In vitro diagnostic system

Abstract

A method and system for identifying the attributes of a cartridge inserted into an electronic reader includes providing identification marks (or multiple marks) on the cartridge label (or on the cartridge itself), thereby allowing one or more LEDs inside the reader to illuminate the identification marks (or multiple marks), and enabling one or more photodetectors inside the reader to measure the optical signature from each identification mark. The cartridge attributes, in combination with the reader, may indicate one or more assays that can be performed using the cartridge.

Description

[Background technology]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 456,444, filed on 331 March 2023, and U.S. Provisional Patent Application No. 63 / 469,294, filed on 26 May 2023 (the disclosures of each of these applications are incorporated herein by reference in their entirety).

[0002] The ability to rapidly diagnose diseases, especially highly infectious diseases, is essential for maintaining human health. For example, the development and widespread use of rapid and accurate COVID-19 diagnostic tests has enabled the rapid identification and isolation of infected individuals, leading to disease containment. The COVID-19 pandemic has catalyzed the development and adoption of rapid tests for use in point-of-care (POC) or home settings worldwide, and the management of many other infectious diseases can be enhanced by improved diagnostic testing. [Overview of the project] [Means for solving the problem]

[0003] This specification provides diagnostic devices, systems, and methods useful for detecting target nucleic acid sequences. The devices provided herein can be used in a point-of-care (POC) or home setting without specialized equipment. The devices disclosed herein are low-cost and easy to use.

[0004] In one embodiment, this embodiment relates to a method and system for identifying the attributes of a cartridge inserted into an electronic reader, wherein identification marks(s) are provided on the cartridge label (or on the cartridge itself), thereby enabling one or more LEDs inside the reader to illuminate the identification marks(s), and enabling one or more photodetectors inside the reader to measure the optical signature from each identification mark. The cartridge attributes, in combination with the reader, may indicate one or more assays that can be performed using the cartridge.

[0005] In one embodiment, this embodiment relates to a method for identifying the attributes of a cartridge configured to be inserted into an electronic reader, comprising: providing an electronic reader comprising at least one internal LED, at least one internal photodetector, and at least one internal microprocessor communicatively coupled to both the at least one photodetector and the at least one LED; providing a cartridge comprising the cartridge comprising at least one identification mark; inserting the cartridge into the reader; illuminating the at least one identification mark with at least one LED to generate an illuminated identification mark; detecting the optical signature of the illuminated identification mark with the photodetector; and identifying at least one attribute of the cartridge based on the illuminated identification mark with at least one microprocessor.

[0006] In some embodiments, illuminating at least one identification mark occurs when the cartridge is inserted into the electronic reader.

[0007] In some embodiments, at least one identification mark includes at least one barcode.

[0008] In some embodiments, at least one LED includes at least a first LED and a second LED, and at least one barcode includes a first barcode used to quantify the speed at which a cartridge is inserted into an electronic reader, and illuminated by the first LED, and a second barcode used to identify at least one attribute of the cartridge, and illuminated by the second LED, and at least one microprocessor enables calibrating the optical signature of the second barcode using the quantified speed, thereby making a proper determination of cartridge identification.

[0009] In some embodiments, at least one internal photodetector includes at least one of a photodiode and a phototransistor.

[0010] In some embodiments, the LED includes at least one of a red LED and a blue LED.

[0011] In some embodiments, at least one internal photodetector includes a dual-mode photodetector configured to measure light emitted from at least one LED in both a first spectrum and a second spectrum, wherein the second spectrum does not overlap with the first spectrum.

[0012] In some embodiments, the first spectrum includes wavelengths in the range of about 600 nm to about 660 nm, and the second spectrum includes wavelengths in the range of about 730 nm to about 900 nm.

[0013] In some embodiments, at least one identification mark includes one or more printed marks on a label placed on the surface of the cartridge.

[0014] In some embodiments, at least one LED comprises about 4 to about 12 identification LEDs, and one or more print marks comprise about 4 to about 12 print marks, each print mark positioned to correspond to one position of an identification LED, so that the presence or absence of a print mark at a certain position can be detected by the identification LED at the corresponding position.

[0015] In some embodiments, the number of identification LEDs is greater than the number of printed marks.

[0016] In some embodiments, a unique combination of marked positions on the label is associated with an attribute of the cartridge.

[0017] In some embodiments, the attributes include one or more types of assays that can be performed using a cartridge.

[0018] In some embodiments, the microprocessor executes one or more preloaded routines based on attributes determined as a result of a method for identifying the cartridge attributes.

[0019] In some embodiments, one or more printed marks include one or more position-detecting feature units.

[0020] In some embodiments, one or more position detection features include a first position detection feature positioned on a label such that, when the cartridge is correctly positioned within the reader, the center line of the first position detection feature is positioned slightly above the center line of a first component of the reader, and a second position detection feature positioned on a label such that, when the cartridge is correctly positioned within the reader, the center line of the second position detection feature is positioned slightly below the center line of a second component of the reader, the method comprising determining by a microprocessor whether the cartridge is correctly positioned within the reader or not, based on 1) the position of the first position detection feature relative to a first component of the reader, and 2) the position of the second position detection feature relative to a second component of the reader, each of the first and second components including at least one of at least one internal photodetector and at least one LED.

[0021] In some embodiments, the reader is configured to identify cartridge attributes based on a plurality of operating modes, the plurality of operating modes including static identification, which involves identifying attributes via at least one identification mark and the reader after the cartridge has been inserted into the reader, and dynamic identification, which involves identifying attributes via at least one identification mark and the reader while the cartridge is inserted into the reader.

[0022] In some embodiments, one or more printed marks are printed in black ink on a label over a background that is at least one of white and gray.

[0023] In some embodiments, the cartridge contains at least one lyophilized bead disposed therein, and the at least one lyophilized bead includes at least one of a lyophilized lysis bead and a lyophilized polymerase chain reaction (PCR) bead.

[0024] In another aspect, the embodiment includes obtaining a biological sample from a subject, incubating the biological sample with at least one of a reagent and a buffer to thereby generate a biological solution, performing a lysis step on the biological solution, passively cooling the biological solution, amplifying one or more target nucleic acids in the biological solution by isothermal amplification, incubating the biological solution with a composition comprising a CRISPR / Cas enzyme having collateral cleavage activity, a guide RNA that specifically hybridizes with one target nucleic acid, and a detectably labeled nucleic acid probe, wherein hybridization of the guide RNA with the target nucleic acid induces or increases the collateral cleavage activity of the CRISPR / Cas enzyme, the CRISPR / Cas enzyme cleaves the detectably labeled nucleic acid probe, and cleavage of the detectably labeled nucleic acid probe results in an increase in the detectable label, incubating, and determining that the target nucleic acid is present in the biological sample based on detecting an increase in the detectable label.

[0025] In some embodiments, the lysis step includes a thermal lysis step performed at a temperature in the range of about 70°C to about 95°C, and the thermal lysis step is performed in a first heating zone.

[0026] In some embodiments, passively cooling the biological solution includes flowing the biological solution through an internal passage of the cartridge, the internal passage being vertically oriented, and flowing the biological solution through the internal passage includes gravity flow.

[0027] In some embodiments, the isothermal amplification step includes amplifying the biological solution at a temperature in the range of about 50°C to about 70°C, and the isothermal amplification step is performed in a second heating zone.

[0028] In some embodiments, the isothermal amplification step includes loop-mediated isothermal amplification (LAMP).

[0029] In some embodiments, detectably labeled nucleic acid probes are labeled with fluorescent labels.

[0030] In some embodiments, the fluorescent label contains a fluorescent group at its 5' end and a quenching group at its 3' end.

[0031] In some embodiments, determining the presence of a target nucleic acid in a biological sample includes irradiating the biological solution with light and detecting at least one fluorescent signature using at least one photodiode and / or phototransistor, wherein the at least one fluorescent signature indicates the presence of at least one target nucleic acid.

[0032] In some embodiments, irradiating a biological solution with light includes irradiating the biological solution with light using light-emitting diodes (LEDs) that emit light in the wavelength range of about 430 nm to about 500 nm.

[0033] In some embodiments, detecting at least one fluorescence signature includes detecting at least one fluorescence signature without amplifying it.

[0034] In some embodiments, the target nucleic acid is from a eukaryote and / or prokaryote.

[0035] In some embodiments, the target nucleic acid is from a protozoan, bacterium, virus, and / or fungus.

[0036] In some embodiments, the target nucleic acid is derived from Chlamydia trachomatis, Neisseria gonorrhoeae, influenza A, influenza B, SARS-CoV-2, polynuclear respiratory virus (RSV), and / or Trichomonas vaginalis.

[0037] In some embodiments, detecting at least one fluorescent signature includes passing at least one fluorescent signature through a gel filter.

[0038] In another embodiment, this embodiment is a method for detecting the presence of at least one target nucleic acid, comprising: obtaining a biological sample from a subject via a sample container; incubating the biological sample via the sample container with at least one of a reagent and a buffer, thereby generating a biological solution; inserting the sample container into a cartridge, thereby allowing the biological solution to flow into an internal chamber of the cartridge, the internal chamber comprising a first heating zone; inserting the cartridge into an electronic reader comprising a plurality of heating elements to create a first heating zone and a second heating zone within the cartridge; performing a dissolution step in the biological solution within the first heating zone; passively cooling the biological solution by opening an internal passage of the cartridge, thereby allowing the biological solution to flow into the internal passage via gravity, the internal passage comprising a plurality of reaction chambers located downstream of the fluid in the internal chamber and vertically below; and passively cooling the biological solution within an internal passage comprising a plurality of reaction chambers located downstream of the fluid in the internal passage. The present invention relates to a method for amplifying one or more target nucleic acids in a biological solution by isothermal amplification within a zone, wherein each of a plurality of reaction chambers comprises a CRISPR / Cas enzyme having collateral cleavage activity, a guide RNA that specifically hybridizes with one target nucleic acid, and a detectably labeled nucleic acid probe, wherein hybridization of the guide RNA with the target nucleic acid induces or increases the collateral cleavage activity of the CRISPR / Cas enzyme, the CRISPR / Cas enzyme cleaves the detectably labeled nucleic acid probe, and the cleavage of the detectably labeled nucleic acid probe results in an increase in detectable labeling; irradiating the biological solution inside each of the plurality of reaction chambers via a plurality of light energy sources, wherein each of the plurality of light energy sources is located near one of the plurality of reaction chambers; and determining the presence of at least one target nucleic acid in the biological solution based on the presence or level of detectable labeling via a detection device.

[0039] In another embodiment, this embodiment relates to a system for performing nucleic acid diagnostic testing, comprising a durable electronic device capable of receiving a consumable cartridge, and a consumable cartridge configured to be attached to the electronic device and containing reagents used for nucleic acid diagnostic testing.

[0040] In some embodiments, the diagnostic test uses one or more reagents for CRISPR / Cas detection.

[0041] In some embodiments, two or more separate amplification reactions occur within a consumable cartridge.

[0042] In some embodiments, eight amplification reactions occur within a consumable cartridge.

[0043] In some embodiments, fluorescence detection is used to measure molecular amplification.

[0044] In some embodiments, excitation is used to assist in fluorescence detection.

[0045] In some embodiments, optical filtration is used to assist fluorescence detection.

[0046] In some embodiments, the heat treatment of the sample is performed in a consumable cartridge.

[0047] In some embodiments, the thermal dissolution of the sample is performed in a consumable cartridge.

[0048] In some embodiments, gravity is used to move fluid within a consumable cartridge.

[0049] In some embodiments, at least one result is displayed as a combination of 1) an illuminated indicator on an electronic device and 2) graphics on a consumable cartridge.

[0050] In some embodiments, the electronic device is configured to operate with multiple types of consumable cartridges.

[0051] In some embodiments, the electronic device automatically detects the configuration of the consumable cartridge.

[0052] In some embodiments, an electronic device uses photoexcitation and detection to determine whether the reaction chamber in a consumable cartridge contains a reagent.

[0053] In some embodiments, the determination is performed continuously.

[0054] In some embodiments, the determination is made in less than one second.

[0055] In some embodiments, an electronic device uses photoexcitation and detection to determine whether the reaction chamber in a consumable cartridge contains a liquid.

[0056] In some embodiments, the liquid includes a gas.

[0057] In some embodiments, the liquid contains the sample to be tested.

[0058] In some embodiments, the determination is performed continuously.

[0059] In some embodiments, the determination is made in less than one second.

[0060] In some embodiments, an electronic device uses photoexcitation and detection to determine whether the reaction chamber contains a gas.

[0061] In another embodiment, this embodiment relates to an electronic device for performing nucleic acid diagnostic testing in combination with a cartridge, comprising: an electronic subsystem for executing a test sequence based on pre-programmed parameters and unique parameters based on the type of cartridge installed; a mechanical subsystem for receiving and positioning the cartridge; a thermal subsystem for heating two reaction zones within the cartridge; an optical subsystem for exciting, filtering, and detecting fluorescence in real time; and a microfluidic control subsystem for activating feature parts on the cartridge to control the flow of fluid within the cartridge.

[0062] In some embodiments, the mechanical subsystem supports each of the following: an electronic subsystem, a thermal subsystem, an optical subsystem, and a microfluidic control subsystem.

[0063] In another embodiment, this embodiment relates to a microfluidic cartridge for performing nucleic acid diagnostic testing in combination with an electronic device, the microfluidic cartridge comprising an outer casing that interfaces with the electronic device, a reagent housed within the casing, a fluid valve operated by the electronic device, a filter element that allows air to pass through the outer casing and holds liquid, and a visual display that transmits identification information to the electronic device by at least one of absorbance and reflectance at a given location.

[0064] In another embodiment, this embodiment relates to a diagnostic cartridge identification method comprising a device for identifying a cartridge, the device comprising an optical module for measuring an optical signature in a first spectrum, and an optical module for measuring separate optical targets in the first spectrum to identify a cartridge type. In some embodiments, the optical module is configured to measure fluorescence in a second spectrum, which is different from the first spectrum, for detecting the presence of at least one nucleic acid from a predetermined group of nucleic acids in the cartridge, the predetermined group comprising nucleic acids each associated with one or more indications.

[0065] In some embodiments, the nucleic acid includes a human sample. In some embodiments, the cartridge uses one or more reagents for CRISPR / Cas detection.

[0066] In some embodiments, the cartridge includes an identification label containing one or more optical targets. In some embodiments, the cartridge includes at least one printed barcode label.

[0067] In some embodiments, the method includes using a dual-mode photodetector to measure both the optical signature in a first spectrum and the fluorescence in a second spectrum. In some embodiments, the dual-mode photodetector utilizes two separate spectra enabled by two different wavelength LEDs. In some embodiments, the LEDs are computer-controlled.

[0068] In some embodiments, at least one printed barcode is a printing ink (e.g., black ink, colored ink, or a combination of inks).

[0069] In some embodiments, the optical target includes a fluorescent ink having specific spectral properties.

[0070] In some embodiments, the method further includes inserting a cartridge into the device, illuminating an identification label with one of two LEDs, detecting an optical signature associated with an optical signature, and identifying the cartridge type based on the detected optical signature.

[0071] In another embodiment, this embodiment relates to a system including a reader and cartridge provided herein.

[0072] In another embodiment, this embodiment relates to a system for detecting the presence of nucleic acids associated with at least one indication in a biological solution, comprising an electronic reader for performing one or more assays and displaying the results, a cartridge including a cartridge assembly configured to be inserted into the reader, and a sample collection container for collecting a biological sample and transferring it to an internal chamber of the cartridge.

[0073] In some embodiments, the system includes an internal assembly, which includes a cartridge assembly, once the cartridge is inserted into the reader, and the internal assembly further includes a heating unit having one or more heating elements, and an optical assembly including at least one LED and at least one photodetector.

[0074] In some embodiments, the cartridge assembly includes a dissolution chamber for receiving a biological sample from a sample collection container, and one or more reaction chambers located downstream of the fluid in the dissolution chamber.

[0075] In some embodiments, one or more heating elements include a first heating element for maintaining a melting chamber at a first temperature, and a second heating element for maintaining one or more reaction chambers at a second temperature.

[0076] In some embodiments, the cartridge assembly includes at least one buffer and reagent placed in the lysis chamber, at least one lyophilized lysis bead placed in the lysis chamber, and lyophilized PCR beads placed inside each of one or more reaction chambers.

[0077] In some embodiments, each of one or more reaction chambers includes a transparent dome.

[0078] In some embodiments, the cartridge assembly includes a polymer casing that forms the rear surface of the cartridge and a film layer that includes the front surface of the cartridge, with the polymer casing and film layer each sandwiching a buffer, a reagent, at least one lyophilized lysis bead, and / or lyophilized PCR beads between them.

[0079] In some embodiments, the film layer includes a polypropylene laminate.

[0080] In some embodiments, at least one LED includes a first LED that optically communicates with one or more reaction chambers, and at least one LED is configured to illuminate the interior of one or more reaction chambers.

[0081] In some embodiments, at least one LED includes a second LED that optically communicates with at least one identification marker located on the surface of the cartridge.

[0082] In some embodiments, at least one photodetector is configured to measure fluorescence emitted from the irradiated interior of one or more reaction chambers.

[0083] In some embodiments, each of at least one LED and at least one photodetector is integrated into a printed circuit board assembly (PCBA).

[0084] In some embodiments, the heating unit is located adjacent to the front of the cartridge, and the PCBA is located adjacent to the rear of the cartridge.

[0085] In some embodiments, the heating unit is integrated into the PCBA.

[0086] In some embodiments, one or more reaction chambers comprise a plurality of reaction chambers, and the type of lyophilized PCR beads in the first reaction chamber of the plurality of reaction chambers is different from the type of lyophilized PCR beads in the second reaction chamber of the plurality of reaction chambers.

[0087] In some embodiments, one or more reaction chambers comprise a plurality of reaction chambers, each of which comprises different types of lyophilized PCR beads configured for use in different reactions.

[0088] In some embodiments, one or more reaction chambers comprise a plurality of reaction chambers, each of which contains the same type of lyophilized PCR beads configured for use in the same reaction.

[0089] In some embodiments, light emitted from at least one LED excites at least one nucleic acid contained within one or more reaction chambers without passing through an optical lens.

[0090] In some embodiments, the system includes a mechanical assembly configured to move the cartridge laterally within the reader after the cartridge has been inserted into the reader and the lid of the reader has been closed, the mechanical assembly comprising at least one link mechanism connecting the lid to an internal device located within the reader, and a cam coupled to both the at least one link mechanism and the internal device, which converts the closing motion of the lid into the lateral motion of the internal device.

[0091] In some embodiments, the cartridge includes at least one identification mark configured to be illuminated by at least one LED. In some embodiments, the at least one identification mark includes at least one printed barcode. In some embodiments, the at least one identification mark includes one or more printed shapes. In some embodiments, the at least one identification mark is printed with ink (e.g., black ink, colored ink, or a combination of inks). In some embodiments, the at least one identification mark is associated with at least one indication.

[0092] In some embodiments, the Disclosure provides a CRISPR-based in vitro detection system. An exemplary detection system is shown in Figure 1. In some embodiments, the Disclosure provides a sample collector. In some embodiments, the sample collector includes a sterile swab and a buffer / reagent container. In some embodiments, the Disclosure provides a cartridge. In some embodiments, the cartridge is disposable. In some embodiments, the Disclosure provides a reader. In some embodiments, the reader is a powered reader. In some embodiments, the powered reader is a USB powered processing device. In some embodiments, the powered reader may be battery-powered.

[0093] The diagnostic devices provided herein are designed for multiplex detection and provide accurate, easy-to-use, at-home testing for detecting target nucleic acids, such as viruses or bacteria. An exemplary in vitro diagnostic system is a molecular test for COVID / Flu multiplex using nasal swab samples. Another exemplary in vitro test is a molecular test for a sexually transmitted infection (STI) panel including multiple positive and negative control results using genital (e.g., vaginal) swabs.

[0094] This disclosure includes a detection device and a method for detecting one or more target nucleic acids that have clear advantages over currently available diagnostic testing products, including, but are not limited to, the accuracy of molecular testing enhanced by CRISPR technology, sensitivity and selectivity comparable to laboratory-based PCR testing, multiplexing capability allowing multiple assays (e.g., eight separate chambers available for assay and control reactions) to be performed on a single sample, rapid assay and test kit development, menu expandability with two programmable heating zones (e.g., thermal dissolution performed separately from amplification reactions), a low-cost, durable reader designed for home use, no calibration or maintenance required, and low-cost operation provided by microfluidic gravity flow. Furthermore, the detection device and method of this disclosure are easy to use. The system of this disclosure runs from start to result without user intervention during the test sequence.

[0095] This disclosure also provides cartridges. In some embodiments, the cartridges are suitable for use in detection systems according to this disclosure. In some embodiments, the cartridges are configured for readers provided herein. The cartridges according to this disclosure offer numerous advantages, including, but are not limited to, reagent cartridge identification with very low-cost components, the ability of a barcode reader to read barcodes using only four additional LED components, shared software and photodetector components providing dual use, spectral coding of the label cartridge ID target being recognizable to a general photodetector via selective LED wavelength excitation while maintaining fluorescence measurement channels in the same photodetector, and a unique label ID pattern providing static and dynamic detection modes as well as positional information. [Brief explanation of the drawing]

[0096] [Figure 1A] This shows a sample collection container for an in vitro diagnostic testing platform according to an embodiment of this model.

[0097] [Figure 1B]A cartridge for an in vitro diagnostic testing platform according to an embodiment of this model is shown.

[0098] [Figure 1C] A leader for an in vitro diagnostic testing platform according to an embodiment of this model is shown.

[0099] [Figure 1D] This shows a sample collection container, which is inserted into a cartridge of an in vitro diagnostic testing platform and then inserted into a reader, according to an aspect of this embodiment.

[0100] [Figure 1E] An exemplary detection device system for an in vitro diagnostic testing platform according to an aspect of this embodiment is shown.

[0101] [Figure 2A] A positive result indicator for the reader according to an embodiment of this model is shown.

[0102] [Figure 2B] A negative result indicator for the reader according to an embodiment of this model is shown.

[0103] [Figure 3] This section outlines the user workflow and assay workflow combined according to an aspect of this embodiment.

[0104] [Figure 4A] This shows a nasal swab collection method according to an aspect of this embodiment.

[0105] [Figure 4B] This shows the elution of the sample in the buffer solution of the sample collection container according to an aspect of this embodiment.

[0106] [Figure 5] This shows the transfer of a sample to a cartridge according to an aspect of this embodiment.

[0107] [Figure 6] This shows the cartridge mounting according to an aspect of this embodiment.

[0108] [Figure 7] The status indicator LED of the reader according to an embodiment of this model is shown.

[0109] [Figure 8A] This shows the cartridge melting and heating according to an aspect of this embodiment.

[0110] [Figure 8B] A cross-sectional view shows the control of the heat of dissolution according to an aspect of this embodiment.

[0111] [Figure 9] This describes the transfer of a biological solution to a reaction chamber according to an aspect of this embodiment.

[0112] [Figure 10A] A cross-sectional view shows the reaction chamber heating according to an aspect of this embodiment.

[0113] [Figure 10B] A cross-sectional view shows the thermal control of the reaction chamber according to an aspect of this embodiment.

[0114] [Figure 11A-1] An exemplary LAMP curve, including a SARS-CoV-2 assay with a target, according to an aspect of this embodiment, is shown. [Figure 11A-2] An exemplary LAMP curve, including a SARS-CoV-2 assay with a target, according to an aspect of this embodiment, is shown.

[0115] [Figure 11B-1] An exemplary LAMP curve, including a negative control, according to an aspect of this embodiment is shown. [Figure 11B-2] An exemplary LAMP curve, including a negative control, according to an aspect of this embodiment is shown.

[0116] [Figure 12-1] This is an example execution overview template according to an aspect of this embodiment. [Figure 12-2] This is an example execution overview template according to an aspect of this embodiment.

[0117] [Figure 13] The leader clamping mechanism in the open position according to this embodiment is shown in a perspective view (left) and a side view (right).

[0118] [Figure 14] The leader clamping mechanism in the closed position according to this embodiment is shown in a perspective view (left) and a side view (right).

[0119] [Figure 15A] This shows an optical module including an optical printed circuit board assembly (PCBA) according to an aspect of this embodiment.

[0120] [Figure 15B] This shows an optical module according to an aspect of this embodiment, including an inserted cartridge and an overlaid optical printed circuit board assembly (PCBA).

[0121] [Figure 16] The emission spectrum of a blue LED according to an embodiment of this model is shown.

[0122] [Figure 17] The emission and excitation spectra of fluorescein according to an embodiment of this model are shown.

[0123] [Figure 18] The transmission spectrum of a Kodak Ratten 2-12 optical filter according to an embodiment of this model is shown.

[0124] [Figure 19A] An optical PCBA equipped with a photodiode according to an aspect of this embodiment is shown.

[0125] [Figure 19B] An optical PCBA equipped with a phototransistor according to an aspect of this embodiment is shown.

[0126] [Figure 20A] The heater temperature profile of the dissolution chamber according to an aspect of this embodiment is shown.

[0127] [Figure 20B] The heater temperature profile of the reaction chamber according to an aspect of this embodiment is shown.

[0128] [Figure 21] The temperature profiles of multiple reaction chambers according to an aspect of this embodiment are shown.

[0129] [Figure 22] The basic system operation sequence according to an aspect of this embodiment is shown below.

[0130] [Figure 23A-1] A system timing diagram of the entire system according to an aspect of this embodiment is shown. [Figure 23A-2] A system timing diagram of the entire system according to an aspect of this embodiment is shown. [Figure 23A-3] A system timing diagram of the entire system according to an aspect of this embodiment is shown.

[0131] [Figure 23B-1] A system timing diagram of the cartridge ID sequence according to an embodiment of this product is shown. [Figure 23B-2] A system timing diagram of the cartridge ID sequence according to an embodiment of this product is shown. [Figure 23B-3] A system timing diagram of the cartridge ID sequence according to an embodiment of this product is shown.

[0132] [Figure 24A-1]The system timing diagram of the cartridge ID sequence (A) in high-speed mode according to an embodiment of this product is shown. [Figure 24A-2] The system timing diagram of the cartridge ID sequence (A) in high-speed mode according to an embodiment of this product is shown. [Figure 24A-3] The system timing diagram of the cartridge ID sequence (A) in high-speed mode according to an embodiment of this product is shown.

[0133] [Figure 24B-1] The system timing diagram of the filling detection algorithm sequence according to an embodiment of this invention is shown. [Figure 24B-2] The system timing diagram of the filling detection algorithm sequence according to an embodiment of this invention is shown. [Figure 24B-3] The system timing diagram of the filling detection algorithm sequence according to an embodiment of this invention is shown.

[0134] [Figure 25A-1] The system timing diagram for the LAMP fluorescence measurement timing (A) according to this embodiment is shown. [Figure 25A-2] The system timing diagram for the LAMP fluorescence measurement timing (A) according to this embodiment is shown. [Figure 25A-3] The system timing diagram for the LAMP fluorescence measurement timing (A) according to this embodiment is shown.

[0135] [Figure 25B-1] A detailed system timing diagram of the LAMP fluorescence measurement A / D timing according to an embodiment of this model is shown. [Figure 25B-2] A detailed system timing diagram of the LAMP fluorescence measurement A / D timing according to an embodiment of this model is shown. [Figure 25B-3] A detailed system timing diagram of the LAMP fluorescence measurement A / D timing according to an embodiment of this model is shown.

[0136] [Figure 26] A cross-sectional view shows the details of the cartridge assembly according to an embodiment of this product.

[0137] [Figure 27] Details of the cartridge assembly according to this embodiment are shown in an exploded perspective view.

[0138] [Figure 28A] Details of the static cartridge identification feature section according to an aspect of this embodiment are shown below.

[0139] [Figure 28B] Details of the static cartridge identification feature section according to an aspect of this embodiment are shown below.

[0140] [Figure 28C] Details of the static cartridge identification feature section according to an aspect of this embodiment are shown below.

[0141] [Figure 28D] Details of the static cartridge identification feature section according to an aspect of this embodiment are shown below.

[0142] [Figure 29] Details of the respiratory cartridge panel according to an embodiment of this model are shown below.

[0143] [Figure 30] Details of the STI cartridge panel according to an embodiment of this model are shown below.

[0144] [Figure 31] Details of the LED circuit according to this embodiment are shown below.

[0145] [Figure 32] Details of a cartridge panel having an ambient light blocking color according to an aspect of this embodiment are shown.

[0146] [Figure 33]An exemplary LED emission spectrum according to an aspect of this embodiment is shown.

[0147] [Figure 34] An exemplary LED emission spectrum according to an aspect of this embodiment is shown.

[0148] [Figure 35] A cross-sectional side view of a reaction chamber assembly according to an aspect of this embodiment is shown.

[0149] [Figure 36] An enlarged cross-sectional side view of the reaction chamber assembly according to an aspect of this embodiment is shown.

[0150] [Figure 37] A virus detection method according to an embodiment of this present invention is shown.

[0151] [Figure 38] A cartridge identification method according to an aspect of this embodiment is shown.

[0152] [Figure 39] A method for detecting filling according to an aspect of this embodiment is shown.

[0153] [Figure 40] The timing sequence for LAMP fluorescence measurement according to an aspect of this embodiment is shown.

[0154] [Figure 41A] An exemplary industrial design concept according to an aspect of this embodiment is shown.

[0155] [Figure 41B] An exemplary industrial design concept according to an aspect of this embodiment is shown.

[0156] [Figure 41C] An exemplary industrial design concept according to an aspect of this embodiment is shown.

[0157] [Figure 41D] An exemplary industrial design concept according to an aspect of this embodiment is shown.

[0158] [Figure 41E] An exemplary industrial design concept according to an aspect of this embodiment is shown.

[0159] [Figure 41F] An exemplary industrial design concept according to an aspect of this embodiment is shown.

[0160] [Figure 42A] An alternative user workflow concept according to an aspect of this embodiment is shown.

[0161] [Figure 42B] An alternative user workflow concept according to an aspect of this embodiment is shown.

[0162] [Figure 42C] An alternative user workflow concept according to an aspect of this embodiment is shown.

[0163] [Figure 43] A cross-sectional side view of an alternative reaction chamber assembly according to an aspect of this embodiment is shown.

[0164] definition Room temperature: As used herein, the term “room temperature” refers to the ambient temperature. Generally, the term room temperature should be understood as the temperature of any object or environment surrounding an article. Measuring room temperature can be achieved by using a thermometer or sensor. The room temperature of an article depends on the ambient temperature of the article. The ambient temperature can be any temperature, e.g., below 95°C, e.g. below 90°C, e.g. below 85°C, e.g. below 80°C, e.g. below 75°C, e.g. below 70°C, e.g. below 65°C, e.g. below 60°C, e.g. below 55°C, e.g. below 50°C, e.g. below 45°C, e.g. below 40°C, e.g. below 35°C, e.g. below 30°C, e.g. below 25°C, e.g. below 24°C, e.g. below 23°C, e.g. below 22°C, e.g. below 21°C, e.g. below 20°C. Examples of room temperature ranges include 5°C to 50°C, for example 10°C to 40°C, for example 15°C to 35°C, for example 20°C to 30°C, for example 20°C to 25°C, and for example 20°C to 22°C.

[0165] Biological Sample: As used herein, the term “biological sample” usually refers to a sample obtained from or derived from the biological source of interest (e.g., tissue or organism or cell culture) as described herein. In some embodiments, the source of interest is or includes an organism such as an animal or human. In some embodiments, the biological sample is or includes biological tissue or fluid. In some embodiments, the biological sample may include bone marrow; blood; blood cells; ascites; tissue or fine-needle biopsy samples; cell-containing fluids; suspended nucleic acids; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; lavage fluids or lavage fluids; e.g., tubal lavage fluid or bronchoalveolar lavage fluid; aspirates; scrapes; bone marrow samples; tissue biopsy samples; surgical samples; other fluids; secretions; and / or excretions; and / or cells from them, and / or combinations or components thereof. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the obtained cells are or include cells derived from the individual from which the sample is obtained. In some embodiments, the sample is a “primary sample” obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine-needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.). In some embodiments, as will be apparent from the context, the term “sample” means a preparation obtained by processing the primary sample (e.g., by removing one or more components and / or adding one or more active ingredients). For example, filtration using a semipermeable membrane may be used. Such a “processed sample” may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to techniques such as mRNA amplification or reverse transcription, isolation and / or purification of specific components.

[0166] Cell Lysate: As used herein, the term “cellular lysate” means a fluid containing the contents of one or more disrupted cells (i.e., cells with disrupted membranes). In some embodiments, the cell lysate contains both hydrophilic and hydrophobic cellular components. In some embodiments, the cell lysate contains primarily hydrophilic components. In some embodiments, the cell lysate contains primarily hydrophobic components. In some embodiments, the cell lysate is a lysate of one or more cells selected from the group consisting of plant cells, microbial (e.g., bacterial or fungal) cells, animal cells (e.g., mammalian cells), human cells, and combinations thereof. In some embodiments, the cell lysate is a lysate of one or more abnormal cells, such as cancer cells. In some embodiments, the cell lysate is a crude lysate in that little or no purification is performed after cell disruption. In some embodiments, such a lysate is called a “primary” lysate. In some embodiments, one or more isolation or purification steps are performed on the primary lysate. However, the term “lysate” means a preparation containing multiple cellular components and not a pure preparation of individual components.

[0167] Composition: As used herein, the term “composition” may be used to mean a physically independent element comprising one or more specific components, as will be understood by those skilled in the art. Generally, unless otherwise specified, a composition may be in any form (e.g., gas, gel, liquid, solid, etc.) or combination of forms.

[0168] Complementary: As used herein, the term “complementary” refers to the overall relationship between polymer molecules, for example, between polynucleotides. In some embodiments, polynucleotides, such as nucleotide sequences (e.g., primer nucleotide sequences or target nucleotide sequences), are considered “complementary” to each other if their sequences are at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical. In some embodiments, polynucleotides are considered “complementary” to each other if their sequences are at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% similar. In some embodiments, polynucleotides are considered “complementary” to each other if they can hybridize to each other.

[0169] Comprising: Any composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that while the named elements or steps are mandatory, other elements or steps may be added to the scope of the composition or method. To avoid redundancy, any composition or method described as "comprising" (or "comprises") one or more named elements or steps also represents a more limited composition or method that "consisting essentially of" (or "consists essentially of") the same named elements or steps, meaning that the composition or method may include additional elements or steps that include the mandatory named elements or steps but do not substantially affect the basic and novel properties (or properties) of the composition or method. Furthermore, any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps is understood to also describe a corresponding, more limited, closed-end composition or method "consisting of" (or "consists of") the named elements or steps, excluding any other elements or steps not named. In any composition or method disclosed herein, a known or disclosed equivalent of any of the named essential elements or steps may be used in place of that element or step.

[0170] Detectable entity: As used herein, the term "detectable entity" refers to any element, molecule, functional group, compound, fragment or moiety that can be detected. In some embodiments, the detectable entity is provided or utilized alone. In some embodiments, the detectable entity is provided and / or utilized in association with (e.g., conjugated to) another agent. Examples of detectable entities include, but are not limited to: various ligands, radionuclides (e.g., 3 H, 14 C, 18 F, 19 F, 32 P, 35 [[ID=I2]]S, 135 I, 125 I, 123 I, 64 Cu, 187 Re, 111 In, 90 Y, 99m Tc, 177 Lu, 89 Zr, etc.), fluorescent dyes (see below for specific exemplary fluorescent dyes), chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes (see below for specific examples of enzymes), colorimetric labels (e.g., dyes, gold colloids, etc.), biotin, digoxigenin, haptens, and proteins for which antiserum or monoclonal antibodies are available.

[0171] Determining: Many methodologies described herein include a “determining” step. Those skilled in the art will understand, upon reading this specification, that such “determining” can be utilized or carried out by using any of the various methods available to those skilled in the art, including, for example, certain methods expressly mentioned herein. In some embodiments, determining involves the manipulation of a physical sample. In some embodiments, determining involves the consideration and / or manipulation of data or information, for example, using a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, determining involves receiving relevant information, data, and / or material from a source. In some embodiments, determining involves comparing one or more features of a sample or entity with a comparable reference.

[0172] Diagnostic Information: As used herein, “diagnostic information” or “information for use in diagnosis” is information useful in determining whether a patient has a disease, disorder, or condition, and / or classifying a disease, disorder, or condition into a meaningful phenotypic category or any category with respect to the prognosis of the disease, disorder, or condition, or the likely response to treatment of the disease, disorder, or condition (treatment in general or any specific treatment). Similarly, “diagnosis” means providing any type of diagnostic information, including but not limited to information on whether a subject has or is likely to develop a disease, disorder, or condition, information on the manifestation, stage, or characteristics of the disease, disorder, or condition in the subject, information on the nature or classification of a tumor, information on the prognosis, and / or information useful in selecting appropriate treatment. Treatment selection may include choices on specific therapeutic agents or other treatment modalities such as surgery or radiation, choices on whether to withhold or deliver therapy, and choices on medication regimens (e.g., the frequency or level of one or more doses of a specific therapeutic agent or combination of therapeutic agents).

[0173] Gel: As used herein, the term “gel” refers to a viscoelastic material whose rheological properties are distinct from those of a solution, solid, etc. In some embodiments, a composition is considered a gel if its storage modulus (G') is greater than its modulus (G”). In some embodiments, a composition is considered a gel if a chemical or physical crosslinking network exists in the solution, distinguishing it from entangled molecules in a viscous solution.

[0174] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reactor, or in a cell culture, rather than within a multicellular organism.

[0175] Isolated: As used herein, the term “isolated” means (1) a substance and / or entity that has been separated from at least a portion of the components to which it was originally produced (whether in a natural or experimental environment), and / or (2) a substance and / or entity that has been designed, produced, prepared, and / or manufactured by human hands. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components to which they were originally produced. In some embodiments, the isolated active ingredient is ultrapure of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99%. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may be considered “isolated” or even “pure” after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.). In such embodiments, the isolation rate or purity of the substance is calculated without including such carriers or excipients. For example, in some embodiments, a naturally occurring biological polymer such as a polypeptide or polynucleotide is considered "isolated" if a) it does not associate with some or all of its components in its natural state due to its origin or source of induction, b) it substantially does not contain other polypeptides or nucleic acids of the same species from which it naturally produces, or c) it is expressed by or otherwise associated with components from cells or other expression systems other than the species that naturally produces it. Therefore, for example, in some embodiments, a chemically synthesized polypeptide, or a polypeptide synthesized in a cell system different from the cell system that naturally produces it, is considered an "isolated" polypeptide.Alternatively or additionally, in some embodiments, a polypeptide subjected to one or more purification techniques may be considered an “isolated” polypeptide insofar as it has been separated from a) other naturally related components and / or b) other components that were related when it was first produced.

[0176] Nucleic Acid: As used herein, the term “nucleic acid” means, in its broadest sense, any compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is any compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain via phosphodiester linkage. As is evident from the context, in some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” is or contains RNA, and in some embodiments, “nucleic acid” is or contains DNA. In some embodiments, a nucleic acid is one or more native nucleic acid residues, or contains or consists of them. In some embodiments, a nucleic acid is one or more nucleic acid analogs, or contains or consists of them. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, contains, or consists of one or more “peptide nucleic acids” known in the art, and peptide nucleic acids having peptide bonds rather than phosphodiester bonds in their backbone are considered to be within the scope of the systems and / or methods provided herein. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite links rather than phosphodiester bonds. In some embodiments, the nucleic acid is, contains, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, includes, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid contains one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in natural nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or protein. In some embodiments, the nucleic acid contains one or more introns. In some embodiments, the nucleic acid is prepared by one or more of the following: isolation from natural sources, enzymatic synthesis by polymerization based on complementary templates (in vivo or in vitro), replication in recombinant cells or systems, and chemosynthesis. In some embodiments, the nucleic acid has a residue length of at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 residues. In some embodiments, the nucleic acid is partially or entirely single-stranded. In some embodiments, nucleic acids are partially or entirely double-stranded.In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is a complement to a polypeptide-encoding sequence. In some embodiments, the nucleic acid has enzymatic activity.

[0177] Pandemic strain: A “pandemic” influenza strain is one that has or has the capacity to cause a pandemic infection in a human population. In some embodiments, the pandemic strain causes a pandemic infection. In some embodiments, such a pandemic infection involves an epidemic infection that spans multiple regions, particularly those separated from one another (e.g., by mountains, bodies of water, or as part of a separate continent), thereby spanning regions where infections do not normally traverse.

[0178] Prognostic and Predictive Information: As used herein, the terms “prognostic information” and “predictive information” are used to mean any information that may be used to indicate any aspect of the course of a disease or condition, either in the absence or in the presence of treatment. Such information may include, but is not limited to, the patient’s life expectancy, the likelihood that the patient will survive over a given period (e.g., 6 months, 1 year, 5 years), the likelihood that the patient’s disease will be cured, or the likelihood that the patient’s disease will respond to a particular therapy (response may be defined in any of the following ways). Prognostic and predictive information is included in the broad category of diagnostic information.

[0179] Polypeptide: As used herein, refers to any polymer chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has a non-natural amino acid sequence. In some embodiments, the polypeptide has an amino acid sequence that is manipulated in the sense that it is designed and / or produced by human action. In some embodiments, the polypeptide may contain or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, the polypeptide may contain or consist of only natural amino acids or only non-natural amino acids. In some embodiments, the polypeptide may contain D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide may contain only D-amino acids. In some embodiments, the polypeptide may contain only L-amino acids. In some embodiments, the polypeptide may include one or more pendant groups or other modifications, e.g., modifications or attachments to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., or combinations thereof. In some embodiments, the polypeptide may be cyclic and / or contain a cyclic portion. In some embodiments, the polypeptide is not cyclic and / or does not contain a cyclic portion. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide is or may contain a staple polypeptide. In some embodiments, the term “polypeptide” may be prepended to the name of a reference polypeptide, activity, or structure. In such cases, the Spectrum is used to refer to polypeptides that share a relevant activity or structure and can therefore be considered members of the same class or family of polypeptides.For each such class, this specification provides exemplary polypeptides within the class whose amino acid sequence and / or function is known, and / or which will be apparent to those skilled in the art; in some embodiments, such exemplary polypeptides are reference polypeptides of the class or family of polypeptides. In some embodiments, members of the class or family of polypeptides exhibit significant sequence homology or identity with the reference polypeptide of the class; and in some embodiments, with all polypeptides within the class, sharing common sequence motifs (e.g., characteristic sequence elements) and / or common activity (in some embodiments, at equivalent levels or within a specified range). For example, in some embodiments, the member polypeptide comprises at least about 30–40% and often exhibits an overall degree of sequence homology or identity with the reference polypeptide exceeding about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and / or includes at least one region (for example, a conserved region that may be, or may contain, a characteristic sequence element) that often exhibits a very high degree of sequence identity exceeding 90%, or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically comprises at least 3–4 amino acids, often up to 20 or more, and in some embodiments, the conserved region comprises at least one interval of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more consecutive amino acids. In some embodiments, the relevant polypeptide may include or consist of a fragment of the parent polypeptide.In some embodiments, the useful polypeptide may contain or consist of multiple fragments, each found in the same parent polypeptide in a different spatial arrangement from that found in the polypeptide of interest (for example, a fragment directly linked to the parent may be spatially separated in the polypeptide of interest, or vice versa, and / or the fragments may be present in the polypeptide of interest in a different order than that of the parent), and thus the polypeptide of interest is a derivative of its parent polypeptide.

[0180] Protein: As used herein, the term “protein” means polypeptide (i.e., a sequence of at least two amino acids linked together by peptide bonds). Proteins may include non-amino acid portions (e.g., glycoproteins, proteoglycans, etc.) and / or may be separately processed or modified. Those skilled in the art will understand that “protein” may be a complete polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those skilled in the art will understand that a protein may include two or more polypeptide chains linked, for example, by one or more disulfide bonds, or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of the various amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may include native amino acids, non-native amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, its biologically active portion, and / or its characteristic portion.

[0181] Reference: As used herein, a reference represents a standard or control for the subject being compared. For example, in some embodiments, a substance, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control of the same substance, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is examined and / or determined substantially simultaneously with the examination or determination of interest. In some embodiments, the reference or control is a past reference or control incorporated into a tangible medium by option. Typically, as understood by those skilled in the art, the reference or control is determined or characterized under conditions or circumstances equivalent to those being evaluated. Those skilled in the art will understand that there is sufficient similarity to justify reliance on and / or comparison with a particular reference or control.

[0182] Sample: In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be a cell or organism such as a microorganism, plant, or animal (e.g., human), or may include them. In some embodiments, the source of interest may be a biological tissue or body fluid, or may include them. In some embodiments, the biological tissue or body fluid may include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chime, semen, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous fluid, vomit, and / or combinations or components thereof. In some embodiments, body fluids may include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, body fluids may also include plant exudates. In some embodiments, biological tissues or specimens may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchoalveolar epithelium, ducts, nose, eye, oral cavity, uterus, vagina, or other washing or lavage). In some embodiments, biological specimens are or contain cells obtained from an individual. In some embodiments, specimens are “primary specimens” obtained directly from the source of interest by any suitable means. In some embodiments, as will be apparent from the context, the term “specimen” means a preparation obtained by processing a primary specimen (e.g., by removing one or more components and / or adding one or more active ingredients). For example, filtration using a semipermeable membrane may be used. Such “processed samples” may include, for example, nucleic acids or proteins extracted from a sample, or obtained by subjecting a primary sample to one or more methods (e.g., nucleic acid amplification or reverse transcription, separation and / or purification of specific components).

[0183] Specificity: As is known in the art, "specificity" is the degree to which a particular ligand can distinguish its binding partner from other potential binding partners.

[0184] Static: As used herein, the term “static” in the context of cartridge identification means that the cartridge ID process is performed after the cartridge has been fully inserted into the reader and / or when the cartridge is not being moved.

[0185] Subject: As used herein, the term “subject” means an organism, e.g., a mammal (e.g., human, non-human mammal, non-human primate, primate, laboratory animal, mouse, rat, hamster, garbill, cat, dog). In some embodiments, a human subject is an adult, adolescent, or child subject. In some embodiments, a subject has a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., cancer or tumor listed herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition, and in some embodiments, a susceptible subject is predisposed to developing a disease, disorder, or condition and / or exhibits increased risk (compared to the mean risk observed in a reference subject or reference population). In some embodiments, a subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, a subject does not exhibit any particular symptoms (e.g., clinical symptoms of a disease) or features of a disease, disorder, or condition. In some embodiments, a subject does not exhibit any symptoms or features of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, the subject is an individual who is being diagnosed and / or receiving treatment. [Modes for carrying out the invention]

[0186] In some embodiments, the disclosure provides readers, cartridges, sample collection devices, detection systems, and methods for detecting one or more target nucleic acids.

[0187] A. Leader and assay workflow Figure 1A shows a sample collection container 18, cap 20, and swab 16 of an in vitro diagnostic testing platform according to an embodiment of this embodiment. Figure 1B shows a cartridge 12 of an in vitro diagnostic testing platform according to an embodiment of this embodiment. Figure 1C shows a reader 14 of an in vitro diagnostic testing platform according to an embodiment of this embodiment. Figure 1D shows a sample collection container inserted into the cartridge and then into the reader of an in vitro diagnostic testing platform according to an embodiment of this embodiment. Figure 1E shows an exemplary detection device system 10 of an in vitro diagnostic testing platform, including the sample collection container 18, cartridge 12, and reader 14, according to an embodiment of this embodiment.

[0188] In some embodiments, the disclosure provides a reader 14. In some embodiments, the reader is an electric reader 14. In some embodiments, the electric reader is a USB electric processing device. As shown in Figure 1, the reader 14 may be part of a system 10 (shown in Figure 1D). The reader is a device intended to be user-friendly with a simple workflow that can be used appropriately by general users (non-experts) without formal training. The system 10 may include the reader 14 (shown in Figure 1C), a swab 16 for obtaining biological samples (e.g., saliva, mucus, nasal swabs, oral swabs, etc.), a fluid container / vessel / or vial 18 for receiving the sample, a cap 20 for fluidly closing the fluid container / vessel / or vial 18 (all shown in Figure 1A), and a cartridge 12 shown in Figure 1B. As shown in Figure 1C, the reader 14 may include a slot 22 for receiving the cartridge 12. Furthermore, the cartridge 12 may include an acceptance port 24 for interface with the vial 18 and / or cap 20 to receive a sample into the cartridge 12. Figure 1E includes a high-level process flow showing that a sample (e.g., saliva, mucus, nasal swab, oral swab, etc.) is introduced into the cartridge 12 (via the vial 18), and then the cartridge 12 is inserted into the reader 14.

[0189] The reader 14 automatically provides fluid control, thermal control, and optical measurements of the cartridge 12 within a test time of 15 to 45 minutes. In some embodiments, the reader 14 can accept multiple types of cartridges 12, including an extended menu after distribution to the end user, allowing the end user to program the reader as needed. In some embodiments, the reader 14 automatically detects the cartridge 12 type and executes one or more indicated test sequences for one or more assays contained therein.

[0190] Figure 2A shows a positive result indicator for the reader according to an embodiment of this embodiment. Figure 2B shows a negative result indicator for the reader according to an embodiment of this embodiment. In some embodiments, the result is indicated by one or more indicators 26 (i.e., illuminated LEDs) on the reader 14. In some embodiments, the result is indicated through an indicator that combines illuminated LEDs on the reader 14 and corresponding labels 28 on the cartridge 12 (e.g., corresponding to diagnoses such as COVID-19, influenza, RSV, negative diagnosis, etc.) (see Figures 2A and 2B).

[0191] In some embodiments, the reader is a durable device that performs the inspection process in combination with consumable elements, including a cartridge and / or sample collector.

[0192] In some embodiments, the reader is operated in an upright position. In some embodiments, the upright position allows for gravity-driven movement of the fluid (see Figure 1C) (i.e., the fluid can be supplied to the cartridge by gravity). In some embodiments, the reader is lightweight. In some embodiments, the reader is portable. In some embodiments, the reader can be powered by a standard USB wall charger, AC adapter, internal battery, disposable battery, and / or rechargeable battery.

[0193] Figure 3 shows a combined overview of the user workflow and assay workflow 40 according to an embodiment of this model. Workflow 40 shows each step of the process, as well as the time associated with simultaneous user actions and chemical processes. For example, in step 32, workflow 40 may include sample collection 32 for a period of about 1 minute or less, during which the user self-collects a biological sample (e.g., from one or both nostrils), saliva, mucus, or other biological sample using a swab 16 (shown in Figure 1A), as shown in Figure 4A. The swab 16 is then rotated in a sample collector (or vial) 18 containing a buffer, as shown in Figure 4B. During this time, the sample is chemically transferred to the buffer. In some embodiments, the buffer may include Tris buffer, e.g., pH about 8.8, or HCl buffer with a pH of about 8.6 to about 9.0. In step 34, workflow 40 may include inserting the sample collector (or vial) 18 into the cartridge 12 for a period of about 1 minute or less, as shown in Figures 1D, 1E, and 5. In the embodiments of Figures 1D and 1E, the cap 20 is placed over the vial 18, and the vial 18 is then inverted and inserted into the cartridge 12. In the embodiment of Figure 5, an aspiration device 48 (such as a syringe or dropper) may be used to transfer the sample from the sample collector (or vial) 18 to the reservoir 52 located on top of the cartridge 12. The cap 20 can then be secured on top of the reservoir 52. During this step (34) of the workflow 40, the user attaches the cartridge 12 to the slot or hub 22 of the reader 14, as shown in Figures 1E and 6, and attaches the sample collector (or vial) 18 to the cartridge 12, or alternatively, transfers the sample to the cartridge as shown in Figure 5 and described above.

[0194] Referring further to Figure 3, workflow 40 may include thermal dissolution in step 36, which in some embodiments may take place for a period of about 3 to 5 minutes, during which time the cells are lysed. Thermal dissolution is further described herein in conjunction with Figures 8A and 8B. In step 38, workflow 40 may include fluid transfer for a period of about 1 minute or less. Fluid transfer is further described herein in conjunction with Figure 9. In step 42, workflow 40 may include allowing the reaction to take place for a period of about 10 to 30 minutes. During this time, workflow 40 may include hydration of lyophilized beads, followed by amplification, Cas-12 activation, and / or reporter cleavage. In step 44, workflow 40 may include providing a readout of assay results for a period of about 1 minute or less. During this time, the user views the readout diagnosis from the base station / reader 14 and cartridge, as shown in Figures 2A, 2B, and 7. The embodiment / system 10 in Figure 7 may include an alternative configuration in which the cartridge 12 is inserted into the reader 14 horizontally (i.e., not vertically). The embodiment / system 10 in Figure 7 may also include a panel 142 on the surface of the reader 14 that includes a status LED. In step 46, the workflow may include disposing of the used cartridge 12 and sampler (or vial) 18 in a conventional household trash can (i.e., no special disposal of the cartridge 12 and sampler (or vial) 18 is required).

[0195] Mechanical subsystem Figures 13 and 14 show the leader clamp mechanism in the open and closed positions, respectively, according to an aspect of this embodiment. In some embodiments, the leader 14 comprises an internal mechanical subsystem or assembly 50. In some embodiments, the mechanical subsystem comprises an internal mechanical assembly and a clamp cover 54. Exemplary mechanical subsystems or assemblies 50 are shown in Figures 13 and 14, which show the internal components of the leader 14. The front outer casing of the leader 14 is not shown in Figures 13 and 14 so as to allow the components of the mechanical subassembly 50 to be visible. In the embodiments of Figures 13 and 14, when the cover or clamp 54 is closed, it pulls the link mechanism 56 upward, thereby rotating the cam 58 around its axis (or first coupling) 62. The follower 88 moves away from the cam 58 by the rotational motion of the cam 58 and then pushes the cartridge 12 (to the right in the side view images of Figures 13 and 14) via the support plate 70. Accordingly, when the lid 54 is closed, the lid 54 ensures that the cartridge 12 is secured in the correct position within the reader 14. The cam 58 can pivot around the first coupling 62. The cam 58 may include a sector-shaped portion 76 at one end, which includes a curved edge and a curved groove 64 located therein. The curved groove 64 has increasing or decreasing depth, and thereafter when it interfaces with the follower 88 (see the side views in Figures 13 and 14), the support plate moves toward and away from the cartridge 12, depending on whether the clamp lid 54 is closed or open. At the end opposite the curved groove 64, the cam is rotatably coupled to a linkage mechanism 56 via a second coupling 64. The linkage mechanism may include a first linear member 72 and a second linear member 74 that are rigidly coupled to each other and / or monolithic, and in some embodiments may be oriented to form an angle between them, the angle ranging from about 125 to 180 degrees, or about 135 to 175 degrees, or about 140 to about 170 degrees, or about 145 to about 165 degrees, or about 150 to 160 degrees, and / or other partial ranges in between.In some embodiments, the cam 58 may include a curved slot 92 that helps support and maintain the correct position of the cam 58 as it rotates around the first coupling 62. A protrusion or projection (not shown) extending from the support wall 70 may extend through the curved slot 92, allowing the cam 58 to rotate around it when the lid 54 is opened and closed. The support wall 70 is part of the internal mechanism (i.e., inside the reader) and helps facilitate the lateral movement of the cartridge within the reader. The cam 58 helps convert the closing (i.e., rotational) motion of the reader lid into lateral motion of the internal mechanism.

[0196] Referring further to Figures 13 and 14, at the end opposite to the coupling with the cam 58, the link mechanism 56 may be rotatably coupled to the lid 54 via a third coupling 66 located within the lid 54. When the lid 54 is opened or closed, the lid 54 rotates around a fourth coupling 68 that connects the lid 54 to the body of the reader 14, and the link mechanism 56 and the cam 58 then act via the third coupling 66 and the second coupling 64. The assembly 50 may include a positioning system or sensor 60 positioned adjacent to and / or near the cap 20 and the vial 18 when they are inserted into the reader 14. The positioning system 60 detects when the cartridge 12 is properly inserted into the reader 14 and positioned within the reader 14, thereby allowing the assay workflow to start and / or continue as described herein in conjunction with Figures 22-25. The support plate 70 may include one or more support tabs 82 on either side to facilitate the movement of the support plate 70 within the leader 14. The support tabs 82 may be firmly coupled to the support plate 70 and may be configured to have one or more through holes positioned in the support tabs 82 to slide on one or more horizontal orientation guides 68. In the open position shown in Figure 13, a horizontal gap 86 between the support tabs 82 and the edge of the outer wall 78 is shown in the side view. In comparison, in the side view of Figure 14, the gap is not visible because the support plate 70 is moved to the right (towards the outer wall 78) in the closed position. In some embodiments, the assembly 50 may include one or more springs 84 positioned around the horizontal guides 68. When the lid 54 is closed, the springs 84 are compressed. When the lid 54 is opened, the springs 84 expand, thereby moving the support plate away from the outer wall (i.e., rear wall) 78 of the leader 14.

[0197] Heating and thermal control Figure 13 shows a perspective view (left) and a side view (right) of the reader clamp mechanism in the open position according to an embodiment of this model. Figure 14 shows a perspective view (left) and a side view (right) of the reader clamp mechanism in the closed position according to an embodiment of this model. In some embodiments, the cartridge 12 is attached to the reader 14 by insertion into the vertical slot 22 when the clamp (i.e., the lid 54) is in the open position (Figure 13). In some embodiments, when the clamp / lid 54 is closed, it closes to a position where it is latched (closed position) (Figure 14). In some embodiments, the action of closing the clamp drives a mechanical linkage mechanism 56 that moves the cartridge 12 to the optical module and positions it precisely.

[0198] In some embodiments, the leader 14 includes a leader thermal subsystem. In some embodiments, the leader thermal subsystem 90 heats the cartridge melting chamber 94 and maintains precise control using an open-loop strategy (Figures 8A and 20).

[0199] In some embodiments, the leader thermal subsystem 90 heats the cartridge reaction chamber 94 and maintains precise control using an open-loop strategy (Figure 20A). This eliminates the need for an on-cartridge sensor. For example, as shown in Figure 8B, the melt heating element 116 includes a temperature sensor 120, which is used in a feedback loop to maintain the heater temperature at approximately 100°C (by repeatedly starting and stopping the heater, as indicated by the melt temperature 110 shown in Figure 20A). Heat is then transferred via a heat spreader 118 (e.g., a first heat spreader 118, see Figure 8B) positioned between the heating element 116 and the melt chamber 94, thereby maintaining the temperature at both the bottom 112 and top 114 of the melt chamber 94 at approximately 90°C (+ / - 1~2°C), as shown in Figure 20A. Therefore, as long as the temperature 110 in the melting heating element 116 is maintained at 100°C or approximately 100°C, the temperature in the melting chamber is maintained at a target temperature of approximately 90°C. Thus, a temperature sensor is not required in the melting chamber itself. Each of the first heating element 116 and the second heating element 126 may be a resistance heater and / or a film heater, or may include them. In some embodiments, each of the first heating element 116 and the second heating element 126 is located on the opposite side of the cartridge 12 from the PCBA 150. In some embodiments, each of the first heating element 116 and the second heating element 126 is directly integrated into the PCBA 150.

[0200] Referring again to Figure 8A, the dissolution chamber 94 may include a bottom tapered section 96 that helps to deliver the biological solution from the dissolution chamber 94 when a ball valve 98 is activated upon completion of dissolution heating. In this case, the biological solution may flow through a vertical passage 104 and eventually into a plurality of reaction chambers 106, each containing at least one freeze-dried bead and fluidly coupled to a vent 109 covered by a permeable membrane 108, both of which are located vertically above each reaction chamber 106 to which it is coupled in the cartridge 12. In some embodiments, the cartridge 12 may include heights of about 70 mm to about 120 mm, or about 75 mm to about 115 mm, or about 80 mm to about 110 mm, or about 85 mm to about 105 mm, or about 90 mm to about 100 mm, or about 95 mm. In some embodiments, the cartridge 12 may include widths of approximately 40 mm to approximately 90 mm, or approximately 45 mm to approximately 85 mm, or approximately 50 mm to approximately 80 mm, or approximately 55 mm to approximately 75 mm, or approximately 60 mm to approximately 70 mm, or approximately 75 mm. The leader thermal subsystem 90 may also include one or more mechanical supports 130 on which a heat spreader 118 (e.g., a first heat spreader 118) and / or a heating element 118 can be mounted.

[0201] Figure 9 illustrates the transfer of a biological solution to the reaction chambers 106 according to an embodiment of this model. In the embodiment of Figure 9, gravity draws the solution through the internal passages into the eight reaction chambers shown. The total volume of fluid may be around 500 microliters, which provides sufficient hydrostatic head to overcome capillary action and deliver the fluid to the reaction chambers. After the biological solution exits the dissolution chamber 94 through the vertical passage 104, it flows into the first horizontal passage 132 and the second horizontal passage 134, each of which is fluidically coupled to a plurality (e.g., 2, 3, 4, 5, 6) reaction chambers 106. The hydrostatic pressure generated by the biological solution or fluid in the vertical passage 104 is sufficient to push out any residual gas in the passage through the permeable membrane 108 and vents 109, and also sufficient to push the biological fluid into each of the reaction chambers 106 via the first horizontal passage 132 and the second horizontal passage 134. In some embodiments, the permeable membrane 108 includes hydrophobic vents that allow each of the reaction chambers 106 to be filled (i.e., by allowing gas to exit the passages when they are filled with liquid). Each of the passages 104, 132, 134 is sized such that the volume is large enough for the entire fluid contained therein to produce a hydrostatic head, and the inner diameter is not so small as to produce excessive flow resistance or surface tension, and is small enough to avoid bubble formation. For example, in some embodiments, the inner diameters of the channels or passages 104, 132, 134 range from about 400 μm to about 1200 μm, or about 500 μm to about 1100 μm, or about 600 μm to about 1000 μm, or about 700 μm to about 900 μm, or about 750 μm to about 850 μm, or about 800 μm. In some embodiments, the volume of each reaction chamber 106 is about 30 μL to about 50 μL, or about 35 μL to about 45 μL, or about 40 μL. In some embodiments, the cartridge 12 contains about 5, 6, 7, 8, 9, or 10 reaction chambers 106. In some embodiments, the total volume (i.e., dead volume) of the passages or channels 104, 132, 134 is about 50 μL to about 150 μL.Therefore, the total amount of fluid required to fill the cartridge may range from approximately 200 μL to approximately 650 μL, or approximately 300 μL to approximately 550 μL, or approximately 400 μL to approximately 500 μL, and / or other partial ranges in between.

[0202] Figure 10A shows a cross-sectional view of the reaction chamber heating according to an aspect of this embodiment. Referring to Figure 10A, the cartridge 12 may include a reaction chamber heating zone 136 according to an aspect of this embodiment. While the biological fluid flows from the dissolution chamber to the reaction chamber, the biological fluid is passively cooled from a temperature of about 90°C to about 60°C, as described herein. The reaction region heating element 126 (i.e., the second heating element, the dissolution region heating element 116 being the first heating element) reaches and maintains a temperature of about 63°C, thereby generating a reaction chamber temperature in the range of about 59°C to about 60.5°C or about 60°C, as shown on the left side of Figure 20B. The right side of Figure 20B shows the dissolution heater temperature 110 and the reaction heater temperature 126, both plotted as a function of time. The dissolution heater is initially heated to a temperature of about 100°C, as described herein. Following dissolution, the dissolution heater is passively cooled, and the reaction heater is heated to a temperature of about 63°C. Figure 10B shows a side view of the reaction heater heating zone according to an embodiment of this model. As shown in Figure 21, the temperature of each of the eight reaction chambers 106 is maintained within the range of approximately 59.0°C to approximately 62.5°C, with an average reaction chamber 106 temperature of approximately 61.25°C. These reaction chamber 106 temperatures (i.e., in the range of 59 to 62.5°C) are achieved by maintaining the reaction chamber heater / heating element 126 in a temperature range of approximately 63 to 64°C (again, in an open-loop configuration with the reaction chamber 106 itself).

[0203] In some embodiments, the reaction temperature is uniform and reproducible across all cartridge chambers (e.g., eight chambers) (Figure 20B left and Figure 21).

[0204] In some embodiments, the leader 14 includes heaters (e.g., a melting heater or heating element 116 and a second heater including a reaction area heater or heating element 126). In some embodiments, the leader 14 includes a film heater. In some embodiments, the leader 14 may heat areas of the cartridge. In some embodiments, the leader includes a film heater for heating areas of the cartridge. In some embodiments, the leader can heat the cartridge melting chamber to about 80°C to about 100°C, for example, about 85°C to about 95°C, as described herein. In some embodiments, the contact pressure between the heating elements 116, 126, the heat spreaders 118, 128, and / or the areas to be heated 94, 106 is supplied by a mechanical clamping mechanism during installation (see Figures 13 and 14). In some embodiments, the heat spreaders are made of aluminum and / or other conductive material and include a thickness of about 0.08 inches to about 0.2 inches.

[0205] In some embodiments, the leader 14 can heat the cartridge reaction chamber region 106 to about 50°C to about 70°C, for example, about 60°C (or about 58°C to about 63°C, or about 58°C to about 62°C, or about 59°C to about 62°C, or about 59°C to about 61°C, and / or other partial ranges between about 57°C and about 64°C) for the duration of the reaction (e.g., during the duration of amplification or the duration of another reaction). In some embodiments, the cartridge reaction chamber region 106 of the cartridge 14 is heated to a target temperature of about 60°C by a flexible heater plate or heat spreader 128 within the leader 14. In some embodiments, contact pressure is supplied by a mechanical clamping mechanism during mounting, as described herein.

[0206] Optical module Figure 15A shows an optical module 140 including an optical printed circuit board assembly (PCBA) according to an aspect of this embodiment. Figure 1B shows the same embodiment as Figure 15A with a cartridge overlaid, according to an aspect of this embodiment. In some embodiments, the reader 14 comprises the optical module 140. The optical module 140 can measure fluorescence. When the cartridge 12 is inserted, the optical module 140 is not yet needed in the operating sequence (Figures 23A and B, and Figure 24A). In some embodiments, the optical module 140 includes one or more photodetectors. The photodetectors of the optical module may be used for other tasks, such as identifying the cartridge 12 when it is inserted into the reader 14, as will be described in more detail below.

[0207] In some embodiments, the optical module is tuned for a specific dye (e.g., a specific excitation and / or emission). In some embodiments, the optical module is tuned for a FAM dye (excitation at 460 nm, emission at approximately 540 nm, or in some embodiments, absorption / excitation wavelength at 495 nm, emission wavelength at approximately 517 nm, and / or in some embodiments, absorption / excitation wavelength in the range of approximately 450 nm and emission wavelength in the range of approximately 500 nm to approximately 550 nm). In some embodiments, the FAM dye comprises a carboxyfluorescein molecule containing a carboxyl group.

[0208] In some embodiments, the optical module 140 includes a filter. In some embodiments, the filter is a gel filter 160 (shown in Figures 19A and 19B). In some embodiments, the filter is a 520 nm long-pass filter. In some embodiments, the gel filter is composed of and / or contains polyester and / or cellulose acetate and can be bonded to the photodetectors 144, 162 by an optical adhesive. The gel filter may have a thickness of about 0.004 inches to about 0.008 inches (i.e., 4 to 8 mils).

[0209] If additional LED excitation wavelengths beyond the 520 nm long-pass filter are used during the insertion of cartridge 12, a photodetector can be used to measure the cartridge label ID intensity reflectance. These reflectances are what can be uniquely printed on the label in various patterns to provide an identification (ID) feature (also referred to herein as a barcode).

[0210] In some embodiments, the labels may have as few as 2, 4, 6, 8, 10, and / or other unique IDs, or as many as 256 or more unique IDs. Label reading by the optical module 140 may be via a static (insertion) mode or a dynamic (in insertion) mode. Label colors and printing materials may vary from simple white labels using black ink to fluorescently tagged inks and other color combinations. Labels may be attached to the cartridge 12 by adhesive, or the optical target may be printed directly onto the cartridge plastic or injection molded.

[0211] In some embodiments, the optical module 140 uses simple and low-cost components. The optical components are aligned with the reaction chamber of the cartridge when installed (see Figure 15B). For example, as shown in Figure 15A, the optical module 140 may include a first row 148 of optical components and a second row 152 of optical components. For example, each of the first row 148 and the second row 152 may include four photodetectors (e.g., four photodiodes 144) and four LEDs 146, for a total of eight photodiodes 144 and eight LEDs 146. Each of the eight LEDs 146 may be spaced apart from one another to match the spacing of the reaction chambers 106 shown in Figures 8A, 9, 10A, and 27, and the LEDs 146 may be used to irradiate and excite the reaction chambers 106. Excitation light from the LEDs on the PCBA reaches the corresponding reaction chamber, and fluorescence from the sample in the reaction chamber is transferred to the photodetectors 144. As shown in Figures 13 and 14, the PCBA (i.e., printed circuit board) 150 is visible through the support wall 70 within the reader 14. Figure 15B shows the cartridge 12 overlaid on top of the PCBA 150, and the reaction chamber 106 of the cartridge 12 aligned with the LED 146 and photodetector 144 of the optical module 140. In the diagram shown in Figure 15B, the heat spreaders 118, 128 and heating elements 116, 126 are positioned in front of the cartridge 12 (i.e., coming out of the page). In other words, once the cartridge 12 is mounted in the reader 14 and positioned in place as described in Figures 13 and 14 above, the cartridge 12 is sandwiched between the heat spreaders 118, 128 and the PCBA 150. Therefore, when the cartridge 12 is installed inside the reader 14, the PCBA assembly is adjacent to the cartridge 12 on the first side, so that the LED 146 can excite the reaction chamber 106 and the photodetector 144 can detect the fluorescence emitted therefrom.At the same time, the heat spreaders 118, 128 and heating elements 116, 126 are adjacent to cartridge 12 on the second side of the cartridge (the second side of the cartridge is opposite to the first side of the cartridge), so that the heat spreaders 118, 128 and heating elements 116, 126 can heat both the dissolution chamber 94 and the reaction chamber 106.

[0212] In some embodiments, during the amplification reaction, the reaction chamber 106 of cartridge 12 is excited by a blue LED 156 (centered on a wavelength of approximately 465 nm, or, for example, in the range of approximately 430 nm to approximately 490 nm), as shown in Figure 16.

[0213] Figure 17 shows the emission and excitation spectra of fluorescein according to an embodiment of this model. In some embodiments, the positive reaction (amplification) in reaction chamber 106 results in a logistic increase in the fluorescence response to excitation using a fluorescent dye (i.e., fluorescein) (Figure 17). For example, as shown in Figure 17, the excitation spectrum 154 may be in the range of about 440 nm to about 520 nm, and may be centered at about 490 nm. On the other hand, the emission spectrum 156 may be in the range of about 480 nm to about 560 nm, and may be centered at about 515 nm.

[0214] Figure 18 shows the transmission spectrum of a Kodak Ratten 2-12 optical filter according to an embodiment of this invention. In some embodiments, reflected fluorescence emission from the reaction chamber is filtered through a low-pass filter 160 (i.e., a Kodak Ratten 2-12 gel filter) (Figure 18). As shown in Figure 18, the transmittance is only about 1% at wavelengths below 500 nm, about 12% at 510 nm, about 46% at 520 nm, about 74% at 530 nm, about 85% at 540 nm, and over 90% (e.g., about 95%) at wavelengths above 550 nm. Therefore, according to an embodiment of this invention, the low-pass filter can be configured such that substantially all of the light in the excitation spectrum 154 is removed, while substantially all of the light in the emission spectrum 156 can be detected. The filter 160 is shown in Figures 19A and 19B.

[0215] Figure 19A shows an optical PCBA with a photodiode according to an embodiment. Figure 19B shows an optical PCBA with a phototransistor according to an embodiment. In some embodiments, two optical architectures have been successfully realized: one using a photodiode and the other using a phototransistor (Figures 19A and B). In some embodiments, a red LED 158 is used for cartridge identification. For example, as shown in Figure 19A, in some embodiments, the PCBA 150 may include a photodiode 144, a first set of LEDs 146 (i.e., blue LEDs), and a second set of LEDs 158 (i.e., red LEDs). In some embodiments, as further described below, the blue LED 146 is used to excite the reaction chamber 106, while the red LED 158 is used to identify the cartridge 12. In some embodiments, the red LED 158 is positioned adjacent to the photodetectors 144, 162 at a position approximately 90 degrees from the position of the blue LED 146 relative to the photodetectors 144, 162. In other words, the red LED 158 and the blue LED 146 may be spaced approximately 90 degrees apart from the photodetectors 144 and 162. The PCBA 150 may also include a filter 160 (i.e., a low-pass filter 160) and an optical shield 336. In some embodiments, the positions of the blue LED 146 and the red LED 158 are reversed (for example, the positions of other feature elements such as identification markers may also be adjusted accordingly to match the position of the red LED 158). In the embodiment of Figure 19B, the PCBA 150 includes a phototransistor 162 instead of the photodiode 144 in Figure 19A. Thus, the PCBA 150 may include multiple photodetectors, each containing a photodiode 144 and / or a phototransistor 162. In some embodiments, the lateral distance (center to center) between each of the blue LED 146 and the red LED 158 and the photodetectors 144 and 162 may be approximately 3 mm to 9 mm, or approximately 4 mm to 8 mm, or approximately 5 mm to 7 mm, or approximately 5 mm, or approximately 6 mm, or approximately 5 mm to 6 mm.

[0216] Software / Firmware In some embodiments, the reader includes software and / or firmware. System software requirements are defined as subsystems of the SRS (Software Requirements Specification).

[0217] Overall sequence control In some embodiments, the basic operation sequence 170 for the inspection is shown in Figure 22. The overall timing and sequence of the inspection are shown in Figure 23A, and the timing and sequence of the "Cartridge ID" function are shown in Figure 23B. The timing and sequence of the Cartridge ID function are shown in Figure 24A, and the timing and sequence of the "Filling Detection" function are shown in Figure 24B. The timing and sequence of the optical fluorescence measurement during amplification are shown in Figure 25A, and the detailed timing and sequence of the optical fluorescence measurement are shown in Figure 25B.

[0218] Referring to Figure 22, which shows an exemplary operation flow according to this embodiment, the basic operation sequence or method 170 may include the following steps: a power-on start step 166, a system boot sequence step 168, and a reader ready state step 172. The basic operation sequence or method 170 may include the following further steps: a cartridge 12 containing a sample (by the user) step 188, a cartridge 12 inserted into the reader 14 step 174, a cartridge identification step 176, a clamping step 178, an auto-start routine run step 178, a test run step 180, a test or assay run and display of results step 182, a removal of the cartridge 12 from the reader 14 and disposal of the cartridge 12 step 184, and a power-off step 186 for the reader 14 (i.e., turning off the reader 14).

[0219] In some embodiments, when configured for unconnected use, the system (i.e., the reader 14) visually reports the test results to the user (for example, each assay lights up with a positive result, as shown in Figure 2A). In some embodiments, the reader 14 does not store detailed test data or transmit detailed test data to another system or device.

[0220] In some embodiments, when configured for connected use, the reader 14 can transmit data via Bluetooth® radio. This data may include inspection programs and parameters, firmware and software upgrades, user input information from mobile phone applications, inspection results, raw inspection data, error codes, and inspection execution metadata.

[0221] In some embodiments, the reader 14 may include alternative forms, for example, to optimize operability and workflow. A viable industrial design concept 700 of the system is shown in Figure 41. For example, the industrial design concept may include a cartridge 702 configured to be inserted horizontally into a corresponding reader 704, and a reader 706 having an upper surface that is at least partially angled and at least partially flat (i.e., at least partially parallel to the horizontal plane).

[0222] Alternative user workflows 800 are shown in Figures 42A, 42B, and 42C. A first alternative workflow 810, shown as Concept A in Figure 42A, may include the following steps: placing the cartridge in the hub 802; removing the foil covering the container holding the buffer 804; mixing the swab in the container 806; pushing the cap onto the tube 808; screwing the tube onto the cartridge and proceeding with the assay according to this specification 812.

[0223] Referring to Figure 42B, a second alternative workflow 830, shown as Concept B, may include the following steps: removing the top foil and placing the tube in the cap on the holder (i.e., the reader) 814; mixing the swab in the container 816; closing the cap on the tube 818; securing the cartridge upright 820; attaching the tube to the cartridge 822; and inserting the tube together with the cartridge into the reader 824. In step 826, the second alternative workflow 830 may include puncturing the tube (i.e., by the cap) to move the fluid. In step 828, the second alternative workflow 830 may include proceeding with the assay according to the present disclosure.

[0224] Referring to Figure 42C, a third alternative workflow 840, shown as Concept C, may include the following steps: step 832 inserting the tube into the cartridge without puncturing the bottom of the tube; step 834 removing the tube foil; step 836 mixing the swab in the container; step 838 pushing the plunger cap into the tube, thereby allowing the fluid to flow into the dissolution chamber. In some embodiments, this may include the use of a measurement function. The third alternative workflow 840 may further include the following steps: step 842 inserting the cartridge and tube into the reader, and step 844 proceeding with the assay after insertion into the reader is complete. In some embodiments, the system 100 may include a puncturable foil.

[0225] Figure 23A shows the overall sequence timing 200 (i.e., the timing sequence for the system to run completely) for the general operation of the cartridge 12, the reader 14, and the system 10. For example, the overall sequence timing 200 includes, among other things, the LED status indicator timing 188, the sequence timing 190 for cartridge insertion, cartridge identification 192, cartridge clamping 194, melting and heating 196, valve activation 198, filling detection 202, LAMP heating 204, and result display 206. The overall sequence timing 200 also includes a time bar 208 (Figure 23A) showing the time corresponding to each step of the sequence. For example, cartridge insertion 190 starts at approximately 1 minute and 25 seconds, and the cartridge remains inserted for approximately 33 minutes until approximately 5 or 10 seconds before the overall sequence ends. In another example, valve activation 198 occurs during a period of approximately 10 seconds following melting and heating 196.

[0226] Figure 23B shows the sequence timing 210 related to the cartridge identification process or algorithm 1700. The cartridge identification process or algorithm 1700 at a higher level is shown in Figure 38. The process 1700 may include the following steps: a system boot step 1702, a system idling step 1704, a cartridge insertion and identification detection step 1706, a step 1708 performing one or more optical checks, a step 1710 executing an identification routine, and a step 1712 sending internal communications to the microprocessor (in the reader) to confirm the identification of the cartridge and terminate. As shown in Figure 23B, the cartridge identification sequence timing 210 may include an LED status indicator timing 188, a cartridge clamp 194, a cartridge position detection 216 (i.e., detecting whether cartridge 12 is fully inserted), activation of a red LED 212, activation of a blue LED 218, and photodetector readings 214 from each of the eight photodetectors (channels 1-8). As shown in the figure, the cartridge identification sequence timing 210 may include sequentially cycling through each of the eight photodetector channels, as described herein, and then repeating this cycle several times (e.g., 3, 4, 5, 6, 7, 8, 9 times, and / or more than 9 times) to read the identification markings on the cartridge 12.

[0227] Figure 24A shows the cartridge identification sequence timing 220 in high-speed mode according to an embodiment of this model. At high levels, the cartridge identification sequence timing 220 in high-speed mode follows the same process flow as the normal mode shown in Figure 38. The cartridge identification sequence timing 220 in high-speed mode is similar to the sequence timing 210 shown in Figure 23B, with the exception of including a first period 222 for the sequential activation of the first four photodetector channels at a higher frequency, followed by a second period 224 for the sequential activation of all eight photodetector channels at a normal frequency, where the normal frequency is approximately half of the higher frequency.

[0228] Figure 24B shows the filling detection algorithm sequence timing 230 according to an aspect of this embodiment. A filling detection algorithm 1800 or process at a high level is shown in Figure 39. The filling detection algorithm or process 1800 may include the following steps: dissolution step 1802 (e.g., via dissolution heating in dissolution chamber 94), followed by a delay step 1804, in some embodiments, simultaneous with or preceding ball valve operation, during the period in which dissolution is taking place. After ball valve operation, as described herein, the biological solution flows from dissolution chamber 94 into reaction chamber 106 (while the biological solution is passively cooled). The process 1800 may further include the following steps: performing the filling detection algorithm 1806, performing loop-mediated isothermal amplification (LAMP) 1808, completion of the filling detection routine, and a microprocessor communicating that the cartridge filling detection routine is complete (filling detected or not detected) 1810. As shown in Figure 24B, in some embodiments, the fill detection 226 is initiated simultaneously with the activation or actuation of the ball valve and is performed at a frequency of 10 Hz (or approximately 5 Hz to approximately 20 Hz, or approximately 3 Hz to approximately 50 Hz, or approximately 2 Hz to approximately 100 Hz, and / or other partial ranges in between). In some embodiments, the actuation of the ball valve 98 is performed by mechanical means. In some embodiments, the system 10 includes thermal means (e.g., melting area heating element 116) for acting the ball valve 98. In some embodiments, simultaneously with the initiation of the fill detection 226, the fill detection algorithm sequence timing 230 may include monitoring a single 228 for fill activity.

[0229] Figure 25A shows a LAMP fluorescence measurement timing sequence 240 according to an aspect of this embodiment. A high-level LAMP fluorescence measurement timing sequence 1900 or process is shown in Figure 40. Process 1900 may include the following steps: step 1902 of performing (or completing) a packing detection routine; step 1904 of acquiring LAMP fluorescence (i.e., via the photodetector and LED described herein); step 1902 of displaying the results following the acquisition of LAMP fluorescence; completion of the LAMP fluorescence acquisition routine; and step 1908 of communicating by / to / within the microprocessor that the LAMP fluorescence acquisition routine has been completed. As shown in Figure 25A (and Figure 24B), in some embodiments, LAMP reaction heating 232 is started simultaneously with the completion of packing detection 226. The timing sequence 240 may also include a LAMP fluorescence acquisition mode 234 which is started slightly after the start of LAMP reaction heating 332 (e.g., 1, 2, 5, 10, 20, or 30 seconds later). Timing sequence 240 may also include the start of signal curve algorithm monitoring 236, concurrent with the start of LAMP fluorescence acquisition mode 234. Finally, as shown in Figure 25A, timing sequence 240 may also include the start of calling algorithm 238, upon completion (i.e., concurrently therewith) of each of the LAMP reaction heating 232, LAMP fluorescence acquisition mode 234, and signal curve algorithm monitoring 236.

[0230] Figure 25B shows a LAMP fluorescence measurement A / D timing sequence 250 according to an embodiment of this model. The LAMP fluorescence measurement A / D timing sequence 250 enables oversampling, thereby improving the resolution of fluorescence acquisition. The LAMP fluorescence measurement A / D timing sequence 250 in Figure 25B is shown for a single channel (i.e., a single reaction chamber 106 (CH1), and the corresponding LED and photodetector), but can be applied with equal force to any channel / reaction chamber 106. The LAMP fluorescence measurement A / D timing sequence 250 may include initiating a LAMP fluorescence acquisition mode 242 (e.g., with a 10-second scan) that includes acquisition in each of the reaction chambers 106 (e.g., channel 1 (CH1) acquisition 244). The LAMP fluorescence measurement A / D timing sequence 250 may also include activating 246 blue LEDs (e.g., at a frequency of 100 ms) in synchronization with the reaction chamber scan 244. Following a 20ms LED delay 252, a CH1 photodetector reading 248 is performed, and a photodetector A / D oversampling acquisition 254 is initiated (e.g., repeated several cycles at 10ms intervals, followed by a pause period (e.g., 30ms-50ms), and then a second and subsequent period of initiation at 10ms intervals). During the second period of the photodetector A / D oversampling acquisition 254, a dark current reading 256 may be performed. A specific A / D acquisition 258 may also be initiated at 50ms intervals following the LED delay 252. Finally, the LAMP fluorescence measurement A / D timing sequence 250 may include signal processing 262 upon completion of the 50ms interval A / D specific acquisition 258 (i.e., to facilitate data storage).

[0231] B. Cartridge Assembly In some embodiments, the disclosure provides a cartridge 12. In some embodiments, the cartridge 12 is assay-specific (independent). In some embodiments, the cartridge 12 is a disposable cartridge. In some embodiments, the cartridge 12 is a low-cost disposable device comprising multiple reagents for detecting a target nucleic acid.

[0232] In some embodiments, the cartridge 12 interfaces with the reader 14.

[0233] Referring to Figures 26 and 27, according to this embodiment, the cartridge assembly 260 may include a cartridge 12, a plurality of permeable membranes 108, a ball valve 98, lyophilized lysis beads 266, a plurality of lyophilized PCR beads 268, and a front film 270. The lyophilized lysis beads 266 may be preloaded into the lysis chamber 94, while each of the plurality (e.g., eight) of lyophilized PCR beads 268 may be preloaded into each of the plurality (e.g., eight) reaction chambers 106. Thus, when the cartridge assembly 260 is fully assembled, each of the lyophilized reaction / PCR beads 268 and lysis beads 266 is positioned between the cartridge 12 and the film 270, thereby partially encapsulating them in their respective reaction chambers 106 or lysis chambers 94, with the film 270 holding them in place. In some embodiments, the lyophilized reaction / PCR beads 268 may include a C7 FAM reporter, RS9 enzyme, DNA polymerase, dNTPs, buffers, and their corresponding primers and guides. In some embodiments, due to the material properties of the film 270, the film 270 functions as a heat spreader 128, thereby helping to enable heat transfer from the reaction area heating element 126 to the reaction chamber 106. As shown in Figure 27, the cartridge assembly 260 may include an additional permeable membrane 272 (i.e., a ninth permeable membrane 272 in addition to the eight permeable membranes 108 used to permeate each of the eight reaction chambers 106 during filling). The additional permeable membrane 272 may be used in conjunction with vents 274 to permeate the melting chamber 94 (i.e., allow air to escape) when the cartridge 12 is first filled through the melting chamber 94. The cartridge assembly 260 may also include a valve film 264 for holding the ball valve 98 in place and facilitating the operation of the ball valve 98, and in some embodiments, the valve film 264 may have a diameter of 3 mm. In some embodiments, the cartridge 12 may be molded (e.g., by injection molding). In some embodiments, the cartridge 12 may be 3D printed or formed by machining, for example, by CNC drilling.In some embodiments, the ball valve 98 can be coated with a lubricant (e.g., a polymer coating (e.g., a parylene coating)) to facilitate the operation of the ball valve 98 and provide a moisture barrier to protect the ball valve 98 from corrosion or degradation due to exposure to biological solutions (i.e., saliva and buffer / reagent mixtures). In some embodiments, the film 270 (i.e., the front film 270) can include or be a microfluidic layer film having a thickness of from about 0.003 inches to about 0.008 inches, such as about 0.003, about 0.004, about 0.005, about 0.006, about 0.007, about 0.008 inches, and various sub-ranges of thickness therebetween. In some embodiments, the front film 270 includes a polypropylene laminate including an adhesive layer. In some embodiments, the front film 270 (i.e., the film layer 270) can be thermally welded, laser welded, and / or ultrasonically welded to the cartridge 12.

[0234] In some embodiments, the cartridge 12 includes an injection molded cartridge body 284. Exemplary cartridge bodies 284 are shown in FIGS. 6 and 29. In some embodiments, the cartridge body 284 is a plastic cartridge body. In some embodiments, the plastic cartridge body is made from a plastic polymer selected from the group consisting of polycarbonate, polymethyl methacrylate (PMMA), cycloolefin polymer (COP), and cycloolefin copolymer (COC).

[0235] In some embodiments, one or more reaction chambers 106 are formed within the cartridge body 284. In some embodiments, 1 to 30 reaction chambers 106 (e.g., 2 to 20, 4 to 16, 5 to 15, 6 to 12, 7 to 10, or about 8) are formed within the cartridge body 284. In some embodiments, the reaction chambers 106 are fluidly connected to the sample dissolution chamber 94 via a valve (not shown). In some embodiments, a plurality of reaction chambers 106 are formed within a plastic body 284. An exemplary cartridge body 284 with 8 reaction chambers 106 is shown in FIG. 29. The number of reaction chambers can be from 1 to 30.

[0236] In some embodiments, the cartridge 12 includes one or more reaction chambers 106. In some embodiments, the cartridge includes two or more reaction chambers 106. In some embodiments, the cartridge includes three or more reaction chambers 106. In some embodiments, the cartridge includes four or more reaction chambers 106. In some embodiments, the cartridge includes five or more reaction chambers 106. In some embodiments, the cartridge includes six or more reaction chambers 106. In some embodiments, the cartridge includes seven or more reaction chambers 106. In some embodiments, the cartridge includes eight or more reaction chambers 106.

[0237] In some embodiments, the volume of the reaction chamber 106 is from about 5 μL to about 100 μL (e.g., about 10 μL to about 60 μL, e.g., about 20 μL to about 50 μL, e.g., about 40 μL). In some embodiments, the reaction chamber 106 contains a lyophilized reagent. In some embodiments, the reaction chamber 106 is sealed by a film layer 270 (shown in FIG. 27).

[0238] In some embodiments, one or more reaction chambers 106 contain lyophilized reagents. In some embodiments, the detection system 10 contains lyophilized beads. In some embodiments, lyophilized beads 268 (shown in Figure 27) are used to verify the filling of each reaction chamber 106. The filling of the reaction chambers 106 with the sample can be verified by measuring the change in fluorescence that occurs when the lyophilized beads 268 are rehydrated.

[0239] In some embodiments, the Disclosure provides a disposable cartridge 12 for detecting a target nucleic acid, comprising a dissolution chamber 94 (e.g., a first chamber or first heating zone) for receiving a sample containing the target nucleic acid, and a reaction chamber 106 connected to the dissolution chamber via a first channel 104 (shown in Figure 9) and connected to a first vent 274 via a second channel 286 (e.g., a vent channel 286 shown in Figures 26 and 27).

[0240] In some embodiments, cartridge 12 is a disposable element that receives the sample, contains the dried reagent, and performs the assay in the reader 14. After sample transfer, cartridge 12 is sealed by cap 20 (shown in Figure 1) and contains all reagents and reaction products during and after the test. Cartridge 12 contains the dried reagent (in the form of lyophilized beads 255, 268) in the reaction chamber and dissolution chamber (Figure 27).

[0241] Referring again to Figure 29, in some embodiments, the cartridge body 284 comprises a sample dissolution chamber 94. In some embodiments, the cartridge body 284 comprises a sample inlet port 288. In some embodiments, the cartridge body 284 comprises a label 290. In some embodiments, the cartridge body 284 comprises a unique device identification (UDI) barcode 292. In some embodiments, the cartridge body 284 comprises a result display 294. Permeable membranes 108 are also shown in Figure 29, each positioned on the cartridge body 284 and spaced vertically above each fluidly coupled reaction chamber 106.

[0242] In some embodiments, the cartridge assembly 260 includes a fluid ball valve 98, hydrophobic vents 101 and 272, and two cover films 264 and 270 (Figure 27). In some embodiments, the cartridge body 284 is the main component and is mainly composed of an optically transparent polymer (such as polycarbonate, COC, or COP).

[0243] Alternatively, the cartridge body 284 may be composed of a material that is optically transparent only in the reaction chamber 106 region, and otherwise composed of a different material (i.e., an optically opaque material) (for example, using a two-shot injection molding process, an insert molding process, or other assembly method).

[0244] In some embodiments, the cartridge 12 may take on an alternative form, for example, to optimize operability and workflow. A viable industrial design concept of the system is shown in Figure 41. Alternative form factors of the cartridge 12, sample collection, and user workflow are shown in Figures 42A, 42B, and 42C.

[0245] In some embodiments, the cartridge 12 comprises a Cas enzyme, a probe, and a guide.

[0246] In some embodiments, the cartridge 12 includes a label 290 indicating the test menu type and the assay and / or analysis results.

[0247] Cartridge ID Optics In some embodiments, as described herein, the reader 14 detects the type of cartridge 12 using optical reading of the cartridge device label. For example, the cartridge 12 type may be detected using either a static ID procedure or a dynamic ID procedure.

[0248] static id In some embodiments, the static ID mode is used as a barcode to identify which type of cartridge 12 is being used (i.e., including the corresponding reagents, buffers, and / or lyophilized beads) so that the reader 14 can perform the appropriate test sequence and routine depending on the type of assay(s) being performed. In some embodiments, the static ID mode is used to identify a specific cartridge 12, such as a specific test panel. In some embodiments, the static identification label 290 may include a printed optical target 296 (or label mark 296, or identification marker 296). In some embodiments, the optical target 296 (or printed mark 296) alters the reflection of an irradiating LED whose wavelength exceeds the cutoff of the long-pass detector. An exemplary optical target 296 (or printed mark 296) is shown printed as a black circle in Figure 29 and as a black rectangle in Figure 30. In some embodiments, photodetectors 144, 162 positioned adjacent to the irradiating LEDs (e.g., 158, 146) detect the presence of the label mark 296 (or printer mark 296). The corresponding optical target array 296 (or printed mark 296) includes a barcode or a unique signature. The reflectance measurement sequence is shown in Figures 23B and 24A.

[0249] In some embodiments, the cartridge 12 connects to a reader 14 to identify a specific cartridge 12 using one or more specific label patterns. An exemplary label pattern for respiratory diseases is shown in Figure 29. Figure 28 shows an exemplary label embodiment that includes a conventional 1D barcode referencing method (Figure 28A) using a conventional barcode 298 (which can be connected to and read by a conventional barcode scanner).

[0250] Location detection In some embodiments, the cartridge 12 incorporates position-sensing feature units 302, 304. An example is shown in Figure 28B. The position-sensing feature units 302, 304 help and are useful in determining whether the cartridge 12 is properly and completely inserted into the reader 14. Such feature units 302, 304 can be used to check that a positive condition is met (when the cartridge 12 is inserted). In some embodiments, the position-sensing feature units 302, 304 are realized by optical targets 302, 304. In some embodiments, the sensing feature units 302, 304 are targets positioned slightly above the centerline of the adjacent reaction chamber 106. In some embodiments, the sensing feature units 302, 304 are positioned slightly below the horizontal axis centerline of the adjacent reaction chamber 106. An example is shown in Figure 28B. During operation, the LEDs in Figures 19A and 19B (e.g., red LED 158) are positioned on the centerline of the reaction chamber 106 and the photodiode 144 (or phototransistor 162). To determine if cartridge 12 is fully inserted into the correct position, system 10 measures the reflections from both optical targets 302 and 304. If cartridge 12 is positioned too high or too low (vertical axis in the figure), only one condition is met, thereby allowing for the detection of an improper insertion. This configuration and system, when properly gain-calibrated, can measure position with a resolution of approximately 0.010 inches.

[0251] Referring again to Figure 28B, the first position-sensing feature 302 is positioned slightly above the centerline of the adjacent reaction chamber 106A. Similarly, the cartridge 12 (relating to the label 290) may include a second position-sensing feature 304 positioned slightly above the centerline (i.e., horizontal centerline) of the adjacent reaction chamber 106B. Each of the first position-sensing feature 302 and the second position-sensing feature 304 includes, or may include, a single shadow or colored geometric shape on the background surface or area of ​​the label 290 of a different shade or color, thereby making the contrast between the position-sensing feature 302, 304 and the background visible or detectable via the photodiode 144 (or phototransistor 162). For example, in the embodiment of Figure 28B, the position-sensing feature 302, 304 includes a black rectangle on a white background. In the embodiments of Figures 28C and 28D, the position detection feature units 302, 304 and the printed optical target 296 (or label mark 296, or identification marker 296) may include a circle, an octagon, a square with rounded corners, and / or other preferred shapes. For the “correct position” signal to be processed by the microprocessor (i.e., within the reader), the first position detection feature unit 302 must be identified as being above the centerline of the reference reaction chamber 106A, while the second position detection feature unit 304 must be identified as being below the centerline of the reference reaction chamber 106B. If both the first position detection feature unit 302 and the second position detection feature unit 304 are either below or above the centerlines of the reference reaction chambers 106A and 106B, the cartridge 12 is not detected as being in the correct position, and the additional algorithms and routines do not proceed. In contrast to the relative positions of the first position detection feature unit 302 and the second position detection feature unit 304, and further referring to Figure 28B, the other printed optical targets 296 (or label marks 296, or identification markers 296) need to be aligned with the respective centerlines of the adjacent reaction chambers 106.

[0252] Figures 28C and 28D show cartridges 12 with two different static ID patterns. In the cartridge of Figure 28C, other printed optical targets 296 (or label marks 296, or identification markers 296) are in different positions than those in Figure 28D. For example, in the embodiment of Figure 28C, the cartridge contains six identification markers 296, while the embodiment of Figure 28D contains five identification markers 296. Furthermore, some of the identification markers 296 in Figure 28C are in different positions than those in Figure 28D, and vice versa. There are eight potential positions for the identification markers 296, and if there are 0 to 8 identification markers 296 that can be included in a given cartridge, then there are 8 factorial (plus 1) or 40,321 potential identifications that can be encoded on the label 290 using the identification markers 296.

[0253] Dynamic ID In some embodiments, the cartridge 12 can be identified in dynamic ID mode, i.e., while the cartridge 12 is moving when it is inserted into the reader 14. In some embodiments, the cartridge 12 comprises an identification ID label 290. An exemplary identification ID label is shown in Figure 30. In some embodiments, the identification ID label 290 includes one or more static identification markers 296, in addition to a first barcode optical target pattern 306 and / or a second barcode optical target pattern 308.

[0254] During operation, the LED (e.g., the red LED 158 shown in Figures 19A and 19B) remains lit throughout the cartridge insertion step. In some embodiments, this is enabled via software control. During insertion (vertical downward movement of cartridge 12 on the vertical axis), the barcode optical target patterns 306, 308 in Figure 30 are illuminated by the LED (e.g., the red LED 158), and the resulting optical signature is detected by photodetectors 144, 162 (as shown in Figures 19A and 19B), thereby forming a dynamic detection system that uses the photodetectors 144, 162 already present in the reader 14 to detect the identification of cartridge 12 while it is moving.

[0255] Figure 31 shows an exemplary design schematic of the LED driver circuit 280 and its components. In some embodiments, the LED driver circuit 280 may include eight LEDs 146 (or possibly 158) arranged in parallel between a voltage source 278 (e.g., a 5-volt source) and ground 282, with each LED 146 positioned downstream of a corresponding resistor 276. In some embodiments, adjacent photodetectors 144, 162 located above the reaction chamber 106 measure the reflectance. In some embodiments, the photodetectors 144, 162 are multiplexed at a frequency high enough to analyze the speed of cartridge 12 insertion. Typically, the upper horizontal rows of the reaction chamber 106 and detectors 144, 162 are used when the horizontal rows first detect the label barcodes 306, 308 (during insertion from above). In some embodiments, the first barcode optical target pattern 306 and the second barcode optical target pattern 308 are identical. In some embodiments, the first barcode optical target pattern 306 and the second barcode optical target pattern 308 are different. Furthermore, it is recognized that one of the patterns 306 or 308 may be a timing marker of equally spaced marks for measuring the speed of the cartridge 12, if it is found to facilitate software identification. In other words, one of the patterns (i.e., either the first barcode optical target pattern 306 or the second barcode optical target pattern 308) may be the same each time and is therefore used to assess how quickly the cartridge 12 is being inserted into the reader 14. The other pattern (i.e., either the first barcode optical target pattern 306 or the second barcode optical target pattern 308) may then be used as an actual identifier representing the type of cartridge 12 being inserted.In some embodiments, the insertion rate information from the first pattern (i.e., the timing pattern or barcode, e.g., the first barcode optical target pattern 306) can be used to calibrate the optical signature from the second pattern (i.e., the identification barcode, e.g., the second barcode optical target pattern 308), whereby the appropriate spacing between bars of the barcode can be adjusted and / or corrected as needed to ensure an accurate interpretation of the cartridge identification. The identification marker 296, the position detection features 302, 304, and the barcode optical target patterns 306, 308 (i.e., shown in FIGS. 28 - 30 and 32) enable the cartridge 12 to be properly identified by the reader 14 and correctly positioned within the reader 14 without the need for a barcode scanner, by using the photodetectors 144, 162 already present within the reader 14.

[0256] Referring further to FIGS. 28 - 30 and 32, when the identification of the cartridge 12 is determined, the reader 14 automatically executes the routine and sequence corresponding to that particular cartridge 12. The cartridge 12 identification indicates which set of internal (software - mediated) sequences are to be executed by the reader. In some embodiments, the reader 14 is Wi - Fi (i.e., network or internal) - compliant, whereby software updates can be implemented (i.e., remotely uploaded and installed to the reader 14) to enable new and / or updated assays to be run on the reader 14 (i.e., using the same reader 14 hardware), and in some cases, using newly developed disposable cartridges 12 (e.g., to enable strain - specific assays of various viruses (i.e., new strains) to be run through the same underlying system).

[0257] Cartridge ID by Ambient Light Blocking Label In some embodiments, the cartridge 12 comprises a label 312 having a colored or shaded background. In some embodiments, the cartridge 12 comprises a dark or opaque label 312 background. An exemplary dark or opaque label background 312 is shown in Figure 32.

[0258] The shaded or colored label 312 provides ambient light blocking properties. If used, the barcode optical target feature portion 296 may be alternating color, transparent, or white 314 to allow for reflection changes that can be measured by the photodetectors 144, 162. The cartridge 12 may also include a cartridge name label 316 (i.e., a second label different from the first labels 290, 312).

[0259] In some embodiments, the LED used for fluorescence excitation has specific spectral emission characteristics. Figure 34 shows the emission spectrum of a fluorescence-excited LED. In some embodiments, it is important that the irradiation pattern includes a narrow beam to maximize energy transfer when exciting the reaction chamber 106 fluid. A typical emission pattern with a half-power angle is in the range of 5 to 10 degrees relative to a typical full field of view of 20 degrees. In some embodiments, the LED may be the SunLED XZCBD78W. There are also several alternative part numbers that can be used, which fall into the categories of surface mount, InGaN light-emitting material, narrow cone angle beam pattern, and high-intensity output.

[0260] In some embodiments, cartridge 12 comprises a filter. In some embodiments, the optical module includes a filter. In some embodiments, the filter is a fluorescence long-pass filter. In some embodiments, the filter has a centerline cutoff of about 520 nm. In some embodiments, the fluorescence long-pass filter is used to measure the FAM-labeled reaction fluid. The optical module 140 uses this filter and / or other gel, film, or plastic filters instead of conventional dichroic glass filters. Conventional dichroic glass filters may be too expensive to be practical in the product concepts developed in connection with Reader 14 and this disclosure. In some embodiments, the filter is a Kodak Ratten 12 filter having a centerline cutoff of about 520 nm. In some embodiments, separate filters may be located on the outer surface of each reaction chamber dome. In some embodiments, filters may be located above multiple reaction chambers.

[0261] Figure 33 shows the emission spectrum of a red-illuminated LED. In some embodiments, the illumination pattern includes a narrow beam to improve performance. A typical emission pattern with a half-angle of maximum power is in the range of 5 to 10 degrees. In some embodiments, the red-illuminated LED is the VLDR1235G LED from Vishay Semiconductor, which can emit light in the wavelength range of approximately 600 nm to approximately 660 nm.

[0262] Figure 34 shows the emission spectrum of an alternative illumination LED emitting light with near-infrared wavelengths centered at approximately 865 nm in the range of approximately 730 nm to approximately 900 nm. In some embodiments, the illumination pattern includes a narrow beam to optimize performance. In some embodiments, the illumination near-infrared spectrum is in the range invisible to the human eye (e.g., the range of approximately 730 nm to approximately 900 nm and / or wavelengths above 700 nm). A typical emission pattern with a half-angle of maximum power is in the range of 5 to 10 degrees. In some embodiments, the LED is the LED SFH 4059-QS by OSRAM Opto Semiconductors GmhH. In some embodiments, the photodetectors 144, 162 described herein include dual-mode or tri-mode photodetectors (i.e., dual / tri-mode photodiodes 144 and / or dual / tri-mode phototransistors 162) that can be calibrated to measure wavelengths in the range shown in Figure 33 (i.e., 600 nm to 660 nm), the range shown in Figure 34 (i.e., 730 nm to 900 nm), and / or the range shown in Figure 17 (i.e., 400 nm to 640 nm). Thus, in some embodiments, the photodetectors 144, 162 described herein can be calibrated to measure wavelengths in the range of about 400 nm to about 900 nm.

[0263] Figure 35 shows an embodiment of the prototype design. Figure 35 shows a cutaway side view of the cartridge 12, as well as its interfaces with the heating element 126, the heater spreader 128, and the optical module 140.

[0264] Figure 19A shows details of the PCBA layout of the optical module. Improvement of the optical signal-to-noise ratio is achieved by positioning the optical components relative to the cartridge reaction chamber 106, as shown in Figure 35. In some embodiments, as shown in Figure 36 (an enlarged view of a portion of Figure 35), the photodiode 144 is positioned directly above the volume of the reaction chamber 106, thereby collecting maximum light energy from the fluorescence emission. In some embodiments, the 460 nm blue excitation LED 146 is positioned as close as feasible, thereby allowing the maximum output energy from a narrow (e.g., 20-degree cone angle) beam to enter and excite the reaction mixture in the reaction chamber 106. An optical shield 122 may be used to prevent excitation stray light from entering the photodiode 144 from the sides or via unwanted reflections. In some embodiments, the photodiode 144 incorporates a filter 160 (shown in Figures 19A and 19B) on its top surface by photo-epoxy or a similar assembly method.

[0265] In the embodiments shown in Figures 35 and 36, the photodetector may include an illustrated photodiode 144 or phototransistor 162. As shown in Figure 36, the optical module 140 may include a recess 164 having a recessed contour that matches the outer shape of the reaction chamber 106 dome (or hemisphere). The recess 164 may be located on a printed circuit board (PCBA) 150. When the cartridge 12 is inserted into the reader 14, in some embodiments, as described in connection with the description of Figures 13 and 14, the cartridge 12 is inserted vertically (i.e., downward, i.e., off-page in the drawings of Figures 35 and 36) and then pushed laterally (i.e., upward in the drawings of Figures 35 and 36), so that the reaction chamber 106 approaches the optical module 140 and the heating elements 116, 126 and heat spreaders 118, 128 approach or come into contact with the cartridge 12. Therefore, when the cartridge 12 is pushed laterally by the mechanical assembly 50, each dome (or hemisphere) of the reaction chamber 106 fits into each of the corresponding recesses 164 located in the PCBA 150. Once the cartridge 12 is correctly positioned within the leader 14, thereby causing the dome of the reaction chamber 106 to protrude at least partially into the arc of the recess 164, a gap 328 of approximately 1 mm to approximately 4 mm (e.g., approximately 2 mm to approximately 3 mm) is maintained between the top of the dome of the reaction chamber 106 and the center point of the recess 164, as shown in Figure 36.

[0266] In some embodiments, the dome (or hemisphere) of the reaction chamber 106 is concentric within the recess 164 when correctly inserted. In some embodiments, the dome (or hemisphere) of the reaction chamber 106 is constructed from a transparent polycarbonate (cyclic olefin copolymer and cyclic olefin polymer) such as COC and / or COP. The arrangement described herein with respect to Figures 35 and 36 has been shown to result in an improved signal-to-noise ratio with respect to fluorescence detection. For example, this embodiment does not include or requires lenses due to the arrangement of the reaction chamber 106 close to both the light source (i.e., LEDs 146, 158) and the photodetectors 144, 162. Therefore, the optical assembly 140 of this embodiment may be less expensive and less complex than other equivalent systems because it does not require optical lenses.

[0267] In the embodiment shown in Figure 36, the optical shield prevents the light emitted by the LED from being directly detected by the photodetector. After activating the biological solution in the reaction chamber, the photodetector detects the activated solution. Due to the photodetector's proximity to the reaction chamber, an optical lens is not required. In some embodiments, the first LED 158 (e.g., a red LED) in Figures 19A and 19B is positioned on one side of the photodiode 144, and the second LED (e.g., a 460 nm (blue) LED 146) is positioned on the other adjacent side of the photodiode 144, so that the first LED 158, the photodiode 144, and the second LED 146 form a 90-degree angle. In some embodiments, the LED (e.g., red LED 158) is also positioned horizontally with the photodiode 144 and between adjacent reaction chambers. This allows the barcoded optical target 296 to be printed on the label 290 in the available space on the surface of the cartridge 12 that does not interfere with the hydrophobic permeable section 108 (i.e., the permeable membrane 108). Furthermore, the position of the red LED 158 allows for alignment for dynamic ID detection during cartridge 12 insertion, which is acceptable and may be advantageous.

[0268] In some embodiments, the photodiode 144 is a photodetector from Vishay Semiconductors' TEMD5020X01. Naturally, other photodiodes 144 or phototransistors 162 (and associated components) can also be used as elements of the photodetectors 144, 162, provided that size and cost constraints are met, thereby improving the feasibility of home testing.

[0269] Figure 43 shows a side view of an alternative configuration reaction chamber assembly according to an aspect of this embodiment. In the embodiment of Figure 43, at least one of the LEDs (e.g., a blue LED 146) is integrated into the cartridge 12, thereby positioning it directly below the reaction chamber 106 to enhance the excitation of the nucleic acid contained therein. The presence of the LED 146 positioned within the cartridge 12 requires space, pores, and / or voids within the cartridge 12, potentially reducing heat transfer to the reaction chamber 106. In some embodiments, the LED 146 has a diameter smaller than the diameter of the reaction chamber 106, thereby allowing the ring 334 to surround the LED 146, which is in direct contact with the cartridge 12 material, thus facilitating heat transfer to the reaction chamber 106. Alternatively, or further, an inefficient LED (i.e., an LED that draws additional current and releases more thermal energy) may be used, thereby allowing the LED itself to act as a heat source in maintaining the reaction chamber 106 temperature at 60°C or approximately 60°C during amplification.

[0270] High-speed cartridge ID mode Figure 24A shows an alternative embodiment of the cartridge ID timing sequence. In some embodiments, the cartridge ID timing sequence utilizes four excitation LEDs instead of eight. This offers the advantage of enabling faster scanning (multiplexing of photodetectors). This can be advantageous when the user inserts the cartridge 12 into the reader 14 very quickly. The high-speed mode can be used for static or dynamic ID detection.

[0271] C. Sample collector In some embodiments, the disclosure provides one or more components for collecting a sample (i.e., a sample collector, e.g., a container or vial 18). In some embodiments, the sample collection container 18 is used to collect and / or contain a sample. In some embodiments, the sample collection container 18 contains one or more target nucleic acids. In some embodiments, the sample collection container 18 comprises one or more components that interface with a cartridge 12. In some embodiments, the sample collection container 18 is sample-specific. In some embodiments, the sample collection container 18 is assay-specific.

[0272] In some embodiments, the sample collection container 18 comprises a sterile swab 16 and a buffer / reagent container.

[0273] D. Detection System In some embodiments, this disclosure provides a detection system 10. An exemplary detection system is shown in Figure 1. System 10 is designed for use by untrained users (consumers) and incorporates simple and familiar tasks wherever possible. Detection system 10 is designed to operate in a home environment. In some embodiments, the detection system uses only standard wall power (e.g., 110V AC using a commercially available USB charger). The architecture of detection system 10 (cartridge 12, reader 14, and assay reagents, etc.) provides a flexible and scalable system that allows for the addition and development of new applications and measurement menus / functions. In some embodiments, system 10 (i.e., reader 14) draws a current of approximately 100mA to approximately 1000mA when powered by a USB charger. In some embodiments, the system includes a lithium-ion battery (e.g., rechargeable battery, non-rechargeable battery) to power the device, thereby making the system portable and usable when AC power is unavailable (e.g., in locations without power access, during a power outage, etc.).

[0274] E. Detection of nucleic acids In some embodiments, the systems, methods, compositions, or devices provided herein detect one or more target nucleic acids. In some embodiments, the target nucleic acid is deoxyribonucleic acid (DNA). In some embodiments, the target nucleic acid is ribonucleic acid (RNA). In some embodiments, the target nucleic acid is single-stranded. In some embodiments, the target nucleic acid is double-stranded.

[0275] In some embodiments, the target nucleic acid is present in the sample. In some embodiments, the sample contains one or more target nucleic acids. In some embodiments, the sample contains one or more target nucleic acids and one or more nucleic acids other than the target nucleic acids. In some embodiments, the target nucleic acid is of eukaryotic origin. In some embodiments, the target nucleic acid is of prokaryotic origin. In some embodiments, the target nucleic acid is parasitic (e.g., protist), bacterial, viral, or fungal. In some embodiments, the target nucleic acid is of human origin.

[0276] In some embodiments, the sample is an environmental sample. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is obtained from or derived from a biological source of interest (e.g., tissue or organism or cell culture). In some embodiments, the source of interest is or includes an organism such as an animal or human. In some embodiments, the biological sample is or includes biological tissue or bodily fluid. In some embodiments, the biological sample may be or include bone marrow, blood, blood cells, ascites, tissue or fine-needle biopsy specimens, cell-containing fluids, suspended nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluids, skin swabs, vaginal swabs, oral swabs, nasal swabs, lavage fluids or washes such as mammary duct lavage fluid or bronchoalveolar lavage fluid, aspirates, scrapes, bone marrow specimens, tissue biopsy specimens, surgical specimens, feces, other bodily fluids, secretions, and / or excretions, and / or cells derived therefrom. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the obtained cells are or include cells derived from the individual from which the sample is obtained. In some embodiments, the sample is a “primary sample” obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine-needle aspiration or tissue biopsy), surgical procedure, and collection of bodily fluids (e.g., blood, lymph, feces, etc.).

[0277] In some embodiments, cells in a sample are lysed to release nucleic acids. In some embodiments, cells in a sample are lysed within a composition or device provided herein. In some embodiments, cells in a sample are lysed by heating the sample. In some embodiments, cells in a sample are lysed by heating the sample to 80°C, 85°C, 90°C, or 95°C.

[0278] In some embodiments, the systems, methods, compositions, or devices provided herein involve amplification of a target nucleic acid. Those skilled in the art will recognize the various methods known in the art for amplifying nucleic acids. In some embodiments, the systems, methods, compositions, or devices provided herein involve isothermal amplification of nucleic acids. In some embodiments, isothermal amplification may be nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand-displacement amplification (SDA), helicase-dependent amplification (HDA), or nicking enzyme amplification (NEAR). In certain exemplary embodiments, non-isothermal amplification methods may be used, including but not limited to polymerase chain reaction (PCR), multiple substitution amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), or branched amplification (RAM). In some embodiments, isothermal amplification is LAMP, as described, for example, in Japanese Patents US9,909,168, US7,374,913, US7,851,186, and US7,846,695. In some embodiments, isothermal amplification is condensed LAMP (cLAMP), as described, for example, in US63 / 470,298 and US63 / 511,491. In some embodiments, isothermal amplification is performed at approximately 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, LAMP is performed at approximately 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, LAMP or cLAMP is performed at room temperature (e.g., room temperature).

[0279] Those skilled in the art are aware of various techniques useful for detecting one or more target nucleic acids. In some embodiments, the detection technique includes, for example, absorbance, CRISPR / Cas detection (e.g., SHERLOCK), FRET, or crosslinking ligation (e.g., INSPECTR as described in WO2020037038A1, the entire contents of which are incorporated herein by reference).

[0280] Certain CRISPR / Cas enzymes have been identified that, when activated by binding to a target site in a target nucleic acid recognized by the guide RNA to which the CRISPR / Cas enzyme is complexed, possess the ability to nonspecifically cleave collateral nucleic acids. Representative examples of Cas12, Cas13, and Cas14 have been shown to possess such collateral cleavage activity. For example, Swarts and Jinek,Mol.Cell.2019 Feb 7;73(3):589-600.e4, Harrington LBet al.,Science 2018;362:839-842, Li SYet al.Cell Res.2018;28:491-493, Chen JSet al.,Science 2018;360:436-439, Abudayyeh OOet al.,Science 2016;353aaf5573, East-Seletsky A.et al.,Nature 2016;538:270-273, Gootenberg JSet al.,Science 2017;356:438-442,Myhrvold C.,et al.,Science See 2018;360:444-448 and Gootenberg JSet al., Science 2018;360:439-444. The collateral cleavage activity of some CRISPR / Cas enzymes digests or cleaves single-stranded nucleic acids. The collateral cleavage activity of some CRISPR / Cas enzymes digests or cleaves double-stranded nucleic acids. The collateral cleavage activity of some CRISPR / Cas enzymes digests or cleaves RNA. The collateral cleavage activity of some CRISPR / Cas enzymes digests or cleaves DNA. The collateral cleavage activity of some CRISPR / Cas enzymes digests or cleaves both RNA and DNA. Collateral activity is utilized in the development of CRISPR / Cas detection (e.g., diagnostics) technologies to achieve the detection of nucleic acids (e.g., Cas target nucleic acids) or their complements containing relevant target sites in biological and / or environmental samples(s).For example, see Gootenberg, J.S. Set al., Science 2017, 356(438-442), WO2019 / 011022, U.S. Patent Nos. 10,494,664, 10,337,051, and 10,266,887, and sherlock.bio / better-faster-affordable-diagnostic-testing.

[0281] SHERLOCK is a detection technique comprising the steps of contacting a CRISPR / Cas complex containing a Cas protein having collateral cleavage activity, a guide RNA selected or manipulated to be complementary to a target nucleic acid (e.g., a Cas target nucleic acid sequence), and a sample potentially containing the Cas target nucleic acid (see, for example, WO2018 / 107129 and WO2019 / 011022, which are incorporated herein by reference). In some embodiments, the CRISPR / Cas-based detection may be a CRISPR / Cas13-based detection system. In some embodiments, the CRISPR / Cas-based detection system is a CRISPR / Cas12-based detection system. In some embodiments, the CRISPR / Cas13 or CRISPR / Cas12-based detection system is a SHERLOCK detection system. Those skilled in the art will recognize a variety of CRISPR / Cas enzymes that may be useful in the systems, compositions, and methods provided herein. For example, CRISPR / Cas enzymes are described in WO2016 / 166340, WO2016 / 205711, WO / 2016 / 205749, WO2016 / 205764, WO2017 / 070605, WO / 2017 / 189308, WO2021 / 154866A1, and WO2023 / 009526, the entire contents of each of these documents being incorporated herein by reference. In some embodiments, the SHERLOCK detection technique also includes a detectably labeled nucleic acid probe. Cleavage of the detectably labeled nucleic acid probe by the collateral cleavage activity of the CRISPR / Cas enzyme can induce or increase the detectably labeled area indicating the presence of the target nucleic acid. In some embodiments, the detectably labeled nucleic acid probe is labeled with a fluorescent label. In some embodiments, the fluorescent label includes a fluorescent group at the 5' end and a quenching group at the 3' end. In some embodiments, the fluorescent group is hexachlorofluorescein (HEX) or carboxyfluorocein (FAM). In some embodiments, the quenching group is a black hole quencher (BHQ).

[0282] In some embodiments, the detection of one or more nucleic acids involves obtaining a biological sample from a subject via a sample container (see, for example, Figures 1-5), incubating the biological sample via the sample container with at least one of a reagent and a buffer to produce a biological solution, inserting the sample container into a cartridge so that the biological solution flows into the cartridge's internal chamber, which includes a first heating zone, inserting the cartridge into an electronic reader equipped with multiple heating elements to create a first heating zone and a second heating zone within the cartridge, performing a dissolution step in the biological solution within the first heating zone, and passively cooling the biological solution by opening an internal passage in the cartridge so that the biological solution flows into the internal passage via gravity feed, which includes a plurality of reaction chambers located downstream of the fluid in the internal chamber and vertically below it. The method comprises amplifying one or more target nucleic acids in a biological solution by isothermal amplification in a second heating zone, each of a plurality of reaction chambers comprising a CRISPR / Cas enzyme having collateral cleavage activity, a guide RNA that specifically hybridizes with one target nucleic acid, and a detectably labeled nucleic acid probe, wherein hybridization of the guide RNA with the target nucleic acid induces or increases the collateral cleavage activity of the CRISPR / Cas enzyme, the CRISPR / Cas enzyme cleaves the detectably labeled nucleic acid probe, and the cleavage of the detectably labeled nucleic acid probe results in an increase in detectable labeling; irradiating the biological solution inside each of the plurality of reaction chambers via a plurality of light energy sources, each energy source of the plurality of light energy sources located near one of the plurality of reaction chambers; and determining the presence of at least one target nucleic acid in the biological solution based on the presence or level of detectable labeling via a detection device (Figure 28).

[0283] F. Method and Use Referring to Figure 37, in some embodiments, the present disclosure provides a method 1600 for detecting one or more target nucleic acids using a reader 14, a cartridge 12, a sample collector, or a combination thereof. In some embodiments, the method according to the present disclosure includes one or more of the following steps: i. To collect a sample, ii. Thermal dissolution of cells in the sample, iii. Passive sample cooling, iv. Transfer to one or more reaction chambers. v. Isothermal amplification, vi.Cas enzyme activation, and vii. Detection. For example, as shown in Figure 37 and as described herein, in step 1602, method 1600 may include preparing a biological sample (e.g., saliva, mucus, etc.) depending on the subject. In step 1604, method 1600 may include incubating the sample with a reagent / buffer. In step 1606, method 1600 may include thermal lysis of cells in the sample-reagent mixture. In step 1608, method 1600 may include passive cooling of the sample-reagent mixture following lysis (e.g., while the sample moves (i.e., flows) between the lysis chamber 94 and the reaction chamber 106). In step 1610, method 1600 may include isothermal amplification in the reaction chamber 106. In step 1612, method 1600 may include detection of a target nucleic acid.

[0284] The entire workflow for this test takes approximately 20 to 40 minutes, depending on the assay being performed. The user steps to initiate the test run take less than 2 minutes, and the rest of the test is performed automatically. Figure 3 shows an overview of the combined user and assay workflows.

[0285] In some embodiments, the sample is collected. In some embodiments, the user collects the sample from themselves using a standard sterile swab 16. Alternatively, an adult may collect a nasal swab sample from a child or other subject.

[0286] In some embodiments, the sample is eluted into a buffer. In some embodiments, the user opens the buffer tube and rotates the swab head to elute the sample into the buffer. Alternatively, a standard extraction tube can be used for elution.

[0287] In some embodiments, the sample is transferred to a cartridge. In some embodiments, the buffer containing the sample is provided through a sample inlet port. An exemplary transfer is shown in Figure 5. In some embodiments, the cartridge is sealed using a cap. An exemplary cap is shown in Figure 5. After the sample is added to the cartridge, the cartridge is inserted into the reader.

[0288] In some embodiments, the reader automatically provides fluid control, thermal control, and optical measurements of cartridge 12 within a test time of 15 to 45 minutes (e.g., 20 to 40 minutes). Once the test is complete, the results are displayed using an easy-to-read LED (e.g., the LED lights up in the case of a positive result). In some embodiments, cartridge 12 includes a label indicating the test type and assay and / or analysis results.

[0289] In some embodiments, the cartridge 12 is inserted into the reader 14. In some embodiments, the insertion of the cartridge 12 into the reader 14 initiates the detection method. An exemplary cartridge 12 mounting is shown in Figure 6.

[0290] In some embodiments, the reader 14 automatically detects the presence of the cartridge 12. In some embodiments, the reader 14 automatically detects the type of cartridge 12.

[0291] In some embodiments, the reader 14 communicates its status (e.g., power on / off, ready / running / completed, invalid result or error) by LEDs. In some embodiments, the LEDs are positioned adjacent to the corresponding label 290. In some embodiments, the label 290 is located on the case (i.e., the outer surface of the reader 14). An example of a status LED scheme is shown in Figure 7.

[0292] In some embodiments, the temperature is configurable via software. In some embodiments, the step time is configurable via software. In some embodiments, the execution sequence includes a heating cycle and an amplification cycle.

[0293] Lysis of cells in the sample In some embodiments, the method according to the present invention includes a step of heating the sample. In some embodiments, the sample is heated to lyse cells in the sample. In some embodiments, a heating cycle brings about cell lysis in the sample. In some embodiments, the sample is heated to at least 80°C, for example, at least 85°C, for example, 90°C. In some embodiments, the sample is heated in the lysis chamber 94 of the cartridge 12.

[0294] In some embodiments, the dissolution chamber 94 region of the cartridge (Figure 8) is heated by the reader 14 for a duration set by the assay sequence (e.g., up to 90°C). In some embodiments, the dissolution chamber 94 region is then passively cooled to below 60°C before the next step.

[0295] In some embodiments, the sample is transferred from the dissolution chamber 94 to the reaction chamber 106. In some embodiments, the sample is divided into individual reaction chambers 106. In some embodiments, the sample is transferred to one or more reaction chambers 106. In some embodiments, the sample is transferred to two or more reaction chambers 106. In some embodiments, the sample is transferred to three or more reaction chambers 106. In some embodiments, the sample is transferred to four or more reaction chambers 106. In some embodiments, the sample is transferred to five or more reaction chambers 106. In some embodiments, the sample is transferred to six or more reaction chambers 106. In some embodiments, the sample is transferred to seven or more reaction chambers 106. In some embodiments, the sample is transferred to eight or more reaction chambers 106. In some embodiments, the reaction chamber 106 may contain sample volumes in the range of about 10 μl to about 100 μl, for example, 15 μl to about 80 μl, for example, 20 μl to about 60 μl, for example, 30 μl to about 50 μl, for example, 40 μl. In some embodiments, sample transfer is driven by gravity flow (for example, when the reader 14 and cartridge 12 are positioned vertically). An exemplary vertical orientation is shown in Figure 9.

[0296] In some embodiments, the detection system 10 verifies the filling of the reaction chambers by using the rehydration of lyophilized beads in each reaction chamber 106. Chamber filling can be detected, for example, by a change in fluorescence that occurs when the lyophilized beads 268 are rehydrated.

[0297] Amplification cycle In some embodiments, the amplification cycle is performed in a temperature range of about 50°C to about 70°C, for example, about 55°C to about 65°C. In some embodiments, the amplification is performed under isothermal conditions.

[0298] In some embodiments, the leader 14 uses a film heater to heat the reaction chamber region 126 of the cartridge 12 to about 50°C to about 70°C, for example, about 55°C to about 65°C, for example, about 60°C, for the duration of amplification.

[0299] In some embodiments, the amplification is LAMP amplification.

[0300] detection In some embodiments, amplification products are detected. In some embodiments, LAMP amplification products are detected. In some embodiments, operating software detects whether amplification occurred in each reaction chamber. In some embodiments, detection is based on raw optical data. Figure 11 shows two examples of raw optical data plotted on a graph. The liquid SARS-CoV-2 sample (Figure 11A) shows amplification of 318 at approximately 15 minutes, while the negative control sample (Figure 11B) shows no amplification after 60 minutes.

[0301] In some embodiments, the raw data is plotted in a standardized format that also captures important metadata from the run, such as identification of the instrument (reader 14), sample type, and cartridge 12 type (see Figure 12), for analysis. The displayed data may include assay reaction results 318 for each of the eight channels, temperature profiles 320 for each of the two heating zones, filling detection signals 322 for each of the eight channels, and ambient light monitoring 324 for each of the eight channels.

[0302] As shown in Figure 11A, when the reaction chamber 106 is filled with fluid, less light from the LEDs reaches the photodetectors 144 and 162. Therefore, when the fluid enters the reaction chamber 106, the filling detection signal 322 may include a decrease in amplitude 330. Furthermore, as shown in Figure 11B, the filling detection signal 322 may increase in magnitude (332) due to the increase in reflectivity when the cartridge is inserted. The filling detection algorithm 230 verifies that a decrease in magnitude 330 is observed in each of the reaction chambers 106 (i.e., all eight), as shown in Figure 24B, thereby confirming that all reaction chambers 106 are filled with fluid. As shown in Legend 326 of Figure 12, each channel (i.e., reaction chamber 106) may hold or contain lyophilized beads corresponding to different indications (including, but not limited to, respiratory viruses such as SARS-CoV-2 (SCV2), FluA-1, FluA-2, FluB, and RSV (multinuclear respiratory virus), as well as sexually transmitted infections (STIs) including Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV), and other potential indications, infections, bacteria, and / or viruses). That is, each lyophilized bead may contain reagents, primers for nucleic acids, enzymes, etc., which are specifically selected to perform a particular assay to detect a particular indication. This arrangement, by having different lyophilized bead compositions in each reaction chamber 106, allows for the simultaneous performance of multiple assays corresponding to multiple viruses or conditions based on a single patient sample.

[0303] In some embodiments, the reader 14 displays the assay results. In some embodiments, the reader 14 displays the assay results via illuminated LEDs. In some embodiments, the illuminated LEDs correspond to labels 290 on cartridges 12. That is, each cartridge 12 may have a different printed label 290 corresponding to the state being tested in cartridge 12, so that the label is visible adjacent to the LEDs on the reader 14, so that the same LEDs on the reader 14 can indicate various different states depending on which cartridge 12 is inserted. In some embodiments, the target nucleotide acid is detected in the reaction chamber 106, resulting in a positive result. In some embodiments, an assay considered positive is shown. Exemplary indications are shown in Figure 2. If none of the assays are positive and the test run was valid (including the control reaction), the LED corresponding to "NEG" (i.e., indicating all assays are negative) illuminates, as shown in Figure 2B. [Examples]

[0304] Example 1: This embodiment demonstrates the detection of the SARS-CoV-2 virus using the detection system according to the present invention. Figure 11 shows two examples of raw optical data plotted on a graph. The vertical axis represents the optical signal at the photodetector in millivolts, and the horizontal axis represents the reaction time in minutes. The liquid sample containing SARS-CoV-2 (Figure 11A) shows amplification (i.e., increase in fluorescence) at approximately 15 minutes, while the negative control sample (Figure 11B) shows no amplification (i.e., no increase in fluorescence) after 60 minutes. The optical signals of the eight channels in Figures 11A and 11B correspond to signals measured by eight separate photodetectors, each corresponding to a different reaction chamber in a cartridge containing eight reaction chambers. In the examples shown here, each of the eight reaction chambers had the same detection assay (i.e., SARS-CoV-2), but in other examples, each reaction chamber may feature a different assay for detecting different target nucleic acids.

[0305] Equal portions Those skilled in the art will recognize numerous equivalents to the specific embodiments of the present invention described herein, or can verify them through routine experimentation alone. The scope of the present invention is not intended to be limited to the above description, but rather as described in the following claims.

Claims

1. A system for detecting the presence of nucleic acids associated with at least one indication in a biological solution, An electronic reader for performing one or more assays and displaying the results. A cartridge configured to be inserted into the reader, including a cartridge assembly, and The system comprises a sample collection container for collecting a biological sample and transferring it to an internal chamber of the cartridge.

2. When the cartridge is inserted into the reader, the internal assembly further comprises the cartridge assembly, and the internal assembly A heating unit comprising one or more heating elements, and The system according to claim 1, further comprising an optical assembly including at least one LED and at least one photodetector.

3. The system according to claim 2, wherein the cartridge assembly comprises a dissolution chamber for receiving the biological sample from the sample collection container, and one or more reaction chambers disposed on the fluid downstream side of the dissolution chamber.

4. The system according to claim 3, wherein the one or more heating elements include a first heating element for maintaining the melting chamber at a first temperature, and a second heating element for maintaining the one or more reaction chambers at a second temperature.

5. The aforementioned cartridge assembly At least one of the buffer and reagents placed in the dissolution chamber, At least one freeze-dried dissolution bead disposed within the dissolution chamber, and The system according to claim 3, further comprising freeze-dried PCR beads disposed inside each of the one or more reaction chambers.

6. The system according to claim 3, wherein each of the one or more reaction chambers is provided with a transparent dome.

7. The system according to claim 3, wherein the cartridge assembly comprises a polymer casing forming the rear surface of the cartridge and a film layer including the front surface of the cartridge, the polymer casing and the film layer each sandwiching a buffer, a reagent, at least one lyophilized lysis bead, and / or lyophilized PCR beads between them.

8. The system according to claim 7, wherein the film layer includes a polypropylene laminate.

9. The at least one LED includes a first LED that communicates optically with one or more reaction chambers, The system according to claim 1, wherein the at least one LED is configured to illuminate the inside of one or more reaction chambers.

10. The system according to claim 9, wherein the at least one LED includes a second LED that optically communicates with at least one identification marker disposed on the surface of the cartridge.

11. The system according to claim 9, wherein the at least one photodetector is configured to measure fluorescence emitted from the irradiated interior of one or more reaction chambers.

12. The system according to claim 1, wherein each of the at least one LED and the at least one photodetector is integrated into a printed circuit board assembly (PCBA).

13. The system according to claim 12, wherein the heating unit is positioned adjacent to the front of the cartridge, and the PCBA is positioned adjacent to the rear of the cartridge.

14. The system according to claim 12, wherein the heating unit is integrated into the PCBA.

15. The one or more reaction chambers include a plurality of reaction chambers, The system according to claim 5, wherein the type of lyophilized PCR beads in the first reaction chamber of the plurality of reaction chambers is different from the type of lyophilized PCR beads in the second reaction chamber of the plurality of reaction chambers.

16. The system according to claim 5, wherein the one or more reaction chambers comprises a plurality of reaction chambers, each of which comprises different types of lyophilized PCR beads configured for use in different reactions.

17. The system according to claim 5, wherein the one or more reaction chambers comprises a plurality of reaction chambers, each of which comprises the same type of lyophilized PCR beads configured for use in the same reaction.

18. The system according to claim 3, wherein light emitted from at least one LED excites at least one nucleic acid contained in one or more reaction chambers without passing through an optical lens.

19. The mechanical assembly further comprises a mechanical assembly configured to move the cartridge laterally within the reader after the cartridge has been inserted into the reader and the lid of the reader has been closed, and the mechanical assembly further comprises a mechanical assembly, At least one link mechanism connecting the lid to an internal device located within the leader, and The system according to claim 2, comprising a cam coupled to both the at least one link mechanism and the internal device, the cam converting the closing motion of the lid into a lateral motion of the internal device.

20. The system according to claim 1, wherein the cartridge comprises at least one identification mark configured to be illuminated by the at least one LED.

21. The system according to claim 20, wherein the at least one identification mark includes at least one printed barcode.

22. The system according to claim 20, wherein the at least one identification mark includes one or more printed shapes.

23. The system according to claim 20, wherein the at least one identification mark is printed with ink (for example, black ink, colored ink, or a combination of inks).

24. The system according to claim 20, wherein the at least one identification mark is associated with the at least one indication.

25. A method for identifying the attributes of a cartridge configured to be inserted into an electronic reader, The electronic reader is provided such that the electronic reader comprises at least one internal LED, at least one internal photodetector, and at least one internal microprocessor that is communicatively coupled to both the at least one photodetector and the at least one LED. The provision of the cartridge, wherein the cartridge is provided with at least one identification mark, Inserting the aforementioned cartridge into the reader, The at least one internal LED illuminates the at least one identification mark, thereby generating the illuminated identification mark. The optical signature of the irradiated identification mark is detected by the at least one internal photodetector, The method comprising using the at least one internal microprocessor to identify at least one attribute of the cartridge based on the illuminated identification mark.

26. The method according to claim 25, wherein the illumination of the at least one identification mark is performed when the cartridge is inserted into the electronic reader.

27. The method according to claim 26, wherein the at least one identification mark includes at least one barcode.

28. The aforementioned at least one internal LED includes at least a first LED and a second LED, The aforementioned at least one barcode, A first barcode used to quantify the speed at which the cartridge is inserted into the electronic reader, the first barcode being illuminated by the first LED, and A second barcode used to identify the at least one attribute of the cartridge, the second barcode being illuminated by the second LED, The method according to claim 27, wherein the at least one microprocessor uses the quantified speed to calibrate the optical signature of the second barcode, thereby enabling the attributes of the cartridge to be identified.

29. The method according to claim 25, wherein the at least one internal photodetector includes at least one of a photodiode and a phototransistor.

30. The method according to claim 25, wherein the at least one internal LED includes at least one of a red LED and a blue LED.

31. The at least one internal photodetector includes a dual-mode photodetector configured to measure light emitted from at least one LED in both a first spectrum and a second spectrum, The method according to claim 25, wherein the second spectrum does not overlap with the first spectrum.

32. The first spectrum includes wavelengths in the range of approximately 600 nm to approximately 660 nm. The method according to claim 31, wherein the second spectrum includes wavelengths in the range of about 730 nm to about 900 nm.

33. The method according to claim 25, wherein the at least one identification mark includes one or more printed marks on a label placed on the surface of the cartridge.

34. The aforementioned at least one LED includes approximately 4 to approximately 12 identification LEDs. The method according to claim 33, wherein the one or more print marks comprise approximately 4 to approximately 12 print marks, and each print mark is positioned to correspond to one position of the identification LED, so that the presence or absence of a print mark at a certain position can be detected by the identification LED at the corresponding position.

35. The method according to claim 34, wherein the number of identification LEDs is greater than the number of printed marks.

36. The method according to claim 34, wherein a unique combination of marked positions on the label is associated with the attribute of the cartridge.

37. The method according to claim 25, wherein the attribute includes one or more types of assays that can be performed using the cartridge.

38. The method according to claim 25, wherein the at least one internal microprocessor executes one or more preloaded routines based on attributes determined as a result of a method for identifying the attributes of a cartridge.

39. The method according to claim 33, wherein the one or more printed marks include one or more position detection feature units.

40. The one or more position detection feature units, A first position detection feature unit, which is positioned on the label such that when the cartridge is correctly positioned within the reader, the center line of the first position detection feature unit is positioned slightly above the center line of the first component of the reader, and The second position detection feature unit is positioned on the label such that, when the cartridge is correctly positioned within the reader, the center line of the second position detection feature unit is positioned slightly below the center line of the second component of the reader. The above method is performed by the microprocessor, 1) The position of the first position detection feature portion relative to the first component of the reader, and 2) Determining whether the cartridge is correctly positioned within the reader or not, based on the position of the second position detection feature portion relative to the second component of the reader, The method according to claim 39, wherein each of the first component and the second component includes at least one of the at least one internal photodetector and the at least one LED.

41. The reader is configured to identify the attributes of the cartridge based on a plurality of operating modes, and the plurality of operating modes are Static identification including the identification of the attribute via the reader and the at least one identification mark after the cartridge has been inserted into the reader, and The method according to claim 25, comprising dynamic identification including the identification of the at least one identification mark and the attribute via the reader while the cartridge is inserted into the reader.

42. The method according to claim 33, wherein one or more printed marks are printed in ink (e.g., colored ink, black ink, and / or other color combinations) on the label on a background which is at least one of white and gray.

43. The method according to claim 25, wherein the cartridge comprises at least one freeze-dried bead disposed therein, and the at least one freeze-dried bead comprises at least one of freeze-dried soluble beads and freeze-dried PCR beads.

44. A system comprising the reader and cartridge described in claim 25.

45. Obtaining (or preparing) a biological sample, The biological sample is incubated with at least one of a reagent and a buffer solution to produce a biological solution. The dissolution step is performed on the aforementioned biological solution, The process involves amplifying one or more target nucleic acids in the biological solution by isothermal amplification, The aforementioned biological solution, CRISPR / Cas enzyme having collateral cleavage activity, One or more guide RNAs, each specifically hybridizing with one or more of the aforementioned target nucleic acids, and Incubating with a composition containing a detectably labeled nucleic acid probe, The hybridization of each of the one or more guide RNAs with each target nucleic acid induces or increases the collateral cleavage activity of the CRISPR / Cas enzyme, and the CRISPR / Cas enzyme cleaves the detectably labeled nucleic acid probe. The cleavage of the detectably labeled nucleic acid probe results in an increase in the detectably labeled substance, and the incubation process is performed. A method comprising determining the presence of the target nucleic acid in the biological sample based on detecting an increase in the detectable label.

46. The dissolution step includes a thermal dissolution step carried out at a temperature in the range of approximately 70°C to approximately 95°C. The method according to claim 45, wherein the thermal melting step is performed in a first heating zone.

47. The method according to claim 45, further comprising passively cooling the biological solution after the dissolution step.

48. Passively cooling the biological solution includes flowing the biological solution through the internal passages of the cartridge. The aforementioned internal passage is oriented vertically, The method according to claim 45, wherein the flow of the biological solution through the internal passage includes gravity flow.

49. The isothermal amplification step includes amplifying the biological solution at a temperature in the range of about 50°C to about 70°C. The method according to claim 45, wherein the isothermal amplification step is performed in a second heating zone.

50. The method according to claim 49, wherein the isothermal amplification step includes loop-mediated isothermal amplification (LAMP).

51. The method according to claim 45, wherein the detectably labeled nucleic acid probe is labeled with a fluorescent label to form a fluorescently labeled nucleic acid probe.

52. The method according to claim 51, wherein the fluorescently labeled nucleic acid probe contains a fluorescent group at its 5' end and a quenching group at its 3' end.

53. Determining that the target nucleic acid is present in the biological sample is The aforementioned biological solution is irradiated with light, The method according to claim 52, comprising detecting at least one fluorescent signature using at least one of a photodiode and a phototransistor, wherein the at least one fluorescent signature indicates the presence of at least one target nucleic acid.

54. The method according to claim 53, wherein the irradiation of the biological solution with light includes irradiating the biological solution with light using a light-emitting diode (LED) that emits light in the range of about 430 nm to about 500 nm.

55. The method according to claim 53, wherein detecting at least one fluorescent signature includes detecting the at least one fluorescent signature without amplifying the at least one fluorescent signature.

56. The method according to claim 45, wherein the target nucleic acid is from a eukaryotic cell or a prokaryotic cell.

57. The method according to claim 56, wherein the target nucleic acid is from a protozoan, a bacterium, a virus, and / or a fungus.

58. The method according to claim 57, wherein the target nucleic acid is derived from Chlamydia trachomatis, Neisseria gonorrhoeae, influenza A, influenza B, SARS-CoV-2, polynuclear respiratory virus (RSV), or Trichomonas virginalis.

59. The method according to claim 53, wherein detecting at least one fluorescent signature includes passing the at least one fluorescent signature through a gel filter.

60. A method for detecting the presence of at least one target nucleic acid in a biological sample, Obtaining the biological sample from the subject, wherein the biological sample is placed in a sample container, The biological sample is incubated with at least one of the reagents and buffers in the sample container, thereby generating a biological solution in the sample container. The insertion of the sample container into the cartridge, thereby causing the biological solution to flow into the internal chamber of the cartridge, the internal chamber including a first heating zone, To heat the first heating zone and the second heating zone within the cartridge, the cartridge is inserted into an electronic reader equipped with a plurality of heating elements. The dissolution step is performed in the first heating zone, The passive cooling of the biological solution by opening the internal passage of the cartridge, thereby allowing the biological solution to flow into the internal passage via gravity, and the internal passage being located downstream and vertically below the fluid in the internal chamber, The method involves amplifying at least one target nucleic acid in the biological solution by isothermal amplification in the second heating zone, which includes a plurality of reaction chambers located downstream of the fluid in the internal passage, Each of the aforementioned plurality of reaction chambers, CRISPR / Cas enzyme having collateral cleavage activity, At least one guide RNA that specifically hybridizes with the at least one target nucleic acid, and Equipped with a detectably labeled nucleic acid probe, Hybridization of the guide RNA with the target nucleic acid induces or increases the collateral cleavage activity of the CRISPR / Cas enzyme, and the CRISPR / Cas enzyme cleaves the detectably labeled nucleic acid probe. The cleavage of the detectably labeled nucleic acid probe results in an increase in the detectable label, and the amplification is performed. Irradiating the biological solution inside each of the plurality of reaction chambers via a plurality of light energy sources, wherein each of the plurality of light energy sources is located near one of the plurality of reaction chambers, The method comprising determining the presence of at least one target nucleic acid in the biological solution based on the presence or level of the detectable label via a detection device.

61. A system for performing nucleic acid diagnostic tests, An electronic device capable of receiving a consumable cartridge, and The system comprises a consumable cartridge configured to be attached to the electronic device and containing reagents used for nucleic acid diagnostic testing.

62. The system according to claim 61, wherein the diagnostic test uses one or more reagents for detecting a target nucleic acid sequence using CRISPR / Cas detection.

63. The system according to claim 61, wherein two or more separate amplification reactions occur within the consumable cartridge.

64. The system according to claim 61, wherein eight separate amplification reactions occur within the consumable cartridge.

65. The system according to claim 61, wherein fluorescence detection is used to measure molecular amplification.

66. The device according to claim 65, wherein photoexcitation is used to excite a sample and generate a fluorescence signal used in the fluorescence detection.

67. The device according to claim 65, wherein optical filtration is used to assist fluorescence detection.

68. The device according to claim 61, wherein the heat treatment of the sample is performed in the consumable cartridge.

69. The device according to claim 68, wherein the thermal dissolution of the sample is performed within the consumable cartridge.

70. The device according to claim 61, wherein gravity is used for the fluid motion within the consumable cartridge.

71. The device according to claim 61, wherein at least one result is displayed as a combination of 1) an illuminated indicator on the electronic device and 2) graphics on the consumable cartridge.

72. The device according to claim 61, wherein the electronic device is configured to receive a plurality of types of consumable cartridges and to perform assays associated with each of the plurality of types of consumable cartridges.

73. The device according to claim 72, wherein the electronic device automatically detects the configuration of the consumable cartridge.

74. The device according to claim 61, wherein the electronic device determines whether the reaction chamber in the consumable cartridge contains a reagent using photoexcitation and detection.

75. The device according to claim 74, wherein the determination is performed in a continuous manner.

76. The device according to claim 74, wherein the determination is performed in less than one second.

77. The device according to claim 61, wherein the electronic device determines whether the reaction chamber in the consumable cartridge contains liquid using photoexcitation and detection.

78. The device according to claim 77, wherein the liquid includes a gas.

79. The device according to claim 77, wherein the liquid contains a sample to be tested.

80. The device according to claim 77, wherein the determination is performed in a continuous manner.

81. The device according to claim 77, wherein the determination is made in less than one second.

82. The device according to claim 61, wherein the electronic device determines whether the reaction chamber contains a gas using photoexcitation and detection.

83. An electronic device for performing nucleic acid diagnostic testing in combination with a cartridge, The mechanical subsystem to which the cartridge is attached and positioned, An electronic subsystem executes a test sequence based on pre-programmed parameters and unique parameters based on the type of cartridge installed. A thermal subsystem that heats the two reaction zones within the cartridge, An optical subsystem for exciting, filtering, and detecting fluorescence in real time, and The electronic device comprises a microfluidic control subsystem that operates a feature portion on the cartridge to control the flow of fluid within the cartridge.

84. The device according to claim 83, wherein the mechanical subsystem supports each of the electronic subsystem, the thermal subsystem, the optical subsystem, and the microfluidic control subsystem.

85. A microfluidic cartridge for performing nucleic acid diagnostic testing in combination with an electronic device, An outer casing that interfaces with the electronic device when the cartridge is attached to the electronic device, The reagents housed within the outer casing, A fluid valve operated by the aforementioned electronic device, A filter element that allows air to pass through the outer casing and holds liquid, and The microfluidic cartridge comprises a visual display that transmits identification information related to the reagent to the electronic device by at least one of absorbance and reflectance at a predetermined position.

86. A diagnostic cartridge identification method, This includes providing a device for identifying the cartridge, and the device is Diagnostic cartridge identification method, comprising: an optical module for measuring an optical signature in a first spectrum, the optical module for measuring separate optical targets in the first spectrum to identify a cartridge type.

87. The method according to claim 86, wherein the optical module is configured to measure fluorescence of a second spectrum, the second spectrum being different from the first spectrum, and for detecting the presence of at least one nucleic acid from a predetermined group of nucleic acids in the cartridge, the predetermined group comprising nucleic acids each associated with one or more indications.

88. The method according to claim 86, wherein the nucleic acid includes a human sample.

89. The method according to claim 86, wherein the cartridge comprises one or more reagents for CRISPR / Cas detection.

90. The method according to claim 86, wherein the cartridge comprises an identification label including one or more optical targets.

91. The method according to claim 86, wherein the cartridge comprises at least one printed barcode label.

92. The method according to claim 86, wherein the optical target comprises a fluorescent ink having specific spectral characteristics.

93. The method according to claim 86, wherein the apparatus comprises a dual-mode photodetector configured to measure both the optical signature in the first spectrum and the fluorescence in the second spectrum, and two LEDs that emit light in two different spectra.

94. The method according to claim 93, wherein the LED is computer-controlled.

95. The method according to claim 91, wherein the at least one printed barcode is a printing ink (for example, black ink, color ink, or a combination of inks).

96. Inserting the aforementioned cartridge into the aforementioned device, The identification label is illuminated by one of the two LEDs, To detect the optical signature associated with the optical signature, The method according to claim 93, further comprising identifying the cartridge type based on the detected optical signature.

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