Fluidic test cartridge
阅读说明:本技术 流控式测试盒 (Fluidic test cartridge ) 是由 唐纳德·J·托马斯 蔡红 罗伯特·B·凯瑞 于 2018-04-20 设计创作,主要内容包括:一种用于检测核酸或执行其他测定的一次性盒。所述盒可在使用期间插入基站中。所述盒具有用于确保装置在重力下正确操作的多个特征,诸如通气口袋,所述通气口袋用于在所述通气口袋未被密封时使得样品流体能够从一个腔室流动到下一个腔室。所述通气口袋具有用于帮助防止意外再密封的突起。所述盒还可具有用于确保打开的通气口袋之间的自由空气移动的垫圈。具有设置在热稳定材料上的图案化金属电子部件的柔性电路可与所述腔室中的流体直接接触,并且具有与所述通气口袋和所述腔室对准的电阻加热元件。盒通道或腔室中的凹槽可具有用于引导流体流动以增强设置在所述凹槽中的冻干试剂的再水化的结构,诸如脊部或沟槽。所述腔室中的偏流器可降低所述样品流体的流速,并增加有效流体流动路径长度,从而实现对所述盒中流体流动的更准确控制。每个腔室的最高处可具有凸出部,所述凸出部防止横跨所述腔室的顶部的毛细管流体流动,从而减少或防止来自大部分反应溶液体积的新再悬浮试剂的截存。(A disposable cartridge for detecting nucleic acids or performing other assays. The cassette may be inserted into a base station during use. The cartridge has a number of features for ensuring proper operation of the device under gravity, such as a vent pocket for enabling sample fluid to flow from one chamber to the next when the vent pocket is not sealed. The vent pocket has a protrusion to help prevent accidental resealing. The case may also have a gasket for ensuring free air movement between the open vent pockets. A flexible circuit having a patterned metallic electronic component disposed on a thermally stable material may be in direct contact with fluid in the chamber and have a resistive heating element aligned with the vent pocket and the chamber. The grooves in the cartridge channel or chamber may have structures, such as ridges or grooves, for directing fluid flow to enhance rehydration of lyophilized reagents disposed in the grooves. Flow diverters in the chamber can reduce the flow rate of the sample fluid and increase the effective fluid flow path length, thereby enabling more accurate control of fluid flow in the cartridge. The uppermost of each chamber may have a projection that prevents capillary fluid flow across the top of the chamber, thereby reducing or preventing the entrapment of fresh resuspended reagent from the bulk of the reaction solution volume.)
1. A cartridge for detecting nucleic acids, the cartridge comprising at least one reaction chamber;
wherein, when the cartridge is vertically oriented, a top of the reaction chamber comprises an inlet and a protrusion extending downward into the reaction chamber to minimize or prevent capillary fluid flow across the top of the reaction chamber.
2. The cartridge of claim 1, wherein the projection is generally triangular in shape.
3. The cartridge of claim 1, wherein a first side of the projection extends substantially vertically adjacent the inlet.
4. The cartridge of claim 3, wherein a second side of the projection extends upward toward the top of the reaction chamber at an angle of less than about 60 degrees from vertical.
5. The cassette of claim 4, wherein the angle is less than about 45 degrees.
6. The cassette of claim 5, wherein the angle is less than about 30 degrees from vertical.
7. The cartridge of claim 6, wherein the second side of the projection extends vertically toward the top of the reaction chamber.
8. The cartridge of claim 1, comprising a recess for containing at least one lyophilized or dried reagent disposed in a channel connected to the inlet of the reaction chamber.
9. The cartridge of claim 8, wherein the projections reduce or prevent the entrapment of freshly resuspended reagent from a majority of reaction solution volume.
10. The cartridge of claim 8, wherein the recess comprises one or more structures for directing a fluid to facilitate rehydration of the at least one lyophilized or dried reagent.
11. The cartridge of claim 10, wherein the structure comprises ridges, grooves, dimples, or a combination thereof.
12. The cartridge of claim 1, wherein the reaction chamber comprises a recess for containing at least one lyophilized or dried reagent.
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 include a disposable portion and a reusable portion.
Background
Disclosure of Invention
The present invention is a cartridge for detecting a target nucleic acid, the cartridge comprising a plurality of chambers; a plurality of vent pockets connected to the chamber; and a heat labile material for sealing one or more of the vent pockets, wherein at least one of the vent pockets includes a protrusion. The protrusions preferably comprise dimples or microprotrusions and are preferably sufficient to prevent molten heat labile material from adhering to heat labile material disposed adjacent to the heat labile material to prevent resealing of the vent pocket after rupture of the heat labile material.
The present invention is also a cartridge for detecting a target nucleic acid, the cartridge comprising a plurality of chambers; a plurality of vent pockets connected to the chamber; a heat labile material for sealing one or more of the vent bags; a thermally stable material; and a gasket disposed between the thermally unstable material and the thermally stable material, the gasket including an opening surrounding the plurality of vent pockets. The gasket is preferably thick enough to provide sufficient air volume to equalize pressure and ensure free air movement between open vent pockets. The cartridge preferably includes a flexible circuit including a patterned metallic electronic component disposed on a thermally stable material. The gasket preferably includes a second opening or is restricted in size so that the flex circuit will be in direct contact with the fluid in at least one of the chambers. The electronic components preferably comprise resistive heating elements or conductive tracks. The resistive heating elements are preferably aligned with the vent pockets and chambers. The cartridge preferably comprises one or more ambient temperature sensors for adjusting the heating temperature, heating time and/or heating rate of one or more of the chambers.
The present invention is also a cartridge for detecting a target nucleic acid, the cartridge comprising a vertically oriented detection chamber; a lateral flow test strip disposed in the test chamber oriented such that a sample receiving end of the test strip is at a bottom end of the test strip; and a space below the lateral flow detection strip in the detection chamber for receiving a fluid comprising amplified target nucleic acids, the space comprising sufficient volume to accommodate the entire volume of fluid at a height that enables the fluid to flow by capillary action onto the detection strip without overflowing or otherwise bypassing a region of the detection strip. The space preferably includes detection particles such as dye polystyrene microspheres, latex, colloidal gold, colloidal cellulose, nanogold, or semiconductor nanocrystals. The detection particles preferably comprise oligonucleotides complementary to the sequence of the amplified target nucleic acid or a ligand capable of binding to the amplified target nucleic acid, such as biotin, streptavidin, a hapten or an antibody. The detection particles are preferably dried, lyophilized, or present as a dry mixture of detection particles in a carrier (such as polysaccharides, detergents, proteins) on at least a portion of the inner surface to facilitate resuspension of the detection particles. A capillary pool of fluid is preferably formed in the space that provides improved mixing and dispersion of the detection particles to facilitate blending of the detection particles with the amplified target nucleic acid. The cartridge optionally performs assays having a volume of less than about 200 μ L, preferably less than about 60 μ L.
The present invention is also a cartridge for detecting a target nucleic acid, the cartridge comprising one or more recesses for containing at least one lyophilized or dried reagent, at least one of the recesses comprising one or more structures for directing a fluid to facilitate rehydration of the at least one dried or lyophilized reagent, the recess disposed in one or more detection chambers or one or more channels connected to a detection chamber. The structure preferably comprises ridges, grooves, dimples or a combination thereof.
The present invention is also a cartridge for detecting a target nucleic acid, the cartridge comprising at least one chamber comprising an outlet for preventing a fluid vertically entering the top of the chamber from directly flowing into the chamber. The feature preferably deflects the fluid to the side of the chamber opposite the outlet. The resulting fluid flow path preferably includes a horizontal component to substantially increase the effective length of the flow path and to substantially decrease the flow rate of the fluid to limit the amount of fluid exiting the outlet. The feature preferably creates a swirling flow of fluid within the chamber, thereby increasing mixing of the reagents within the fluid. The shape of the features is preferably triangular or trapezoidal. The outlet is optionally tapered. The channel downstream of the outlet preferably comprises a bend for increasing the effective length of the channel. The feature is preferably located near the bottom of the chamber, or at the bottom of the chamber, or near the middle of the chamber.
The present invention is also a method of controlling the vertical flow of fluid through a chamber in a cartridge for detecting a target nucleic acid, the method comprising deflecting the flow of fluid into the top of the chamber, thereby preventing the direct flow of fluid into an outlet of the chamber. The method preferably comprises reducing the flow rate of the fluid, thereby reducing the distance the fluid flows down the channel connected to the outlet before the fluid stops. The method preferably comprises dividing the fluid flow into the chamber into a first fluid flow in contact with the chamber wall and directed upwardly and a second fluid flow into the outlet. The first fluid flow is preferably rotated in the chamber to increase mixing of the reagents within the fluid. The second fluid flow preferably forms a meniscus that increases the pressure in the closed air space in the fluid downstream channel until the pressure stops the fluid flow in the channel and travels through the channel connected to the outlet. The outlet is optionally tapered, increasing the volume of compressible air at the inlet of the outlet. The method optionally includes providing a bend in the channel connected to the outlet, thereby increasing the effective path length of the channel and reducing the flow rate of the fluid in the channel.
The present invention is also a cartridge for detecting nucleic acids comprising at least one reaction chamber, wherein, when the cartridge is oriented vertically, the top of the reaction chamber comprises an inlet and a projection that extends downwardly into the reaction chamber to minimize or prevent capillary fluid flow across the top of the reaction chamber. The shape of the projection is preferably substantially triangular. The first side of the projection preferably extends substantially vertically adjacent the inlet. The second side of the projection preferably extends upwardly toward the top of the reaction chamber at an angle of less than about 60 degrees from vertical, more preferably less than about 45 degrees from vertical, even more preferably less than about 30 degrees from vertical, and optionally vertically. The cartridge preferably comprises a recess for containing at least one lyophilized or dried reagent, said recess being provided in a channel connected to the inlet of the reaction chamber. The projections preferably reduce or prevent the entrapment of fresh resuspended reagent from the bulk of the reaction solution volume. The recess preferably comprises one or more structures for directing a fluid to facilitate rehydration of the at least one lyophilized or dried reagent. The structure preferably comprises ridges, grooves, dimples or a combination thereof. Alternatively or additionally, the reaction chamber comprises a recess for containing at least one lyophilized or dried reagent.
The 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.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention. These 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 figure:
FIG. 1A is a diagram showing an embodiment of a test cartridge of the present invention.
FIG. 1B is an exploded view of one embodiment of a test cartridge showing the sliding seal, sampling port, sampling cup, and the interior region of the expansion chamber.
Fig. 2A is a schematic representation of a fluidic network in one embodiment of a test cartridge of the present invention.
Fig. 2B-2C are schematic diagrams of how a thermally triggered vent may be employed to vent an expansion chamber to achieve fluid flow control in the context of a hermetically sealed test cartridge before and after the vent is opened, respectively.
Fig. 2D is a diagram of one embodiment of a disposable test cartridge showing the placement of a Printed Circuit Assembly (PCA) including a resistive heating element and a temperature sensor.
Fig. 2E is a photograph of an injection molded plastic test cartridge that includes the features depicted in fig. 2A.
Fig. 3A is an illustration of the operating principle of an embodiment of an expansion chamber.
Figure 3B is a cross section of a piston-based expansion chamber in a test cassette prior to expansion of the gas.
Figure 3C is a cross section of the piston-based expansion chamber after expansion of the gas within the test cassette.
Fig. 4A is an illustration of a method of forming an inflation chamber in which an inflatable bladder is employed to provide an inflated internal volume.
Figure 4B is a cross section of the bladder-based expansion chamber prior to expansion of the gas within the test cassette.
Figure 4C is a cross section of the bladder-based expansion chamber after gas expansion within the test cassette.
Fig. 5A is an illustration of a method of forming an expansion chamber in which an expandable bellows is employed to provide an expanded internal volume.
Figure 5B is a cross section of a bellows-based expansion chamber prior to expansion of the gas within the test cassette.
Figure 5C is a cross section of a bellows-based expansion chamber after expansion of the gas within the test cassette.
Fig. 6A shows the use of a semi-permeable barrier, membrane or material that allows gas to pass freely while particles such as bacteria, viruses or macromolecules (such as DNA or RNA, for example) remain within the device.
Figure 6B is a cross section of the semipermeable barrier used in place of the expansion chamber to equalize or reduce the internal pressure to ambient pressure.
FIG. 7 is an exploded view of the cartridge design, wherein the expansion chamber is formed by a spacer between a layer of Biaxially Oriented Polystyrene (BOPS) film.
Fig. 8A is a diagram of an embodiment of a flexible circuit including resistive heating elements for two fluid chambers, a test strip chamber, and three vents, and electrical contact pads.
Fig. 8B is an embodiment of a flexible circuit including a resistive heating element for two fluid chambers, a test strip chamber, and three vents, and electrical contact pads for energizing the resistive heating element.
Figure 8C is an exploded view of an embodiment of a test cartridge.
FIG. 8D is a view of the assembled test cartridge of FIG. 8C.
Fig. 9 depicts a lateral flow strip of a device with and without a capillary cell at the sample receiving end of the strip. When a capillary cell was present, a more uniform distribution of detection particles and a more uniform signal were observed on the strip.
Fig. 10 is a diagram showing a layering method of sample separation.
Fig. 11 is an illustration of a multi-channel fluidic network for multiplexing and sample subdivision showing the fluid flow paths for each test. Additional fluidic paths or channels may be incorporated into the network to further increase the number of parallel tests that can be simultaneously performed in a single disposable test cartridge.
FIG. 12 is a diagram of a fluidic network in one embodiment of a test cartridge of the present invention in which samples are separated after introduction into a sampling cup through a sampling port to enable parallel independent testing of the same input sample. The bifurcated fluid path from the sampling cup allows the sample solution to be separated into two different fluidic channels or paths of the test cartridge to allow the test to run concurrently on separate samples.
Fig. 13A is a diagram of an assembled sample preparation subsystem, showing the internal component arrangement.
Fig. 13B is an exploded view of the sample preparation subsystem showing components of a nucleic acid purification device configured for integration with a test cartridge.
FIG. 14 is a cross section through the sample preparation subsystem showing part movement that occurs during processing of a sample.
Fig. 15 is an exploded view showing a sample preparation subsystem with hermetic sealing components, injection molded fluidic subsystems, corresponding cartridge backing, and PCA.
FIG. 16 is a photograph of an embodiment of a test cartridge with an integrated sample preparation subsystem.
Fig. 17A is an exploded view showing a sample preparation subsystem with a hermetic seal and injection molded fluidic subsystem design.
Fig. 17B is a diagram of a test cartridge embodiment with an integrated sample preparation subsystem shown interfaced to a PCA.
Fig. 17C is a cross-sectional view of a cartridge embodiment depicting fluidic pathways, interfacing electronics, and sample preparation components.
Fig. 18A is a diagram of an embodiment of a docking unit of the present invention shown with the lid in an open position and an inserted test cartridge.
Fig. 18B is a diagram of the docking unit shown with the lid in the closed position.
FIG. 19 is a photograph of one embodiment of a docking unit shown with the lid in an open position and the test cartridge inserted. The LCD display indicates detection of insertion of the influenza A/B virus test cassette.
Fig. 20 shows an embodiment of the cartridge sealing mechanism of the present invention.
Fig. 21A is a diagram of a cartridge seal sensor placed in a docking unit with an inserted cartridge and a seal in an open position.
FIG. 21B is a cross-sectional view of a cartridge seal sensor placed in a docking unit with an inserted cartridge and seal in an open position.
Fig. 21C is a diagram of a cartridge seal sensor placed in a docking unit with an inserted cartridge and seal in a closed position.
FIG. 21D is a cross-sectional view of a cartridge seal sensor placed in a docking unit with an inserted cartridge and seal in a closed position.
Fig. 22 is a diagram of an embodiment of a cartridge sealing mechanism in which a drive gear is employed to regulate sealing closure using a rotary valve.
Figure 23A is a diagram showing an embodiment of a test cartridge in which the lid is a hinged lid that includes an O-ring seal and an empty air volume that acts as an expansion chamber. In this figure, the lid is in the open position.
Fig. 23B is a diagram showing the lid in a closed position, with the O-ring forming a hermetic seal with the rim of the sampling port.
Fig. 24A is an exploded view of the heater plate and the cartridge seat components of the docking unit that form the cartridge receiving subassembly.
Fig. 24B is a diagram of an embodiment of a cartridge receiving subassembly of a docking unit.
FIG. 25 is a side view of the test cartridge bay and heater plate mounting system in an engaged position and a disengaged position.
Fig. 26 is a diagram depicting infrared temperature sensors placed in one embodiment of the docking unit to monitor the temperature of the first and second heated fluidic chambers.
Fig. 27A is a diagram showing an optical sensor placed within an embodiment of a docking unit to allow reading of a bar code positioned near the bottom of a test cartridge.
Fig. 27B is a detail of fig. 27A.
Fig. 28A and 28B are exploded and assembled views, respectively, of a dual heating plate configuration with a test cartridge sandwiched between two heater plate assemblies.
Fig. 29A and 29B are a physical and transparent view, respectively, of an embodiment of a docking unit in which a pivoting door is used to receive a test cartridge. The closing of the pivoting door brings the rear of the test cartridge into contact with the heater plate mounted within the docking unit.
Fig. 30A and 30B are front and side sectional views, respectively, of the internal components of the docking unit, including the servo motor for actuating sample preparation and the optical system for test result collection.
Fig. 31A and 31B are front and side view photographs of an optical subsystem for an embodiment of a docking unit, the optical subsystem incorporating a test reader.
Fig. 32A and 32B are photographs of a docking unit embodiment with a pivoting test cartridge receiving door in an open position and a closed position, respectively.
Fig. 33 shows a reusable subassembly for the docking unit of the present invention.
Fig. 34 shows the test results obtained in example 1 described herein.
Fig. 35 shows the test results obtained in example 2 described herein.
Fig. 36 shows the test results obtained in example 3 described herein.
Fig. 37A is a perspective view of a cartridge including three chambers.
Fig. 37B is an exploded view of the cartridge of fig. 37A.
Fig. 38 is a transparent view of the cartridge of fig. 37A showing fluidic features.
Fig. 39 shows an embodiment of a chamber of the present invention comprising a triangular convex flow feature and a tapered outlet.
Fig. 40 shows an embodiment of a chamber of the present invention comprising triangular convex flow features and parallel outlets.
Fig. 41 shows an embodiment of a chamber of the present invention comprising trapezoidal convex flow features and parallel outlets.
Fig. 42 shows an embodiment of a chamber of the present invention comprising stacked triangular flow features and parallel outlets.
Fig. 43 shows an embodiment of a chamber of the present invention that includes a convex flow feature located approximately in the middle of the chamber.
Fig. 44 shows a reagent recess including internal features for directing fluid flow.
Figure 45 shows an embodiment of a vent pocket of the present invention that includes a dimple arrangement.
Fig. 46A shows a diagram of an embodiment of a fluidic layer of a cartridge of the present invention that includes a lyophilized reagent recess disposed in a fluid flow path. Fig. 46B is an enlarged view of the grooves and reaction chamber showing vertical projections extending into the chamber.
Fig. 47A shows a diagram of an embodiment of a fluidic layer of a cartridge of the present invention that includes lyophilized reagent wells disposed in one or more reaction chambers. Fig. 47B is an enlarged view of a groove in the reaction chamber showing a vertical projection extending into the chamber.
Fig. 48 shows a reaction chamber without vertical projections.
Detailed Description
One embodiment of the present invention is a sealable disposable platform for detecting a target nucleic acid, the disposable platform preferably comprising: a sample chamber for receiving a sample comprising the target nucleic acid; an amplification chamber connected to a sample chamber by a first channel and to a first vent pocket by a second channel; a labeling chamber connected to an amplification chamber through a third channel and to a second vent pocket through a fourth channel; a detection subsystem connected to the labeling chamber by a fifth channel and to a third vent pocket by a sixth channel; a plurality of resistive heating elements; and one or more temperature measuring devices, wherein the vent pockets are each sealed from communication with the air chamber by a thermally unstable material in a suitable form, such as a diaphragm, film or plastic sheet, located adjacent to one or more of the resistive heating elements. The disposable platform optionally includes a seal for sealing the platform prior to detection of assay initiation. The disposable platform preferably includes grooves along the channels between the chambers to incorporate dried or lyophilized reagents into the disposable platform. These grooves may optionally include structures on one or more of the surfaces facing the one or more reagents, preferably using capillary or surface tension effects to help direct fluid to the enclosed dry reagents to promote rehydration of the dry reagents. Such features may include ridges (such as
The disposable platform optionally further comprises a sample preparation station comprising an output in direct fluid communication with the input of the sample chamber. The substantially planar surface of the amplification chamber is preferably about the same size as the substantially planar surface of the resistive heating element in thermal contact with the amplification chamber. The amplification chamber optionally contains an amplification solution, and the recess located in the channel from the sample chamber to the amplification chamber optionally comprises a lyophilized amplification reagent mixture, and preferably there is a recess comprising dried or lyophilized detection particles in the channel from the amplification chamber to the labeling chamber. The amplification chamber and the labeling chamber are preferably capable of being heated using a resistive heating element. The detection subsystem preferably includes a lateral flow strip that includes detection particles. The chambers, channels and vent pockets are preferably positioned on a fluidic component layer, and the electronic components of the device are preferably positioned on a separate layer comprising a printed circuit board that is bonded to or placed in contact with the fluidic component layer by a docking unit. The detection subsystem is preferably positioned on the fluidic component layer or optionally on the second fluidic component layer. The volume of at least one of the chambers is preferably between about 1 microliter and about 150 microliters. The disposable platform preferably further comprises a connector for docking the disposable platform with the docking unit, which preferably maintains the disposable platform in a vertical or tilted orientation, and optionally provides electrical contacts, components and/or a power source.
One embodiment of the present invention is a method for detecting one or more target nucleic acids, preferably comprising: dispensing a sample comprising the target nucleic acid in a sample chamber of a disposable platform; orienting the disposable platform vertically or obliquely; opening a first vent pocket connected to an amplification chamber to a closed volume of air, thereby enabling the sample to flow into the amplification chamber; reacting the sample with a previously lyophilized amplification reagent mixture located in a recess of a channel between a sample chamber and an amplification chamber; amplifying the target nucleic acid in the amplification chamber; opening a second vent bag connected to the labeling chamber to a closed volume of air, thereby enabling flow of amplified target nucleic acid into the labeling chamber; labeling the amplified target nucleic acids with detection particles located in a recess in a channel between the amplification chamber and the labeling chamber; opening a third vent pocket connected to the detection subsystem to a closed volume of air, thereby enabling flow of labeled target nucleic acids into the detection subsystem; and detecting the amplified target nucleic acid. The amplification step preferably comprises: a resistive heating element located near the amplification chamber within the disposable platform is used to amplify the target nucleic acid. The method preferably further comprises: passively cooling the amplification chamber. The method preferably further comprises: a resistive heating element located near the marking chamber within the disposable platform is used to heat the marking chamber during the marking step. The method preferably further comprises: the operation of the disposable platform is controlled by using a docking unit that is not an external instrument.
Embodiments of the present invention include a disposable platform that integrates an external instrument-independent device that performs all the necessary steps of nucleic acid molecule assays, and complements current immuno-lateral flow rapid assays, with a new generation of nucleic acid tests providing more informative and sensitive assays. Embodiments of the present invention facilitate the more widespread use of rapid nucleic acid testing in small clinics and in harsh or remote environments, where infectious diseases, bio-threat factors, agriculture, and environmental testing are most likely to have the greatest impact. Certain embodiments of the present invention are fully self-contained and disposable, achieving "surge capacity" as demand increases by allowing parallel tests to be run without bottlenecks imposed by external instruments. In addition, in those application areas where low cost disposable cartridges coupled with inexpensive battery powered or AC adapter powered docking units are preferred, embodiments of the present invention that employ simple docking units further reduce testing costs by placing reusable components in reusable but inexpensive bases. The platform technology disclosed herein provides sensitivity similar to laboratory nucleic acid amplification based methods, minimal user intervention and training requirements, sequence specificity conferred by both amplification and detection, multiplexing capability, stable reagents, compatibility with low cost mass manufacturing, battery or solar powered operation allowing use in harsh environments, and flexible platform technology allowing incorporation of additional or alternative biomarkers without redesign of the device.
Embodiments of the present invention adapt systems and methods for low cost, point-of-use nucleic acid detection and identification for analysis at a location remote from the laboratory environment in which testing is typically conducted. Advantageously, the nucleic acid amplification reaction volume may be within the same volume range (e.g., 5 μ L-150 μ L) commonly used in conventional laboratory testing. Thus, the reactions performed in embodiments of the invention directly correspond to approved laboratory assays and allow for the containment of the same sample volumes typically employed in conventional molecular testing. Furthermore, the amplification of the nucleic acid is preferably performed in a hermetically sealed test cartridge, which is preferably permanently sealed before the amplification is initiated. Retaining the amplified nucleic acids within the sealed system prevents contamination of the test environment and surrounding areas with amplified products and thus reduces the likelihood that subsequent tests will produce false positive results. The integration of the sealing system into the test cartridge enables the use of a corresponding sealing engagement system in the docking unit to force the formation of a seal upon initiation of an assay. In one embodiment of the invention, a rack and pinion mechanism is used to slide the sealing mechanism of the integrated test cartridge into place to ensure the seal is closed prior to amplification. Prior to initiating the test reaction, a sensor placed in the docking unit interrogates the test cartridge to confirm that a seal has been formed.
Embodiments of the present invention may be produced using an injection molding process and ultrasonic welding to achieve high throughput manufacturing and low cost disposable parts. In some embodiments, one or more recesses are provided in the flow control member for individually receiving the dried reagent pellets. The grooves enable the use of lyophilized or otherwise dried material to be present in the fluidic components during final assembly, while ultrasonic welding can be used without the pellets being destroyed by any energy introduced into the system during welding.
Embodiments of the invention can be used to detect the presence of one or more target nucleic acid sequences in a sample. The target sequence can be DNA (such as chromosomal DNA or extrachromosomal DNA (e.g., mitochondrial DNA, chloroplast DNA, plasmid DNA, etc.)) or RNA (e.g., rRNA, mRNA, small RNA, or viral RNA). Similarly, embodiments of the invention can be used to identify nucleic acid polymorphisms, including single nucleotide polymorphisms, deletions, insertions, inversions, and sequence repeats. In addition, embodiments of the invention can be used to detect gene regulatory events, such as gene up-and down-regulation at the transcriptional level. Thus, embodiments of the invention may be used in such applications as: 1) detection and identification of pathogen nucleic acids in agricultural, clinical, food, environmental and veterinary samples; 2) detection of genetic biomarkers for disease; and 3) diagnosis of the presence of a disease or metabolic state by detecting biomarkers associated with the disease or metabolic state, such as gene regulatory events (mRNA up-or down-regulated, or small RNA or other nucleic acid molecules induced to be produced or inhibited during the disease or metabolic state) that occur in response to pathogens, toxins, other pathogens, environmental stimuli, or the presence of a metabolic state.
Embodiments of the invention include means for target nucleic acid sample preparation, amplification and detection after addition of a nucleic acid sample, including all aspects of fluid control, temperature control and reagent mixing. In some embodiments of the invention, the device provides a means for conducting nucleic acid tests using a portable power source, such as a battery, and is completely disposable. In other embodiments of the invention, the disposable nucleic acid test cartridges work in conjunction with simple, reusable electronic components that can perform all the functions of a laboratory instrument (such as the external instruments typically required for nucleic acid testing) without the use of such laboratory instruments or external instruments.
Embodiments of the invention provide nucleic acid amplification and detection devices including, but not limited to, housings, circuit boards, and fluidic or microfluidic components. In certain embodiments, the circuit board may include various surface mount components, such as resistors, thermistors, Light Emitting Diodes (LEDs), photodiodes, and microcontrollers. In certain embodiments, the circuit board may comprise a flexible circuit board comprising a thermally stable substrate such as polyimide. In some embodiments, the flexible circuit may include a copper or other conductive coating or layer deposited onto or bonded to the thermally stable substrate. These coatings may be etched or otherwise patterned to include resistive heating elements for biochemical reaction temperature control and/or conductive traces for housing such heaters and/or surface mount components such as resistors, thermistors, Light Emitting Diodes (LEDs), photodiodes, and microcontrollers. Fluidic or microfluidic components are part of a device that receives, contains, and moves aqueous samples, and can be made from a variety of plastics and a variety of manufacturing techniques, including ultrasonic welding, bonding, fusing or laminating, laser cutting, water jet cutting, and/or injection molding. The fluidic component and the circuit board component are reversibly or irreversibly held together, and their thermal coupling may be enhanced by thermally conductive materials or compounds. The housing preferably acts in part as an aesthetic and protective sheath, hiding delicate components of the microfluidic layer and circuit board layers, and may also be used to facilitate initiation of processes required for sample input, buffer release, nucleic acid elution, seal formation, and device function. For example, the housing may incorporate a sample input port, a mechanical system for sealing formation or engagement, buttons or similar mechanical features that allow for user activation, buffer release, sample flow initiation, nucleic acid elution, and thermal or other physical interface formation between the electronic and fluidic components.
In some embodiments of the invention, the fluidic or microfluidic component comprises a series of chambers in controlled fluidic communication, wherein the chambers are optionally temperature controlled, thereby subjecting the fluid contained therein to a programmable temperature regime. In some embodiments of the invention, the fluidic or microfluidic component comprises five chambers, preferably an expansion chamber, a sample input chamber, a reverse transcription chamber, an amplification chamber, and a detection chamber. The sample input chamber preferably comprises a conduit leading to an expansion chamber, a sample input orifice into which a nucleic acid-containing sample can be added, a first recess into which material dried during manufacture can be placed for mixing with the input sample, an outlet conduit leading to a second recess into which material dried during manufacture can be placed, and a conduit leading from the outlet conduit to the reverse transcription chamber. In other embodiments, the functions of two or more of the chambers are combined into a single chamber, enabling the use of fewer chambers.
The first and second wells may also include lyophilization reagents, which may include, for example, suitable buffers, salts, deoxyribonucleotides, ribonucleotides, oligonucleotide primers, and enzymes (such as DNA polymerases and reverse transcriptases). Such lyophilized reagents preferably dissolve when the nucleic acid sample enters the recess. In some embodiments of the invention, the first recess comprises salts, chemicals and buffers present in the input sample that can be used to lyse biological agents and/or stabilize nucleic acids. In some embodiments of the invention, the input sample is heated in the sample input chamber to effect lysis of cells or viruses present in the sample. In some embodiments of the invention, the second recess includes a lyophilization reagent and an enzyme, such as a reverse transcriptase that can be used to synthesize cDNA from RNA. In one embodiment of the invention, the second recess is sufficiently isolated from the sample input chamber to allow the material within the second recess to maintain a temperature lower than the temperature of the sample input chamber during heating. In some embodiments of the invention, the reverse transcription chamber comprises a conduit comprising a third recess comprising lyophilized reagents for nucleic acid amplification. The sample input chamber, reverse transcription chamber, amplification chamber and detection chamber are preferably positioned in alignment with and sufficiently close to the heater element on the heater circuit board to provide thermal conduction when mounted to the heater board, either directly or by inserting fluidic or microfluidic components or cartridges into the docking unit. Similarly, the electronic components present on the heater circuit board are preferably placed in physical contact with or in proximity to the vent pockets in the fluidic components to enable electronic control by opening the vent ports. The heater circuit board physical layout is designed to provide alignment with elements of the fluidic or microfluidic component such that the resistive heating elements of the heater circuit board for lysing, reverse transcription, amplification, hybridization, and/or fluid flow control are positioned to form a thermal interface with elements of the fluidic component and the resistive heating elements that interact.
In embodiments of the invention, the fluidic or microfluidic component preferably comprises five chambers, including a sample input chamber, a lysis chamber, a reverse transcription chamber, an amplification chamber and a detection chamber, and a well for dried or lyophilized reagents located along the channel between each chamber. In this embodiment, reverse transcription of RNA to cDNA and amplification of cDNA occur in separate chambers. In this embodiment, a first recess located along a conduit leading from the sample input cup to the lysis chamber comprises a salt, a chemical (e.g., dithiothreitol), and a buffer (e.g., for stabilizing, raising, or lowering the pH) present in the input sample that can be used to lyse the biological agent and/or stabilize the nucleic acid. In an embodiment of the invention, the input sample is heated in the thermal cracking chamber such that it first flows from the sample input cup through the first recess where it has been optionally blended with the material comprising the first recess. In other embodiments of the invention, lysis is achieved by chemical treatment due to the sample being blended with chemicals in the first recess and the sample being incubated in the lysis chamber in the presence of these chemicals.
After the process in the lysis chamber is substantially complete, the sample solution is released by electronic control of the heater, which non-mechanically ruptures the vent to allow the sample solution to flow through the channel, through the second recess and into the reverse transcription chamber. The second recess may optionally include lyophilization reagents that may include suitable buffers, salts, deoxyribonucleotides, ribonucleotides, oligonucleotide primers, and enzymes (such as DNA polymerase and reverse transcriptase needed to effect reverse transcription of RNA to cDNA in the sample). After the reverse transcription reaction is substantially complete, the second vent is opened to release sample solution to flow through the channel and a third well containing reagents required for nucleic acid amplification, such as lyophilization reagents, which may include suitable buffers, salts, deoxyribonucleotides, ribonucleotides, oligonucleotide primers, and enzymes (such as a DNA polymerase), and into the amplification chamber.
After amplification of nucleic acid in the amplification chamber is substantially complete, the third vent is opened to release the sample solution to the channel leading to the detection chamber. The channel may optionally but preferably comprise a fourth recess comprising dried or lyophilized detection reagents (such as chemicals) and/or detection particle conjugates that can be used to detect nucleic acids in the detection chamber. The detection chamber preferably comprises a capillary cell, reagents for detecting amplified nucleic acid and a lateral flow detection strip. The capillary cell preferably provides a space of sufficient capacity to accommodate the entire volume of fluid in the detection chamber at a height that enables fluid to flow by capillary action onto the detection strip without overflowing or otherwise bypassing regions of the detection strip that are designed to receive fluid to effect proper capillary migration onto the detection strip. In some embodiments of the invention, the detection reagent is a lyophilized reagent. In some embodiments of the invention, the detection reagent comprises dyed polystyrene microspheres, colloidal gold, semiconductor nanocrystals, or cellulose nanoparticles. The sample solution is blended with the detection reagent in the detection chamber and flows by capillary action onto the detection strip. A micro-heater aligned with the detection chamber may optionally be used to control the temperature of the solution as it migrates onto the detection strip.
In some embodiments of the invention, the amplification reaction is an asymmetric amplification reaction in which one primer of each primer pair is present in a different concentration than the other primer of a given pair in the reaction. Asymmetric reactions can be used for the generation of single-stranded nucleic acids to facilitate hybridization detection. Asymmetric reactions can also be used to generate amplicons in linear amplification reactions, allowing quantitative or semi-quantitative analysis of target levels in a sample.
Other embodiments of the invention include a nucleic acid reverse transcription, amplification and detection device integrated with a sample preparation device. Embodiments that include a sample preparation device provide a means for communicating fluid between a sample preparation subsystem output port or valve and one or more input ports of a fluidic or microfluidic component of the device.
Other embodiments of the invention include devices that separate an input sample into two or more fluidic paths in a fluidic or microfluidic component. The apparatus for separating an input sample includes a branch conduit for delivering an input fluid to a metering chamber having a volume designed to divide the volume across a plurality of fluid paths. Each metering chamber includes a channel conduit leading to a vent pocket and a channel conduit leading to the next chamber in the fluid path (e.g., a lysis chamber or a reverse transcription chamber or an amplification chamber).
Unless defined otherwise, all technical terms, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those skilled in the art to which this invention belongs. Many of the techniques and procedures described or referenced herein are generally well known and can often be employed by those skilled in the art by using conventional methods, such as the widely used molecular cloning methods described in: sambrook et al, Molecular Cloning, A Laboratory Manual, 3 rd edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology (edited by Ausubel et al, John Wiley & Sons, Inc.2001). Where appropriate, procedures involving the use of commercial kits and reagents are typically performed according to manufacturer-defined protocols and/or parameters, unless otherwise indicated.
As used throughout the specification and claims, the term 'target nucleic acid' or 'template nucleic acid' means a single-or double-stranded DNA or RNA fragment or sequence intended for detection.
As used throughout the specification and claims, the term 'microparticle' or 'detection particle' means any compound used to label nucleic acid products produced during an amplification reaction, including fluorochromes specific for double-stranded nucleic acids, fluorescently modified oligonucleotides and oligonucleotide-conjugated quantum dots or solid phase elements (such as polystyrene, latex, cellulose or paramagnetic particles or microspheres).
As used throughout the specification and claims, the term 'chamber' means a fluidic compartment in which fluid resides for a period of time. For example, the chamber may be a sample chamber, an amplification chamber, a labeling chamber, or a detection chamber.
As used throughout the specification and claims, the term 'cartridge' is defined as a disposable or consumable cartridge, housing, component, or cartridge for performing assays or other chemical or biochemical analyses. The cartridge may be single use or multiple use.
As used throughout the specification and claims, the term 'pocket' means a compartment that acts as a discharge mechanism. The pocket is preferably adjacent to or overlying a resistor or other mechanism that opens the pocket. For example, unlike the fluidic chambers described above, the pockets formed in the fluidic components of the cartridge can have an open face that is aligned with the resistors on the PCA. This open face is preferably covered by a thin membrane, film or other material to form a sealed cavity that is easily broken by energizing the underlying resistor.
As used throughout the specification and claims, the term 'channel' means a narrow conduit within a fluidic assembly that typically connects two or more chambers and/or pockets or combinations thereof, including, for example, an inlet channel, an outlet channel, or a vent channel. In the case of an inlet channel or an outlet channel, the fluid sample migrates through the channel. The conduit preferably remains free of fluid with respect to the vent channel and connects the fluidic chamber to the vent pocket.
As used throughout the specification and claims, the term "external instrument" means a reusable instrument having one or more of the following characteristics: performing mechanical actions on the disposable assay or cartridge other than sealing the cartridge, including but not limited to piercing a buffer pocket and/or pumping or otherwise actively providing a transport force for the fluid; movable parts including other components for controlling valves and fluid flow control in a cartridge or disposable assay; controlling fluid flow rather than measured by selective heating; or require periodic calibration.
As used throughout the specification and claims, the term "docking unit" means a reusable device that controls an assay but does not have any of the characteristics listed above for an external instrument.
Embodiments of the present invention are devices for low cost, point-of-use nucleic acid testing that are suitable for analysis at a location remote from the laboratory environment in which testing is typically performed. Some devices include fluidic components or layers and electronic components or layers, optionally encapsulated by a protective housing. In an embodiment of the invention, the fluidic component is constructed of plastic and includes a series of chambers and pockets connected by narrow channels in which the chambers are vertically oriented relative to each other during operation. The fluidic components are coated with or otherwise in physical contact with electronic components, preferably controlled by a microcontroller, such as a printed circuit board (SMD) comprising off-the-shelf surface mount devices, and/or a flexible circuit comprising etched conductive material to form resistive heating elements and optionally comprising SMDs. In an embodiment of the device, the entire assembly is disposable. In other embodiments, the fluidic layer and the physically bonded electronic layer are disposable, while the small, inexpensive control unit is reusable. In another embodiment, the fluidic component is disposable and the mini-control docking unit or docking unit is reusable. For all embodiments, the present invention may be integrated with a Nucleic Acid Sample Preparation device such as that disclosed in International publication No. WO 2009/137059A 1 (incorporated herein by reference) entitled "high throughput simple Flow-Based Nucleic Acid Sample Preparation and Passive Flow control", and/or using the methods described in that publication.
Embodiments of the present invention include integrated nucleic acid testing devices that can be inexpensively manufactured using established manufacturing methods. The present invention provides molecular test data while retaining the simplicity of the end-user perspective of a widely recognized handheld immunoassay, overcoming the following challenges: regulating the temperature of the fluid within the device, delivering small sample volumes in successive steps, reagent addition, reagent mixing, and nucleic acid detection. In some embodiments of the invention, subsystems for collecting, interpreting, reporting and/or transmitting assay results are incorporated into the invention. Embodiments of the present invention are uniquely suited for utilizing off-the-shelf electronic components that can be constructed by standard assembly techniques and require no or little 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 that is capable of nucleic acid isolation, amplification, and detection without the need for dedicated laboratory infrastructure.
Existing nucleic acid testing devices typically use complex heating elements, such as deposited film heaters and Peltier (Peltier) devices, which add significant cost and/or require specialized manufacturing methods. In embodiments of the invention, heating of the reaction solution is preferably accomplished by using a simple resistive surface mount device, which can be purchased with a few pennies or less, and assembled and tested by common manufacturing standards. By layering fluidic chambers on these resistive elements and associated sensor elements, the fluidic temperature of the reaction solution can be conveniently regulated. The widespread use of SMD resistors and flexible circuits in the electronics industry ensures that the present invention can employ sophisticated quality control methods. In other embodiments of the invention, resistive heating is achieved using heating elements formed by patterns fabricated in the conductive layer of the flexible circuit substrate. Many nucleic acid amplification techniques, such as PCR, require not only rapid heating of the reaction solution, but also rapid cooling. The reaction chamber in the present invention is preferably heated on one side and the ambient temperature across the opposite side is used to help reduce the fluid temperature. In addition, the vertical orientation of the embodiments of the device allows for faster cooling by passive convection than if the device were oriented horizontally, thus reducing thermal cycling periods without the use of expensive 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 low cost nucleic acid testing device design. Devices known in the art typically employ electromechanical, electric, or piezoelectric pumping mechanisms to manipulate fluid during operation of the device. These pumping elements add both complexity and cost to the device. Similarly, valves using complex micromechanical designs or moving parts may increase manufacturing costs and reduce reliability due to complications such as moving part failure or biofouling. Unlike the previously described nucleic acid testing devices, embodiments of the present invention utilize hydrostatic pressure, as well as capillary and surface tension forces under microcontroller control to manipulate fluid volume. The vertical orientation of some embodiments of the invention allows for cascading of reaction solutions from one chamber to another under microcontroller control to accommodate the desired assay manipulation. The fluid may be maintained in each reaction chamber by a balance of channel size, hydrostatic pressure and surface tension, which inhibit fluid advancement by gas displacement. The sample is preferably advanced to the lower chamber only after activation of a simple discharge mechanism under the control of the microcontroller. Once opened, the vent allows fluid to move from the first chamber to the second chamber in a manner that provides a path for displaced air to escape from the second chamber upon fluid entry. Each chamber within the fluidic component (or each channel between chambers) is preferably connected to a sealed vent pocket through a narrow vent channel. The vent pocket is preferably sealed to one side with a thin, thermally unstable plastic membrane or sheet that is easily ruptured by heating a small surface mounted resistor that underlies, is near, or is adjacent to the membrane or sheet. Once the vent of the lower chamber is opened, fluid advancement continues even at low hydrostatic pressure.
As described in more detail below, the fluidic or microfluidic discharge mechanisms used in some embodiments of the invention preferably employ a heating element in thermal and (optionally) physical contact with a thermally labile seal to enable electronically controlled fluid movement by venting the lower-height chambers while allowing fluid from the higher-height chambers to flow into the lower chambers. In one embodiment, the resistor is mounted on a printed circuit board using widely used and well-established electronic device manufacturing methods and placed in physical contact with a channel seal comprising a thermally unstable material. When the energized surface mount resistor generates sufficient heat to rupture the seal, this results in venting of the chamber to allow the area or chamber in which the fluid is moving to equalize with the pressure in the area or chamber in which the fluid resides prior to venting. The pressure balance between the chambers allows fluid to move from chambers of higher elevation to chambers of lower elevation. Preferably no direct seal between the higher level chamber and the lower level chamber is employed. The channel and vent seal can be positioned away from the fluid chamber to facilitate a fluidic device layout that is structurally efficient for manufacturing. The sealing material may comprise any material capable of sealing the vent passage and rupturing as described by heating, such as a thin plastic sheet. Such methods for fluid movement control in a device benefit from low material costs, manufacturing applicability using established manufacturing techniques, while providing the ability to move fluid through a series of chambers under the control of an electronic control circuit such as a microprocessor or microcontroller. The use of vents, thermally labile materials to seal the vents (but not the fluid chambers or the fluid microchannels themselves) and the use of heat to break the seals of the electronic device provides a means of controlling fluid flow through the device so that the fluid can move after a predetermined time or upon completion of a particular event (e.g., reaching a certain temperature, a temperature change or a series of temperature changes, or completion of one or more incubation times or other events). In some embodiments, when gas phase water must be isolated from chambers connected by the channels, plugs may be introduced into the channels between the chambers. The plug may be a soluble material that dissolves when contacted by liquid water after the vent is opened, or a readily meltable material (such as paraffin) that is removable by introducing heat to the plug site.
Furthermore, the vent approach has many advantages over sealing the fluid chamber itself. The vent pockets can be located anywhere on the fluidic layout and simply communicate with their regulated chambers through vent channels. From a manufacturing perspective, the vent pockets may be positioned such that only a single sealing membrane (which may include a vent pocket manifold) for all vent pockets is attached to the fluidic component, preferably by established methods such as adhesives, heat lamination, ultrasonic welding, laser welding, and the like. In contrast, directly sealing the fluid chambers requires the sealing material to be placed at different locations corresponding to each chamber location, which makes manufacturing more difficult. This presents a more challenging situation during manufacture than a single vent pocket manifold sealed by a single septum. Additionally, if the chambers are directly sealed, the molten sealing material may remain in the channels between the chambers, blocking flow. The viscosity of the sealing material may require a pressure in the fluid column that is greater than the pressure obtained in a miniaturized gravity driven device.
In embodiments of the invention, reagent mixing does not require much more complexity than other systems. Reagents necessary for nucleic acid amplification, such as buffers, salts, deoxyribonucleotides, oligonucleotide primers, and enzymes, are preferably stably incorporated by using a lyophilized pellet or cake. These lyophilized reagents sealed in fluidic chambers, recesses in fluidic chambers, or recesses in channels can be readily dissolved upon contact with aqueous solutions. The vertical orientation of embodiments of the present invention provides an opportunity for a novel method of mixing solutions where additional mixing is required. By utilizing a heater underlying the fluidic chamber, the gas can be heated to deliver bubbles to the reactive solution in the chamber when the solution contains a heat sensitive component. Alternatively, in the case where the solution does not contain a heat sensitive component, the solution may be directly heated to the extent that boiling occurs using a heater. Bubbles are generally undesirable in the previously disclosed fluidic and microfluidic devices because they may accumulate in the fluidic chambers and channels and displace the reaction solution or impede fluid movement within the device. The vertical design of the embodiments of the invention presented herein allows bubbles to rise to the surface of the fluid, resulting in only minimal and transient fluid displacement, effectively ameliorating any adverse effects of the bubbles on the fluidic or microfluidic system. Mixing by boiling is also convenient for this vertical design, since after turning off the heating element, the displaced fluid returns only to the original fluidic chamber during the process due to gravity.
In embodiments of the invention, a colorimetric detection strip is used to detect the amplified nucleic acids. Lateral flow assays are commonly used for immunoassay testing due to ease of use, reliability, and low cost. The prior art contains descriptions of the use of porous materials as sample receiving regions for detecting lateral flow strips of nucleic acids at or near a labeling zone that also contains porous material and placed at or near one end of a lateral flow assay device. In these prior inventions, the marker portion is located in the marker region. The use of a porous material as the sample receiving zone and the label zone results in some of the sample solution and detection particles remaining in the porous material. Although label zones comprising a porous material with a reversibly immobilized portion required for detection may be used in embodiments of the invention, embodiments of the invention preferably utilize detection particles, or portions that are retained in a region of the device other than the sample receiving zone of the lateral flow strip and comprise a non-porous material with low fluid retention properties. This method allows for labeling of a sample containing nucleic acid targets prior to introduction into the porous section of the sample receiving end of the lateral flow section of the device, and thereby eliminates retention and/or loss of sample material and detection particles in the porous labeling zone. This method further enables the use of various treatments (such as high temperature treatment) to the sample in the presence of the detection moiety to effect denaturation of secondary structures within the double stranded target or single stranded target, regardless of the effect of temperature on the porous sample receiving or labeling zone material or lateral flow strip material. Furthermore, the use of a label zone that is not in lateral flow contact with the sample receiving zone but is controlled by a flow control means (such as a vent) allows the target and label to remain in contact for a period of time controlled by the fluid flow control system. Thus, embodiments of the present invention may differ from conventional lateral flow test strips in that the interaction time and conditions of the sample and detection particles are determined by the capillary transport properties of the material. By incorporating detection particles into the temperature-regulated chamber, denaturation of double-stranded nucleic acids is possible, allowing for efficient hybridization-based detection. In an alternative embodiment, fluorescence is used to detect nucleic acid amplification using a combination of LEDs, photodiodes, and filters. These optical detection systems can be used for real-time nucleic acid detection and quantification during amplification and post-amplification endpoint detection.
Embodiments of the invention include providing a low cost point-of-use system in which nucleic acid samples can be selectively amplified and detected. Additional embodiments include integration with a nucleic Acid Sample Preparation device such as that described in International publication No. WO 2009/137059A 1 entitled "high simple Flow-based nucleic Acid Sample Preparation and Passive Flow Control". One embodiment of the device preferably includes both a plastic fluidic component and a Printed Circuit Assembly (PCA) and/or a flexible circuit, and is optionally enclosed in a housing that protects the active components. Temperature regulation, fluid and reagent mixing are preferably coordinated by a microcontroller. The reaction cartridge is preferably vertically oriented and runs in a vertical direction such that gravity, hydrostatic pressure, capillary force, and surface tension in conjunction with the microcontroller triggered vent controls fluid movement within the device.
In embodiments of the invention, prepared or crude sample fluid enters the sampling port and fills or partially fills the sampling cup. The sample may be retained in the sampling cup for different periods of time, and the dried or lyophilized reagents in the sampling cup may be mixed with the sample. Such reagents (such as positive control reagents, control templates, or chemical reagents beneficial to the performance of the test) can be introduced into the sample solution by inclusion in the sample cup in dry, liquid, or lyophilized form. Other processes, such as controlled temperature incubation or thermal cleavage of bacterial or viral analytes, can optionally be implemented in the sample cup by an underlying micro-heater and temperature sensor system interfaced with temperature control electronics. The fluidic network includes a sampling port through which samples are introduced to the cartridge by a user, either manually or through an automated system (e.g., a subsystem integral with the docking unit or a sample processing subsystem); a sampling cup in which the sample is held for facilitating accumulation during sample introduction and for adding reagents, components for desired processing (e.g., thermal treatment for lysis of bacterial cells or viruses) before the sample is moved further into the downstream portion of the fluidic network; a recirculation drain passage for equalizing air, gas or solution pressure of the fluidic channel and/or chamber with pressure of the expansion chamber of the cartridge; a bead recess in which reagent beads (e.g., beads or pellets made of materials, reagents, chemicals, biologicals, proteins, enzymes or other substances or mixtures of such substances) in a dried/dehydrated or lyophilized or semi-dried state can be rehydrated by the sample solution, or a buffer solution is introduced to the cartridge prior to addition of the sample to rehydrate the beads or pellets contained therein and thus blend the materials therein with the sample solution; a set of one or more vents that can be opened to control fluidic movement within the cartridge; a first chamber in which a sample may be subjected to a temperature regime; an optional barrier located within a fluidic channel connecting the first and second chambers to avoid premature intrusion of liquid and/or gas into the second chamber or to temporarily control movement of solution or gas into the second chamber; a second chamber in which the sample solution may optionally be subjected to a further temperature regime after addition of reagents from an optional reagent bead well optionally located between the first and second chambers; a test strip recess forming a chamber in which a test strip is mounted for detection of an analyte or reporter or other substance indicative of the presence of an analyte. In some embodiments, the cartridge is inserted into a docking unit that performs the functions of sealing the cartridge, elution, detection, and data transfer. Preferably, no user intervention is required once the cartridge is inserted into the docking unit, the sample is loaded and the lid is closed.
Referring to the representative drawings of the
After nucleic acid amplification, the
Fluid movement from the
The face opposite the open face of the vent pocket may optionally include dimples, protrusions, microprotrusions or other similar structures (such as
The sealed
The expansion chamber may be incorporated as an empty air volume, such as the included volume shown in the
In embodiments in which the second chamber is an amplification chamber, the chamber is preferably in contact with a heater element to provide the means for temperature regulation necessary to support nucleic acid amplification. In some embodiments of the invention, the amplification chamber may comprise an oligonucleotide on at least a portion of the inner surface. At the interface between the
Placing the heating element and, in some embodiments, the corresponding temperature sensor or sensors on the disposable component enables the manufacture of highly reproducible thermal coupling between the temperature control subsystem and the amplification and detection chambers to which they are interfaced. This approach enables a highly reliable means of coupling the fluidic subsystem to the electronic thermal control subsystem by forming a thermally conductive interface during manufacturing. The resulting excellent thermal contact between the electronic temperature control component and the fluidic subsystem results in a fast temperature equilibration and, therefore, a fast assay. The use of a flexible circuit to provide a disposable resistive heating element fused to the rear of the fluidic component backing, either directly or through an intermediate gasket, allows for excellent thermal contact, rapid temperature cycling, and a low cost device that is reproducibly manufactured. Resistive heating elements for reverse transcription, amplification and fluidic flow port control can be formed directly on the flex circuit by: the conductive layer of the flex circuit is etched to form a geometry that exhibits the desired resistance. This approach eliminates the need for additional electronic components and simplifies manufacturing while reducing costs.
In one embodiment of the invention, a
In this embodiment, the
Embodiments of the cartridge chamber preferably include materials capable of withstanding repeated heating and cooling to temperatures in the range of about 30 ℃ to about 110 ℃. Even more preferably, the chamber comprises a material capable of withstanding repeated heating and cooling to a temperature in the range of about 30 ℃ to about 110 ℃, with a rate of temperature change of about 10 ℃ to about 50 ℃ per second. The chamber is preferably capable of maintaining the solution therein at a temperature suitable for heat-mediated lysis and biochemical reactions, such as reverse transcription, thermocycling or isothermal amplification protocols, which is preferably controlled by programming of a microcontroller. In some nucleic acid amplification applications, it is desirable to provide an initial incubation at elevated temperatures (e.g., temperatures between about 37 ℃ and about 110 ℃) for a period of 1 second to 5 minutes to denature the target nucleic acid and/or activate the hot-start polymerase. Subsequently, the reaction solution is maintained in the amplification chamber at an amplification temperature for isothermal amplification, or for thermal cycle-based amplification, such that the temperature of the reaction solution varies between at least two temperatures, including but not limited to a temperature that results in denaturation of the nucleic acid duplex and a temperature suitable for primer annealing by hybridization to the target and extension of the primer by polymerase-catalyzed nucleic acid polymerization. The duration of incubation at each necessary temperature in the thermocycling protocol can vary with the sequence composition of the target nucleic acid and the composition of the reaction mixture, but is preferably between about 0.1 second and about 20 seconds. Repeated heating and cooling is typically performed for about 20 cycles to about 50 cycles. In embodiments involving isothermal amplification methods, the temperature of the reaction solution is maintained at a constant temperature (in some cases, after initial incubation at elevated temperature) for about 3 minutes to about 90 minutes, depending on the amplification technique used. Once the amplification reaction is complete, further manipulation of the amplified nucleic acid is accomplished by opening a vent port in communication with the chamber below the chamber for amplification, delivering the amplification reaction solution to the lower chamber. In some embodiments of the invention, the manipulation comprises denaturation of the amplified nucleic acids and hybridization to detection oligonucleotides conjugated to detection particles. In some embodiments of the invention, the amplified nucleic acids are hybridized to detection oligonucleotides conjugated to detection particles and to capture probes immobilized on detection strips.
In some embodiments, additional biochemical reactions may be performed in the amplification chamber before, during, or after the amplification reaction. Such processes may include, but are not limited to, reverse transcription, wherein RNA is transcribed into cDNA, multiplexing, wherein multiple primer pairs simultaneously amplify multiple target nucleic acids, and real-time amplification, wherein amplification products are detected during the course of the amplification reaction. In the latter regard, the amplification chamber may be free of valves or outlet channels, and the amplification chamber will preferably include an optical window or otherwise be configured to enable interrogation of amplicon concentration during the course of an amplification reaction. In one real-time amplification embodiment, the fluorescence intensity of a fluorescently labeled oligonucleotide complementary to a target nucleic acid or a fluorescent dye specific for duplex DNA is monitored by an excitation light source (such as an LED or one or more diode lasers) and a detector (such as a photodiode) and appropriate optical components (including but not limited to optical filters).
Detection of
Embodiments of the
Suitable detection particles include, but are not limited to, fluorescent dyes specific for double-stranded nucleic acids, fluorescently modified oligonucleotides, or oligonucleotide-conjugated dyed microparticles or colloidal gold or colloidal cellulose. Detection of amplicons involves a 'detection oligonucleotide' or other 'detection probe' that is complementary to, or capable of specifically binding to, the amplicon to be detected. Conjugation of the detection oligonucleotide to the microparticle may occur by using 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 detector oligonucleotide to the detectable moiety can occur internally or at the 5 'end or the 3' end. The detection oligonucleotide may be attached to the microparticle directly or more preferably via a spacer moiety such as ethylene glycol or a polynucleotide. In some embodiments of the invention, the detection particles can bind to a plurality of amplified nucleic acids resulting from a process such as multiplex amplification. In these embodiments, specific detection of each amplified nucleic acid can be achieved by performing detection on a detection band using methods specific to each species to be detected. In such embodiments, the tag introduced to the target nucleic acid during amplification can be used to label all amplified species present, while subsequent hybridization of the labeled nucleic acid to the species-specific capture probe immobilized on the detection strip is used to determine which specific species of amplified DNA is present.
For double stranded DNA amplification products, heating the reaction solution after introduction into the detection chamber can facilitate detection. Melting double-stranded DNA or denaturing the secondary structure of single-stranded DNA, and then cooling in the presence of the detection oligonucleotide results in sequence-specific labeling of the amplified target nucleic acid. The fluid volume may be heated for about 1 second to about 120 seconds using a heating element underlying the detection chamber to melt the double stranded DNA or initiate denaturation of the single stranded DNA secondary structure. When the solution is allowed to cool to room temperature, the amplified target nucleic acid can specifically hybridize to the detection particles. The reaction volume is then preferably directed to the area of the detection chamber below the labeling chamber by opening the vent of the detection chamber.
For efficient labeling, it is preferable to mix the dissolved detection particles well with the reaction solution. In embodiments of the invention, detection particles may be positioned in the
Embodiments of the detection chamber of the present invention provide for specific detection of amplified target nucleic acids. In certain embodiments of the invention, detection is achieved by capillary wicking of a solution comprising labeled amplicons through an absorbent strip comprising a porous material (such as cellulose, nitrocellulose, polyethersulfone, polyvinylidene fluoride, nylon, charge-modified nylon, or polytetrafluoroethylene) patterned with lines, dots, microarrays, or other visually discernable elements, comprising binding moieties capable of specifically binding directly or indirectly to labeled amplicons. In some embodiments, the absorbent strip component of the device comprises up to three porous substrates in physical contact: a surfactant pad comprising an amphiphilic agent to enhance wicking; a detection zone comprising a porous material (such as cellulose, nitrocellulose, polyethersulfone, polyvinylidene fluoride, nylon, charge modified nylon, or polytetrafluoroethylene) to which at least one binding moiety capable of selectively binding the labeled amplicon is immobilized; and/or an absorbent pad for providing additional absorbent capacity. Although the detection particles may optionally be incorporated within the lateral flow porous material in the detection chamber, unlike the lateral flow detection devices previously described, the detection particles are preferably held upstream of a capillary cell where substantially enhanced formation of binding complexes between amplicons and detection particles can occur prior to or simultaneously with introduction of the resulting labeled nucleic acids into the porous component of the device.
The 'capture oligonucleotide' or 'capture probe' is preferably immobilised to the detection strip elements 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 designed to capture the labeled nucleic acid when a solution comprising the labeled nucleic acid core passes through the capture zone resulting in an increase in the concentration of label at the capture probe immobilization site, thereby generating a detectable signal indicative of the presence of one or more labeled target nucleic acid amplicons. A single detection strip can be patterned with one or more capture probes to enable multiplex detection of multiple amplicons, determination of amplicon sequences, amplicon quantification by extending linearity of detection signal, and assay quality control (positive and negative controls).
Fluidic component
Embodiments of the fluidic components preferably comprise plastics such as acrylic, polycarbonate, PETG, polystyrene, polyester, polypropylene, and/or other similar materials. These materials are readily available and can be manufactured by standard methods. The fluidic component includes both chambers and channels. The fluidic chamber comprises a wall, two faces, and is connected to one or more channels (such as inlets, outlets, grooves, or vents). The channel may connect two fluidic chambers or one fluidic chamber and one recess and comprise a wall and two faces. The fluidic chamber design preferably maximizes the surface area to volume ratio to facilitate heating and cooling. The internal volume of the chamber is preferably between about 1 μ L and about 200 μ L. The area of the chamber face that is in contact with the solution preferably corresponds to the area to which the heating element is interfaced to ensure a uniform fluid temperature during heating. The shape of the fluidic chamber can be selected to cooperate with the heating element and provide a favorable geometry for solution ingress and egress. In some embodiments, the volume of the chamber may be greater than the volume of the fluid to provide space for bubbles to occur during operation of the device. The fluidic chamber may have an enlarged extension to the vent channel to ensure that fluid does not invade the channel by capillary action or otherwise clog the venting mechanism.
In some embodiments, it may be desirable to reduce or eliminate the intrusion of liquid or gas phase water into the chamber prior to the release of the solution. The elevated temperatures used in the processes of some embodiments generate vapors (e.g., vapor phase water) that can cause premature intrusion of moisture into the channels, chambers, or grooves. It may be desirable to reduce liquid or gas phase intrusion to maintain a dry state of, for example, a dried or lyophilized reagent present in a chamber or well. In some embodiments, the channels may be temporarily completely or partially blocked with a material that can be removed by an external force, such as heat, moisture, and/or pressure. Materials suitable for temporarily blocking the channels include, but are not limited to, latex, cellulose, polystyrene, thermal glue, paraffin, wax, and oil.
In some embodiments, the test cartridge includes a preferably injection molded fluidic component including a sampling cup, a chamber, a channel, a vent pocket, and an energy director. The injection molded test cartridge fluidic components are preferably constructed of a plastic suitable for ultrasonic welding to a backing plastic of similar composition. In one embodiment of the invention, the test cartridge fluidic components comprise a single injection molded piece ultrasonically welded to a backing material. The energy director is an optional feature of the fluidic component that directs ultrasonic energy to only those areas of the thermally labile layer that are intended to be bonded to the fluidic component. The injection molded fluidic components may optionally be contained in a housing. Fig. 7 shows a cartridge comprising a preferred injection molded fluidic component 400 (preferably comprising a polymer such as High Impact Polystyrene (HIPS), polyethylene, polypropylene or
In some embodiments, the adhesive spacer includes a
In some embodiments of the invention, the thickness of the fluidic chambers and channel walls is in the range of about 0.025mm to about 1mm, and preferably in the range of about 0.1mm to about 0.5 mm. This thickness preferably both meets the structural integrity requirements of the fluidic component and supports sealing of the closed chamber at elevated temperatures and associated pressures. The thickness of the channel walls, in particular the vent channel walls, is preferably less than the thickness of the chamber and is in the range of about 0.025mm to about 0.25 mm. The width of the inlet and outlet channels is preferably selected to enhance capillary action. The shallow vent channels impart improved rigidity to the fluidic components without adversely affecting emissions. The plastic forming the face of the flow control member is preferably thinner than the plastic forming the walls in order to maximize heat transfer. An optional thermal break cuts through some components of the fluidic component and surrounds the amplification and detection chambers, thereby facilitating thermal isolation of the temperature-controlled chambers.
In some embodiments of the invention, additional components of the test cartridge, such as
Referring to fig. 2 and 7, the vent pocket preferably differs from the other chambers in its structure. After construction of the fluidic component as described above, the vent bag has an open face on the side of the fluidic component that will directly or in some embodiments indirectly interface with the PCA75 through the
Additional components of fluidic components
As mentioned above, several additional components are preferably incorporated into the fluidic components of the present invention prior to final bonding. Reagents including buffers, salts, dntps, NTPs, oligonucleotide primers and enzymes (such as DNA polymerases and reverse transcriptases) may be lyophilized or freeze-dried into pellets, spheres or cakes prior to assembly of the device. Reagent lyophilization is well known in the art and involves dehydrating frozen reagent aliquots by sublimation under an applied vacuum. By adding specific formulations of lyoprotectants, such as sugars (disaccharides and polysaccharides) and polyols, to the reagents prior to freezing, the activity of the enzyme can be preserved and the rate of rehydration can be increased. The lyophilized reagent pellets, spheres, or cakes are manufactured by standard methods and, once formed, are quite durable and can be easily placed into a particular chamber of the fluidic component prior to lamination of the final face. More preferably, recesses are incorporated into the fluidic network to allow pellets, spheres, or cakes of lyophilized reagents to be placed in the fluidic component prior to bonding the fluidic component to the backing material. By selecting the fluidic network geometry and the groove locations and sequence, the sample can react with the desired lyophilized reagents at the desired time to optimize performance. For example, by depositing lyophilized (or dried) Reverse Transcription (RT) and amplification reagent spheres into two separate wells in the fluid paths of the RT reaction chamber and the amplification chamber, optimal reverse transcription reactions can be achieved without interference from the amplification enzymes. Furthermore, to minimize the interference of RT enzymes with subsequent amplification reactions, RT enzymes after RT reactions present in the RT reactions can be heat inactivated prior to introduction of amplification reagents to minimize their interference with amplification. Optionally, other salts, surfactants, and other enhancing chemicals can be added to different wells to modulate the performance of the assay. In addition, these grooves facilitate blending of the lyophilized reagent with the liquid as it passes through the grooves, and also serve to isolate the lyophilized material from ultrasonic energy during ultrasonic welding, as well as to isolate the lyophilized reagent from extreme temperatures during the heating step of the test prior to dissolution. Furthermore, the grooves ensure that the freeze-dried pellets are not compressed or crushed during manufacture, so that they remain porous to minimize rehydration time.
In some embodiments of the invention, the detection particle is another additional component of the fluidic component. In some embodiments, the microparticles may be lyophilized, as described above for the reaction reagents. In other embodiments, the microparticles in the liquid buffer may be applied directly to the interior face of the fluidic chamber and dried prior to final assembly of the test cartridge. The liquid buffer containing the microparticles preferably also contains a sugar or polyol to aid rehydration. Incorporating the microparticles in the aqueous buffer directly into the fluidic components prior to drying can simplify manufacturing and reduce the ultimate cost of manufacturing, and complete blending of the lyophilized particles with the reaction solution and denaturation of double-stranded nucleic acids or double-stranded regions of nucleic acids into single-stranded nucleic acids can be facilitated by heating or nucleate boiling. In some embodiments, the lyophilized detection particles are placed in a recess in a fluidic network. In other embodiments, lyophilized or dried test particles are placed in the
In some embodiments of the invention, a lateral flow detection strip assembly is also incorporated into the fluidic component. The test strip preferably includes a membrane assembly that includes at least one porous member, and optionally may include an absorbent pad, a test membrane, a surfactant pad, and a backing film. The detection membrane is preferably made of nitrocellulose, cellulose, polyethersulfone, polyvinylidene fluoride, nylon, charge modified nylon, or polytetrafluoroethylene, and may be backed with a plastic film. As described above, the capture probes may be deposited and irreversibly immobilized on the detection membrane in a line, spot, microarray, or any pattern that is visible to the naked eye or an automated detection system such as an imaging system. After the capture probe deposition, the membrane can be examined by UV irradiation to permanently fix the deposited oligonucleotides. The surfactant pad may comprise a porous substrate, preferably having minimal nucleic acid binding and fluid retention properties, which allows for unimpeded migration of nucleic acid products and detection particles. The surfactant pad may comprise materials such as glass fibers, cellulose, or polyester. In an embodiment of the invention, the formulation comprising at least one amphiphilic agent is dried on a pad of surfactant to allow uniform migration of the sample through the detection membrane. The absorbent pad may comprise an absorbent material and help induce wicking of the sample through the detection membrane assembly. Using an adhesive backing film (such as a double-sided adhesive film) as a substrate, the test membrane components are assembled by first placing the test membrane, followed by bringing the optional absorbent pad and/or surfactant pad into physical contact with the test membrane with an overlap of between about 1mm and about 2 mm. In some embodiments of the invention, the detection membrane may be in indirect capillary communication with the surfactant pad, wherein there is a physical separation between the surfactant pad and the detection pad, the intermediate space comprising a capillary space in which fluid can traverse the space by capillary action. In some embodiments, the surfactant pad or region of the surfactant pad can comprise detection particles, dried detection particles, or lyophilized detection particles.
Three-chamber box
In some embodiments of the invention, additional reaction chambers and/or additional recesses may be incorporated for dried or lyophilized reagents. In some embodiments, this design facilitates testing where it is desirable to provide an initial separate cleavage reaction prior to reverse transcription and amplification. As shown in fig. 37A, 37B, and 38, the
The opening of vent valve 5009 connected to the top of
Opening of the next vent valve 5009 connected to the top of the
Subsequently, the opening of final vent valve 5009 connected to the distal end of
Flow control feature
The design of the fluidic component may optionally include flow control features within the reaction chamber or at its outlet. These features deflect the flow entering the chamber to the side of the chamber opposite the outlet prior to the flow entering the outlet. As a result, the flow enters the outlet channel at a lower velocity, thereby reducing the distance the fluid flows down the channel before stopping. Furthermore, the horizontal component of the flow path increases the length of the channel without increasing the vertical spacing between the chambers, thereby increasing the effective length of the flow path, and thus sufficient to stop the flow at a desired location based on the reduced flow rate. This allows for tighter vertical spacing between chambers of the cartridge because fewer vertical channels are required. In addition, the redirection of the flow across the reaction chamber creates a swirling effect in the flow within the chamber, thereby improving the mixing of the reagent and sample fluids. The flow control feature may comprise any shape.
In the embodiment shown in fig. 39, fluid enters
In the embodiment shown in fig. 40, fluid enters the
In the embodiment shown in fig. 41, fluid enters the
In the embodiment shown in fig. 42, fluid enters the
In the embodiment shown in fig. 43, fluid enters the
To facilitate effective blending of the reaction solution with the lyophilized reagents, embodiments of the cartridge portion that include a fluid flow path may include dedicated reagent grooves 4600 incorporated in the fluid flow path between the chambers, as shown in fig. 46A and 46B. In an alternative embodiment,
In applications where it is desirable to place reagent grooves within a fluidic channel (such as shown in the standard embodiment shown in fig. 48), improved reagent resuspension and blending of reagents with reaction solutions can be achieved by incorporating projections (such as projections 4605 shown in fig. 46B, or
Multiplexing of assays
In some embodiments of the invention, multiple independent assays may be performed in parallel by employing a fluidics design that enables the input fluid sample to be split into two or more parallel fluidics paths through the device. Fig. 10 is a schematic of the separation of a fluid volume of, for example, 80uL, first into two separate 40uL volumes and then into four 20uL volumes in two consecutive steps. The presented protocol can be used to enable individual independent manipulations (such as biochemical reactions) to be performed on separate volumes. By facilitating multiplex detection of multiple targets (such as nucleic acid sequences in multiplex nucleic acid reverse transcription and/or amplification reactions), such a configuration can be used to increase the number of analytes that can be detected in a single device. Similarly, the use of multiple detection strips at the ends of independent fluid paths may provide enhanced readability of the strips for detecting multiple targets or distinguishing sequence differences or mutations in nucleic acid analytes. In addition, providing additional detection bands for independently interrogating multiple amplification reaction products can enhance specificity by reducing the likelihood of false cross-reactions (e.g., cross-hybridization) during the detection step of the test. FIG. 11 shows a test cartridge including two fluid paths in a single test cartridge. Each fluid path may be independently controlled in terms of time, type of reaction, etc. Referring now to fig. 12, a sample introduced into
After completion of a biochemical reaction such as reverse transcription, nucleic acid amplification, or concomitant increased reverse 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, the seals for the vented
Sample preparation
In embodiments of the invention, it may be desirable to incorporate the sample preparation system into a cartridge. A sample preparation system (such as a nucleic acid purification system) may contain an encapsulation solution for completing sample preparation and eluting purified molecules (such as purified DNA, RNA, or proteins) into a test cartridge. Fig. 13 depicts a nucleic acid
Upon initiation of sample preparation,
Movement of sample preparation subsystem components that occurs during sample preparation is shown in fig. 14, which fig. 14 depicts a cross section of a sample preparation subsystem embodiment before and after sample processing. Prior to elution, the binding
Referring now to fig. 15,
Electronic device
In some embodiments, it is desirable to place the electronic components in the reusable component such that the heaters, sensors, and other electronics interface to the disposable test cartridge by means that can establish a favorable thermal interface and accurate alignment of the electronics with the overlying elements of the disposable test cartridge to which they must interface. In other embodiments, it may be desirable to use a combination of reusable and disposable components for temperature control. For example, off-target temperature monitoring may be accomplished with an infrared sensor placed in the reusable docking unit, while resistive heaters for temperature control and fluid control are placed in a flexible circuit integrated into a disposable test cartridge.
In some embodiments, a Printed Circuit Board (PCB) comprises a standard 0.062 inch thick FR4 copper clad laminate, although other standard board materials and thicknesses may also be used. The electronic components, such as resistors, thermistors, LEDs, and microcontrollers, preferably comprise off-the-shelf Surface Mount Devices (SMDs) and are placed according to industry standard methods.
In an alternative embodiment, the PCA may be integrated with the cartridge wall and include a flexible plastic circuit. Flexible circuit materials such as PET and polyimide may be used as shown in fig. 8. The use of flexible plastic circuits is well known in the art. In another embodiment, the heating elements and temperature sensors can be screen printed onto the plastic fluidic components using techniques developed by companies such as Soligie corporation.
In some embodiments of the invention, the PCB thickness and the amount and placement of copper in the area around the resistive heater are tailored for thermal management of the reaction solution in the fluidic component. This can be achieved by using standard manufacturing techniques already mentioned.
In some embodiments of the invention, the resistor is a thick film 2512 package, but other resistors may be used. The heating chamber in the fluidic component preferably has dimensions similar to those of the resistor to ensure uniform heating of the entire chamber. Assuming a fluidic component thickness of 0.5mm, a single resistor of this size is sufficient to heat about 15 μ L of solution. The graph in fig. 2D shows two
In some embodiments of the present invention, the
In some embodiments of the invention, vent
In some embodiments of the invention, the microcontroller is microchip technology PIC16F 1789. The microcontroller is preferably matched to the complexity of the fluidic system. For example, with multiplexing, the number of individual vents and heaters is comparable to the number of microcontroller I/O lines. The memory size may be selected to accommodate the program size.
In certain embodiments of the present invention, the current load of the vent and heater resistors is modulated using N-channel MOSFETs in the SOT-23 package operating in the ON-OFF mode. The modulated signal is sent through the microcontroller. In alternative embodiments, pulse width modulation schemes and/or other control algorithms may be used to achieve more advanced thermal management of flow control. This is typically handled by a microcontroller and may require additional hardware and/or software features known to those skilled in the art.
Depending on the application, some embodiments include devices in which the docking unit is small controlled or operates a smaller disposable unit that includes a fluidic system (referred to as a cartridge) in contact with the biological material. In one such embodiment, the docking unit includes an electronic component. Eliminating the electronic components from the disposable test cartridge reduces cost and, in some cases, reduces environmental impact. In another embodiment, some electronic components are included in both the docking unit and the test cartridge. In this particular embodiment, the test cartridge preferably comprises a low cost PCA or preferably a flex circuit to provide some electrical functions (such as temperature control, fluid flow control and temperature sensing) that are activated, controlled and/or interrogated by the docking unit through a suitable interface. As mentioned above, the electrical function of such a device is preferably separated into two separate sub-assemblies. The
Where
Referring now to fig. 33, the docking unit preferably includes a
To minimize operating costs, another embodiment of the device may be used in a manner that reduces the cost of the consumable parts of the system by eliminating all the electronic circuitry located on the disposable parts. The microcontroller, heaters, sensors, power supply, and all other circuitry are located on multiple PCAs and are electrically connected to each other by high conductor count industry standard ribbon cables. A display may also be added to assist the user in operating the device. An optional serial control port may also be utilized to allow the user to upload changes in test parameters and monitor the progress of any test. A version of this embodiment includes five different PCAs. The motherboard PCA contains control circuitry, serial ports, power supplies and connectors for connecting to other boards in the system. The heater board PCA contains heating resistor elements, temperature sensors, and exhaust combustion heating elements. To facilitate the thermal interface between this heater plate and the disposable fluidic cartridge, this plate is mounted on a spring-loaded carrier that is moved toward the back side of the fluidic cartridge by the closing action of the cover until contact is made with the fluidic cartridge. Thin, thermally conductive heating pads are attached on top of the chamber heater resistors and temperature sensors, improving heat transfer between the heater board and the fluidic cartridge. A durable exhaust combustion heating element can be realized using nichrome wire wrapped around a small ceramic carrier. The IR sensor board PCA is mounted a small distance from the other side of the cartridge for monitoring the heating chamber temperature. This enables closed loop temperature control of the heating and cooling process and accommodates ambient temperature changes. A plurality of reflective sensing optical couplers are also mounted on the IR sensor board, which enable sensing of the presence of the cassette and can be used to identify the type of cassette represented by the configurable reflective pattern located on the cassette. The display panel PCA may be located generally behind the IR panel to allow a user to see the display from the front of the device. The final PCA, a shutter plate, is positioned across the top edge of the cassette and contains a switch and reflective optical coupler to detect whether the cassette has been used and, when the lid is closed, to hold the cassette in place for testing.
System cooling is optionally enhanced using a fan, such as a noise reduction fan (noise fan), which is turned on by the microcontroller only during the cooling phase of the test. The ventilation system is preferably used to direct the cooler outside air towards the heating chamber and out the side of the device.
To provide complete sample-result molecular testing, any of the above-described embodiments of the invention can be interfaced with a
Docking unit
The reusable docking unit includes the necessary electronic components for implementing the functionality of the test cartridge. Various docking unit embodiments have been invented to interface with corresponding variations in test cartridge design. In one embodiment, the docking unit shown in fig. 18 and 19 includes all of the electronic components needed to run the test, thereby eliminating the need for electronic components in the test cartridge. Referring now to fig. 18, prior to adding a sample, the
In some embodiments of the docking unit, the mechanism is incorporated into a hinge of the
In some docking unit embodiments, a set of components preferably facilitate proper cartridge insertion while ensuring that electronic components that must interface with the cartridge do not physically interfere with cartridge insertion, but rather form a reliable thermal interface during testing. These components form a mechanism for holding PCA75 away from the cassette insertion path until
In some embodiments, the docking unit includes additional sensors for applications such as temperature sensing, detecting the presence or removal of a test cartridge, and detecting a particular test cartridge to enable automated selection of test parameters. Referring now to fig. 26, an
In some embodiments of the invention, it is desirable to heat both sides of the cartridge. A dual heater PCA configuration is depicted in fig. 28A and 28B, with a test cartridge inserted between the two heater PCAs.
In another embodiment, the docking unit includes a servo actuator, an optical subsystem for automated result readout, a wireless data communication subsystem, a touch screen user interface, a rechargeable battery power source, and a cartridge receiver that accepts a test cartridge that includes an integrated sample preparation subsystem. Referring now to fig. 29A, 29B and 30,
Optionally, the digitized results may be transmitted for offline analysis, storage, and/or visualization through a wireless communication system incorporated in the docking unit using a standard WiFi or cellular communication network. Photographs of this docking unit embodiment are shown in fig. 32A and 32B.