Systems and methods for performing bioassays using heat-sealed valves

Abstract

Systems and methods for performing biological assays using heat-sealed valves and / or incubation chambers are provided herein. The systems and methods determine one or more characteristics of a nucleic acid amplified sample based on modified optical properties of the sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 351,427, filed on June 12, 2022, the entire contents of which are incorporated herein by reference in their entirety for all purposes. Background Art

[0003] Biological sample determination is used to evaluate one or more characteristics of biological samples. Such determinations can qualitatively evaluate and / or quantitatively measure the presence, amount and / or functional activity of one or more analytes in a biological sample. Such assessments can be performed based on changes or lack of changes that occur in the determination. For example, changes in the color and / or transmittance of a biological sample or its aspects that occur under specific conditions during the determination can be used as an indicator of one or more characteristics of the sample being determined. Summary of the invention

[0004] Provided herein are systems and methods for performing bioassays on cells that require lysis. The systems and methods herein utilize heat-sealed valves to isolate and fluidically connect an incubation chamber and a downstream reaction chamber. The systems and methods determine one or more characteristics of the sample based on the modified optical properties of the nucleic acid amplification sample.

[0005] In one aspect, the present disclosure provides a system for performing a bioassay, the system comprising one or more of the following:

[0006] a. A thermal mixing module, the thermal mixing module comprising:

[0007] i. a sample receiving module for receiving a sample solution comprising a liquid sample (eg, a biological sample) and a preparation solution; and

[0008] ii. an incubation chamber in fluid communication with the sample receiving module;

[0009] b. a wax valve channel in fluid communication with the incubation chamber;

[0010] c. an optical property modification (OPM) module, the OPM module being operatively coupled to the incubation chamber via the wax valve channel, the OPM module comprising one or more reaction chambers, each reaction chamber comprising an assay reagent;

[0011] d. a heat-sealed valve disposed i) within the wax valve channel and ii) between the sample receiving module and the OPM module;

[0012] e. A mixing heater configured to supply heat to the incubation chamber.

[0013] f. valve heater; and

[0014] g. Reaction heater.

[0015] In some embodiments, the incubation chamber comprises a reagent or a lysing agent. As described herein, where a lysing agent is envisioned, a reagent is also envisioned. In some embodiments, the mixing heater is configured to align with at least a portion of the thermal mixing module and offset from a central portion of the incubation chamber.

[0016] In some embodiments, the system further comprises a sample preparation device configured to cooperate with the sample receiving module. In some embodiments, the sample receiving module comprises a puncture element configured to puncture a rupturable seal on the sample preparation device, thereby achieving fluid communication between a sample preparation chamber in the sample preparation device and the incubation chamber.

[0017] In some embodiments, the rupturable seal comprises a foil. In some embodiments, the sample receiving module comprises a luer connector for coupling a sample preparation tube to an assay device. In some embodiments, the sample preparation tube comprises a collar of the luer connector contacting the sample receiving module so as to form a leakproof seal between the sample preparation device and the sample receiving module when mated together. In some embodiments, the incubation chamber comprises a vent. In some embodiments, the vent is a selective vent element. In some embodiments, the selective vent element is a rigid or semi-rigid porous matrix comprising (e.g., embedded therein) a material that swells when in contact with a liquid. In some embodiments, the selective vent element is a self-sealing polymer comprising (e.g., embedded therein) a material that swells when in contact with a liquid. In some embodiments, the selective vent element is a self-sealing sintered polymer vent plug. In some embodiments, the selective vent element is a self-sealing fiber material comprising (e.g., embedded therein) a material that swells when in contact with a liquid. In some embodiments, the selective vent element is a self-sealing porous polyethylene vent comprising an embedded hydrogel. In some embodiments, the selective vent element is thermoplastic (e.g., temperature resistant). In some embodiments, the selective vent element is polytetrafluoroethylene or polyethersulfone. In some embodiments, the selective vent element comprises a hot melt material. In some embodiments, the selective vent element comprises a hydrophobic porous membrane. In some embodiments, the vent comprises a sensing channel in fluid communication with a fill detection chamber, the fill detection chamber being configured to detect liquid filling thereof.

[0018] In some embodiments, the thermal mixing module further comprises one or more sensors configured to i) detect the initial presence of liquid in the incubation chamber, ii) detect the liquid level in the incubation chamber, iii) or both. In some embodiments, the one or more sensors comprise a capacitive sensor disposed below the incubation chamber. In some embodiments, the one or more sensors comprise one or more bottom electrodes operatively connected to a circuit board and configured to penetrate a bottom wall of the incubation chamber so as to be exposed within the incubation chamber.

[0019] In some embodiments, the thermal mixing module comprises a light source, optionally comprising a light emitting diode (LED) or a laser.

[0020] In some embodiments, the system further comprises one or more top electrodes in operative communication with the circuit board and configured to penetrate a wall other than the bottom wall of the incubation chamber so as to be exposed within the incubation chamber and thereby detect a liquid level within the incubation chamber. In some embodiments, the thermal mixing module comprises an electrode socket comprising an electrode (e.g., a top electrode; see e.g., Figure 5 ). In some embodiments, a sensor of the one or more sensors is disposed within or otherwise operatively connected to the filling detection chamber (e.g., the sensor may be positioned within a sample receiving module but not within the filling detection chamber, however, the sensor may still detect light transmitted from and / or through the filling detection chamber). In some embodiments, the filling detection chamber is in fluid communication with the incubation chamber via a sensing channel, and the one or more sensors are configured to detect changes in light within the filling detection chamber. In some embodiments, the one or more sensors include a thermocouple coupled to the mixing heater and / or a portion of the incubation chamber so as to detect changes in the temperature of the mixing heater and / or the incubation chamber, thereby being associated with the presence of liquid within the incubation chamber. In some embodiments, the one or more sensors are operatively connected to the mixing heater. In some embodiments, the one or more sensors serve as an interlock device for the mixing heater such that the mixing heater is configured to be activated and / or deactivated based on detection of liquid and / or liquid level within the incubation chamber by the one or more sensors.

[0021] In some embodiments, the incubation chamber comprises a light source (eg, a light emitting diode (LED), a laser, etc.). As used herein, a "light source" refers to an element that is capable of solely illuminating a surface.

[0022] In some embodiments, the lysing agent comprises a lyophilized pellet. In some embodiments, the lysing agent comprises dithiothreitol (DTT), proteinase K, mutanolysin, lysostaphin, lysozyme, or a combination thereof. In some embodiments, the lysing agent comprises one or more surfactants. In some embodiments, the one or more surfactants comprise polysorbate. In some embodiments, the lysing agent comprises one or more components of a buffer solution. In some embodiments, the mixing heater is configured to heat the sample solution within the incubation chamber, thereby enabling the sample solution to be mixed with the lysing agent therein to form a prepared sample solution.

[0023] In some embodiments, the incubation chamber has one or more rounded edges. In some embodiments, the one or more rounded edges enable the sample solution to circulate or substantially circulate within the incubation chamber when receiving heat from the mixing heater. In some embodiments, the height of the incubation chamber is specified relative to the width of the incubation chamber to minimize and / or reduce the dead volume area within the incubation chamber. In some embodiments, the length of the incubation chamber is about 0.5 times to about 3 times the height of the incubation chamber. In some embodiments, the length of the incubation chamber is about 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, or 3.5 times the height of the incubation chamber. In some embodiments, the width of the incubation chamber is about 1 / 8 times to about 1.0 times the average of the length and width of the incubation chamber. In some embodiments, the width of the incubation chamber is about 1 / 8, 1 / 7, 1 / 6, 1 / 5, ¼, 1 / 3, 1 / 2 to about 1.0 times the average of the length and width of the incubation chamber. In some embodiments, the volume of the incubation chamber is about 0.1 mL to about 10 mL, such as about 0.5 mL to about 5 mL. In some embodiments, the volume of the incubation chamber is about 0.1 mL to 0.2 mL, 0.1 mL to 0.3 mL, 0.1 mL to 0.4 mL, 0.1 mL to 0.5 mL, 0.1 mL to 0.6 mL, 0.1 mL to 0.7 mL, 0.1 mL to 0.8 mL, or 0.1 mL to 0.9 mL. In some embodiments, the volume of the incubation chamber is about 0.1 mL to 1 mL, 0.1 mL to 2 mL, 0.1 mL to 3 mL, 0.1 mL to 4 mL, 0.1 mL to 5 mL, 0.1 mL to 6 mL, 0.1 mL to 7 mL, 0.1 mL to 8 mL, 0.1 mL to 9 mL, 0.1 mL to 10 mL. In some embodiments, the volume of the incubation chamber is about 0.5 mL to 1 mL, 0.5 mL to 2 mL, 0.5 mL to 3 mL, 0.5 mL to 4 mL, 0.5 mL to 5 mL, 0.5 mL to 6 mL, 0.5 mL to 7 mL, 0.5 mL to 8 mL, 0.5 mL to 9 mL, or 0.5 mL to 10 mL.In some embodiments, the volume of the incubation chamber is about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3 mL, 3.1 mL, 3.2 mL, 3.3 mL, 3.4 mL, 3.5 mL, 3.6 mL, 3.7 mL, 3.8 mL, 3.9 mL, 4 mL, 4.1 mL, 4.2 mL, 4.3 mL, 4.4 mL, 4.5 mL, 4.6 mL, 4.7 mL, 4.8 mL, 4.9 mL, 5 mL, 5.1 mL, 5.2 mL, 5.3 mL, 5.4 mL, 5.5 mL, 5.6 mL, 5.7 mL, 5.8 mL, 5.9 mL, 6 mL, 6.1 mL, 6.2 mL, 6.3 mL, 6.4 mL, 6.5 mL, 6.6 mL, 6.7 mL, 6.8 mL, 6.9 mL, 7 mL, 7.1 mL, 7.2 mL, 7.3 mL, 7.4 mL, 7.5 mL, 7.6 mL, 7.7 mL, 7.8 mL, 7.9 mL, 8 mL, 8.1 mL, 8.2 mL, 8.3 mL, 8.4 mL, 8.5 mL, 8.6 mL, 8.7 mL, 8.8 mL, 8.9 mL, 9 mL, 9.1 mL, 9.2 mL, 9.3 mL, 9.4 mL, 9.5 mL, 9.6 mL, 9.7 mL, 9.8 mL, 9.9 mL or 10 mL.

[0024] In some embodiments, the mixing heater is configured to heat the sample solution within the incubation chamber for a specified amount of time. In some embodiments, the specified amount of time is about 5 minutes. In some embodiments, the specified amount of time is about 30 seconds to about 20 minutes. In some embodiments, the specified amount of time is about 1 minute to about 10 minutes. In some embodiments, the specified amount of time is about 1 minute to 5 minutes or about 5 minutes to 10 minutes. In some embodiments, the prescribed amount of time is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 seconds. In some embodiments, the prescribed amount of time is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the prescribed amount of time is about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, or 25 minutes. In some embodiments, the prescribed amount of time is about 1 minute to 2 minutes, 1 minute to 3 minutes, 1 minute to 4 minutes, 1 minute to 5 minutes, 1 minute to 6 minutes, 1 minute to 7 minutes, 1 minute to 8 minutes, 1 minute to 9 minutes, 1 minute to 10 minutes, 1 minute to 11 minutes, 1 minute to 12 minutes, 1 minute to 13 minutes, 1 minute to 14 minutes, 1 minute to 15 minutes, 1 minute to 16 minutes, 1 minute to 17 minutes, 1 minute to 18 minutes, 1 minute to 19 minutes, or 1 minute to 20 minutes. In some embodiments, the prescribed amount of time is about 5 minutes to 6 minutes, 5 minutes to 7 minutes, 5 minutes to 8 minutes, 5 minutes to 9 minutes, 5 minutes to 10 minutes, 5 minutes to 11 minutes, 5 minutes to 12 minutes, 5 minutes to 13 minutes, 5 minutes to 14 minutes, 5 minutes to 15 minutes, 5 minutes to 16 minutes, 5 minutes to 17 minutes, 5 minutes to 18 minutes, 5 minutes to 19 minutes, or 5 minutes to 20 minutes. In some embodiments, the prescribed amount of time is about 10 minutes to 11 minutes, 10 minutes to 12 minutes, 10 minutes to 13 minutes, 10 minutes to 14 minutes, 10 minutes to 15 minutes, 10 minutes to 16 minutes, 10 minutes to 17 minutes, 10 minutes to 18 minutes, 10 minutes to 19 minutes, or 10 minutes to 20 minutes.

[0025] In some embodiments, the wax valve channel is located at the end of the incubation chamber opposite to the sample receiving module. In some embodiments, the heat seal valve comprises wax. In some embodiments, the wax is water soluble. In some embodiments, the wax comprises a water soluble polymer. In some embodiments, the heat seal valve comprises a polymer. In some embodiments, the polymer is water soluble. In some embodiments, the polymer comprises polyethylene glycol (PEG). In some embodiments, the molecular weight of the heat seal valve is about 1,300 g / mol to about 10,000 g / mol. In some embodiments, the molecular weight of the heat seal valve is about 6,000 g / mol.

[0026] In some embodiments, the melt temperature of the heat-sealed valve is about 40° C. to about 75° C. In some embodiments, the melt temperature of the heat-sealed valve is about 40° C. to about 45° C., about 40° C. to about 50° C., about 40° C. to about 55° C., about 40° C. to about 60° C., about 40° C. to about 65° C., about 40° C. to about 70° C., about 40° C. to about 75° C., or about 40° C. to about 80° C. In some embodiments, the melt temperature of the heat-sealed valve is about 35° C. to about 40° C., about 35° C. to about 45° C., about 35° C. to about 50° C., about 35° C. to about 55° C., about 35° C. to about 60° C., about 35° C. to about 65° C., about 35° C. to about 70° C., about 35° C. to about 75° C., or about 35° C. to about 80° C. In some embodiments, the melt temperature of the heat-sealed valve is about 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C. In some embodiments, the melt temperature of the heat-sealed valve is about 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C.

[0027] In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 2 uL to about 6 uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 3 uL to about 5 uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 3 uL to about 4 uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 2 uL to about 5 uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 2 uL to about 4 uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 2 uL to about 3 uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 2uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 3 uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 4 uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 5 uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 6 uL. In some embodiments, the volume of the heat-sealed valve within the wax valve channel is about 7 uL. In some embodiments, the wax valve channel comprises a valve fill port for receiving the valve therein. In some embodiments, the heat-sealed valve is solid or substantially solid at a first temperature to help prevent the sample solution from flowing through the wax valve channel. In some embodiments, the heat-sealed valve is configured to transition from a solid or substantially solid configuration to a soft, dissolving and / or molten configuration upon receiving sufficient heat.

[0028] In some embodiments, the system further comprises a valve heater, the valve heater being configured to heat the heat-sealed valve, thereby enabling the heat-sealed valve to be softened, dissolved and / or melted to allow the sample solution to flow therethrough. In some embodiments, the entrance from the incubation chamber to the wax valve channel comprises one or more converging walls. In some embodiments, the system further comprises one or more thermally conductive pads, the thermally conductive pads being operatively coupled to the mixing heater, the valve heater, or both. In some embodiments, the one or more thermally conductive pads (e.g., thermal gap pads) comprise valve thermally conductive pads, the valve thermally conductive pads being configured to transfer heat from the valve heater to the wax valve channel to heat the heat-sealed valve. The thermally conductive pads (e.g., thermal gap pads) allow the distance between the substrate and the heat-sealed valve or incubation chamber to be changed by filling space and conducting heat.

[0029] In some embodiments, the liquid level of the sample solution in the incubation chamber is located at a higher height than the wax valve channel, so that the heat-sealed valve is under hydrostatic pressure from the sample solution. In some embodiments, the sample preparation tube is positioned at a higher height than the heat-sealed valve or the wax valve channel, so that the heat-sealed valve is under hydrostatic pressure from the sample solution. In some embodiments, the heat-sealed valve is configured to dissolve into the sample solution. In some embodiments, the prepared sample solution is configured to enter at least one of the one or more reaction chambers after passing through the wax valve channel. In some embodiments, the dissolved heat-sealed valve enters at least one reaction chamber together with the prepared sample solution.

[0030] In some embodiments, the system further comprises an isolation chamber located downstream of the wax valve channel and upstream of the one or more reaction chambers, wherein the isolation chamber is configured to receive an initial flow of the prepared sample solution and the heat-sealed valve dissolved therein so as to reduce the amount of the dissolved heat-sealed valve found in the one or more reaction chambers. In some embodiments, the isolation chamber comprises an exhaust port. In some embodiments, the chamber has an outlet and is indirectly connected to the exhaust port.

[0031] In some embodiments, the system further comprises one or more mixing chambers (eg, shuttle mixing chambers) to improve distribution of the dissolved heat-sealed valve across the one or more reaction chambers.

[0032] In some embodiments, the system further comprises a substrate operatively connected to the thermal mixing module, the wax valve channel and / or the OPM module. In some embodiments, the substrate comprises a printed circuit board. In some embodiments, the mixing heater and / or the valve heater are placed on the substrate. In some embodiments, the substrate further comprises a power supply operatively connected to the mixing heater and / or the valve heater. In some embodiments, the substrate comprises a controller to regulate the power supplied to the mixing heater and / or the valve heater so as to substantially maintain the mixing heater and / or the valve heater at a predetermined temperature. In some embodiments, the power supply is configured to supply power to the mixing heater and / or the valve heater at a substantially constant rate. In some embodiments, the substrate comprises a thermal gap pad.

[0033] In some embodiments, the preparation solution is a nucleic acid amplification preparation solution. In some embodiments, the preparation solution further comprises an optical property modification agent. In some embodiments, the liquid sample (e.g., a biological sample) comprises human saliva, urine, human mucus, vaginal fluid, semen, blood, oral lavage or solid tissue such as cheek tissue, bacteria, one or more spores, one or more viruses, or a combination thereof and / or a concentrate thereof.

[0034] In some embodiments, the OPM module comprises a reaction chamber channel in fluid communication with the wax valve channel, wherein the one or more reaction chambers are in fluid communication with the reaction chamber channel through corresponding branches. In some embodiments, each reaction chamber is substantially equidistant from a single sensing region disposed in the OPM module. In some embodiments, the OPM module further comprises a first plurality of light pipes, each first light pipe being capable of transmitting light between one of the one or more reaction chambers and the single sensing region. In some embodiments, the OPM module further comprises a reaction heater configured to heat the one or more reaction chambers.

[0035] In some embodiments, the assay reagent comprises a dried or lyophilized reagent. In some embodiments, the assay reagent comprises a nucleic acid amplification enzyme and a DNA primer.

[0036] In another aspect, the present disclosure provides a method for determining one or more characteristics of a nucleic acid amplification sample based on modified optical properties of a liquid sample (e.g., a biological sample), the method comprising:

[0037] a. providing the liquid sample (eg, a biological sample) comprising nucleic acid;

[0038] b. combining the liquid sample (eg, a biological sample) with a preparation solution comprising a buffer solution and / or an optical property modifying reagent solution to produce a sample solution;

[0039] c. dispensing the sample solution into the incubation chamber;

[0040] d. mixing the sample solution with the lysing agent using a mixing heater to apply heat to the incubation chamber to achieve thermal mixing, thereby forming a prepared sample solution;

[0041] e. heating a heat-sealed valve disposed in a wax valve channel in fluid communication with the incubation chamber so as to allow the prepared sample solution to flow through the wax valve channel to one or more reaction chambers containing assay reagents, wherein the prepared sample solution is mixed with the assay reagents to form a reaction mixture;

[0042] f. heating the reaction mixture to promote a nucleic acid amplification reaction using the nucleic acid and the assay reagent present in the liquid sample (eg, a biological sample), the reaction producing an amplified nucleic acid and a plurality of protons;

[0043] g. reacting the protons with the optical property modifying agent, wherein the reaction is capable of modifying an optical property of the optical property modifying agent to allow detection of the modified optical property, thereby indicating the presence of a suspected analyte in the liquid sample (eg, a biological sample); and

[0044] h. causing a plurality of light emitting elements to emit light in a repeating pattern at a repetition frequency so as to determine one or more characteristics of the liquid sample (eg, a biological sample) using a photosensor based on the modified optical properties.

[0045] In some embodiments, the method further comprises displaying the determined characteristic using an electronic results display mechanism.

[0046] In some embodiments, providing the liquid sample (e.g., a biological sample) comprises performing a nasal swab on the subject, performing a tonsil and / or throat swab on the subject, performing a vaginal swab on the subject, obtaining a hair sample from the subject, drawing blood from the subject, obtaining a urine sample from the subject, or a combination thereof.

[0047] In some embodiments, the liquid sample (eg, biological sample) and the preparation solution are combined within a sample preparation device.

[0048] In some embodiments, the method further comprises providing a system as described in the present disclosure.

[0049] In some embodiments, dispensing the sample solution into the incubation chamber comprises coupling the sample preparation device to the sample receiving module to establish a fluid pathway between the sample preparation device and the incubation chamber.

[0050] In some embodiments, the method further comprises rupturing and / or breaking a rupturable seal on the sample preparation device to enable the sample solution to flow from the sample preparation device to the incubation chamber.

[0051] In some embodiments, the method further comprises maintaining the mixing heater in a deactivated state until the sample solution is detected within the incubation chamber and / or until a minimum level of the sample solution within the incubation chamber is detected.

[0052] In some embodiments, a sensor is used to detect the sample solution in the incubation chamber and / or until a minimum level of the sample solution in the incubation chamber is detected. In some embodiments, the sample solution is mixed with the lysing agent for a specified amount of time. In some embodiments, the specified amount of time is about 1 minute to about 20 minutes.

[0053] In some embodiments, the method further comprises regulating the mixing heater based on i) a constant or substantially constant power supplied to the mixing heater by a power supply, or ii) maintaining a constant or substantially constant temperature of the mixing heater or a portion of the incubation chamber. In some embodiments, heating the heat-sealed valve comprises activating the valve heater after the specified amount of time. In some embodiments, heating the heat-sealed valve causes the heat-sealed valve to soften, melt, and / or dissolve, wherein the dissolving is performed within the prepared sample solution. In some embodiments, the heat-sealed valve is any valve described herein.

[0054] In some embodiments, the method further comprises isolating an initial volume of the prepared sample solution and a dissolved heat-sealed valve in an isolation chamber upstream of the one or more reaction chambers.

[0055] Another aspect of the present disclosure provides a method for preparing a heat-sealed valve system, the method comprising:

[0056] a. dispensing molten or dissolved wax into the wax valve channel through the valve fill port;

[0057] b. sealing the valve fill port; and

[0058] c. Drying or cooling the wax valve passage to solidify or substantially solidify the wax.

[0059] In some embodiments, the wax is dispensed into the wax valve channel using a wax dispenser. In some embodiments, sealing the valve fill port comprises using a polymer, a stopper, a plug, a hot melt and / or a pressure sensitive adhesive of the port.

[0060] Another aspect of the present disclosure provides a kit for performing a biological assay, the kit comprising:

[0061] a. Sample preparation equipment; and

[0062] b. A measuring device, comprising:

[0063] i. A thermal mixing module, the thermal mixing module comprising:

[0064] a sample receiving module for receiving a sample solution comprising a liquid sample (eg, a biological sample) and a preparation solution; and

[0065] An incubation chamber in fluid communication with the sample receiving module and containing a lysing agent;

[0066] ii. A wax valve channel in fluid communication with the incubation chamber;

[0067] iii. an optical property modification (OPM) module, the OPM module being operatively coupled to the incubation chamber via the wax valve channel, the OPM module comprising one or more reaction chambers, each reaction chamber comprising an assay reagent;

[0068] iv. a heat-sealed valve disposed i) within the wax valve channel and ii) between the sample receiving module and the OPM module; and

[0069] v. A mixing heater configured to supply heat to the incubation chamber.

[0070] In some embodiments, the assay device further comprises a valve heater. In some embodiments, the assay device further comprises a reaction heater.

[0071] In some embodiments, the sample preparation device comprises a sample collector configured to obtain a liquid sample (eg, a biological sample), and a tube containing a preparation solution and configured to receive at least a portion of the sample collector.

[0072] In some embodiments, the sample collector comprises a nasal swab, a throat and / or tonsil swab, a vaginal swab, or a combination thereof. In some embodiments, the assay device comprises any of the features described in the systems or methods of the present disclosure.

[0073] In one aspect, the present disclosure provides a kit comprising an assay device and / or a sample preparation device as disclosed herein or a component thereof. In some embodiments, the kit comprises instructions for use or a QR code or a bar code to provide instructions for use to the user. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1A and 1B A perspective view of an exemplary system for performing a biological assay having a sample preparation device and an assay device according to embodiments described herein is provided. Figure 1C A side view of an exemplary assay device having a thermal mixing module, an optical property modification (OPM) module, and a printed circuit board (PCB) according to embodiments described herein is provided. Figure 1DA perspective view of an exemplary system for performing a biological assay having a sample preparation device and an assay device according to embodiments described herein is provided. Figure 1E A side view of an exemplary assay device having a thermal mixing module, an optical property modification (OPM) module, and a printed circuit board (PCB) according to embodiments described herein is provided.

[0075] Figure 2A A top view of the bottom of an assay device according to embodiments described herein is provided. Figure 2B A top view of the bottom of an assay device according to an alternative embodiment described herein is provided.

[0076] Figure 3A A side cross-sectional view of an image depicting a sample preparation device coupled to an assay device is provided according to embodiments described herein. Figure 3B A side cross-sectional view of an image depicting a sample preparation device coupled to an assay device is provided according to an alternative embodiment described herein.

[0077] Figure 4 An exemplary side cross-sectional view of an image depicting a coupled sample receiving module and sample preparation device is provided according to embodiments described herein.

[0078] Figure 5 Another exemplary side cross-sectional view of an image depicting a sample preparation device coupled to an assay device showing top and bottom electrodes according to embodiments described herein is provided.

[0079] Figure 6 An exemplary side cross-sectional view of an image depicting an assay device showing an incubation chamber, wax valve channel, and thermal pad of a valve heater is provided according to embodiments described herein.

[0080] Figure 7 Graphs of chamber heater and wall temperature profiles based on various fill conditions are provided.

[0081] Figure 8 An exemplary front view of an image of a wax dispenser device according to embodiments described herein is provided.

[0082] Fig. 9 An exemplary process flow for filling a wax valve channel with wax according to embodiments described herein is provided.

[0083] Fig.10 An exemplary process flow for adding fluid to an incubation chamber and dissolving a heat seal valve material upon application of heat according to embodiments described herein is provided.

[0084] Fig.11An exemplary flow chart of a method for performing a bioassay using the system described herein is provided according to embodiments described herein.

[0085] Fig.12 A graphical representation of concentrations of dissolved valve material detected in a plurality of reaction chambers according to embodiments described herein is provided.

[0086] Fig.13 A graphical representation of dissolved valve material detected in multiple reaction chambers with and without an isolation chamber is provided.

[0087] Fig.14 A top view of a test apparatus used to evaluate the actuation temperature and movement of heat-sealed valve materials in a channel is provided.

[0088] Fig.15 A graphical representation of the actuation temperature of a heat seal valve material (eg, PEG) under different environmental conditions is provided.

[0089] Fig.16 and 17 Each provides a top view of a sample receiving module according to embodiments described herein, showing two different wax valve channel geometries.

[0090] Fig.18 A top view of an exemplary fill detection chamber having a light source and a photosensor to detect liquid entry / chamber filling is provided.

[0091] Fig.19 A top view of an aluminum cylinder secured by three extruded ribs is provided. DETAILED DESCRIPTION

[0092] Provided herein are systems and methods for performing bioassays. The systems and methods determine one or more characteristics of a nucleic acid amplification sample based on the modified optical properties of the eluted sample. The systems and methods herein utilize a heat-sealed valve comprising wax to isolate the contents of an incubation chamber and a downstream reaction chamber. The wax is dissolved to allow the fluid between the incubation chamber and the downstream reaction chamber to be connected.

[0093] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, because these specific embodiments can certainly vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be restrictive, because the scope of the present invention will only be limited by the appended claims.

[0094] Where a range of values ​​is provided, it is understood that, unless the context clearly indicates otherwise, each intervening value (to one-tenth of the unit of the lower limit) between the upper and lower limits of the range, as well as any other stated or intermediate values ​​within the range, are encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also encompassed within the disclosure, subject to any explicitly excluded limits within the stated ranges. Where the stated range includes one or both of the limits, ranges excluding either or both of the included limits are also encompassed within the disclosure.

[0095] Certain ranges can be presented herein with numerical values ​​guided by the term "about". Unless otherwise noted, as used in this specification and claims, the terms "about" and "approximately" refer to a change of less than or equal to + / -1%, + / -2%, + / -3%, + / -4%, + / -5%, + / -6%, + / -7%, + / -8%, + / -9%, + / -10%, + / -11%, + / -12%, + / -14% or + / -15%, depending on the embodiment. As a non-limiting example, depending on the embodiment, about 100 meters represents a range of 95 to 105 meters, 90 to 110 meters or 85 to 115 meters.

[0096] The term "substantially" refers to a variation of less than or equal to + / -1%, + / -2%, + / -3%, + / -4%, + / -5%, + / -6%, + / -7%, + / -8%, + / -9%, + / -10%, + / -11%, + / -12%, + / -14%, or + / -15%. As non-limiting examples, depending on the embodiment, substantially parallel means a range of -1 to 1 degree, -5 to 5 degrees, or -15 to 15 degrees from parallel.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials will now be described.

[0098] All publications and patents cited in this specification are incorporated herein by reference, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in conjunction with the cited publications. Reference to any publication is for its disclosure prior to the filing date, and should not be construed as an admission that the present invention is not entitled to antedate such publication by prior invention. Further, the publication date provided may be different from the actual publication date, which may need to be independently confirmed.

[0099] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for using such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements or for using a "negative" limitation.

[0100] In addition, certain embodiments of the disclosed devices and / or related methods may be represented by drawings that may be included in the present application. Embodiments of the device and their specific spatial characteristics and / or capabilities include embodiments and characteristics and / or capabilities shown or substantially shown in the drawings or that can be reasonably inferred from the drawings. Such characteristics include, for example, one or more of the following (e.g., one, two, three, four, five, six, seven, eight, nine, or ten, etc.): symmetric about a plane (e.g., a cross section) or an axis (e.g., an axis of symmetry), an edge, a periphery, a surface, a specific direction (e.g., a proximal side; a distal side) and / or a number (e.g., three surfaces; four surfaces) or any combination thereof. Such spatial characteristics also include, for example, lacking (e.g., specifically not existing) one or more of the following (e.g., one, two, three, four, five, six, seven, eight, nine, or ten, etc.): symmetric about a plane (e.g., a cross section) or an axis (e.g., an axis of symmetry), an edge, a periphery, a surface, a specific direction (e.g., a proximal side) and / or a number (e.g., three surfaces) or any combination thereof.

[0101] As will be clear to those skilled in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the invention. Any enumerated method can be executed in the order of the enumerated events or in any other logically possible order.

[0102] In further describing the present invention, the inventive apparatus for practicing the inventive system will be discussed in more detail, followed by a review of related methods.

[0103] definition

[0104] Unless otherwise indicated, the terms used in the claims and specification are defined as follows. Unless otherwise defined, all technical terms, symbols and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art. In some cases, for clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein indicates a difference from the meanings commonly understood in the art.

[0105] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "including," "such as," and the like are intended to express inclusion rather than limitation, unless specifically stated otherwise.

[0106] As used herein, the term "comprising" also specifically includes embodiments of "consisting of" and "consisting essentially of" the recited elements, unless specifically stated otherwise.

[0107] The term "colorimetry" or "colorimetry" refers to a technique for quantifying or otherwise observing the concentration of a colored compound in a solution. "Colorimetric detection" refers to any method for detecting such colored compounds in solution and / or color changes of said compounds. Methods may include visual observation, absorbance measurement, or fluorescence measurement, among others.

[0108] The term "acid developer" refers to a composition that changes color in a chemical reaction. Specifically, an acid developer may refer to a composition that changes color as pH changes. Different acid developers may change color within different pH transition ranges.

[0109] The term "transition pH range" or "pH transition range" refers to the pH range over which the color of a particular sample or compound changes. The specific transition pH range of a sample may depend on the acid-added chromogenic agent in the sample (see above).

[0110] The term "nucleic acid amplification" or "amplification reaction" refers to a method of amplifying DNA, RNA, or modified versions thereof. Nucleic acid amplification includes several techniques, such as isothermal reactions or thermal cycling reactions. More specifically, nucleic acid amplification includes methods such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), and nucleic acid sequence-based amplification (NASBA). The term "isothermal amplification" refers to amplification methods that are performed without changing the temperature of the amplification reaction. Protons are released during the amplification reaction: During the amplification reaction, a proton (H) is released for each deoxynucleotide triphosphate (dNTP) added to the single-stranded DNA template. + ).

[0111] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to modulate protein aggregation in a cell.

[0112] As used herein, liquid sample can refer to biological sample." biological sample " refers to a sample containing a certain amount of organic matter, such as one or more organic molecules, such as one or more nucleic acids, such as DNA and / or RNA or parts thereof, which can be collected from a subject. Therefore, "biological sample determination" is to test a biological sample to assess one or more characteristics of the sample. In some aspects, a biological sample is a nucleic acid amplification sample, which is a sample comprising or suspected of comprising one or more nucleic acids or parts thereof that can be amplified according to an embodiment.

[0113] A biological sample can be provided by a subject and include one or more cells, such as tissue cells of the subject. As used herein, the term "tissue" refers to one or more cell aggregates having similar functions and structures in a subject (e.g., a living organism, such as a mammal (e.g., a human)), or to multiple different types of such aggregates. Tissues can include, for example, organ tissue, muscle tissue (e.g., cardiac muscle, smooth muscle and / or skeletal muscle), connective tissue, neural tissue, and / or epithelial tissue. In some versions, tissues can include cells from the inside of a subject's cheek and / or cells in the subject's saliva. In some embodiments, when biological samples are referred to herein, liquid samples are contemplated.

[0114] In some embodiments, the biological sample is a swab of body fluid (mucus, vaginal fluid, semen, urine, saliva, etc.). In some embodiments, the biological sample is a tonsil / throat swab. In some embodiments, the biological sample is a nasal swab. In some embodiments, the biological sample is a nasopharyngeal swab. In some embodiments, the biological sample is a vaginal swab.

[0115] As mentioned above, biological samples can be provided by subjects. In certain embodiments, subjects are "mammals" or "mammal-like" subjects, wherein these terms are widely used to describe organisms within mammals, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs and rats) and primates (e.g., humans, chimpanzees and monkeys). In certain embodiments, subjects are people. The term "people" can include human subjects of both sexes and in any growth stage (e.g., fetuses, newborns, infants, teenagers, adolescents and adults), wherein in certain embodiments, the human subjects are teenagers, adolescents or adults. Although the device and method described herein can be used in conjunction with human subjects, it should be understood that the device and method of the present invention can also be used in conjunction with other subjects, i.e., applied to "non-human subjects".

[0116] Liquid samples (eg, biological samples) may include, for example, human saliva, urine, human mucus, blood, oral lavage, or solid tissue such as cheek tissue. Biological samples may also include viruses, bacteria, and / or spores.

[0117] In some embodiments, the biological sample is mixed with a preparation solution. In some embodiments, the preparation solution comprises a nucleic acid amplification preparation solution. In some embodiments, the preparation solution comprises an elution buffer. In some embodiments, the preparation solution may alternatively or additionally include an optical property modification reagent. According to some embodiments, when the biological sample or one or more aspects thereof are exposed to an optical property modification reagent, the optical property of the optical property modification reagent changes due to the presence or absence of a specific marker in the biological sample. Examples of optical properties that can be changed include color and absorbance as measured by spectrophotometry. Changes in optical properties can be detected and used to identify the properties of the biological sample. In some embodiments, cells of the sample are lysed and / or nucleic acids are extracted from the sample using a lysing agent of the preparation solution. Such extracted nucleic acids can be released into the resulting prepared sample solution. In some embodiments, a step of extracting genomic deoxyribonucleic acid (DNA) from the biological sample is included. If desired, the preparation solution is a nucleic acid amplification preparation solution, and the nucleic acid of the sample exposed to the solution is prepared for amplification, such as isothermal amplification.

[0118] Furthermore, as used herein, the phrase "optical property" refers to one or more optically discernible characteristics, such as color, absorbance, reflectance, scattering, fluorescence, phosphorescence, etc., resulting from the wavelength and / or frequency of radiation (e.g., light) emitted or transmitted by a sample before, during, or after an assay reaction performed using the sample. Thus, modifying an optical property refers to changing such a property.

[0119] system

[0120] Aspects of the present disclosure include systems for performing biological assays by modifying optical properties of biological samples and detecting these modified properties.Such systems can include various devices, including sample preparation devices and assay devices.

[0121] In some embodiments, the sample preparation device is configured to prepare a biological sample before being dispensed into an assay device. In some embodiments, a sample collector (e.g., a nasal swab, etc.) is used to collect the biological sample. In some embodiments, the biological sample is mixed with a preparation solution to form a sample solution. In some embodiments, the preparation solution comprises a nucleic acid amplification preparation solution. In some embodiments, the preparation solution comprises an elution buffer. In some embodiments, the preparation solution comprises an optical property modification reagent. In some embodiments, when the biological sample or one or more aspects thereof is exposed to the optical property modification reagent, the optical property of the optical property modification reagent changes due to the presence or absence of a specific marker in the biological sample. In some embodiments, the sample solution is configured to be dispensed into an assay device.

[0122] In some embodiments, the assay device comprises one or more thermal mixing modules and / or one or more optical property modification modules. In some embodiments, the thermal mixing module and the optical property modification module are fluidly connected via a wax valve channel therebetween. In some embodiments, the biological assay is performed on a biological sample received by the thermal mixing module (e.g., the biological sample can be provided in a sample solution). In some embodiments, the assay device is configured to incubate the biological sample for a specified period of time before transferring it to the optical modification module for one or more reactions, as described herein.

[0123] In some embodiments, a heat-sealed valve located within a valve channel provides a barrier to prevent a biological sample (and a corresponding sample solution) from flowing toward the optical modification module. In some embodiments, the heat-sealed valve is configured to soften, melt, and / or dissolve upon receiving sufficient heat so that the sample solution can be pushed through the valve and flow through the channel into the optical property modification module. In some embodiments, as described herein, such incubation of the sample allows the sample to be mixed with a lysing agent, wherein such incubation and mixing helps to lyse cells and release biological material to help promote reactions in the optical modification module. In some embodiments, the mixing of the sample with the lysing agent is performed by thermal mixing. The heat-sealed valve described herein can also be used in alternative mixing chambers that do not involve cell lysis (e.g., eluting the sample with a reagent).

[0124] In some embodiments, the optical property modification module comprises one or more reaction chambers, which may optionally have assay reagents (e.g., optical property modification reagents, nucleic acid amplification reagents, or both) therein to mix and react with the sample in the prepared sample solution. In some embodiments, the reaction (e.g., an amplification reaction) provides a reaction product that reacts with the optical property modification reagent to produce a detectable change in the optical property of the biological sample to indicate the presence, absence, or amount of an analyte suspected of being present in the sample.

[0125] In some embodiments, the assay reagents are present in a preparation solution. In some embodiments, the assay reagents are present in an incubation chamber. In some embodiments, the assay reagents are present in one or more reaction chambers.

[0126] In some embodiments, each reaction chamber may include a sample receiving opening for receiving a biological sample from a sample inlet and / or a conduit. The sample receiving opening may be operatively (e.g., fluidly) connected to the sample inlet. In some versions, each reaction chamber includes one or more (e.g., two) additional openings, such as an "exhaust" and a "replenishment," or a "first" and a "second" opening. In some embodiments, a channel downstream of the wax valve channel 110 leads to one or more conduits that branch from the channel (e.g., like spokes) and connect the channel to the reaction chamber fluid. In some embodiments, each conduit is connected to a different reaction chamber.

[0127] In certain embodiments, each reaction chamber comprises a selective exhaust element as described herein. These selective exhaust elements are configured to allow any gas (e.g., air) and / or steam to be discharged from the reaction chamber when the sample solution enters the corresponding reaction chamber. Once the selective exhaust element is made into an impermeable fluid, the method includes preventing the fluid from further flowing through the device, or once the fluid contacts the selective exhaust element, preventing it from flowing back out of the reaction chamber, making it impermeable to the fluid. Therefore, after such flow stops, diffusion is the only way to transport any pollutants into and / or out of the reaction chamber. Therefore, if the inlet and / or conduit have sufficient length, the pollutant diffusion time is substantially longer than the reaction and / or read time, and the result is not affected by the pollutants.

[0128] When miniaturizing and automating biochemical protocols into microfluidic systems, a challenge is how to accurately divide samples into multiple reaction chambers while minimizing cross contamination. In some embodiments, this is achieved by using multiple reaction chambers at the end of the conduit branching from the main fluid channel. Before reactions can occur in the reaction chambers, the aliquots must be isolated so that there is no crosstalk between the reactions. In some embodiments, sample isolation can be achieved by placing input and / or output valves between each chamber. The valve seals the chamber to prevent any crosstalk. Although it is feasible to a certain extent to use multiple valves, such schemes require active control of the opening and closing of the valves, which in turn requires energy, infrastructure and expenses for implementation, and thereby complicates the system design of the OPM module. In addition, some valve structures work best when filling, and therefore need to fill the microfluidic system with an initial filling liquid. Such filling steps complicate the system workflow. Therefore, according to a version of the method of the present invention, the method does not include filling.

[0129] In contrast, the devices and methods of the present invention can fully provide automatic fluid flow control by passively aliquoting through one or more parts of the device so that the assay can be performed. For example, one or more fluids (e.g., air and / or biological samples) can move through and / or be prevented from moving through one or more parts of the device with little or no specific user interaction. The passive sealing of the device or its parts eliminates the need for active control and minimizes the complexity of the entire device and the user steps required to run the device. Therefore, the present disclosure provides a simple and easy-to-use assay device. Such devices and methods of the present invention do not require valves or complex valve control schemes. Therefore, the present disclosure provides a simple and robust implementation of an on-chip aliquoting function without moving parts. According to embodiments of the present invention, the aliquoted sample volume and number are controlled by the channel and chamber geometry (e.g., the length and cross-section of the channel between the sample receiving openings of the reaction chamber are long enough and small enough, respectively, to prevent crosstalk between the contents of the reaction chamber). For example, in some embodiments, the cross-sectional area in each reaction chamber channel does not exceed 1 / 10 of the reaction chamber.

[0130] In some embodiments, the measuring device comprises a substrate. The substrate may include one or more control units, such as a central processing unit (CPU) or a field programmable gate array (FPGA). Such units may include a memory and / or a processor, such as a microprocessor, which is configured to generate one or more outputs (e.g., electrical signals) based on one or more sets of inputs (e.g., inputs from a user and / or a sensor and / or a timer) and / or instructions stored in the memory. The device may also include a user interface for receiving inputs and operatively connected to the control unit. In some embodiments, the substrate includes a printed circuit board (PCB) disposed below a thermal mixing module, a channel, and / or an optical property modification module. In some embodiments, the substrate includes one or more heaters to supply heat to the measuring device. For example, the substrate may also include one or more thermal gap pads to connect a heating element on the PCB to an appropriate fluid position.

[0131] In some embodiments, the sample preparation device is configured to be operatively coupleable with an assay device.

[0132] As used herein, "operably coupled," "operably connected," and "operably attached" mean connected in a particular manner that allows the disclosed device to effectively operate and / or perform the method in the manner described herein. For example, operationally coupled can include removably coupling or fixedly coupling two or more aspects. Operationally coupled can also include fluidically and / or electrically and / or matably and / or adhesively coupling two or more components. Thus, an operationally coupleable device is a device that is capable of being operationally coupled. In addition, as used herein, "removably coupled" means coupled in a manner, such as physically and / or fluidly and / or electrically coupled, wherein two or more coupled components can be decoupled and then repeatedly recoupled.

[0133] Measuring device

[0134] Figure 1A and 1B An exemplary embodiment of a system 100 for performing a biological assay on a biological sample as described herein is depicted. In some embodiments, the system includes an assay device 102 coupled to a sample preparation device 104. As used herein, "assay device" and test cartridge may be used interchangeably. In some embodiments, the assay device includes a thermal mixing module 106 and an optical property modification ("OPM") module 108, as described herein. In some embodiments, the sample preparation device 104 is used to elute the biological sample to prepare a sample solution 107. In some embodiments, the thermal mixing module 106 and the OPM module 108 are formed as a single integral piece on the assay device 102. In some embodiments, the assay device 102 includes a top portion 103 and a bottom portion 105 configured to be operatively coupled. In some embodiments, the top portion 103 and the bottom portion 105 are spaced apart along a parting line of the assay device (see, e.g., Figure 6 In some embodiments, the top and bottom of the device may be sandwiched with a gasket made of an elastomer or other soft material to form a fluid seal. Figure 1C A side view of an exemplary embodiment of an assay device 102 is depicted. Figure 1D and 1E , which has a flat or low-profile exhaust port on the incubation chamber of the thermal mixing module 106. Figure 2A A top view of a bottom portion 105 of an embodiment of an exemplary assay device 102 is depicted. As described herein, in some embodiments, the assay device 102 includes a thermal mixing module 106 having an incubation chamber 112 therein, an OPM 108, and a channel 110 disposed therebetween. The term "incubation chamber" may be used interchangeably herein with "mixing chamber." Figure 2B A similar assay device 102 is depicted without the isolation chamber 158 and the filled detection chamber 126 .

[0135] In some embodiments, Figure 1C As shown, the assay device 102 further includes a substrate 119 operatively coupled to the bottom 105 of the thermal mixing module 106, the channel 110, and the OPM module 108. In some embodiments, as described herein, the substrate 119 includes a PCB 120. In some embodiments, the substrate 119 includes one or more heaters, each heater being operatively coupled to a power source (e.g., a single power source can power all heaters, or one or more power sources can power the heaters). In some embodiments, when there are multiple heaters, only one heater is energized at a time. In some embodiments, as described herein, the substrate 119 includes a mixing heater aligned with the thermal mixing module 106, a valve heater aligned with the channel 110, and / or a reaction heater aligned with the OPM module 108. For example, in some embodiments, the substrate 119 includes one or more thermal gap pads to couple a heating element on the PCB to an appropriate fluid location.

[0136] refer to Figure 3A and 3B , depicts a side cross-sectional image of an assay device 102, according to embodiments described herein. In some embodiments, the thermal mixing module 106 includes a sample receiving module 114 and an incubation chamber 112. In some embodiments, the sample receiving module 112 is configured to be operatively coupled to the sample preparation device 104 to receive a sample therefrom (as described herein). In some embodiments, the sample receiving module 112 includes a Luer connector 116 defining an opening therein to a sample inlet 118, which is in fluid communication with the incubation chamber 112. In some embodiments, the Luer connector 116 is configured to be operatively coupled to the sample preparation device 104 to facilitate receiving a sample into the assay device, such as through or around a puncture element. For example, Figure 3A and 3B An exemplary connection of the sample preparation device 104 to the Luer connector 116 is provided. The sample preparation device can be envisioned as a tube, vial, syringe, or other container. In some embodiments, the piercing element 121 is configured to rupture a rupturable seal of the sample preparation device to allow fluid to flow therefrom. As used herein, a "rupturable seal" refers to a member used to seal an opening at the sample receiving module end that can be ruptured. Other members for rupturing the rupturable seal are envisioned in the devices of the present disclosure, for example, the rupturable seal can be punctured, pierced, penetrated, prodded, sliced, cut, etc. Reference Figure 4, shows an exemplary depiction of the interaction between the Luer connector 116 and the sample preparation device 104, wherein the Luer connector 116 is configured to cooperate with the collar 122 of the sample preparation device, thereby forming a leak-proof interface. The collar 122 can further include ribs that form a stronger seal between the Luer connector 116 and the collar 122. In some embodiments, the sample receiving module further includes a puncture element 121 to rupture the rupturable seal of the sample preparation device 104 to allow fluid to flow therefrom. In some embodiments, as described herein, the incubation chamber 112 is configured to receive a sample solution from the sample receiving module.

[0137] The puncture element ruptures the rupturable seal, thereby connecting the sample preparation device 104 to the assay device or incubation chamber fluid. In certain embodiments, the puncture element provides minimal deformation of the rupturable seal and / or the puncture element when the puncture element punctures the rupturable seal. PCT / US2021 / 049178 (the relevant disclosure of which is incorporated herein by reference) describes a bubble-free puncture mechanism that can be used to rupture a rupturable seal, which allows for a clear puncture of the rupturable seal in a repeatable geometry, thereby minimizing the size and / or number of bubbles or preventing bubble formation entirely. As used, the term "bubble-free puncture" refers to a mechanism that reduces (minimizes) the size and / or number of bubbles, or prevents bubble formation entirely, when, for example, puncturing a sample collection tube occurs. The bubble-free free puncture mechanism described herein minimizes or prevents the introduction of bubbles into the punctured chamber (e.g., a sample collection tube).

[0138] The "seal" referred to in the "breakable seal" can be a layer of material, such as a polymer and / or a metallic material as described herein. In some embodiments, the seal is a foil made of aluminum and / or other metals.

[0139] In some embodiments, the sample preparation device comprises a valve rather than a rupturable seal to be pierced. Examples of sample preparation devices comprising rupturable seals or valves are provided in PCT / US2017 / 022304, the relevant disclosure of which is incorporated herein by reference.

[0140] In some embodiments, the assay device may further include one or more filters for filtering fluid discharged through any portion of the assay device or fluid discharged from the sample preparation device. The filter may be configured to filter the sample fluid prior to discharging the sample fluid through the valve.

[0141] In some embodiments, the interface comprises a hydrophobic material. In some embodiments, the puncture element comprises a hydrophilic material. In some embodiments, the outlet port comprises a hydrophobic material. In some embodiments, the breakable seal comprises a hydrophilic material. In some embodiments, the interface comprises a plastic material. In some embodiments, the puncture element comprises a metal material. In some embodiments, the outlet port comprises a plastic material. In some embodiments, the breakable seal comprises a metal material. In some embodiments, the puncture element comprises a material having a hardness at least 2X times the hardness of the material comprising the breakable seal, so that the puncture element provides a clear puncture of the breakable seal with a repeatable geometry without deforming the puncture element. In some embodiments, the hardness of the puncture element is at least about 1X times, about 2X times, about 3X times, about 4X times, about 5X times, about 6X times, about 7X times, about 8X times, about 9X times, about 10X times, about 11X times, about 12X times, about 13X times, about 14X times, about 15X times, or about 20X times the hardness of the material comprising the breakable seal.

[0142] In some embodiments, the end of the second portion of the puncture element that is configured to contact the rupturable seal comprises a blunt tip such that the surface area of ​​the end of the second portion is orthogonal to the length of the puncture element. In some embodiments, the end of the second portion of the puncture element that is configured to contact the rupturable seal comprises a sharp tip.

[0143] Incubation room exhaust vent

[0144] In some embodiments, the incubation chamber 112 includes a vent 124 configured to allow any gas (e.g., air) and / or vapor to escape from the incubation chamber 112 as the sample solution enters the incubation chamber 112. In some embodiments, the vent 124 is open to the surrounding environment, wherein the sample solution overflows from the top of the vent 124 as the incubation chamber 112 is filled. In some embodiments, the vent 124 includes a selective vent element configured to seal when in direct contact with a liquid, thereby preventing the liquid (e.g., the sample solution) from passing therethrough.

[0145] The selective vent element can be porous and thus have a plurality of holes extending therefrom. Such elements can have a passively adjustable porosity and / or can control the flow of one or more fluids (e.g., gases such as air and / or liquids such as biological samples) within the device. For example, in some embodiments, the vent 124 comprises a self-sealing sintered polymer vent plug or a hydrophobic porous membrane. In some embodiments, the selective vent element is a porous hydrophobic membrane having holes that allow gas (e.g., air) to pass therethrough but do not allow water droplets or other polar liquids to pass therethrough.

[0146] As used herein, the phrase "passively adjustable porosity" refers to the ability to have a first conformation in which one or more gases (e.g., air) can pass therethrough (e.g., through the pores) and a second conformation in which a fluid including one or more gases and a liquid (e.g., a liquid including a biological sample) is blocked or reduced from passing therethrough (e.g., through the pores), and automatically advances from the first conformation to the second conformation upon contact with the liquid. In addition, in the second conformation, the selective vent element prevents the liquid from evaporating therethrough (e.g., through the pores). In addition, in the second conformation, the selective vent element can fluidly seal the fluid channel (e.g., a reaction chamber at an end) by covering an opening (e.g., a vent opening) of the reaction chamber and prevent the fluid from passing therethrough, including evaporation. In addition, the selective vent element is configured to passively (e.g., automatically without user interaction) advance from the first conformation to the second conformation when one or more liquids (e.g., a liquid including a liquid sample) are brought into contact with the selective vent element or a portion thereof (e.g., a surface, such as a surface forming a wall of an incubation chamber or a reaction chamber). Thus, in some embodiments, the selective vent element can be self-sealing to liquids and gases when contacted by liquids.

[0147] The use of selective exhaust elements can minimize or eliminate the need for input valves and / or output valves within the device (or device channels). Although the use of multiple valves is feasible to a certain extent, such schemes require active control of the opening and closing of the valves, which in turn requires energy and infrastructure for implementation and thus complicates the system design. Passive sealing of the device or its parts eliminates the need for active control and minimizes the complexity of the entire device and the user steps required to operate the device. The use of at least one selective exhaust element in each reaction chamber can facilitate passive control of fluid flow within the device; and therefore, in some embodiments, the device does not require valves to operate and / or control the flow of fluid to a chamber (e.g., a reaction chamber).

[0148] refer to Figure 2AIn some embodiments, the assay device comprises a fill detection chamber 126 in fluid communication with the incubation chamber 112 via a sensing channel 128, optionally wherein the fill detection chamber 126 is operatively coupled to a light conduit operatively coupled to an LED. For example, in some embodiments, the sensing channel 128 is in fluid communication with the incubation chamber 112 such that when fluid enters the incubation chamber 112, it also flows into the fill detection chamber through the sensing channel 128. An opening at the end of the sensing channel may allow fluid communication between the sensing channel 128 and the incubation chamber 112. In some embodiments, the fill detection chamber 126 comprises (or is operatively coupled to) a light sensor to detect light from the light conduit, wherein filling the fill detection chamber 126 with a sample solution (by filling the incubation chamber 112) causes the light sensor to detect a decrease in light (due to the sample, which may be dark depending on the liquid used (e.g., buffer)). The position of the opening at the end of the sensing channel 128 that connects the sensing channel 128 to the incubation chamber 112 may determine when liquid filling is detected and the output (e.g., activation of heating and / or stirring) produced thereby. For example, the opening of the sensing channel 128 can be placed at the top of the incubation chamber 112 so that liquid begins to enter the fill detection chamber 126 only when the incubation chamber 112 is at or near maximum liquid capacity. The liquid then flows into the fill detection chamber 126, thereby attenuating the light detected by the light sensor.

[0149] In some embodiments, the light sensor includes one of a complementary metal oxide semiconductor (CMOS) chip, a photodiode, a phototransistor, a photocell, and a photomultiplier tube.

[0150] Incubation room liquid testing

[0151] In some embodiments, the assay device includes one or more sensors configured to detect i) the entry of a liquid (e.g., a sample solution) into an incubation chamber (e.g., initial entry of a sample solution), ii) an incubation chamber filled with liquid, or iii) both. In some embodiments, the one or more sensors operate as an interconnect device and help prevent the activation of a heater (e.g., a mixing heater) before the liquid enters the incubation chamber. For example, premature activation of the mixing heater before the sample solution enters the incubation chamber 112 may overheat the assay device 102 and / or unnecessarily shorten the battery life of a power supply that powers the mixing heater. In some embodiments, the sensor is operatively coupled to the power supply for activating the heater. For example, in some embodiments, a substrate 119 that may include a PCB and an optional thermal gap pad is operatively connected to a controller and a power supply, wherein once the one or more sensors detect a fluid and / or a specified liquid level within the incubation chamber 112, the controller will allow the power supply to power the mixing heater.

[0152] In some embodiments, the incubation chamber may include one or more light sources configured to emit light. Such light sources may be operatively coupled to one or more sensors and / or PCBs and / or controllers / control units such that the sensors are positioned (e.g., opposite the light sources within the same chamber) to receive / detect light from the light sources. See, e.g., Fig.18 In some embodiments, one or more sensors may be configured to detect an optical property, such as a wavelength of light, such as color, and / or a change in an optical property, such as a wavelength of light emitted from the contents of the chamber. In some embodiments where the light source and one or more sensors are located in the same chamber (e.g., an incubation chamber, a reaction chamber, a filling detection chamber, etc.), the light source emits light before the liquid enters the chamber, and the light signal detected by the sensor (e.g., a light sensor) is attenuated when the liquid enters and fills the chamber. Once the light signal decays to a threshold amount, the input from the sensor indicates that there is liquid in the chamber. In some embodiments, the controller may respond by generating an output that initiates an action (e.g., activating a heating element or a mixing element). The light source according to various embodiments may also include one or more light emitting diodes (LEDs). In some embodiments, this may also be achieved by using one or more light pipes, wherein the one or more light pipes enable light to be transmitted between sensors. In some embodiments, the one or more light pipes are operatively connected to the incubation chamber, and it transmits light passing through the incubation chamber (e.g., an incubation chamber, a reaction chamber, a filling detection chamber, etc.) to one or more sensors. In some embodiments, light emitted by the light source is transmitted through the one or more light pipes using at least one of the one or more reflective surfaces and / or one or more refractive surfaces located within the one or more light pipes.

[0153] In some embodiments, once fluid is detected in the incubation chamber, the system initiates the incubation heating step. An indicator on the display can then signal that sample processing has begun. If no fluid is detected, the system can remain in a "ready to load sample" state (e.g., similar to the indicator used when the device is first powered on or loaded). This will enable the device to flag an error if the reagent has been exposed to air for too long.

[0154] In some embodiments, the sensor will provide a signal indicating the detection of fluid within the incubation chamber 112, where the signal may be an audible signal (eg, a beep that may originate from a PCB) and / or a visual signal (eg, a flashing light).

[0155] In some embodiments, the one or more sensors include a capacitive sensor disposed below the incubation chamber 112. In some embodiments, the capacitive sensor is configured to measure the increased dielectric constant in the sample solution (e.g., including a buffer) compared to air. In some embodiments, the capacitive sensor at the bottom (of the incubation chamber) is configured to detect the entry of the sample solution into the incubation chamber 112 or the level of the sample solution within the incubation chamber 112.

[0156] In some embodiments, the one or more sensors include a conductive sensor including one or more electrodes. In some embodiments, the one or more electrodes are operatively coupled to a substrate (e.g., a PCB) and configured to penetrate a bottom wall of the incubation chamber such that the electrodes are exposed within the incubation chamber (see, e.g., Figure 2A , 3A and 5). Thus, in some embodiments, one or more electrodes 130 are configured to contact a liquid (e.g., a sample) and thus detect the initial entry of a sample solution into the incubation chamber 112. In some embodiments, the electrodes 130 require a liquid in order to conduct a charge between the two electrodes 130. For example, in some embodiments, the sample solution includes an ion buffer having ions configured to conduct electricity between the two electrodes 130. Thus, when there is no fluid in the incubation chamber 112, there may not be any charge conduction between the electrodes 130. In some embodiments, the charge conduction between the electrodes 130 enables activation of the mixing heater 134 via the PCB. In some embodiments, the electrodes 130 comprise a metal, a polymer, or an elastomer in which conductive particles are embedded to make the entire electrode conductive.

[0157] refer to Figure 5 In some embodiments, in addition to or as an alternative to having an electrode 130 at the bottom of the incubation chamber 112, the assay device further comprises an electrode 132 located at the top of the incubation chamber. In some embodiments, similar to the two electrodes located at the bottom of the incubation chamber, the bottom electrode 130 and the top electrode 132 will conduct charge therebetween when exposed to a liquid (e.g., a sample). Thus, in some embodiments, the top 132 and bottom 130 electrode configurations enable detection of when the incubation chamber 112 is filled with liquid (thereby forming a conductive path through the height of the incubation chamber). In some embodiments, the top electrode 132 is operatively connected to a substrate (e.g., a PCB) via a conductive clip or wire. In some embodiments, the mixing heater 134 is activated when the incubation chamber is filled with a sample solution. In some embodiments, the assay device comprises two bottom electrodes 130 and one top electrode 132 so as to be able to detect the initial presence (or entry) of liquid in the incubation chamber 112 and the level of liquid (e.g., a sample) in the incubation chamber 112.

[0158] In some embodiments, the one or more sensors include a light sensor as described herein, wherein the fill detection chamber 126 is used to detect the liquid level in the incubation chamber 1112, such that a decrease in the detection of light by the light sensor indicates that the incubation chamber is filled with liquid (e.g., sample solution) (e.g., see Figure 2A and 18 ). In some embodiments, such detection allows activation of the hybrid heater 134.

[0159] In some embodiments, one or more sensors include a thermocouple that is operatively coupled to the mixing heater 134 and configured to measure the temperature of the mixing heater 134 or another area of ​​the PCB that is in close thermal contact with the incubation chamber. In some embodiments, a constant power source is provided to the mixing heater 134 (via one or more power sources as described herein) such that a delay in the temperature increase of the mixing heater 134 as detected by the thermocouple indicates the presence of a liquid within the incubation chamber 112. For example, in some embodiments, the sample solution includes a buffer having a high heat capacity such that when the sample solution is present in the incubation chamber 1121, the temperature of the mixing heater 134 increases more slowly than when it is not present. Figure 7 An exemplary comparison of the temperature increase of the mixing heater 134, the side walls, and the bottom wall of the incubation chamber 112 when the incubation chamber 112 i) is empty and ii) is filled with buffer (e.g., provided with a sample solution) is provided. As shown, the temperature increase is much slower when the incubation chamber 112 is full compared to an empty incubation chamber 112. In some embodiments, such detection of the temperature increase by the thermocouple allows the mixing heater 134 to be activated.

[0160] Sample solution mixing

[0161] In some embodiments, the incubation chamber is configured to mix the sample solution with a lysing agent so as to lyse the cells obtained by the biological sample. In some embodiments, such lysing allows the nucleic acid in the cell to be released for amplification in the OPM module 108. In some embodiments, the lysing agent is placed in the incubation chamber 112 before the sample solution is dispensed into the incubation chamber. In some embodiments, the lysing agent is provided to the incubation chamber 112 after the sample solution has been dispensed into the incubation chamber 112. In some embodiments, the lysing agent is dispensed into the incubation chamber 112 substantially simultaneously with the sample solution. In some embodiments, the lysing agent is suspended in the sample inlet, just past the puncture element, to facilitate faster and more complete mixing.

[0162] In some embodiments, the lysing agent comprises a lyophilized pellet 136 that rehydrates when exposed to a sample solution. In some embodiments, the lysing agent is a liquid. In some embodiments, the lysing agent comprises dithiothreitol (DTT), proteinase K, mutanolysin, lysostaphin, lysozyme, or any combination thereof. In some embodiments, the lysing agent further comprises one or more surfactants, such as Tween. In some embodiments, the lysing agent further comprises one or more components of a buffer provided with the sample.

[0163] In some embodiments, where a cleaving agent is used, the use of an RNase inhibitor or some other agent that prevents nucleic acid degradation is also contemplated.

[0164] refer to Figure 3A and 3B In some embodiments, the sample solution is mixed with the lysing agent 136 by thermal mixing, as described herein. In some embodiments, the mixing heater 134 is configured to supply heat to the incubation chamber 112 so as to heat a portion of the sample solution located near the mixing heater 134 more quickly (compared to the remaining sample elsewhere in the incubation chamber 112) and thereby raise the temperature of the portion of the sample solution more quickly. Such an increase in temperature reduces the density of the portion of the sample solution, thereby increasing the buoyancy of the portion compared to the surrounding sample solution, causing the portion of the sample solution to rise within the incubation chamber 112 and causing another portion of the sample solution to move to its position near the mixing heater 134 (e.g., see Figure 3A and 3B 114. In some embodiments, the continuous supply of heat to the incubation chamber 112 gradually heats the sample solution. In some embodiments, the heating of the sample solution helps to further lyse the cells in the sample solution.

[0165] In some embodiments, the incubation chamber is configured to mix the liquid sample in the absence of a lysing agent. In some embodiments, the incubation chamber is configured to mix the liquid sample and a reagent as contemplated by one of ordinary skill in the art.

[0166] like Figure 3A and 3B As depicted, in some embodiments, the incubation chamber 112 is configured with one or more rounded edges 138 to facilitate circulation of the sample solution therein, wherein such circulation is provided by movement of the fluid through the mixing heater 134. In some embodiments, the one or more rounded edges 138 facilitate circulation of the fluid in an approximately circular motion. In some embodiments, the corners of the incubation chamber 112 near the mixing heater 134 may not be rounded 139.

[0167] In some embodiments, the incubation chamber 112 is sized to optimize sample solution mixing therein and to help reduce or minimize dead volume areas where sample solution may be more stagnant and therefore have reduced mixing. In some embodiments, the length of the incubation chamber 112 is between 0.75 and 2 times the chamber height. In some embodiments, the width of the chamber is between 1 / 6 and 2 / 3 of the average of the channel length and width.

[0168] In some embodiments, the thickness of one or more walls of the incubation chamber 112 is specified to optimize sample solution mixing by shearing as the sample solution passes through the wall. If the chamber is too narrow and the walls are too close together, wall friction will slow mixing. The wider the wall, the lower the ratio of the boundary wall to the entire cross-section, and the shear stress caused by wall friction will not significantly affect mixing.

[0169] In some embodiments, the volume of the incubation chamber 112 is about 0.5 mL to 5 mL, about 0.1 mL to about 100 mL, about 0.5 mL to about 50 mL, about 0.75 mL to about 25 mL, or about 1 mL to about 10 mL.

[0170] like Figure 3A and 3B As depicted, in some embodiments, the mixing heater 134 is positioned offset from the center position 140 of the incubation chamber 112 (e.g., along the width of the incubation chamber). In some embodiments, positioning the mixing heater 134 offset from the center position 140 of the incubation chamber 112 (e.g., along the width) helps promote uneven buoyancy mixing of the sample solution (as opposed to a more central location of heating). Figure 3A and 3B As shown, the portion of the sample solution located near the mixing heater 134 (where the incubation chamber may have corners 139 that are not rounded) will receive more heat than the sample solution elsewhere in the incubation chamber 112 so as to promote fluid movement within the incubation chamber 112 through density differences (as described herein) and thereby promote mixing.

[0171] Various sizes are envisioned for the mixing heater. In some embodiments, the mixing heater is 1 / 50 of the size of the incubation chamber. In various embodiments, the length of the mixing heater is no more than half the length of the incubation chamber. In some embodiments, the size of the mixing heater is no more than half the size of the incubation chamber.

[0172] In some embodiments, as described herein, the substrate includes a controller in operative communication with a power source that provides power to the mixing heater 134 to heat it. In some embodiments, the power supplied to the mixing heater 134 is regulated by the controller based on the temperature of the mixing heater 134, so that a sensor (e.g., a thermocouple) sends a feedback signal to the controller to change the power supplied so as to maintain the mixing heater 134 at a specified temperature (e.g., between 30° C. and 65° C.). In some embodiments, the power supplied to the mixing heater 134 is a constant or substantially constant rate regardless of the temperature of the mixing heater 134. In some embodiments, because thermal mixing is promoted by non-uniformity of temperature, such a constant or substantially constant power supply rate is sufficient. In some embodiments, the mixing heater 134 is a resistive heater.

[0173] In some embodiments, the thermal gap pad 109 conducts heat from the PCB heater to different parts of the device. In some embodiments, the metal element conducts heat from the PCB heater to the heat seal valve and / or the incubation chamber. In some embodiments, the metal element is cylindrical. In some embodiments, the metal element is an aluminum cylinder. The metal element can be made of any heat-conducting metal, such as silver, copper, aluminum or gold. In some embodiments, the metal element is a metal alloy, for example, it contains at least one heat-conducting metal. In some embodiments, the metal element is press-fitted to a position between the PCB and the chamber or device assembly to receive heat (e.g., adjacent to the heat seal valve). In some embodiments, the metal element is press-fitted to the extrusion rib. As used herein, "extrusion rib" refers to a group of very small protrusions that deform when the part is press-fitted therein. Without being limited to the mechanism of action, the use of press-fit (and optionally press-fitting of the extrusion rib) minimizes heat loss to the rest of the assay device and / or the environment. Fig.19 An illustration is provided of an aluminum cylinder secured by three extruded ribs 1901. The aluminum cylinder conducts heat from the PCB heater to the heat seal valve.

[0174] As described herein, in some embodiments, the wax valve channel 110 serves as an outlet from the incubation chamber 112 to the OPM module 108. In some embodiments, the wax valve channel 110 includes a heat-sealed valve, as described herein, that provides a barrier to prevent the sample solution from flowing through the channel 110 so as to retain the sample solution within the incubation chamber 112 for the mixing and / or incubation to proceed for a specified amount of time. In some embodiments, for an incubation chamber having a volume of 1 mL, the specified amount of time for mixing and / or incubation (to lyse cells in the sample solution) is about 1 minute to 20 minutes, about 3 minutes to 10 minutes, about 2 minutes to 15 minutes, or about 5 minutes to 10 minutes.

[0175] Heat seal valves and passages

[0176] In some embodiments, as described herein, the wax valve channel 110 connects the thermal mixing module 106 with the OPM module 108. In some embodiments, the wax valve channel is provided with a valve to provide a barrier that helps prevent the sample solution from prematurely entering the OPM module 108 before the cells in the sample solution are adequately mixed and lysed. In some embodiments, the valve is a heat-sealed valve (e.g., see Fig. 9 142 in the heat-sealed valve), which may initially be solid at a first temperature, but upon receiving sufficient heat, the wax valve softens, melts, and / or dissolves, thereby allowing a liquid (e.g., a prepared sample solution, which is a sample solution after sufficient mixing and / or incubation) to pass through the valve to the OPM 108. In some embodiments, the heat-sealed valve comprises a water-soluble wax. In some embodiments, the heat-sealed valve comprises a polymer. In some embodiments, the polymer is water-soluble. In some embodiments, the valve comprises a wax, optionally comprising a water-soluble wax. In some embodiments, the heat-sealed valve comprises a polymer, such as polyethylene glycol (PEG). In some embodiments, the wax (e.g., PEG) dissolves with the sample solution after softening and / or melting, thereby reducing or eliminating the risk of the wax re-solidifying when removed from the heat source (and thus cooled). Therefore, this helps prevent blockage (partial or complete) of flow within the wax valve channel 110 or further downstream of the assay device. In some embodiments, the dissolved valve material has minimal or no detrimental effect on the sample solution with respect to the reaction (e.g., amplification) within the OPM module 108.

[0177] In some embodiments, the heat-sealed valve comprises other types of waxes and materials known in the art for use as heat-sealed valve materials. Non-limiting examples of wax materials that can be used in the devices of the present disclosure include polyethylene glycol (PEG), HYDROSOL™, Aquasol, and Cerita. In some embodiments, the wax can be an insoluble wax and an emulsifier, such as polysorbate.

[0178] In some embodiments, the actuation temperature of the heat-sealed valve depends on the molecular weight of its material. In the context of the molecular weight of the heat-sealed valve material, the term "about" also indicates that in the preparation of a polymer (e.g., PEG), some molecules will weigh more than the molecular weight, while some molecules will weigh less than the molecular weight. As used herein, "actuation temperature" refers to the temperature at which the wax dissolves. For example, in some embodiments, the molecular weight of the wax is about 1,305 g / mol to about 10,000 g / mol, which corresponds to an actuation temperature range of about 43°C to about 63°C. In some embodiments, increasing the molecular weight of the valve increases the actuation temperature. In some embodiments, the molecular weight of the valve is about 500 g / mol to about 20,000 g / mol, about 1,000 g / mol to about 15,000 g / mol, or about 1305 g / mol to about 10,000 g / mol. In some embodiments, the molecular weight of the heat-sealed valve is about 1,300 g / mol to about 10,000 g / mol. In some embodiments, the molecular weight of the heat-sealed valve is about 6,000 g / mol. In some embodiments, the heat seal valve material is PEG.

[0179] In some embodiments, the heat-sealed valve material is selected based on its ability to remain stable (including solid state) at room temperature and standard shipping conditions. For example, the higher the molecular weight, the higher the transportation and / or storage temperature that the heat-sealed valve material can withstand. The standard shipping condition requirements of the device are known to those of ordinary skill in the art. For example, the required standard shipping state can be based on ASTM D4332 conditions (e.g., ASTM D4332-14). In short, the ASTM D4332 test describes a plan for adjusting containers, packages, or packaging components. The purpose is to make the containers approach or reach a balance with the atmosphere to which they may be exposed. In some embodiments, the heat-sealed valve material must maintain stability (e.g., maintain solid) at up to about 60°C and optionally at about 15% relative humidity. In some embodiments, the heat-sealed valve material must maintain stability (e.g., maintain solid) at up to about 40°C and optionally at about 90% relative humidity. In some embodiments, the heat-sealed valve material must maintain stability at a temperature as low as about -30°C. In some embodiments, the heat seal valve material must remain stable (e.g., remain solid) at temperatures up to about 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, or 65°C. In some embodiments, the heat seal valve material must remain stable (e.g., remain solid) between about 40°C and 60°C or 40°C and 65°C.

[0180] In some embodiments, the heat seal valve material must maintain stability (e.g., remain solid) at a relative humidity of about 15%. In some embodiments, the heat seal valve material must maintain stability (e.g., remain solid) at a relative humidity of about 90%. In some embodiments, the heat seal valve material must maintain stability (e.g., remain solid) at a relative humidity of about 15% and 90%. In some embodiments, the heat seal valve material must maintain stability (e.g., remain solid) at a relative humidity of about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0181] In some embodiments, the heat-sealed valve material must maintain stability (e.g., remain solid) at any of the temperature and / or relative humidity conditions disclosed herein for at least 6 hours, 12 hours, 24 hours, 36 hours, 38 hours, or 72 hours. In some embodiments, the heat-sealed valve material must maintain stability (e.g., remain solid) at any of the temperatures disclosed herein for at least 6 hours. In some embodiments, the heat-sealed valve material must maintain stability (e.g., remain solid) at any of the temperatures disclosed herein for at least 12 hours. In some embodiments, the heat-sealed valve material must maintain stability (e.g., remain solid) at any of the temperatures disclosed herein for at least 24 hours. In some embodiments, the heat-sealed valve material must maintain stability (e.g., remain solid) at any of the temperatures disclosed herein for at least 36 hours. In some embodiments, the heat-sealed valve material must maintain stability (e.g., remain solid) at any of the temperatures disclosed herein for at least 48 hours. In some embodiments, the heat-sealed valve material must maintain stability (e.g., remain solid) at any of the temperatures disclosed herein for at least 72 hours.

[0182] In some embodiments, the heat seal valve material remains stable (eg, remains solid) at up to 60°C for 72 hours.

[0183] In certain embodiments, the preferred material for heat seal valve also has an actuation time between about 30 seconds and 50 seconds on average. In certain embodiments, the actuation time is no more than about 30 seconds. In certain embodiments, the actuation time is no more than about 25 seconds, 26 seconds, 27 seconds, 28 seconds, 29 seconds, 30 seconds, 31 seconds, 32 seconds, 33 seconds, 34 seconds, 35 seconds, 36 seconds, 37 seconds, 38 seconds, 39 seconds, 40 seconds, 41 seconds, 42 seconds, 43 seconds, 44 seconds, 45 seconds, 46 seconds, 47 seconds, 48 ​​seconds, 49 seconds or 50 seconds. In certain embodiments, the actuation time is between about 25 seconds and 30 seconds, 25 seconds and 35 seconds, 25 seconds and 40 seconds, 25 seconds and 45 seconds or 25 seconds and 50 seconds. In certain embodiments, the actuation time is between about 30 seconds and 35 seconds, 30 seconds and 40 seconds, 30 seconds and 45 seconds, 30 seconds and 50 seconds. In some embodiments, the actuation time is between about 30 seconds and 35 seconds, 30 seconds and 40 seconds, 30 seconds and 45 seconds, or 30 seconds and 50 seconds. In some embodiments, the actuation time is between about 35 seconds and 40 seconds, 35 seconds and 45 seconds, or 35 seconds and 50 seconds. In some embodiments, the actuation time is between about 40 seconds and 45 seconds, or 40 seconds and 50 seconds.

[0184] In some embodiments, PEG with a molecular weight of 6,000 g / mol is used because it maintains stability (including remaining in the solid state) during standard shipping conditions.

[0185] In some embodiments, wax is introduced through a wax fill port (see Figure 3A , 3B , 6 and 9) is provided in the wax valve channel 110. In some embodiments, the wax is provided in a molten state, which is then solidified in the wax valve channel 110. In some embodiments, the amount of wax provided in the wax valve channel 110 is about 1 uL to about 10 uL, about 2 uL to about 8 uL, about 3 uL to about 6 uL, or about 4 uL to about 5 uL. In some embodiments, no specific pattern or configuration of wax is required in the wax valve channel 110.

[0186] In some embodiments, the substrate includes a valve heater 135 configured to supply heat to the heat-sealed valve to soften, melt, and / or dissolve it, as described herein. In some embodiments, the wax valve channel 110 is spaced farther in height from the substrate 109 than, for example, the bottom of the incubation chamber 112 aligned with the mixing heater 134. Thus, reference Figure 6In some embodiments, the assay device further comprises a thermally conductive material 137 (e.g., a thermal pad) disposed between the substrate and the heat-sealed valve (in the wax valve channel 110) to transfer heat therebetween. For example, in some embodiments, the thermally conductive material comprises GAP PAD®, 3M thermally conductive adhesive transfer tape, ARTIC thermally conductive pad, Laird Tflex, Fujipoly CARCON gap filling pad, or equivalent material.

[0187] In some embodiments, as described herein, the substrate includes a power source that supplies power to the valve heater 135 to heat it. In some embodiments, the power source is the same as the power source of the hybrid heater 134. In some embodiments, the power source is different from the power source of the hybrid heater 134. In some embodiments, the power source is external to the substrate. In some embodiments, the valve heater is a resistive heater. The heater can be adjusted to control a given temperature, or the heater can be heated at a constant power until fluid is detected in the reaction well (which will indicate that the heat seal valve is open).

[0188] In some embodiments, Figure 6 As depicted, one or more walls 148 from the incubation chamber 112 to the entrance 146 of the wax valve channel 110 are converging (e.g., sloped). In some embodiments, having converging walls at the entrance 146 of the wax valve channel 110 helps reduce or eliminate the formation of bubbles in the wax valve channel and / or along the valve fluid interface (e.g., see Fig.10 150 in). As described herein (see, e.g., Example 1), in some embodiments, the heat-sealed valve is configured to dissolve (e.g., slowly dissolve) when in contact with certain liquids (e.g., a buffer containing a sample solution). In some embodiments, hydrostatic pressure from the sample solution helps provide a force to push the softened, melted, and / or dissolved valve forward through the wax valve channel 110, wherein shearing of the valve caused by movement against the walls of the wax valve channel 110 may further increase dissolution (of the valve). Thus, in some embodiments, such dissolution of the valve by contact with the sample solution, and hydrostatic pressure applied to the heat-sealed valve by the sample solution within the wax valve channel, helps reduce the pressure and time required to open the valve (e.g., by softening and / or melting) to allow the prepared sample solution to flow through the wax valve channel and reach the OPM module 108. In some embodiments, such opening of the valve can occur at a temperature above or about a wax melting point value known in the art, or in some cases, at a temperature below a wax melting point value known in the art.

[0189] The solubility of the heat seal valve material also increases with temperature. Therefore, when the temperature near the assay device or wax valve channel is higher, actuation (opening) of the heat seal valve (i.e., dissolution of the heat seal valve material) occurs faster.

[0190] In some embodiments, the amount of time that the heat-sealed valve can prevent sample solution from flowing through the wax valve channel 110 is a function of the sample solution temperature (e.g., within the incubation chamber 112), the valve actuation temperature, the cross-sectional area of ​​the wax valve channel, and / or the length of the wax valve channel that is blocked by the valve. In some embodiments, the channel diameter is between about 100 μm and 2 mm. In some embodiments, the wax mass within the channel is between about 100 μm and 10 mm—it can vary depending on the length of the wax valve channel.

[0191] In some embodiments, when the sample solution temperature approaches the valve melting temperature, the integrity of the valve may be compromised and, therefore, the sample solution cannot be reliably prevented from flowing through the wax valve channel 110. Therefore, in some embodiments, the assay device includes a thermocouple that detects the temperature of the sample solution within the incubation chamber 112 during the incubation / mixing phase, such that if the temperature of the sample solution approaches a specified range of valve melting temperatures, a controller coupled to the mixing heater 134 is configured to stop powering the heater. In some embodiments, the thermocouple is present and helps prevent the assay in the incubation chamber from reaching a specific temperature or temperature range.

[0192] In some embodiments, the presence of bubbles (e.g., air bubbles) causes the valve to open at temperatures above literature values ​​due to limited or lack of prior dissolution through contact with the sample solution, and / or due to limited or reduced hydrostatic pressure, thereby reducing the force received by the valve to be pushed open and through the wax valve channel 110.

[0193] In some embodiments, as described herein, the assay device is provided with a valve that is solidified therein. For example, in some embodiments, the assay device can be assembled and packaged with the valve therein and thus only contact liquid when used by a subject when performing an assay, so as to prevent potential slow and eventual dissolution of the valve prior to introduction of a sample solution into the incubation chamber 112.

[0194] In some embodiments, once the prepared sample solution flows through the wax valve channel, additional unmixed sample solution may flow out of the sample inlet and / or sample preparation device 104 (which may remain coupled to the assay device 102 after the mixing / incubation phase). Thus, in some embodiments, the inlet 146 of the wax valve channel 110 is spaced as far as possible from the sample inlet 118 to help reduce the risk and / or amount of unmixed (and therefore potentially unlysed) sample entering the OPM module.

[0195] Optical property modification module

[0196] As described herein, in some embodiments, after mixing and / or incubation for a sufficient period of time (thus, a prepared sample solution), once the heat-sealed valve is opened, the sample solution will flow through the wax valve channel 110 and enter the optical property modification (OPM) module 108. In some embodiments, the OPM module 108 comprises an apparatus as described in PCT / US2018 / 044044 ("the '044 Application"), which is incorporated herein in its entirety. In some embodiments, the common sample receiving inlet in the '044 Application is in fluid communication with the wax valve channel 110 to receive the sample therefrom. In some embodiments, the OPM module 108 comprises a reaction chamber channel 152 ( Figure 2A and 2B ), in place of or in addition to the common sample receiving inlet of the '044 application. In some embodiments, the reaction chamber channel 152 extends along the OPM module 108 around the central portion 156, with one or more branches leading to corresponding reaction chambers 154. In some embodiments, the first and second components as described in the '044 application are each a portion of the top and bottom of the assay device 102 as described herein. In some embodiments, the OPM module 108 is a detachable device that is connectable to the wax valve channel 110 and the thermal mixing module 106.

[0197] In some embodiments, the wax is dissolved when the prepared sample solution enters the reaction chamber channel 152 in the OPM module 108. In some embodiments, the prepared sample solution is distributed to one or more reaction chambers 154 through one or more branches of the reaction chamber channel 152.

[0198] In some embodiments, the OPM module 108 further comprises an isolation chamber 158 coupled to the first branch of the reaction chamber channel 152. Figure 2A In some embodiments, the isolation chamber is vented. As described herein in Example 2, in some cases, a large amount of dissolved valve material will be found in the initial volume of prepared sample solution entering the OPM module 108, and thus the isolation chamber 158 will acquire a large amount (e.g., an excess) of dissolved valve material, thereby reducing the amount of dissolved valve material distributed within the one or more reaction chambers 154.

[0199] In some embodiments, the OPM module 108 includes a series of one or more mixing chambers (e.g., shuttle mixing chambers) configured to help distribute the dissolved heat seal valve material more evenly throughout the reaction chamber. In some embodiments, such one or more shuttle mixing chambers are arranged to be connected in series with each other using narrow shuttle mixing channels, wherein at least one shuttle mixing chamber is configured to accommodate the entire sample solution volume. In some embodiments, one or more shuttle mixing chambers are disposed upstream of the reaction chamber.

[0200] In some embodiments, the wax valve channel tapers at the proximal end of the wax fill port. In some embodiments, the wax fill port is narrower at the proximal end of the wax fill port (e.g., approximately 500 um) and wider at the distal end of the wax fill port (e.g., 1 mm at the distal end). In some embodiments, the ratio of the narrowest end of the wax valve channel to the widest end of the wax valve channel is approximately 1:2 or less (e.g., 1:2.5, 1.3, etc.). When dispensed within the channel, this wax valve channel geometry allows the heat seal valve to maintain a consistent length (i.e., the same or smaller length than the heated areas with different volumes of dispensed heat seal valve material), and any excess heat seal valve material is dispensed to the wider section of the channel without significantly increasing the overall length of the valve. This can also minimize excess heat seal valve material entering the OPM. See, for example, Fig.16 A wax valve channel geometry is shown that does not taper proximal to the wax fill port, or widen distal to the wax fill port, and may allow excess heat seal valve material to enter the OPM. Alternatively, Fig.17 The wax valve passage 110 is shown to be narrow at the proximal end of the wax fill port and widens at the distal end of the wax fill port.

[0201] Sample preparation device

[0202] In some embodiments, the sample preparation device comprises any of the sample preparation devices described in any of US Pat. Nos. 11,123,736 and 11,125,661, the disclosures of which are incorporated herein in their entireties.

[0203] Method for preparing a heat-sealed valve

[0204] refer to Fig. 9 , depicts a method for preparing a heat-sealed valve within a wax valve channel 110 of an assay device. In some embodiments, as described herein, an assay device is provided having a thermal mixing module, a wax valve channel 110, and an OPM module (or optionally, an OPM module detachably coupled to the wax valve channel 110). In some embodiments, the wax valve channel 110 includes a valve filling port 144 that allows a heat-sealed valve to be inserted into the wax valve channel 110. In some embodiments, the heat-sealed valve material is dispensed into the wax valve channel 110 in a molten or dissolved state. Figure 8An exemplary device 200 for dispensing heat-sealed valve materials is depicted, which has a pressurized wax reservoir 202, a precision dispensing valve 204, a heater block 206 and / or a wax dispenser needle 208. In some embodiments, the wax dispensing device 200 is a pneumatic drive system. Using the device 200, heat-sealed valve materials can be dispensed onto an integrated test chamber 210. Many other systems in the art may require heating of plastic devices deposited with heat-sealed valve materials. In the device of the present disclosure, in order to dispense heat-sealed valve materials, the measuring device does not have to be heated. In some embodiments, the wax dispenser device and / or consumables are configured to dispense about 1 uL to about 10 uL, about 2 uL to about 7 uL, about 3uL to about 5 uL or about 4 uL of heat-sealed valve materials into the wax valve channel 110.

[0205] In some embodiments, once a sufficient amount of heat-sealed valve material has been dispensed into the wax valve channel 110, the valve filling port 144 is sealed. In some embodiments, additional polymer is melted around the filling port 144, which solidifies after cooling and thereby seals the wax filling port 144 opening. In some embodiments, a plug is introduced into the wax filling port 144 opening. In some embodiments, a pressure-sensitive adhesive is placed on the wax filling port 144 opening to seal it. In some embodiments, the wax filling port opening remains open. In some embodiments, the plastic of the wax filling port is melted and hot-melt sealed. For example, a material such as device plastic is melted on the wax filling port to seal it.

[0206] In some embodiments, the molten or dissolved heat seal valve material (also referred to as dissolved valve material) will flow into the wax valve channel 110 and solidify accordingly without the need to shape and / or position the heat seal valve material within the wax valve channel 110. In some embodiments, the heat seal valve material and / or the wax valve channel are dried and / or cooled prior to packaging to ensure solidification of the heat seal valve material within the wax valve channel 110.

[0207] Method for performing a bioassay in a system

[0208] Fig.11 is a flow chart of a method 1100 for performing a bioassay using a system (eg, 100) according to embodiments described herein. In other embodiments, the method may include: Fig.11 In addition, in various embodiments, the steps of the method may be combined with Fig.11 The order in which they are described is different from the order in which they are executed.

[0209] A subject provides 1101 a biological sample. As described herein, a biological sample is a sample containing an amount of organic matter, such as one or more organic molecules, such as one or more nucleic acids, such as DNA and / or RNA, or portions thereof, which can be collected from a subject. In some aspects, the biological sample is a nucleic acid amplification sample, which is a sample suspected of including one or more nucleic acids, or portions thereof, that can be amplified.

[0210] The provided biological sample may include one or more cells, such as tissue cells of a subject. As used herein, the term "tissue" refers to one or more cell aggregates having similar functions and structures in a subject (e.g., a living organism, such as a mammal (e.g., a human)), or to multiple different types of such aggregates. Tissue may include, for example, organ tissue, muscle tissue (e.g., cardiac muscle, smooth muscle and / or skeletal muscle), connective tissue, neural tissue and / or epithelial tissue. In some versions, tissue may include cells from the inside of a subject's cheek and / or cells in a subject's saliva.

[0211] The provided liquid sample (e.g., biological sample) may include, for example, human saliva, urine, human mucus, blood, oral lavage, or solid tissue such as cheek tissue. The biological sample may also include viruses or spores. The biological sample may be provided by a sample collector. Providing may include contacting (e.g., rubbing and / or scraping) the sample collector with one or more surfaces of the subject and / or the surface of the subject's biological sample, such as a liquid sample extracted from the subject, such as a saliva and / or blood sample. Thus, in some versions, providing includes extracting one or more biological samples from the subject. In some versions, providing the biological sample may include instructing the subject to produce the biological sample, such as by spitting on and / or in the sample collector. Providing the biological sample may also include retaining the biological sample or a portion thereof (e.g., one or more cells) on the sample collector while, for example, transferring the sample collector to a sample preparation device (e.g., 104) or an assay device (e.g., 102). In some cases, the sample collector is a swab, and providing the biological sample includes swabbing the inside of the subject's mouth and / or nose to obtain the biological sample on the collector. In some versions, the sample collector is a nasopharyngeal, middle turbinate, genital, and / or nasal swab. After providing the biological sample, method 1100 can include mixing the biological sample with a preparation solution to form a sample solution 1102. In some embodiments, the biological sample is mixed with the preparation solution in a sample preparation device (e.g., 104) as described herein.

[0212] In some embodiments, the prepared solution comprises an optical property modifying reagent solution. In some embodiments, the optical property modifying reagent solution comprises an optical property modifying reagent and a liquid buffer.

[0213] Optical property modifying agents may include, for example, pH sensitive dyes, fluorescent dyes, FRET dyes, microparticles and nanoparticles, fluorescent proteins, colorimetric substrates, enzymes and reagents, plasmonic structures, precipitating reagents and substrates, or any combination thereof.

[0214] In some versions, the optical property modification reagent is or includes an enzyme-linked immunosorbent assay (ELISA) reagent. In some aspects, the ELISA reagent is selected from the group consisting of alkaline phosphatase, horseradish peroxidase, β-galactosidase, BCIP / NBT (5-bromo-4-chloro-3-indolyl-phosphate / nitro blue tetrazolium), TMB (3,3',5,5'tetramethylbenzidine), DAB (3,3',4,4'diaminobenzidine) and 4CN (4-chloro-1-naphthol). TMB (bifunctional substrate), ABTS (2,2'-azino-di[3-ethylbenzothiazoline]sulfonate), OPD (o-phenylenediamine), MUG (4-methylumbelliferyl galactoside), HPA (hydroxyphenylacetic acid) and HPPA (3-p-hydroxyphenylpropionic acid).

[0215] In various cases, the optical property modifying agent may include one or more optical property modifying substances and, therefore, may be configured to modify one of its optical properties, such as color. Thus, method 1100 includes modifying one or more optical properties of the optical property modifying agent.

[0216] Modifying an optical property refers to changing one or more optically identifiable characteristics of an aspect (e.g., a sample), such as characteristics resulting from the wavelength and / or frequency of radiation (e.g., light) emitted from an aspect, such as color, fluorescence, phosphorescence, etc. For example, in some versions, the optical property is color, and modifying the optical property includes changing the color. In some aspects, such optical property changes (e.g., color changes) can be detected by an unaided human eye, for example, under ambient light. In alternative aspects, such as method 1100, the optical property modification can be detected using a photoelectric sensor. Modifying the optical property can also include changing the transmittance and / or opacity of the substance, and can include causing the substance to substantially change from transparent to opaque or from opaque to transparent. Thus, method 1100 can include detecting such changes in transmittance with a photoelectric sensor.

[0217] The user dispenses 1103 the sample solution into the assay device via the sample receiving module (eg, 114), or specifically into the incubation chamber (eg, 112) of the thermal mixing module (see, e.g., Fig.10). In some embodiments, the incubation chamber 112 has a lysing agent (e.g., 136) therein for mixing with the sample solution to form a prepared sample solution. Such mixing helps prepare the biological sample within the sample solution so that it reacts with, for example, an assay reagent and / or an optical property modifying reagent when exposed thereto. The mixing may include lysing cells of the biological sample with the lysing agent and / or extracting nucleic acids therefrom. Such extracted nucleic acids may be released into the resulting prepared sample solution. In some embodiments, a step of extracting genomic deoxyribonucleic acid (DNA) from the biological sample is included. In some embodiments, the lysing agent is provided in the form of a pellet that can be lyophilized and is hydrated once exposed to the sample solution.

[0218] Then, the incubation chamber is heated 1104 using a mixing heater 134 placed below the incubation chamber so that the sample solution can be thermally mixed with the lysing agent. In some embodiments, the mixing heater 134 is aligned to deviate from the center of the incubation chamber 112 to help promote the uneven buoyancy of the sample solution, thereby promoting the movement of the fluid in the chamber 112 (for example, the temperature of the portion of the sample solution near the mixing heater will become high, thereby reducing the density and allowing it to rise and move in the chamber). In some embodiments, the incubation chamber includes one or more circular walls 138 to help promote the cyclic movement in the incubation chamber 112, and in some cases, to promote circular or similar movements. In some embodiments, the mixing heater 134 is based on receiving power from a power supply at a constant rate or by regulating power to keep the mixing heater temperature 134 constant or substantially constant to provide heat. In some embodiments, the sample solution is mixed and / or incubated for a specified time. In some embodiments, the specified time is about 1 minute to about 20 minutes.

[0219] After the sample solution has been mixed for a prescribed time, heat is then applied to the heat-sealed valve 1105 using the valve heater 135 (see, e.g., Fig.10). In some embodiments, the heat-sealed valve is located within a wax valve channel 110 that extends from an end of the incubation chamber 110 opposite the entrance to the sample receiving module 114. In some embodiments, the heat-sealed valve provides a barrier to prevent the sample solution from passing through the wax valve channel 110. In some embodiments, the heat-sealed valve is solid at a first temperature, but is configured to soften and / or melt once exposed to sufficient heat (e.g., the valve heater 135). In some embodiments, the heat-sealed valve comprises wax or a water-soluble polymer (e.g., PEG). In some embodiments, the valve heater heats the heat-sealed valve, and in some cases, the melting temperature of the heat-sealed valve is about 40°C to about 65°C. In some embodiments, the heat-sealed valve softens, melts, and / or dissolves sufficiently to be opened by the prepared sample solution, and thereby allows the prepared sample solution to flow through the wax valve channel 110 to the optical property modification (OPM) module 108. In some embodiments, the heat-sealed valve dissolves in the prepared sample solution.

[0220] The prepared sample solution enters the reaction chamber channel 152 in the OPM module 108, which is in fluid communication with one or more reaction chambers 154. The prepared sample solution is then transferred to the reaction chamber 154 (step 1106). Specifically, the prepared sample solution travels along the reaction chamber channel 152 and branches into one or more reaction chambers 154, wherein the reaction chamber 154 contains assay reagents, thereby generating a nucleic acid reaction mixture.

[0221] As described herein, in some embodiments, each of the reaction chambers 154 contains an assay reagent. Thus, transferring the prepared sample solution to one or more reaction chambers can include mixing the prepared sample solution with the assay reagent and thereby generating a nucleic acid reaction mixture including the prepared sample solution and the assay reagent for performing a nucleic acid amplification reaction.

[0222] The assay reagent comprises an enzyme and a nucleic acid primer capable of reacting with the biological sample so that one or more nucleic acids suspected to be present in the sample (if present) can be amplified, such as isothermal amplification. In some embodiments, the assay reagent comprises a nucleic acid amplification enzyme and a DNA primer. For example, the assay reagent may include one or more primers, deoxynucleotides (dNTPs) and / or polymerases, Trizma preset crystals (Tris buffer, pH 8.8; Sigma, catalog number T9443), potassium chloride (KCl; Wako Pure Chemicals, catalog number 163-03545), magnesium sulfate heptahydrate (MgS04; Wako Pure Chemicals, catalog number 137-00402), ammonium sulfate ((H4)2S04; Kanto Chemical, catalog number 01322-00), Tween 20 (Tokyo Chemical Industry Co., Ltd., catalog number 01322-00), Chemical Industry, catalog number T0543), betaine solution (betaine, 5M; Sigma, catalog number B0400), calcein (DOJINDO, catalog number 340-00433) plus one or more optical modification agents as discussed above, manganese (II) chloride tetrahydrate (MnCl2; Wako Pure Chemical Industries, catalog number 133-00820), agarose S, EtBr solution, template nucleic acid, or any combination thereof. In addition, in some versions, the assay reagent can be stored in a dry (e.g., lyophilized) form in the fluid chamber 205. Thus, preparing the reaction mixture can include mixing the prepared sample solution and the assay reagent and / or hydrating the assay reagent.

[0223] The assay reagents may include one or more reagents capable of amplifying nucleic acids present in a biological sample by an isothermal amplification scheme, including: transcription-mediated amplification, strand displacement amplification, nucleic acid sequence-based amplification, rolling circle amplification, loop-mediated isothermal amplification, isothermal multiple displacement amplification, helicase-dependent amplification, ring helicase-dependent amplification, single primer isothermal amplification, loop-mediated amplification, or any combination thereof.

[00202] In certain embodiments, the amplification reaction performed is LAMP. In the LAMP reaction, two or three pairs of primers are used to amplify a double-stranded or single-stranded DNA template in dynamic equilibrium at high temperature. Primers are designed based on the DNA template using primer design software such as LAMPDesigner (Premier Biosoft, Palo Alto, CA). In the first step of the LAMP reaction, the F2 region of the FIP (forward inner primer) anneals to the single-stranded DNA at the corresponding complementary (F2c) position. Next, a polymerase with strand displacement activity incorporates dNTPs along the template from the 3' end of F2. The incorporation of nucleotides releases protons, thereby lowering the pH of the reaction mixture. The F3 forward primer then anneals to the F3c region upstream of the F2 region and on the template. The F3 forward primer begins to amplify the template strand, which releases additional protons and displaces the previously synthesized FlP-incorporated strand. This single strand contains the Fl sequence (within the target sequence) and its complementary Flc sequence (within FIP). This forms a stem-loop because Flc anneals to Fl at the 5' end. At the same time, BIP (reverse inner primer) anneals to the other end of the strand, and nucleotides extend from B2, releasing more protons. The reverse primer B3 then binds to the B3c region downstream of the B2 region, displacing the strand amplified by BIP and promoting extension to produce a double strand. This displaced strand now contains the Bl sequence (within the target sequence) and its complementary Blc sequence (within BIP), forming another stem-loop at the 3' end. The structure now has two stem-loop structures at each end, from which continuous shifts and extensions occur to amplify the template. The LAMP reaction can be amplified by adding additional forward and reverse loop primers to generate more amplicons with stem-loop structures.

[0224] The LAMP procedure can be performed at a fixed temperature, thereby minimizing the need for any expensive thermal cycling equipment. Typically, isothermal methods require a set temperature determined by the selected reagents. For example, in the LAMP method, the enzyme works best between 60-65°C. Amplification according to embodiments of the present invention can also be performed by applying PCR.

[0225] In some embodiments, the system (e.g., 100) heats 1107 a reaction mixture (e.g., using a reaction heater) to produce amplified nucleic acids and a plurality of protons. Specifically, heating the reaction mixture with the reaction heater promotes a nucleic acid amplification reaction using nucleic acids from a biological sample and an assay reagent. The reaction produces amplified nucleic acids and a plurality of protons.

[0226] In some embodiments, the heating step 1107 includes transferring heat energy from the reaction heater to a thermal gap pad and then to one or more reaction chambers. Heating the reaction mixture promotes a nucleic acid amplification reaction between the nucleic acid of the biological sample and the assay reagent. The nucleic acid amplification reaction produces amplified nucleic acid and a plurality of protons.

[0227] The protons then react with the optical property modifying reagent 1108. Reacting the reaction product or aspects thereof with the optical property modifying reagent can include chemically modifying the reaction product and / or the optical property modifying reagent, such as by combining one or more protons with the optical property modifying reagent. In some embodiments, such reaction of the protons with the optical property modifying reagent sufficiently modifies the optical property of the optical property modifying reagent to allow detection of the modified optical property indicative of the presence of a suspected analyte in the biological sample.

[0228] The assay device causes 1109 the light emitting element to emit light. Specifically, the system's microprocessor (as described in the '044 application) instructs the plurality of light emitting elements to emit light in a repeating pattern at a repetition frequency. During the repeating pattern, each of the plurality of light emitting elements emits light at a different point in time, such that only one of the plurality of reaction chambers is illuminated at any time. Exposure to light can provide a change in conditions such that an optical property can be measured. In this way, during each repeating pattern, the optical property of the contents of each reaction chamber can be continuously monitored by the photosensor.

[0229] Based on the optical properties detected in step 1109, the system (e.g., 100) can determine 1110 one or more characteristics of the sample contained in the reaction chamber using a photoelectric sensor and a microprocessor that performs an optical property analysis on the reaction mixture in one or more reaction chambers, as described in the '044 application. Performing the optical property analysis can include determining whether an optical property of one or more contents of the reaction chamber has changed.

[0230] Optical property analysis can be carried out in real time during the whole process of the amplified reaction described about step 1106, or after carrying out the amplified reaction.The detection of the modified optical property of the reaction mixture can be associated with the digital indication of the presence or absence of the amplified reaction product.In other words, the modified optical property of the detection reaction mixture can provide information about whether the amplified reaction product exists.In certain embodiments, the modified optical property of the detection reaction mixture shows that the exponential phase or the plateau phase of the amplified reaction has been obtained.

[0231] In certain embodiments, relative to the amplification reaction of the reaction mixture that does not contain the acid-adding color developer, the detection of the amplification reaction product is accelerated.In other embodiments, from the starting time of the amplification reaction, the optical property modification of the reaction mixture is detected in less than 60 minutes.Because the acid-adding color developer (weak acid or weak base) in the reaction mixture absorbs the proton produced during the amplification reaction, and the recombination of the free proton accelerates the detection of the amplification reaction, the accelerated detection of the amplification reaction product is obtained.Reaction can be designed so that the minimum amplification is required to produce the pH conversion that is enough to make the acid-adding color developer change the optical property.The conventional amplification techniques using fluorescent intercalating dyes, molecular beacons, hybridization probes, detection based on dyes, UV-Vis or other detection methods need to have a certain threshold amount of amplification before the amplification signal can be detected.However, before the optical property change of the acid-adding color developer can be detected, the method of the present disclosure requires the amplification of a relatively small threshold amount, and therefore relative to conventional amplification methods, the detection of the amplification reaction product is accelerated.

[0232] In some embodiments, the system is configured to display the determined characteristics using an electronic results display mechanism (such as described in the '044 application). The results provided may be in the form of a visual output on a display and / or in the form of an audio output.

[0233] With respect to method 1100, it is noteworthy that in various embodiments, the system includes one or more (e.g., three) assay controls: a sample adequacy control, a positive control (e.g., an internal positive control), and / or a negative control. The sample adequacy control detects, for example, abundant human nucleic acid markers, such as housekeeping genes, RNA, and / or human β-actin deoxyribonucleic acid (DNA), to ensure that sufficient swab samples are provided. The positive control amplifies synthetic oligonucleotides that can be co-packaged and / or co-lyophilized in the reaction chamber. Such synthetic oligonucleotides can be included in, for example, an optical property modification reagent solution and / or an assay reagent. Such controls ensure that the system is operated under conditions that allow amplification of the genetic marker of interest. The negative control also amplifies the positive control, but without the co-lyophilized synthetic oligonucleotides. Such controls ensure that there are no contaminating self-amplified amplicons.

[0234] Reagent test kit

[0235] Embodiments disclosed herein also include kits that include the inventive device and can be used according to the inventive method. According to any embodiment or any combination of the embodiments described herein, the inventive kit can include two or more, such as multiple, three or less, four or less, five or less, ten or less, or fifteen or less, or fifteen or more assay devices or components thereof.

[0236] The kit may include one or more compositions and / or reagents, such as any of those described herein, such as optical property modification reagents, amplification compositions, preparation solutions and / or buffers, which may be stored in a kit in a container separate from the device. In addition, the kit may include any device or other element that may facilitate the operation of any aspect of the kit. For example, the kit may further include one or more devices for preparing samples and / or analyzing one or more characteristics of a sample (e.g., a prepared sample). In some embodiments, the kit comprises a sample preparation tube having one or more compositions and / or reagents. The kit may also include packaging, such as packaging for transporting the device without breaking.

[0237] In certain embodiments, the kit disclosed herein includes instructions, such as instructions for using the device. In some aspects, the instructions for using the device are recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Therefore, the instructions can be present in the kit as a package insert, in a label of a kit container or its components (i.e., associated with a package or sub-package, etc.). In other embodiments, the instructions are present as an electronic storage data file on a suitable computer-readable storage medium, such as a portable flash drive, a CD-ROM, a disk, a cloud, etc. The instructions can be stored and / or reproduced in one or more programs (such as computer applications). In some embodiments, the instructions can be obtained by scanning a QR code or barcode printed on a device component, a substrate (such as paper or plastic, etc.). The instructions can take any form, including a complete description of how to use the device, or as a website address, through which the instructions posted on the World Wide Web can be accessed.

[0238] Examples

[0239] Below is the example of the specific embodiment of the present invention.These examples are provided for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.Efforts have been made to ensure the accuracy of the numbers (e.g., amounts, temperatures, etc.) used, but of course some experimental errors and deviations should be allowed.

[0240] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, which are within the skill of the art. Such techniques are fully explained in the literature. See, e.g., TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg, Advanced Organic Chemistry, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); 3rd edition (Plenum Press), Volumes A and B (1992).

[0241] Example 1 - Distribution of dissolved valve material in the reaction chamber

[0242] Fig.12 The distribution of heat-seal valve material (e.g., polyethylene glycol) dissolved in a sample solution in multiple reaction chambers (Ch 1 to Ch 8) in an assay device is depicted. Two cases are shown, wherein the first case represents the sample solution (water) without thermal mixing (as described herein) (unmixed group), while the second case represents the sample solution (water) after thermal mixing (mixed group). In both cases, it is apparent that the maximum concentration of dissolved heat-seal valve material occurs in the first chamber, while the remaining chambers have a relatively uniform and significantly smaller volume of heat-seal valve material. This highlights the importance of having an isolation chamber (a collection chamber for excess dissolved heat-seal valve material, e.g., Figure 2A), so as to remove a large amount (e.g., a large portion) of the dissolved valve material before distributing the prepared sample solution into the reaction chamber. This also has the advantage of minimizing the variation of the assay results from different reaction chambers, because the isolation chamber allows for a relatively uniform distribution of the dissolved valve material in each chamber.

[0243] Fig.13 Included are illustrations of a system with an isolation chamber (e.g., Figure 2A 158) and an assay device without an isolation chamber (e.g., Figure 2B ). Ch W represents an isolation chamber. Ch 1 to Ch 8 represent reaction chambers. The results show that dissolved valve material is present in all reaction chambers regardless of whether an isolation chamber is used, and that the distribution of dissolved valve material between Ch 2 to Ch 8 is comparable without an isolation chamber. In the assay device set without an isolation chamber, a disproportionate but still small amount of dissolved valve material is present in the first reaction chamber (Ch 1).

[0244] Example 2 – Effect of Liquid Dissolution and Hydrostatic Pressure on the Actuation Temperature of Heat Seal Valve Materials

[0245] like Fig.14 As shown, a test apparatus having some of the assay elements described herein (eg, mixing chamber and wax valve passage) is used to test the actuation temperature of heat seal valve materials. Fig.15 A graph is included depicting the actuation temperature of a heat seal valve (e.g., PEG) under different assembly conditions. Literature values ​​for PEG with a molecular weight of 1305 to 1595 are about 44°C to about 46°C. Under the first condition, the wax valve channel on the assay device is not provided with an angled inlet from the incubation chamber, wherein the heat seal valve is located in the center of the channel, and there is air between the fluid (e.g., liquid, such as a buffer) and the heat seal valve material, so that the dissolution caused by the liquid on the heat seal valve material is reduced or no dissolution, and the hydrostatic pressure exerted by the liquid is limited or no hydrostatic pressure. The resulting actuation temperature is 77°C-79°C.

[0246] In the second condition, there is an inclined entrance to the wax valve channel, where the liquid is in contact with the heat seal valve, but the liquid exerts limited or no hydrostatic pressure on the heat seal valve. Therefore, the resulting actuation temperature is 61°C-63°C, indicating that the dissolution of the heat seal valve caused by exposure to the liquid reduces the actuation temperature by 16°C compared to no contact with the liquid.

[0247] In the third condition, there was an inclined inlet to the wax valve channel, and a vertically oriented vial providing the liquid, so that the liquid exerted hydrostatic pressure on the heat seal valve. The resulting actuation temperature was therefore 38°C-39°C, indicating that dissolution of the heat seal valve by exposure to the liquid and hydrostatic pressure further reduced the actuation temperature, in this case below the literature value.

[0248] Table 1 provides the injection parameters for the PEG-based heat-sealed valve in the assembly of the present disclosure. The molecular weight of the PEG used was 6,000 g / mol.

[0249] Table 1: Injection parameters for PEG 6000 MW.

[0250] The actuation times for 6000 g / mol PEG were measured and averaged between 30 and 50 seconds. See Table 2.

[0251] Table 2: Actuation time of loaded PEG 6000 MW.

[0252] All publications and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated herein by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0253] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

Claims

1. A system for performing a bioassay, the system comprising: a. A thermal mixing module, the thermal mixing module comprising: i. a sample receiving module for receiving a sample solution comprising a biological sample and a preparation solution; and ii. an incubation chamber in fluid communication with the sample receiving module; b. a wax valve channel in fluid communication with the incubation chamber; c. an optical property modification (OPM) module, the OPM module being operatively coupled to the incubation chamber via the wax valve channel, the OPM module comprising one or more reaction chambers, each reaction chamber comprising an assay reagent; d. a heat-sealed valve disposed i) within the wax valve channel and ii) between the sample receiving module and the OPM module; and e. A mixing heater configured to supply heat to the incubation chamber.

2. The system of claim 1, wherein the incubation chamber comprises a lysing agent.

3. The system of claim 1 or 2, wherein the mixing heater is configured to be aligned with at least a portion of the thermal mixing module and offset from a central portion of the incubation chamber.

4. The system of any one of claims 1 to 3, further comprising a sample preparation device configured to cooperate with the sample receiving module.

5. The system according to any one of claims 1 to 4, wherein the sample receiving module comprises a puncturing element, which is configured to puncture a breakable seal on the sample preparation device, thereby achieving fluid communication between the sample preparation chamber within the sample preparation device and the incubation chamber.

6. The system of any one of claims 1 to 5, wherein the rupturable seal comprises a foil.

7. The system of any one of claims 1 to 6, wherein the sample receiving module comprises a Luer connector for coupling a sample preparation tube to an assay device.

8. The system of any one of claims 4 to 7, wherein the sample preparation tube comprises a collar that contacts the Luer connector of the sample receiving module to form a leak-proof seal between the sample preparation device and the sample receiving module when mated together.

9. The system of any one of claims 1 to 8, wherein the incubation chamber comprises an exhaust port.

10. The system of claim 9, wherein the exhaust port is a selective exhaust element.

11. The system of claim 9 or 10, wherein the selective venting element comprises a self-sealing polymer vent plug.

12. The system of claim 9 or 10, wherein the selective venting element comprises a self-sealing porous polymer vent, the vent further comprising an embedded hydrogel.

13. The system of claim 9 or 10, wherein the selective vent element comprises a self-sealing porous polyethylene vent, the vent further comprising an embedded hydrogel.

14. The system of claim 9 or 10, wherein the selective venting element comprises polytetrafluoroethylene or polyethersulfone.

15. The system of claim 9, wherein the selective venting element comprises a hydrophobic porous membrane.

16. The system of claim 9, wherein the vent comprises a sensing channel in fluid communication with a fill detection chamber, the fill detection chamber being configured to detect liquid filling thereof.

17. The system of any one of claims 1 to 16, wherein the thermal mixing module comprises a light source.

18. The system of claim 17, wherein the light source is a light emitting diode (LED) or a laser.

19. The system of any one of claims 1 to 16, wherein the thermal mixing module further comprises one or more sensors configured to i) detect the initial presence of liquid in the incubation chamber, ii) detect the liquid level in the incubation chamber, iii) or both.

20. The system of claim 19, wherein the one or more sensors are configured relative to the light source such that the one or more sensors detect light emitted by the light source, and when the incubation chamber is filled with liquid, the light detected by the sensors is attenuated.

21. The system of claim 19, wherein the one or more sensors comprise a capacitive sensor disposed below the incubation chamber.

22. The system of claim 19 or 21, wherein the one or more sensors comprise one or more bottom electrodes operatively connected to a circuit board and configured to penetrate a bottom wall of the incubation chamber so as to be exposed within the incubation chamber.

23. The system of claim 22, further comprising one or more top electrodes operatively connected to the circuit board and configured to penetrate a wall other than the bottom wall of the incubation chamber so as to be exposed within the incubation chamber and thereby detect a liquid level within the incubation chamber.

24. The system of claim 19, wherein a sensor of the one or more sensors is disposed within a fill detection chamber that is in fluid communication with the incubation chamber via a sensing channel, wherein the sensor is configured to detect light changes within the fill detection chamber.

25. The system of claim 19, wherein the one or more sensors comprise a thermocouple coupled to the mixing heater and / or a portion of the incubation chamber so as to detect a change in the temperature increase of the mixing heater and / or the incubation chamber, thereby correlating to the presence of liquid within the incubation chamber.

26. The system of any one of claims 19 to 24, wherein the one or more sensors are in operative communication with the hybrid heater.

27. A system according to claim 26, wherein the one or more sensors serve as an interlock device for the mixing heater, so that the mixing heater is configured to be activated and / or deactivated based on the detection of liquid and / or liquid level in the incubation chamber by the one or more sensors.

28. The system of any one of claims 2 to 27, wherein the lysis agent comprises a lyophilized pellet.

29. The system of any one of claims 2 to 28, wherein the lysing agent comprises dithiothreitol (DTT), proteinase K, mutanolysin, lysostaphin, lysozyme, or a combination thereof.

30. The system of any one of claims 2 to 29, wherein the lysing agent comprises one or more surfactants.

31. The system of claim 30, wherein the one or more surfactants comprises a polysorbate.

32. The system of any one of claims 2 to 31, wherein the lysing agent comprises one or more components of a buffer solution.

33. The system of any one of claims 2 to 32, wherein the mixing heater is configured to heat the sample solution within the incubation chamber, thereby enabling mixing of the sample solution with the lysing agent therein to form a prepared sample solution.

34. The system of any one of claims 1 to 33, wherein the incubation chamber has one or more rounded edges.

35. The system of claim 34, wherein the one or more rounded edges enable the sample solution to circulate or substantially circulate within the incubation chamber when receiving heat from the mixing heater.

36. The system of any one of claims 1 to 35, wherein the height of the incubation chamber is specified relative to the width of the incubation chamber to minimize and / or reduce dead volume areas within the incubation chamber.

37. The system of claim 36, wherein the length of the incubation chamber is about 0.5 to about 3 times the height of the incubation chamber.

38. The system of claim 36 or 37, wherein the width of the incubation chamber is from about 1 / 8 to about 1.0 times the average of the length and width of the incubation chamber.

39. The system of any one of claims 1 to 38, wherein the volume of the incubation chamber is from about 0.1 mL to about 10 mL, such as from about 0.5 mL to about 5 mL.

40. The system of any one of claims 1 to 39, wherein the mixing heater is configured to heat the sample solution within the incubation chamber for a specified amount of time.

41. The system of claim 40, wherein the specified amount of time is from about 30 seconds to about 20 minutes.

42. The system of claim 41, wherein the prescribed amount of time is about 1 minute to 5 minutes or about 5 minutes to 10 minutes.

43. The system of any one of claims 1 to 42, wherein the wax valve channel is located at an end of the incubation chamber opposite the sample receiving module.

44. A method according to any one of claims 1 to 43 wherein the heat seal valve comprises wax.

45. The system of claim 44, wherein the wax is water soluble.

46. ​​The system of any one of claims 1 to 45, wherein the wax comprises a water soluble polymer.

47. The system of claim 46, wherein the polymer comprises polyethylene glycol (PEG).

48. The system of any one of claims 1 to 47, wherein the heat-sealed valve has a molecular weight of about 1,300 g / mol to about 10,000 g / mol.

49. The system of any one of claims 1 to 47, wherein the heat-sealed valve has a molecular weight of about 6,000 g / mol.

50. The system of any one of claims 1 to 48, wherein the heat sealed valve has a melt temperature of about 40°C to about 75°C.

51. The system of any one of claims 1 to 50, wherein the volume of the heat seal valve within the wax valve channel is about 2 uL to about 6 uL.

52. The system of any one of claims 1 to 51, wherein the wax valve channel includes a valve fill port for receiving the valve therein.

53. The system of any one of claims 1 to 52, wherein the heat-sealed valve is solid or substantially solid at a first temperature to help prevent the sample solution from flowing through the wax valve channel.

54. The system of claim 53, wherein the heat-sealed valve is configured to transition from a solid or substantially solid configuration to a soft, molten, and / or melted configuration upon receiving sufficient heat.

55. The system according to any one of claims 1 to 54, further comprising a valve heater, which is configured to heat the heat-sealed valve, thereby enabling the heat-sealed valve to be softened, dissolved and / or melted to allow the sample solution to flow therethrough.

56. The system of any one of claims 1 to 55, wherein an entrance from the incubation chamber to the wax valve channel comprises one or more converging walls.

57. The system of claim 55 or 56, further comprising one or more thermally conductive pads operatively coupled to the mixing heater, the valve heater, or both.

58. The system of claim 57, wherein the one or more thermally conductive pads include a valve thermally conductive pad configured to transfer heat from the valve heater to the wax valve channel to heat the heat-sealed valve.

59. The system of any one of claims 1 to 58, wherein the level of the sample solution within the incubation chamber is located at a higher elevation than the wax valve channel such that the heat-sealed valve is under hydrostatic pressure from the sample solution.

60. The system of any one of claims 1 to 59, wherein the sample preparation tube is positioned at a higher elevation than the heat seal valve or the wax valve channel such that the heat seal valve is under hydrostatic pressure from the sample solution.

61. The system of any one of claims 1 to 60, wherein the heat-sealed valve is configured to dissolve into the sample solution.

62. The system of any one of claims 1 to 61, wherein the prepared sample solution is configured to enter at least one reaction chamber of the one or more reaction chambers after passing through the wax valve channel.

63. The system of claim 61 or 62, wherein a dissolved heat-sealed valve enters at least one reaction chamber along with the prepared sample solution.

64. A system according to any one of claims 61 to 63, further comprising an isolation chamber located downstream of the wax valve channel and upstream of the one or more reaction chambers, wherein the isolation chamber is configured to receive an initial flow of the prepared sample solution and the dissolved heat-sealed valves therein so as to reduce the amount of the dissolved heat-sealed valves found in the one or more reaction chambers.

65. The system of any one of claims 61 to 64, further comprising one or more mixing chambers (eg, shuttle mixing chambers) to improve distribution of the dissolved heat-sealed valve across the one or more reaction chambers.

66. The system of any one of claims 1 to 65, further comprising a substrate operatively coupled to the thermal mixing module, the wax valve channel, and / or the OPM module.

67. The system of claim 66, wherein the substrate comprises a printed circuit board.

68. The system of claim 66 or 67, wherein the mixing heater and / or the valve heater are disposed on the substrate.

69. The system of any one of claims 66 to 68, wherein the substrate further comprises a power source operatively connected to the mixing heater and / or the valve heater.

70. The system of claim 69, wherein the substrate comprises a controller to regulate power supplied to the mixing heater and / or the valve heater so as to maintain the mixing heater and / or the valve heater substantially at a predetermined temperature.

71. The system of claim 69, wherein the power source is configured to provide power to the mixing heater and / or the valve heater at a substantially constant rate.

72. The system of any one of claims 66 to 71, wherein the substrate comprises a thermal gap pad.

73. The system of any one of claims 1 to 71, wherein the preparation solution is a nucleic acid amplification preparation solution.

74. The system of any one of claims 1 to 73, wherein the preparation solution further comprises an optical property modifying reagent.

75. The system of any one of claims 1 to 74, wherein the biological sample comprises human saliva, urine, human mucus, vaginal fluid, semen, blood, oral lavage or solid tissue such as cheek tissue, bacteria, one or more spores, one or more viruses, or a combination thereof.

76. The system of any one of claims 1 to 75, wherein the OPM module comprises a reaction chamber channel in fluid communication with the wax valve channel, wherein the one or more reaction chambers are in fluid communication with the reaction chamber channel via corresponding branches.

77. The system of any one of claims 1 to 76, wherein each reaction chamber is substantially equidistant from a single sensing region disposed in the OPM module.

78. The system of any one of claims 1 to 77, wherein the OPM module further comprises a first plurality of light pipes, each first light pipe being capable of transmitting light between one of the one or more reaction chambers and the single sensing region.

79. The system of any one of claims 1 to 78, wherein the OPM module further comprises a reaction heater configured to heat the one or more reaction chambers.

80. The system of any one of claims 1 to 79, wherein the assay reagents comprise dried or lyophilized reagents.

81. The system of any one of claims 1 to 80, wherein the assay reagents comprise a nucleic acid amplification enzyme and a DNA primer.

82. A method for determining one or more characteristics of a nucleic acid amplification sample based on modified optical properties of a biological sample, the method comprising: a. providing said biological sample comprising nucleic acid; b. combining the biological sample with a preparation solution comprising a buffer solution and / or an optical property modifying reagent solution to produce a sample solution; c. dispensing the sample solution into the incubation chamber; d. mixing the sample solution with the lysing agent using a mixing heater to apply heat to the incubation chamber to achieve thermal mixing, thereby forming a prepared sample solution; e. heating a heat-sealed valve disposed in a wax valve channel in fluid communication with the incubation chamber so as to allow the prepared sample solution to flow through the wax valve channel to one or more reaction chambers containing assay reagents, wherein the prepared sample solution is mixed with the assay reagents to form a reaction mixture; f. heating the reaction mixture to promote the use of the nucleic acid present in the biological sample and the assay reagents for nucleic acid amplification reaction, the reaction produces amplified nucleic acid and a plurality of protons; g. reacting the protons with the optical property modifying agent, wherein the reaction is capable of modifying the optical property of the optical property modifying agent to allow detection of the modified optical property, thereby indicating the presence of a suspected analyte in the biological sample; as well as h. causing a plurality of light emitting elements to emit light in a repeating pattern at a repetition frequency so as to determine one or more characteristics of the biological sample using a photosensor based on the modified optical properties.

83. The method of claim 82, further comprising displaying the determined characteristics using an electronic results display mechanism.

84. The method of claim 82 or 83, wherein providing the biological sample comprises performing a nasal swab on the subject, performing a tonsil and / or throat swab on the subject, performing a vaginal swab on the subject, obtaining a hair sample from the subject, drawing blood from the subject, obtaining a urine sample from the subject, obtaining an oral lavage, or a combination thereof.

85. The method of any one of claims 82 to 84, wherein the biological sample and the preparation solution are combined within a sample preparation device.

86. The method of any one of claims 82 to 85, further comprising providing a system of any one of claims 1 to 81.

87. The method of claim 86, wherein dispensing the sample solution into the incubation chamber comprises coupling the sample preparation device to the sample receiving module to establish a fluid pathway between the sample preparation device and the incubation chamber.

88. The method of claim 87, further comprising rupturing and / or breaking a rupturable seal on the sample preparation device to enable the sample solution to flow from the sample preparation device to the incubation chamber.

89. The method of any one of claims 82 to 88, further comprising maintaining the mixing heater in a deactivated state until the sample solution is detected in the incubation chamber and / or until a minimum level of the sample solution in the incubation chamber is detected.

90. The method of claim 89, wherein the sample solution is detected within the incubation chamber using a sensor of one or more sensors of any one of claims 19 to 27 and / or until a minimum level of the sample solution in the incubation chamber is detected.

91. The method of any one of claims 82 to 90, wherein the sample solution is mixed with the lysing agent for a specified amount of time.

92. The method of claim 91, wherein the prescribed amount of time is from about 1 minute to about 20 minutes.

93. The method of any one of claims 82 to 92, further comprising regulating the mixing heater based on i) a constant or substantially constant power supplied to the mixing heater by a power supply, or ii) maintaining a constant or substantially constant temperature of the mixing heater or a portion of the incubation chamber.

94. The method of any one of claims 91 to 93, wherein heating the heat-sealed valve comprises activating the valve heater after the specified amount of time.

95. The method of any one of claims 82 to 94, wherein heating the heat-sealed valve causes the heat-sealed valve to soften, melt and / or dissolve, wherein the dissolution occurs within the prepared sample solution.

96. The method of claim 95, further comprising isolating an initial volume of the prepared sample solution and a dissolved heat-sealed valve in an isolation chamber upstream of the one or more reaction chambers.

97. A method according to any one of claims 82 to 96, wherein the heat-sealed valve comprises any valve of claims 44 to 51.

98. A method for preparing a heat-sealed valve in a system according to any of claims 1 to 66, the method comprising: a. dispensing molten or dissolved heat-sealed valve material into the wax valve channel through the valve filling port; b. sealing the valve fill port; and c. Drying or cooling the wax valve channel to cure or substantially cure the heat seal valve material.

99. The method of claim 98, wherein the heat seal valve material is dispensed into the wax valve channel using a wax dispenser.

100. The method of claim 98 or 99, wherein sealing the valve filling port comprises using a polymer, a stopper, a plug, a hot melt and / or a pressure sensitive adhesive of the port.

101. A kit for performing a biological assay, the kit comprising: a. Sample preparation equipment; and b. A measuring device, comprising: i. A thermal mixing module, the thermal mixing module comprising: i. a sample receiving module for receiving a sample solution comprising a biological sample and a preparation solution; and ii. an incubation chamber in fluid communication with the sample receiving module and comprising a lysing agent; ii. A wax valve channel in fluid communication with the incubation chamber; iii. an optical property modification (OPM) module, the OPM module being operatively coupled to the incubation chamber via the wax valve channel, the OPM module comprising one or more reaction chambers, each reaction chamber comprising an assay reagent; iv. a heat-sealed valve disposed i) within the wax valve channel and ii) between the sample receiving module and the OPM module; and v. A mixing heater configured to supply heat to the incubation chamber.

102. The kit of claim 101, wherein the sample preparation device comprises: a. a sample collector configured to obtain a biological sample; and b. A tube containing a preparation solution and configured to receive at least a portion of the sample collector.

103. The kit of claim 101 or 102, wherein the sample collector comprises a nasal swab, a throat and / or tonsil swab, a vaginal swab, or a combination thereof.

104. The kit of any one of claims 101 to 103, wherein the assay device further comprises a valve heater.

105. The kit of any one of claims 101 to 104, wherein the assay device further comprises a reaction heater.

106. The kit according to any one of claims 101 to 105, wherein the assay device comprises any of the features described in any one of claims 1 to 81.

Images