Automated point-of-care device for complex sample processing and method for using the same

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 by reference in its entirety.

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

[0003] Point-of-care (POC) devices allow for convenient and rapid testing at the site of patient care. Thus, 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 on 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 had issues with contamination control, especially in steps that involve human interaction during processing. Thus, there is a need to automate sample processing within the sample-to-answer LOC that minimizes human interaction. These sample-to-answer and LOCs are typically a few square millimeters to a few square centimeters in size and are often of the Microelectromechanical Systems (MEMS) type. Here, MEMS that can detect and analyze 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 these guidelines. One of these conditions is if the device or equipment meets certain risk, error, and complexity requirements. For a POC diagnostic test to be eligible for CLIA exemption, 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 bottles, syringes, or reservoirs filled with external reagents. These systems are not only difficult to transport, but are also complex due to the large number of parts 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 by the state of the art on the market, and thus have 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 the sample-to-answer sequence. Low-cost, low-power, miniaturized instrumentation to automate sample-to-answer sequences has not yet been realized, and thus point-of-care microfluidic devices to perform sample-to-answer sequences rely on additional instrumentation in the form of standalone benchtop or portable devices to perform the analysis on the microfluidic device. Implementing a separate instrumentation that can automate the sample processing steps on the 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 with solenoid plungers, linear actuators, microcontrollers, and electronics to automate the sample processing sequence. This instrumentation provides user control of the sample processing sequence, but 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 environments where laypeople do not need or can purchase expensive instruments for testing, or are not trained to operate the instruments that accompany the tests. Thus, 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 sequences is seen as an obstacle to developing single-use test devices that can perform complex multi-step 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 that include 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 a sample preparation step. 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 the way the test is performed. Furthermore, if the reagents are not entirely contained within the device, there is an additional risk of contamination. Some strong reagents that are harmful if handled without proper laboratory protocols, gloves and equipment (e.g. fume hoods and laboratory infrastructure such as contained biosafety equipment) cannot be performed with these kit tests unless the tests are performed by skilled technicians in a sealed facility.

[0007] When tests are automated rather than simple, non-experts perform the tests inaccurately. As the complexity of the tests 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 cations) reduce the burden of instrumentation for heating / cooling thermal cycling since these tests only need to be held at a single temperature (usually between 60-70°C). However, these tests still require multiple steps to be initiated by the user to complete the sample-to-answer sequence, which requires a skilled operator or additional automated instrumentation.

[0008] Sample preparation is essential for many diagnostic analyses, including the processing of biological samples. Biological samples generally 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, as well as to remove substances in the sample that may interfere with the desired analysis. Sample processing steps often include precise conditions for temperature, reagent volumes, and incubation times that must be performed in precise sequences and in tightly controlled environments, such as laboratory settings. Conventional automated systems for sample processing require highly complex and expensive instrumentation and skilled personnel to operate them. These systems are often located in centralized laboratories, so unprocessed samples often have to be properly stored at different locations and transported to the laboratory for processing. These factors are associated with several limitations, including high costs, delayed results, and reduced sample integrity due to transportation and improper storage.

[0009] US Patent Application Publication No. 2016 / 0136666, filed on July 25, 2016, relates to a sample processing device including magnetic and mechanical actuation elements using linear or rotational motion and a method for using the same. US Patent Application Publication No. 2016 / 0136666, filed on July 25, 2016, relates to a sample extraction device and a method for 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 instrument-free, non-laboratory settings. [Means for solving the problem]

[0012] In accordance with the present invention, various embodiments of a sampling device and methods of use thereof 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) on the microfluidic devices, etc. The present invention includes a portable analytical automation device and a microfluidic cartridge containing reagents stored in liquid and dry forms 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 associated processing methods for maximizing sample elution efficiency during transfer of the sample from a sample collection device (e.g., a swab, etc.) to a medium or buffer on a fluidic device, as well as during integration of the sample into a medium or buffer on the 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 description of the drawings]

[0016] Having described the subject matter of the present disclosure in general terms, reference is now 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 bag. [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] FIG. 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 a locking mechanism prior to actuation. [Figure 2B] FIG. 1 illustrates a cross-sectional view of an exemplary microfluidic device with an integrated RDU, including a plunger and a 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] 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. FIG. [Figure 4B] FIG. 1 is a schematic top view of the microfluidic device showing the microfluidic cartridge position prior to the magnetic bead-based sample preparation step. [Figure 4C]FIG. 13 is a top view of the microfluidic device showing the microfluidic cartridge position at the end of magnetic bead-based sample preparation, where the beads have been 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. 13 is a top view of a microfluidic device showing the position of the microfluidic cartridge at a moment prior to actuation of a sharp object by an upper actuation element to facilitate uptake of amplified products by the lateral flow strip for detection. [Figure 7A] FIG. 1 is a schematic diagram of an exemplary sampling device for extracting and processing a raw sample attached to a swab, showing different parts within the assembly. [Figure 7B] FIG. 1 is an assembled sample extraction device for processing an unprocessed sample attached to a swab, showing the swab rotating within a washing insert to facilitate mechanical washing 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 unprocessed sample attached to a swab, illustrating a step-by-step sequence for processing the elution from the swab prior to transfer to a microfluidic cartridge. [Figure 9A] ~ [Figure 9B] 1 is an illustration of an exemplary sample processing unit having a rotational actuator element, showing perspective and exploded views. [Figure 10A] ~ [Figure 10C] 13 shows an example of a sequence of movements as a rotary actuator element rotates relative to a reagent pallet in a sample processing unit. [Figure 11] FIG. 1 shows an exploded schematic view of a rotating shaft-based sample processing unit. [Figure 12] FIG. 2 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-B 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] FIG. 1 shows top and perspective views of an exemplary sample-to-answer microfluidic device for nucleic acid amplification testing (NAAT) with lateral induction-based readout. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The subject matter of the present disclosure 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 subject matter of the present disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the subject matter disclosed herein 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 subject matter of the present disclosure is not to be limited to the specific 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 FOR USE THEREOF 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, which is the volume retained in a reagent pouch or fluid conduit / channel, can greatly impede the reproducibility and reliability of the analysis performed on the cartridge. In particular, for analytical steps that depend on high pipetting accuracy to be performed successfully, such as amplification steps where even small changes in the system's volume can greatly affect the concentration of the reagent and the performance of the analysis, and steps that require precise pH control, it is essential to have a metering system that can deliver precise volumes of reagent to the desired reaction chamber. 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, but this type of system requires precisely shaped metering chambers to control the accuracy of reagent dispensing on the microfluidic device. In addition, systems that use metering chambers can be more susceptible to air bubbles in the system that can affect the reliability of fluid dispensing. Such additional debubbling mechanisms can require pumps and valves that add 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. The system efficiently delivers all the aqueous reagents necessary to accurately perform a microfluidic cartridge-based assay, 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 to press on the pouches to rupture a frangible seal layer on the pouch and expel their contents when sufficient actuation force is applied. In some embodiments, the RDU may include a sharp object or protrusion capable of rupturing the frangible seal on the RDU when sufficient actuation force is applied thereto. The sharp object or protrusion may be located within the pouch or adjacent to the frangible seal of the RDU such that when an actuation force is applied, 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 aqueous reagents 103 and non-aqueous immiscible reagents 102 in a single reagent pouch that are sealed with a frangible seal layer 104 that can be broken upon actuation to allow 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 that is used to squeeze the reagent pouch and rupture the frangible seal layer 104 assembled on the microfluidic device. The RDU is assembled on the microfluidic device 108 such that the frangible seal 104 is at the interface of an inlet conduit 107 into a fluidic reagent well 109 on the microfluidic device. The microfluidic device includes one or more reagent wells 109 and a waste well 110 that aids in the collection of excess immiscible reagent 102 that overflows from the reagent wells 109.

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

[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 to rupture the frangible seal 104 and fluidly connect 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 flows first from the inlet conduit and into the fluid well, followed by the immiscible fluid 102, which acts to effectively push out any aqueous reagent that would otherwise occupy dead volume space in the fluid conduit and the RDU. The excess immiscible non-aqueous fluid 102 overflows the reagent well and is collected in the waste well 110. Using this system, precise amounts of aqueous reagents 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 to prevent evaporation of the aqueous reagent during heating steps such as thermal cycling or thermal incubation. This type of system greatly reduces the complexity of the system since it does not require the use of complex valves or pumps for proper operation. In addition, the precise shaping of the reaction chambers that is typically required to meter precise volumes of reagent is not required since the system does not depend on the volume of the reaction chamber to meter precise volumes. Rather, aqueous reagents that are pre-loaded into the RDU using well-known precision pipetting processes with immiscible non-aqueous fluids are delivered completely to the desired fluid wells upon actuation because there is water-immiscible fluid to completely push out the aqueous reagent and occupy all 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 it does not require precise actuation control on the instrument to ensure run-to-run reproducibility with aqueous reagent delivery from single-dose reagent packs, because once all aqueous reagents are loaded into the device, the immiscible non-aqueous fluids will overflow, pushing the aqueous reagents out to occupy all dead space and ensure delivery to the desired fluidic wells, ensuring that all aqueous reagents are pushed out of the RDU and into the microfluidic system during run.

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

[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 a fixed position with a barbed pin 203 captured within a locking hole 208 and oriented to restrict the movement of the plunger 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 disposed on the microfluidic device. The reagent bag includes a frangible seal layer 205 that is broken when a sufficient actuation force is applied to the plunger, as shown in FIG. 2B. Upon actuation, the frangible seal 205 is broken 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 pouch.

[0028] One aspect of the invention is a microfluidic device that includes 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) that contain stored liquid reagents that are separated from an inlet into the fluid wells by a frangible seal. The reagent pouches may be filled with an aqueous fluid, a water-immiscible fluid, or a combination thereof. Upon actuation, the frangible seal is broken and the contents of the reagent dispensing unit 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 a primary channel filled with a non-aqueous fluid.

[0029] For a care environment, a self-contained system is advantageous since 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. The 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 bursts, 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 when the pouch bursts, its contents are forced into the fluid conduit, filling the fluid well.

[0030] Each fluid well volume is designed to be only partially filled with miscible liquid reagents so that the miscible liquids in each fluid well do not overflow and mix with each other through the upper fluid conduit of each fluid well. 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 to form an immiscible oil phase on top of the aqueous reagent filled in the fluid well. 2) All the 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 avoid mixing with each other. The primary fluid conduit exits into a waste well to collect the excess oil.

[0031] Although it is possible to pre-fill the fluid wells with buffers separated by an oil phase and 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 over extended periods of time and therefore need to be stored in lyophilized form and hydrated prior to use. Additionally, there are challenges with introducing samples into such pre-filled systems. The disclosed invention provides methods and devices that solve the challenges associated with sample introduction, reagent delivery and analysis 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] The sample-to-answer microfluidic cartridge includes one or more reagent wells 309 connected to each other via a primary fluidic channel 305. The RDU assembled to the microfluidic cartridge includes multiple reagent bags 302 separated from the inlet conduits to the fluidic 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 reagents into the reagent bags. In this embodiment, the plunger element 303 is designed to contact all of the reagent bags at the same instant, depressing and releasing all of the individual reagents from the reagent bags in parallel from a single actuation step. In other embodiments, the plunger element may include spatially oriented protrusions with various depths to contact the desired reagent bags in a preferred sequence as the plunger is depressed to facilitate sequential delivery of reagents to the microfluidic cartridge. Upon actuation of the RDU, the reagent wells are filled with aqueous reagents and the fluidic circuit between the reagent wells is completed through the primary fluidic 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 fluidic channel 305. To perform NAAT, the reagent bag 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, etc.).

[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 may be hydrated during use by either the sample or the dispensed buffer. The cartridge includes a sample inlet port to transfer the sample into 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, including but 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, making it susceptible to deformation upon actuation such that the frangible seal layer breaks and the amplified product is squeezed 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, in a single actuation step involving rotating the microfluidic cartridge between the actuator elements, the spatially oriented magnets capture, resuspend, and transport the magnetic beads between different reagent wells to transfer a sample preparation sequence (e.g., binding, washing, elution sequences on the sample, etc.) to the microfluidic cartridge. In the exemplary embodiment shown in FIG. 3B, the top actuator element includes a protrusion 316 designed to contact the microfluidic cartridge at a predetermined time in the analysis sequence to actuate a sharp object in a pouch 307 thereon, rupturing the frangible seal layer and introducing the amplified products into the lateral flow piece 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 cycle time.

[0039] Sample-to-answer NAAT An exemplary sample-to-answer NAAT is described herein as a microfluidic analytical automation platform that uses a single actuator to provide rotational motion, such as a servo or stepper motor, a wind-up spring, a hand crank, or actuation by a user's finger. The wind-up spring mechanism provides the ability to automate the analysis 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 the operation of the system. 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 the nucleic acids are eluted from the surface without the need for time-consuming precipitation steps. A unique advantage is that the Chargeswitch technology uses aqueous buffers and does not require the use of ethanol, chaotropic salts, or organic solvents that can inhibit downstream applications such as amplification.

[0041] Magnetic beads are a highly effective and simple solid-phase capture support 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 many samples need to be purified quickly. Magnetic beads coated with ionizable (switchable) coatings 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 a 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 which contains the actuator elements, motor, electronics and display. 3. Close the instrument lid and begin 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. By way of 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. The cells in the raw sample are lysed. The lid of the device is then closed. In this embodiment, closing the lid distributes the actuation force of the plunger 303 and the reagents from the 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 smooth rotation of the microfluidic cartridge between the actuator elements.

[0044] Referring to FIG. 4A, an upper view of a microfluidic device showing the position of the microfluidic cartridge after actuating 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 capture the beads in the desired wells. The beads captured on the upper 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 into 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 upper view of the microfluidic device showing 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. In isothermal amplification reactions, which require incubation at a single temperature, no additional heating elements are utilized and a single heater element can deliver the thermal energy for amplification. In 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 cycle time.

[0046] Referring to FIG. 5, a schematic top view of the microfluidic device (top actuator element not shown) is shown with the amplification well 404 positioned above a heater element set at temperature T1 in FIG. 5A, T2 in FIG. 5B, and T3 in FIG. 5C. This shows how a sample-to-answer NAAT with rapid thermal cycling is achieved by switching / actuating the microfluidic cartridge in a precise timed sequence using three fixed thermal zones on the actuator element and also using a single motor that is used to run the entire analysis 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 that includes 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 products is performed colorimetrically on the integrated lateral flow strip. The lateral flow strip is separated from the amplification wells containing the amplified products by a frangible seal layer that is coupled to a pouch containing a sharp object 307 such that upon actuation, the frangible seal can be ruptured to deliver the amplified products to the lateral flow strip 308 for detection. In some embodiments, the amplification wells themselves may contain a frangible layer, susceptible to deformation upon actuation such that the frangible seal layer is broken and the amplified products are squeezed onto the lateral flow strip.

[0048] Referring to Figure 6, a top view of the microfluidic device at the time of reaching the position of the lateral flow detection step is shown. A is a close-up 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 housing 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 piece.

[0049] Sample Collection and Extraction Equipment Swabs are primarily used as biological sample collection devices. Swabs, such as COPAN FLOQSwabs™, are manipulated so that the entire sample remains close to 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 out and further processed depending on the type of analysis.

[0050] In point-of-care (POC) and low-resource environments, vortexing samples is not a convenient method to elute the sample in a liquid medium, and manual shaking or stirring can lead to operator variability. Furthermore, because the swab is absorbent, a finite amount of sample in solution remains on the swab, resulting in loss. If the analyte is present in a very low concentration, an insufficient amount of 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 a user to deliver multiple reagents directly to a sample in a sample extraction device using a simple user-activated process. The disclosed invention can greatly 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, a schematic diagram of an exemplary sample extraction device for extracting and processing an unprocessed sample attached to a swab is shown. The sample extraction device, in some embodiments, comprises 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 lid 702 of the swab. When the swab is inserted into the container 705, the swab contacts a washing insert 715, 716 having one or more protrusions 713 that contact the swab head 704, washing the head when the lid is closed and the swab rotates / pivots within the insert. This washing 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 number of small bristles 713 that are spatially oriented to contact the swab head when spatially 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, which dictates the number of turns or full revolutions that the swab head 704 makes within the cleaning insert, can be optimized to maximize sample recovery from the swab.

[0054] Similarly, the type and design of mechanical elements can be optimized for the 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 a plunger 708 and a plunger tip 709. The syringe may include one or more grooved recesses 710 that may contain stored reagents in dry, liquid capsules or pelletized 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 pushed 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 buffers, neutralization buffers, binding buffers, wash buffers, pH control buffers, 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 recovering an unprocessed sample attached to a swab prior to transfer to a microfluidic cartridge.

[0057] Step 1- Insert the swab sample into the container and rotate 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 an exemplary embodiment, the container 705 is pre-filled with a suitable swab transport medium, such as phosphate buffered saline (PBS), Amies medium, etc. The swab is inserted into the container and the lid is rotated "n" times to close (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, and as the swab rotates in 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 in the container. Then, by withdrawing the plunger on the attached syringe sample processing unit, the sample from the container is filtered through the 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, dry, liquid, or gel, present in the barrel in a continuous manner.

[0059] In an exemplary embodiment for performing NAAT, the stored dry reagents include pellets of dried lysis buffer that are 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 reagent and dried neutralization buffer reagent, which are sequentially introduced to the sample such that cells in the sample are first lysed and then the lysate is neutralized by introduction to 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 preloaded 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 sample extraction device can be used for 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, 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 such 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 acid 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 through to the next stage into the sample processing unit, where the purified lysate is neutralized by the neutralizing reagent present therein.

[0065] Sample Processing Unit 9A and 9B, a perspective view and an exploded view, 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 in the container 902 in a precisely timed predetermined sequence. FIG. 9B is a schematic exploded view of an exemplary sample processing unit showing 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 that contain 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 container 902 in a precisely timed sequence when the rotary actuator element 907 rotates in proximity to the reagent pallet 905. In some embodiments, the reagent is guided from the reagent dispensing conduit 908 into the sample collection container 902. In some embodiments, the reagent is 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 wind-up spring, a motor, etc. In some implementations, 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 the kitchen egg timer. These mechanisms generate a steady rotational motion until fully uncoiled. Wind-up spring mechanisms can be designed to fully uncoil in a fixed amount of time by appropriate selection of the spring and gear mechanism used. Sequential reagent delivery can be powered by a wind-up spring mechanism that actuates an actuator to deliver reagents to the system in a precise timed sequence. In the present invention, the rotating actuating element includes a spatially oriented mechanical element that interacts with a reagent-filled bag on a reagent pallet at a given instance along the rotational path of the rotating actuating element, thereby deforming and squeezing the reagent bag and thereby delivering the reagent in a precise predetermined 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 the reagents required 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 reduces the risk of erroneous results due to user-generated errors by eliminating complex and time-consuming pipetting steps and enabling reagent delivery using simple and universal twist, slide or rotate movements that do not require a skilled operator to perform, and can 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 motion sequence is shown as a rotating actuator element 907 rotates relative to a reagent pallet 905. FIG. 10A shows the position of the rotating actuator element before any reagent delivery has occurred. In FIG. 10B, the rotating actuator element has moved to a position where a mechanical element 906 present 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 rotating actuator element has moved to a position along the 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] The exemplary embodiment depicted in Figure 9 utilizes rotational motion to perform the 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 such that they can sequentially interface with different spatial dimensions of a sample processing device or microfluidic cartridge.

[0070] 11, an exploded schematic diagram 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 analysis 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 to dispense their contents in a predetermined sequence.

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

[0072] Each reagent may be packaged in an individual reagent pouch and assembled onto the microfluidic cartridge, however, 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 is preferable to create a reagent card that includes 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 showing individual reagent pouches 1202 and a flow-through reagent pouch 1203 is shown. The reagent card may be designed with a shape that easily mates and aligns with a mating groove on the cartridge during assembly. The reagent card may be filled manually or using multiple automated pipetters 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 features molded into 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, either individually, in a sequential manner, or in parallel with 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 with each other. In the past, mixing has been done in microfluidic devices using active mixers, where external energy is applied to agitate the fluids, or passive mixers, where specially designed geometries and channel configurations are used to increase the contact area and contact time of the mixing fluids. In some embodiments, it may be necessary to mix two or more reagents. For example, it may be necessary for at least one reagent to be in solid form, or to contain solid particles in a low or high viscosity liquid medium, or to 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 liquid reagents, 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 with a known volume of reconstitution liquid. In some cases, it is desirable to dry or lyophilize the reagents and store them directly in the reaction chamber of the 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 potency or even destroying them permanently. 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 non-functional when dried. It is therefore important to store the beads in a liquid matrix to maintain their functionality.Although it may be possible to develop a custom drying process using custom chemistry that helps preserve the functional coating on the beads, developing a custom process is often expensive and requires extensive testing to ensure there is no loss of functionality. Therefore, storing functionalized particles such as magnetic beads in a liquid matrix is ​​preferred. However, on-chip storage of magnetic beads in liquid form presents many challenges of its own. Specifically, the magnetic particles are stored in the liquid matrix at very high concentrations (often in the range of 5mg / ml to 50mg / ml) and then diluted with sample and buffer to meet the binding capacity requirements. Manufacturers have provided protocols that require very small amounts of magnetic bead reagent, typically in the range of 10μl to 40μl per test, but due to manufacturing process limitations (minimum volume of 50μl), it is difficult to package in a foil-sealed reagent pouch 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 750μm×750μm and a length of 1 inch has a dead volume of 15μl. 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 reagents by being trapped in dead spaces is undesirable. The invention in this disclosure uses a flow-through based approach to facilitate efficient transfer and mixing of reagents on a microfluidic device. The flow-through system utilizes a fluid medium, either liquid or gas, present in large quantities used 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 all of the 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 effect of dead volumes in the reagent bag or the microfluidic device by filling them with transfer reagent. Since the volume of 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 bag. In some embodiments, the transfer medium may be the liquid sample being analyzed. During the interaction of the transfer medium with the contents of the flow-through reagent bag, 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, decreasing the channel cross section, adding physical barriers to impede the flow rate, increasing the fluid pressure, and creating turbulence in the fluid flow are few of the approaches 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 the particles / beads to facilitate binding of analytes present in solution to the functionalized particles / beads.

[0077] Referring to FIG. 13A, a cross-sectional schematic of an exemplary microfluidic device is shown with a reagent card including 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 an inlet of the flow-through reagent bag via a transfer fluid conduit 1308 on the microfluidic device, which is capped with a frangible seal 1304. The flow-through reagent bag includes a rupture element (ball) 1305, which is connected to a reaction chamber on the microfluidic device through an outlet fluid conduit 1309. As shown in FIG. 13B, when an actuation force is applied, 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 on the microfluidic device rather than inside the flow-through reagent bag.

[0078] Working 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, the fluid conduit, and the crushed transfer reagent bag) of 60 μl, the transfer reagent bag contains 360 μl of binding buffer to offset losses due to dead volume. This dead volume is inherent to the design of the microfluidic device and can be easily calculated from the geometry of the device and confirmed using experimental methods. When an actuation force is applied, the frangible seal is ruptured so that the binding buffer enters 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 the outlet fluid conduit 1309 to complete the binding protocol.

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

[0080] Oil / Immiscible Phase Dispensing System In some embodiments, an oil-filled reagent bag can be used to store the dispensable oil phase upon application of the actuation force. However, reagent bags are very difficult to manufacture and fill, and difficult to seal without dead air zones. Typical manufacturing tolerances for dead air in the bag can be on the order of 10% to 20% of the total bag volume. Especially with viscous oil phase reagents, trapped air can generate bubbles in the oil phase, which leads to reproducibility issues and problems with the movement of magnetic particles in the immiscible oil phase or between miscible water-soluble oil phase and immiscible oil phase. In addition, the flow of the oil phase from the reagent bag during dispensing can be turbulent, forming air pockets in the microfluidic device as the oil phase fills each reaction well and the primary channel. Furthermore, another unique embodiment herein describes preventing the formation of air bubbles in 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 so that liquid does not escape. In this unique embodiment, smooth laminar flow is generated by utilizing the pressure head of the oil phase. This can be supplemented 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 be able to generate fluid flow by bursting the frangible seal during dispensing. Furthermore, the presence of air in the oil phase container is not affected since air is too light to displace the oil phase.

[0081] 14, an exemplary microfluidic device showing an oil / immiscible phase dispensing system 1401 is shown before application of an actuation force in FIG. 14A and after application of an actuation force in FIG. 14B. 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 that contains 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 that 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 outlet are fractured and the fracture ball 1408 pierces the frangible seals, connecting the air vent and oil / reagent outlet to the vent hole on the microfluidic device and the reaction chamber 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 piece 1505 assembled on a microfluidic chip. The microfluidic chip itself includes multiple reaction chambers 1502 connected to each other by primary channels 1503. Inlet channels 1504 connect the reagent bags 1303 on the reagent card to the individual reaction chambers. An actuation element on the lid of the device with a plunger having a spatial topography that matches the spatial location of the individual reagent bags and the deformable lid element 1407 on the oil dispensing system 1401 is used to provide the actuation force to rupture the frangible seal.

[0083] A typical sequence of operations would be 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 containing covalently linked subunits called nucleotides. A "nucleotide" is a molecule or individual unit within a larger nucleic acid molecule that contains 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 may be of any length, including RNA, DNA, or RNA / DNA hybrid sequences. Polynucleotides for use in the present invention may be naturally occurring, synthetic, recombinant, ex vivo produced, or combinations thereof, and may 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 thus has undergone molecular biological manipulation from its natural 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", "upper", "lower" and "upper" 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 convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to a "subject" includes a plurality of subjects unless the context clearly indicates the contrary (e.g., a plurality of subjects).

[0091] Throughout this specification and claims, the term "comprising" is used in a non-exclusive sense, unless the context dictates otherwise. Similarly, the term "including" 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 the appended claims are to be understood as being modified in all instances by the term "about," even if the value, amount, or range is not expressly stated. Thus, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are not precise, and need not be precise, but can be approximate and / or larger or smaller, as desired, to reflect tolerances, conversion factors, rounding, measurement errors, 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, in some embodiments, 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 conjunction 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., the 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 incorporated by reference herein to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. Although a number of 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. [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. 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 sequence corresponding to steps of 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 is 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. 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 when an actuation force is applied to the amplification well bag to dispense an amplification product contained within the amplification well.

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 bag at a predetermined time in an analysis sequence to actuate at least one of the sharp objects or protrusions to rupture the sealing layer.

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 bag sealing layer, and rupture of the amplification well bag 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 technologies.

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

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

8. A microfluidic device as described in any one of claims 1 to 7, wherein the at least one amplification well comprises at least a first amplification well and a second amplification well, and rupture of the amplification well bag allows the amplification product to flow from the first amplification well to the second amplification well for further amplification.

9. A microfluidic device as described in any one of claims 1 to 8, wherein 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. 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 to rotate continuously about a central axis, the continuous rotation being powered by a take-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 via 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. A microfluidic device as described in any one of claims 1 to 12, 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. The microfluidic device of claim 13, wherein the locking mechanism comprises a hooked pin in a locking hole configured to limit movement of the at least one plunger in a direction that facilitates pressing against the at least one reagent bag upon application of an actuation force.

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. A microfluidic device as described in any one of claims 1 to 15, comprising a plurality of plungers and a plurality of reagent bags, the plurality of plungers being 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. A microfluidic device as described in any one of claims 1 to 16, comprising a plurality of plungers and a plurality of reagent bags, the plurality of plungers having spatially oriented protrusions of various depths for contacting desired reagent bags among the plurality of reagent bags in a predetermined sequence and sequentially dispensing the one or more reagents into the microfluidic cartridge.

18. A microfluidic device as described in any one of claims 1 to 17, further comprising a sample inlet port through which a reagent to be introduced into the microfluidic device can be injected.

19. A microfluidic device as claimed in any one of claims 1 to 18, wherein the microfluidic cartridge comprises one or more freeze-dried or gelled reagents.

20. The reagent dispensing unit includes a plurality of reagent bags including at least one flow-through reagent bag; the at least one flow-through reagent bag comprises a reagent, a sealing layer, and a rupture element configured to rupture the sealing layer; 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. The microfluidic device of claim 20, wherein the transfer reagent comprises an immiscible fluid that flows into the flow reagent bag upon activation of the transfer reagent source, pushing the reagent in the flow reagent bag out and causing the reagent to flow into the reaction chamber.

22. A microfluidic device as described in any one of claims 1 to 21, comprising a single motor configured to power the microfluidic device and complete a sequence of analytical processes.

23. A microfluidic device comprising: A reagent dispensing unit comprising at least one reagent bag having one or more reagents and a sealing layer, the at least one reagent bag comprises at least one first non-compartmentalized reagent bag, the at least one first non-compartmentalized reagent bag comprising an aqueous reagent and a non-aqueous immiscible reagent sealed together in the first non-compartmentalized reagent bag; The reagent dispensing unit; At least one plunger; at least one sharp object or protrusion configured to rupture the sealing layer; at least one actuator element comprising a spatially oriented magnet; a microfluidic cartridge comprising a plurality of wells, the microfluidic cartridge being disposed proximate to the at least one actuator element and configured to receive magnetic particles and to rotate about a central axis; A microfluidic device comprising:

24. The microfluidic device of claim 23, wherein the at least one actuator element is a single actuator element.

25. A microfluidic device as described in claim 23 or 24, wherein the microfluidic cartridge is configured such that the multiple spatially oriented magnets capture, resuspend and transport the magnetic beads through the microfluidic cartridge in a single operating step that includes rotating the microfluidic cartridge.

26. The microfluidic device of claim 23, wherein the at least one actuator element comprises a plurality of mechanical elements configured to contact the microfluidic cartridge at a predetermined time in a test sequence and actuate the sharp object or protrusion in the reagent bag to rupture the sealing layer.

27. ​​The microfluidic device of claim 23, wherein the at least one actuator element further comprises one or more spatially oriented heating elements configured to provide constant, single-temperature heat or thermal cycling for isothermal or polymerase chain reaction (PCR)-based nucleic acid amplification.

28. The microfluidic device of claim 27, wherein the at least one actuator element comprises a plurality of spatially oriented heater elements, each set to a constant single temperature, and the amplification well contacts or is in proximity to a desired heating element for a desired cycle time.

29. The reagent dispensing unit includes a plurality of reagent bags including at least one flow-through reagent bag; the at least one flow-through reagent bag comprises a reagent, a sealing layer, and a rupture element configured to rupture the sealing layer; 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; 29. A microfluidic device according to any one of claims 23 to 28.

30. The microfluidic device of claim 29, wherein the transfer reagent comprises an immiscible fluid that flows into the flow reagent bag upon activation of the transfer reagent source, pushing the reagent in the flow reagent bag out and causing the reagent to flow into the reaction chamber.

31. A microfluidic device as described in any one of claims 23 to 30, comprising a single motor configured to power the microfluidic device and complete a sequence of analytical processes.

32. A microfluidic device comprising: a microfluidic cartridge comprising a reaction chamber; a reagent dispensing unit comprising at least one flow-through reagent bag, the at least one flow-through reagent bag comprising a reagent, a sealing layer, and a rupture element configured to rupture the sealing layer; 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; Microfluidic devices.

33. The microfluidic device of claim 32, wherein the transfer reagent comprises an immiscible fluid that flows into the flow reagent bag upon activation of the transfer reagent source, pushing the reagent in the flow reagent bag out and causing the reagent to flow into the reaction chamber.

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