System and method for performing a bioassay using a heat-sealing stop valve

Abstract

Provided herein is a system and method for performing a bioassay using a heat-sealable valve and / or an incubation chamber. This system and method determine one or more characteristics of a nucleic acid amplification sample based on altered optical characteristics of the sample.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 351,427, filed on Jun. 12, 2022, the entire content of which is incorporated herein by reference for all purposes.

Background Art

[0002] Introduction Assays of biological samples are used to evaluate one or more characteristics of a biological sample. Such assays can qualitatively evaluate and / or quantitatively measure the presence, amount, and / or functional activity of one or more analytes in a biological sample. Such evaluations can be performed based on the presence or absence of changes that occur in the assay. For example, a change in the color and / or transmittance of a biological sample or a portion thereof under specific conditions during an assay can function as an indicator of one or more characteristics of the biological sample.

Summary of the Invention

[0003] Systems and methods are provided herein for performing bioassays on cells that require lysis. The systems and methods described herein isolate and fluidly connect an incubation chamber and a downstream reaction chamber using a heat - seal valve. The systems and methods determine one or more characteristics of a nucleic acid amplification sample based on the changed optical properties of the sample.

[0004] In one aspect, the present disclosure provides a system for performing a bioassay, the system comprising: a. a thermal mixing module comprising: i. a sample receiving module for receiving a sample solution comprising a liquid sample (e.g., 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 operably coupled to the incubation chamber via a wax valve channel, the OPM module including one or more reaction chambers each containing an assay reagent, d. i) A thermal seal valve disposed within the wax valve channel and ii) between the sample receiving module and the OPM module, e. A mixing heater configured to supply heat to the incubation chamber, f. A valve heater, and g. A reaction heater comprising one or more of the above.

[0005] In some embodiments, the incubation chamber contains a reagent or a solvent. When a solvent is contemplated as described herein, a reagent is also contemplated. In some embodiments, the mixing heater is configured to be aligned with at least a portion of the thermal mixing module and offset from the central portion of the incubation chamber.

[0006] In some embodiments, the system further comprises a sample preparation device configured to mate with the sample receiving module. In some embodiments, the sample receiving module includes a piercing element configured to penetrate a frangible seal on the sample preparation device, thereby enabling fluid communication between a sample preparation chamber within the sample preparation device and the incubation chamber.

[0007] In some embodiments, the frangible seal comprises a foil. In some embodiments, the sample receiving module comprises a luer for connecting a sample preparation tube to the assay device. In some embodiments, the sample preparation tube comprises a collar that contacts the luer of the sample receiving module, thereby forming a leak-free seal between the sample preparation device and the sample receiving module when they are mated. In some embodiments, the incubation chamber comprises a vent hole. In some embodiments, the vent hole is a selective venting element. In some embodiments, the selective venting element comprises a rigid or semi-rigid porous matrix (e.g., embedded therein) of a material that swells upon contact with a liquid. In some embodiments, the selective venting element is a self-sealing polymer that comprises a material that swells upon contact with a liquid (e.g., embedded therein). In some embodiments, the selective venting element is a self-sealing sintered polymer exhaust plug. In some embodiments, the selective venting element is a self-sealing fibrous material that comprises a material that swells upon contact with a liquid (e.g., embedded therein). In some embodiments, the selective venting element is a self-sealing porous polyethylene exhaust plug that comprises an embedded hydrogel. In some embodiments, the selective venting element is a thermoplastic (e.g., heat resistant). In some embodiments, the selective venting element is polytetrafluoroethylene or polyethersulfone. In some embodiments, the selective venting element comprises a heat-sealable material(s). In some embodiments, the selective venting element comprises a hydrophobic porous membrane. In some embodiments, the vent hole comprises a detection channel that is in fluid communication with a filling detection chamber configured to detect its liquid filling.

[0008] In some embodiments, the thermal mixing module further comprises one or more sensors configured to: i) detect an initial presence of liquid in the incubation chamber, ii) detect a liquid level in the incubation chamber, or iii) detect both. In some embodiments, the one or more sensors comprise a capacitance sensor disposed below the incubation chamber. In some embodiments, the one or more sensors comprise one or more bottom electrodes configured to operably communicate with a circuit board and to be exposed into the incubation chamber through the bottom wall of the incubation chamber.

[0009] In some embodiments, the thermal mixing module comprises a light source, optionally a light emitting diode (LED) or a laser.

[0010] In some embodiments, the system further comprises one or more upper electrodes that are operably communicable with the circuit board and are exposed into the incubation chamber through a wall other than the bottom wall of the incubation chamber, thereby configured to detect the liquid level in the incubation chamber. In some embodiments, the thermal mixing module comprises an electrode socket that includes an electrode (e.g., an upper electrode; see, e.g., FIG. 5). In some embodiments, among the one or more sensors, one sensor is disposed within the filling detection chamber or is otherwise operably connected to the filling detection chamber (e.g., the sensor may be disposed within the sample receiving module, but even if not disposed within the filling detection chamber, the sensor can 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 one or more sensors are configured to detect a change in light within the filling detection chamber. In some embodiments, the one or more sensors comprise a mixing heater and / or a thermocouple coupled to a portion of the incubation chamber, thereby correlating the presence of liquid in the incubation chamber by detecting a temperature change in the mixing heater and / or the incubation chamber. In some embodiments, the one or more sensors are operably communicable with the mixing heater. In some embodiments, the one or more sensors function as an interlock for the mixing heater, whereby the mixing heater is configured to operate and / or stop based on the detection of liquid and / or the liquid level in the incubation chamber by the one or more sensors.

[0011] In some embodiments, the incubation chamber comprises a light source (e.g., a light emitting diode (LED), a laser, etc.). As used herein, a "light source" refers to an element capable of illuminating a surface individually.

[0012] In some embodiments, the lysing agent comprises lyophilized pellets. 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. In some embodiments, the mixing heater is configured to heat the sample solution within the incubation chamber, thereby enabling mixing of the sample solution and the lysing agent therein to form a prepared sample solution.

[0013] 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 defined relative to the width of the incubation chamber, thereby minimizing and / or reducing the dead volume region within the incubation chamber. In some embodiments, the length of the incubation chamber is from 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 time, 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 from 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 from about 1 / 8 times, 1 / 7 times, 1 / 6 times, 1 / 5 times, 1 / 4 times, 1 / 3 times, 1 / 2 times to about 1.0 times the average of the length and width of the incubation chamber. In some embodiments, the incubation chamber has a volume of about 0.1 mL to about 10 mL, such as about 0.5 mL to about 5 mL. In some embodiments, the incubation chamber has a volume of 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 incubation chamber has a volume of 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 incubation chamber has a volume of 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 incubation chamber has a volume of 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.

[0014] In some embodiments, the mixing heater is configured to heat the sample solution in the incubation chamber for a predetermined time. In some embodiments, the predetermined time is about 5 minutes. In some embodiments, the predetermined time is from about 30 seconds to about 20 minutes. In some embodiments, the predetermined time is from about 1 minute to about 10 minutes. In some embodiments, the predetermined time is from about 1 minute to 5 minutes or from about 5 minutes to 10 minutes. In some embodiments, the predetermined time is about 20 seconds, 21 seconds, 22 seconds, 23 seconds, 24 seconds, 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, 50 seconds, 51 seconds, 52 seconds, 53 seconds, 54 seconds, 55 seconds, 56 seconds, 57 seconds, 58 seconds, or 59 seconds. In some embodiments, the predetermined time is about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes. In some embodiments, the predetermined 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 predetermined time is from about 1 minute to 2 minutes, from about 1 minute to 3 minutes, from about 1 minute to 4 minutes, from about 1 minute to 5 minutes, from about 1 minute to 6 minutes, from about 1 minute to 7 minutes, from about 1 minute to 8 minutes, from about 1 minute to 9 minutes, from about 1 minute to 10 minutes, from about 1 minute to 11 minutes, from about 1 minute to 12 minutes, from about 1 minute to 13 minutes, from about 1 minute to 14 minutes, from about 1 minute to 15 minutes, from about 1 minute to 16 minutes, from about 1 minute to 17 minutes, from about 1 minute to 18 minutes, from about 1 minute to 19 minutes, or from about 1 minute to 20 minutes. In some embodiments, the predetermined time is from about 5 minutes to 6 minutes, from about 5 minutes to 7 minutes, from about 5 minutes to 8 minutes, from about 5 minutes to 9 minutes, from about 5 minutes to 10 minutes, from about 5 minutes to 11 minutes, from about 5 minutes to 12 minutes, from about 5 minutes to 13 minutes, from about 5 minutes to 14 minutes, from about 5 minutes to 15 minutes, from about 5 minutes to 16 minutes, from about 5 minutes to 17 minutes, from about 5 minutes to 18 minutes, from about 5 minutes to 19 minutes, or from about 5 minutes to 20 minutes. In some embodiments, the predetermined time is from about 10 minutes to 11 minutes, from about 10 minutes to 12 minutes, from about 10 minutes to 13 minutes, from about 10 minutes to 14 minutes, from about 10 minutes to 15 minutes, from about 10 minutes to 16 minutes, from about 10 minutes to 17 minutes, from about 10 minutes to 18 minutes, from about 10 minutes to 19 minutes, or from about 10 minutes to 20 minutes.

[0015] In some embodiments, the wax valve channel is disposed at the end of the incubation chamber opposite the sample receiving module. In some embodiments, the heat-sealing 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-sealing 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 heat-sealing valve has a molecular weight of from about 1,300 g / mol to about 10,000 g / mol. In some embodiments, the heat-sealing valve has a molecular weight of about 6,000 g / mol.

[0016] In some embodiments, the heat-sealing valve has a melting temperature of from about 40°C to about 75°C. In some embodiments, the heat-sealing valve has a melting temperature of from about 40°C to 45°C, 40°C to 50°C, 40°C to 55°C, 40°C to 60°C, 40°C to 65°C, 40°C to 70°C, 40°C to 75°C, or 40°C to 80°C. In some embodiments, the heat-sealing valve has a melting temperature of from about 35°C to 40°C, 35°C to 45°C, 35°C to 50°C, 35°C to 55°C, 35°C to 60°C, 35°C to 65°C, 35°C to 70°C, 35°C to 75°C, or 35°C to 80°C. In some embodiments, the heat-sealing valve has a melting temperature of 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 heat-sealing valve has a melting temperature of about 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C.

[0017] In some embodiments, the heat-sealing valve has a volume of about 2 μL to about 6 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 3 μL to about 5 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 3 μL to about 4 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 2 μL to about 5 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 2 μL to about 4 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 2 μL to about 3 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 2 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 3 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 4 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 5 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 6 μL within the wax valve channel. In some embodiments, the heat-sealing valve has a volume of about 7 μL within the wax valve channel. In some embodiments, the wax valve channel comprises a valve filling port for accommodating the valve therein. In some embodiments, the heat-sealing valve is solid or substantially solid at a first temperature, which helps prevent the sample solution from flowing through the wax valve channel. In some embodiments, the heat-sealing valve is configured to transition from a solid or substantially solid configuration to a soft, dissolved, and / or melted configuration after receiving sufficient heat.

[0018] In some embodiments, the system further comprises a valve heater configured to heat the heat-sealing valve, thereby softening, melting, and / or fusing the heat-sealing valve to allow the sample solution to pass therethrough. In some embodiments, the inlet 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 heat conduction pads operably coupled to the mixing heater, the valve heater, or both. In some embodiments, the one or more heat conduction pads (e.g., thermal gap pads) comprise a valve heat conduction pad configured to transfer heat from the valve heater to the wax valve channel to heat the heat-sealing valve. The heat conduction pads (e.g., thermal gap pads) fill the space and conduct heat, thereby allowing for variations in the distance between the substrate and the heat-sealing valve or the incubation chamber.

[0019] In some embodiments, the liquid level of the sample solution in the incubation chamber is at a higher position than the wax valve channel, whereby the heat-sealing valve is subjected to the hydrostatic pressure of the sample solution. In some embodiments, the sample preparation tube is disposed at a higher position than the heat-sealing valve or the wax valve channel, whereby the heat-sealing valve is subjected to the hydrostatic pressure of the sample solution. In some embodiments, the heat-sealing valve is configured to dissolve in 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-sealing valve enters the at least one reaction chamber together with the prepared sample solution.

[0020] In some embodiments, the system further comprises an isolation chamber that is downstream of the wax valve channel and upstream of the one or more reaction chambers, the isolation chamber being configured to receive therein an initial flow rate of the prepared sample solution and the dissolved heat-sealing valve, thereby reducing the amount of the dissolved heat-sealing valve contained within the one or more reaction chambers. In some embodiments, the isolation chamber comprises a vent hole. In some embodiments, the chamber has an outlet and is indirectly connected to the vent hole.

[0021] In some embodiments, the system further comprises one or more mixing chambers (e.g., shuttle mixing chambers), thereby improving the distribution of the dissolved heat-sealing valve across the one or more reaction chambers.

[0022] In some embodiments, the system further comprises a substrate operably coupled 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 disposed on the substrate. In some embodiments, the substrate further comprises a power source operably connected to the mixing heater and / or the valve heater. In some embodiments, the substrate includes a controller that regulates the power supplied to the mixing heater and / or the valve heater, thereby maintaining the mixing heater and / or the valve heater at a substantially predetermined temperature. In some embodiments, the power source 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.

[0023] In some embodiments, the preparation solution is a preparation solution for nucleic acid amplification. In some embodiments, the preparation solution further includes an optical property modification reagent. In some embodiments, the liquid sample (e.g., a biological sample) includes human saliva, urine, human mucus, vaginal secretion, semen, blood, oral rinse fluid, or solid tissue such as buccal tissue, bacteria, one or more spores, one or more viruses, or a combination thereof, and / or concentrates thereof.

[0024] In some embodiments, the OPM module includes a reaction chamber channel that is in fluid communication with the wax valve channel, and one or more reaction chambers are in fluid communication with the reaction chamber channel via corresponding branch portions. In some embodiments, each reaction chamber is approximately equidistant from a single sensing region disposed within the OPM module. In some embodiments, the OPM module further includes a first plurality of optical pipes, and each first optical pipe can transmit light between one of the one or more reaction chambers and the single sensing region. In some embodiments, the OPM module further includes a reaction heater configured to heat one or more reaction chambers.

[0025] In some embodiments, the assay reagent includes a dry reagent or a lyophilized reagent. In some embodiments, the assay reagent includes a nucleic acid amplification enzyme and a DNA primer.

[0026] In another aspect, the present disclosure provides a method for determining one or more properties of a nucleic acid amplification sample based on modified optical properties of a liquid sample (e.g., a biological sample), the method including the following. a. Providing a liquid sample (e.g., a biological sample) containing nucleic acid; b. Combining the liquid sample (e.g., a biological sample) with a preparation solution containing a buffer and / or an optical property modification reagent solution to generate a sample solution; c. Dispensing the sample solution into an incubation chamber; d. Using a mixing heater that applies heat to the incubation chamber, mix the sample solution with the solvent, thereby enabling thermal mixing and forming a prepared sample solution thereby. e. Heating a heat-sealing valve disposed within a wax valve channel that is in fluid communication with the incubation chamber, thereby enabling the prepared sample solution to flow through the wax valve channel into one or more reaction chambers containing assay reagents, and heating such that the prepared sample solution is mixed with the assay reagents to form a reaction mixture. f. Heating the reaction mixture to promote a nucleic acid amplification reaction using the nucleic acid present in the liquid sample (e.g., biological sample) and the assay reagents, and generating amplified nucleic acid and a plurality of protons by that reaction. g. Reacting the protons with an optical property modifying reagent, which reaction modifies the optical properties of the optical property modifying reagent and enables detection of the modified optical properties, thereby being able to indicate the presence of a suspected analyte in the liquid sample (e.g., biological sample), reacting, and h. Repeatedly emitting light from a plurality of light emitting elements in a repeating pattern at a constant repetition frequency, and using a photosensor to determine one or more properties of the liquid sample (e.g., biological sample) based on the modified optical properties.

[0027] In some embodiments, the method further includes displaying the determined properties using an electronic result display mechanism.

[0028] In some embodiments, providing a liquid sample (e.g., biological sample) includes performing a nasal swab on a subject, performing a tonsil and / or throat swab on a subject, performing a vaginal swab on a subject, obtaining a hair sample from a subject, performing a blood draw from a subject, obtaining a urine sample from a subject, or a combination thereof.

[0029] In some embodiments, combining the liquid sample (e.g., biological sample) and the preparation solution is performed within a sample preparation device.

[0030] In some embodiments, the method further includes providing the system described in this disclosure.

[0031] In some embodiments, dispensing a sample solution into an incubation chamber includes coupling a sample preparation device to a sample receiving module, thereby forming a fluid path between the sample preparation device and the incubation chamber.

[0032] In some embodiments, the method further includes breaking and / or rupturing a frangible seal on the sample preparation device, thereby enabling the sample solution to flow from the sample preparation device into the incubation chamber.

[0033] In some embodiments, the method further includes maintaining the mixing heater in a non-operating state until the sample solution is detected within the incubation chamber and / or until a minimum liquid level of the sample solution within the incubation chamber is detected.

[0034] In some embodiments, the sample solution is detected within the incubation chamber using a sensor and / or until a lowest liquid level of the sample solution within the incubation chamber is detected. In some embodiments, the sample solution is mixed with a solvent for a predetermined time. In some embodiments, the predetermined time is from about 1 minute to about 20 minutes.

[0035] In some embodiments, the method further includes adjusting the mixing heater based on i) a constant or substantially constant power supplied to the mixing heater via a power supply device, or ii) maintaining a constant or substantially constant temperature of a portion of the mixing heater or the incubation chamber. In some embodiments, heating the heat-sealing valve includes activating the valve heater after a predetermined time. In some embodiments, heating the heat-sealing valve causes the heat-sealing valve to become soft, melt, and / or dissolve, and such dissolution occurs within the prepared sample solution. In some embodiments, the heat-sealing valve is any valve described herein.

[0036] In some embodiments, the method further includes isolating a prepared sample solution and an initial amount of the dissolved heat-sealable valve within an isolation chamber located upstream of one or more reaction chambers.

[0037] Another aspect of the disclosure provides a method of preparing a heat-sealable valve system, the method including the following. a. Dispensing molten or dissolved wax into a wax valve channel via a valve filling port, b. Sealing the valve filling port, and c. Drying or cooling the wax valve channel to solidify or substantially solidify the wax.

[0038] In some embodiments, the wax is dispensed into the wax valve channel using a wax dispenser. In some embodiments, sealing the valve filling port includes using a polymer, barrier, stopper, heat welding of the port, and / or a pressure-sensitive adhesive.

[0039] Another aspect of the disclosure provides a kit for performing a bioassay, the kit including the following. a. A sample preparation device, and b. An assay device, comprising i. A thermal mixing module, comprising · A sample receiving module for receiving a sample solution containing a liquid sample (e.g., a biological sample) and a preparation solution, and · An incubation chamber containing a solubilizing agent and in fluid communication with the sample receiving module. ii. A wax valve channel in fluid communication with the incubation chamber, iii. An optical property modification (OPM) module operably coupled to the incubation chamber via the wax valve channel, the OPM module including one or more reaction chambers each containing an assay reagent. iv.i) within the wax valve channel, and ii) heat-sealed valves dispensed between the sample receiving module and the OPM module, and v. A mixing heater configured to supply heat to the incubation chamber, an assay device comprising.

[0040] In some embodiments, the assay device further comprises a valve heater. In some embodiments, the assay device further comprises a reaction heater.

[0041] In some embodiments, the sample preparation device comprises a sampling tool configured to collect a liquid sample (e.g., a biological sample) and a tube containing a preparation solution therein and configured to receive at least a portion of the sampling tool.

[0042] In some embodiments, the sampling tool includes a nasal swab, a pharyngeal swab and / or a tonsil swab, a vaginal swab, or a combination thereof. In some embodiments, the assay device comprises any feature described in the systems or methods of the present disclosure.

[0043] In one aspect, the present disclosure provides a kit comprising an assay device and / or a sample preparation device as disclosed herein or components thereof. In some embodiments, the kit includes instructions for use or a QR code or barcode that provides the user with access to the instructions for use.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0062] Systems and methods for performing a bioassay are provided herein. These systems and methods determine one or more characteristics of a nucleic acid amplification sample based on modified optical properties of an eluted sample. The systems and methods herein utilize a heat-sealing valve containing wax to isolate the contents of an incubation chamber from one or more downstream reaction chambers. By melting the wax, a fluid connection between the incubation chamber and one or more downstream reaction chambers is enabled.

[0063] Before describing the present invention(s) in detail, it should be understood that the present invention(s) is not limited to the specific embodiments described, as such embodiments may vary. Also, since the scope of the present invention(s) is limited only by the appended claims, it should be understood that the terms used in this specification are used only for the purpose of describing specific embodiments and are not intended to be limiting.

[0064] Where a range of values is provided, each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limits of that range and other specified intervening values within that range are to be understood as being included herein. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are included herein unless the limit values within the specified range are explicitly excluded arbitrarily. Where the specified range includes one or both of the limit values, ranges excluding either or both of those included limit values are also included herein.

[0065] A particular range may be presented in this specification by prefixing the numerical value with the term "about". As used in this specification and the claims, unless otherwise specified, the terms "about" and "approximately" refer to variations within ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±14%, or ±15% depending on the embodiment. By way of non-limiting example, about 100 meters represents a range of 95 meters to 105 meters, 90 meters to 110 meters, or 85 meters to 115 meters depending on the embodiment.

[0066] The term "substantially" refers to variations within ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±14%, or ±15%. By way of non-limiting example, substantially parallel represents a range of difference from the state of being parallel of -1 degree to 1 degree, -5 degrees to 5 degrees, or -15 degrees to 15 degrees depending on the embodiment.

[0067] 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 this invention belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative, exemplary methods and materials are described below.

[0068] All documents and patents cited herein are hereby incorporated by reference as if each individual document or patent were specifically and individually indicated to be incorporated by reference, disclosing and describing the methods and / or materials related to the cited documents. Any citation of a document is for the purpose of disclosure prior to its filing date and should not be construed as an admission that the present invention does not have the right to antedate such document by virtue of a prior invention. Further, the disclosed dates may be different from the actual publication dates, which may need to be independently confirmed.

[0069] It should be noted that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Further, it should be noted that the claims can be drafted to exclude any element. Accordingly, this description is intended to function as a basis for the use of exclusive terms such as "solely", "only", etc. in connection with the recitation of claim elements and as a preamble for "negative" limitations.

[0070] Also, certain embodiments of the disclosed apparatus and / or related methods may be represented by the drawings included in this application. Embodiments of the apparatus and their specific spatial characteristics and / or functions include those shown in the drawings or substantially shown in the drawings, or those reasonably inferable from the drawings. Such characteristics include, for example, symmetry with respect to a plane (e.g., a cross-sectional plane) or an axis (e.g., a symmetry axis), edges, peripheries, surfaces, a particular direction (e.g., proximal, distal), and / or a number (e.g., three surfaces, four surfaces), or one or more of any combination thereof (e.g., one, two, three, four, five, six, seven, eight, nine, or ten, etc.). Such spatial characteristics include, for example, the absence (e.g., explicitly absent) of symmetry with respect to a plane (e.g., a cross-sectional plane) or an axis (e.g., a symmetry axis), edges, peripheries, surfaces, a particular direction (e.g., proximal), and / or a number (e.g., three surfaces), or one or more of any combination thereof (e.g., one, two, three, four, five, six, seven, eight, nine, or ten, etc.).

[0071] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has individual components and features that can be readily separated or combined with the features of other multiple embodiments without departing from the scope or spirit of the invention. Any described method can be performed in the described order or in any other logically possible order.

[0072] In further describing the subject invention, the subject apparatus used in the implementation of the subject system will be described in detail, followed by an overview of the related method.

[0073] Definitions The terms used in the claims and the specification are defined as follows, unless otherwise specified. Unless otherwise defined, all technical terms, notations, and other scientific terms used in this specification are intended to have the meanings commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings may be clarified and / or defined in this specification for the sake of clarity and / or ease of reference, but including these definitions in this specification should not necessarily be construed as meaning something different from what is commonly understood in the art.

[0074] As used in this specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "include", "such as", and the like are intended to be inclusive without limitation unless otherwise expressly stated.

[0075] As used in this specification, the term "comprising" specifically includes embodiments consisting of the recited elements and embodiments consisting essentially of the recited elements, unless otherwise expressly stated.

[0076] The term "colorimetric analysis" or "colorimetric" refers to techniques for quantifying or otherwise observing the concentration of a colored compound in a solution. "Colorimetric detection" refers to any method for detecting such a colored compound and / or a change in the color of a compound in a solution. The methods can include, among other things, visual observation, absorbance measurement, fluorescence measurement, and the like.

[0077] The term "color-changing agent" refers to a compound whose color changes due to some chemical reaction. In particular, a color-changing agent may refer to a compound whose color changes due to a change in pH. Different color-changing agents can change color in different pH transition ranges.

[0078] 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 color-changing agent in the sample (see above).

[0079] The term "nucleic acid amplification" or "amplification reaction" refers to methods for amplifying DNA, RNA, or modified forms thereof. Nucleic acid amplification includes several techniques, such as isothermal reactions and 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 an amplification method that is carried out without changing the temperature of the amplification reaction. Protons are released during the amplification reaction. That is, for each deoxynucleotide triphosphate (dNTP) added to a single-stranded DNA template during the amplification reaction, one proton (H + ) is released.

[0080] The term "sufficient amount" means an amount sufficient to produce the desired effect, for example, an amount sufficient to regulate protein aggregation in cells.

[0081] As used herein, a liquid sample may refer to a biological sample. A "biological sample" is an amount of sample that contains one or more organic molecules, such as one or more nucleic acids (e.g., DNA and / or RNA or portions thereof), and can be taken from a subject. Thus, an "assay of a biological sample" is a test performed on a biological sample that evaluates one or more characteristics of the sample. In some embodiments, the biological sample is a nucleic acid amplification sample, which is a sample that contains or is presumed to contain one or more nucleic acids or portions thereof and is amplifiable according to one embodiment.

[0082] A biological sample is provided from a subject and can contain one or more cells such as the subject's tissue cells. As used herein, the term "tissue" refers to an aggregate of one or more cells having similar functions and structures within a subject (e.g., a living organism such as a mammal like a human), or an aggregate of such a plurality of different types. Tissues can include, for example, organ tissue, muscle tissue (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle), connective tissue, nerve tissue, and / or epithelial tissue. Tissues can, in some types, include cells from the inside of the subject's cheek and / or cells in the subject's saliva. In some embodiments, when a biological sample is referred to herein, a liquid sample is contemplated.

[0083] In some embodiments, the biological sample is a swab of body fluid (such as 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.

[0084] As described above, a biological sample can be provided from a subject. In certain embodiments, the subject is a subject that is a "mammal" or "mammalian", where these terms are widely used to refer to organisms belonging to the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the subject is a human. The term "human" can include human subjects of both genders at all stages of development (e.g., fetus, newborn, infant, child, adolescent, adult), and in certain embodiments, the subject is an infant, adolescent, or adult. It should be understood that the devices and methods described herein can be applied in relation to human subjects, but these devices and methods can also be applied in relation to other subjects, i.e., "non - human subjects".

[0085] Liquid samples (e.g., biological samples) can include, for example, human saliva, urine, human mucus, blood, oral rinse fluid, or solid tissues such as buccal tissue. Biological samples can also include viruses, bacteria, and / or spores.

[0086] In some embodiments, the biological sample is mixed with a preparation solution. In some embodiments, the preparation solution includes a preparation solution for nucleic acid amplification. In some embodiments, the preparation solution includes an elution buffer. In some embodiments, the preparation solution alternatively or additionally includes an optical property modifying reagent. According to some embodiments, the optical property modifying reagent changes its optical properties depending on the presence or absence of a specific marker in the biological sample when the side of the biological sample or one or more of them is exposed to the optical property modifying reagent. Examples of optical properties that can change include color and absorbance measured by spectrophotometry. The change in optical properties can be detected and used to identify the characteristics of the biological sample. In some embodiments, the cells of the sample are lysed with a solvent of the preparation solution and / or nucleic acids are extracted therefrom. The nucleic acids so extracted can be released into the resulting prepared sample solution. In some embodiments, the step of extracting genomic deoxyribonucleic acid (DNA) from the biological sample is included. Optionally, the preparation solution is a preparation solution for nucleic acid amplification, and by being exposed to this solution, the nucleic acids of the sample are prepared for amplification, for example, isothermal amplification.

[0087] Also, as used herein, the phrase "optical property" refers to one or more optically recognizable features, for example, features resulting from the wavelength and / or frequency of radiation (e.g., light) emitted from or transmitted through the sample before, during, or after an assay reaction performed using such a sample, and this includes color, absorbance, reflectance, scattering, fluorescence, phosphorescence, etc. Thus, modifying an optical property refers to changing such a feature.

[0088] System Aspects of the present disclosure include systems for performing bioassays by modifying the optical properties of a biological sample and detecting these modified properties. Such systems can include various devices, such as sample preparation devices and assay devices.

[0089] In some embodiments, the sample preparation device is configured to prepare a biological sample before being dispensed into the bioassay device. In some embodiments, the biological sample is collected using a sample collector (e.g., nasal swab, etc.). In some embodiments, the biological sample is mixed with a preparation solution to form a sample solution. In some embodiments, the preparation solution includes a preparation solution for nucleic acid amplification. In some embodiments, the preparation solution includes an elution buffer. In some embodiments, the preparation solution includes an optical property modifying reagent. In some embodiments, the optical properties of the optical property modifying reagent change depending on the presence or absence of a specific marker in the biological sample when the biological sample or one or more of its aspects is exposed to the optical property modifying reagent. In some embodiments, the sample solution is configured to be dispensed into the assay device.

[0090] In some embodiments, the assay device includes one or more thermal mixing modules and / or one or more optical property modifying modules. In some embodiments, the thermal mixing module and the optical property modifying module are fluidly connected via a wax valve channel disposed therebetween. In some embodiments, the bioassay is performed on the biological sample received in the thermal mixing module (e.g., the biological sample may be provided in the sample solution). In some embodiments, as described herein, the assay device is configured to perform a predetermined time incubation before transferring the biological sample to the optical modification module to perform one or more reactions.

[0091] In some embodiments, a heat-sealing valve disposed within the valve channel provides a barrier that prevents the biological sample (and corresponding sample solution) from flowing into the optical modification module. In some embodiments, the heat-sealing valve is configured to soften, melt, and / or dissolve after receiving sufficient heat, whereby the sample solution can push through the valve and flow across the channel into the optical property modification module. In some embodiments, such incubation of the sample causes the sample to be mixed with the lysing agent, and as described herein, such incubation and mixing helps to lyse cells and release biological materials, facilitating the reaction in the optical modification module. In some embodiments, the mixing of the sample and the lysing agent is performed via thermal mixing. The heat-sealing valves described herein can also be used in alternative mixing chambers (e.g., elution of the sample with a reagent) that do not involve cell lysis.

[0092] In some embodiments, the optical property modification module includes one or more reaction chambers, one or more of which can optionally contain assay reagents (e.g., optical property modification reagents, nucleic acid amplification reagents, or both), where they are mixed and reacted with the sample in the conditioned sample solution. In some embodiments, the reaction (e.g., amplification reaction) provides a reaction product that reacts with the optical property modification reagent to produce a detectable change in the optical properties of the biological sample, indicating the presence, absence, or amount of the analyte suspected to be present in the sample.

[0093] In some embodiments, the assay reagents are present in the preparation solution. In some embodiments, the assay reagents are present in the incubation chamber. In some embodiments, the assay reagents are present in one or more reaction chambers.

[0094] In some embodiments, each reaction chamber can include a sample receiving opening for receiving a biological sample from a sample inlet and / or conduit. The sample receiving opening can 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, which can include “venting” and “auxiliary” openings, or “first” and “second” openings, etc. In some embodiments, the channels downstream from the wax valve channel 110 lead to one or more conduits (e.g., like spokes) branching from the channel and fluidly connecting the channel to the reaction chamber. In some embodiments, each conduit connects to a different reaction chamber.

[0095] In some embodiments, each reaction chamber is provided with a selective venting element as described herein. These selective venting elements are configured to release gas (e.g., air) and / or vapor from the reaction chamber when the sample solution flows into the respective reaction chamber. When the selective venting element becomes impermeable to liquid, these methods include preventing further flow of liquid through the device and / or preventing backflow of liquid from the reaction chamber after contacting and becoming impermeable to the selective venting element. Therefore, after such flow has stopped, diffusion is the only way to transport any contaminants into and / or out of the reaction chamber. Thus, if the inlets and / or conduits are of sufficient length, the diffusion time of contaminants will be substantially longer than the reaction and / or readout time and will not be affected by contaminants as a result.

[0096] One of the challenges in miniaturizing and automating biochemical protocols in a microfluidic system is the method of accurately aliquoting samples into multiple reaction chambers while minimizing cross - contamination. In some embodiments, this is achieved by using multiple reaction chambers at the ends of conduits branching from a main fluid channel. Before the reaction takes place within the chamber, this aliquot must be isolated so that cross - talk between reactions does not occur. In some embodiments, sample isolation can be achieved by installing input valves and / or output valves placed between each chamber. These valves seal the chambers and prevent any cross - talk. Although using multiple valves functions to some extent, in such protocols, it is necessary to actively control the opening and closing of the valves, which requires energy, infrastructure, and implementation costs, thus complicating the system design of the OPM module. Also, some valve structures function optimally when primed, so it is necessary to fill the microfluidic system with an initial priming liquid. Such a priming step complicates the system workflow. Therefore, according to versions of the method in question, these methods do not include priming.

[0097] In contrast, the apparatus and method in question can provide sufficient automatic fluid flow control to enable an assay to be performed by passive aliquoting through one or more parts of the apparatus. For example, one or more fluids (e.g., air and / or biological samples) can be moved through, or prevented from moving through, one or more parts of the apparatus with little or no specific user operation required. Passively sealing the apparatus or a part thereof eliminates the need for active control and minimizes the complexity of the overall apparatus and the user procedures required for operation of the apparatus. Thus, the present disclosure provides a simple and easy-to-use assay device. Such an apparatus and method in question do not require valves or complex valve control protocols. Thus, the present disclosure provides for a simple and robust implementation of an on-chip aliquoting function without using moving parts. According to embodiments of the subject matter, the amount and number of aliquots are controlled by the shape of the channels and chambers (e.g., the length and cross-sectional area of the channel between the sample receiving openings of the reaction chamber are set to be sufficiently long and sufficiently small, respectively, to prevent crosstalk between the contents of the reaction chamber). For example, in some embodiments, the cross-sectional area of each reaction chamber channel is 1 / 10 or less of that of the reaction chamber.

[0098] In some embodiments, the assay device includes a substrate. The substrate can include one or more control units (e.g., a central processing unit (CPU) or a field programmable gate array (FPGA)). Such units can include a memory and / or a processor (e.g., a microprocessor) and are configured to generate one or more outputs (e.g., electrical signals) based on one or more inputs (e.g., inputs from a user and / or a sensor, and / or a timer, and / or instructions stored in a memory). The device can also include a user interface for receiving inputs, which is operably coupled to the control unit. In some embodiments, the substrate includes a printed circuit board (PCB), which is disposed under 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 assay device. The substrate can also include one or more thermal gap pads, e.g., to couple a heating element on the PCB to an appropriate fluid location.

[0099] In some embodiments, the sample preparation device is configured to be operably coupled to the assay device.

[0100] As used herein, "operably coupled," "operably connected," and "operably attached" mean that the disclosed device is connected in a particular way that enables the device to operate in the manner described herein and / or be effectively implemented. For example, operably coupling can include removably coupling or fixedly coupling two or more elements. Operably coupling can also include coupling two or more components fluidly and / or electrically and / or in a mating and / or adhering manner. Thus, an operably couplable device is a device that can be operably coupled. Also, as used herein, "removably connected" means, for example, being physically and / or fluidly and / or electrically coupled, and two or more coupled components can repeatedly uncouple and then recouple.

[0101] Assay device Figures 1A and 1B show exemplary embodiments of the system 100 described herein for performing a bioassay on a biological sample. In some embodiments, the system includes an assay device 102 coupled to a sample preparation device 104. As used herein, the term "assay device" can be used interchangeably with test cartridge. In some embodiments, the assay device comprises 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 a biological sample to create a sample solution 107. In some embodiments, the thermal mixing module 106 and the OPM module 108 are formed as a single integrated component on the assay device 102. In some embodiments, the assay device 102 comprises an upper portion 103 and a lower portion 105 configured to be operably coupled. In some embodiments, the upper portion 103 and the lower portion 105 are joined along a dividing line of the assay device (see, e.g., 160 in FIG. 6). In some embodiments, the upper and lower portions of the device can form a fluid seal by sandwiching a gasket made of an elastomer or other flexible material. FIG. 1C shows a side view of an exemplary embodiment of the assay device 102. See also FIGS. 1D and 1E, which have flat or low-profile vents in the incubation chamber of the thermal mixing module 106. FIG. 2A shows a top view of the lower portion 105 in one embodiment of an exemplary assay device 102. As described herein, in some embodiments, the assay device 102 comprises a thermal mixing module 106 having an incubation chamber 112 therein, an OPM 108, and a channel 110 disposed therebetween. The term "incubation chamber" can be used interchangeably with "mixing chamber" herein. FIG. 2B shows a similar assay device 102 without an isolation chamber 158 and a fill detection chamber 126.

[0102] In some embodiments, as shown in FIG. 1C, assay device 102 further comprises a lower portion 105 of thermal mixing module 106, a channel 110, and a substrate 119 operably connected to OPM module 108. In some embodiments, as described herein, substrate 119 comprises a PCB 120. In some embodiments, substrate 119 comprises one or more heaters, each operably in communication with a power source (e.g., a single power source can supply power to all heaters, or one or more power sources can supply power to the heaters). In some embodiments, where there are multiple heaters, only one heater is powered at a time. In some embodiments, substrate 119 comprises, as described herein, a mixing heater aligned with thermal mixing module 106, a valve heater aligned with channel 110, and / or a reaction heater aligned with OPM module 108. In some embodiments, substrate 119 comprises one or more thermal gap pads, for example, used to couple a heating element on a PCB to an appropriate fluid location.

[0103] Referring to FIGS. 3A and 3B, a side cross-sectional view of the assay device 102 according to the embodiments described herein is shown. 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 operably coupled to the sample preparation device 104 and configured to receive a sample therefrom (as described herein). In some embodiments, the sample receiving module 112 includes a luer 116 that defines an opening to the sample inlet 118 therein, and the sample inlet 118 is in fluid communication with the incubation chamber 112. In some embodiments, the luer 116 is operably coupled to the sample preparation device 104 and configured to readily receive a sample into the assay device, for example, through or around a piercing element. For example, FIGS. 3A and 3B provide an example of coupling the sample preparation device 104 to the luer 116. The sample preparation device is contemplated as a tube, vial, syringe, or other container. In some embodiments, the piercing element 121 is configured to break a frangible seal of the sample preparation device, thereby allowing fluid flow therefrom. As used herein, "frangible seal" refers to a means that can be broken to seal an opening at the end of the sample receiving module. Other means for breaking a frangible seal are also contemplated within the apparatus of the present disclosure. For example, the frangible seal can be pierced, stabbed, penetrated, poked, torn, cut, etc. Referring to FIG. 4, an example of the interaction between the luer 116 and the sample preparation device 104 is shown, and the luer 116 is configured to fit with a collar 122 of the sample preparation device, thereby forming a leak-free joint. The collar 122 can further include a tube rib, thereby further strengthening the seal between the luer 116 and the collar 122. In some embodiments, the sample receiving module further includes a piercing element 121, thereby breaking the frangible seal of the sample preparation device 104 and allowing fluid flow therefrom.In some embodiments, as described herein, the incubation chamber 112 is configured to receive a sample solution from the sample receiving module.

[0104] The piercing element fluidly connects the sample preparation device 104 to the assay device or the incubation chamber by breaking a frangible seal. In certain embodiments, when the piercing element penetrates the frangible seal, deformation of the frangible seal and / or the piercing element is minimized. PCT / US2021 / 049178 (the related disclosure of which is incorporated herein by reference) describes a bubble-free piercing mechanism that can be used to break a frangible seal, which allows for clear piercing of the frangible seal in a repeatable shape, thereby minimizing the size and / or number of bubbles or completely preventing the generation of bubbles. As used herein, the term "bubble-free piercing" refers to a mechanism that reduces (minimizes) the size and / or number of bubbles or completely prevents the generation of bubbles when, for example, piercing a sample collection tube. The bubble-free piercing mechanism(s) described herein minimize or prevent the entry of bubbles into the chamber being pierced (e.g., a sample collection tube).

[0105] The "seal" referred to in "frangible seal" can be a layer of material such as a polymeric material and / or a metallic material as described herein. In some embodiments, the seal is a foil sheet made of aluminum and / or other metal.

[0106] In some embodiments, the sample preparation device includes a valve instead of a frangible seal to be pierced. Examples of sample preparation devices with frangible seals or valves are described in PCT / US2017 / 022304, the related disclosure of which is incorporated herein by reference.

[0107] In some embodiments, the assay device can also include one or more filters for filtering fluid discharged from any part of the assay device or the sample preparation device. The filter can be configured to filter the sample fluid before it is discharged through the valve.

[0108] In some embodiments, the contact portion includes a hydrophobic material. In some embodiments, the piercing element includes a hydrophilic material. In some embodiments, the discharge port includes a hydrophobic material. In some embodiments, the frangible seal includes a hydrophilic material. In some embodiments, the contact portion includes a plastic material. In some embodiments, the piercing element includes a metallic material. In some embodiments, the discharge port includes a plastic material. In some embodiments, the frangible seal includes a metallic material. In some embodiments, the piercing element includes a material having a hardness at least twice the hardness of the material including the frangible seal, whereby the piercing element clearly pierces the frangible seal in a repeatable shape without deformation of the piercing element. In some embodiments, the piercing element has a hardness of at least about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, or about 20 times the hardness of the material including the frangible seal.

[0109] In some embodiments, the end of the second portion of the piercing element configured to contact the frangible seal has a blunt tip, whereby the surface portion of the end of the second portion is orthogonal to the length of the piercing element. In some embodiments, the end of the second portion of the piercing element configured to contact the frangible seal has a sharp tip.

[0110] Vent holes of the incubation chamber In some embodiments, the incubation chamber 112 includes a vent hole 124 and is configured to discharge gas (e.g., air) and / or vapor from the incubation chamber 112 when the sample solution flows into the incubation chamber 112. In some embodiments, the vent hole 124 is open to the surrounding environment, and when the incubation chamber 112 is filled, the sample solution overflows from above the vent hole 124. In some embodiments, the vent hole 124 includes a selective ventilation element that forms a seal when in direct contact with a liquid and is configured to prevent the liquid (e.g., the sample solution) from passing through.

[0111] The selective ventilation element may be porous, so that a plurality of pores extend therethrough. Such an element can have a passively adjustable porosity and / or can control the flow of one or more fluids (e.g., a gas such as air and / or a liquid such as a biological sample) within the device. For example, in some embodiments, the vent hole 124 includes a vent plug made of a sintered polymer having self-sealing properties or a hydrophobic porous membrane. In some embodiments, the selective ventilation element is a hydrophobic porous membrane having pores through which gas (e.g., air) can pass but through which water droplets and other polar liquids cannot pass.

[0112] As used herein, the expression "passively adjustable porosity" has a first form in which one or more gases (e.g., air) can pass through pores, for example, and a second form in which a fluid containing one or more gases and liquids (e.g., a liquid containing a biological sample) is prevented or reduced from passing through the pores, and automatically transitions from the first form to the second form when in contact with a liquid. Also, in the second form, the selective ventilation element prevents, for example, evaporation of the liquid through the pores. Further, in the second form, the selective ventilation element fluidly seals a fluid passage, such as a reaction chamber, at its end by covering an opening of the reaction chamber, such as a ventilation opening, and can prevent the passage of a fluid including evaporation therethrough. Further, the selective ventilation element is configured to passively, i.e., automatically, transition from the first form to the second form without the need for user operation when one or more liquids (e.g., a liquid containing a liquid sample) come into contact with the selective ventilation element or a part thereof (e.g., a surface forming the wall of an incubation chamber or a reaction chamber). Thus, in some embodiments, the selective ventilation element can self-seal against liquids and gases when in contact with a liquid.

[0113] Using a selective ventilation element can minimize or eliminate the need for input and / or output valves within the device (or within the channels of the device). Using multiple valves functions to some extent, but such a protocol requires active control of the opening and closing of the valves, resulting in the need for the introduction of energy and infrastructure, and thus complicating the system design. Passively sealing the device or a part thereof eliminates the need for active control and minimizes the complexity of the entire device and the user procedures required for device operation. Using at least one selective ventilation element in each reaction chamber makes it easier to passively control the flow of fluids within the device. Thus, in some embodiments, no valves are required to operate the device and / or to control the flow of fluid into a chamber (e.g., a reaction chamber).

[0114] Referring to FIG. 2A, in some embodiments, the assay device includes a fill detection chamber 126 that is in fluid communication with the incubation chamber 112 via a detection channel 128. Optionally, this fill detection chamber 126 is operably coupled to a light pipe that is operably connected to an LED. For example, in some embodiments, the detection channel 128 is in fluid connection with the incubation chamber 112 such that when fluid enters the incubation chamber 112, it also flows into the fill detection chamber through the detection channel 128. The opening at the end of the detection channel allows for fluid communication between the detection channel 128 and the incubation chamber 112. In some embodiments, the fill detection chamber 126 includes (or is operably connected to) a light sensor that detects light from the light pipe, and when the fill detection chamber 126 is filled with the sample solution (via filling of the incubation chamber 112), as a result, the light sensor detects a decrease in light (this decrease is due to the sample that may darken by the liquid used (e.g., buffer)). The position of the opening at the end of the detection channel 128 (which couples the detection channel 128 to the incubation chamber 112) can determine the timing at which liquid filling is detected and the associated output (e.g., activation of heating and / or stirring). For example, the opening of the detection channel 128 can be placed at the top of the incubation chamber 112 such that liquid only begins to flow into the fill detection chamber 126 when the incubation chamber 112 is at or near its maximum liquid volume. Thereafter, the liquid flows into the fill detection chamber 126, thereby attenuating the light detected by the light sensor.

[0115] In some embodiments, the light sensor includes any one of a complementary metal oxide semiconductor (CMOS) chip, a photodiode, a phototransistor, a photocell, or a photomultiplier tube.

[0116] Liquid Detection in the Incubation Chamber In some embodiments, the assay device includes one or more sensors configured to detect i) the inflow of a liquid (e.g., a sample solution) into the incubation chamber (e.g., an initial inflow of the sample solution), ii) an incubation chamber filled with the liquid, or iii) both. In some embodiments, the one or more sensors function as an interlock and help prevent a heater (e.g., a mixing heater) from activating before the liquid enters the incubation chamber. For example, if the mixing heater is prematurely activated before the sample solution enters the incubation chamber 112, the assay device 102 may overheat or the battery life of the power supply that powers the mixing heater may be unnecessarily shortened. In some embodiments, the sensor is operably coupled to a power source for activating the heater. For example, in some embodiments, a substrate 119 that includes a PCB and optionally includes thermal gap pads is in operative communication with a controller and a power source such that when one or more sensors detect a fluid and / or a predetermined fluid level within the incubation chamber 112, the power source is enabled to supply power to the mixing heater.

[0117] In some embodiments, the incubation chamber can include one or more light sources configured to emit light. Such light sources are operably coupled to one or more sensors and / or PCBs and / or controllers / control units such that the sensors are disposed (e.g., on the opposite side of the light source within the same chamber) and can receive / detect light from the light source. See, for example, FIG. 18. In some embodiments, one or more sensors can be configured to detect optical properties (e.g., the wavelength of light (e.g., color)) and / or changes in optical properties (e.g., the wavelength of light emitted from the contents of the chamber). In embodiments where the light source and one or more sensors are within the same chamber (e.g., an incubation chamber, a reaction chamber, a fill detection chamber, etc.), the light source emits light before the liquid enters the chamber, and when the liquid flows into and fills the chamber, the light signal detected by the sensor (e.g., a light sensor) attenuates. When the light signal attenuates to a threshold value, the input from the sensor indicates the presence of liquid within the chamber. In some embodiments, the controller can respond by generating an output to initiate an operation (e.g., activate a heating element or a mixing element). Light sources according to various embodiments can also include one or more light-emitting diodes (LEDs). In some embodiments, this can also be achieved by using one or more light pipes. In this case, the one or more light pipes enable the transmission of light between the light source and the sensor. In some embodiments, one or more light pipes are operably connected to the incubation chamber and transmit light passing through the chamber (e.g., an incubation chamber, a reaction chamber, a fill detection chamber, etc.) to one or more sensors. In some embodiments, the light emitted from the light source is transmitted through one or more light pipes using at least one of one or more reflective surfaces and / or one or more refractive surfaces disposed within the one or more light pipes.

[0118] In some embodiments, when fluid is detected within the incubation chamber, the system initiates an incubation heating step. Thereafter, an indicator on the display can indicate that sample processing has begun. If no fluid is detected, the system can maintain a "ready to load sample" state (e.g., similar to an indicator used when first powering on the device or when loading a sample). This enables the device to warn of an error if the reagent has been exposed to air for an extended period of time.

[0119] In some embodiments, the sensor provides a signal indicative of the detection of fluid within the incubation chamber 112, and this signal can be an audible signal (e.g., a beep emitted from the PCB) and / or a visual signal (e.g., a blinking light).

[0120] In some embodiments, one or more sensors comprise a capacitance sensor disposed beneath the incubation chamber 112. In some embodiments, the capacitance sensor is configured to measure an increase in the dielectric constant of a sample solution (e.g., including a buffer) compared to air. In some embodiments, the capacitance sensor disposed at the bottom (of the incubation chamber) is configured to detect the inflow of sample solution into the incubation chamber 112 or the level of sample solution within the incubation chamber 112.

[0121] In some embodiments, the one or more sensors comprise a conductive sensor having one or more electrodes. In some embodiments, the one or more electrodes are operably coupled to a substrate (e.g., a PCB) and configured to penetrate the bottom wall of the incubation chamber, such that the electrode(s) are exposed within the incubation chamber (e.g., see electrode 130 in FIGS. 2A, 3A, and 5). Thus, in some embodiments, the one or more electrodes 130 are configured to contact a liquid (e.g., a sample), thereby enabling detection of an initial influx of the sample solution into the incubation chamber 112. In some embodiments, the electrodes 130 require a liquid to conduct charge between the two electrodes 130. For example, in some embodiments, the sample solution includes an ionic buffer, and the ions in this buffer are configured to conduct electricity between the two electrodes 130. Thus, when there is no liquid present within the incubation chamber 112, charge conduction between the electrodes 130 may not occur. In some embodiments, conduction of charge between the electrodes 130 enables activation of the mixing heater 134 via the PCB. In some embodiments, the electrodes 130 comprise a metal, polymer, or elastomer embedded with conductive particles, such that the entire electrode is conductive.

[0122] Referring to FIG. 5, in some embodiments, in addition to or instead of providing electrode 130 at the bottom of incubation chamber 112, the assay device further comprises an electrode 132 disposed at the top of the incubation chamber. In some embodiments, similar to the two electrodes disposed at the bottom of the incubation chamber, bottom electrode 130 and top electrode 132 conduct charge therebetween when exposed to a liquid (e.g., a sample). Thus, in some embodiments, the configuration of top electrode 132 and bottom electrode 130 enables detection that the incubation chamber 112 is filled with liquid (thereby forming a conductive path through the height of the incubation chamber). In some embodiments, top electrode 132 is operably connected to a substrate (e.g., a PCB) via a conductive clip or wire. In some embodiments, when the incubation chamber is filled with a sample solution, mixing heater 134 is activated. In some embodiments, the assay device comprises two bottom electrodes 130 and one top electrode 132, thereby enabling detection of the initial presence (or inflow) of liquid within incubation chamber 112 and the level of liquid (e.g., a sample) within incubation chamber 112.

[0123] In some embodiments, one or more sensors comprise the optical sensors described herein, and fill detection chamber 126 is used to detect the liquid level within incubation chamber 112. In this case, a decrease in the detection of light by the optical sensor indicates that the incubation chamber is filled with a liquid (e.g., a sample solution) (see, e.g., FIGS. 2A and 18). In some embodiments, such detection enables activation of mixing heater 134.

[0124] In some embodiments, one or more sensors comprise thermocouples, are operably coupled to the mixing heater 134, and are configured to measure the temperature of another region where the mixing heater 134 or the PCB is in close thermal contact with the incubation chamber. In some embodiments, a constant power is supplied to the mixing heater 134 (by one or more power supplies described herein), whereby the delay in the temperature rise of the mixing heater 134 detected by the thermocouple indicates the presence of liquid within the incubation chamber 112. For example, in some embodiments, the sample solution includes a buffer with a high heat capacity, such that when the sample solution is present within the incubation chamber 112, the temperature rise of the mixing heater 134 is slower than when there is no temperature rise. FIG. 7 shows an exemplary comparison of the temperature rise of the mixing heater 134, side wall, and bottom wall of the incubation chamber 112 when the incubation chamber 112 is i) empty and ii) filled with a buffer (e.g., provided with the sample solution). As shown, the temperature rise when the incubation chamber 112 is filled is significantly slower compared to the case of an empty incubation chamber 112. In some embodiments, by detecting such a temperature rise with the thermocouple, it becomes possible to operate the mixing heater 134.

[0125] Mixing of Sample Solution In some embodiments, the incubation chamber is configured to mix the sample solution with a lysing agent to lyse the cells obtained from the biological sample. In some embodiments, such lysis releases the nucleic acids within these cells and enables amplification within the OPM module 108. In some embodiments, the lysing agent is disposed within the incubation chamber 112 before the sample solution is dispensed into the incubation chamber 112. In some embodiments, the lysing agent is provided into the incubation chamber 112 after the sample solution is dispensed into the incubation chamber 112. In some embodiments, the lysing agent is dispensed into the incubation chamber 112 at approximately the same time as the sample solution. In some embodiments, the lysing agent is suspended within the sample inlet immediately distal to the piercing element, facilitating more rapid and complete mixing.

[0126] In some embodiments, the lysing agent includes a lyophilized pellet 136 that is rehydrated by contacting the sample solution. In some embodiments, the lysing agent is liquid. In some embodiments, the lysing agent includes dithiothreitol (DTT), proteinase K, mutanolysin, lysostaphin, lysozyme, or any combination thereof. In some embodiments, the lysing agent further includes one or more surfactants such as Tween. In some embodiments, the lysing agent further includes one or more components of the buffer provided with the sample.

[0127] In some embodiments, when a lysing agent is used, RNase inhibitors and other agents that prevent nucleic acid degradation are also contemplated.

[0128] Referring to FIGS. 3A and 3B, in some embodiments, the sample solution is mixed with the solvent 136 via thermal mixing as described herein. In some embodiments, the mixing heater 134 is configured to supply heat to the incubation chamber 112, thereby heating a portion of the sample solution located in the vicinity of the mixing heater 134 faster (compared to the remaining sample solution located elsewhere in the incubation chamber 112), and thereby enabling the temperature of that portion of the sample solution to rise faster. Such a temperature rise causes the density of that portion of the sample solution to decrease, thereby increasing the buoyancy of that portion of the sample solution compared to the surrounding sample solution. This causes a portion of the sample solution to rise within the incubation chamber 112 and a portion of another sample solution to move to and occupy the vicinity of the mixing heater 134 (see, for example, the movement of the arrows in FIGS. 3A and 3B which represent the approximate movement of the sample solution). In some embodiments, by continuing to supply heat to the incubation chamber 112, the sample solution is gradually heated. In some embodiments, this heating of the sample solution serves to further dissolve the cells within the sample solution.

[0129] In some embodiments, the incubation chamber is configured to mix a liquid sample that does not contain a solvent. In some embodiments, the incubation chamber is configured to mix a liquid sample and a reagent as would be contemplated by one of ordinary skill in the art.

[0130] As shown in FIGS. 3A and 3B, in some embodiments, the incubation chamber 112 is configured to include one or more rounded edges 138, thereby promoting the circulation of the sample solution inside. Such circulation is caused by the movement of the liquid via the mixing heater 134. In some embodiments, the one or more rounded edges 138 assist the liquid to circulate with a generally circular movement. In some embodiments, the corners of the incubation chamber 112 located in the vicinity of the mixing heater 134 may not be rounded (139).

[0131] In some embodiments, the dimensions of the incubation chamber 112 are set to optimize the mixing of the sample solution therein and to reduce or minimize dead volume regions where the sample solution is prone to stagnation and mixing may decline. In some embodiments, the length of the incubation chamber 112 is between 0.75 times and 2 times the height of the chamber. In some embodiments, the width of the chamber is between 1 / 6 and 2 / 3 of the average value of the channel length and width.

[0132] In some embodiments, the thickness of one or more walls of the incubation chamber 112 is set to optimize the mixing of the sample solution via shear as the sample solution passes through the wall. If the chamber is too narrow and the walls are too close together, mixing is slowed by friction with the walls. The wider the walls, the smaller the proportion of the boundary walls to the overall cross-sectional area, and as a result, the shear stress caused by wall friction contributes less significantly to mixing.

[0133] 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.

[0134] As shown in FIGS. 3A and 3B, in some embodiments, the mixing heater 134 is disposed at a position offset from the central position 140 of the incubation chamber 112 (e.g., a position along the width direction of the incubation chamber). In some embodiments, disposing the mixing heater 134 at a position offset from the central position 140 (e.g., the width direction) of the incubation chamber 112 helps to promote non-uniform buoyancy mixing of the sample solution (as opposed to heating at a more central location). Thus, as shown in FIGS. 3A and 3B, the portion of the sample solution located within the vicinity of the mixing heater 134 (when the incubation chamber has rounded corners 139) receives more heat than the sample solution in other locations within the incubation chamber 112 and promotes fluid movement and thereby mixing within the incubation chamber 112 by taking advantage of density differences (as described herein).

[0135] A variety of sizes are contemplated for the mixing heater. In some embodiments, the size of the mixing heater is 1 / 50 of the incubation chamber. In various embodiments, the length of the mixing heater is less than or equal to half the length of the incubation chamber. In some embodiments, the size of the mixing heater is less than or equal to half the size of the incubation chamber.

[0136] In some embodiments, as described herein, the substrate includes a controller that operably communicates with a power source, and the power source supplies power to the mixing heater 134 to heat it. In some embodiments, the power supplied to the mixing heater 134 is adjusted by the controller based on the temperature of the mixing heater 134. Accordingly, a sensor (e.g., a thermocouple) transmits a feedback signal to the controller to vary the supplied power and maintain the mixing heater 134 at a predetermined temperature (e.g., 30°C to 65°C). In some embodiments, the power supplied to the mixing heater 134 is constant or substantially constant regardless of the temperature of the mixing heater 134. In some embodiments, such a constant or substantially constant power supply rate is sufficient because thermal mixing is promoted by non-uniformity in temperature. In some embodiments, the mixing heater 134 is a resistive heater.

[0137] 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 member conducts heat from the PCB heater to the heat-sealing valve and / or the incubation chamber. In some embodiments, the metal member is cylindrical. In some embodiments, the metal member is a cylinder made of aluminum. The metal member can be made of any thermally conductive metal, such as, for example, silver, copper, aluminum, or gold. In some embodiments, the metal member is a metal alloy, such as, for example, at least one thermally conductive metal. In some embodiments, the metal member is press-fitted and disposed between the PCB and a chamber or device component that receives heat (e.g., a portion adjacent to the heat-sealing valve). In some embodiments, the metal member is press-fitted into a crush rib. As used herein, "crush rib" refers to a collection of very small protrusions that deform when a component is press-fitted. By using press-fitting (and optionally press-fitting by crush ribs), heat loss to other parts of the assay device and / or the surrounding environment can be minimized, without being limited to the mechanism of action. FIG. 19 shows a diagram of a cylinder made of aluminum fixed by three crush ribs 1901. The cylinder made of aluminum conducts heat from the PCB heater to the heat-sealing valve.

[0138] As described herein, in some embodiments, the wax valve channel 110 functions as an outlet from the incubation chamber 112 to the OPM module 108. In some embodiments, the wax valve channel 110 includes a heat-sealing valve as described herein, providing a barrier to prevent the sample solution from flowing through the channel 110. This keeps the sample solution within the incubation chamber 112 and allows the mixing and / or incubation to occur for a predetermined time. In some embodiments, for an incubation chamber with a volume of 1 mL, the predetermined 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.

[0139] Heat-sealing valve and channel In some embodiments, as described herein, the wax valve channel 110 connects the thermal mixing module 106 and the OPM module 108. In some embodiments, the wax valve channel is provided with a valve that provides a barrier useful for preventing premature flow of cells in the sample solution into the OPM module 108 before the cells are well mixed and lysed. In some embodiments, the valve is a heat-sealing valve (see, e.g., 142 in FIG. 9) and may be solid at a first temperature in an initial state, but upon receiving sufficient heat, the wax valve softens, melts, or dissolves, thereby allowing a liquid (e.g., a conditioned sample solution that is the sample solution after being well mixed and / or incubated) to pass through the valve and flow to the OPM 108. In some embodiments, the heat-sealing valve comprises a water-soluble wax. In some embodiments, the heat-sealing valve comprises a polymer. In some embodiments, the polymer is water-soluble. In some embodiments, the valve comprises wax and optionally a water-soluble wax. In some embodiments, the heat-sealing 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 that the wax will re-solidify when it moves away from (and thus cools) the heat source. Thus, this helps to prevent (partially or completely) blockage of the flow within the wax valve channel 110 or further downstream in the assay device. In some embodiments, the dissolved valve material has little or no adverse effect on the sample solution with respect to reactions (e.g., amplification) within the OPM module 108.

[0140] In some embodiments, the heat-sealing valve includes other types of waxes and materials for use as heat-sealing valve materials known in the art. 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 may be a water-insoluble wax and an emulsifier such as polysorbate.

[0141] In some embodiments, the operating temperature of the heat-sealing valve depends on the molecular weight of its material. In the context of the molecular weight of the heat-sealing valve material, the term "about" indicates that in the preparation of the polymer (e.g., PEG), some of the molecules are heavier than the stated molecular weight and some are lighter. As used herein, "operating temperature" refers to the temperature at which the wax melts. For example, in some embodiments, the molecular weight of the wax is from about 1,305 g / mol to about 10,000 g / mol, and the corresponding operating temperature range is from about 43°C to about 63°C. In some embodiments, increasing the molecular weight of the valve increases the operating temperature. In some embodiments, the molecular weight of the valve is from about 500 g / mol to about 20,000 g / mol, from about 1,000 g / mol to about 15,000 g / mol, or from about 1,305 g / mol to about 10,000 g / mol. In some embodiments, the heat-sealing valve has a molecular weight of from about 1,300 g / mol to about 10,000 g / mol. In some embodiments, the heat-sealing valve has a molecular weight of about 6,000 g / mol. In some embodiments, the material of the heat-sealing valve is PEG.

[0142] In some embodiments, the material of the heat-sealing valve is selected based on its ability to maintain a stable state (including the solid state) at room temperature and standard transportation conditions. For example, the higher the molecular weight, the higher the transportation and / or storage temperature that the material of the heat-sealing valve can withstand. The requirements for standard transportation conditions for the device are well known to those skilled in the art. For example, the required standard transportation conditions may be based on ASTM D4332 conditions (e.g., ASTM D4332-14). Briefly, the ASTM D4332 test describes a plan for conditioning containers, packages, or packaging components. The purpose is to bring the container closer to or reach equilibrium with the atmosphere to which it may be exposed. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., maintain the solid state) up to about 60°C, optionally at a relative humidity of about 15%. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., maintain the solid state) up to about 40°C, optionally at a relative humidity of about 90%. In some embodiments, the material of the heat-sealing valve needs to maintain stability even at low temperatures such as about -30°C. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., maintain the solid state) 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 material of the heat-sealing valve needs to maintain stability (e.g., maintain the solid state) between about 40°C and 60°C or between about 40°C and 65°C.

[0143] In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain in a solid state) at a relative humidity of about 15%. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain in a solid state) at a relative humidity of about 90%. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain in a solid state) at relative humidities of about 15% and about 90%. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain in a solid state) at relative humidities of about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0144] In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain solid) for at least 6 hours, 12 hours, 24 hours, 36 hours, 38 hours, or 72 hours at any of the temperatures and / or relative humidities disclosed herein. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain solid) for at least 6 hours at any of the temperatures disclosed herein. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain solid) for at least 12 hours at any of the temperatures disclosed herein. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain solid) for at least 24 hours at any of the temperatures disclosed herein. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain solid) for at least 36 hours at any of the temperatures disclosed herein. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain solid) for at least 48 hours at any of the temperatures disclosed herein. In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain solid) for at least 72 hours at any of the temperatures disclosed herein.

[0145] In some embodiments, the material of the heat-sealing valve needs to maintain stability (e.g., remain solid) at up to 60 °C for 72 hours.

[0146] In some embodiments, the preferred material of the heat-sealing valve also has an average operating time between about 30 seconds and 50 seconds. In some embodiments, the operating time is about 30 seconds or less. In some embodiments, the operating time is 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 or less. In some embodiments, the operating 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 some embodiments, the operating 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 operating 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 operating time is between about 35 seconds and 40 seconds, 35 seconds and 45 seconds, or 35 seconds and 50 seconds. In some embodiments, the operating time is between about 40 seconds and 45 seconds or 40 seconds and 50 seconds.

[0147] In some embodiments, PEG having a molecular weight of 6,000 g / mol is used to maintain stability (including maintaining a solid state) during standard shipping conditions.

[0148] In some embodiments, as described herein, wax is supplied to the wax valve channel 110 via a wax filling port (see 144 in FIGS. 3A, 3B, 6, 9). In some embodiments, the wax is supplied in a molten state and then solidifies within the wax valve channel 110. In some embodiments, the amount of wax supplied into the wax valve channel 110 is from about 1 uL to about 10 uL, from about 2 uL to about 8 uL, from about 3 uL to about 6 uL, or from about 4 uL to about 5 uL. In some embodiments, no specific patterning or configuration of the wax within the wax valve channel 110 is required.

[0149] In some embodiments, as described herein, the substrate includes a valve heater 135 configured to supply heat for softening, melting, and / or dissolving the thermal sealing valve. In some embodiments, the wax valve channel 110 is disposed further away from the substrate 109 than, for example, the bottom of the incubation chamber 112 aligned with the mixing heater 134, with respect to height. Thus, referring to FIG. 6, in some embodiments, the assay device is disposed between the substrate and the thermal sealing valve (within the wax valve channel 110) and further includes a heat conducting material 137 (e.g., a thermal pad) for transferring heat therebetween. For example, in some embodiments, the heat conductive material includes GAP PAD (registered trademark), 3M thermal conductive adhesive transfer tape, ARTIC Thermal Pad, Laird Tflex, Fujipoly CARCON gap filler pad, or equivalent materials.

[0150] In some embodiments, as described herein, the substrate comprises a power source for supplying power to and heating the valve heater 135. In some embodiments, the power source is the same as the power source of the mixing heater 134. In some embodiments, the power source is different from the power source of the mixing heater 134. In some embodiments, the power source is external to the substrate. In some embodiments, the valve heater is a resistive heater. This heater can be adjusted to control a predetermined temperature or can be heated at a constant power until a fluid is detected within the reaction well (which indicates that the thermal sealing valve is open).

[0151] In some embodiments, as shown in FIG. 6, one or more walls 148 of the inlet 146 from the incubation chamber 112 to the wax valve channel 110 converge (e.g., are inclined). In some embodiments, providing a wall converging at the inlet 146 of the wax valve channel 110 helps reduce or eliminate the formation of air bubbles within the wax valve channel and / or at the fluid junction of the valve (e.g., see 150 in FIG. 10). As described herein (e.g., see Example 1), in some embodiments, the heat-sealing valve is configured to dissolve (e.g., gradually dissolve) upon contact with a particular liquid (e.g., a buffer solution containing a sample solution). In some embodiments, the hydrostatic pressure from the sample solution serves to provide a force that pushes the softened, melted, and / or dissolved valve forward through the wax valve channel 110, and the shearing of the valve caused by its movement against the walls of the wax valve channel 110 may further facilitate (the) dissolution. Thus, in some embodiments, such dissolution of the valve upon contact with the sample solution, along with the hydrostatic pressure exerted by the sample solution within the wax valve channel on the heat-sealing valve, helps reduce the pressure and time required to open the valve (e.g., through softening and / or melting). This enables the prepared sample solution to flow through the wax valve channel and to the OPM module 108. In some embodiments, such valve opening may occur at a temperature higher than or corresponding to a value known in the art for the melting point of the wax, or in some cases, at a temperature lower than a value known in the art for the melting point of the wax.

[0152] The solubility of the heat-sealing valve material also increases with temperature. Thus, the operation (opening) of the heat-sealing valve (i.e., the dissolution of the heat-sealing valve material) occurs more rapidly as the temperature in the vicinity of the assay device or the wax valve channel is higher.

[0153] In some embodiments, the time that the heat-sealing valve can prevent the sample solution from passing through the wax valve channel 110 is a function of the temperature of the sample solution (e.g., the temperature within the incubation chamber 112), the operating temperature of the valve, the cross-sectional area of the wax valve channel, and / or the length of the wax valve channel blocked by the valve. In some embodiments, the diameter of the channel 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, which varies according to the length of the wax valve channel.

[0154] In some embodiments, when the temperature of the sample solution approaches the melting temperature of the valve, the integrity of the valve is compromised, and as a result, it may not be possible to reliably prevent the sample solution from flowing through the wax valve channel 110. Accordingly, 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 stage, such that when the temperature of the sample solution approaches a predetermined range of the melting temperature of the valve, a controller coupled to the mixing heater 134 is configured to stop the power supply to the heater. In some embodiments, the presence of the thermocouple facilitates preventing the assay from reaching a specific temperature or temperature range within the incubation chamber.

[0155] In some embodiments, the presence of bubbles (e.g., air bubbles) causes insufficient or absent dissolution due to prior contact with the sample solution, and / or the hydrostatic pressure is limited or reduced, whereby, as a result, the force received from the valve to open the valve and allow passage through the wax valve channel 110 is reduced, resulting in the valve opening at a temperature higher than the literature value.

[0156] In some embodiments, as described herein, the assay device is provided with a solidified valve. For example, in some embodiments, the assay device may be assembled and packaged with the valve inside, such that the subject is only brought into contact with the liquid when performing the assay, preventing the valve from gradually dissolving before the sample solution is introduced into the incubation chamber 112.

[0157] In some embodiments, as the prepared sample solution passes through the wax valve channel, additional unmixed sample solution may flow from the sample inlet and / or the sample preparation device 104 (it may remain coupled to the assay device 102 even after the mixing / incubation step). Thus, in some embodiments, the inlet 146 of the wax valve channel 110 is positioned as far as possible from the sample inlet 118, which helps to reduce the risk and amount of unmixed (and thus potentially undissolved) sample entering the OPM module.

[0158] Optical property modification module As described herein, in some embodiments, after the sample solution has been mixed and / or incubated for a sufficient time (and thus becomes a prepared sample solution), when the heat-sealing valve is opened, it flows into the optical property modification module (OPM) 108 through the wax valve channel 110. In some embodiments, the optical property modification module 108 comprises the apparatus described in PCT / US2018 / 044044 (the " '044 application"), the entirety of which is incorporated herein by reference. In some embodiments, the common sample inlet in the '044 application is in fluid communication with the wax valve channel 110 and is adapted to receive the sample therefrom. In some embodiments, the optical property modification module 108 comprises a reaction chamber channel 152 in fluid communication with the wax valve channel 110, instead of or in addition to, the common sample inlet of the '044 application (Figures 2A and 2B). In some embodiments, the reaction chamber channel 152 extends within the OPM module 108 to near the central portion 156 and has one or more branches leading to the corresponding reaction chamber 154. In some embodiments, the first and second pieces described in the '044 application are, respectively, part of the upper and lower portions of the assay apparatus 102 described herein. In some embodiments, the OPM module 108 is a separable device that can be coupled to the wax valve channel 110 and the thermal mixing module 106.

[0159] In some embodiments, when the prepared sample solution enters the reaction chamber channel 152 within the OPM module 108, the wax dissolves. In some embodiments, the prepared sample solution is distributed to one or more reaction chambers 154 via one or more branches branching off from the reaction chamber channel 152.

[0160] In some embodiments, the OPM module 108 further includes an isolation chamber 158 coupled to a first branch that branches from the reaction chamber channel 152. See FIG. 2A. In some embodiments, the isolation chamber is vented. As described in Example 2 herein, in some cases, a large amount of dissolved valve material may be included in the initial volume of the prepared sample solution entering the OPM module 108, so that the isolation chamber 158 takes in a large amount (e.g., an excess amount) of the dissolved valve material, reducing the amount of dissolved valve material distributed across one or more reaction chambers 154.

[0161] In some embodiments, the OPM module 108 includes a series of one or more mixing chambers (e.g., shuttle mixing chambers) and is configured to help distribute the dissolved heat-sealing valve material more uniformly throughout the reaction chamber. In some embodiments, such one or more shuttle mixing chambers are arranged in series connected to each other by narrow shuttle mixing channels, and at least one shuttle mixing chamber is configured to accommodate the entire volume of the sample solution. In some embodiments, the one or more shuttle mixing chambers are disposed upstream of the reaction chamber.

[0162] In some embodiments, the wax valve channel tapers near the wax filling port. In some embodiments, the wax filling port is narrow near the wax filling port (e.g., about 500 um) and widens distally (e.g., 1 mm at the distal end). In some embodiments, the ratio of the narrowest end to the widest end of the wax valve channel is about 1:2 or less (e.g., 1:2.5, 1:3, etc.). This shape of the wax valve channel enables the heat-sealing valve to maintain a constant length when dispensed into the channel (i.e., even if the volume of the heat-sealing valve material dispensed changes, the length is the same as, or less than, the length of the heating region, and the excess heat-sealing valve material is distributed to the wider part of the channel, so the overall length of the valve does not increase significantly). This minimizes the entry of excess heat-sealing valve material into the OPM. For example, see Figure 16. This figure shows the shape of a wax valve channel that does not taper near the wax filling port and does not widen distally, indicating that excess heat-sealing valve material may enter the OPM. Alternatively, Figure 17 shows a wax valve channel 110 that is narrow near the wax filling port and widens at the distal end from the wax filling port.

[0163] Sample preparation device In some embodiments, the sample preparation device comprises a sample preparation device as described in either U.S. Patent Nos. 11,123,736 and 11,125,661, the contents of which disclosures are incorporated herein by reference in their entirety.

[0164] Method for preparing a heat-sealing valve Referring to FIG. 9, a method for preparing a heat-sealing valve within the wax valve channel 110 of an assay device is shown. In some embodiments, an assay device is provided that has a thermal mixing module, a wax valve channel 110, and an OPM module (or, optionally, the OPM module is removably coupled to the wax valve channel 110), as described herein. In some embodiments, the wax valve channel 110 includes a valve filling port 144, which allows for insertion of the heat-sealing valve into the wax valve channel 110. In some embodiments, the heat-sealing valve material is dispensed into the wax valve channel 110 in a molten or dissolved state. FIG. 8 shows an exemplary device 200 for dispensing a heat-sealing valve material, having a pressurized wax reservoir 202, a precision dispensing valve 204, a heater block 206, and / or a wax dispensing needle 208. In some embodiments, the wax dispensing device 200 is a pneumatic drive system. The heat-sealing valve material can be dispensed into the integrated test chamber 210 by using the device 200. In many other systems in the prior art, it may be necessary to heat a plastic device in which the heat-sealing valve material is disposed. In the devices of the present disclosure, it is not necessary to heat the assay device to dispense the heat-sealing valve material. In some embodiments, the wax dispensing device and / or consumables are configured to dispense from about 1 μL to about 10 μL, from about 2 μL to about 7 μL, from about 3 μL to about 5 μL, or about 4 μL of heat-sealing valve material into the wax valve channel 110.

[0165] In some embodiments, when a sufficient amount of heat-sealable valve material is dispensed into the wax valve channel 110, the valve filling port 144 is sealed. In some embodiments, excess polymer melts around the filling port 144 and solidifies after cooling, thereby sealing the opening of the wax filling port 144. In some embodiments, a stopper is inserted into the opening of the wax filling port 144. In some embodiments, a pressure-sensitive adhesive is placed over the opening of the wax filling port 144 to seal it. In some embodiments, the opening of the wax filling port remains open. In some embodiments, the plastic of the wax filling port is melted and closed by heat welding. For example, a material such as the plastic of the device is melted over the wax filling port and thereby sealed.

[0166] In some embodiments, the melted or dissolved heat-sealable valve material (also referred to as the dissolved valve material) flows into the wax valve channel 110 and solidifies accordingly, so there is no need to mold and / or place the heat-sealable valve material within the wax valve channel 110. In some embodiments, the heat-sealable valve material and / or the wax valve channel are dried and / or cooled prior to packaging, thereby ensuring solidification of the heat-sealable valve material within the wax valve channel 110.

[0167] Method of performing a bioassay within a system FIG. 11 is a flowchart of a method 1100 of performing a bioassay using a system (e.g., 100) in accordance with embodiments described herein. In other embodiments, the method may include different and / or additional steps than those shown in FIG. 11. Further, the steps of this method may be executed in a different order than the order described in connection with FIG. 11 in various embodiments.

[0168] The subject provides a biological sample (1101). As described herein, a biological sample is a sample containing organic substances, such as one or more organic molecules (one or more nucleic acids, such as DNA and / or RNA, or a part thereof), and can be collected from the subject. In some embodiments, the biological sample is a nucleic acid amplification sample, which is a suspected sample containing one or more nucleic acids or a part thereof that can be amplified.

[0169] The provided biological sample can contain one or more cells, such as tissue cells of the subject. As used herein, the term "tissue" refers to an aggregate of one or more cells having similar functions and structures within a subject (such as a living body like a mammal such as a human), or an aggregate of such a plurality of different types. Tissues can include, for example, organ tissues, muscle tissues (such as cardiac muscle, smooth muscle, and / or skeletal muscle), connective tissues, nerve tissues, and / or epithelial tissues. Tissues can, in some embodiments, include cells collected from the inner side of the subject's cheek and / or cells in the subject's saliva.

[0170] The provided liquid sample (e.g., biological sample) can include, for example, human saliva, urine, human mucus, blood, oral rinse, or solid tissue such as buccal mucosa tissue. The biological sample can also include bacteria and spores. The biological sample may be collected by a sampling device. The act of providing can include contacting, e.g., rubbing or scraping, a sampling device onto one or more surfaces of a subject and / or onto the surface of a biological sample of the subject (e.g., a sample extracted from the subject, i.e., a liquid such as saliva and / or blood). Thus, in some cases, the act of providing includes extracting one or more biological samples from the subject. In some cases, the act of providing a biological sample can include instructing the subject to generate a biological sample (e.g., instructing the subject to spit saliva onto or into a sampling device). The act of providing a biological sample can also include moving the sampling device to a sample preparation device (e.g., 104) or an assay device (e.g., 102) while holding the biological sample or a portion thereof (e.g., one or more cells) on the sampling device. In some examples, the sampling device is a swab, and the act of providing a biological sample includes obtaining a biological sample on the sampler by swabbing the oral cavity and / or nasal cavity of the subject. In some cases, the sampling device is a nasopharyngeal, middle turbinate, genital, and / or nasal swab. After the biological sample is provided, 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 within a sample preparation device (e.g., 104) as described herein.

[0171] In some embodiments, the preparation solution includes an optical property modifying reagent solution. In some embodiments, the optical property modifying reagent solution includes an optical property modifying reagent and a liquid buffer.

[0172] Optical property modifying reagents can include, for example, pH-sensitive dyes, fluorescent dyes, FRET dyes, micro and nanoparticles, fluorescent proteins, chromogenic substrates, enzymes and reagents, plasmonic structures, precipitation reagents and substrates, or any combination thereof.

[0173] In some versions, the optical property modifying reagent is or includes a reagent for enzyme-linked immunosorbent assay (ELISA). In some embodiments, 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 / nitroblue tetrazolium), TMB (3,3’,5,5’-tetramethylbenzidine), DAB (3,3’,4,4’-diaminobenzidine), 4CN (4-chloro-1-naphthol), TMB (bifunctional substrate), ABTS (2,2’-azinodi[3-ethylbenzothiazoline] sulfonic acid), OPD (o-phenylenediamine), MUG (4-methylumbelliferyl galactoside), HPA (hydroxyphenylacetic acid), and HPPA (3-p-hydroxyphenylpropionic acid).

[0174] The optical property modifying reagent can, in various cases, include one or more optical property modifying substances and is thus configured such that one of the optical properties, such as color, is modified. Thus, method 1100 includes modifying one or more optical properties of the optical property modifying reagent.

[0175] Modification of optical properties refers to changing one or more optically recognizable properties of a side surface (e.g., a sample), such as properties resulting from the wavelength and / or frequency of radiation (e.g., light) emitted from the side surface, such as color, fluorescence, phosphorescence, etc. For example, in some cases, the optical property is color, and modification of the optical property includes changing that color. In some embodiments, such modification of the optical property (e.g., change in color) is detectable by the unaided human eye under natural light, for example. In another embodiment, for example, in method 1100, modification of the optical property is detectable using a light detector. Modification of the optical property can also include changing the transmittance and / or opacity of a substance, and can include changing the substance from substantially transparent to opaque, or from opaque to transparent. Thus, method 1100 can include detecting such a change in transmittance using a light detector.

[0176] The user dispenses (1103) a sample solution into an assay device, or specifically, an incubation chamber (e.g., 112) of a thermal mixing module, via a sample receiving module (e.g., 114) (see, for example, FIG. 10). In some embodiments, the incubation chamber 112 has a lysing agent (e.g., 136) therein, which is mixed with the sample solution to form a prepared sample solution. Such mixing prepares the biological sample in the sample solution and helps it react with, for example, assay reagents and / or optical property modifying reagents when exposed to them. Mixing can include lysing the cells of the biological sample with a lysing agent and / or extracting nucleic acids therefrom. The nucleic acids thus extracted can be released into the resulting prepared sample solution. In some embodiments, the step of extracting genomic deoxyribonucleic acid (DNA) from the biological sample is included. In some embodiments, the lysing agent is provided as a lyophilized pellet and is hydrated when exposed to the sample solution.

[0177] Thereafter, the incubation chamber is heated (1104) using a mixing heater 134 disposed at its lower part to enable thermal mixing of the sample solution and the solvent. In some embodiments, the mixing heater 134 is offset and aligned from the center of the incubation chamber 112 to promote non-uniform buoyancy of the sample solution, thereby promoting the movement of the liquid within the chamber 112 (e.g., the temperature of the portion of the sample solution near the mixing heater increases, thereby reducing its density and enabling it to rise and move within the chamber). In some embodiments, the incubation chamber comprises one or more rounded walls 138, which helps to promote a circulating motion, possibly a circular motion or a somewhat similar motion, within the incubation chamber 112. In some embodiments, the mixing heater 134 supplies heat based on receiving power from a power source at a constant rate, or heat is supplied by adjusting the power such that the temperature of the mixing heater 134 is maintained constant or substantially constant. In some embodiments, the sample solution is mixed and / or incubated for a predetermined time. In some embodiments, the predetermined time is from about 1 minute to about 20 minutes.

[0178] After the sample solution has been mixed for a predetermined time, heat is applied to the heat-sealing valve 1105 using the valve heater 135 (see, e.g., FIG. 10). In some embodiments, the heat-sealing valve is disposed within the wax valve channel 110, which extends from the end of the incubation chamber 110, the end of which is on the opposite side of the inlet of the sample receiving module 114. In some embodiments, the heat-sealing valve provides a barrier to prevent the sample solution from passing through the wax valve channel 110. In some embodiments, the heat-sealing valve is configured to be solid at a first temperature but to soften and / or melt when exposed to sufficient heat (e.g., the valve heater 135). In some embodiments, the heat-sealing valve comprises wax or a water-soluble polymer (e.g., PEG). In some embodiments, the valve heater heats a heat-sealing valve having a melting temperature of about 40° C. to about 65° C. in some cases. In some embodiments, the heat-sealing valve becomes sufficiently softened, melted and / or dissolved to be opened by the prepared sample solution, such that the prepared sample solution can flow through the wax valve channel 110 to the optical property modification (OPM) module 108. In some embodiments, the heat-sealing valve dissolves in the prepared sample solution.

[0179] The prepared sample solution enters the reaction chamber channel 152 in the OPM module 108 that is in fluid communication with one or more reaction chambers 154. The prepared sample solution is then transferred to the reaction chambers 154 (step 1106). Specifically, the prepared sample solution moves along the reaction chamber channel 152 and branches into one or more reaction chambers 154. The reaction chambers 154 contain assay reagents, thereby producing a nucleic acid reaction mixture.

[0180] As described herein, in some embodiments, each reaction chamber 154 contains assay reagents. Thus, the act of transferring the prepared sample solution to one or more reaction chambers can include mixing the prepared sample solution with the assay reagents, thereby producing a nucleic acid reaction mixture comprising the prepared sample solution and the assay reagents and performing a nucleic acid amplification reaction.

[0181] The assay reagent contains an enzyme and nucleic acid primers, and by reacting with a biological sample, one or more nucleic acids presumed to be present in the sample can be amplified (e.g., amplified isothermally) if actually present. In certain embodiments, the assay reagent contains a nucleic acid amplification enzyme and DNA primers. For example, the assay reagent may include one or more primers, deoxynucleotides (dNTPs), and / or polymerase, Trizma preset crystal (Tris buffer, pH 8.8; Sigma, product number T9443), potassium chloride (KCl; Wako Pure Chemicals, product number 163 - 03545), magnesium sulfate heptahydrate (MgSO; Wako Pure Chemicals, product number 137 - 00402), ammonium sulfate ((NH4)2SO4; Kanto Chemical, product number 01322 - 00), Tween 20 (Tokyo Chemical Industry, product number T0543), betaine solution (betaine, 5 M; Sigma, product number B0400), calcein (DOJINDO, product number 340 - 00433), and one or more of the optical property modifying reagents described above, manganese(II) chloride tetrahydrate (MnCl; Wako Pure Chemicals,, product number 133 - 00820), agarose S, EtBr solution, template nucleic acid, or any combination thereof can be included. Further, in some versions, the assay reagent can be stored in a dried form, e.g., lyophilized form, within the fluid chamber 205. Therefore, the act of preparing the reaction mixture can include mixing the prepared sample solution and the assay reagent and / or hydrating the assay reagent.

[0182] The assay reagent can include one or more reagents capable of amplifying nucleic acids present in a biological sample via an isothermal amplification protocol. This protocol can include 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, circular helicase-dependent amplification, single primer isothermal amplification, loop-mediated amplification, or any combination thereof.

[0202] In certain embodiments, the amplification reaction performed is LAMP. In the LAMP reaction, double-stranded or single-stranded DNA templates in a dynamic equilibrium state at high temperature are amplified using two or three pairs of primers. The primers are designed based on the DNA template using primer design software such as LAMP Designer (Premier Biosoft, Palo Alto, California). In the first stage of the LAMP reaction, the F2 region of the FIP (Forward Inner Primer) anneals to single-stranded DNA at their respective complementary positions (F2c). Next, a polymerase with strand displacement activity incorporates dNTPs along the template from the 3' end of F2. The incorporation of this nucleotide releases protons, lowering the pH of the reaction mixture. Subsequently, the F3 forward primer anneals to the F3c region on the template upstream of the F2 region. The F3 forward primer initiates amplification of the template strand, further releasing protons and displacing the strand incorporating the previously synthesized FIP. This single strand contains the Fl sequence (within the target sequence) as well as its complementary Flc sequence (within the FIP). Thereby, a stem-loop is formed by the annealing of Flc to Fl at the 5' end. At the same time, the BIP (Backward Inner Primer) anneals to the other end of the strand, and nucleotides extend from B2, releasing more protons. Next, the backward primer B3 binds to the B3c region located downstream of the B2 region, promotes elongation by displacing the strand amplified by the BIP, and generates a double strand. This displaced strand now contains the Bl sequence (within the target sequence) along with its complementary Blc sequence (within the BIP), forming another stem-loop at the 3' end. This structure at this stage has two stem-loop structures at both ends respectively, from which continuous displacement and elongation occur, promoting amplification of the template.The LAMP reaction is amplified by further adding a Forward Loop primer and a Backward Loop primer, and more unit replication arrays with stem-loop structures can be generated.

[0183] Since the LAMP method can be carried out at a fixed temperature, the need for an expensive thermal cycling device can be minimized. Usually, in an isothermal method, a set temperature is required, which is determined by the selected reagents. For example, in the LAMP method, the enzyme functions best between 60 - 65 °C. The amplification according to this embodiment can also be carried out by applying PCR.

[0184] In some embodiments, the system (e.g., 100) heats (1107) the reaction mixture (e.g., using a reaction heater) to generate amplified nucleic acid and a plurality of protons. Specifically, by heating the reaction mixture using a reaction heater, a nucleic acid amplification reaction between the nucleic acid derived from the biological sample and the assay reagent is promoted. This reaction generates amplified nucleic acid and a plurality of protons.

[0185] In some embodiments, the heating step 1107 includes transferring thermal energy from the reaction heater to one or more reaction chambers via a thermal gap pad. By heating the reaction mixture, the nucleic acid amplification reaction between the nucleic acid of the biological sample and the assay reagent is promoted. This nucleic acid amplification reaction generates amplified nucleic acid and a plurality of protons.

[0186] Thereafter, the protons react with the optical property modifying reagent (1108). Reacting the reaction product or an aspect thereof with the optical property modifying reagent can include chemically modifying the reaction product and / or the optical property modifying reagent, for example, binding one or more protons to the optical property modifying reagent. In some embodiments, this reaction between the protons and the optical property modifying reagent sufficiently modifies the optical properties of the optical property modifying reagent, and the modified optical properties can be detected to indicate the presence of the suspected analyte in the biological sample.

[0187] The assay device causes a light-emitting element to emit light (1109). Specifically, the system's microprocessor (described in the '044 application) instructs the plurality of light-emitting elements to emit light in a repeated pattern at a repetition frequency. During the repeated pattern, each light-emitting element of the plurality of light-emitting elements emits light at different timings so that only one of the plurality of reaction chambers is irradiated at any given time. By exposing to light, the conditions can be changed and the optical properties can be measured. In this way, during each repeated pattern, the optical properties of the contents in each reaction chamber can be continuously monitored by a light sensor.

[0188] Based on the optical properties detected in step 1109, the system (e.g., 100) can use a photodetector and a microprocessor to identify one or more properties of the sample contained in the reaction chamber (1110). This microprocessor performs an optical property analysis of the reaction mixture in one or more reaction chambers as described in the '044 application. The act of performing the optical property analysis can include identifying whether a change has occurred in the optical properties of one or more of the contents of the reaction chamber.

[0189] The optical property analysis can be performed in real time throughout the amplification reaction described in relation to step 1106 or after the amplification reaction is performed. The detection of the modified optical properties of the reaction mixture can be associated with a digital display indicating the presence or absence of the amplification reaction product. That is, by detecting the modified optical properties of the reaction mixture, information regarding whether the amplification reaction product is present can be obtained. In certain embodiments, the detection of the modified optical properties of the reaction mixture indicates that the exponential growth phase or the stationary phase of the amplification reaction has been obtained.

[0190] In some embodiments, the detection of the amplification reaction product is accelerated compared to an amplification reaction using a reaction mixture that does not contain a halochromic agent. In yet another embodiment, the modification of the optical properties of the reaction mixture is detected in less than 60 minutes from the start time of the amplification reaction. The detection of the amplification reaction product is accelerated because the halochromic agent (weak acid or weak base) in the reaction mixture absorbs the protons generated during the amplification reaction, and the recombination of the free protons serves to accelerate the detection of the amplification reaction. The reaction can be designed to minimize the amplification necessary to produce a pH transition sufficient for the halochromic agent to change its optical properties. In conventional amplification techniques using fluorescent intercalating dyes, molecular beacons, hybridization probes, dye-based detection, UV-Vis, and other detection methods, a certain threshold amount of amplification needs to occur before the amplification signal becomes detectable. However, in the method of the present disclosure, the detection of the amplification reaction product is accelerated compared to conventional amplification methods because the threshold amount of amplification required before the modification of the optical properties of the halochromic agent becomes detectable is relatively small.

[0191] In some embodiments, the system is configured to display the identified properties using an electronic result display mechanism (described in the '044 application). The results provided can be presented in a visual output format on a display and / or in an audio output format.

[0192] Regarding Method 1100, it should be noted that in various embodiments, the system can include one or more, for example, three assay controls. This includes a sample suitability control, a positive control (e.g., an internal positive control), and / or a negative control. The sample suitability control can, for example, detect abundant human nucleic acid markers such as housekeeping genes, RNA, and / or human β-actin deoxyribonucleic acid (DNA) to confirm that a sufficient swab sample has been provided. The positive control amplifies synthetic oligonucleotides and can thus be co-packaged and / or co-lyophilized within the reaction chamber. Such synthetic oligonucleotides can be included, for example, in an optical property-modifying reagent solution and / or an assay reagent. Such a control ensures that the system operates under conditions that enable amplification of the target gene marker. The negative control also amplifies the positive control but does not include co-lyophilized synthetic oligonucleotides. Such a control ensures that there is no contamination by self-amplifying unit replication sequences.

[0193] Kit The embodiments disclosed herein also include kits that contain the subject device and can be used according to the subject method. The kits of interest can include two or more, for example, a plurality, three or less, four or less, five or less, ten or less, fifteen or less, or fifteen or more assay devices or components thereof, according to any of the embodiments described herein, or any combination thereof.

[0194] The kit can include one or more compositions and / or reagents, such as any of those described herein, e.g., an optical property modifying reagent, an amplification composition, a preparation solution, and / or a buffer solution, which can be stored in a container separate from the device. Further, the kit can include any device or other element that can facilitate the operation of any aspect of the kit. For example, the kit can further include one or more devices for analyzing one or more characteristics of a sample, such as sample preparation and / or a prepared sample. In some embodiments, the kit comprises a sample preparation tube containing one or more compositions and / or reagents. The kit can also include packaging, such as a packing material for transporting the device without damage.

[0195] In certain embodiments, the kits disclosed herein include instructions, such as instructions regarding the method of use of the device. In some aspects, the instructions regarding the method of use of the device are recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Thus, the instructions can be present as an enclosure of the kit, within the label of the kit's container or its components (i.e., related to the packaging or sub-packaging, etc.). In other embodiments, the instructions are present as an electronic data storage file on a suitable computer-readable recording medium (e.g., a portable flash drive, CD-ROM, diskette, on the cloud, etc.). The instructions can be stored and / or reproducible within one or more programs, such as a computer application. In some embodiments, the instructions are accessible by scanning a QR code or barcode printed on a substrate such as a component of the device, paper, or plastic. The instructions can take any form, such as complete instructions regarding the method of use of the device or the address of a website where instructions posted on the World Wide Web can be accessed.

Examples

[0196] Examples of specific embodiments for carrying out the present invention are shown below. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Although efforts are made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), of course, some experimental errors and deviations should be tolerated.

[0197] In the practice of the present invention, unless otherwise specified, within the scope of the relevant art, conventional protein chemistry, biochemistry, recombinant DNA technology, and pharmacological methods are used. Such techniques are well described in the literature. For example, see the following references: T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993), A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition), 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 3rd Ed. (Plenum Press) Vols A and B (1992).

[0198] Example 1: Distribution of Dissolved Valve Material between Reaction Chambers Figure 12 shows the distribution of a heat-sealing valve material (e.g., polyethylene glycol) dissolved in a sample solution across a plurality of reaction chambers (Ch1 to Ch8) in an assay device. Two cases are shown. The first case shows a sample solution (water) in which no heat mixing (as described herein) has been performed (unmixed group), while the second case shows the sample solution (water) after heat mixing (mixed group). In both cases, it is clear that the maximum concentration of the dissolved heat-sealing valve material is present in the first chamber, and the remaining chambers contain a relatively uniform and significantly smaller amount of the heat-sealing valve. This demonstrates the technical advantage of installing one or more isolation chambers (chambers for recovering the over-dissolved heat-sealing valve material, e.g., Figure 2A) upstream of the reaction chambers, which enables removal of a significant amount of the dissolved valve material (e.g., most of it) before distributing the prepared sample solution into the reaction chamber(s). This also has the advantage of minimizing the variation in assay results obtained from different reaction chambers because the isolation chamber can distribute the dissolved valve material in each chamber relatively uniformly.

[0199] Figure 13 includes plots showing the distribution of the dissolved valve material in the reaction chambers in an assay device equipped with an isolation chamber (e.g., 158 in Figure 2A) and an assay device not equipped with an isolation chamber (e.g., Figure 2B). ChW represents the isolation chamber. Ch1 to Ch8 represent the reaction chambers. The results show that the dissolved valve material was present in all reaction chambers whether or not the isolation chamber was used, and that when the isolation chamber was not used, the distribution of the dissolved valve material in Ch2 to Ch8 was approximately the same. In the group of assay devices without an isolation chamber, a small but unbalanced amount of the dissolved valve material was present in the first reaction chamber (Ch1).

[0200] Example 2: Influence of Liquid Dissolution and Hydrostatic Pressure on the Operating Temperature of the Heat-Sealing Valve Material As shown in FIG. 14, the operating temperature of the heat-sealing valve material was tested using a test device equipped with some of the assay elements described in this specification (e.g., mixing chambers and wax valve channels). FIG. 15 includes plots showing the operating temperatures of heat-sealing valves (e.g., PEG) under different assembly conditions. The literature values for PEG with a molecular weight of 1305 to 1595 are approximately 44°C to 46°C. Under the first condition, no inclined inlet from the incubation chamber was provided to the wax valve channel on the assay device, the heat-sealing valve was centrally located within the channel, air was present between the liquid (e.g., a liquid such as a buffer solution) and the heat-sealing valve material, dissolution of the heat-sealing valve material by the liquid was suppressed or did not occur at all, and the hydrostatic pressure exerted by the liquid was limited or did not occur at all. As a result, the operating temperature was 77°C to 79°C.

[0201] Under the second condition, an inclined inlet to the wax valve channel was provided and the liquid was in contact with the heat-sealing valve, but the hydrostatic pressure exerted by the liquid was limited or did not occur at all. Accordingly, as a result, the operating temperature was 61°C to 63°C, indicating that dissolution of the heat-sealing valve due to exposure to the liquid decreased the operating temperature by 16°C compared to the case where it was not in contact with the liquid.

[0202] Under the third condition, an inclined inlet to the wax valve channel was provided and a vertically arranged vial for supplying the liquid was installed, such that the hydrostatic pressure exerted by the liquid was applied to the heat-sealing valve. Accordingly, as a result, the operating temperature was 38°C to 39°C, indicating that exposure to the liquid and the application of hydrostatic pressure caused the heat-sealing valve to dissolve and further decreased the operating temperature, in this case below the literature value.

[0203] Table 1 shows the injection parameters of the PEG-based heat-sealing valve in the assembly of the present disclosure. The molecular weight of the PEG used was 6,000 g / mol.

Table 1

[0204] The operating time of PEG with 6000 g / mol was measured, and its average value was between 30 seconds and 50 seconds. See Table 2.

Table 2

[0205] All publications and patent applications cited in this specification are hereby incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Any citation of a document is for the purpose of disclosing prior to its filing date and should not be construed as an admission that the present invention does not have the right to antedate such document by virtue of prior invention.

[0206] The above invention has been described in some detail using figures and examples for clarity of understanding. However, in light of the teachings of the present invention, it will be readily apparent to those skilled in the art that certain changes and modifications can be made without departing from the spirit and scope of the appended claims.

Claims

1. A system for performing a bioassay, the system comprising: a. A thermal mixing module, comprising: i. A sample receiving module for receiving a sample solution containing a biological sample and a preparation solution; and ii. An incubation chamber fluidly connected to the sample receiving module, the thermal mixing module comprising the incubation chamber; b. A wax valve channel fluidly connected to the incubation chamber; c. An optical property modification (OPM) module operably coupled to the incubation chamber via the wax valve channel, the OPM module comprising one or more reaction chambers each containing an assay reagent; d. i) A heat-sealing valve dispensed 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 according to claim 1, wherein the incubation chamber contains a solvent.

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

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

5. The system according to any one of claims 1 to 4, wherein the sample receiving module includes a piercing element configured to penetrate a breakable seal of the sample preparation device, thereby enabling fluid communication between a sample preparation chamber within the sample preparation device and the incubation chamber.

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

7. The system according to any one of claims 1 to 6, wherein the sample receiving module includes a luer for coupling the sample preparation tube to the assay device.

8. The system according to any one of claims 4 to 7, wherein the sample preparation tube includes a collar that contacts the luer of the sample receiving module, thereby forming a leak-free sealed connection between the sample preparation device and the sample receiving module when they are fitted together.

9. The system according to any one of claims 1 to 8, wherein the incubation chamber is provided with a vent hole.

10. The system according to claim 9, wherein the vent hole is a selective vent element.

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

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

13. The system according to claim 9 or REF_Ref105406534 $r$h 10, wherein the selective vent element comprises a self-sealing porous polyethylene vent and further comprises an embedded hydrogel.

14. The system according to claim 9 or 10, wherein the selective vent element comprises polytetrafluoroethylene or polyethersulfone.

15. The system according to claim 9, wherein the selective vent element comprises a hydrophobic porous membrane.

16. The system according to claim 9, wherein the vent hole comprises a sensing channel that is in fluid communication with a filling detection chamber configured to detect its liquid filling.

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

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

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

20. The system according to claim 19, wherein the one or more sensors are configured with respect to the light source, whereby the one or more sensors detect light emitted by the light source and the light detected by the sensors attenuates as the incubation chamber is filled with liquid.

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

22. The system according to claim 19 or 21, wherein the one or more sensors include one or more lower electrodes that communicate operatively with a circuit board and are configured to penetrate a bottom wall of the incubation chamber and expose into the incubation chamber.

23. The system according to claim 22, further comprising one or more upper electrodes that communicate operatively with the circuit board and penetrate a wall of the incubation chamber other than the bottom wall to expose into the incubation chamber, thereby being configured to detect a liquid level in the incubation chamber.

24. The system according to claim 19, wherein one of the one or more sensors is disposed in a filling detection chamber that communicates fluidically with the incubation chamber via a sensing channel, and the sensor is configured to detect a change in light in the filling detection chamber.

25. The system according to claim 19, wherein the one or more sensors comprise a thermocouple coupled to a part of the mixing heater and / or the incubation chamber, and by detecting a change in temperature rise of the mixing heater and / or the incubation chamber, correlate with the presence of liquid in the incubation chamber.

26. The system according to any one of claims 19 to 24, wherein the one or more sensors communicate operatively with the mixing heater.

27. The system according to claim 26, wherein the one or more sensors function as an interlock for the mixing heater, and thereby the mixing heater is configured to operate and / or stop based on detection of liquid and / or liquid level in the incubation chamber by the one or more sensors.

28. The system according to any one of claims 2 to 27, wherein the solubilizing agent comprises lyophilized pellets.

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

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

31. The system according to claim 30, wherein the one or more surfactants comprise polysorbate.

32. The system according to any one of claims 2 to 31, wherein the solvent contains one or more components of the buffer solution.

33. The system according to any one of claims 2 to 32, wherein the mixing heater is configured to heat the sample solution in the incubation chamber, thereby mixing the sample solution and the solvent to form a prepared sample solution.

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

35. The system according to claim 34, wherein the one or more rounded edges are capable of circulating or substantially circulating the sample solution in the incubation chamber when receiving heat from the mixing heater.

36. The system according to any one of claims 1 to 35, wherein the height of the incubation chamber is set with respect to the width of the height of the incubation chamber, and the dead volume region in the incubation chamber is minimized and / or reduced.

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

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

39. The system according to any one of claims 1 to 38, wherein the incubation chamber has a volume of about 0.1 mL to about 10 mL, for example about 0.5 mL to about 5 mL.

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

41. The system according to claim 40, wherein the predetermined time ranges from about 30 seconds to about 20 minutes.

42. The system according to claim 41, wherein the predetermined time is about 1 minute to 5 minutes or about 5 minutes to 10 minutes.

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

44. The system according to any one of claims 1 to 43, wherein the heat-sealing stop valve comprises wax. **Claim 45** The system according to claim 44, wherein the wax is water-soluble. **Claim 46** The system according to any one of claims 1 to 45, wherein the wax comprises a water-soluble polymer. **Claim 47** The system according to claim 46, wherein the polymer comprises polyethylene glycol (PEG). **Claim 48** The system according to any one of claims 1 to 47, wherein the heat-sealing stop valve has a molecular weight of about 1,300 g / mol to about 10,000 g / mol. **Claim 49** The system according to any one of claims 1 to 47, wherein the heat-sealing stop valve has a molecular weight of about 6,000 g / mol. **Claim 50** The system according to any one of claims 1 to 48, wherein the heat-sealing stop valve has a melting temperature of about 40 °C to about 75 °C. **Claim 51** The system according to any one of claims 1 to 50, wherein the heat-sealing stop valve has a volume of about 2 μL to about 6 μL within the wax valve channel. **Claim 52** The system according to any one of claims 1 to 51, wherein the wax valve channel comprises a valve filling port for accommodating the valve. **Claim 53** The system according to any one of claims 1 to 52, wherein the heat-sealing stop valve is solid or substantially solid at a first temperature, thereby helping to prevent the sample solution from flowing through the wax valve channel. **Claim 54** The system according to claim 53, wherein the heat-sealing stop valve is configured to transition from the solid or substantially solid state to a soft state, a dissolved configuration, and / or a melted configuration after receiving sufficient heat. **Claim 55** The system according to any one of claims 1 to 54, further comprising a valve heater configured to heat the heat-sealing stop valve, thereby enabling the heat-sealing stop valve to be softened, dissolved, and / or melted to allow the sample solution to pass through. **Claim 56** The system according to any one of claims 1 to 55, wherein an inlet from the incubation chamber to the wax valve channel comprises one or more converging walls. **Claim 57** The system according to claim 55 or 56, further comprising one or more heat conduction pads operably coupled to the mixing heater, the valve heater, or both. **Claim 58** The system according to claim 57, comprising a valve heat conduction pad configured such that the one or more heat conduction pads transfer heat from the valve heater to the wax valve channel, thereby heating the heat seal valve.

59. The system according to any one of claims 1 to 58, wherein a liquid level of the sample solution in the incubation chamber is at a position higher than the wax valve channel, whereby the heat seal valve receives a hydrostatic pressure from the sample solution.

60. The system according to any one of claims 1 to 59, wherein the sample preparation tube is disposed at a position higher than the heat seal valve or the wax valve channel, whereby the heat seal valve receives a hydrostatic pressure from the sample solution.

61. The system according to any one of claims 1 to 60, wherein the heat seal valve is configured to dissolve in the sample solution.

62. The system according to any one of claims 1 to 61, wherein 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.

63. The system according to claim 61 or 62, wherein the dissolved heat seal valve enters into at least one reaction chamber together with the prepared sample solution.

64. The 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, the isolation chamber being configured to receive therein the initial inflow of the prepared sample solution and the dissolved heat seal valve, thereby reducing the amount of the dissolved heat seal valve contained in the one or more reaction chambers.

65. The system according to any one of claims 61 to 64, further comprising one or more mixing chambers (e.g., shuttle mixing chambers), thereby distributing and improving the dissolved heat seal valve throughout the one or more reaction chambers.

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

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

68. The system according to claim 66 or 67, wherein the mixing heater and / or the valve heater is disposed on the substrate.

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

70. The system according to claim 69, wherein the substrate includes a control device for adjusting the power supplied to the mixing heater and / or the valve heater, thereby maintaining the mixing heater and / or the valve heater at a substantially predetermined temperature.

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

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

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

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

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

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

77. The system according to any one of claims 1 to 76, wherein each reaction chamber is approximately equidistant from a single sensor region disposed within the OPM module.

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

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

80. The system according to any one of claims 1 to 79, wherein the assay reagent comprises a dry reagent or a lyophilized reagent.

81. The system according to any one of claims 1 to 80, wherein the assay reagent comprises 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 characteristics of a biological sample, the method comprising: a. providing a biological sample containing nucleic acid; b. combining the biological sample with a preparation solution containing a buffer and / or an optical property modifying reagent solution, thereby generating a sample solution; c. dispensing the sample solution into an incubation chamber; d. using a mixing heater that applies heat to the incubation chamber to mix the sample solution with a solvent, thereby enabling thermal mixing and forming a prepared sample solution; e. heating a heat-sealing valve disposed in a wax valve channel that is in fluid communication with the incubation chamber, thereby enabling the prepared sample solution to flow through the wax valve channel into one or more reaction chambers containing assay reagents, and heating so that the prepared sample solution is mixed with the assay reagents to form a reaction mixture; f. heating the reaction mixture to promote a nucleic acid amplification reaction using the nucleic acid present in the biological sample and the assay reagents, thereby generating amplified nucleic acid and a plurality of protons by the reaction; g. reacting the protons with an optical property modifying reagent, making it possible to modify the optical properties of the optical property modifying reagent by the reaction, and making it possible to detect the modified optical properties, thereby indicating the presence of a suspected analyte in the biological sample; h. emitting light from a plurality of light-emitting elements in a repeating pattern at a repeating frequency, and thereby determining one or more characteristics of the biological sample using a photosensor based on the modified optical characteristics.

83. The method according to claim 82, further comprising displaying the determined characteristics using an electronic result display mechanism.

84. Providing the biological sample comprises performing a nasal swab on a subject, performing a tonsil and / or throat swab on the subject, performing a vaginal swab on the subject, collecting a hair sample from the subject, collecting blood from the subject, collecting a urine sample from the subject, collecting oral rinse fluid, or a combination thereof, the method according to claim 82 or 83.

85. Combining the biological sample and the preparation solution is performed within a sample preparation device, the method according to any one of claims 82 to 84.

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

87. Dispensing the sample solution into the incubation chamber comprises coupling the sample preparation device to the sample receiving module, thereby forming a fluid path between the sample preparation device and the incubation chamber, the method according to claim 86.

88. Further comprising breaking and / or rupturing a frangible seal on the sample preparation device to allow the sample solution to flow from the sample preparation device into the incubation chamber, the method according to claim 87.

89. Maintaining the mixing heater in an inactive state until the sample solution is detected within the incubation chamber and / or until the minimum liquid level of the sample solution within the incubation chamber is detected, the method according to any one of claims 82 to 88.

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

91. The sample solution is mixed with the solvent for a predetermined time, the method according to any one of claims 82 to 90.

92. The predetermined time is from about 1 minute to about 20 minutes, the method according to claim 91.

93. The method according to any one of claims 82 to 92, further comprising adjusting the mixing heater based on: i) a constant or substantially constant power supplied to the mixing heater via a power source, or ii) maintaining a constant or substantially constant temperature of the mixing heater or a part of the incubation chamber.

94. The method according to any one of claims 91 to 93, wherein heating the heat-sealing valve includes operating the valve heater after a predetermined time.

95. The method according to any one of claims 82 to 94, wherein heating the heat-sealing valve results in softening, melting and / or dissolving the heat-sealing valve, and the dissolving is performed in the prepared sample solution.

96. The method according to claim 95, further comprising isolating an initial amount of the prepared sample solution and the dissolved heat-sealing valve in an isolation chamber located upstream of the one or more reaction chambers.

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

98. A method for preparing a heat-sealing valve in any system according to claims 1 to 66, the method comprising: a. dispensing a molten or dissolved heat-sealing valve material into a wax valve channel via a valve filling port; b. sealing the valve filling port; c. drying or cooling the wax valve channel, thereby solidifying or substantially solidifying the heat-sealing valve material.

99. The method according to claim 98, wherein the heat-sealing valve material is dispensed into the wax valve channel using a wax dispenser.

100. The method according to claim 98 or 99, wherein sealing the valve filling port includes using a polymer, an obstacle, a stopper, heat welding of the port, and / or a pressure-sensitive adhesive.

101. A kit for performing a bioassay, the kit comprising: a. a sample preparation device; b. an assay device, comprising: i. a thermal mixing module, comprising: i. a sample receiving module for receiving a sample solution containing a biological sample and a preparation solution; ii. a thermal mixing module comprising an incubation chamber in fluid communication with the sample receiving module and containing a solvent; ii. a wax valve channel in fluid communication with the incubation chamber iii. An optical property modification (OPM) module operably coupled to an incubation chamber via a wax valve channel, the OPM module including one or more reaction chambers each containing an assay reagent, iv. i) A heat-sealing valve dispensed 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, an assay device comprising: a kit.

102. The kit according to claim 101, wherein the sample preparation device a. A sampler configured to collect a biological sample, and b. A tube containing a preparation solution therein and configured to accommodate at least a portion of the sampler.

103. The kit according to claim 101 or 102, wherein the sampler includes a nasal swab, a throat and / or tonsil swab, a vaginal swab, or a combination thereof.

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

105. The kit according to 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 includes any feature according to any one of claims 1 to 81.

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