Automated point-of-care device for complex sample processing and method of use

Abstract

To provide a method and a device for simple, low power, automated processing of a biological sample through multiple sample preparation and assay steps.SOLUTION: A reagent pouch in a reagent dispensing unit (RDU) (Fig. 1A) comprises an aqueous reagent 103 and a non-aqueous immiscible reagent 102 in a single reagent pouch, and these are sealed with a frangible seal layer 104 that can be ruptured upon actuation to enable reagent delivery into a microfluidic device. Fig. 1B depicts the RDU assembled on a microfluidic device 108. The RDU comprises a filled reagent pouch, and plunger elements 106 for squeezing the reagent pouch and for actuating a sharp object 105 that is used to rupture the frangible seal layer 104 assembled on the microfluidic device. The RDU is integrated into the microfluidic device 108 such that the frangible seal 104 is present at an interface of an inlet conduit 107 into a fluidic reagent well 109 on the microfluidic device.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 428,976, filed December 1, 2016, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to an automated point-of-care device for complex sample processing and methods for its use. [Background technology]

[0003] Point-of-care (POC) devices enable convenient and rapid testing at the point of patient care. Therefore, sample-to-answer and lab-on-a-chip (LOC) systems, a type of POC device that integrates microfluidics technology, are becoming increasingly popular. These LOCs integrate various laboratory functions, such as extraction, amplification, detection, interpretation, and reporting, that were previously performed manually and / or off-site, all onto the same device. Because sample-to-answer and LOC testing is performed at the point of patient care and not in a laboratory facility, these types of tests have presented challenges with contamination control, especially in processes that involve human interaction during processing. Therefore, there is a need to automate sample processing within sample-to-answer LOCs to minimize human interaction. These sample-to-answer and LOC devices are typically a few square millimeters to several square centimeters in size and are often microelectromechanical systems (MEMS). Here, MEMS capable of detecting and analyzing biological materials are commonly referred to as Bio-MEMS.

[0004] Most POC diagnostic devices on the market are classified as high or moderate complexity under the Clinical Laboratory Improvement Amendments (CLIA). These federal guidelines generally apply to human clinical laboratory testing devices, except for certain conditions that exempt them. One of these conditions is if the device or equipment meets certain risk, error, and complexity requirements. For a POC diagnostic test to qualify as CLIA-exempt, sample preparation and fluid handling steps must be minimized. One way to minimize these steps is to store reagents in a sealed device, such as a releasable blister or burst bag. Reagent delivery to the microfluidic chip typically involves the use of pumps, such as syringe pumps or peristaltic pumps, and external reagent-filled bottles, syringes, or reservoirs. These systems are not only difficult to transport but also complex due to the numerous components that must be integrated and the need for a leak-free fluid interface to the microfluidic chip. Simple, miniaturized, and low-power automated fluid handling methods have not yet been successfully implemented with the current state of the art on the market, and this has therefore been seen as an obstacle preventing the implementation of POC in the majority of multi-stage bioassay tests still performed in large clinical centers.

[0005] Complex biological assays requiring multiple processing steps, including but not limited to pipetting, heating, cooling, mixing, washing, incubation, labeling, binding, and elution, rely on expensive laboratory automation equipment to perform sample-to-answer sequencing. Low-cost, low-power, and miniaturized instrumentation for automating sample-to-answer sequencing has not yet been realized, and therefore, point-of-care microfluidic devices for performing sample-to-answer sequencing rely on additional instrumentation in the form of stand-alone benchtop or portable devices to perform the analysis on the microfluidic device. Implementing a separate instrumentation capable of automating sample processing steps on a microfluidic cartridge has been considered as a way to keep the cost per test, and therefore the cartridge cost, low. Systems developed for point-of-care applications can take the form of portable benchtop devices equipped with solenoid plungers, linear actuators, microcontrollers, and electronic circuitry to automate the sample processing sequence. While this instrumentation provides user control of the sample processing sequence, it requires a controlled environment and a significant amount of power to run. These point-of-care systems are not feasible in low-resource environments where the infrastructure to run the instrument does not exist, or in home or non-hospital settings where laypeople do not need or can afford to purchase expensive testing equipment or are not trained to operate the equipment involved. Therefore, the development of methods to enable low-power, stand-alone, inexpensive, and disposable instrumentation that can be integrated directly onto microfluidic devices and perform automated sample-to-answer sequencing is seen as an obstacle to developing single-use test devices capable of performing complex multistep nucleic acid, protein, and immunoassays from sample-to-answer.

[0006] Disposable tests that do not require instrumentation to perform them are limited to simple single-step and multi-step assays. In simple single-step assays, the sample is the only liquid and no reagents are used. These tests typically include urine test strips and dipstick tests such as pregnancy tests. Multi-step assays are sold in kit form, including reagent vials and instruction sets that allow the user to follow instructions and dispense the reagents into different areas of a disposable test cartridge. These devices typically perform immunoassays that do not require sample preparation steps. Some examples of these devices include, but are not limited to, Chembio Diagnostic Systems Inc.'s DPP® HIV 1 / 2 Assay, SURE CHECK® HIV 1 / 2, HIV 1 / 2 STAT-PAK®, and HIV 1 / 2 STAT-PAK® dipstick tests. These tests rely on the user to manually perform a series of steps to complete the sequence. If the user lacks skill or the test is not performed as instructed, there is a risk that the test will be performed inaccurately, and therefore the results may vary depending on how the test is performed. Furthermore, if the reagents are not completely contained within the device, there is an additional risk of contamination. Some strong reagents are hazardous if handled without proper laboratory protocols, gloves, and equipment (e.g., fume hoods and laboratory infrastructure such as contained biosafety equipment), and these kit tests cannot be performed unless the tests are performed by skilled technicians in a sealed facility.

[0007] When tests are automated rather than simple, non-experts perform them imprecisely. As test complexity increases beyond two or three steps, these manual, kit-based tests become impractical. Advances in nucleic acid amplification assays (e.g., isothermal assays such as loop-mediated amplification assays) reduce the instrumentation burden for heating / cooling thermal cycles because these tests only need to be held at a single temperature (usually between 60 and 70°C). However, these tests still require multiple user-initiated steps to complete sample-to-answer sequencing, which requires a skilled operator or additional automated instrumentation.

[0008] Sample preparation is essential for many diagnostic analyses, including biological sample processing. Biological samples typically must undergo multiple complex processing steps before they are suitable for use in an analysis. These steps are necessary to separate, concentrate, and / or purify the analytes of interest from the raw sample and to remove substances in the sample that may interfere with the desired analysis. Sample processing steps often involve precise conditions for temperature, reagent volumes, and incubation times that must be performed in a precise sequence and in a tightly controlled environment, such as a laboratory setting. Traditional automated systems for sample processing require highly complex and expensive instrumentation and skilled personnel to operate them. Because these systems are often located in a centralized laboratory, unprocessed samples often must be properly stored at a different location and transported to the laboratory for processing. These factors result in several limitations, including high costs, delayed results, and compromised sample integrity due to transportation and improper storage.

[0009] U.S. Patent Application Publication No. 2016 / 0129997, filed July 25, 2016, relates to a sample processing device including magnetic and mechanical actuation elements using linear or rotary motion and methods of using the same. U.S. Patent Application Publication No. 2016 / 0129997, filed July 25, 2016, relates to a sample extraction device and methods of using the same. The entire contents of both of these applications are incorporated herein by reference in their entirety. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Patent Application No. PCT / US16 / 43911 [Patent Document 2] International Patent Application No. PCT / US16 / 43855 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention provides methods and devices for simple, low-power, automated processing of biological samples through multiple sample preparation and analysis steps. The described methods and devices facilitate point-of-care performance of complex diagnostic assays in equipment-free, non-laboratory settings. [Means for solving the problem]

[0012] In accordance with the present invention, various embodiments of a sampling device and methods for its use are disclosed.

[0013] In accordance with the present invention, analytical automation devices and methods are disclosed for performing automated analyses, such as sample-to-answer microfluidic devices, nucleic acid amplification tests (NAATs), etc., on microfluidic devices. The present invention includes a portable analytical automation device and a microfluidic cartridge containing reagents stored in liquid and dry form that are dispensed in a predefined sequence to perform a sample-to-answer NAAT.

[0014] The present disclosure also includes various embodiments of sample processing devices and related processing methods for maximizing sample elution efficiency during transfer of a sample from a sample collection device (e.g., a swab, etc.) to a medium or buffer on a fluidic device, and during integration of the sample into a medium or buffer on a fluidic device.

[0015] Certain aspects of the subject matter disclosed herein, referred to in whole or in part by the subject matter disclosed herein, will become apparent as the description proceeds in conjunction with the accompanying examples and drawings, which are most particularly described below. [Brief explanation of the drawings]

[0016] Having described the subject matter of the present disclosure in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale. [Figure 1A] FIG. 2 is a cross-sectional view of an exemplary filled reagent pouch. [Figure 1B] FIG. 1 is a cross-sectional view of an exemplary microfluidic device with an integrated reagent dispensing unit (RDU), showing the RDU prior to actuation. [Figure 1C] 1 is a cross-sectional view of an exemplary microfluidic device with an integrated reagent dispensing unit (RDU), showing the RDU after actuation. [Figure 2A] FIG. 1 is a cross-sectional view of an exemplary microfluidic device with an integrated RDU, including a plunger and locking mechanism prior to actuation. [Figure 2B] FIG. 1 is a cross-sectional view of an exemplary microfluidic device with an integrated RDU, including a plunger and locking mechanism after actuation. [Figure 3A] FIG. 1 is a perspective view of an exemplary sample-to-answer microfluidic cartridge for performing a nucleic acid amplification test (NAAT). [Figure 3B] FIG. 1 is an exploded view of a schematic microfluidic device including a microfluidic cartridge that rotates between a top actuator element and a bottom actuator element. [Figure 4A] FIG. 1 is a schematic top view of a microfluidic device showing the position of the microfluidic cartridge relative to the actuator elements after actuation of the RDU. [Figure 4B] FIG. 1 is a schematic top view of the microfluidic device showing the microfluidic cartridge position before the magnetic bead-based sample preparation step. [Figure 4C]FIG. 10 is a top view of the microfluidic device showing the microfluidic cartridge position at the end of magnetic bead-based sample preparation, with the beads transported into the amplification wells. [Figure 5A] ~ [Figure 5C] FIG. 1 is a schematic top view of a microfluidic device showing an amplification well on three separate heating elements on a bottom actuator element and having different temperature zones T1, T2, and T3 to facilitate rapid thermal cycling with rotational position control. [Figure 6] FIG. 10 is a top view of a microfluidic device showing the position of the microfluidic cartridge at a moment before actuation of a sharp object by an upper actuation element to facilitate wicking of amplified products by the lateral flow strip for detection. [Figure 7A] FIG. 1 is a schematic diagram of an exemplary sample extraction device for extracting and processing a raw sample attached to a swab, showing different parts within the assembly. [Figure 7B] 1 is an assembled sample extraction device for processing an unprocessed sample attached to a swab, showing the swab rotating within a cleaning insert to facilitate mechanical cleaning and squeezing of the swab head to maximize sample elution from the swab. [Figure 8] FIG. 1 shows an exemplary sample processing protocol for recovering raw sample from a swab, showing a step-by-step sequence for processing the elution from the swab before transfer to a microfluidic cartridge. [Figure 9A] ~ [Figure 9B] 1A-1C are illustrations of an exemplary sample processing unit having a rotational actuator element, showing perspective and exploded views. [Figure 10A] ~ [Figure 10C] 10 shows an example of a sequence of movements when a rotary actuator element rotates relative to a reagent pallet in a sample processing unit. [Figure 11] 1 shows an exploded schematic view of a rotating shaft-based sample processing unit. [Figure 12] FIG. 1 is a perspective view of an exemplary reagent pouch card. [Figure 13]1A-1C are cross-sectional schematic diagrams of an exemplary microfluidic device including a reagent card including a transfer reagent bag and a flow-through reagent bag before and after application of an actuation force. [Figure 14A] ~ [Figure 14B] 14A and 14B are cross-sectional views of an exemplary microfluidic device showing an oil / immiscible phase dispensing system before (FIG. 14A) and after (FIG. 14B) application of an actuation force. [Figure 15] Top and perspective views of an exemplary sample-to-answer microfluidic device for nucleic acid amplification testing (NAAT) with lateral guidance-based readout. DETAILED DESCRIPTION OF THE INVENTION

[0017] The presently disclosed subject matter will now be described more fully hereinafter with reference to the accompanying drawings, which illustrate some, but not all, embodiments of the disclosed subject matter. Like numerals refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the particular embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0018] Automated point-of-care device for complex sample processing and method of use The present invention relates to an apparatus, assay, and method for sample preparation, nucleic acid amplification, and detection in an integrated sample-to-answer microfluidic device. The assay is simple in design and can be performed by the end user with minimal implementation time and equipment requirements. A typical manual sample preparation protocol involves multiple pipetting / fluid transfer and bead capture / resuspension steps, followed by binding, washing, and elution cycles to obtain purified DNA as the final product in a final volume of eluent.

[0019] Dead volume, the volume retained within a reagent pouch or fluid conduit / channel, can significantly hinder the reproducibility and reliability of analyses performed on the cartridge. Particularly for analytical processes that rely on high pipetting accuracy to be successful, such as amplification processes where even small changes in the system's volume can significantly affect reagent concentration and analytical performance, and processes requiring precise pH control, it is essential to have a metering system that can deliver precise volumes of reagent to the desired reaction chamber. While it is possible to include a reaction chamber with a fixed volume capacity to meter liquid reagents and configure any excess reagent delivered to the chamber to overflow and be discarded, this type of system requires precisely shaped metering chambers to control the accuracy of reagent dispensing on the microfluidic device. Additionally, systems that use metering chambers may be more susceptible to air bubbles within the system, which can affect the reliability of fluid dispensing. Such additional debubbling mechanisms may require pumps and valves, adding complexity to the microfluidic cartridge and instrument.

[0020] This disclosure describes a reagent dispensing unit (RDU) that overcomes the problems associated with dead volumes held in microfluidic conduits and stored in reagent pouches. This system efficiently delivers all aqueous reagents necessary to accurately perform microfluidic cartridge-based assays, thereby eliminating the need for sophisticated metering systems to meter precise volumes of aqueous reagents.

[0021] The reagent dispensing unit includes one or more reagent pouches containing miscible and immiscible liquid reagents packaged in separate pouches or together in a single pouch, and one or more plungers for pressing on the pouches to rupture a frangible seal layer on the pouch and expel their contents upon application of sufficient actuation force. In some embodiments, the RDU may include a sharp object or protrusion capable of rupturing the frangible seal on the RDU upon application of sufficient actuation force. The sharp object or protrusion may be located within the pouch or adjacent to the frangible seal of the RDU such that upon application of actuation force, the sharp object contacts and ruptures the frangible seal.

[0022] Referring to FIG. 1, a cross-sectional view of an exemplary microfluidic device having an integrated reagent dispensing unit (RDU) 101 is shown showing a filled reagent bag (1A), the RDU before actuation (1B), and the RDU after actuation (1C).

[0023] The reagent pouches in the RDU (FIG. 1A) contain an aqueous reagent 103 and a non-aqueous, immiscible reagent 102 in a single reagent pouch, sealed with a frangible seal layer 104 that can be broken upon actuation to enable reagent delivery to the microfluidic device. FIG. 1B shows the RDU assembled on a microfluidic device 108. The RDU includes a filled reagent pouch and a plunger element 106 that actuates a sharp object 105 used to squeeze the reagent pouch and rupture the frangible seal layer 104 assembled on the microfluidic device. The RDU is integrated into the microfluidic device 108 such that the frangible seal 104 resides at the interface of an inlet conduit 107 into fluidic reagent wells 109 on the microfluidic device. The microfluidic device includes one or more reagent wells 109 and a waste well 110 to help collect excess immiscible reagent 102 that overflows from the reagent wells 109.

[0024] The immiscible reagents are selected so that the aqueous reagent is closest to the interface between the frangible seal and the inlet conduit of the fluid well when the device is in operation. In some embodiments, an immiscible fluid less dense than the aqueous reagent, such as mineral oil, may be used in the system to suspend them and form an immiscible layer on top of the aqueous reagent. In other embodiments, an immiscible fluid more dense than the aqueous reagent, such as a fluorine-containing compound, e.g., a fluorocarbon such as Fluorinert (3M), may be used so that the less dense aqueous reagent floats on top of the immiscible Fluorinert fluid. The immiscible non-aqueous fluid is selected so that the aqueous reagent is closest to the interface between the frangible seal and the inlet conduit of the fluid well when the device is in its operating position.

[0025] FIG. 1C shows an actuated RDU assembled on a microfluidic device 108. When an actuation force is applied to the device, an integrated plunger squeezes the reagent pouch, rupturing the frangible seal 104 and fluidly connecting with the reagent well 109 through the inlet fluid conduit 107. Referring to FIG. 1C, the aqueous fluid 103 closest to the inlet fluid conduit 107 first flows from the inlet conduit into the fluid well, followed by the water-immiscible fluid 102, which effectively pushes out any aqueous reagent that would otherwise occupy dead volume space within the fluid conduit and the RDU. Excess immiscible non-aqueous fluid 102 overflows the reagent well and is collected in a waste well 110. Using this system, precise amounts of aqueous reagent can be effectively delivered onto a microfluidic device while eliminating dead volume issues by filling the space with a non-reactive immiscible non-aqueous fluid. The water-immiscible fluid 102 also acts to form a barrier on top of the aqueous fluid in the fluid wells, preventing evaporation of the aqueous reagent during heating processes such as thermal cycling or thermal incubation. This type of system significantly reduces system complexity because it does not require the use of complex valves or pumps for proper operation. Additionally, because the system does not rely on the volume of the reaction chamber to meter an accurate volume, the precise shaping of the reaction chamber, typically required to meter an accurate volume of reagent, is not required. Rather, aqueous reagents pre-filled into the RDU using well-known precision pipetting processes with immiscible, non-aqueous fluids are delivered completely to the desired fluid wells upon actuation, as there is water-immiscible fluid to completely push out the aqueous reagent and occupy any dead space that would otherwise be filled with the aqueous reagent. The amount of immiscible reagent dispensed into the system is not critical and does not require precise delivery.Therefore, the primary advantage of this type of system is that precise operational control on the instrument is not required to ensure run-to-run reproducibility of aqueous reagent delivery from single-dose reagent packs, because once all aqueous reagents are loaded into the device, the immiscible non-aqueous fluid overflows, pushing the aqueous reagents out and occupying all dead space to ensure delivery to the desired fluid wells, ensuring all aqueous reagents are pushed out of the RDU and into the microfluidic system during operation.

[0026] In some embodiments, the RDU may include a locking mechanism, such as, but not limited to, a pin capture mechanism such as a ball lock pin, a rivet, or a barbed pin, that functions to lock the plunger element in its depressed position to prevent backflow of reagent into the reagent pouch.

[0027] 2A and 2B, cross-sectional views of an exemplary microfluidic device with an integrated RDU are shown, including a plunger and locking mechanism 201 before and after actuation, respectively. A plunger element 202 is assembled adjacent to a reagent bag 204. The plunger element is secured in place by a barbed pin 203 that is captured within a locking hole 208 and oriented to restrict plunger movement in a direction that favors compressing the bag upon application of an actuation force. In this exemplary embodiment, the captured barbed pin 203 can only move downward along a locking hole 208 located on the microfluidic device. The reagent bag includes a frangible seal layer 205 that is broken when sufficient actuation force is applied to the plunger, as shown in FIG. 2B. Upon actuation, the frangible seal 205 breaks, and the contents of the reagent bag 204 are transferred to a fluid well 207 through an inlet fluid conduit 206 on the microfluidic device. The barbed pin 203 descends into the locking hole 208 to lock the plunger in its depressed position to prevent backflow of reagent into the reagent bag.

[0028] One aspect of the present invention is a microfluidic device including two or more fluid wells connected to each other via a primary channel. The fluid wells are connected to one or more reagent dispensing units (RDUs) containing stored liquid reagents separated from inlets into the fluid wells by frangible seals. The reagent pouches may be filled with aqueous fluids, water-immiscible fluids, or combinations thereof. Upon actuation, the frangible seals are broken and the contents of the reagent dispensing units are transferred to the fluid wells. At the end of the RDU actuation sequence, the stored reagents are successfully transferred to the fluid wells on the microfluidic device. The fluid wells are filled with their respective aqueous reagents and connected to each other via primary channels filled with non-aqueous fluids.

[0029] For care environments, a self-contained system is advantageous because it does not require complicated user-driven pipetting or injection steps. In one exemplary embodiment, reagents may be stored on the fluidic device in a reagent pouch. Reagents include, but are not limited to, buffers, salts, acids, bases, labels, tags, markers, water, alcohol, solvents, waxes, oils, gases, gels, and the like. When sufficient pressure is applied to the pouch, the pouch ruptures, thereby dispensing the pouch contents into a fluid conduit that adequately conducts the intended reaction. The pouch is designed with a frangible seal aligned with the inlet of the fluid conduit, such that upon rupture, the pouch forces its contents into the fluid conduit and fills the fluid well.

[0030] Each fluid well volume is designed to be only partially filled with miscible liquid reagents, preventing miscible liquids from overflowing and intermixing through each fluid well's upper fluid conduit. A reagent bag containing an immiscible liquid, such as mineral oil, is connected to the primary fluid conduit during operation. 1) The contents of the reagent bag containing the immiscible liquid are released, forming an immiscible oil phase above the aqueous reagent filled in the fluid well. 2) All miscible liquids in the fluid wells are connected to the sequencing process to form a fluid circuit, but are separated from each other by the oil phase to prevent intermixing. The primary fluid conduit exits into a waste well to collect excess oil.

[0031] While it is possible to pre-fill the fluid wells with buffers separated by an oil phase and then seal and store the cartridge for later use, some reagents, including but not limited to enzymes, oligos, dNTPs, and buffers, are not stable in their liquid form at room temperature or for extended periods of time and therefore must be stored in lyophilized form and hydrated prior to use. Additionally, challenges exist with introducing samples into such pre-filled systems. The disclosed invention provides methods and devices that address challenges associated with sample introduction, reagent delivery, and analytical automation for sample processing on microfluidic devices.

[0032] Referring now to FIG. 3A, a perspective view of an exemplary sample-to-answer microfluidic cartridge 301 for performing a nucleic acid amplification test (NAAT) is shown.

[0033] A sample-to-answer microfluidic cartridge includes one or more reagent wells 309 connected to each other via a primary fluid channel 305. The RDU assembled to the microfluidic cartridge includes multiple reagent bags 302 separated from the inlet conduits to the fluid wells 309 on the microfluidic cartridge by frangible seals and an integrated plunger element 303 with a locking pin 304 that locks the plunger in its depressed position after actuation, preventing backflow of reagent into the reagent bags. In this embodiment, the plunger element 303 is designed to contact all reagent bags at the same time, depressing and expelling all individual reagents from the reagent bags in parallel from a single actuation step. In other embodiments, the plunger element may have spatially oriented protrusions with varying depths to contact desired reagent bags in a preferred sequence as the plunger is depressed, facilitating sequential reagent delivery to the microfluidic cartridge. Upon actuation of the RDU, the reagent wells fill with aqueous reagents, and the fluidic circuit between the reagent wells is completed through a primary fluid channel filled with a water-immiscible fluid. The cartridge includes a waste well 306 that captures excess immiscible reagents that overflow from the reagent well 309 through the primary fluid channel 305. To perform a NAAT, the reagent pouch may contain a lysis buffer, a binding buffer, magnetic beads, a wash buffer, a hydration buffer, and an immiscible fluid (e.g., mineral oil, wax, or a fluorocarbon-based compound such as Fluorinert).

[0034] The reagents and reagent delivery sequences may be designed differently depending on the type of analysis being automated on the microfluidic device. The cartridge may also contain dried and lyophilized reagents that can be hydrated during use by either the sample or the dispensed buffer. The cartridge includes a sample inlet port through which the sample is transferred to the cartridge for processing. The sample inlet port may include a quick connect fitting such as a Luer 311 through which the sample can be injected into the cartridge. Other embodiments may include an access port through which the sample can be pipetted into the cartridge.

[0035] In some embodiments, the cartridge includes one or more filter membranes 310 at the interface between the inlet port and the cartridge, where impurities and inhibitors from the sample are filtered out prior to sample delivery to the cartridge. The filter membrane material and pore size can be selected depending on the type of analysis to be performed and includes, but is not limited to, nitrocellulose, nylon, PTFE, PES, glass fiber, PVDF, MCE, polycarbonate, etc. Depending on the type of detection method used, the cartridge may include further downstream analytical units such as DNA hybridization microarrays, protein arrays, lateral flow strips, etc.

[0036] In some embodiments, the amplification products may be detected using fluorescent, electrochemical, or colorimetric-based detection techniques. The exemplary microfluidic cartridge shown in FIG. 3 employs colorimetric detection using a lateral flow strip 308 that can be read digitally through an optical readout or visually by the end user. The lateral flow strip is separated from the amplification well by a frangible seal layer that interfaces with a pouch containing a sharp object 307, which, upon actuation, ruptures the frangible seal and delivers the amplified product to the lateral flow strip 308 for detection. In some embodiments, the amplification well itself may include a frangible layer that is susceptible to deformation upon actuation, causing the frangible seal layer to break and squeeze the amplified product onto the lateral flow strip.

[0037] Referring now to FIG. 3B, an exploded view of a schematic microfluidic device is shown that includes a microfluidic cartridge 301 that rotates between a top actuator element 318 and a bottom actuator element 313 .

[0038] By providing the top and bottom actuator elements with spatially oriented magnets 317 and 312, respectively, a single actuation step involving rotating the microfluidic cartridge between the actuator elements allows the spatially oriented magnets to capture, resuspend, and transport magnetic beads between different reagent wells to transfer a sample preparation sequence (e.g., a binding, washing, and elution sequence on the sample) to the microfluidic cartridge. In the exemplary embodiment shown in FIG. 3B, the top actuator element includes protrusions 316 designed to contact the microfluidic cartridge at predetermined times during the analysis sequence and actuate a sharp object in a pouch 307 thereon, rupturing the frangible seal layer and introducing amplified products into the lateral flow strip 308. The bottom actuator element includes one or more spatially oriented heating elements 314 that help provide the stable single-temperature heat or thermal cycling required for isothermal or PCR-based amplification of nucleic acids, respectively. The spatially oriented heating elements 314 can also help provide heat for sample preparation steps or downstream post-amplification steps, depending on the analysis being performed on the system. When thermal cycling is performed, the microfluidic cartridge is cyclically rotated between three heater elements set to a constant single temperature such that the amplification wells are in contact with or in close proximity to the desired heating element for the desired cycling time.

[0039] Sample-to-answer NAAT This specification describes an exemplary sample-to-answer NAAT, a microfluidic analytical automation platform that uses a single actuator to provide rotational motion, such as a servo or stepper motor, a winding spring, a hand crank, or actuation by a user's finger. Winding spring mechanisms offer the ability to automate analyses without using electrical / battery power, which is particularly advantageous for applications in low-resource environments. However, motors such as servo motors and stepper motors are inexpensive and easy to control for system operation. The system may be configured to incorporate different methods and steps depending on the type of analysis and analytical operation sequence.

[0040] Chargeswitch® technology (Invitrogen) is an extremely simple and effective method for purifying nucleic acids. It uses a unique ionizable coating that can be covalently attached to solid supports such as magnetic or non-magnetic beads, membranes, or plastic tubes and plates. The charge of the ionizable coating can be switched by changing the pH of the surrounding buffer. At low pH, the surface is positively charged, allowing negatively charged nucleic acids to bind to the solid support, while proteins and other contaminants can be easily washed away. At higher pH, the surface charge is neutralized, and nucleic acids are eluted from the surface without the need for time-consuming precipitation steps. A unique advantage is that chargeswitch technology uses aqueous buffers and does not require the use of ethanol, chaotropic salts, or organic solvents, which can inhibit downstream applications such as amplification.

[0041] Magnetic beads are highly effective and simple solid-phase capture supports for nucleic acid extraction and purification. Magnetic bead-based DNA purification does not rely on centrifugation and can be easily automated, reducing hands-on time. They are the method of choice when rapid purification is required, and when considering semi-automated or fully automated systems, magnetic DNA purification is a clear improvement over centrifugation-dependent isolation techniques. These systems are used when large numbers of samples need to be purified quickly. Magnetic beads coated with an ionizable (switchable) coating can be used to rapidly and efficiently purify nucleic acids from raw biological samples. Described herein is a unique analytical automation platform that allows magnetic beads to be captured, resuspended, and transported through a series of reagent-filled chambers via an oil-filled primary fluid conduit in a single rotational motion. This platform may be used to extract and purify nucleic acids from raw biological samples in a two-minute sequence.

[0042] The microfluidic cartridge contains a reagent pouch containing aqueous reagents such as binding buffer, magnetic beads in suspension, wash buffer, hydration buffer, and non-aqueous mineral oil as an overlay, as well as transport fluid. The microfluidic cartridge also contains dried lysis buffer reagents and lyophilized amplification mix present in respective reagent wells. When a test is ready to be run, the following steps are performed: 1. An unprocessed biological sample is pipetted and dropped or injected into the cartridge through the sample inlet port. 2. The cartridge is inserted into a handheld device containing an actuator element, a motor, electronics and a display. 3. Close the instrument lid and start the test.

[0043] System Operation The system may be configured to incorporate different methods and steps depending on the type of analysis, the biological sample and sample processing steps required for the sample, and the analytical operational sequence. As an example, the operational sequence for performing a NAAT on a swab sample, such as a urogenital swab or oral swab, is described. The swab is expressed in a buffer to extract cells from the collected swab sample. The sample is then transferred to the microfluidic cartridge, where the dried lysis buffer reagent present in the lysis / binding well 402 is hydrated. Cells in the raw sample are lysed. The lid of the device is then closed. In this embodiment, closing the lid applies an actuation force to the plunger 303 and dispenses reagents from a stored reagent pouch into the respective wells. During successful operation, magnetic beads in suspension and binding buffer are dispensed into the lysis / binding well containing the raw sample lysate, wash buffer is dispensed into the wash well 403, hydration buffer is dispensed into the amplification well 404 containing the lyophilized amplification mix, and mineral oil is dispensed forming a continuous overlay over the wells, filling the primary fluid channel 305 and completing the fluidic circuit. A locking barbed pin present on the cartridge holds the plunger element in its depressed position on the microfluidic cartridge, preventing backflow and preventing interference with the smooth rotation of the microfluidic cartridge between the actuator elements.

[0044] Referring to FIG. 4A, a top view of a microfluidic device showing the position of the microfluidic cartridge after activating the RDU is shown. Following the reagent loading step, the microfluidic cartridge begins to rotate proximate to the upper actuator element and the bottom actuator element, as shown in FIG. 4B. As the cartridge rotates, the spatially oriented magnets present on the upper actuator element and the bottom actuator element act to capture, resuspend, and transport magnetic beads through different reagent-filled wells. Nucleic acids bind to the magnetic beads in the presence of a binding buffer that changes the pH of the solution surrounding the beads to <pH 6. The magnetic beads are then captured by the first permanent magnet on the upper actuator element, moved through the main channel, and transported into the first wash well. The main channel includes obstacles that prevent the beads from freely moving under the influence of the magnetic field and trap the beads in the desired wells. The beads captured on the top surface of the wash well in the oil phase are affected by the second permanent magnet on the bottom actuator element, which pulls them down from the oil phase into the aqueous phase of the wash buffer reagent. This pulling magnetic force on the beads effectively resuspends them in the wash buffer (pH 7) present in the second well. As the microfluidic cartridge continues to rotate, this sequence of capture, transport, and resuspension of the beads continues to occur, effectively purifying the nucleic acids from the proteins and inhibitors present in the sample. The entire sequence from binding to elution can be completed in 2 minutes using the described automated analysis platform. At the end of the sample preparation stage, the beads are transported and resuspended in the amplification well 404 containing the hydrated amplification mixture. The pH of the amplification mixture is -8.5, which neutralizes the charge on the magnetic beads, thereby directly eluting all of the purified nucleic acids into the amplification mixture. FIG. 4C shows a schematic top view of the microfluidic device indicating the cartridge position at the end of the sample preparation stage.

[0045] During the amplification phase, the cartridge rotates to a position where the amplification wells are in close proximity to the spatially oriented heating elements 314 on the actuator elements. The heating elements function to provide the thermal energy required for nucleic acid amplification. For isothermal amplification reactions that require incubation at a single temperature, no additional heating elements are utilized; a single heater element can deliver the thermal energy for amplification. For applications involving polymerase chain reaction (PCR), which involves required thermal cycling, the microfluidic cartridge rotates in a cyclical manner between three heater elements set at a constant single temperature, such that the amplification wells are in contact with or in close proximity to the desired heating element for the desired cycling time.

[0046] Referring to Figure 5, a schematic top view of the microfluidic device (top actuator element not shown) shows the amplification well 404 positioned above a heater element set at temperatures T1 in Figure 5A, T2 in Figure 5B, and T3 in Figure 5C. This shows how a sample-to-answer NAAT with rapid thermal cycling is achieved by switching / actuating the microfluidic cartridge in a precisely timed sequence using three fixed thermal zones on the actuator element and a single motor that is also used to execute the entire analytical automation sequence. The motor rotates back and forth between the three heating zones, thereby cycling the amplification chamber between the three heating zones set at temperatures corresponding to denaturation, extension, and annealing cycles. This continues until a predefined number of cycles are completed. In some embodiments, rapid dual-temperature PCR can be performed using only two of the three heating elements. In some embodiments, a heating element comprising a custom aluminum block with integrated resistive elements can be used as a heat sink to easily quench the reaction to the desired temperature. In some embodiments, the heating element can also help provide heat for sample preparation steps or downstream post-amplification steps such as DNA hybridization on a microarray, depending on the analysis being performed on the system.

[0047] Following the amplification step, detection of the amplified product is performed colorimetrically on the integrated lateral flow strip. The lateral flow strip is separated from the amplification well containing the amplified product by a frangible seal layer that is coupled to a pouch containing a sharp object 307 that, upon actuation, can rupture the frangible seal and deliver the amplified product to the lateral flow strip 308 for detection. In some embodiments, the amplification well itself may include a frangible layer, susceptible to deformation upon actuation, causing the frangible seal layer to break and squeeze the amplified product onto the lateral flow strip.

[0048] Referring to Figure 6, a top view of the microfluidic device at the point where it reaches the lateral flow detection step is shown. A is a magnified image showing the protrusions 316 that contact and deform the pouch containing the sharp object 307 to rupture the frangible seal layer. The upper actuator element has spatially oriented protrusions 316 thereon that squeeze and deform the pouch containing the sharp object 307 when the microfluidic cartridge is rotated into contact with the protrusions. This deformation force ruptures the frangible seal layer, thereby wicking the amplified product through the lateral flow strip.

[0049] Sample Collection and Extraction Device Swabs are primarily used as biological sample collection devices. Swabs, such as COPAN FLOQSwabs™, are manipulated to ensure the entire sample remains near the surface for rapid and complete elution, but physical force must be used to maximize elution of the sample into the transport medium or buffer. Manual agitation by vigorously rotating the swab in the transport medium, or vortexing, is typically used in laboratories to maximize elution of the sample from the swab into solution. The swab is manually expressed, and then the solution containing the sample is pipetted and further processed depending on the type of analysis.

[0050] In point-of-care (POC) and low-resource settings, vortexing samples is not a convenient method for eluting samples in a liquid medium; manual shaking or stirring can lead to operator variability. Furthermore, because swabs are absorbent, a finite amount of sample in solution remains on the swab, resulting in loss. If the analyte is present at a very low concentration, insufficient analyte can be eluted from the swab into solution, reducing sensitivity.

[0051] Therefore, there is a need for improved devices and methods for sample extraction that minimize operator variability, are simple to use, do not consume power, and do not rely on laboratory equipment such as vortexers or centrifuges to operate.

[0052] The invention disclosed below is a mechanism, device, and method that can be used at the point of care to replace laboratory protocols to maximize sample recovery from swab samples. The disclosed invention also allows users to deliver multiple reagents directly to a sample in a sample extraction device using a simple, user-activated process. The disclosed invention can significantly simplify laboratory-based sample processing protocols and eliminate the need for sophisticated equipment required to perform laboratory-based sample processing protocols.

[0053] 7A and 7B, schematic diagrams of an exemplary sample extraction device for extracting and processing a raw sample attached to a swab are shown. The sample extraction device, in some embodiments, includes a sample collection container 705 and a sample processing unit 707 that is removable from the sample collection container. The device in this exemplary embodiment is for swab sample processing and includes a swab 704 having a swab shaft 703 and a screw-top lid 702 attached to the sample collection container 705. The container includes threads 706 that engage with the swab lid 702. When the swab is inserted into the container 705, the swab contacts a cleaning insert 715, 716 having one or more protrusions 713 that contact the swab head 704, cleaning the head as the lid is closed and the swab rotates / pivots within the insert. This cleaning action serves to release the sample attached to the swab head, thereby dissolving it into a buffer or medium 714 contained within the container. In some embodiments, the cleaning insert may have a plurality of small bristles 713 spatially oriented to contact the swab head when inserted. In other embodiments, the cleaning insert may have mechanical elements 716, such as ridges, O-rings, etc., that can function to scrape and squeeze the swab head within the insert. The number of threads dictates the number of turns or full rotations that the swab head 704 makes within the cleaning insert and can be optimized to maximize sample recovery from the swab.

[0054] Similarly, the type and design of mechanical elements can be optimized for each swab type to maximize sample recovery. In some embodiments, the container may include one or more filter membranes 712 selected to remove unwanted impurities and inhibitors from the sample. The container includes a quick-connect connector 711, such as a Luer connector, that connects to a removable sample processing unit 707. In some embodiments, the removable sample processing unit 707 may be a syringe including a barrel and plunger 708 and plunger tip 709. The syringe may include one or more grooved recesses 710 that can contain stored reagents in dry, liquid capsule, or pelleted form. The grooved recesses containing the stored reagents may be spatially oriented to be continuously introduced into the sample as the plunger tip 709 is withdrawn. In some embodiments, the syringe plunger may be aspirated and repeatedly depressed to release biological material present in the sample collection container using a forced flow.

[0055] Stored reagents include, but are not limited to, lyophilized or dried buffers such as lysis buffer, neutralization buffer, binding buffer, wash buffer, pH control buffer, solid phase capture supports such as magnetic beads, enzymes, antibodies, aptamers, conjugation buffers, functionalized particles such as gold nanoparticles, latex particles, magnetic particles, etc., chemiluminescent or colorimetric detection reagents, etc.

[0056] Referring now to FIG. 8, there is shown a step-by-step sequence of an exemplary sample processing protocol for collecting a raw sample attached to a swab prior to transfer to a microfluidic cartridge.

[0057] Step 1 - Insert the swab sample into the container and twist the lid closed. Step 2 - The plunger is retracted, thereby introducing the eluted sample from the container into the reagent stored in dry form present in the grooved recess of the syringe barrel. Step 3 - Remove the syringe from the quick connect on the container and discard the container with the swab. Step 4 - Connect the syringe to the quick connect sample inlet port on the microfluidic cartridge and depress the plunger to transfer the sample into the microfluidic cartridge.

[0058] In the exemplary embodiment, the container 705 is pre-filled with a suitable swab transport medium, such as phosphate-buffered saline (PBS), Amies medium, or the like. A swab is inserted into the container, and the lid is closed by rotating it "n" times (where n is the number of rotations determined by the threads 706 on the container). When the swab is inserted into the container, it contacts the protrusions and mechanical elements on the washing insert present in the container. As the swab rotates within the washing insert, the swab head is washed and squeezed by the mechanical elements, releasing the sample attached to the swab head and eluting it into the solution / medium contained within the container. Then, by withdrawing the plunger on the attached syringe sample processing unit, the sample from the container is filtered through a filter membrane to remove impurities and inhibitors and collected in the syringe barrel below. As the syringe plunger is withdrawn, the sample is introduced into one or more stored reagents, whether dry, liquid, or gel, present in succession within the barrel.

[0059] In an exemplary embodiment for performing NAAT, the stored dry reagents include a pellet of dried lysis buffer that is hydrated and activated when the sample is introduced therein and stored magnetic beads in liquid form that are resuspended in the lysate present in the syringe barrel of the sample processing unit. Alternatively, the stored reagents in the sample processing unit may include dried lysis buffer and dried neutralization buffer reagents that are sequentially introduced to the sample such that cells in the sample are first lysed and then the lysate is neutralized by introduction of a second neutralization reagent.

[0060] The neutralized sample may then be sequentially introduced into a third grooved well containing magnetic beads also stored in the sample processing unit prior to transfer of the processed contents to the microfluidic cartridge. Alternatively, the microfluidic cartridge may contain magnetic beads pre-loaded in a reagent well present therein, and the neutralized sample lysate may be introduced to the magnetic beads for sample purification as it is transferred to the microfluidic cartridge.

[0061] The described sampling device can be used in any biological analysis that involves multiple steps involving multiple reagents that need to be delivered to the sample in a predetermined sequence, where the reagents and steps may be selected and designed based on the analysis being performed.

[0062] Although the exemplary sample collection containers and associated sample processing units described herein are for processing swab samples where the sample must be attached to a swab and eluted into a solution for downstream processing, the containers can be adapted for different sample types not collected on a swab, including, but not limited to, biological samples such as saliva, blood, plasma, serum, urine, sputum, CSF, tissue, feces, plant matter, food, soil, small organisms, etc. The types of stored buffers / media and filters used within the containers can also be adapted for downstream analyses and sample types.

[0063] In an exemplary embodiment, a sample collection container may be used to collect and process a urine sample. The sample collection container may contain a lysis buffer reagent in a dried form that can be hydrated and activated when urine introduced into the container causes cell lysis to occur on the urine sample present in the sample collection container. The sample processing unit may contain a dried neutralization reagent therein so that the lysate is neutralized as it is drawn into the sample processing unit. The filter 712 may be selected to retain inhibitors and proteins, allowing only purified nucleic acids to pass through.

[0064] In an exemplary embodiment, an alkaline lysis buffer may be used that changes the sample pH to a range between 9-13. The pH 9-13 sample is then filtered through a membrane filter 712, such as a nitrocellulose or mixed cellulose ester (MCE) membrane with a pore size of 0.45 μm to 0.8 μm. Due to the selected pore size and highly alkaline pH of the sample, proteins and inhibitors present in the sample are retained or bind to the filter membrane, and only the purified nucleic acids pass on to the next stage into the sample processing unit, where the purified lysate is neutralized by a neutralizing reagent present therein.

[0065] Sample Processing Unit 9A and 9B, perspective and exploded views, respectively, of an exemplary sample processing unit are shown. The sample processing unit includes a sample collection container 902 and a lid actuator 903 that facilitates automated, sequential reagent delivery to samples within the container 902 in a precisely timed sequence. FIG. 9B is a schematic exploded view of the exemplary sample processing unit illustrating the functional components of the lid actuator 903 as a sequential reagent delivery system. In this exemplary embodiment, the lid actuator includes: a unique reagent dispensing unit that includes a reagent pallet 905; one or more reagent bags 904 containing reagents stored in dry, liquid, or gelled form; and one or more rotary actuation elements 907, spatially oriented mechanical elements 906 including, but not limited to, protrusions, valves, ridges, etc., that function to actuate the reagent bags 904 to dispense contents into the sample collection containers 902 in a precisely timed sequence when the rotary actuator element 907 rotates adjacent to the reagent pallet 905. In some embodiments, reagents are guided from the reagent dispensing conduit 908 into the sample collection container 902. In some embodiments, reagents are dispensed into the sample collection container under force or gravity. In some embodiments, the sample processing unit includes a mechanism for providing rotational motion, such as a winding spring, a motor, etc. In some embodiments, the reagent dispensing unit may be manually actuated by a user's finger.

[0066] In an exemplary embodiment, a wind-up spring is used. Wind-up spring mechanisms are well known and commonly used as mechanical timer devices. A well-known mechanical spring timer is a kitchen egg timer. These mechanisms generate a steady rotational motion until fully uncoiled. By appropriately selecting the spring and gear mechanism used, the wind-up spring mechanism can be designed to fully uncoil in a set amount of time. Sequential reagent delivery can be powered by the wind-up spring mechanism actuating an actuator to deliver reagents to the system in a precisely timed sequence. In the present invention, the rotary actuation element includes a spatially oriented mechanical element that interacts with a reagent-filled bag on a reagent pallet at a predetermined instance along the rotational path of the rotary actuation element, thereby deforming and squeezing the reagent bag, thereby delivering the reagent in a precisely timed sequence.

[0067] This sample processing unit has advantages over typical kits that employ manual reagent delivery protocols using pipettes or droppers because it is a self-contained system with all reagents necessary for sample processing packaged in a single unit, including a simple, pre-dispensed reagent delivery mechanism. Particularly in non-laboratory environments where only CLIA-waived tests can be performed (simple and easy tests without the risk of the user generating erroneous results), this self-contained sample processing unit eliminates complex, time-consuming pipetting steps and reduces the risk of erroneous results due to user-generated error by enabling reagent delivery using simple, universal twist, slide, or rotate motions that do not require a skilled operator to perform. It can also be automated using a single motor or self-powered winding spring actuator to further reduce hands-on time.

[0068] 10A, 10B, and 10C, an instance in the operational sequence is shown as a rotary actuator element 907 rotates relative to a reagent pallet 905. FIG. 10A shows the position of the rotary actuator element before reagent delivery occurs. In FIG. 10B, the rotary actuator element has moved to a position where a mechanical element 906 residing thereon interferes with a first reagent bag in its path, thereby deforming it and forcing its contents through a reagent dispensing conduit 908 and into a sample collection container. In FIG. 10C, the rotary actuator element has moved to a position along its path where a mechanical element 906 interferes with a second reagent bag, deforming it and forcing the contents of the second reagent bag into a sample collection container.

[0069] 9 utilizes rotational motion to perform actuation steps. However, other embodiments can utilize linear motion to accomplish the same task, for example, using one or more linear sliding actuator elements. The actuation elements may be oriented in different spatial dimensions to allow for sequential interference with different spatial dimensions of a sample processing device or microfluidic cartridge.

[0070] 11, an exploded schematic view of a rotating shaft-based sample processing unit is shown. This unique embodiment of the present invention uses a rotating shaft actuator element 1103 that provides additional dimensional control for analytical automation. The rotating shaft actuator element comprises one or more spatially oriented mechanical elements 1102 that interact with and actuate reagent pouches 1105 on a reagent pallet 1104, dispensing their contents in a predetermined sequence.

[0071] In some embodiments, a detection unit may be integrated into the sample processing unit to facilitate direct detection of analytes within a self-contained system without the need to transfer the analyte from the container to the detection unit. This detection unit may be visual, using a colorimetric reagent that produces a color change in the container depending on the presence or absence of the analyte, or it may be an immunochromatographic detection device that uses a dipstick or lateral flow device. In some embodiments, the lateral flow device may be integrated onto the surface of the sample collection container or into the lid of the sample processing unit.

[0072] While each reagent may be packaged in an individual reagent pouch and assembled onto the microfluidic cartridge, this approach results in a more complicated assembly process, requiring each reagent pouch to be individually assembled and sealed onto the cartridge. In some embodiments, it may be preferable to create a reagent card containing multiple reagent pouches that can be assembled onto the microfluidic cartridge as a single unit. Referring to FIG. 12 , a perspective view of an exemplary reagent card 1201 is shown, showing individual reagent pouches 1202 and a flow-through reagent pouch 1203. The reagent card may be designed with a shape that easily mates and aligns with mating grooves on the cartridge during assembly. The reagent card can be loaded manually or using multiple automated pipettors into a custom fixture to dispense the desired fluid volume into each reagent pouch prior to frangible foil sealing. In some embodiments, the reagent card may include molded features on the pouch to aid in placing and aligning the reagent pouch card onto the microfluidic cartridge. When an actuation force is applied to the reagent bags individually, in a sequential manner, or in parallel to multiple reagent bags on the card, the bottom frangible foil seal ruptures, allowing reagents to flow through the fluidic channels into the appropriate reaction chambers in the fluidic cartridge. The actuation force may be distributed to the reagent card via a plunger with spatially oriented protrusions that contact one or more reagent bags in succession during actuation.

[0073] In some embodiments, it may be necessary to mix or combine one or more reagents present in a reagent pouch. Mixing in microfluidic devices has traditionally been achieved using active mixers, in which external energy is applied to agitate the fluids, or passive mixers, which use specially designed geometries and channel configurations to increase the contact area and time of the mixing fluids. In some embodiments, it may be necessary to mix two or more reagents. For example, at least one reagent may need to be in solid form, contain solid particles in a low- or high-viscosity liquid medium, or be a high-viscosity liquid or gel. In microfluidic cartridges, solid reagents are often stored by drying them directly in the reaction chamber, in which case they may be reconstituted during use with a liquid reagent, which may be, for example, a reconstitution buffer or an unprocessed or processed liquid sample being analyzed. These dried reagents are brought to the desired concentration using a known volume of reconstitution fluid. In some cases, it may be desirable to dry or lyophilize reagents and store them directly in the reaction chamber of a microfluidic device. For example, lyophilized master mixes for nucleic acid amplification tests (NAATs) eliminate the need for cryogenic storage, allowing microfluidic cartridges to be stored at room temperature. In other cases, the drying process can adversely affect reagents, reducing their efficacy or even permanently destroying them. For example, charge-switch magnetic beads for nucleic acid sample preparation supplied by Thermofisher Scientific (Carlsbad, CA) lose functionality once dried and must be kept in solution at all times. Some magnetic beads contain functional coatings, such as the cellulose-coated magnetic beads from Promega's Magazorb® DNA extraction kit, which irreversibly aggregate and become nonfunctional when dried. Therefore, it is important to store the beads in a liquid matrix to maintain their functionality.While it may be possible to develop a custom drying process using custom chemistries that helps preserve the functional coating on the beads, developing a custom process is often expensive and requires extensive testing to ensure no loss of functionality. Therefore, storing functionalized particles, such as magnetic beads, in a liquid matrix is ​​preferable. However, on-chip storage of magnetic beads in liquid form presents its own set of challenges. Specifically, magnetic particles are stored in a liquid matrix at very high concentrations (often in the range of 5 mg / ml to 50 mg / ml) and then diluted with sample and buffer to meet binding capacity requirements. Manufacturers provide protocols requiring very small amounts of magnetic bead reagent per test, typically in the range of 10 μl to 40 μl. However, manufacturing process limitations (minimum volume of 50 μl) make it difficult to package these in foil-sealed reagent pouches without significant dead volume issues. Additional dead volume in the channels or fluid conduits leading to the reaction chamber results in significant loss of reagent during dispensing. For example, a channel with a cross section of 750 μm x 750 μm and a length of 1 inch has a dead volume of 15 μl. Compounding this problem is that as much as 20% of the reagent may still be present in the crushed reagent pouch during the dispensing process.

[0074] Losing 20% ​​of the concentrated essential reagent due to entrapment in dead space is undesirable. The invention disclosed herein uses a flow-through-based approach to facilitate effective reagent transfer and mixing on a microfluidic device. The flow-through system utilizes a fluid medium, either liquid or gas, present in large quantities as a transfer reagent to effectively transfer / displace the reagent present in the flow-through reagent bag into the reaction chamber on the microfluidic device. Here, the transfer reagent can be an immiscible fluid, such as mineral oil (liquid) or air (gas), or a miscible liquid, such as an aqueous buffer. When the immiscible liquid and gas enter the flow-through reagent bag, they effectively displace the entire contents of the reagent bag into the reaction chamber of the microfluidic device. When a miscible fluid, such as a buffer, enters the flow through the chamber, it mixes with the reagent present in the flow through the chamber, and the contents entering the reaction chamber of the microfluidic device are a mixture of the transfer reagent and the reagent in the flow-through reagent bag. This method counteracts the effects of dead volume in the reagent bag or microfluidic device by filling them with transfer reagent. Since the volume of the transferred reagent is not essential or critical to the reaction occurring within the reaction chamber of the fluidic chip, this method helps to effectively transfer reagents present in a flow-through reagent bag whose volume is critical to the proper functioning of the assay.

[0075] Alternatively, the transfer medium may be in the form of a reconstitution buffer that rehydrates the lyophilized reagent pellets that may be present in the flow-through reagent pouch. In some embodiments, the transfer medium may be the liquid sample being analyzed. During interaction between the transfer medium and the contents of the flow-through reagent pouch, mixing of the two occurs. This mixing can be further assisted by increasing the contact area and contact time. Many approaches, such as increasing the channel length, reducing the channel cross-section, adding physical barriers to the flow rate, increasing the fluid pressure, or introducing turbulence into the fluid flow, are just a few that can be used to facilitate mixing.

[0076] In one embodiment, this method mitigates loss of functionalized particles, such as magnetic beads, and aids in flow-based mixing and homogenization of particles / beads to promote binding of analytes present in solution to the functionalized particles / beads.

[0077] Referring to FIG. 13A, a cross-sectional schematic diagram of an exemplary microfluidic device is shown with a reagent card containing a transfer reagent bag 1303 filled with transfer reagent 1306 and a flow-through reagent bag 1302 containing magnetic beads / particles in a liquid medium. The transfer reagent bag is connected to the flow-through reagent bag inlet via a transfer fluid conduit 1308 on the microfluidic device, which is capped with a frangible seal 1304. The flow-through reagent bag contains a rupture element (ball) 1305 and is connected to a reaction chamber on the microfluidic device through an outlet fluid conduit 1309. As shown in FIG. 13B, upon application of an actuation force, the rupture element breaks the underlying frangible seal, thereby opening a path for the transfer reagent to enter the flow-through reagent bag and displace its contents. In some embodiments, the rupture element may be located on the microfluidic device rather than within the flow-through reagent bag.

[0078] Example The manufacturer provided a protocol for adding 40 μl of Charge Switch® magnetic beads and 300 μl of the provided binding buffer to 600 μl of bacterial cell lysate. To perform this protocol on the automated microfluidic device described herein, 600 μl of cell lysate is first dispensed into the reaction chamber on the microfluidic device using a dispensing dropper or syringe. The flow-through reagent bag contains 40 μl of magnetic beads. Assuming a total dead volume in the system (i.e., the volume remaining in the crushed flow-through reagent bag, fluid conduit, and crushed transfer reagent bag) of 60 μl, the transfer reagent bag contains 360 μl of binding buffer to offset the loss due to dead volume. This dead volume is inherent in the design of the microfluidic device and can be easily calculated from the device geometry and confirmed using experimental methods. When an actuation force is applied, the frangible seal is ruptured, allowing binding buffer to enter the flow-through reagent bag containing the magnetic beads. The turbulent flow of the binding buffer as it enters the flow-through reagent bag initiates resuspension of the magnetic beads that may have settled due to storage. The product of resuspended magnetic beads in binding buffer then enters the reaction chamber containing the cell lysate through outlet fluid conduit 1309, completing the binding protocol.

[0079] This system avoids the use of complex systems such as metering pumps which add to the cost and complexity of the device.

[0080] Oil / Immiscible Phase Dispensing System In some embodiments, an oil-filled reagent pouch can be used to contain the dispensable oil phase upon application of an actuation force. However, reagent pouches are very difficult to manufacture and fill, and they are difficult to seal without dead air zones. Typical manufacturing tolerances for dead air within the pouch can be as little as 10% to 20% of the total pouch volume. Particularly with viscous oil-phase reagents, trapped air can create bubbles in the oil phase, leading to reproducibility issues and problems with magnetic particle movement in the immiscible oil phase or between miscible aqueous and immiscible oil phases. In addition, the flow of the oil phase from the reagent pouch during dispensing can be turbulent, forming air pockets within the microfluidic device as the oil phase fills each reaction well and primary channel. Furthermore, another unique embodiment described herein prevents bubble formation within the system by optimizing channel and well geometries to promote laminar flow and by implementing an in-line debubbling mechanism, such as a microporous hydrophobic / oleophobic PTFE membrane, which selectively releases trapped air without allowing liquid to escape. In this unique embodiment, smooth laminar flow is generated by utilizing the pressure head of the oil phase. This can be complemented with optimized channel and well geometries to create a completely bubble-free oil phase in the primary channel without complicating the system by using a degassing mechanism. The pressure head-based approach requires a vent hole to allow fluid flow to be generated by rupturing the frangible seal during dispensing. Furthermore, the presence of air in the oil phase container is not affected, as air is too light to displace the oil phase.

[0081] Referring to FIG. 14, an exemplary microfluidic device showing an oil / immiscible phase dispensing system 1401 is shown in FIG. 14A before the application of an actuation force and in FIG. 14B after the application of an actuation force. The microfluidic chip-based oil / immiscible phase dispensing system 1401 includes an oil storage container 1402 that holds a desired volume of an oil / immiscible or liquid reagent 1404. The oil / immiscible phase storage container includes an air vent conduit 1403 and an oil / reagent conduit 1405 that function to connect the container 1402 to a vent hole and a reaction chamber on the microfluidic device, respectively. A deformable lid 1407 containing a burst ball 1408 is present at the vent and oil / reagent outlet. The vent 1409 and oil / reagent outlet 1410 are sealed by a frangible seal 1406 and are isolated from the microfluidic device by the same frangible seal, which functions as a one-time valve. Upon application of an actuation force, as shown in Figure 14B, the deformable caps on the air vent and oil / reagent outlets are fractured, causing the burst balls 1408 to pierce the frangible seals, connecting the air vent and oil / reagent outlets to the vent holes on the microfluidic device and the reaction chambers on the microfluidic device, respectively.

[0082] Referring to FIG. 15, an exemplary embodiment of a sample-to-answer microfluidic device for nucleic acid amplification testing (NAAT) is shown. The microfluidic device includes a detection reagent card 1201, an oil dispensing system 1401, and a lateral flow strip 1505 assembled on a microfluidic chip. The microfluidic chip itself contains multiple reaction chambers 1502 connected to each other by primary channels 1503. Inlet channels 1504 connect reagent pouches 1303 on the reagent card to the individual reaction chambers. An actuation element on the instrument lid, comprising a plunger with a spatial topography that matches the spatial location of the individual reagent pouches and a deformable lid element 1407 on the oil dispensing system 1401, is used to provide the actuation force that ruptures the frangible seal.

[0083] A typical sequence of operations is as follows: 1. The sample is injected into the cartridge or dispensed through the sample inlet. 2. The cartridge is inserted into the instrument and the lid is closed, providing the actuation force to rupture the frangible seals in the reagent card and oil dispensing system. 3. The user enters a start command by pressing a button to begin the magnetic bead-based sample processing and amplification sequence. 4. Results are displayed on the lateral flow strip after the test is completed.

[0084] General definition Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention described herein belongs.

[0085] As used herein, "nucleic acid" refers to a polymeric compound comprising covalently linked subunits called nucleotides. A "nucleotide" is a molecule or individual unit within a larger nucleic acid molecule that comprises a nucleoside (i.e., a compound containing a purine or pyrimidine base linked to a sugar, usually ribose or deoxyribose) linked to a phosphate group.

[0086] "Polynucleotide" or "oligonucleotide" or "nucleic acid molecule" are used interchangeably herein to mean the polymeric form of a phosphate ester of ribonucleosides (adenosine, guanosine, uridine, or cytidine, "RNA molecule" or simply "RNA") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine, "DNA molecule" or simply "DNA") or any phosphoester analog thereof, in either single- or double-stranded form.

[0087] Polynucleotides can be of any length and contain RNA, DNA, or RNA / DNA hybrid sequences. Polynucleotides used in the present invention can be naturally occurring, synthetic, recombinant, ex vivo produced, or combinations thereof, and can be purified using any purification method known in the art. Thus, the term "DNA" includes, but is not limited to, genomic DNA, plasmid DNA, synthetic DNA, semi-synthetic DNA, complementary DNA ("cDNA" is DNA synthesized from a messenger RNA template), and recombinant DNA (DNA that is artificially designed and therefore has undergone molecular biological manipulation from its naturally occurring nucleotide sequence).

[0088] "Amplify," "amplification," "nucleic acid amplification," and the like refer to the production of multiple copies of a nucleic acid template (e.g., a template DNA molecule) or the production of multiple copies of a nucleic acid sequence that is complementary to a nucleic acid template (e.g., a template DNA molecule).

[0089] The terms "top," "bottom," "up," "down," and "above" are used throughout the description to refer to the relative positions of components of the described devices, such as the relative positions of an upper substrate and a lower substrate within the device. It will be understood that the devices will function regardless of their orientation in space.

[0090] Following long-standing patent law practice, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, a reference to a "subject" includes a plurality of subjects unless the context clearly dictates otherwise (e.g., a plurality of subjects).

[0091] Throughout this specification and claims, the term "comprises" is used in a non-exclusive sense unless the context dictates otherwise. Similarly, the term "include" and its grammatical variations are intended to be open-ended, such that the recitation of items in a list does not exclude other similar items that may be substituted for or added to the listed items.

[0092] For purposes of this specification and the appended claims, all numbers expressing quantities, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, amounts, properties, and other numerical values ​​used in the specification and claims, even if the value, amount, or range does not explicitly state otherwise, are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are not precise and need not be exact, but may be approximate and / or larger or smaller, as desired, and may reflect tolerances, conversion factors, rounding, measurement error, and the like, as well as other factors known to those of ordinary skill in the art depending upon the desired properties to be obtained by the subject matter disclosed herein. For example, the term "about" when referring to a value is meant to include a range of ±100%, in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% of the specified amount, as appropriate for practicing the disclosed methods or using the disclosed compositions.

[0093] Furthermore, the term "about," when used in connection with one or more numbers or numerical ranges, should be understood to mean all numbers, including all numbers within the range, and modifying that range by extending the boundaries above and below the numerical values ​​set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., all integers, including fractions subsumed within that range (e.g., recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc.), and all ranges within that range.

[0094] All publications, patent applications, patents, and other references mentioned in this specification are indicative of the level of skill of those skilled in the art to which the subject matter disclosed herein pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, or other reference was specifically and individually indicated to be incorporated by reference. Although numerous patent applications, patents, and other references are referenced herein, it will be understood that such references are not an admission that any of these documents form part of the general knowledge in the art.

[0095] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will understand that certain changes and modifications may be practiced within the scope of the appended claims. [Appendix 1] 1. A microfluidic device comprising a reagent dispensing unit, the reagent dispensing unit comprising: at least one reagent pouch comprising one or more reagents and a frangible seal layer; A microfluidic device comprising at least one plunger and at least one sharp object or protrusion configured to rupture the frangible seal layer and deliver the one or more reagents to the microfluidic device when an actuation force is applied to the reagent dispensing unit. [Appendix 2] The microfluidic device further comprises: at least one inlet conduit; at least one reagent well; at least one waste well; 2. The microfluidic device of claim 1, wherein the inlet conduit, the reagent well, and the waste well are fluidly connected and configured such that an interface exists between the frangible seal and the inlet conduit, and when an actuation force is applied to the reagent dispensing unit and excess reagent overflowing from the reagent well is collected in the waste well, one or more reagents are delivered to the reagent well via the inlet conduit. [Appendix 3] 3. The microfluidic device of claim 2, wherein at least two reagents are packaged in separate pouches. [Appendix 4] 3. The microfluidic device of claim 2, wherein at least two reagents are packaged together in a single pouch. [Appendix 5] 5. The microfluidic device of claim 4, wherein one reagent is an aqueous reagent and one reagent is a non-aqueous, immiscible reagent. [Appendix 6] 6. The microfluidic device of claim 5, wherein the aqueous reagent is closest to the interface between the frangible seal and the inlet conduit. [Appendix 7] 6. The microfluidic device of claim 5, wherein the non-aqueous, immiscible reagent has a lower density than the aqueous reagent and floats on top of the aqueous reagent, thereby forming an immiscible layer on top of the aqueous reagent. [Appendix 8] 6. The microfluidic device of claim 5, wherein the aqueous reagent has a lower density than the non-aqueous immiscible reagent and floats on top of the non-aqueous immiscible reagent, thereby forming an aqueous layer on top of the non-aqueous immiscible reagent. [Appendix 9] 6. The microfluidic device of claim 5, wherein when an actuation force is applied to the reagent dispensing unit, the aqueous reagent first flows out of the inlet conduit and into the reagent well, followed by the non-aqueous, immiscible reagent. [Appendix 10] 3. The microfluidic device of claim 2, further comprising a locking mechanism configured to lock the plunger in a depressed position, thereby preventing backflow of the reagent into the reagent bag. [Appendix 11] 11. The microfluidic device of claim 10, wherein the locking mechanism comprises a barbed pin within a locking hole configured to limit movement of the plunger in a direction that facilitates depressing the bag during application of an actuation force. [Appendix 12] 3. The microfluidic device of claim 2, comprising two or more reagent wells connected to each other and to one or more reagent dispensing units via a primary channel. [Appendix 13] 13. The microfluidic device of claim 12, wherein at the end of an actuation sequence, the reagent wells are filled with aqueous reagents and connected to each other via the primary channels filled with non-aqueous fluids. [Appendix 14] 13. The microfluidic device of claim 12, configured such that at the end of an actuation sequence, an immiscible oil phase is formed over the aqueous reagents in the fluid wells, and the aqueous reagents in the fluid wells are separated from each other by the oil phase but are fluidly connected during the sequence to form a fluid circuit. [Appendix 15] 13. The microfluidic device of claim 12, further comprising a plurality of reagent bags separated from the inlet conduits to the fluid wells by frangible seals, and an integrated plunger element having a locking pin that locks the plunger in a depressed position after actuation, thereby preventing backflow of reagent into the reagent bags. [Appendix 16] 16. The microfluidic device of claim 15, wherein the plunger is configured to contact all of the reagent bags at the same moment and push and expel all of the reagents from the reagent bags in parallel in a single actuation step. [Appendix 17] 16. The microfluidic device of claim 15, wherein the plunger has spatially oriented protrusions of varying depths configured to facilitate sequential reagent delivery to the microfluidic device when the plunger is depressed in contact with desired reagent bags in a preferred sequence. [Appendix 18] 3. The microfluidic device of claim 2, further comprising a sample inlet port through which a sample can be injected into the microfluidic device. [Appendix 19] 19. The microfluidic device of claim 18, wherein the sample inlet port further comprises one or more filter membranes. [Appendix 20] 3. The microfluidic device of claim 2, further comprising a microfluidic cartridge configured to rotate between a top actuator element and a bottom actuator element, the top and bottom actuator elements comprising spatially oriented magnets, the spatially oriented magnets capturing, resuspending, and transporting magnetic beads between different reagent wells in a single actuation step comprising rotating the microfluidic cartridge between the top and bottom actuator elements. [Appendix 21] 21. The microfluidic device of claim 20, wherein the top actuator element comprises a protrusion configured to contact the microfluidic cartridge at a predetermined time during an analysis sequence and actuate a sharp object or protrusion in the reagent pouch to rupture the frangible seal layer and deliver amplified material to the lateral flow strip, and the bottom actuator element comprises one or more spatially oriented heater elements configured to provide a stable single temperature heat or thermal cycle for isothermal or polymerase chain reaction (PCR)-based amplification of nucleic acids. [Appendix 22] 22. The microfluidic device of claim 21, wherein the spatially oriented heater elements are configured to provide thermal cycling, and the microfluidic cartridge is cyclically rotated between multiple heater elements, each set to a constant single temperature, so that the amplification wells contact or are in close proximity to a desired heater element for a desired cycling time. [Appendix 23] 1. A sample extraction device comprising a sample collection container and a sample processing unit, wherein the sample collection container comprises a swab having a swab head and a swab shaft attached to a screw-top lid, the sample collection container having threads that mate with the lid, the sample collection container configured such that when the swab is inserted into the container, the swab contacts a cleaning insert having one or more protrusions that contact the swab head and cleans the swab head, and the lid is closed. [Appendix 24] 24. The sampling device of claim 23, wherein the cleaning insert comprises a plurality of bristles spatially oriented to contact the swab head when pulled within the insert. [Appendix 25] 24. The sample extraction device of claim 23, wherein the cleaning insert comprises a mechanical element. [Appendix 26] 26. The sampling device of claim 25, wherein the mechanical element comprises a ridge or an O-ring. [Appendix 27] 26. The sample extraction device of claim 25, wherein the container comprises one or more filter membranes. [Appendix 28] 26. The sample extraction device of claim 25, wherein the container is provided with a quick connect connector for connection to a removable sample processing unit. [Appendix 29] 29. The sample extraction device of claim 28, wherein the removable sample processing unit is a syringe having a barrel, a plunger, and a plunger tip. [Appendix 30] 30. The sample extraction device of claim 29, wherein the syringe comprises one or more grooved recesses containing stored reagents. [Appendix 31] 31. The sampling device of claim 30, wherein the grooved recess is spatially oriented such that the stored reagent is continuously introduced into the sample as the plunger tip is withdrawn. [Appendix 32] A sample processing unit comprising: a sample collection container; a lid actuator configured for automated sequential delivery of reagents to the samples in the containers in a predetermined timed sequence; The lid actuator is a reagent dispensing unit including a reagent pallet with one or more reagent bags containing stored reagents; A sample processing unit comprising one or more rotary actuator elements comprising spatially oriented mechanical elements configured to actuate the reagent bags to dispense contents into the sample collection containers in a predetermined timing sequence when the rotary actuator elements rotate in proximity to the reagent pallets. [Appendix 33] 33. The sample processing unit of claim 32, wherein the rotational actuation element comprises a mechanism for providing rotational movement. [Appendix 34] 34. The sample processing unit of claim 33, wherein the mechanism for providing the rotational motion is a winding spring. [Appendix 35] 33. The sample processing unit of claim 32, wherein the rotary actuation element is configured to be manually actuated by a user's finger. [Appendix 36] 33. The sample processing unit of claim 32, wherein the rotary actuation element comprises a rotating shaft including one or more spatially oriented mechanical elements that interact with and actuate the reagent bags on the reagent pallet to dispense their contents in a predetermined sequence. [Explanation of symbols]

[0096] 101 Reagent dispensing unit 102 Non-aqueous immiscible reagents 103 Aqueous Reagents 104 Frangible seal layer 105 Sharp Objects 106 Plunger element 108 Microfluidic Devices

Claims

1. 1. A microfluidic device comprising: a reagent dispensing unit including at least one reagent bag having one or more reagents and a sealing layer; at least one actuator element comprising a spatially oriented magnet; a microfluidic cartridge comprising a plurality of wells; the microfluidic cartridge is configured to receive magnetic particles; the microfluidic cartridge is configured to: a) rotate incrementally forward and reverse about a central axis in a predetermined order corresponding to steps in an analytical processing sequence; or b) rotate continuously about a central axis through 360 degrees or less; the microfluidic cartridge and the at least one actuator element are positioned proximate to and substantially parallel to one another, and rotation of the microfluidic cartridge causes the spatially oriented magnets to transport the magnetic particles between the wells; at least one plunger configured to contact the at least one reagent bag and apply an actuation force to the at least one reagent bag to break the sealing layer and dispense the one or more reagents into the plurality of wells. Microfluidic devices.

2. 10. The microfluidic device of claim 1, wherein the plurality of wells comprises at least one amplification well coupled to an amplification well bag including a sealing layer and a sharp object or protrusion, the sealing layer and the sharp object or protrusion configured to rupture the sealing layer and dispense an amplification product contained in the amplification well when an actuation force is applied to the amplification well bag.

3. 3. The microfluidic device of claim 2, wherein the at least one actuator element comprises a plurality of spatially oriented mechanical elements configured to apply the actuation force to the amplification well pouch at a predetermined time in an analysis sequence to actuate at least one of the sharp object or protrusion to rupture the sealing layer.

4. 4. The microfluidic device of claim 3, wherein the microfluidic cartridge further comprises an integrated detection element separated from the amplification well by the amplification well sack sealing layer, wherein rupture of the amplification well sack causes the amplification product to flow to the integrated detection element.

5. The microfluidic device of claim 4 , wherein the detection element is selected from fluorescent, electrochemical, or colorimetric based detection techniques.

6. The microfluidic device of claim 5 , wherein the detection element comprises a lateral flow strip.

7. 7. The microfluidic device of claim 1, wherein the amplification well comprises a lyophilized amplification mix and the at least one reagent pouch comprises a hydration buffer configured to hydrate the lyophilized amplification mix to generate an amplification master mix.

8. 8. The microfluidic device of claim 1, wherein the at least one amplification well comprises at least a first amplification well and a second amplification well, and rupturing the amplification well sack allows the amplification product to flow from the first amplification well to the second amplification well for further amplification.

9. 9. The microfluidic device of claim 1, wherein the at least one actuator element comprises one or more spatially oriented heater elements configured to provide stable, single-temperature heat or thermal cycling for isothermal or polymerase chain reaction (PCR)-based amplification of nucleic acids.

10. 10. The microfluidic device of claim 9, wherein the at least one actuator element comprises a first actuator element having the spatially oriented magnet and a second actuator element having the one or more spatially oriented heater elements, and the microfluidic cartridge is sandwiched between the first actuator element and the second actuator element.

11. The microfluidic device of claim 1 , wherein the microfluidic cartridge is configured for continuous rotation about a central axis, the continuous rotation being powered by a wind-up spring.

12. at least one inlet conduit; at least one waste well; the plurality of wells comprises at least one reagent well; 12. The microfluidic device of claim 1, wherein the inlet conduit, the reagent well, and the waste well are fluidly connected and configured such that when an actuation force is applied to the at least one reagent bag of the reagent dispensing unit, one or more reagents are supplied to the reagent wells through the inlet conduit and an interface exists between the sealing layer of the at least one reagent bag and the inlet conduit such that excess reagent overflowing from the reagent wells is collected in the waste well.

13. 13. The microfluidic device of claim 1, further comprising a locking mechanism configured to lock the at least one plunger in a depressed position, thereby preventing the one or more reagents from flowing back into the at least one reagent bag.

14. 14. The microfluidic device of claim 13, wherein the locking mechanism comprises a barbed pin in a locking hole configured to limit movement of the at least one plunger in a direction that tends to compress the at least one reagent bag upon application of an actuation force.

15. 15. The microfluidic device of claim 1, wherein the at least one reagent well comprises a plurality of reagent wells, and the microfluidic cartridge comprises a primary channel connecting the plurality of reagent wells.

16. 16. The microfluidic device of claim 1, comprising a plurality of plungers and a plurality of reagent bags, the plurality of plungers configured to simultaneously apply the actuation force to each of the plurality of reagent bags so as to simultaneously dispense the one or more reagents from the plurality of reagent bags.

17. 17. The microfluidic device of claim 1, comprising a plurality of plungers and a plurality of reagent bags, the plurality of plungers having spatially oriented protrusions of various depths that contact desired reagent bags from the plurality of reagent bags in a predetermined order to sequentially dispense the one or more reagents into the microfluidic cartridge.

18. 18. The microfluidic device of claim 1, further comprising a sample inlet port through which a reagent can be injected to be introduced into the microfluidic device.

19. 19. The microfluidic device of claim 1, wherein the microfluidic cartridge comprises one or more lyophilized or gelled reagents.

20. the reagent dispensing unit includes a plurality of reagent bags, each of which includes at least one reagent bag for distribution; the at least one reagent bag includes a reagent; Optionally, the at least one flow-through reagent bag is fluidly connected to a transfer reagent source that, upon activation, causes transfer reagent to flow into the flow-through reagent bag.

20. A microfluidic device according to any one of claims 1 to 19.

21. 21. The microfluidic device of claim 20, wherein the transfer reagent comprises an immiscible fluid that flows into the flow-through reagent bag upon activation of the transfer reagent source and pushes the reagent in the flow-through reagent bag so that the reagent flows into the reaction chamber.

22. 22. The microfluidic device of claim 1, comprising a single motor configured to power the microfluidic device and complete a sequence of analytical processes.

23. The microfluidic device described in Claim 20, wherein the flow-through reagent bag comprises a venting member including a sealing layer and a rupturing element, and the venting member and the rupturing element are configured to receive an actuation force that ruptures the sealing layer and allows the reagent to flow to a designated cartridge element.

24. A microfluidic device as described in claims 2 to 10, 15, and 23, wherein the predetermined cartridge element comprises the primary channel or the integrated detection element.

25. A microfluidic device as described in claims 2 to 10, 20, 23, and 24, wherein the amplification well bag is a flow-through reagent bag.

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