temperatures required for different target and/or primer sequences.

Following c acid ication, vent pocket 150 is opened to allow the amplification reaction product to flow via channel 135 into chamber 230. Detection strip 235 situated in chamber 230 enables the detection of target nucleic acids labeled by detection particles located on a region of detection strip 235 or optionally in capillary pool 93.

Fluid movement from the sample cup 10 to first chamber 30 occurs because chamber 30 is vented to expansion chamber 52 via opening 51. Fluid movement from the first chamber to the second chamber of the device is preferably accomplished by the opening of a vent connected to the second r. When fluid enters first r 30, vent pocket 50, connected to the downstream chamber, is sealed, and thus fluid will not pass h channel 35 connecting the two chambers.

Referring now to , movement of fluid from chamber 30 to chamber 90 can be accomplished by allowing air within chamber 90 to communicate with air in expansion chamber 52 by rupturing a seal overlying vent pocket 50. Rupture of the seal at vent pocket 50 allows communication of air in chamber 90 via vent channel 60 with air in ion chamber 52, which is connected via opening 51 to vent pocket 54. The seal at vent pocket 54 is preferably open, or was previously ed, as shown in FIG. ZB. As shown in , e of the seal of vent pocket 50 allows vent pocket 50 (and thus chamber 90) to communicate with vent pocket 54 (and thus expansion chamber 52). This method of fluid movement is preferably embodied within a ically sealed space to contain bio- hazardous samples and amplified nucleic acids within the test cassette. To enable a hermetically sealed te, selectively heat resistant and heat labile materials are layered in the manner represented schematically in cross section in FIGS. 28-20. Referring now to FIGS. 28-20, heat source 70, which preferably comprises a resistor, on printed circuit board or PCA 75 is placed in register with vent pockets 50, 54 and in proximity to heat labile vent pocket seal material 80. The vent pocket seal may comprise a heat labile material such as polyolefin or polystyrene. A heat stable material (such as polyimide) 72 is preferably disposed between heat source 70 and heat labile vent pocket seal material 80 to form a hermetic barrier. In some embodiments the sealed space 55 between or surrounding vent pockets is ted by the inclusion of an optional gasket or spacer 56 comprising an adhesive layer that bonds heat stable material 72 to heat labile material 80 and/or fluidic component 5 and maintains a hermetic seal of the test cassette in the region of the vents after one or more of the vents are opened, while preferably also providing an air gap for the communication of air between opened vents and/or the optional expansion chamber. In this embodiment, heat is erred from heat source 70 through heat stable material 72 and sealed space 55 to the heat labile vent pocket seal material 80, rupturing it and opening vent pocket 50. A microcontroller is preferably responsible for sending electrical current to heat source 70. Vent pocket 50 preferably opens to an enclosed space such that the gas within the test cassette may remain sealed with respect to the environment outside of the test cassette. The enclosed space may comprise the air within the test cassette, ally including a vacant air chamber to allow for gas expansion, such as an expansion chamber. As shown in , opening of vent pocket 50 results in the communication of gases in the vented fluid chamber with the gas of the expansion chamber, since vent pocket 54 was previously ed by heat source 71, and vent pocket 54 is in gaseous communication with the expansion chamber. The resulting reduced pressure in the vented fluid chamber allows fluid to flow by gravity into the vented chamber from a chamber situated above. Other embodiments of the vent pocket may comprise seals other than a heat-sensitive membrane, and may utilize other s of ng the seals, such as puncturing, tearing, or dissolving. A photograph of such a te is shown in .

The face opposite the open face of the vent pocket may optionally comprise a dimple, protrusion, asperity, or other similar structure, such as dimple 7004 of , to facilitate the formation of an opening during rupture of the vent seal al. Such a structure also preferably prevents resealing of the vent after rupture of the seal. This can occur in embodiments comprising a circuit board with surface mount components. In such embodiments the e mount resistors can stretch the ide film, pushing it into the opening in the gasket and against the heat labile al.

Once the seal ruptures, the molten seal material can form a secondary seal with that polyimide, y closing the vent. |n embodiments with a flex circuit comprising metallic traces forming heating elements, the heater can cause the polyimide flex circuit to deform locally, often g a protrusion (often comprising the heater al) extending into the opening in the gasket, possibly ing the vent opening due to the molten seal al. Dimple 7004 can help prevent these occurrences.

Sealed space 55 ally es a conduit to other vents, vent pockets or chambers (such as expansion chamber 52). Following vent opening, fluidic component 5 remains sealed from the al environment 59. Expansion chamber 52 preferably odates gas expansion during heating by ing the air/water vapor volume either by providing a sufficiently large volume so that gas expansion from temperature changes does not significantly impact the pressure of the system, or by accommodating gas expansion by displacement of a piston (, a flexible bladder (, a bellows (, or a hydrophobic barrier that allows gas but not macromolecules to pass free across the barrier (. In the expansion chamber makes use of a piston that is displaced by increasing re within the sealed fluidic system. The expansion chamber serves to reduce or eliminate the lation of pressure within the sealed system. Displacement of the piston occurs in response to increased re within a hermetically sealed test cassette, reducing internal pressure within the test cassette resulting from such processes as gas expansion during heating. In deflection of the bladder occurs in response to increased pressure within a hermetically sealed test cassette. Displacement of the bladder reduces internal pressure within the test cassette resulting from such processes as gas expansion during heating. In stretching of the bellows occurs in response to increased pressure within a hermetically sealed test cassette. Stretching of the bellows reduces internal pressure within the test cassette resulting from such processes as gas expansion during heating.

Expansion chambers may be incorporated as a vacant air volume, such as the included volume shown in expansion chamber 52 at the top of the test cassette illustrated in As illustrated in to facilitate the fabrication of a cassette of minimum thickness, expansion chambers may also be incorporated into air gap 440 formed by a suitably ed gasket 420 when sealed to heat labile material 410 and heat stable material 430 to form the backing of fluidic component 400. Minimization of test te physical dimensions is desirable to reduce shipping costs, reduce thermal mass and e an aesthetically pleasing and convenient design. In addition to forming air volume for gas expansion, gasket 420 generates a space between the heat stable material 430 and heat labile material 410 to facilitate free movement of air through open vents while maintaining a sealed system to prevent re to the environment. Gasket 420 is preferably thick enough to provide a sufficient air gap to equilibrate pressures between open vents, but is also sufficiently thin to not substantially impact the interface between the heaters and the corresponding vent pockets or the sealing of the cassette by the heat stable material. In embodiments of the present invention which se a flex circuit, the flex circuit may comprise a heat stable material such as polyimide, in which case the separate sheet of heat stable material 430 is not ed, for example as shown in . The use of an expansion chamber to reduce or equilibrate pressure within the sealed test cassette ensures that pressure disequilibria do not result in unfavorable or premature on movements within the test cassette and that pressure accumulation does not adversely impact desired fluid movement, such as nt between chambers or through channels. This re control, i.e. the establishment of ated pressure distributions throughout the device, enables the system to work as designed regardless of atmospheric pressure. The expansion chamber therefore enables controlled fluid movements which are dependent upon stable pressure within the system to be employed, and also enables use of a hermetically sealed test cassette, thereby avoiding the disadvantages of the venting the test cassette to atmosphere, for e the potential release of amplicon to the atmosphere. Furthermore, the method of enabling fluid flow by reducing pressure ream of a fluid, such as by opening a vent to the ion chamber, eliminates the need for pumps, such as those that create a positive pressure upstream of the fluid, or other devices with moving parts. Similar advantages are le by venting an area downstream of a fluid to a relatively larger reservoir (such as the expansion chamber) at substantially the same pressure as the downstream area, thereby enabling the fluid to flow under the force of gravity (provided the device is in the appropriate orientation). The size of the expansion chamber is preferably sufficiently large to accommodate reaction vapors produced during the assay without sing the pressure of the system to a point where it overcomes the capillary force or gravitational force ary for the fluid to flow.

In ments where the second chamber is an amplification chamber, the chamber is preferably in contact with heater elements to provide a means for the temperature regulation necessary to support nucleic acid amplification. In some embodiments of the invention, the ication chamber may contain oligonucleotides on at least a portion of the interior surface. At the interface n wall 95 of r 30 and one or more heating elements 100, as illustrated in , it may be advantageous to place a thermally conductive material such as a thermal grease or compound. A microcontroller preferably tes current to the resistive g element(s), preferably by means of metal oxide semiconductor field effect transistors (MOSFETs), based upon data collected from temperature sensor 110 on PCA 75, using simple on/off or proportional integral derivative (PID) temperature control s or other algorithmic temperature l known to those skilled in the art.

Placing the heating elements, and in some embodiments the corresponding temperature sensor(s), on the able component enables the manufacture of highly reproducible thermal coupling between the temperature control subsystem and the amplification and detection chambers to which they interface. This approach enables a highly reliable means of coupling the fluidic subsystem to the electronic thermal control subsystem by forming the thermally conductive interface during manufacture. The ing superior thermal contact between the electronic temperature control components and the fluidic subsystem results in rapid temperature equilibration, and therefore rapid assays. The use of a flexible circuit to provide disposable resistive heating elements that are fused to the rear of the c component g either directly or with an intervening , allows for a low cost means of attaining excellent thermal t, rapid temperature cycling and reproducible manufacture. Resistive heating elements for reverse transcription, amplification and fluid flow vent control can be formed ly on the flex circuit by etching the conductive layer of the flex circuit to form geometries exhibiting the required resistance. This approach ates the need for additional electronic components and simplifies manufacture while reducing cost.

In an embodiment of the present invention, flexible circuit 799 for resistive heating and vent opening is shown in The use of a flexible heater as a component of able cassette allows the cassette backing to be configured to enable fluid in the heated fluid chambers to make direct contact with the material comprising the flexible heater circuit. For example, as shown in , windows 806 in thermally labile material 807 (which preferably comprises BOPS) that forms the rear of WO 95493 the te may be situated over the fluid chambers to allow direct contact of fluid with le circuit 799. Direct contact between the flexible circuit layer and the fluid to be temperature controlled by heaters on the flexible circuit provides for a low thermal mass system capable of rapid temperature changes. To enable collection of temperature data for use in temperature regulation a temperature sensor may be optionally incorporated into the le circuit, andlor a non-contact means of temperature monitoring such as an infrared sensor may be employed. Resistive heating elements, such as heating element 800, in a flexible circuit can be utilized for vent rupture when they are situated in er with a vent pocket. Electrical pads 812 provide current to g ts 800. Similarly, the flexible circuit or circuits may se resistive heating elements 802 and 803 for heating the fluid chambers, and al ive heating element 804 for regulating the ature of the detection strip.

In this embodiment flexible circuit 799 also preferably serves as a heat stable seal to maintain a hermetically sealed cassette, similar to heat stable al 72 described above. Optionally an additional heat stable layer (for example comprising polyimide) can be placed between flexible circuit 799 and rear housing or panel 805. A spacer or gasket 808 is preferably placed around vent ors 800 between lly labile material 807 and flexible circuit 799 to ensure free air movement through open vents while maintaining a sealed cassette. Rear housing or panel 805 preferably comprises thin plastic and is preferably placed over the exposed surface of the flexible circuit to protect it during handling. Rear housing or panel 805 may comprise windows over the heater elements on flexible circuit 799 to facilitate cooling and temperature monitoring. Electrical contact with controlling electronics of the docking unit (described below) may optionally be provided by a set of electrical pads 810, preferably comprising an edge connector or connector pins such as spring loaded pins.

Embodiments of the test cassette chambers preferably comprise materials capable of withstanding ed heating and cooling to 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 to temperatures in the range of approximately 30 "C to approximately 110 °C at a rate of temperature change on the order of imately 10 "C to approximately 50 °C per second. The chambers are preferably capable of maintaining solutions therein at temperatures le for heat mediated lysis and biochemical reactions such as reverse transcription, thermal cycling or isothermal amplification protocols, preferably controlled by programming of the microcontroller. In some nucleic acid amplification applications, it is ble to provide an initial incubation at an elevated ature, 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 to activate a hot start polymerase. Subsequently, the reaction solution is held at the amplification temperature in the amplification chamber for isothermal amplification or, for thermocycling-based amplification, is varied in temperature between at least two temperatures including, but not limited to, a temperature that results in nucleic acid duplex denaturation and a temperature suitable to primer annealing by hybridization to the target and extension of the primer through polymerase catalyzed nucleic acid polymerization. The duration of incubations at each requisite temperature in a thermal cycling regimen may vary with the sequence composition of the target nucleic acid and the composition of the reaction mix, but is preferably between approximately 0.1 s and imately 20 s. Repeated heating and cooling is typically performed for approximately 20 cycles to approximately 50 cycles. In embodiments involving isothermal amplification methods, the temperature of the reaction solution is maintained at a constant temperature (in some cases following an initial incubation at an elevated temperature) for between imately 3 minutes and approximately 90 minutes depending on the ication que used. Once the amplification reaction is complete, the amplification reaction solution is transported, by opening the vent that is in communication with a chamber below the chamber employed for amplification, to the lower chamber to accomplish r manipulations of the amplified nucleic acids. In some ments of the invention manipulations comprise denaturation of the amplified nucleic acids and hybridization to ion oligonucleotides conjugated to detection particles. In some embodiments of the invention, amplified nucleic acids are hybridized to detection oligonucleotides conjugated to detection particles and to e probes immobilized on a ion strip.

In some embodiments, additional biochemical reactions may be conducted in the ication chamber prior to, during, or after the ication reaction. Such processes may include but are not d to reverse transcription wherein RNA is transcribed into cDNA, lexing wherein multiple WO 95493 primer pairs aneously amplify multiple target nucleic acids, and real time amplification wherein ication 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 would preferably comprise an optical window or othen/vise ured to enable interrogation of amplicon concentration during the amplification reaction process. In one real-time amplification embodiment, either fluorescently labeled oligonucleotides complementary to the target nucleic acid or fluorescent dyes specific for duplex DNA are monitored for fluorescence intensity by means of an excitation light source such as LEDs or diode |aser(s) and a detector such as a photodiode, and appropriate optical components including but not d to optical filters.

Detection Embodiments of the detection chamber 230 preferably provide for the specific ng of amplified target nucleic acids ted in the amplification chamber. As shown in , detection r 230 preferably comprises a capillary pool or space 93 and a detection strip 235. Detection particles comprising dye polystyrene microspheres, latex, colloidal gold, colloidal cellulose, nanogold, or semiconductor nanocrystals are preferably present in the capillary pool 93. Said detection particles may comprise oligonucleotides complementary to the target e or may comprise ligands capable of binding to the amplified target nucleic acid such as biotin, streptavidin, a hapten or an antibody ed against a label such as a hapten present on the target amplified nucleic acids. Detection chamber 230 may contain detection particles that are dried, |ized, or present on at least a portion of the interior surface as a dried mixture of detection particles in a carrier such as a ccharide, detergent, protein or other compound known to those skilled in the art to facilitate resuspension of the detection particles. In some embodiments the l flow detection strip may comprise detection particles. In other embodiments a reagent recess channel 135 leading to the detection chamber my 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 c acid, fluorescently modified oligonucleotides, or ucleotide-conjugated dyed articles or colloidal gold or colloidal cellulose. Detection of amplicon involves a ‘detection oligonucleotide’ or other ‘detection probe’ that is mentary or otherwise able to bind specifically to the amplicon to be detected. Conjugation of a detection oligonucleotide to a microparticle may occur by use of streptavidin coated particles and biotinylated oligonucleotides, or by carbodiimide chemistry whereby carboxylated particles are activated in the presence of carbodiimide and react specifically with primary amines present on the detection oligonucleotide. Conjugation of the detection oligonucleotide to the detectable moiety may occur internally or at the 5’ end or the 3’ end. Detection oligonucleotides may be attached directly to the microparticle, or more preferably through a spacer moiety such as ethyleneglycol or polynucleotides. In some embodiments of the invention, detection particles may bind to multiple species of amplified nucleic acids resulting from such ses as multiplexed amplification. In these embodiments the specific detection of each species of amplified nucleic acid can be realized by detection on the detection strip using a method specific for each s to be detected. In such an embodiment, a tag introduced to the target nucleic acids during ication may be used to label all amplified species present while subsequent hybridization of the labeled c acids to species ic capture probes immobilized on the detection strip is employed to determine which specific species of amplified DNA are t.

In the case of a duplex DNA amplification t, heating the reaction solution following introduction to the detection chamber may facilitate detection. Melting duplex DNA or denaturing the secondary structure of single stranded DNA and then cooling in the presence of detection oligonucleotide s in the sequence-specific labeling of the amplified target nucleic acid. The g element underlying the detection chamber may be used to heat the fluid volume for approximately 1 to approximately 120 seconds to te duplex DNA melting or denaturation of single stranded DNA secondary structure. As the solution is allowed to cool to room temperature, the amplified target nucleic acid may ically hybridize to detection microparticles. The on volume is then preferably directed to a region of the detection chamber below the labeling chamber by g the vent of the detection chamber.

For efficient labeling to occur, the solubilized detection les are preferably well mixed with the reaction solution. In embodiments of the invention, detection particles may be localized in capillary pool 93 at the outlet of channel 135 to facilitate mixture with solution as it enters chamber 230.

Detection les in capillary pool 93 may optionally be lyophilized detection particles. The capillary pool provides improved mixing and dispersion of particles to facilitate comingling of the detection particles with the nucleic acids to which the detection particles bind. The ary pool also increases the uniformity of particle migration on the detection strip, as shown in A capillary pool is especially advantageous for low volume , such as those less than 200 uL, or more specifically less than about 100 uL, or even more particularly less than about 60 uL, or even more particularly about 40 "L in .

Embodiments of the detection chamber of the present invention provide for the specific detection of amplified target nucleic acids. In certain embodiments of the invention, ion is accomplished by capillary wicking of solution containing labeled on through an absorbent strip comprised of a porous material (such as cellulose, nitrocellulose, polyethersulfone, polyvinylidine fluoride, nylon, charge-modified nylon, or polytetrafluoroethylene) patterned with lines, dots, microarrays, or other visually nable ts comprising a binding moiety capable of specifically binding to the d 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 tant pad sing amphipathic reagents to enhance wicking, a ion zone comprising a porous material (such as cellulose, nitrocellulose, hersulfone, polyvinylidine fluoride, nylon, charge-modified nylon, or polytetrafluoroethylene) to which at least one binding moiety capable of selectively binding labeled amplicon is immobilized, and/or an absorbent pad to provide additional absorbent capacity.

Although detection particles may ally be incorporated within the lateral flow porous materials in the detection chamber, unlike previously described lateral flow detection devices the detection particles preferably are instead held upstream in a capillary pool where substantially enhanced the formation of binding complexes between amplicon and detection particles may be conducted prior to or concomitant with the introduction of the resultant labeled nucleic acids to the porous components of the device.

A ‘capture oligonucleotide’ or re probe’ is preferably immobilized to the detection strip element of the device by any of a variety of means known to those skilled in the art, such as UV irradiation. The capture probe is ed to capture the labeled nucleic acid as solution containing the labeled nucleic acid wicks through the capture zone resulting in an increased concentration of label at the site of capture probe immobilization, thus producing a detectable signal indicative of the presence of the labeled target c acid on(s). A single detection strip may be patterned with one or multiple capture probes to enable multiplexed detection of multiple amplicons, determination of amplicon sequence, quantification of an amplicon by extending the linearity of the detection signal, and assay quality control ive and negative controls).

Fluidic component Embodiments of the fluidic component preferably se plastic, such as acrylic, polycarbonate, PETG, polystyrene, polyester, polypropylene, andior other like materials. These materials are readily available and able to be manufactured by standard methods. Fluidic components comprise both chambers and channels. Fluidic chambers comprise walls, two faces, and connect to one or more ls such as an inlet, an outlet, a recess, or a vent. Channels can connect two fluidic chambers or a fluidic chamber and a recess, and comprise of walls and two faces. Fluidic chamber design preferably maximizes the surface area to volume ratio to facilitate heating and cooling. The internal volume of a chamber is preferably between approximately 1 uL and approximately 200 uL.

The area of a chamber face in t with on preferably corresponds with the area to which heating elements are interfaced to ensure uniform fluid temperature during heating. The shape of the fluidic chambers may be selected to mate with heating elements and to provide favorable ries for solution ingress and egress. In some embodiments, the volume of the chamber may be larger than the fluid volume in order to e a space for s that appear during the course of device operation. Fluidic chambers may have enlarged extensions leading to vent channels, to ensure that fluid does not encroach upon the channel by capillary action or ise block the g mechanism.

In some embodiments, it may be desirable to reduce or eliminate the on of liquid or gas phase water into a chamber prior to the time of solution release. The elevated temperatures employed in processes of some embodiments generate vapors (e.g. gas phase water) that can result in premature invasion of moisture into a channel, chamber or recess. Reduction of liquid phase or gas phase invasion may be desirable to retain, for e, the dried state ofdried reagents or lyophilized reagents present in a chamber or recess. In some embodiments, channels may be temporarily blocked, tely or partially, with a material that can be d by external forces such as heat, moisture, and/or pressure. Materials suitable for the temporary ge of channels e but are not limited to latex, cellulose, polystyrene, hot glue, paraffin, waxes, and oils.

In some embodiments, the test cassette comprises a preferably injection molded fluidic component comprising sample cup, chambers, channels, vent pockets, and energy directors. The injection molded test cassette fluidic component is preferably comprised of a plastic suitable for ultrasonic welding to a backing c of similar composition. In one embodiment of the invention the test cassette fluidic component comprises a single injection molded piece that is ultrasonically welded to a backing material. The energy ors are optional features of the fluidic component that direct the ultrasonic energy to only those areas of the heat labile layer which are ed to bond to the fluidic component. The injection molded c component may ally be housed in a housing. illustrates a cassette sing a preferably injection molded fluidic component 400 (preferably comprising a polymer such as high-impact polystyrene , polyethylene, polypropylene, or NAS , a styrene acrylic copolymer), a heat labile material 410 (comprising, for example, BOPS, which has relatively low melting temperature of about 239°C and a glass transition temperature of about 100°C, which is sufficient to withstand the elevated atures during denaturation, or polycarbonate, which has a 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 polyester carrier, or any adhesive that can withstand the elevated temperatures of the device), and heat ant layer 430. Heat labile material 410 ruptures via heat which is preferably transmitted through overlying heat resistant layer 430 (comprising, for example, polyimide or another polymer having high heat resistance). Melting of the heat labile material over the vent features of the cassette opens the vent and vent channel to the expansion r, thereby allowing pressure equalization within the cassette. Overlying heat resistant layer 430 preferably remains intact, thereby enabling the cassette to maintain a hermetic seal after vent opening.

In some embodiments, the adhesive spacer comprises a vacant region 440 that may serve as an ion chamber to buffer the ion of gases during heating to reduce the internal pressure of a sealed cassette. Heat labile layer 410 is bonded to fluidic component 400 by a bonding method or process such as ultrasonic welding or employing adhesive. The resulting part is then bonded to a spacer and a heat resistant layer. In some embodiments the heat resistant layer is constructed in such a manner that it is not present over heated chambers. In other embodiments, the heat resistant layer is present over heater chambers. In yet other embodiments, the adhesive spacer and heat resistant layers are present only over a region that is in register with the vent pocket features of the fluidic ent. In this embodiment a heat resistant layer may optionally be placed directly over the heat labile material in the regions in register with the heated chambers.

In some embodiments of the invention the thickness of the fluidic chambers and 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. This thickness preferably meets requirements of both structural ity of the c component and to support sealing of the closed chamber under high temperatures and associated pressures. The thickness of channel walls, particularly vent channel walls, are preferably less than that of the rs and in the range of approximately 0.025 mm to imately 0.25 mm. The width of inlet and outlet channels is preferably chosen to enhance capillarity. A shallow vent channel imparts improved rigidity to the fluidic component with no adverse effect on venting. Plastic forming faces of the fluidic component is preferably thinner than that forming the walls in order to maximize heat transfer. Optional thermal breaks out through some components of the fluidic component and surround the amplification and detection rs, contributing to the thermal ion of the temperature-controlled chambers.

In some embodiments of the invention, before the c component 400 is bonded to the heat labile backing material 410 additional components of the test cassette such as lyophilized reagents 16, detection strip assembly 230, and detection particles may be orated. In some embodiments, the components may be laminated by applying pressure to ensure good adhesion. In some embodiments the components may be bonded by a combination of methods such as pressure sensitive adhesives and ultrasonic welding. Adhesives known or found to negatively impact performance of nucleic acid ication reactions must be avoided. Acrylic- or silicon-based adhesives have been successfully used in the invention. One red adhesive film is Sl7876 supplied by Advanced Adhesives ch. Other adhesives may be used if found to be chemically compatible with employed buffers, plastics and reaction chemistries while providing robust sealing over the temperatures encountered during device ion.

Referring to and 7, vent s are preferably entiated from other chambers in their construction. After construction of the fluidic component as bed above, vent pockets possess an open face on the side of the fluidic component that will meet with PCA 75 either directly or in some embodiments indirectly through an intervening air gap 420 or vent pocket 54 and heat resistant material 430. To form the vent pocket, an additional plastic component is bonded to seal the chamber, preferably comprising a thin, heat labile membrane 410 adjacent to vent resistor 70 of the PCA. Film 410 comprises a material le for ultrasonic welding to the injection molded fluidic component such as polystyrene although other r materials may be used. This film is well suited to both seal the vent pocket and allow for easy perforation and, thus, venting to a lower pressure chamber when current is passed through the vent resistor generating a rapid temperature increase.

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 ication. Use of a material with stability in the temperature ranges employed for denaturation, labeling, reverse transcription, nucleic acid amplification, and detection but with a melting temperature readily attained by resistor 70 allows a single material to be employed for the backing of the injection molded fluidic component 400 to serve as both a face of the chambers and a face of the vent pocket. In some embodiments, onal temperature stability in the areas of the temperature controlled rs can be ed 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 in register with the temperature controlled chambers to allow direct contact between fluids in the chamber and the substrate of a flexible circuit fused to the rear of the test cassette.

Additional ents of the Fluidic Component As described above, l additional components are preferably incorporated within the fluidic component of the present invention before final bonding. Reagents including buffers, salts, dNTPs, NTPs, oligonucleotide s, and s such as DNA polymerase and reverse transcriptase may be lyophilized, or freeze-dried, into pellets, spheres or cakes prior device assembly.

Reagent lyophilization is well known in the art and involves dehydration of frozen reagent aliquots by sublimation under an applied vacuum. By adding specific formulations of lyoprotectants such as sugars (di- and polysaccharides) and polyalcohols to the reagents prior to freezing, the activity of enzymes may be preserved and the rate of rehydration may be increased. Lyophilized reagent pellets, spheres, or cakes are manufactured by standard methods and, once formed, are ably durable and may be easily placed into specific chambers of the fluidic component prior to laminating the final face. More preferably, recesses are incorporated into the c k to allow s, spheres, or cakes of lyophilized reagents to be placed in the fluidic ent prior to bonding of the fluidic component to the backing material. By selecting the fluidic k ry and recess location and order, the sample can react with the desired lyophilized reagent at the desired time to optimize performance. For instance, by depositing lyophilized (or dried) reverse transcription (RT) and amplification reagent spheres into two separate es in the flow paths of RT reaction chamber and amplification chamber s optimal reverse transcription reaction without the interference of amplification enzymes. In addition, to minimize the interference of RT enzymes to subsequent amplification reaction, RT s post RT reaction presented in the RT reaction could be heat inactivated prior introduction to amplification reagents to minimize their interference to amplification.

Optionally, other salt, surfactants and other enhancing chemicals could be added to different recesses to modulate the performance of a assay. er, these recesses facilitate comingling of the lyophilized reagents with liquids as they pass through the recess and also serve to isolate the lyophilized materials from onic energy during ultrasonic g and to isolate lyophilized reagents from temperature extremes during heating steps of a test prior to their solubilization. In addition, the recesses ensure that the lyophilized pellets aren’t compressed or crushed during manufacture, enabling them to remain porous to ze rehydration times.

WO 95493 In some embodiments of the invention, detection articles are another additional component of the fluidic ent. In some embodiments, these microparticles may be lyophilized as bed for the reaction reagents above. In other embodiments, microparticles in liquid buffer may be ly applied to an interior face of a fluidic chamber and dried before final assembly of the test te. The liquid buffer containing the microparticles preferably also comprises sugars or polyalcohols that aid in rehydration. Incorporation of microparticles in aqueous buffer directly into the fluidic component prior to drying may simplify and reduce the final cost of manufacturing, and complete comingling of lyophilized particles with reaction solution and the denaturation of double- ed nucleic acids or double-stranded regions of a nucleic acid into single-stranded c acid may be tated by heating or nucleate 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 ly below the detection strip. In other embodiments ion particles are dried or lyophilized into a bibulous substrate in capillary communication with the detection strip or are dried or lyophilized directly on the detection strip. Capillary communication may be direct physical contact of the said bibulous substrate with the detection strip or indirect wherein capillary communication is over an intervening distance comprised of a channel or chamber region through which capillary transport is achieved to transport fluid from the detection particle laden bibulous substrate to the detection strip.

In some embodiments of the present invention, a lateral flow ion strip assembly is also orated into the fluidic component. The detection strip preferably comprises a membrane assembly sed of at least one porous component and optionally may comprise an absorbent pad, a detection membrane, a tant pad, and a backing film. The detection membrane is preferably made of nitrocellulose, cellulose, polyethersulfone, polyvinylidine fluoride, nylon, charge- modified nylon, or polytetrafluoroethylene and may be backed with a plastic film. As described above, capture probe may be ted and irreversibly immobilized on the detection membrane in lines, spots, microarrays or any pattern that can be visualized by the unaided human eye or an automated detection system such as an imaging system. Deposited oligonucleotides may be permanently immobilized by UV-irradiation of the detection membrane ing capture probe deposition. The tant pad may comprise a porous substrate, preferably with minimal nucleic acid binding and fluid retention properties, that permits unobstructed migration of the c acid t and detection microparticles. The surfactant pad may comprise materials such as glass fiber, cellulose, or ter.

In embodiments of the invention, formulations including at least one amphipathic reagent are dried on the surfactant pad to allow uniform migration of sample through the detection membrane. The absorbent pad may comprise any absorbent material, and helps to induce sample wicking through the detection membrane ly. Using an adhesive backing film, such as a double-sided adhesive film as a base, the detection membrane component is assembled by first placing the detection membrane, followed by optional absorbent pad and/or surfactant pad in physical contact with the detection membrane with between 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 wherein there is a al tion between the tant pad and the detection pad with the intervening space comprised of a capillary space wherein fluids may se the space by means of capillary action. In some embodiments, the tant pad or a region of the surfactant pad may comprise detection particles, dried detection particles or lyophilized detection particles.

Three Chamber Cassette In some embodiments of the invention, additional reaction chambers and/or additional recesses for dried or lyophilized reagents may be incorporated. In some embodiments such a design facilitates tests in which it is desirable to provide for an initial separate lysis reaction prior to reverse transcription and amplification. As shown in FIGS. 37A, 37B, and 38, cassette 5000 ses cover 5020 sealing ion chamber 5021, flexible heater circuit 5022 preferably disposed in intimate contact with fluidic component 5023, and rear cover 5024 which conceals the circuitry from the user. A sample containing nucleic acids is introduced into sample cup 5002 h sample port 5001. The sample flows freely into recess 5003 where it reconstitutes the first lyophilized bead 5004, preferably comprising lysis reagents, prior to flowing down channel 5005 into first reaction chamber 5006. This free flow is facilitated by vent channel 5007 which connects to the top of the sample cup 5002. Vent channel 5007 may additionally connect to expansion chamber 5021 via hole 5008. The sealed air WO 95493 space below first reaction chamber 5006 pressurizes slightly due to the fluid flow and causes the flow to stop just below first on chamber 5006. First reaction chamber 5006 is then preferably heated to a temperature to facilitate proper reaction with the lysis ts, lysing the biological les and/or cells in the sample, exposing any nucleic acids present therein.

Opening of the vent valve 5009 connected to the top of second reaction chamber 5011 then tates sample flow into a second recess where second lyophilized bead 5010, preferably comprising reagents for reverse transcription, is tituted. The fluid then enters second reaction chamber 5011 where it’s flow stops as a result of increased air pressure in the closed air volume below the flow. Second reaction chamber 5011 is then preferably subsequently heated to an appropriate temperature to facilitate the reverse transcription s.

Opening of the next vent valve 5009 connected to the top of third reaction chamber 5013 tes flow of the sample from second reaction chamber 5011 through a third recess where lyophilized bead 5012, preferably comprising lyophilized PCR amplification reagents, is tituted.

The sample then flows into third reaction chamber 5013, where it oes thermal cycling to amplify targeted analytes present in the sample.

Subsequently, opening of the final vent valve 5009 connected to the far end of lateral flow strip 5014 enables the sample which now contains amplified analytes to flow to lateral flow strip 5014 for detection of the analytes as previously described.

Flow l Features The design of the fluidic component may optionally comprise flow control features within, or at the outlets of, the reaction chambers. These features deflect the flow ng the chamber to the side of the chamber opposite from the outlet, prior to the flow entering the outlet. As a result the flow enters the outlet channel at a lower velocity, reducing the distance the fluid flows down the channel before it stops. Furthermore, 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 it is ient to stop the flow at the desired location based on the reduced velocity of the flow. This enables closer vertical spacing between chambers of the cassette since less vertical channel is required. In addition, the redirection of the flow across the reaction chamber creates a swirling action in the flow within the r, improving mixing of the reagents with the sample fluid. The flow control feature may comprise any shape.

In the embodiment shown in , the fluid enters reaction r 4003 from inlet channel 4002 and flows to the bottom of the reaction chamber, where it is redirected to the side of reaction chamber 4003 te the opening to inlet channel 4002 by triangular flow control feature 4001. As the flow proceeds to opposite corner 4004, the flow divides, with some entering outlet 4005, while the rest contacts the wall and is directed upward, creating a swirling effect which improves mixing. The flow into the outlet preferably forms a meniscus and travels through outlet channel 4006 towards the next reaction r or lyophilized bead recess. Since outlet channel 4006 is sealed below reaction chamber 4003, as the fluid travels along outlet channel 4006 the air pressure increases in the channel below the flow until reaching equilibrium with the fluidic pressure head, thus stopping 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 pressure in the closed air space downstream of the flow. This larger opening to the outlet channel preferably provides increased compressible air volume so that a meniscus may be reliably formed at the wider opening.

In the embodiment shown in , the fluid enters reaction chamber 4103 from inlet channel 4102 and flows to the bottom of the reaction chamber, where it is redirected to the side of reaction chamber 4103 opposite the opening to inlet channel 4102 by triangular flow control feature 4101. As the flow proceeds to opposite corner 4104, the flow divides, with some entering outlet 4105, while the rest contacts the wall and is directed , creating a swirling effect which improves mixing. The flow into the outlet preferably forms a meniscus and travels through outlet channel 4106 s the next reaction chamber or lyophilized bead recess. Since outlet channel 4106 is sealed below on chamber 4103, as the fluid travels along outlet channel 4106 the air pressure increases in the l below the flow until reaching equilibrium with the c re head, thus stopping the flow. In this embodiment outlet 4105 and outlet channel 4106 have uniform width. In this ment ion of a meniscus at the on chamber may be somewhat more reliable as a result of the narrower channel. The meniscus subsequently increases pressure in the closed air space downstream of the flow.

In the embodiment shown in , the fluid enters reaction chamber 4103 from inlet channel 4202 and flows to the bottom of the reaction chamber, where it is redirected to the side of reaction chamber 4203 opposite the opening to inlet channel 4202 by trapezoidal flow control feature 4201. As the flow proceeds to opposite corner 4204, the flow divides, with some entering outlet 4205, while the rest contacts the wall and is directed upward, creating a swirling effect which improves mixing. In this embodiment outlet 4205 is ed substantially ally. The flow into the outlet preferably forms a meniscus and travels through outlet channel 4206 towards the next reaction chamber or lyophilized bead recess. Since outlet channel 4206 is sealed below reaction chamber 4203, as the fluid travels along outlet channel 4206 the air pressure increases in the channel below the flow until ng equilibrium with the fluidic pressure head, thus ng the flow. In this embodiment outlet 4205 and outlet channel 4206 have uniform width. In this embodiment formation of a meniscus at the reaction chamber may be somewhat more reliable as a result of the narrower channel. The meniscus subsequently increases pressure in the closed air space downstream of the flow.

In the embodiment shown in , the fluid enters on chamber 4305 from inlet channel 4304 and flows to the bottom of the reaction chamber, where it is redirected to the side of reaction chamber 4305 opposite the opening to inlet channel 4304 by triangular flow control feature 4303. As the flow proceeds to opposite corner 4306, the flow divides, with some entering outlet 4307, while the rest contacts the wall and is directed upward, creating a swirling effect which improves mixing. The flow into the outlet preferably forms a meniscus and travels through outlet channel 4306 towards the next reaction r or lized bead recess. In this embodiment outlet channel 4306 travels h stacked serial flow control features 4303, 4302, and 4301 which provide a tortuous route for the fluid to flow, providing an increased outlet channel length in a small vertical space.

In the ment shown in , the fluid enters on chamber 4403 from inlet channel 4402 and is redirected to the side of on r 4403 te the opening to inlet channel 4402 by flow control e 4401 disposed above the bottom of the reaction chamber, ably imately halfway along the length of reaction chamber 4403. In contrast to the previous embodiments, flow control e 4401 does not form the outlet of reaction chamber 4403.

Flow control feature 4401 deflects the flow away from outlet l 4405 into opposite corner 4404, thereby reducing the flow velocity prior to exiting the chamber. Similar to the previous embodiments the fluidic ction promotes turbulence and reagent mixing.

To facilitate ive co-mingling of reaction solution with lized reagents, embodiments of the test cassette portion comprising the fluid flow path may comprise dedicated t recesses 4600 incorporated into the fluid flow path between chambers, as shown in FIGS. 46A and 468. In alternative embodiments, t recesses 4700 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 the resuspension of the lyophilized reagent or reagents placed within the recess during manufacture. Placing lyophilized reagent(s) within a recess of the fluid chamber may be preferable to placing reagent(s) in a reagent recess in the fluid channel when t resuspension requires longer resuspension times than afforded by the transient e of fluid through a channel-localized recess during solution flow from one chamber to another chamber. By harboring lyophilized reagent within the fluid chamber, the dwell time of the reaction solution with the reagent is prolonged, sing the exposure of the lyophilized material to the fluid and ensuring more complete resuspension of the lyophilized reagents and more complete co-mingling of the reagents with the reaction solution. In addition, lyophilized ts disposed in recesses in the flow path can be susceptible to seepage (or capillary trickle) from the chamber above before the reaction there is complete. This can impart a syrupy consistency to the reagents, causing fluid flow problems when the bulk of the solution is transported through the recess from the upper chamber.

This problem is preferably avoided when the recess is disposed in the lower chamber itself.

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 , improvements to t resuspension and co-mingling of reagent with the reaction solution may be ed by the incorporation of a projection into the fluid chamber, such as projection 4605 shown in 8, or projection 4705 shown in 8. The sharp al rise on the side of the projection within the chamber discourages capillary fluid flow across the top or roof of the fluid chamber, reducing or ting sequestration of newly resuspended reagent from the bulk of the reaction solution volume.

Multiplexing of Assays In some embodiments of the invention, multiple independent assays may be performed in parallel by employing a fluidic design that enables splitting an input fluid sample into two or more parallel fluidic paths through the device. is a schematic representation of splitting a fluid , for example 80 uL, in two sequential steps into first two te 40 uL volumes and uently into four 20 uL s. The rated scheme is useful to enable separate ndent manipulations such as biochemical reactions to be conducted on the split s. Such configurations are useful for increasing the number of analytes that can be detected in a single device by facilitating the multiplexed detection of multiple targets such as nucleic acid sequences in multiplexed nucleic acid reverse transcription and/or amplification reactions. Similarly, the use of multiple detection strips at the end of the ndent fluid paths can afford enhanced readability of strips for the detection of multiple targets or distinguishing sequence differences or mutations in c acid analytes. Furthermore, providing additional detection strips for independent interrogation of multiple amplification reaction products can enhance specificity by reducing the likelihood of spurious cross-reactivity such as cross hybridization during the detection step of the test. illustrates a test cassette comprising two fluid paths in a single test cassette. Each fluid path may be independently controlled with respect to timing, on type, etc. Referring now to , a sample introduced to sample cup 1000 is divided into approximately equal volumes and flows into volume splitting chambers 1001 and 1002, flow into which is regulated by vent valves 1003 and 1004.

Splitting chambers 1001 and 1002 control the volume of sample in each test path by passively equilibrating the amount of sample in each chamber. After volume splitting, solution is allowed to flow h reagent recesses 1007 and 1008 by opening of vents 1005 and 1006. Reagents such as lyophilized reagents are disposed in recesses 1007, 1008 and comingled with the sample as it flows through the recesses and into a first set of preferably temperature controlled chambers 1009 and 1010. Reactions such as heat lysis, e ription, and/or nucleic acid amplification are conducted in each of the first set of heated chambers facilitated by reagents provided in the reagent recesses 1007, 1008. Such reagents may include but are not limited to lyophilized positive control agent (e.g. nucleic acid, virus, bacterial cells, etc.), lyophilized reverse transcriptase and associated accessory reagents such as nucleotides, buffers, DTT, salts, etc. required for reverse transcription of RNA to DNA, and/or DNA amplification using lyophilized DNA polymerase or thermostable lized DNA rase and required accessory reagents such as nucleotides, buffers, and salts.

Following the tion of biochemical reactions such as reverse transcription, nucleic acid amplification or concomitant e transcription and nucleic acid amplification (e.g. single tube reverse transcription-polymerase chain reaction (RT-PCR) or one-step RT-PCR or one-step RT- Oscar) in the first set of chambers, seals for vent pockets 1011 and 1012 are ruptured to allow fluid to flow from the first set of chambers through a second set of reagent recesses 1013 and 1014 and into a second set of preferably ature controlled chambers 1015 and 1016. ts such as lized reagents may be disposed in recesses 1013 and 1014 such that they comingle with the sample solution as fluid flows from chambers 1009 and 1010 to rs 1015 and 1016. Reagents such as lyophilized reagents for nucleic acid amplification or dried or lyophilized detection particles such as probe conjugated dyed polystyrene microspheres or probe conjugated colloidal gold may optionally be placed in reagent recesses 1013 and/or 1014. Following completion of reactions or other manipulations such as binding or hybridization to probe conjugated detection particles in heated rs, on is d to flow into detection strip chambers 1017 and 1018 by opening vent valves 1019 and 1020. In some embodiments, a third set of reagent recesses may be placed in the fluid paths from chambers 1015 and 1016 such that additional reagents, such as detection ts comprising detection particles, salts and/or surfactants and other substances useful to facilitate hybridization or other detection modalities, may be comingled with the solution flowing into strip chambers 1017 and 1018. Detection strip chambers 1017 and 1018 may be heated and preferably comprise detection strips such as lateral flow strips for the detection of analytes such as amplified nucleic acids. ion strips may comprise a series of absorbent materials doped or patterned with dried or Iyophilized detection reagents such as detection particles (e.g. dyed microsphere conjugates and/or colloidal gold conjugates), capture probes for the capture of analytes such as hybridization e ucleotides for the e of nucleic acid analytes by sequence specific ization, ligands such as biotin or streptavidin for the capture of appropriately modified analytes, and absorbent materials to provide an absorbent capacity sufficient to ensure complete migration of the sample solution volume through the detection strip by such means as capillary action or wicking.

Sample Preparation In some embodiments of the invention, it may be desirable to incorporate a sample preparation system into the te. A sample preparation system, such as a c acid purification , may comprise encapsulated solutions for lishing sample preparation and elution of purified les such as purified DNA, RNA or proteins into the test cassette. depicts a nucleic acid sample preparation subsystem 1300 designed for integration with a test cassette. The sample preparation subsystem comprises a main housing 1302 and housing lid 1301 to house components of the subsystem. A solution tmentalization component 1303 comprises crude sample reservoir 1312 which is preferably open on the upper face but sealed underneath by lower seal 1305. Solution compartmentalization component 1303 also preferably comprises reservoir 1314 ning a first wash buffer and reservoir 1315 containing a second wash buffer, both of which are preferably sealed by means of upper seal 1304 and lower seal 1305. A nucleic acid binding matrix 1306 is placed in the on capillary flow path provided by absorbent als 1307 and 1308.

Glass fiber or silica gel exhibiting nucleic acid binding properties and wicking properties are examples of materials le for use as binding matrix 1306. A wide range of absorbent materials may comprise the absorbent materials 1307 and 1308, including polyester, glass fiber, nitrocellulose, polysulfone, cellulose, cotton or combinations thereof as well as other wicking materials provided they offer adequate capillarity and minimal binding to the molecules to be purified by the subsystem. Any readily ruptured or frangible material capable of being sealed to solution compartmentalization component 1303 and chemically compatible with the encapsulated solutions is suitable for use as seal material 1304 and 1305. Seal material 1305 comes in contact with sample or sample lysate and must WO 95493 onally be chemically compatible with the sample or sample lysate on. Examples of suitable seal material are heat sealable metallic film and plastic film. Seal material 1305 is ruptured at the time of use by displacement of the solution compartmentalization component 1303 such that the seal 1305 is pierced by structures 1311 present in housing 1302. Crude sample or crude sample mixed with a lysis buffer such as a buffer comprised ofa chaotropic agent is introduced at the time of use to the sample reservoir 1312 via the sample port 1309 in lid 1301. In some embodiments, lysis buffer may optionally be encapsulated in reservoir 1312 by extending seal 1304 to cover the upper orifice of reservoir 1312. In such ments it may be desirable to include a tab or other means for the partial l of that region of seal 1304 covering reservoir 1312 to allow the addition of crude sample to reservoir 1312 such that crude sample may comingle or mix with the lysis buffer contained therein.

Sample solution or lysate containing sample material is introduced to the sample on port 1309 and retained in sample reservoir 1312 of buffer reservoir 1303 until the initiation of the sample preparation process.

At the time of sample preparation initiation, solution compartmentalization ent 1303 is pushed onto the seal ng structures 1311 resulting in the simultaneous release of sample solution or lysate in reservoir 1312 and first and second wash buffers in reservoirs 1314 and 1315 tively.

Mechanical displacement of component 1303 may be accomplished manually or by the use of an actuator or ors present in a reusable instrument into which the disposable test cassette is placed at the time of use. Actuator access or manual displacement mechanism access to reservoir 1303 is preferably provided through access port 1310 of housing lid 1301. Sample or lysate solution and first and second wash buffers are moved through materials 1307, 1306 and 1308 by capillary action. The physical arrangement of the reservoirs and the ric configuration of absorbent material 1307 ensure sequential flow of the crude lysate, first wash buffer and second wash buffer through the binding matrix 1306. Additional absorbent capacity to ensure continued capillary transport of all solution volumes through the system is provided by absorbent pad 1313 placed in contact with wick 1308. At the completion of solution transport through the ent materials, spent solutions come to rest in absorbent pad 1313. Following exhaustion of capillary transport of all solutions through the system, purified nucleic acids are bound to binding matrix 1306, from which the nucleic acids may be eluted into the sample cup 1402 of the integrated test cassette, as shown in .

Movement of the sample ation subsystem components occurring during the sample preparation process are shown in , which depicts the sample preparation tem embodiment in cross-section before and after sample processing. Elution is preceded by displacement of binding matrix 1306 out of the capillary flow path and through seal component 1316 by the action of an actuator in the associated reusable test instrument. Seal component 1316 forms a seal with a n of elution buffer t 1318 to allow the injection of elution buffer through binding matrix 1306 and into sample cup 1402 t solution loss to the ary flow path of the sample preparation subsystem. t 1318 is attached to or part of the elution buffer injector component comprised of elution buffer reservoir 1317 and plunger 1319. Plunger 1319 may optionally comprise 0- rings to facilitate g of elution buffer within reservoir 1317. During elution of purified nucleic acid, an actuator moves elution reservoir component 1317 such that ed conduit 1318 forms a seal with seal component 1316 and displaces binding matrix 1306 into chamber 1321. Mechanical access to depress elution reservoir 1317 is provided through or access port 1320. Following displacement of g matrix 1306 out of the main capillary solution flow path of the sample preparation subsystem, binding matrix 1306 resides in elution chamber 1321. Elution of purified nucleic acid into sample cup 1402 is accomplished by forcing n bufferfrom elution buffer reservoir 1317 by an actuator acting through actuator port 1322 to move plunger 1319 through reservoir 1317 in a syringe-like action. Elution buffer proceeds via conduit 1318 through binding matrix 1306, resulting in the injection of elution buffer containing eluted purified nucleic acids into sample cup 1402.

Referring now to , sample preparation subsystem 1300 is preferably bonded to fluidic component 1403 of cassette 1500 by widely used manufacturing s such as onic g to form an integrated single use sample-to-result test cassette. In some embodiments, it is desirable to hermetically seal the test cassette fluidics following the introduction of eluate containing purified nucleic acid in order to reduce the likelihood of amplified nucleic acid escape from the cassette. A sliding seal 1404 may optionally but preferably be placed between the sample preparation subsystem WO 95493 and the test cassette fluidic g 1403 to seal the cassette at the entrance to sample cup 1402.

Sliding seal 1404 is moved to the sealed position by the action of an or to form a hermetic seal comprising o-ring 1405. Cassette backing 1406 is bonded to the fluidic housing ing the introduction of dried reagents and test strips. As described above for the backing of other test te embodiments, backing 1406 comprises materials for vent functionality, hermetic seal maintenance, thermal interface, expansion chamber(s) and may optionally comprise a printed circuit board or flexible circuit layer carrying fluid and temperature control electronic components. Electronic ents may optionally be housed in a reusable g unit. PCA 1501 comprising electronic components is preferably ucted of low thermal mass materials and surface mount electronic components.

Arrays of surface mount resistors and proximally situated temperature s provide one means of regulating r temperatures in the test cassette. Surface mount resistors and temperature sensor arrays of PCA 1501 are situated to be in er with the test cassette when the test cassette in loaded into the docking unit. A sample-to-result integrated cassette is shown in . illustrates the integrated cassette with an underlying electronics layer based on traditional printed circuit board and surface mount components. In some embodiments a flexible circuit may be bonded to the rear of the test cassette.

Electronics In some embodiments it is desirable to place electronic components in a reusable component such that heaters, sensors and other electronics are interfaced to the disposable test cassette by a means capable of establishing a favorable thermal interface and accurate registration of electronics with overlying elements of the able test te with which they must interface. In other ments it is desirable to use a combination of reusable and able components for temperature control. For example, stand-off temperature monitoring can be accomplished with infrared sensors placed in a reusable docking unit, while resistive heaters for temperature control and fluidics control are placed in a flexible circuit integrated into the disposable test cassette.

In some embodiments, the printed circuit board (PCB) comprises a standard 0.062 inch thick FR4 copper clad te material, although other standard board materials and thicknesses may be used. Electronic components such as resistors, stors, LEDs, and the microcontroller preferably se off-the-shelf surface mount devices (SMDs) and are placed according to industry standard methodology.

In alternative embodiments, the PCA could be integrated with the cassette wall and comprise a flexible plastic circuit. Flex circuit materials such as PET and polyimide may be used as shown in The use of flexible plastic circuitry is well known in the art. In another embodiment, heating elements and temperature sensors may be screen printed onto the c fluidic component with technology developed by companies such as Soligie, Inc.

In some embodiments of the invention, the PCB thickness as well as the amount and placement of copper in regions surrounding the resistive heaters are tailored for l management of the reaction solution in the fluidic ent. This can be accomplished by use of standard manufacturing techniques y mentioned.

In some embodiments of the invention, the resistor is a thick film 2512 package, although other resistors may be used. Heating rs in the fluidic component are preferentially 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 uL of solution, ng a fluidic component thickness of 0.5 mm. The drawing in shows two resistors 100 forming a heater sufficient to heat approximately 30 uL of solution, assuming a fluidic component thickness of 0.5 mm. In this case, the resistors are preferably 40 ohm each and arranged in a parallel uration.

In some embodiments of the invention, ature sensor 110 preferably comprises a stor, such as a 0402 NTC device, or a temperature sensor such as the Atmel AT30TS750, each of which has a height similar to that of the 2512 resistor package. The thermistor is preferably aligned either adjacent to or in between the resistor heaters in the case of a one resistor or two resistor , respectively. By closely aligning these onic ts, only a very thin air gap results between them. Furthermore, application ofa thermal compound before assembling the fluidic with the electronic layer ensures good thermal t between the fluidic component, resistor, and thermistor.

In some embodiments of the invention, vent resistors 70, 71 comprise a thick film 0805 package, although similar ors may be used. In place of a resistor, a small gauge nichrome wire heating element, such as a 40 gauge nichrome wire may also be used.

In some ments of the invention, the microcontroller is a hip Technologies PIC16F1789. The microcontroller is preferably matched to the complexity of the fluidic system. For example, with multiplexing, the number of individual vents and heaters is commensurate with the number of microcontroller I/O lines. Memory size can be chosen to accommodate program size.

In certain embodiments of the invention, N-channel MOSFETs in the SOT-23 package operating in an ON-OFF mode are used to modulate current load to vent and heater ors.

Modulation signals are sent via the microcontroller. In alternative embodiments, a pulse-width- modulation scheme and/or other control algorithms could be used for more advanced thermal management of fluidics. This would typically be handled by the microcontroller and may require additional hardware and/or software features known to those skilled in the art.

Depending on the ation, some embodiments comprise a device in which a small controlling docking unit or docking unit operates a smaller disposable unit comprising fluidic systems which come in contact with biological materials, referred to as the test cassette. In one such embodiment, the docking unit comprises the electronic components. Elimination of electric components from the disposable test cassette s costs and in some cases environmental impact. In another embodiment, some electronic components are included in both the g unit and the test cassette. In this ular embodiment, the test te preferably comprises a low cost PCA or preferably a flexible t to provide some electrical functions such as temperature control, fluid flow l and ature sensing, which are energized, controlled and/or interrogated by the docking unit through an appropriate interface. As described above, the electronic functions of such a device is preferably split into two separate subassemblies. Disposable cassette 2500 preferably comprises a rear surface designed to interface with resistive heating and sensing elements of the docking unit. Materials comprising the rear face of the test te are preferably selected to provide suitable thermal conductivity and ity while ng fluid flow control via vent rupture. In some embodiments, the rear face of the test cassette or a portion f comprises a flexible circuit manufactured on a substrate such as polyimide. Flexible circuits can be employed to provide low cost ive heating elements with low l mass. Flexible t substrates may preferably be placed in direct contact with solutions present in the fluid network of the test cassette to enable highly ent and rapid heating and cooling. Connector 810 as shown in preferably provides current to the resistive heaters along with a power and signal line to the optional thermistor(s).

If flexible circuit 799 is used, one or more IR sensors located in the docking unit can monitor the temperature of the heated chambers (e.g. ication or detection chambers) by g the signal h a window in backing 805 or directly off the rear of flexible circuit 799. Optionally, thermistors on the PCA or flexible circuit 799 can be used to monitor the temperatures. Optionally performing 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, enabling the system to correct for it to ensure that the sample fluid brates rapidly to the desired temperatures.

Referring now to , the g unit preferably comprises a reusable component subassembly 3980 comprising the microcontroller, MOSFETs, switches, power supply or a ack and/or battery, optional g fan 903, al user interface, infrared temperature sensors 901, 902 and connector 900 compatible with connector 810 of cassette 2500. When the subassemblies are mated via connectors 810 and 900, the docking unit preferably supports disposable cassette 2500 in a substantially vertical or ertical orientation. Although a substantially vertical orientation is preferable in some of the embodiments described herein, similar results may be obtained if the device is operated at a tilt, especially if certain ys are coated to reduce the wetting angle of solutions used.

Another embodiment of the device may be used in order to minimize the operational costs, by reducing the cost of the consumable part of the system by eliminating all onic circuitry located on the disposable part. The microcontroller, heaters, sensors, power supply, and all other circuitry are located on multiple PCA's and electrically connected to each other via high conductor count industry standard ribbon cables. A display may also be added to aid the user in operation of the device. An optional serial l port may also be utilized in order to allow the user to upload changes in test parameters, and to monitor the progress of any testing. One version of this embodiment comprises five different PCA's. The Main Board PCA contains the control circuitry, serial port, power supply, and connectors to connect to the other boards in the system. The Heater Board PCA contains the heating or elements, temperature sensors, and vent burn g elements. In order to facilitate the thermal interface n this heater board and the disposable fluidic cassette, this board is mounted on a spring loaded carrier which is moved towards the backside of the fluidic cassette by the closing action of the lid, until contact with the fluidic cassette is made. A thin lly conductive heating pad is d on top of the chamber heater resistors and temperature senor, improving heat transfer between the heater board and the fluidic cassette. A durable vent burning heating element may be realized using nichrome wire wrapped around a small c carrier. The IR sensor board PCA is mounted some small distance from the opposite side of the cassette and is used for monitoring the heating chamber temperatures. This allows closed loop temperature control of the heating and cooling process, and accommodates ambient temperature ions. Also mounted on the IR sensor board are multiple reflective sensing optical couplers which allow the sensing of the presence of the te, and may be used to fy the type of cassette denoted by the configurable reflective pattern located on the cassette. A Display Board PCA may be located approximately behind the IR Board to allow the user to see the display from the front of the device. A final PCA, the shutter board is located across from the top edge of the cassette and contains a switch and reflective l coupler which is used to sense whether or not the cassette has already been used, and when the lid closes, holding the cassette in place for testing.

System cooling is ally augmented using a fan such as a muffin style fan which is turned on by the microcontroller only during the cooling phase of testing. A system of vents is preferably used to direct cooler outside air against the heating chambers and expel it out the sides of the device.

In order to provide a te sample-to-result molecular test, any of the above embodiments of the invention may be interfaced to a sample preparation system 1300 that es nucleic acids as output to sample chamber 1402. This has been demonstrated using the sample preparation technology described in International Publication No.

Lateral Flow-Based Nucleic Acid Sample Preparation and Passive Fluid Flow Control". An embodiment of the resulting integrated device is illustrated in and .

Docking unit The reusable docking unit comprises requisite electronic components to e test cassette functionality. Various docking unit embodiments have been invented to interface with corresponding variations in test cassette . In one embodiment, the docking unit, shown in and , comprises all electronic components required to run a test, eliminating the need for electronic components in the test cassette. Referring now to , prior to sample addition, cassette 2500 is inserted into docking unit 2501. Docking unit 2501 comprises a display such as LCD display 2502 to communicate information such as test protocols and test status to the user. Following cassette insertion into docking unit 2501, sample is introduced to sample port 20 of cassette 2500 and docking unit lid 2503 is closed to initiate the test. A docking unit with inserted test cassette is shown in , and Docking unit 2501 with lid in the closed position is illustrated in 8.

In some embodiments of the docking unit, a mechanism is incorporated into the hinge of lid 2503 which moves sliding seal 91 of the test cassette to the closed position. A sealed test cassette is helpful to ensure amplified nucleic acids remain contained within the test cassette. ing now to , either a manual or an automated method may be employed to slide a valve over the sample port to seal the cassette. In some embodiments the slide seals the sample port by engaging an o-ring.

The expansion chamber cover holds the valve slide in place above the sample port o-ring. The seal is moved into on by a servo motor or by a manual action such as closing the le docking unit lid, which in turn actuates a mechanism to close the te seal. In the pictured embodiment rack and pinion mechanism 2504 employs slide seal actuator 3979 to move sliding seal 91 to the closed position. Rack and pinion ism 2504 may be motorized or moved by the action of e of docking unit lid 2503 through a mechanical coupling to lid hinge. Optionally, a sensor such as optical sensor 2505 may be ed to interrogate the position of sliding seal 91 to ensure proper seal ent prior to assay initiation as illustrated in . The optical sensor detects the state (i.e. on) of the cassette sample port seal. The optical sensor allows the docking unit to be programed to detect accidental insertion of a previously used test cassette and to detect the successful closure of the test cassette seal. An error message ting seal malfunction may be displayed on display 2502 and the test program aborted should sensor 2505 fall to detect seal e. In other ments of the test cassette and docking unit, the sealing mechanism may comprise other means of mechanically sealing the chamber such as a rotating valve as illustrated in . In yet another embodiment, a test cassette seal may be placed in a hinged cassette lid placed such that insertion into the docking unit is not possible without first closing the cassette lid and thus seating the seal. In this embodiment, sample is added to the test cassette prior to insertion into the docking unit. A test cassette comprising a hinged lid with seal is illustrated in . 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 lid of the docking unit both seals the te automatically and initiates the assay, preferably without the use of servos or other mechanical devices.

In some docking unit embodiments a set of components preferably facilitate proper test cassette insertion while ensuring electronic components that must interface with the test cassette do not al interfere with cassette insertion, yet form a reliable thermal interface during testing. These ents form a mechanism for holding PCA 75 away from the cassette ion path until closure of lid 2503. Referring now to , within the g unit the heater board is mounted on PCA holder 2506, which preferably serves as a low thermal mass scaffold, while the test cassette is loaded into a low thermal mass cassette holder 2507, wherein rails 2509 guide the cassette into the docking unit and hold it in the correct position, such as parallel to the heater board surface, for acing with PCA 75 mounted on PCA holder 2506. In the lid open position, prominences 2508 on cassette holder 2507 ere with PCA holder 2506 to maintain an open path along rails 2509 for te insertion.

Preferably, a sloped surface spans the distance between the surface of prominences 2508 and the lower elevation of component 2507 to facilitate smooth movement of prominences 2508 into depressions 2511 on PCA holder 2506 during closure of lid 2503. Upon closure of the docking unit lid, prominences 2508 engage with depressions 2511, thereby moving the heater board mount closer to the rear surface of test cassette 2500. Closure of lid 2503 exerts rd force on cassette holder 2507 thereby moving te holder 2507 to a position where prominences 2508 come to rest in depressions 2511 resulting in movement of PCA holder 2506 such that PCA 75 is pressed against the rear of cassette 2500. Preferably PCA holder 2506 is under constant force, such as spring force, to enable the exertion of reproducible pressure t the rear of the cassette by PCA 75 after lid closure. Placement of PCA 75 against the rear of cassette 2500 forms the thermal interface which conducts heat from resistive heater elements on the PCA to the temperature controlled chambers and vents of the test cassette. Preferably components 2506 and 2507 are ucted to contribute l l mass to the system and provide access to test cassette surfaces for cooling apparatus, such as fans, and temperature monitoring by sensors, such as infrared sensors. After lid closure the heater board is thus preferably pressed firmly against the rear of the test cassette, forming a thermal interface that enables the microheaters on the heater board to heat solutions in the fluid chambers of the test cassette and to melt the lly labile vent films of the test te, preferably in accordance with ontroller or microprocessor control. illustrates the cassette-PCA interfacing mechanism in cross-section in both disengaged (lid open) and engaged (lid closed) positions.

In some embodiments, the docking unit comprises additional sensors for such applications as ature sensing, detecting the presence of or removal of a test cassette and detecting specific test tes for enabling ted selection of testing parameters. Referring now to , infrared sensors 2600 detect the ature of the test cassette in regions overlying temperature controlled chambers, such as chambers 30 and 90. The sensors enable the collection of temperature data in addition to or in lieu of temperature data collected by PCA 75 localized temperature sensors, such as sensor 110. Optical s may ally but preferably be employed to detect specific test cassettes to identify cassettes for specific diseases or conditions and allow automated ion of temperature profiles suitable for a specific test. Referring now to FIGS. 27A and 278, an optical sensor or l sensor array such as optical senor array 2601 may be employed in conjunction with barcode or barcode-like features 2602 on the test cassette to determine the type of test cassette and to confirm complete insertion and correct seating of the test cassette. Sensor array 2602 in concert with sensor 2505 may be employed to detect the insertion of a previously used test cassette by detecting a closed seal prior to lid closure. The docking unit may comprise sensors to detect the type of test cassette ed into the docking unit andior to confirm the correct insertion, positioning, and alignment of the te within the g unit. Detection of an influenza A/B test cassette is illustrated in the docking unit and test cassette system depicted in . The docking unit can preferably also read a barcode or other symbol on each te and change its programming in accordance with stored programs for ent .

In some ments of the invention it is desirable to heat both sides of a test cassette. A dual heater PCA configuration wherein the test cassette is ed between two heater PCAs is depicted in FIGS. 28A and 288.

In another embodiment, the docking unit comprises servo actuators, an optical subsystem for automated result readout, a wireless data communication subsystem, a touch screen user interface, a rechargeable battery power source, and a test cassette receiver which accepts a test cassette comprising an integrated sample preparation subsystem. Referring now to FIGS. 29A, 298, and 30, docking unit 2700 accepts test cassette 1500 and places the test cassette in thermal contact with PCA 1501 to enable temperature control and fluid flow control of the test cassette. Test te 1500 is inserted into cassette receiver slot 3605 of pivoting docking unit door 2702. Following the addition of crude sample or lysate to the test cassette, closure of docking unit door places the rear of the test cassette in register and in contact with PCA 1501 and in alignment with servo actuators. Servo actuator 3602 is situated to access solution compartmentalization component 1303 h actuator port 1310 of cassette 1500 and provide mechanical force required to rupture sealing material 1305.

Rupture of mechanical seal 1305 releases crude lysate and wash buffers to flow through the sample preparation capillary materials of the sample preparation subsystem as described above. Following completion of ary fluid transport, servo actuator 3601 which is situated to access elution reservoir 1317 through or port 1320 of cassette 1500 provides mechanical force to move component 1317 such that attached conduit 1318 forms a seal with seal 1316 and ces binding matrix 1306 into elution compartment 1321. Servo actuator 3604 ed to access elution plunger 1319 through actuator port 1322 of cassette 1500 then provides mechanical force to plunger 1319 to expel elution buffer from elution reservoir 1317 though binding matrix 1306, resulting in the elution of nucleic acids into sample cup 1402 of cassette 1500. Servo actuator 3603 seals the cassette after elution as described above. Actuator control is preferably provided by a microcontroller or microprocessor on control electronics PCA 3606 ing firmware or software instructions. Similarly, temperature and fluid flow control within the test cassette is according to instructions ed in firmware or software routines stored in microcontroller or microprocessor memory. Optical subsystem 3607 comprising LED light source 3608 and CMOS sensor 3609, shown in , digitizes detection strip signal data.

Collected detection strip images are stored into memory within the docking unit where result interpretation can be accomplished using an on-board processor and reported to LCD display 2701. A ring of LEDs provides uniform nation during image collection with a CMOS-based digital .

Images collected with the postage stamp-sized device can provide high-resolution data (5 megapixels, 10 bit) suitable for metric lateral flow signal analysis. A ably low profile design (~1 cm) er with short working distance optics enables the system to be integrated into a thin device housing.

Optionally, digitized results may be transmitted for off-line analysis, storage and/or visualization via a wireless communication system incorporated into the docking unit employing either standard WiFi or cellular communications networks. Photographs of this docking unit embodiment are shown in FIGS. 32A and 328.

Examples Example 1: Method of lexed amplification and detection of purified viral RNA (ian/B) and an internal positive control virus An nza A and B test cassette was placed into the docking unit. 40 uL of a sample solution was added to the sample port. Sample solutions comprised either purified A/Puerto Rico influenza RNA at a concentration equivalent to 5000 mL, purified B/Brisbane nza RNA at a concentration equivalent to 500 TCleo/mL or molecular grade water (no template l sample).

Upon entering the sample port, the 40 uL sample comingles with a lyophilized bead as it flows to a first r of the test cassette. The lyophilized bead was comprised of M82 phage viral particles as a 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 then cooled to 50 "’0 prior to opening the vent connected a second chamber. Opening the vent connected to the second chamber allows the sample to flow into the second chamber by ng the cement of the air in the second chamber to an expansion chamber. As the sample moved to the second r it comingled with oligonucleotide amplification primers to influenza A, influenza B and M82 phage, and e transcription and nucleic acid amplification ts and s present as a lyophilized pellet in a recess of the fluid path between first and second rs.

The amplification chamber was heated to 47 °C for 6 minutes, during which time RNA te was reverse transcribed into cDNA. After completion of reverse transcription, 40 cycles of thermal cycle amplification was conducted in the second r. After thermal cycling was complete, 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 bead comprising three blue-dyed polystyrene phere conjugates employed as detection particles. Conjugates were comprised of 300 nm polystyrene microspheres covalently linked to oligonucleotide probes complementary to amplified sequences of nza A, or influenza B or M82 phage. 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 they were: A negative control oligonucleotide not complementary to any assayed targets; a capture probe complementary to the amplification product of influenza B; a capture probe complementary to the amplification product of influenza A; and a oligonucleotide complementary to the amplification product of M82 phage. The lateral flow strip was allowed to develop for six minutes prior to visual interpretation of the results.

Upon development of the l flow strip, influenza A positive samples displayed the formation of blue test lines at the influenza A and M82 phage positions, influenza B positive samples displayed the formation of blue test lines at the influenza B and M82 phage positions, negative samples displayed the formation of blue test lines only at the M82 phage position as shown in .

Example 2: Method of multiplexed amplification and detection of viral lysate in buffer and an internal ve control virus An nza A and B test cassette was placed into the docking unit. 40 uL of a sample solution was added to the sample port. Sample solutions comprised either A/Puerto Rico nza virus at a concentration lent to 5000 TCleo/mL, B/Brisbane influenza virus at a concentration equivalent to 500 TCIDso/mL or molecular grade water (no template control sample). Upon entering the sample port, the 40 uL sample comingles with a lyophilized bead as it flows to a first chamber of the test cassette. The lyophilized bead was sed of M82 phage viral particles as a 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 then cooled to 50 "C prior to opening the vent connected a second chamber. Opening the vent connected to the second chamber allows the sample to flow into the second chamber by enabling the displacement of the air in the second chamber to an expansion chamber. As the sample moved to the second chamber it comingled with oligonucleotide amplification primers to influenza A, influenza B and M82 phage, and reverse transcription and nucleic acid amplification reagents and enzymes t as a lyophilized pellet in a recess of the fluid path between first and second chambers.

The amplification chamber was heated to 47 "C for 6 minutes, during which time RNA template was reverse transcribed into cDNA. After completion of reverse transcription, 40 cycles of thermal cycle ication was conducted in the second r. After thermal cycling was te, a vent connected to a third r was opened to allow the reaction solution to flow into the third chamber. The third chamber comprised a test strip and a lyophilized bead comprising three blue-dyed polystyrene microsphere conjugates ed as detection particles. Conjugates were comprised of 300 nm polystyrene microspheres covalently linked to oligonucleotide probes complementary to amplified sequences of influenza A, or influenza B or M82 phage. 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 they were: A negative control oligonucleotide not complementary to any assayed s; a capture probe complementary to the amplification product of influenza B; a capture probe complementary to the amplification product of influenza A; and a oligonucleotide complementary to the ication t of M82 phage. The lateral flow strip was allowed to develop for six minutes prior to visual interpretation of the results.

Upon development of the lateral flow strip, influenza A positive samples displayed the formation of blue test lines at the influenza A and M82 phage positions, nza B positive samples displayed the formation of blue test lines at the nza B and M82 phage positions, negative samples displayed the formation of blue test lines only at the M82 phage position as shown in .

Exam le 3: Method of multi lexed am lification and detection of influenza virus urified s iked into negative clinical nasal samples and an al positive control virus Nasal swab samples collected from human subjects were placed into 3 mL of a 0.025% Triton X-100, 10 mM Tris, pH 8.3 solution and tested for the presence of influenza A and influenza B using an FDA approved real-time RT-PCR test. Samples were confirmed to be negative for nza A and influenza B prior to use in this study. Confirmed influenza negative nasal sample was spiked with AfPuerto Rico influenza virus at a concentration equivalent to 5000 TCleo/mL or employed t the addition of virus as a negative control. 40 uL of the resulting spiked or ve control s were added to the sample port of a influenza A and B test cassette. Upon entering the sample port, the 40 pL sample comingles with a lyophilized bead as it flows to a first chamber of the test te.

The lyophilized bead was comprised of M82 phage viral particles as a 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 then cooled to 50 "C prior to opening the vent connected a second chamber. Opening the vent connected to the second chamber allows the sample to flow into the second chamber by enabling the cement of the air in the second r to an expansion chamber. As the sample moved to the second chamber it comingled with oligonucleotide amplification primers to influenza A, influenza B and M82 phage, and reverse transcription and nucleic acid amplification reagents and enzymes present as a lyophilized pellet in a recess of the fluid path n first and second chambers.

The amplification chamber was heated to 47 °C for 6 minutes, during which time RNA template was reverse transcribed into cDNA. After tion of reverse transcription, 40 cycles of thermal cycle amplification was conducted in the second chamber. After l cycling was complete, 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 bead comprising three blue-dyed polystyrene microsphere ates employed as detection particles. Conjugates were comprised of 300 nm yrene microspheres ntly linked to ucleotide probes complementary to amplified sequences of influenza A, or influenza B or M82 phage. 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 they were: A ve control oligonucleotide not complementary to any assayed targets; a capture probe complementary to the amplification product of influenza B; a capture probe complementary to the amplification product of influenza A; and a oligonucleotide complementary to the ication product of M82 phage. The lateral flow strip was allowed to develop for six minutes prior to visual interpretation of the results.

Upon development of the lateral flow strip, influenza A positive samples displayed the formation of blue test lines at the influenza A and M82 phage positions, negative control samples displayed the formation of blue test lines only at the M82 phage position as shown in .

Note that in the specification and claims, "about" or "approximately" means within twenty percent (20%) of the numerical amount cited. As used herein, the singular forms "a, 71 :5 an," and "the" include plural referents unless the context clearly dictates othenNise. Thus, for e, reference to "a functional group" refers to one or more functional groups, and 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 forth.

Although the invention has been bed in detail with particular nce to the disclosed ments, other ments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover all such modifications and equivalents. The entire sures of all patents and publications cited above are hereby incorporated by reference.

Claims (12)

1. A cassette for detecting a nucleic acid, the cassette comprising 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 downward into said reaction chamber to minimize or t capillary fluid flow across said top of said reaction chamber.

2. The cassette of claim 1 wherein said projection is generally triangular in shape.

3. The cassette of claim 1 wherein a first side of said projection s substantially ally adjacent to said inlet.

4. The cassette of claim 3 wherein a second side of said projection extends upward toward said top of said on chamber at an angle less than approximately 60 degrees from vertical.

5. The te of claim 4 wherein said angle is less than approximately 45 degrees 20 from al.

6. The cassette of claim 5 wherein said angle is less than approximately 30 degrees from vertical. 25

7. The cassette of claim 6 wherein said second side of said projection extends vertically toward said top of said reaction chamber. WO 95493

8. The te of claim 1 comprising a recess for containing at least one Iyophilized or dried reagent, said recess disposed in a channel connected to said inlet of said on chamber.

9. The cassette of claim 8 wherein said projection reduces or prevents sequestration of newly resuspended reagent from the bulk of the reaction solution volume.

10. The cassette of claim 8 wherein said recess comprises one or more structures for directing fluids to facilitate rehydration of the at least one dried or Iyophilized reagent. 10

11. The cassette of claim 10 n said structures comprise ridges, grooves, dimples, or combinations thereof.

12. The cassette of claim 1 wherein said reaction chamber comprises a recess for containing at least one Iyophilized or dried reagent. WO 95493 fie“. Vanna” a fifififiti ‘. 11144111 w‘ \\\‘k\\\\ 5 9 § \\ ”In”... III/ml”;”2”»»»»; 4 p, .i,,‘i 11111111114 {Im/I/I/I/I/I/IW/I/I/I/n»»»»”0”»;nnnv” 4l”mtg: a1 aaalallllllllIlluulnuuuuuuu IIm/l/II/I/I/Illm////I/Il»»~»»»»»””»”»»» —$5 - “WWW“«<«««<\\§ I.I.I.5. LU E,5%”III/k I “—4 ,145,