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

Abstract

The present invention provides a method and apparatus for the simple, low-power, and automated processing of biological samples through multiple sample preparation and analysis steps. [Solution] A microfluidic apparatus comprising a reagent dispensing unit, the reagent dispensing unit comprising at least one reagent bag having one or more reagents and a frangible seal layer, and at least one plunger and at least one sharp object or projection configured to rupture the frangible seal layer and deliver one or more reagents to the microfluidic apparatus when an operating force is applied to the reagent dispensing unit. The present invention includes a microfluidic apparatus comprising a reagent dispensing unit, a sample extraction device and a sample processing unit.

Description

Technical Field

[0004] ,

[0003] , ,

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 428,976, filed on December 1, 2016, the entire content of which is incorporated herein by reference.

[0002] The present invention relates to an automated point - of - care device for complex sample processing and a method of using the same.

Background Art

[0003] Point - of - care (POC) devices enable convenient and rapid testing at the patient care site. Therefore, sample - to - answer and lab - on - a - chip (LOC) systems, which are types of POC devices integrating microfluidics technology, have been increasingly popular. These LOCs integrate various laboratory functions such as extraction, amplification, detection, interpretation, and reporting, which were previously performed manually and / or off - site, all on the same device. Since sample - to - answer and LOC tests are performed at the patient care site rather than in a laboratory facility, these types of tests have had problems regarding contamination control, especially in processes involving human interaction during processing. Therefore, there is a need to automate sample processing within sample - to - answer LOCs that minimize human interaction. These sample - to - answer and LOCs are generally on the order of a few square millimeters to a few square centimeters in size and are often of the micro - electro - mechanical systems (MEMS) type. Here, MEMS that can detect and analyze biological substances are generally called Bio - MEMS.

[0004] Most point-of-care (POC) diagnostic devices on the market are classified as highly or moderately complex under the Clinical Laboratory Improvement Amendments (CLIA). These federal guidelines generally apply to human clinical laboratory testing devices, with the exception of certain conditions that exempt them. One of these conditions is that the device or instrument meets specific risk, error, and complexity requirements. To qualify a POC diagnostic test for CLIA exemption, sample preparation and fluid handling steps must be minimized. One way to minimize these steps is to store reagents in sealed devices such as openable blister or burst bags. Reagent delivery to microfluidic chips 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 complex not only because they are difficult to carry, but also because they require numerous components that must be integrated and a leak-free fluid interface to the microfluidic chip. Modern technologies on the market have yet to successfully implement a method for easily automating fluid handling in a compact and low-power manner. Consequently, this has been seen as an obstacle to conducting proof-of-concept (POC) trials in the vast majority of multi-stage biological assays still performed in large clinical settings.

[0005] Complex biological assays requiring multiple processing steps, including but not limited to pipetting, heating, cooling, mixing, washing, culturing, labeling, binding, and elution, rely on expensive laboratory automation equipment to perform sample-to-answer sequencing. Low-cost, low-power, and miniaturized instrumentation for automating sample-to-answer sequencing has not yet been realized, and therefore, point-of-care microfluidic systems for performing sample-to-answer sequencing rely on additional instrumentation in the form of standalone benchtop or portable devices for performing analysis on the microfluidic system. Implementing separate instrumentation capable of automating the sample preparation process on microfluidic cartridges has been considered a way to keep the cost per test, and therefore the cost of the cartridge, low. Systems developed for point-of-care applications can take the form of portable benchtop devices equipped with solenoid plungers, linear actuators, microcontrollers, and electronic circuits to automate the sample preparation sequence. This instrumentation configuration allows 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 instruments does not exist, or in non-hospital settings such as homes where laypeople do not need, cannot afford, or are not trained to operate instruments involving testing. Therefore, the development of methods to enable low-power, standalone, inexpensive, and disposable instrumentation configurations that can be directly integrated onto microfluidic systems and perform automated sample-to-answer sequences is considered an obstacle to the development of single-use test devices capable of performing complex multi-step nucleic acid, protein, and immunological assays from sample-to-answer.

[0006] Disposable tests that do not require an instrumentation setup to perform them are limited to simple single-step and multi-step analyses. In simple single-step analyses, the sample is a single liquid, and no reagents are used. These tests typically include dipstick tests such as urine strips and pregnancy tests. Multi-step analyses are sold in the form of kits that include reagent vials and indicator sets, allowing the user to dispense reagents into different areas of the disposable test cartridge according to the instructions. These devices typically perform immunological assays 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 the necessary skills or the test is not performed as instructed, there is a risk of the test being performed inaccurately, and therefore, the results may vary depending on how the test is performed. Furthermore, there is an additional risk of contamination if the reagent is not completely contained within the device. Some strong reagents that are hazardous if handled without appropriate laboratory protocols, gloves, and equipment (e.g., fume hoods and laboratory infrastructure such as biosafety equipment) cannot be used with these kit tests unless they are performed by skilled technicians in a sealed facility.

[0007] When tests are not simple and are automated, non-experts may 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 analysis (e.g., isothermal analysis of loop-amplified cations) have reduced the burden of equipment setup for heating / cooling thermal cycles, as these tests only require being held at a single temperature (typically between 60 and 70°C). However, these tests still require multiple steps initiated by the user to complete sample-to-answer sequencing, which may necessitate a skilled operator or additional automated equipment setup.

[0008] Sample preparation is essential for many diagnostic analyses, including the processing of biological samples. Biological samples generally require several complex processing steps before they are suitable for use in analysis. These steps are necessary to separate, concentrate, and / or purify the analyte of interest from the raw sample, and to remove substances in the sample that may interfere with the desired analysis. Sample processing steps often involve precise conditions regarding temperature, reagent volume, and incubation time, which must be carried out in a strictly controlled environment, including accurate sequencing and laboratory settings. Conventional automated systems for sample processing require highly complex and expensive equipment configurations and skilled personnel to operate them. Because these systems are often located in centralized laboratories, raw samples often have to be properly stored in different locations and transferred to the laboratory for processing. These factors result in several limitations, including high costs, delayed results, and a decrease in sample integrity due to transportation and improper storage.

[0009] Patent Document 1, filed on July 25, 2016, relates to a sample processing apparatus including magnetic and mechanical operating elements using linear or rotational motion, and a method of using the same. Patent Document 2, filed on July 25, 2016, relates to a sample extraction apparatus and a method of using the same. The entire contents of both of these applications are incorporated herein by reference. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Patent Application PCT / US16 / 43911 [Patent Document 2] International Patent Application PCT / US16 / 43855 [Overview of the project] [Problems that the invention aims to solve]

[0011] This invention provides a method and apparatus for simply, low-power, and automated processing of biological samples through multiple sample preparation and analysis steps. The described method and apparatus facilitate the implementation of point-of-care for complex diagnostic analyses in non-laboratory settings where equipment is unavailable. [Means for solving the problem]

[0012] According to the present invention, various embodiments of a sample extraction apparatus and a method of using the same are disclosed.

[0013] The present invention discloses analytical automation devices and methods for performing automated analyses, such as sample-to-answer microfluidic devices and nucleic acid amplification tests (NAAT) on microfluidic devices. The present invention includes portable analytical automation devices and microfluidic cartridges that contain reagents stored in liquid and dry forms, which are dispensed in a predefined order for performing sample-to-answer NAAT.

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

[0015] Some aspects of the subject matter disclosed herein, which are referred to in whole or in part by the subject matter disclosed herein, will become apparent as the description progresses in connection with the appended examples and drawings, which are described in the most detail below. [Brief explanation of the drawing]

[0016] The subject matter of this disclosure has been described using general terminology; hereafter, refer to the attached drawings. However, the drawings are not necessarily drawn to scale. [Figure 1A] This is an example cross-sectional view of a filled reagent bag. [Figure 1B] This is a cross-sectional view of an exemplary microfluidic apparatus having an integrated reagent dispensing unit (RDU) showing the RDU before operation. [Figure 1C] This is a cross-sectional view of an exemplary microfluidic apparatus having an integrated reagent dispensing unit (RDU) that shows RDU after operation. [Figure 2A] This is a cross-sectional view of an exemplary microfluidic device having an integrated RDU, including a plunger and locking mechanism before operation. [Figure 2B] This is a cross-sectional view of an exemplary microfluidic device having an integrated RDU, including the plunger and locking mechanism after operation. [Figure 3A] This is a perspective view of an exemplary sample-to-answer microfluidic cartridge for performing nucleic acid amplification testing (NAAT). [Figure 3B] This is a schematic exploded view of a microfluidic device, including a microfluidic cartridge that rotates between an upper actuator element and a lower actuator element. [Figure 4A] This is a schematic top view of the microfluidic apparatus showing the position of the microfluidic cartridge relative to the actuator element after the RDU is activated. [Figure 4B] This is a schematic top view of a microfluidic apparatus showing the position of the microfluidic cartridge before the sample preparation stage, which is based on magnetic beads. [Figure 4C]It is a top view of a microfluidic device showing the position of a microfluidic cartridge at the end of sample preparation based on magnetic beads when the beads are transported into the amplification wells. [Figure 5A] to [Figure 5C] It is a schematic top view of a microfluidic device showing amplification wells on three separate heating elements on a bottom actuator element to facilitate rapid thermal cycling by rotational position control, having different temperature zones T1, T2 and T3. [Figure 6] It is a top view of a microfluidic device showing the position of a microfluidic cartridge at the moment before the actuation of a sharp object by an upper actuating element to facilitate the suction of amplified products by a lateral flow piece for detection. [Figure 7A] It is a schematic diagram of an exemplary sample extraction device for extracting and processing an untreated sample attached to a swab, showing different parts within the assembly. [Figure 7B] It is an assembled sample extraction device for processing an untreated sample attached to a swab, showing a swab rotating within a cleaning insert to facilitate mechanical cleaning and squeezing of the swab head to maximize sample elution from the swab. [Figure 8] It is a diagram showing a step - by - step sequence of processing elution from a swab before transferring an exemplary sample processing protocol for recovering an untreated sample attached to the swab to a microfluidic cartridge. [Figure 9A] to [Figure 9B] It is an explanatory diagram of an exemplary sample processing unit having a rotary actuator element, showing a perspective view and an exploded view. [Figure 10A] to [Figure 10C] It shows an example of an operation sequence when a rotary actuator element rotates with respect to a reagent pallet within a sample processing unit. [Figure 11] It shows an exploded schematic view of a sample processing unit based on a rotary shaft. [Figure 12] It is a perspective view of an exemplary reagent bag card. [Figure 13]This is a schematic cross-sectional view of an exemplary microfluidic apparatus including a reagent card containing moving reagent bags and flowing reagent bags before and after the application of an operating force. [Figure 14A] ~ [Figure 14B] This is a cross-sectional view of an exemplary microfluidic device showing an oil / immiscible phase dispensing system before (Figure 14A) and after (Figure 14B) the application of activating force. [Figure 15] These are a top view and perspective view of an exemplary sample-to-answer microfluidic apparatus for nucleic acid amplification testing (NAAT) with lateral lead-based readout. [Modes for carrying out the invention]

[0017] The subject matter of this disclosure is described in more detail below with reference to the accompanying drawings, which show only a portion of the disclosed subject matter and not all embodiments. Similar figures refer to similar elements throughout. The subject matter of this disclosure may be carried out in many different forms and should not be construed as being limited to the embodiments described herein, but rather these embodiments are provided so that this disclosure satisfies applicable legal requirements. In fact, many modifications and other embodiments of the subject matter disclosed herein will be recalled by those skilled in the art who are interested in the teachings presented in the above description and the accompanying drawings. Accordingly, it should be understood that the subject matter of this disclosure should not be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the accompanying claims.

[0018] Automated point-of-care device for complex sample handling and method of use thereof This invention relates to apparatus, analysis, and methods for sample preparation, nucleic acid amplification, and detection in an integrated sample-to-answer type microfluidic apparatus. This analysis is of a simple design and can be performed by an end user with minimal implementation time and equipment requirements. A typical manual sample preparation protocol involves multiple pipetting / fluid transfer and bead capture / resuscitation steps, performing 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 held within reagent bags and fluid conduits / channels, can significantly impair the reproducibility and reliability of analyses performed on cartridges. In particular, analytical processes that rely on high pipetting precision for successful execution, such as amplification processes where even slight changes in system volume can greatly affect reagent concentration and analytical performance, and processes requiring precise pH control, require a metering system capable of delivering the exact volume of reagent to the desired reaction chamber. While it is possible to include a reaction chamber with a fixed volume for metering liquid reagents, configured to overflow and discard any excess reagent delivered to the chamber, this type of system requires precisely molded metering chambers to control reagent distribution accuracy on the microfluidic apparatus. Additionally, systems using metering chambers may be more susceptible to air bubbles within the system, which can affect the reliability of fluid distribution. Such additional degassing mechanisms may require pumps and valves, adding complexity to the microfluidic cartridge and instrument.

[0020] This disclosure describes a reagent dispensing unit (RDU) that overcomes the problems associated with dead volume held in a microfluidic conduit and stored in a reagent bag. This system eliminates the need for a sophisticated weighing system to measure precise volumes of aqueous reagents by efficiently delivering all the aqueous reagents necessary to accurately perform microfluidic cartridge-based analysis.

[0021] The reagent dispensing unit comprises one or more reagent bags, each containing miscible and miscible liquid reagents packaged together in separate bags or together in a single bag, and one or more plungers for pressing on the bag to rupture the frangible seal layer on the bag and, when sufficient working force is applied, to push out the contents. In some embodiments, the RDU may include a sharp object or projection that, when sufficient working force is applied thereto, can rupture the frangible seal on the RDU. The sharp object or projection may be located inside the bag or close to the frangible seal of the RDU so that when working force is applied, the sharp object comes into contact with the frangible seal and ruptures it.

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

[0023] The reagent bag within the RDU (Figure 1A) contains an aqueous reagent 103 and a non-aqueous, immiscible reagent 102 in a single reagent bag, which are sealed with a frangible seal layer 104 that bursts upon operation, allowing the reagents to be delivered to the microfluidic device. Figure 1B shows the RDU assembled on the microfluidic device 108. The RDU includes the filled reagent bag and a plunger element 106 that acts on a sharp object 105 used to squeeze the reagent bag and rupture the frangible seal layer 104 assembled on the microfluidic device. The RDU is incorporated into the microfluidic device 108 such that the frangible seal 104 is located at the interface of an inlet conduit 107 into a fluid reagent well 109 on the microfluidic device. The microfluidic device includes one or more reagent wells 109 and waste wells 110 to help collect excess immiscible reagent 102 that overflows from the reagent well 109.

[0024] The immiscible reagent is selected such that, when the apparatus is operating, the aqueous reagent is closest to the interface between the frangible seal and the fluid well inlet conduit. In some embodiments, an immiscible fluid with a lower density 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 with a higher density than the aqueous reagent, such as a fluorine-containing compound or a fluorocarbon such as Fluorinert (3M), may be used so that the lower-density aqueous reagent suspends on top of the immiscible Fluorinert fluid. The immiscible non-aqueous fluid is selected such that, when the apparatus is in its operating position, the aqueous reagent is closest to the interface between the frangible seal and the fluid well inlet conduit.

[0025] Figure 1C shows an actuated RDU assembled on a microfluidic device 108. When actuating force is applied to the device, an integrated plunger compresses the reagent bag, rupturing the frangible seal 104 and creating a fluid connection to the reagent well 109 through the inlet fluid conduit 107. Referring to Figure 1C, the aqueous fluid 103 closest to the inlet fluid conduit 107 flows first out of the inlet conduit into the fluid well, and then the non-aqueous miscible fluid 102 functions to effectively push out all aqueous reagent that would occupy dead volume space in the fluid conduit and within the RDU. The excess immiscible non-aqueous fluid 102 overflows from the reagent well and is collected in the waste well 110. Using this system, precise amounts of aqueous reagent can be effectively delivered onto the microfluidic device while eliminating the dead volume problem by filling the space with a non-reactive, immiscible non-aqueous fluid. The non-aqueous miscible fluid 102 also acts to form a barrier over the aqueous fluid in the fluid well, preventing evaporation of the aqueous reagent during heating processes such as thermal cycling or thermal culture. This type of system significantly reduces system complexity because it does not require the use of complex valves or pumps for proper operation. In addition, since the system does not depend on the volume of the reaction chamber for accurate volume measurement, the precise molding of the reaction chamber, typically required for accurate reagent volume measurement, is not necessary. Rather, the aqueous reagent, pre-filled into the RDU using a well-known precision pipetting process along with the immiscible non-aqueous fluid, is completely delivered to the desired fluid well during operation because the non-aqueous miscible fluid completely pushes out the aqueous reagent and occupies all the dead space that would otherwise be filled with the aqueous reagent. The amount of immiscible reagent distributed to the system is not critical and does not require precise delivery.Therefore, the main advantage of this type of system is that it does not require precise operational control on the instrument to ensure run-to-run reproducibility through aqueous reagent delivery from a single-dose reagent pack, because once all aqueous reagent is filled into the instrument, the immiscible non-aqueous fluid overflows, pushing out the aqueous reagent and occupying all dead space, thereby ensuring delivery to the desired fluid well, and during operation, all aqueous reagent is pushed out of the RDU and reliably delivered to the microfluidic system.

[0026] In some embodiments, the RDU may include a locking mechanism that functions to lock the plunger element in its pressed position to prevent backflow of reagent into the reagent bag. This locking mechanism is not limited to pin-retaining mechanisms such as ball-lock pins, rivets, or barbed pins.

[0027] Referring to Figures 2A and 2B, cross-sectional views of an exemplary microfluidic apparatus with an integrated RDU, including the plunger and locking mechanism 201, before and after operation, are shown, respectively. The plunger element 202 is assembled in close proximity to the reagent bag 204. The plunger element is fixed in place by a locking pin 203 that is trapped within a locking hole 208 and oriented to restrict the movement of the plunger in a direction that facilitates pressing the bag when an operating force is applied. In this exemplary embodiment, the trapped locking pin 203 can only move downward along the locking hole 208 located on the microfluidic apparatus. The reagent bag includes a frangible seal layer 205 that is broken when sufficient operating force is applied to the plunger, as shown in Figure 2B. Upon operation, the frangible seal 205 is broken, and the contents of the reagent bag 204 are transferred to the fluid well 207 through an inlet fluid conduit 206 on the microfluidic apparatus. The locking pin 203 descends into the locking hole 208 to prevent reagents from flowing back into the reagent bag, locking the plunger in its depressed position.

[0028] One aspect of the present invention is a microfluidic apparatus comprising two or more fluid wells connected to each other via primary channels. The fluid wells are connected to one or more reagent dispensing units (RDUs) containing stored liquid reagents separated from the inlet into the fluid wells by a frangable seal. The reagent bags may be filled with aqueous fluid, a non-aqueous miscible fluid, or a combination thereof. Upon activation, the frangable seal is broken and the contents of the reagent dispensing unit are transferred to the fluid wells. At the end of the RDU activation sequence, the stored reagents are successfully transferred to the fluid wells on the microfluidic apparatus. The fluid wells are each filled with their respective aqueous reagents and connected to each other via primary channels filled with a non-aqueous fluid.

[0029] For the care environment, a self-contained system is advantageous because it does not require complex user-driven pipetting or injection processes. In one exemplary embodiment, reagents may be stored on a fluid apparatus in a reagent bag. The bag reagents include, but are not limited to, buffers, salts, acids, bases, labels, tags, markers, water, alcohols, solvents, waxes, oils, gases, gels, etc. When sufficient pressure is applied to the bag, the bag bursts, thereby dispensing the contents of the bag into a fluid conduit that fully leads to the intended reaction. The bag is designed to have a frangible seal aligned with the inlet of the fluid conduit, so that when the bag bursts, its contents are forced into the fluid conduit and fill the fluid well.

[0030] Each fluid well volume is designed to be 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 conduits of each fluid well. Reagent bags containing immiscible liquids, such as mineral oil, are connected to the primary fluid conduit during operation. 1) The contents of the reagent bags containing immiscible liquids are released, forming an immiscible oil phase on top of the aqueous reagents filled in the fluid wells. 2) All miscible liquids in the fluid wells are connected to the sequencer to form a fluid circuit, but are separated from each other by the oil phase to prevent mixing. The primary fluid conduit exits into the waste well to collect excess oil.

[0031] While it is possible to pre-fill fluid wells with buffers separated by an oil phase and seal and store cartridges for later use, some reagents, not limited to enzymes, oligos, dNTPs, and buffers, are not stable in their liquid form at room temperature or over long periods and therefore need to be stored in a lyophilized and hydrated form before use. Furthermore, there are challenges in introducing samples into such pre-filled systems. The disclosed invention provides a method and apparatus for solving problems related to sample introduction, reagent delivery, and analytical automation for sample processing on microfluidic devices.

[0032] Next, referring to Figure 3A, an exemplary sample-to-answer microfluidic cartridge 301 for performing nucleic acid amplification testing (NAAT) is shown.

[0033] A sample-to-answer type microfluidic cartridge comprises one or more reagent wells 309 connected to each other via a primary fluid channel 305. The RDU assembled into the microfluidic cartridge comprises a plurality of reagent bags 302 separated from inlet conduits to the fluid wells 309 on the microfluidic cartridge by a frangable seal, and an integrated plunger element 303 with a locking pin 304 that locks the plunger in its depressed position after activation, preventing backflow of reagent into the reagent bags. In this embodiment, the plunger element 303 is designed to contact all reagent bags at the same moment, pushing and releasing all individual reagents from the reagent bags in parallel from a single actuation stroke. In other embodiments, the plunger element may have spatially oriented projections of varying depths to contact desired reagent bags in a preferred sequence to facilitate continuous delivery of reagents to the microfluidic cartridge when the plunger is depressed. When the RDU is actuated, the reagent wells are filled with aqueous reagents, and the fluid circuits between the reagent wells are completed through a primary fluid channel filled with a non-aqueous miscible fluid. The cartridge includes a waste well 306 to capture excess immiscible reagent that overflows from the reagent well 309 through the primary fluid channel 305. To perform NAAT, the reagent bag may also contain a lysis buffer, binding buffer, magnetic beads, washing buffer, hydration buffer, and an immiscible fluid (e.g., a mineral oil, wax, or fluorocarbon-based compound, e.g., Fluorinert).

[0034] The reagents and reagent delivery sequences may be designed to differ depending on the type of analysis being automated on the microfluidic apparatus. The cartridge may also contain dried and lyophilized reagents that can be hydrated during use by either the sample or the dispensed buffer. The cartridge includes a sample inlet port for transferring the sample into the cartridge for processing. The sample inlet port may include a quick-connect fitting, such as a Luer 311, into which the sample can be injected into the cartridge. Other embodiments may include an access port into 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, so that impurities and inhibitors from the sample are filtered out before the sample is delivered to the cartridge. The filter membrane material and pore size can be selected depending on the type of analysis to be performed and are not limited to nitrocellulose, nylon, PTFE, PES, glass fiber, PVDF, MCE, polycarbonate, etc. Depending on the type of detection method used, the cartridge may also include further downstream analysis units such as DNA hybridization microarrays, protein arrays, or lateral flow strips.

[0036] In some embodiments, fluorescence, electrochemical, or colorimetric-based detection techniques may be used to detect the amplified product. An exemplary microfluidic cartridge shown in Figure 3 employs colorimetric detection using a lateral flow piece 308 that can be read digitally through optical readout or visually by the end user. The lateral flow piece is separated from the amplification well by a frangible seal layer connected to a bag containing a sharp object 307, so that the frangible seal ruptures upon operation, delivering the amplified product to the lateral flow piece 308 for detection. In some embodiments, the amplification well itself may include a frangible layer that is easily deformable upon operation so that the frangible seal layer ruptures and the amplified product is compressed onto the lateral flow piece.

[0037] Next, referring to Figure 3B, a schematic exploded view of a microfluidic device is shown, which includes a microfluidic cartridge 301 rotating between an upper actuator element 318 and a bottom actuator element 313.

[0038] By providing spatially oriented magnets 317 and 312 on the upper and lower actuator elements, respectively, the spatially oriented magnets capture, resuspend, and transport magnetic beads between different reagent wells in a single operating step that includes rotating the microfluidic cartridge between the actuator elements, thereby transferring the sample preparation sequence (e.g., binding, washing, and elution sequences on the sample) to the microfluidic cartridge. In the exemplary embodiment shown in Figure 3B, the upper actuator element includes a projection 316 designed to contact the microfluidic cartridge at a predetermined time during the analysis sequence to actuate a sharp object in the bag 307 above it, rupturing the frangible seal layer and introducing the amplified product into the lateral flow piece 308. The lower actuator element includes one or more spatially oriented heating elements 314 that help provide the stable single-temperature heat or thermal cycle required for isothermal or PCR-based amplification of nucleic acids, respectively. The spatially oriented heating element 314 can also help provide heat for sample preparation steps or downstream post-amplified steps, depending on the analysis being performed on the system. When a thermal cycle is performed, the microfluidic cartridge rotates periodically between three heater elements set to a constant single temperature so that the amplification well is in contact with or in close proximity to the desired heating element for the desired cycle time.

[0039] Sample-to-Answer NAAT This specification describes an exemplary sample-to-answer type NAAT, a microfluidic analysis automation platform that uses a single actuator providing rotational motion, such as a servo motor or stepper motor, a retractable spring, a hand crank, or a user's finger. A retractable spring mechanism offers the ability to automate analysis without using electric / 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 processes 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 bonded to a solid support such as magnetic or non-magnetic beads, membranes, or plastic tubes and plates. The charge of the ionizable coating can be switched by changing the pH of the surrounding buffer. At low pH, the surface is positively charged, allowing negatively charged nucleic acids to bind to the solid support, while proteins and other contaminants can be easily washed away. At higher pH, the surface charge is neutralized, and nucleic acids are eluted from the surface without the need for a time-consuming precipitation process. A unique advantage is that Chargeswitch technology uses an aqueous buffer and does not require the use of ethanol, chaotropic salts, or organic solvents, which can hinder downstream applications such as amplification.

[0041] Magnetic beads are highly effective and simple solid-phase capture supports for nucleic acid extraction and purification. Magnetic bead-based DNA purification is centrifugation-independent, easily automated, and reduces implementation time. These are methods of choice when rapid purification is required, and when considering semi-automated or fully automated systems, magnetic DNA purification represents a clear improvement over centrifugation-dependent isolation techniques. These systems are used when a large number of samples need to be purified rapidly. Using magnetic beads coated with an ionizable (switchable) coating, nucleic acids can be rapidly and efficiently purified from raw biological samples. This specification describes a unique analytical automation platform that allows magnetic beads to be captured, resuspended, and transferred through a series of reagent-filled chambers via oil-filled primary fluid conduits in a single rotational motion. Using this platform, nucleic acids may be extracted and purified from raw biological samples in a 2-minute sequence.

[0042] The microfluidic cartridge includes a reagent bag containing a binding buffer, magnetic beads in suspension, a washing buffer, a hydration buffer, and a non-aqueous mineral oil as an overlay, as well as an aqueous reagent such as a transport fluid. The microfluidic cartridge also contains dried dissolution buffer reagents and a lyophilized amplification mixture present in each reagent well. When the test is ready to be performed, the following steps are carried out. 1. Remove the untreated biological sample with a pipette and dispense or inject it into the cartridge through the sample inlet port. 2. The cartridge is inserted into a handheld device that includes actuator elements, a motor, electronic equipment, and a display. 3. Close the device lid and start the test.

[0043] System operation The system may be configured to incorporate different methods and procedures depending on the type of analysis, the required biological sample and sample preparation steps, and the analytical operation sequence. As an example, an operational sequence for performing 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 a microfluidic cartridge, where it is hydrated with the dry lysis buffer reagent present in the lysis / binding well 402. Cells in the untreated sample are lysed. The instrument lid is then closed. In this embodiment, closing the lid provides the force to press down the plunger 303 and distributes the reagent from the stored reagent bag into the respective wells. When the operation is successful, the magnetic beads in the suspension and binding buffer are distributed into the dissolution / binding well containing the untreated sample solution, the washing buffer is distributed into the washing well 403, the hydration buffer is distributed into the amplification well 404 containing the freeze-dried amplification mixture, and the mineral oil is distributed to form a continuous overlay on the well, filling the primary fluid channel 305 and completing the fluid circuit. The locking barbed pins present on the cartridge hold the plunger element in its depressed position on the microfluidic cartridge, preventing backflow and preventing interference with the smooth rotation of the microfluidic cartridge between the actuator elements.

[0044] Referring to FIG. 4A, a top view of the 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 <pH6. 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 (pH7) 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 top 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 well is in close proximity to a spatially oriented heating element 314 on the actuator element. The heating element functions to provide the thermal energy required for nucleic acid amplification. In isothermal amplification reactions requiring single-temperature culture, no additional heating elements are used, and a single heater element can deliver thermal energy for amplification. In applications involving polymerase chain reactions (PCR) with required thermal cycles, the microfluidic cartridge rotates periodically between three heater elements set to a constant single temperature so that the amplification well is in contact with or in close proximity to the desired heating element for the desired cycle time.

[0046] Referring to Figure 5, a schematic top view of the microfluidic apparatus (upper actuator element not shown) is shown above an amplification well 404 positioned above a heater element set to temperature Tl in Figure 5A, T2 in Figure 5B, and T3 in Figure 5C. This illustrates how a sample-to-answer type NAAT with rapid thermal cycling is achieved by switching / actuating the microfluidic cartridge in a precise timing sequence using three fixed thermal zones on the actuator element and a single motor also used to run the entire automated analysis sequence. The motor rotates back and forth between the three heating zones, thereby circulating the amplification chamber between the three heating zones set to temperatures corresponding to the denaturation, extension, and annealing cycles. This continues until a predetermined number of cycles are completed. In some embodiments, rapid two-temperature PCR can be performed using only two of the three heating elements. In some embodiments, the heating element, including a custom aluminum block with an integrated resistive element, can be used as a heat sink to easily rapid cool the reaction to the desired temperature. In some embodiments, the heating element can also assist in providing heat during the analysis being performed on the system, or in downstream post-amplification steps such as sample preparation or DNA hybridization on a microarray.

[0047] Following the amplification stage, detection of the amplified product is performed colorimetrically on an integrated lateral flow piece. The lateral flow piece is separated from the amplification well containing the amplified product by a frangible seal layer, which is coupled to a bag containing a sharp object 307, thereby allowing the frangible seal to rupture upon operation, delivering the amplified product to the lateral flow piece 308 for detection. In some embodiments, the amplification well itself may include a frangible layer and be easily deformable upon operation so that the frangible seal layer is ruptured and the amplified product is compressed onto the lateral flow piece.

[0048] Referring to Figure 6, a top view of the microfluidic apparatus is shown at the point where it reaches the position of the lateral flow detection step. A is a magnified image showing a projection 316 that deforms a bag containing a sharp object 307 by contacting it in order to rupture the frangible seal layer. The upper actuator element has a spatially oriented projection 316 on which, when the microfluidic cartridge rotates so as to contact the projection, it compresses and deforms the bag containing the sharp object 307. This deformation force causes the frangible seal layer to rupture, thereby drawing up the amplified product through the lateral flow piece.

[0049] Sample collection and extraction device Swabs are primarily used as biological sample collection devices. Swabs, such as Copan FloqSwabs (trademark), are manipulated to keep the entire sample close to the surface for rapid and complete elution, but physical force should be used to maximize the elution of the sample into the transport medium or buffer. Typically, manual agitation by vigorously rotating the swab in the transport medium, or using a vortex in the laboratory, maximizes the elution of the sample from the swab into the solution. The swab is manually extruded, and then the solution containing the sample is pipetted and further processed depending on the type of analysis.

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

[0051] Therefore, there is a need for improved apparatus and methods for sample extraction that minimize operator variability, are easy to use, do not consume electricity, and do not rely on laboratory equipment such as operating squirrels or centrifuges.

[0052] The present invention, disclosed below, is a point-of-care mechanism, apparatus, and method for replacing laboratory protocols to maximize sample recovery from swab samples. The disclosed invention also enables a user to directly deliver multiple reagents to a sample in a sample extraction device using a simple, user-operated process. The disclosed invention significantly simplifies laboratory-based sample handling protocols and eliminates the need for sophisticated equipment required to perform laboratory-based sample handling protocols.

[0053] Referring to Figures 7A and 7B, schematic diagrams of an exemplary sample extraction apparatus for extracting and processing an untreated sample attached to a swab are shown. In some embodiments, the sample extraction apparatus comprises a sample collection container 705 and a sample processing unit 707 removable from the sample collection container. The apparatus 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 a screw thread 706 that engages with the swab lid 702. When the swab is inserted into the container 705, the swab comes into contact with cleaning inserts 715, 716 having one or more protrusions 713 that contact the swab head 704, and the lid is closed, cleaning the head as the swab rotates / rotates within the insert. This cleaning action functions to release the sample attached to the swab head, thereby eluting it into the buffer or medium 714 contained in the container. In some embodiments, the cleaning insert may have a plurality of small bristles 713, which 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 or O-rings, that are capable of scraping and compressing the swab head within the insert. The number of threads defines the number of turns or full rotations the swab head 704 forms within the cleaning insert and can be optimized to maximize sample recovery from the swab.

[0054] Similarly, the type and design of mechanical elements can be optimized for swab types to ensure maximum sample recovery. In some embodiments, the container may include one or more filter membranes 712 selected to remove undesirable impurities and inhibitors from the sample. The container is equipped with a quick-connect connector 711, such as a Luer connector, for connection 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 can contain stored reagents in the form of dry liquid capsules or pelletized reagents. The grooved recesses containing the stored reagents may be spatially oriented so that they are continuously introduced into the sample when 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] The stored reagents are not limited to lyophilized or dry buffers such as lysis buffers, neutralization buffers, binding buffers, washing buffers, and pH control buffers; solid-phase capture supports such as magnetic beads; enzymes, antibodies, aptamers, conjugation buffers; functionalized particles such as gold nanoparticles, latex particles, and magnetic particles; chemiluminescent or colorimetric detection reagents.

[0056] Next, referring to Figure 8, a step-by-step sequence of an exemplary sample preparation protocol is shown for recovering the untreated sample attached to the swab before transferring it to the microfluidic cartridge.

[0057] Step 1 - Insert the swab sample into the container and rotate the lid to close it. Step 2 - Retract the plunger, thereby introducing the eluted sample from the container into the reagent, which is stored in a dry form in the grooved recess of the syringe barrel. Step 3 - Remove the syringe from the quick connector 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 press 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) or Amies medium. The swab is inserted into the container and the lid is closed by turning it "n" times (where n is the number of turns determined by the threads 706 on the container). Once the swab is inserted into the container, it comes into contact with projections and mechanical elements on a cleaning insert present in the container. As the swab rotates within the cleaning insert, the swab head is cleaned and compressed by the mechanical elements, releasing the sample attached to the swab head and eluting it into the solution / medium contained in the container. The sample from the container is then filtered through a filter membrane to remove impurities and inhibitors by withdrawing a plunger on an attached syringe sample processing unit and collected in the syringe barrel below. Once the syringe plunger is withdrawn, the sample is introduced into one or more stored reagents, which are dry, liquid, or gel-like, and are continuously present in the barrel.

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

[0060] The neutralized sample may then be continuously introduced into a third grooved recess containing magnetic beads, which are stored in the sample processing unit before the processed contents are transferred to the microfluidic cartridge. Alternatively, the microfluidic cartridge may contain magnetic beads pre-loaded in reagent wells therein, and the neutralized sample solution may be introduced into the magnetic beads for sample purification when it is transferred to the microfluidic cartridge.

[0061] The sample extraction apparatus described may be used for any biological analysis involving multiple steps involving multiple reagents that need to be delivered to the sample in a predetermined sequence. The reagents and steps may be selected and designed based on the analysis performed.

[0062] The exemplary sample collection containers and accompanying sample processing units described herein are for processing swab samples that need to be attached to a swab and eluted into a solution for downstream processing. However, the containers can be applied to different sample types that are not collected on a swab, including, but not limited to, biological samples such as saliva, blood, plasma, serum, urine, sputum, CSF, tissue, feces, plants, food, soil, and fossils. The types of stored buffers / mediums and filters used in the containers can also be applied to downstream analyses and sample types.

[0063] In exemplary embodiments, a sample collection container may be used to collect and process urine samples. The sample collection container may contain a lysis buffer reagent in a dry form that can be hydrated and activated when the 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 dry neutralizing reagent internally so that the lysate is neutralized when it is drawn into the sample processing unit. The filter 712 may be selected to retain inhibitors and proteins that allow only purified nucleic acids to pass through.

[0064] In exemplary embodiments, an alkaline solubilizing buffer may be used to change the sample pH to a range between 9 and 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 pore dimensions of 0.45 μm to 0.8 μm. Due to the selected pore dimensions of the sample and the high alkaline pH, proteins and inhibitors present in the sample are retained by or bound to the filter membrane, and only purified nucleic acids pass through to the next stage in the sample processing unit, where the purified lysate is neutralized by the neutralizing reagent present therein.

[0065] Sample processing unit Referring to Figures 9A and 9B, perspective and exploded views, respectively, of an exemplary sample processing unit are shown. The sample processing unit includes a sample collection container 902 and a lid actuator 903 that facilitates automated, continuous reagent delivery to the sample in the container 902 in a predetermined, precise timing sequence. Figure 9B is a schematic exploded view of an exemplary sample processing unit showing the functional components of the lid actuator 903 as a continuous reagent delivery system. In this exemplary embodiment, the lid actuator includes: a proprietary reagent dispensing unit including a reagent palette 905; one or more reagent bags 904 containing reagents stored in dry, liquid, or gelled form; and one or more rotary actuator elements 907, including but not limited to, spatially oriented mechanical elements 906 that function to operate the reagent bags 904 to distribute their contents into the sample collection container 902 in a precise timing sequence when the rotary actuator elements 907 rotate in close proximity to the reagent palette 905, including projections, valves, bumps, etc. 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 winding spring or motor. In some embodiments, the reagent dispensing unit may be manually operated by the user's finger.

[0066] In exemplary embodiments, a retractable spring is used. Retractable 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 steady rotational motion until they become completely uncoiled. Retractable spring mechanisms can be designed to become completely uncoiled in a certain amount of time by appropriately selecting the spring and gear mechanism used. Sequential reagent delivery can be powered by a retractable spring mechanism that acts on an actuator to deliver reagents to the system in a precise timing sequence. In this invention, the rotary actuating element includes a spatially oriented mechanical element that interferes with reagent-filled bags on a reagent pallet in predetermined instances along the rotational path of the rotary actuating element, thereby deforming and compressing the reagent bags, and thereby delivering reagents in a predetermined precise timing sequence.

[0067] This sample processing unit offers advantages over typical kits that employ manual reagent delivery protocols using pipettes or injectors. This is because it is a self-contained system that has all the reagents necessary for sample processing packaged in a single unit, including a simple dispensing and pre-reagent delivery mechanism. Particularly in non-laboratory environments where only CLIAwaived tests (simple and easy tests with no risk of user error) can be performed, this self-contained sample processing unit reduces the risk of user-generated errors by eliminating complex and time-consuming pipetting steps and enabling reagent delivery using simple and universal twist, slide, or rotational movements that do not require a skilled operator. It can also be automated using a single motor or self-powered retractable spring actuator to further reduce hand-on time.

[0068] Referring to Figures 10A, 10B, and 10C, instances of the operation sequence of a rotary actuator element 907 rotating relative to the reagent palette 905 are shown. Figure 10A shows the position of the rotary actuator element before reagent delivery. In Figure 10B, the rotary actuator element has moved to a position where the mechanical element 906 above it interferes with the first reagent bag in its path, thereby deforming it and pushing its contents into the sample collection container through the reagent dispensing conduit 908. In Figure 10C, the rotary actuator element has moved along a path where the mechanical element 906 interferes with the second reagent bag, deforming it and pushing the contents of the second reagent bag into the sample collection container.

[0069] The exemplary embodiment shown in Figure 9 utilizes rotational motion to perform the actuation process. However, other embodiments may utilize linear motion to accomplish the same task, for example, by using one or more linear sliding actuator elements. The actuation elements may be oriented in different spatial dimensions so as to be able to continuously interfere with the different spatial dimensions of the sample processing apparatus or microfluidic cartridge.

[0070] Referring to Figure 11, an exploded schematic diagram of a rotary shaft-based sample processing unit is shown. This unique embodiment of the present invention uses a rotary shaft actuator element 1103 that provides additional dimensional control for analytical automation. The rotary shaft actuator element comprises one or more spatially oriented mechanical elements 1102 that interfere with reagent bags 1105 on a reagent palette 1104 to actuate them and distribute their contents in a predetermined sequence.

[0071] In some embodiments, the detection unit may be integrated into the sample processing unit to allow for easy direct detection of the analyte within the built-in system without the need to move it from the container to the detection unit. This detection unit may be visual, using a colorimetric reagent that changes color in the container depending on the presence or absence of the analyte, or it may be an immunochromatographic detection device using a dipstick or a lateral flow device. In some embodiments, the lateral flow device may be located on the surface of the sample collection container or within the lid of the sample processing unit.

[0072] Each reagent may be packaged in an individual reagent bag and assembled on a microfluidic cartridge; however, this method results in a more complex assembly process, requiring each reagent bag to be assembled and sealed individually in the cartridge. In some embodiments, it is preferable to create a reagent card containing multiple reagent bags that can be assembled as a single unit on a microfluidic cartridge. Referring to Figure 12, a perspective view of an exemplary reagent card 1201 showing individual reagent bags 1202 and a flowing reagent bag 1203 is shown. The reagent card may be designed with a shape that readily fits and aligns with the fitting grooves on the cartridge during assembly. The reagent card can be filled into a custom fixture manually or using multiple automated pipettes to distribute the desired fluid volume into each reagent bag before frangable foil sealing. In some embodiments, the reagent card may have molded features in addition to the bags to help place and align the reagent bag card on the microfluidic cartridge. When an actuation force is applied to the reagent bags individually, in a continuous manner, or in parallel to multiple reagent bags on the card, the frangible foil seal at the bottom bursts, allowing the reagents to flow through the fluid channels into the appropriate reaction chamber in the fluid cartridge. The actuation force may be distributed to the reagent card via a plunger having spatially oriented projections that continuously contact one or more reagent bags during operation.

[0073] In some embodiments, it may be necessary to mix or combine one or more reagents present in a reagent bag. Historically, this mixing has been done in microfluidic devices using active mixers that apply external energy to agitate the fluid, or passive mixers that increase the contact area and contact time of the fluids being mixed through the use of specially designed geometric shapes and channel configurations. In some embodiments, it may be necessary to mix two or more reagents. For example, at least one reagent may need to be in solid form, or contain solid particles in a low-viscosity or high-viscosity liquid medium, or be a high-viscosity liquid or gel. In microfluidic cartridges, solid reagents are often stored by drying them directly in the reaction chamber, in which case they may be reconstituted during use with liquid reagents, such as a reconstitution buffer or an untreated or treated liquid sample during analysis. These dried reagents are brought to the desired concentration using a known volume of reconstitution solution. In some cases, it is preferable to dry or freeze-dry the reagents and store them directly in the reaction chamber of the microfluidic device. For example, freeze-dried master mixes for nucleic acid amplification tests (NAAT) eliminate the need for low-temperature storage, allowing microfluidic cartridges to be stored at room temperature. In other cases, the drying process can adversely affect reagents, reducing their efficacy or even permanently destroying them. For instance, charge-switched magnetic beads for nucleic acid sample preparation supplied by Thermo Fisher Scientific (Carlsbad, CA) lose their functionality once dried and must always be kept in solution. Some magnetic beads contain functional coatings, such as cellulose-coated magnetic beads from Promega's Magazorb® DNA extraction kit, which irreversibly aggregate and become non-functional when dried. Therefore, it is important to store the beads in a liquid matrix to maintain their functionality.While it might be possible to develop a custom drying process using custom chemistry to help preserve the functional coating on the beads, developing a custom process is often expensive and requires extensive testing to ensure no loss of functionality. Therefore, it is preferable to store functionalized particles such as magnetic beads in a liquid matrix. However, on-chip storage of liquid magnetic beads presents many challenges in itself. Specifically, the magnetic particles are stored in the liquid matrix at very high concentrations (often in the range of 5 mg / ml to 50 mg / ml) and then diluted with samples and buffers to meet binding volume requirements. Manufacturers have provided protocols requiring very small amounts of magnetic bead reagent, typically in the range of 10 μl to 40 μl per test, but due to manufacturing process limitations (minimum volume of 50 μl), it is difficult to package them in foil-sealed reagent bags without significant dead volume issues. Further dead volume in the channels or fluid conduits leading to the reaction chamber significantly reduces the amount of reagent lost during distribution. For example, a channel with a cross-section of 750 μm × 750 μm and a length of 1 inch has a dead volume of 15 μl. What complicates this problem is that, during the dispensing process, approximately 20% of the reagent may still be present in the crushed reagent bag.

[0074] It is undesirable for 20% of concentrated essential reagents to be lost due to being trapped in dead space. The present invention in this disclosure uses a flow-based approach to facilitate the effective movement and mixing of reagents on a microfluidic apparatus. The flow system utilizes a large amount of a fluid medium, either liquid or gas, used as a moving reagent, to effectively move / displace the reagents present in the moving reagent bag into the reaction chamber on the microfluidic apparatus. The moving reagent may be an immiscible fluid such as mineral oil (liquid) or air (gas), or a miscible liquid such as an aqueous buffer. When an immiscible liquid or gas enters the moving reagent bag, it effectively replaces all the contents of the reagent bag into the reaction chamber of the microfluidic apparatus. When a miscible fluid such as a buffer enters the flow through the chamber, it mixes with the reagents present in the flow through the chamber, and the contents entering the reaction chamber of the microfluidic apparatus become a mixture of the moving reagent from the moving reagent bag and the reagents. This method offsets the effect of dead volume in the reagent bag or microfluidic apparatus by filling them with the moving reagent. Since the volume of the transfer reagent is not essential or important for the reaction occurring within the reaction chamber of the fluid tip, this method helps to effectively transfer reagents present in a transfer reagent bag, where its volume is important for the proper functioning of the analysis.

[0075] Alternatively, the transfer medium may be in the form of a reconstitution buffer that rehydrates lyophilized reagent pellets that can be present in the flow reagent bag. In some embodiments, the transfer medium may be the liquid sample being analyzed. Two mixing processes occur during the interaction between the transfer medium and the contents of the flow reagent bag. This mixing can be further facilitated by increasing the contact area and contact time. Many approaches, such as increasing the channel length, decreasing the channel cross-section, adding physical barriers that hinder flow velocity, increasing fluid pressure, and introducing turbulence into the fluid flow, are not effective in facilitating mixing.

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

[0077] Referring to Figure 13A, a schematic cross-sectional view of an exemplary microfluidic apparatus is shown, comprising a reagent card including a transfer reagent bag 1303 filled with transfer reagent 1306 and a flow reagent bag 1302 containing magnetic beads / particles in a liquid medium. The transfer reagent bag is connected to the inlet of the flow reagent bag via a transfer fluid conduit 1308 on the microfluidic apparatus, which is capped with a frangible seal 1304. The flow reagent bag includes a bursting element (ball) 1305 and is connected to a reaction chamber on the microfluidic apparatus through an outlet fluid conduit 1309. As shown in Figure 13B, when an actuation force is applied, the bursting element ruptures the frangible seal located beneath it, thereby opening a path for the transfer reagent to enter the flow reagent bag and replace its contents. In some embodiments, the bursting element may be located on the microfluidic apparatus rather than inside the flow reagent bag.

[0078] Examples The manufacturer provided a protocol for adding 40 μl of ChargeSwitch® magnetic beads and 300 μl of the provided binding buffer to 600 μl of bacterial cell lysate. To implement this protocol on the automated microfluidic apparatus described herein, 600 μl of cell lysate is first distributed into the reaction chamber on the microfluidic apparatus using a dispensing dropper or syringe. The flowing reagent bag contains 40 μl of magnetic beads. Assuming a total dead volume in the system (i.e., the volume remaining in the crushed flowing reagent bag, fluid conduit and crushed transfer reagent bag) of 60 μl, the transfer reagent bag contains 360 μl of binding buffer to offset the loss due to the dead volume. This dead volume is inherent to the design of the microfluidic apparatus and can be easily calculated from the geometric shape of the apparatus and can be verified using experimental methods. When an operating force is applied, the frangible seal is ruptured, and the binding buffer enters the flowing reagent bag containing the magnetic beads. The turbulence of the binding buffer as it enters the fluid reagent bag initiates the resuspending of magnetic beads that may settle during storage. The product of the resuspended magnetic beads in the binding buffer then enters the reaction chamber containing the cell lysate through the outlet fluid conduit 1309, completing 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, reagent bags filled with oil can be used to store a distributable oil phase when an activating force is applied. However, reagent bags are very difficult to manufacture and fill, and are difficult to seal without a dead air zone. A typical manufacturing tolerance for dead air in the bag is about 10% to 20% of the total bag volume. In particular, with viscous oil phase reagents, trapped air can generate bubbles in the oil phase, which leads to reproducibility issues and problems regarding the movement of magnetic particles in the immiscible oil phase or between the miscible water-soluble oil phase and the immiscible oil phase. In addition, the flow of the oil phase from the reagent bag during dispensing can be turbulent, and air pockets may form in the microfluidic apparatus as the oil phase fills each reaction well and primary channel. Furthermore, in another specific embodiment of this specification, it is described that the formation of bubbles in the system can be prevented by optimizing the channel and well geometry to promote laminar flow, and by implementing an in-line defoaming mechanism such as a microporous hydrophobic / oleophobic PTFE membrane that selectively releases trapped air so as not to leak liquid. In this specific embodiment, a smooth laminar flow is generated by utilizing the pressure head of the oil phase. This can be supplemented by optimized channel and well geometry to create a completely bubble-free oil phase within the primary channel without complicating the system by using a defoaming mechanism. The pressure head-based approach requires a vent to allow fluid flow to be generated by rupturing a frangible seal during dispensing. Furthermore, the presence of air in the oil phase container is unaffected because air is too light to displace the oil phase.

[0081] Referring to Figure 14, an exemplary microfluidic apparatus demonstrating an oil / immiscible phase dispensing system 1401 is shown in Figure 14A before the application of activating force and in Figure 14B after the application of activating force. The microfluidic tip-based oil / immiscible phase dispensing system 1401 includes an oil storage container 1402 that holds a desired volume of 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 functions to connect the container 1402 to a vent and reaction chamber on the microfluidic apparatus, 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 separated from the microfluidic apparatus by the same frangible seal that functions as a one-time valve. As shown in Figure 14B, when an actuation force is applied, the deformable lids at the air vent and oil / reagent outlet are shattered, and the bursting sphere 1408 penetrates the frangible seal. This burst connects the air vent and oil / reagent outlet to the vent hole and reaction chamber on the microfluidic apparatus, respectively.

[0082] Referring to Figure 15, an exemplary embodiment of a sample-to-answer microfluidic apparatus for nucleic acid amplification testing (NAAT) is shown. The microfluidic apparatus includes a reagent card 1201 for detection, an oil dispensing system 1401, and a lateral flow piece 1505 assembled on a microfluidic chip. The microfluidic chip itself includes a plurality of reaction chambers 1502 connected to each other by a primary channel 1503. An inlet channel 1504 connects reagent bags 1303 on the reagent card to individual reaction chambers. An actuation element on the lid of the apparatus, with a plunger having a spatial topography that matches the spatial position of the individual reagent bags and deformable lid elements 1407 on the oil dispensing system 1401, is used to provide an actuation force to rupture the frangible seal.

[0083] A typical sequence of actions would be as follows: 1. The sample is either injected into the cartridge or dispensed through the sample inlet. 2. The cartridge is inserted into the instrument, and the lid is closed. Closing the lid provides the necessary force to rupture the frangible seal of the reagent card and the 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. The results will be displayed on the lateral flow piece after the test is completed.

[0084] General definition This specification uses specific terms, but these are used only in a general and descriptive sense and not for limiting purposes. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by a person of ordinary skill in the art to which the inventions described herein pertain.

[0085] As used herein, “nucleic acid” means a macromolecule containing covalently bonded subunits called nucleotides. A “nucleotide” is a molecule or individual unit within a larger nucleic acid molecule, containing 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," "oligonucleotide," or "nucleic acid molecule" are used interchangeably herein to mean single-stranded or double-stranded phosphate ester polymer forms 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.

[0087] Polynucleotides containing RNA, DNA, or RNA / DNA hybrid sequences of any length are possible. The polynucleotides used in this invention may be naturally occurring, synthetic, recombinant, exovivoically produced, or a combination thereof, and may be purified using any purification method known in the art. Accordingly, the term "DNA" includes, but is not limited to, genomic DNA, plasmid DNA, synthetic DNA, semi-synthetic DNA, complementary DNA ("cDNA" being DNA synthesized from a messenger RNA template), and recombinant DNA (DNA that is artificially designed and therefore subjected to molecular biological manipulation from its natural nucleotide sequence).

[0088] Terms such as "amplify," "amplify," and "nucleic acid amplification" refer to the generation of multiple copies of a nucleic acid template (e.g., a template DNA molecule), or the generation of multiple copies of a nucleic acid sequence that are complementary to the nucleic acid template (e.g., a template DNA molecule).

[0089] The terms “up,” “down,” “up,” “down,” and “up” are used throughout the description to refer to the relative positions of components of the described device, such as the relative positions of the upper and lower substrates within the device. It will be understood that the device functions regardless of its orientation in space.

[0090] According to long-standing patent law convention, the terms “a,” “an,” and “the,” when used in this application including the claims, refer to “one or more.” Therefore, a reference to “subjects,” for example, includes multiple subjects unless the context explicitly states otherwise (e.g., multiple subjects).

[0091] Throughout this specification and the claims, the term “equipped with” is used in a non-exclusive sense unless the context otherwise indicates otherwise. Similarly, the term “contains” and its grammatical variations are intended to be non-exclusive, so as to ensure that the enumeration of items in a list does not exclude other similar items that may be substituted for or added to the enumerated items.

[0092] For the purposes of this specification and the accompanying claims, all numbers used herein to represent quantities, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values ​​should be understood in all examples to be modified by the term “approximately,” even if not explicitly stated in the value, quantity, or range. Accordingly, unless otherwise indicated, the numerical parameters described herein and in the accompanying claims are not exact, do not need to be exact, and may be approximately and / or greater or less, as desired, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and may reflect other factors known to those skilled in the art, depending on the desired characteristics to be obtained by the subject matter disclosed herein. For example, when referring to a certain value, the term “approximately” means that, in some embodiments, it includes a range of ±100% of the specified amount, 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%, where appropriate for carrying out the disclosed method or using the disclosed composition.

[0093] Furthermore, the term "approximately," when used in relation to one or more numbers or ranges of numbers, should be understood to mean all numbers, including all numbers within the range, and modifying the range by extending the upper and lower boundaries of the numbers listed. Enumeration of numerical ranges by endpoints includes all numbers, such as all integers including decimals contained within that range (for example, the enumeration of 1-5 includes 1, 2, 3, 4, and 5, as well as their decimals, 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 referenced herein represent the level of skill of those skilled in the art to which the subject matter disclosed herein relates. All publications, patent applications, patents, and other references are incorporated herein by reference to the same extent that each individual publication, patent application, patent, and other reference is specifically and individually indicated as being incorporated herein by reference. While numerous patent applications, patents, and other references are referenced herein, it should be understood that none of these references constitute part of the general knowledge in the art.

[0095] For the purpose of clarifying understanding, the above subject matter has been described in some detail by examples and embodiments, but those skilled in the art will understand that certain changes and modifications can be made within the scope of the attached claims. [Explanation of Symbols]

[0096] 101 Reagent dispensing unit 102 Non-aqueous, immiscible reagents 103 Aqueous reagent 104 Frangible seal layer 105 Sharp objects 106 Plunger elements 108 Microfluidic Devices

Claims

1. A microfluidic apparatus comprising a reagent dispensing unit, wherein the reagent dispensing unit is One or more reagents and at least one reagent bag having a frangible seal layer, A microfluidic device comprising at least one plunger and at least one sharp object or projection configured to rupture the frangible seal layer and deliver one or more reagents to the microfluidic device when an operating force is applied to the reagent dispensing unit.

2. The aforementioned microfluidic device further, At least one inlet conduit, At least one reagent well, Equipped with at least one waste well, The microfluidic apparatus according to claim 1, wherein the inlet conduit, the reagent well, and the waste well are fluidly connected such that an interface exists between the frangible seal and the inlet conduit, and when an operating force is applied to the reagent dispensing unit and excess reagent overflowing from the reagent well is collected in the waste well, one or more reagents are delivered to the reagent well via the inlet conduit.

3. The microfluidic apparatus according to claim 2, wherein at least two reagents are packaged in separate bags.

4. The microfluidic apparatus according to claim 2, wherein at least two reagents are packaged together in a single bag.

5. The microfluidic apparatus according to claim 4, wherein one reagent is an aqueous reagent and one reagent is a non-aqueous, miscible reagent.

6. The microfluidic apparatus according to claim 5, wherein the aqueous reagent is closest to the interface between the frangible seal and the inlet conduit.

7. The microfluidic apparatus according to claim 5, wherein the non-aqueous, immiscible reagent has a lower density than the aqueous reagent and floats above the aqueous reagent, thereby forming an immiscible layer on top of the aqueous reagent.

8. The microfluidic apparatus according to claim 5, wherein the aqueous reagent has a lower density than the non-aqueous, immiscible reagent and floats above the non-aqueous, immiscible reagent, thereby forming an aqueous layer on top of the non-aqueous, immiscible reagent.

9. The microfluidic apparatus according to claim 5, wherein when an operating force is applied to the reagent dispensing unit, the aqueous reagent first flows out from the inlet conduit and into the reagent well, and then the non-aqueous, immiscible reagent flows in.

10. The microfluidic apparatus according to claim 2, further comprising a locking mechanism configured to lock the plunger in the pressed position, thereby preventing the reagent from flowing back into the reagent bag.

11. The microfluidic apparatus according to claim 10, wherein the locking mechanism comprises a barbed pin in a locking hole configured to restrict the movement of the plunger in a direction that facilitates pressing down the bag while an operating force is applied.

12. The microfluidic apparatus according to claim 2, comprising two or more reagent wells that are connected to each other and connected to one or more reagent dispensing units via a primary channel.

13. The microfluidic apparatus according to claim 12, configured such that at the end of the operating sequence, the reagent wells are filled with aqueous reagents and connected to each other via the primary channels filled with a non-aqueous fluid.

14. The microfluidic apparatus according to claim 12, configured such that at the end of the operating sequence, an immiscible oil phase is formed covering the aqueous reagent in the fluid well, and the aqueous reagent in the fluid well is separated from each other by the oil phase, but is fluidly connected during the sequence to form a fluid circuit.

15. The microfluidic apparatus according to claim 12, comprising a plurality of reagent bags separated from the inlet conduit to the fluid well by a frangible seal, and an integrated plunger element having a locking pin that locks the plunger in the pressed position after operation, thereby preventing backflow of reagent into the reagent bags.

16. The microfluidic apparatus according to claim 15, wherein the plunger is configured to contact all of the reagent bags at the same moment and to push and release all of the reagents from the reagent bags in parallel with a single operating step.

17. The microfluidic apparatus according to claim 15, wherein the plunger is provided with spatially oriented projections having various depths, and is configured such that when the plunger is pressed in contact with a desired reagent bag in a preferred sequence, reagents are easily delivered continuously to the microfluidic apparatus.

18. The microfluidic apparatus according to claim 2, further comprising a sample inlet port into which a sample can be injected.

19. The microfluidic apparatus according to claim 18, wherein the sample inlet port further comprises one or more filter membranes.

20. The microfluidic apparatus according to claim 2, further comprising a microfluidic cartridge configured to rotate between an upper actuator element and a lower actuator element, wherein the upper and lower actuator elements are equipped with spatially oriented magnets, and in a single operating step comprising rotating the microfluidic cartridge between the upper and lower actuator elements, the spatially oriented magnets capture, resuspend, and transport magnetic beads between different reagent wells.

21. The microfluidic apparatus according to claim 20, wherein the upper actuator element is configured to contact the microfluidic cartridge at a predetermined time during the analysis sequence, activate a sharp object or protrusion in the reagent bag to rupture the frangible seal layer and deliver amplified material to the lateral fluid piece, and the lower actuator element comprises one or more spatially oriented heater elements configured to provide stable single-temperature heat or thermal cycles for isothermal or polymerase chain reaction (PCR) based amplification of nucleic acids.

22. The microfluidic apparatus according to claim 21, wherein the spatially oriented heater elements are configured to provide a thermal cycle, and the microfluidic cartridge rotates periodically between a plurality of heater elements, each set to a constant single temperature, so that the amplification well is in contact with or in close proximity to a desired heater element for a desired cycle time.

23. A sample extraction apparatus comprising a sample collection container and a sample processing unit, wherein the sample collection container comprises a swab having a swab head and a swab shaft attached to a screw-top lid, the sample collection container has a screw thread that fits with the lid, and the sample extraction apparatus is configured such that when the swab is inserted into the container, the swab comes into contact with the swab head and with a cleaning insert having one or more protrusions for cleaning the swab head, and the lid is closed.

24. The sample extraction apparatus according to claim 23, wherein the cleaning insert comprises a plurality of bristles spatially oriented so as to come into contact with the swab head when pulled within the insert.

25. The sample extraction apparatus according to claim 23, wherein the cleaning insert comprises a mechanical element.

26. The sample extraction apparatus according to claim 25, wherein the mechanical element comprises a raised portion or an O-ring.

27. The sample extraction apparatus according to claim 25, wherein the container comprises one or more filter membranes.

28. The sample extraction apparatus according to claim 25, wherein the container is provided with a quick connection connector that connects to a removable sample processing unit.

29. The sample extraction apparatus according to claim 28, wherein the removable sample processing unit is a syringe comprising a barrel, a plunger, and a plunger tip.

30. The sample extraction apparatus according to claim 29, wherein the syringe is provided with one or more grooved recesses containing stored reagents.

31. The sample extraction apparatus according to claim 30, wherein the grooved recess is spatially oriented so that the stored reagent is continuously introduced into the sample when the plunger tip is withdrawn.

32. A sample processing unit, Sample collection container, The system comprises a lid actuator configured for automated, continuous reagent delivery to the sample in the container in a predetermined timing sequence, The aforementioned cover actuator is A reagent dispensing unit comprising a reagent palette containing one or more reagent bags containing stored reagents, A sample processing unit comprising one or more rotary actuator elements, each having a spatially oriented mechanical element configured to operate the reagent bag so as to distribute its contents into the sample collection container in a predetermined timing sequence when the rotary actuator element rotates near the reagent pallet.

33. The sample processing unit according to claim 32, wherein the rotational operating element is provided with a mechanism for providing rotational motion.

34. The sample processing unit according to claim 33, wherein the mechanism for providing the rotational motion is a winding spring.

35. The sample processing unit according to claim 32, wherein the rotating operating element is configured to be operated manually by the user's finger.

36. The sample processing unit according to claim 32, further comprising a rotating shaft including one or more spatially oriented mechanical elements, wherein the rotating actuation element interferes with the reagent bags on the reagent palette to actuate them and distribute their contents in a predetermined sequence.

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