Rotatable disks, systems, and methods for sample analysis

Abstract

This invention describes a method and device that enables simultaneous sample processing of multiple targets, rapid polymerase chain reaction amplification, and real-time readings while using reaction volumes compatible with molecular assays. The invention can be part of a cartridge or used as a standalone device, enabling a sample to respond to a nucleic acid analysis system (NAAT). In one embodiment, this disclosure provides a clinical practice method and system for performing rapid PCR on a biological sample, e.g., a patient fluid.

Description

[Background technology]

[0001] (Cross-reference of related applications) This application claims the benefit of U.S. Provisional Application No. 63 / 453,724, filed on 21 March 2023, which is incorporated herein by reference.

[0002] Nucleic acid amplification for pathogen detection and tumor analysis is becoming the new optimal standard, replacing other techniques, such as cell culture. With advances in microfluidics, some of these systems are moving from laboratories regulated by the Clinical Laboratory Improvement Amendment (CLIA) to CLIA-exempt facilities closer to patients. Reducing the total turn-to-analysis time (TAT) of these devices is paramount, for example, to deliver results within an average waiting time of less than 15 minutes in a physician's office.

[0003] In parallel, another trend in nucleic acid amplification assay (NAAT) systems provides a holistic view of the causes of specific syndromes, helping physicians provide more accurate diagnoses. These syndrome panel assays typically look for 20 or more nucleic acid targets simultaneously. Current high-speed NAAT systems available in CLIA-exempt facilities can only perform single or low-multiplex nucleic acid analyses and therefore cannot be used for syndrome panels.

[0004] Several challenges need to be addressed to bring a system to market that enables 20 or more multiplexing in less than 15 minutes. First, nucleic acid amplification is preferably performed in 10 minutes or less, using a volume that has the sensitivity and suitability required for diagnostics. Second, the phosphor reader is capable of reading 20 or more targets simultaneously, preferably without delaying the amplification time. Finally, the high-speed amplification process is preferably compatible with a fluid cartridge designed to automate sample preparation.

[0005] Isothermal nucleic acid amplification methods such as EXPAR or RPA can provide NAAT within 10 minutes. However, these techniques cannot be easily multiplexed and lack polymerase chain reaction (PCR) sensitivity and robustness.

[0006] PCR is a robust NAAT amplification method that requires thermal cycling to achieve amplification. The overall reaction time is linked to the time required for the PCR reagents to change temperature. Historically, poor heat transfer and unoptimized chemical properties made PCR a slow process. However, with advances in faster polymerases, mass production of DNA primers, and faster thermal cycling, PCR amplification in under 10 minutes has been achieved. Still, however, the integration of such fast PCR with a sample processing / analysis response integrated system that would allow for reading 20 or more multiplexes remains lacking. [Overview of the Initiative] [Means for solving the problem]

[0007] In one embodiment, the Disclosure provides a clinical practice method and system for performing rapid PCR on a biological sample, such as a patient fluid. In one embodiment, the system and method of the Disclosure simultaneously and rapidly detect multiple target nucleic acid sequences in a biological sample.

[0008] In one aspect, the present disclosure provides an ultrafast real-time polymerase chain reaction (PCR) method for detecting the presence or absence of a target nucleic acid in a biological sample. In some embodiments, the method includes the steps of (a) loading a sample into a loading chamber on a rotatable disk, the rotatable disk further comprising a plurality of reaction chambers and channels that fluidly connect the loading chamber to the plurality of reaction chambers, the reaction chambers being loaded with a PCR reagent mixture comprising primers and a fluorescent probe, the sample flowing through the channels into the plurality of reaction chambers, thereby filling the reaction chambers following the loading of the sample; (b) bringing the channels in the rotatable disk into contact with a sealer, thereby sealing the channels after filling and preventing fluid communication between the plurality of reaction chambers; and (c) rotating the rotatable disk, the plurality of reaction chambers being maintained at a first temperature. The process includes (c) placing the reaction chambers adjacent to a heating element, thereby denaturing the target nucleic acid in the sample, if present, thereby producing a denatured target nucleic acid; (d) rotating a rotatable disk to place the plurality of reaction chambers adjacent to a second heating element maintained at a second temperature, thereby annealing the primers to the denatured target nucleic acid and replicating the denatured target nucleic acid; (e) exposing the plurality of reaction chambers to excitation light of a first wavelength, thereby exciting a phosphor probe; and (f) repeating steps (c)-(e) over a plurality of cycles, measuring the emission light of a second wavelength from the plurality of reaction chambers, wherein if emission light of a second wavelength is detected, the sample comprises the target nucleic acid. In some embodiments, the primers comprise an oligonucleotide sequence complementary to at least a portion of the target nucleic acid. In some embodiments, the phosphor probes are fluoroceine amidite (FAM), SUN TMThe sealer comprises a phosphor selected from the group consisting of TEX615 and cyanine-5 (Cy5). In some embodiments, the sealer is housed within an analytical device for real-time PCR. In some embodiments, the sealer is a thermal sealer. In some embodiments, the channel defines a lumen with a diameter ranging from about 1 μm to about 1 mm. In some embodiments, the channel comprises a thermoplastic material that seals the channel when heated to a target temperature and compressed by the sealer. In some embodiments, the target temperature is about 110°C to about 300°C. In some embodiments, the sealer comprises a first element configured to provide thermal energy and pressure onto a rotatable disk, and a second element configured to provide a reaction force to the pressure. In some embodiments, the step of bringing the rotatable disk and the sealer into contact includes the step of applying a pressure opposite to the reaction force at one or more locations along the channel on the rotatable disk such that the channel deforms and creates multiple reaction chambers. In some embodiments, the excitation light or emitted light comprises two or more wavelengths. In some embodiments, the first and second wavelengths are independently selected from about 400 nm to about 750 nm. In some embodiments, the sample is mixed with primers that are complementary to at least a portion of the target nucleic acid, which are pre-loaded into a plurality of reaction chambers. In some embodiments, the plurality of reaction chambers further include probes, and the sample is further mixed with the probes.

[0009] In one aspect, the present disclosure provides a method for multiplexed real-time amplification and detection of multiple different target nucleic acids in a biological sample, comprising: (a) loading a sample into a loading chamber on a rotatable disk, the rotatable disk further comprising a plurality of reaction chambers and channels that fluidly connect the loading chamber to the plurality of reaction chambers, the reaction chambers being loaded with a PCR reagent mixture comprising a plurality of primers and a plurality of phosphor probes, the sample being mixed with the plurality of primers, each of the plurality of primers being complementary to a corresponding portion of the plurality of different target nucleic acids, the sample flowing through the channels into the plurality of reaction chambers, thereby filling the reaction chambers following the loading of the sample; (b) bringing the channels in the rotatable disk into contact with a sealer, thereby sealing the channels after filling and preventing fluid communication between the plurality of reaction chambers; and (c) rotating the rotatable disk to move the plurality of reaction chambers The present invention provides a method comprising the steps of: (d) placing a sample adjacent to a first heating element maintained at a first temperature, thereby denaturing each of several target nucleic acids in the sample, if present, thereby producing several denatured target nucleic acids; (e) rotating a rotatable disk to place several chambers adjacent to a second heating element maintained at a second temperature, thereby annealing several primers to the corresponding several denatured target nucleic acids and replicating several different target nucleic acids; (f) exposing several reaction chambers to excitation light comprising several excitation wavelengths, thereby exciting several phosphor probes; and (g) repeating steps (c)-(e) over several cycles, measuring several emitted light of several emission wavelengths from several reaction chambers, each of which emission wavelengths corresponds to the presence of each of several different target nucleic acids, and if emitted light of an emission wavelength is detected, the sample comprises the corresponding target nucleic acid. In some embodiments, the sealer is a thermal sealer. In some embodiments, the sealer is housed within an analytical device for multiplexed real-time amplification. In some embodiments, the channel defines a lumen with a diameter ranging from approximately 1 μm to approximately 1 mm.In some embodiments, multiple excitation wavelengths and multiple emission wavelengths are each independently selected from approximately 400 nm to approximately 750 nm. In some embodiments, multiple primers are stored in multiple reaction chambers. In some embodiments, multiple primers are provided as lyophilized powders. In some embodiments, the sample comprises a body sample selected from the group consisting of blood samples, tear samples, saliva samples, mucus samples, sputum samples, fecal samples, cerebrospinal fluid samples, and urine samples. In some embodiments, the phosphorescent probes in the multiple phosphorescent probes are fluoroceine amidite (FAM), SUN. TMThe system comprises a phosphor selected from the group consisting of TEX615 and cyanine-5 (Cy5). In some embodiments, the first, second, or third heating element comprises a heating block. In some embodiments, the probe is provided as a lyophilized powder. In some embodiments, the rotatable disk comprises a rigid plastic. In some embodiments, the components of the PCR reagent mixture are pre-mixed before contacting the sample or loading into the multiple reaction chambers. In some embodiments, the PCR reagent mixture is provided in a reagent mixing chamber, and the sample is mixed with a second PCR reagent mixture in the reagent mixing chamber. In some embodiments, the PCR reagent mixture is provided as a lyophilized powder. In some embodiments, the sample is mixed with the PCR reagent mixture prior to being transferred into the multiple reaction chambers in (a). In some embodiments, each of the multiple reaction chambers is aligned at the same radial distance from the center of the rotatable disk and equally spaced apart from one another. In some embodiments, the multiple reaction chambers occupy a 360-degree arc on the rotatable disk. In some embodiments, the reaction chambers occupy a section of arc on a rotatable disk ranging from approximately 10 to 100 degrees. In some embodiments, each of the reaction chambers has a volume of approximately 5 μL to approximately 100 μL and a depth of approximately 0.1 mm to approximately 1.0 mm. In some embodiments, the reaction chambers have a depth of approximately 0.1 mm to approximately 0.7 mm. In some embodiments, the reaction chambers have a maximum depth of approximately 0.25 mm. In some embodiments, a first temperature is maintained over a first period, and a second temperature is maintained over a second period. In some embodiments, the first period is selected from approximately 500 milliseconds to approximately 2 seconds, and the second period is selected from approximately 3 seconds to approximately 18 seconds. In some embodiments, the first temperature is selected from approximately 90°C to approximately 99°C, and the second temperature is selected from approximately 50°C to approximately 74°C. In some embodiments, the first heating element has a first radial length, and the second heating element has a second radial length. In some embodiments, the second radial length is about 6 to about 9 times the first radial length.In some embodiments, the third temperature is selected from about 65°C to about 75°C. In some embodiments, the third temperature is maintained for a third period. In some embodiments, the third period is selected from about 1 second to about 6 seconds. In some embodiments, the third heating element has a third radial length. In some embodiments, the third radial length is about 2 to about 3 times the first radial length. In some embodiments, the emitted light is measured on a first fluorescence detector or a plurality of fluorescence detectors. In some embodiments, the first detector or a plurality of fluorescence detectors has one or more wavelength channels for measuring the emission intensity of a second wavelength or a plurality of emission wavelengths.

[0010] In one aspect, the present disclosure provides a rotatable disk comprising a loading chamber, a plurality of reaction chambers, and channels connecting the loading chamber to the plurality of reaction chambers, wherein the channels are made of a thermoplastic material that seals the channels when heated to a temperature of 110 to 300°C. In some embodiments, the plurality of reaction chambers are each aligned at the same radial distance from the center of the rotatable disk. In some embodiments, each of the plurality of reaction chambers contains a first PCR reaction mixture comprising primers and a phosphor probe. In some embodiments, the loading chamber contains a second PCR reaction mixture comprising polymerase and a plurality of nucleotides.

[0011] In one aspect, the present disclosure provides a rotatable disk comprising a loading chamber, a plurality of reaction chambers, each aligned at substantially the same radial distance from the center of the rotatable disk, and each of the plurality of reaction chambers comprising a first PCR reaction mixture comprising primers, and a channel connecting the loading chamber to the plurality of reaction chambers. In some embodiments, the loading chamber comprises a second PCR reaction mixture comprising polymerase and a plurality of nucleotides. In some embodiments, the channel comprises a thermoplastic material that seals the channel when heated to a temperature of 110 to 300°C. In some embodiments, each of the plurality of reaction chambers comprises a volume of about 5 μL to about 100 μL. In some embodiments, each of the plurality of reaction chambers comprises a depth of about 0.1 mm to about 1.0 mm. In some embodiments, each of the plurality of reaction chambers comprises a depth of about 0.2 to about 0.7 mm. In some embodiments, each of the plurality of reaction chambers comprises a maximum depth of 0.25 mm. In some embodiments, each of the plurality of reaction chambers is equally spaced apart from one another. In some embodiments, the multiple reaction chambers occupy a 360-degree arc on a rotatable disk. In some embodiments, the multiple reaction chambers occupy a segment of an arc of about 10 to 100 degrees on a rotatable disk. In some embodiments, the multiple reaction chambers comprise 1 to 100 reaction chambers. In some embodiments, the thermoplastic material is selected from polycarbonate, polypropylene, polyethylene terephthalate, and cyclic olefin copolymers. In some embodiments, the thermoplastic material has a thickness of about 10 μm to about 400 μm. In some embodiments, the channel has a z dimension of about 1 μm to about 1 mm, a width of about 1 mm to about 5 mm, and a depth of up to about 200 μm. In some embodiments, the channel has a width of about 2 mm to about 4 mm and a depth of up to about 100 μm. In some embodiments, the aspect ratio is at least 10:1. In some embodiments, the aspect ratio is at least 20:1. In some embodiments, each of the multiple reaction chambers has an interior and an exterior.In some embodiments, each of the multiple reaction chambers is adapted to transmit excitation light of multiple wavelengths from outside the multiple reaction chambers, and the multiple reaction chambers are adapted to transmit emitted light of multiple wavelengths from inside the multiple reaction chambers. In some embodiments, the rotatable disk comprises a thermoformed film and a sealing film that are coupled together to form the multiple reaction chambers. In some embodiments, the thermoformed film forms the shape and size for the multiple reaction chambers and the shape and size for the channels. In some embodiments, the sealing film is sealed to the thermoformed film. In some embodiments, the sealing film is sealed to the thermoformed film via a framework, along with two surfaces co-extruded with a heat-sealed compliant material. In some embodiments, the thermoformed film has a thickness of about 50 μm to about 500 μm. In some embodiments, the ratio of the channel width to the thickness of the thermoformed film is greater than 2. In some embodiments, the sealing film has a thickness of about 10 μm to about 500 μm. In some embodiments, the thermoformed film has a glass transition temperature (Tg) higher than about 100°C. In some embodiments, the sealing film and the thermoformed film comprise a resin. In some embodiments, the resin comprises a single polymer. In some embodiments, the resin comprises an inner polymer and an outer polymer, the inner polymer having a lower Tg than the outer polymer. In some embodiments, the single polymer, the inner polymer, and the outer polymer are each independently selected from polyolefins, polycarbonates, polystyrene, polymethylmethylacrylates, polyethylene, and polypropylene. In some embodiments, the resin for the sealing film and the resin for the thermoformed film can be heat-sealed together. In some embodiments, the resin for the sealing film and the thermoformed film are identical.

[0012] In one aspect, the present disclosure provides a rotatable disk comprising a loading chamber, a plurality of reaction chambers, each aligned at the same radial distance from the center of the rotatable disk, and a channel connecting the loading chamber to the plurality of reaction chambers, wherein the channel comprises a thermoformed thermoplastic film sealed to a sealing thermoplastic film, and the channel has a depth of 10 micrometers to 500 micrometers.

[0013] In one aspect, the present disclosure provides a rotatable disk comprising a loading chamber, a plurality of reaction chambers, each aligned at the same radial distance from the center of the rotatable disk, and a channel connecting the loading chamber to the plurality of reaction chambers, the channel being heat-sealed by compressing a thermoformed film on the ceiling of the channel into contact with a sealing film on the floor of the channel.

[0014] In one aspect, the present disclosure provides a rotatable disk comprising a loading chamber, a plurality of reaction chambers, each aligned at the same radial distance from the center of the rotatable disk, and a channel connecting the loading chamber to the plurality of reaction chambers, wherein the channel comprises a co-extruded thermoplastic film having an inner thermoplastic film layer having a lower Tg than an outer thermoplastic film layer, the inner layer being configured to seal the channel when heated to a temperature of 110° to 300° Celsius.

[0015] In one aspect, the present disclosure provides a rotatable disk comprising: a loading chamber configured to seal a sample under high pressure to a temperature above 100°C; a plurality of reaction chambers, each aligned at the same radial distance from the center of the rotatable disk; and a channel connecting the loading chamber to the plurality of reaction chambers. In some embodiments, the loading chamber is configured to seal a sample under high pressure to a temperature between 101°C and 160°C. In some embodiments, the channel is heat-sealed by compressing a thermoformed film on the ceiling of the channel into contact with a sealing film on the floor of the channel. In some embodiments, the loading chamber is configured to be heated by induction heating. In some embodiments, the loading chamber comprises a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent. In some embodiments, the loading chamber further comprises a stabilizer.

[0016] In one aspect, the present disclosure is a system for a real-time polymerase chain reaction (PCR), comprising: (a) a rotatable disk comprising a loading chamber, a plurality of reaction chambers, and channels connecting the loading chamber to the plurality of reaction chambers; (b) a sample holder for holding the rotatable disk in a substantially horizontal plane, the sample holder configured to rotate the rotatable disk in a substantially horizontal plane; (c) a sealer coupled to an actuator that moves the sealer, thereby changing the distance between the sealer and the rotatable disk; (d) a first heating element located in close proximity to a second portion of the rotatable disk and maintained at a first temperature; (e) a second heating element located in close proximity to a second portion of the rotatable disk and maintained at a second temperature; and (f) a light source oriented to generate excitation light of a first wavelength in a horizontal plane occupied by the reaction chambers of the rotatable disk. The system provides a rotatable disk comprising (g) a first photodetector oriented to detect emitted light of a second wavelength emitted from the reaction chambers of the rotatable disk, wherein when a liquid sample comprising nucleic acid flows from a loading chamber through channels into a plurality of reaction chambers, the rotatable disk is brought into contact with a thermal sealer, thereby sealing the channels and preventing fluid communication between the plurality of reaction chambers, and the rotatable disk is rotated in a substantially horizontal plane, thereby (i) positioning the plurality of reaction chambers adjacent to a first heating element, thereby denaturing the nucleic acid in the sample, (ii) positioning the plurality of reaction chambers adjacent to a second heating element, thereby annealing primers to the denatured nucleic acid and replicating the nucleic acid, and (iii) oriented the rotatable disk so that excitation light generated by a light source flows into the reaction chambers, thereby generating emitted light emitted from the reaction chambers and detected by the photodetector. In some embodiments, the liquid sample from the loading chamber is mixed in a mixing chamber with a PCR reagent mixture comprising polymerase and multiple nucleotides, prior to flowing through channels into multiple reaction chambers. In some embodiments, each of the multiple reaction chambers is pre-loaded with PCR reagent comprising primers and probes.In some embodiments, the loading chamber is configured to be heated by induction heating, and (b) the analysis device includes: (i) a sample holder for receiving a rotatable disk in a substantially horizontal plane and configured to rotate the rotatable disk within the substantially horizontal plane; (ii) a first heating element that contacts the reaction chambers and is configured to heat the plurality of reaction chambers to a first temperature; and (iii) a second heating element that contacts the reaction chambers and is configured to heat the plurality of reaction chambers to a second temperature. In some embodiments, the loading chamber is located at the center on the rotatable disk. In some embodiments, the plurality of reaction chambers are located at the periphery on the rotatable disk.

[0017] In one aspect, the present disclosure provides a system for real-time polymerase chain reaction (PCR), comprising: (a) a rotatable disk including a loading chamber, a plurality of reaction chambers, and a channel connecting the loading chamber to the plurality of reaction chambers; and (b) an analysis device including: (i) a sample holder for receiving the rotatable disk in a substantially horizontal plane and configured to rotate the rotatable disk within the substantially horizontal plane; (ii) a first heating element configured to heat the loading chamber to a first temperature above 100° Celsius; (iii) a second heating element that contacts the reaction chambers and is configured to heat the plurality of reaction chambers to a second temperature; and (iv) a third heating element that contacts the reaction chambers and is configured to heat the plurality of reaction chambers to a third temperature. In some embodiments, the first heating element is configured to heat the loading chamber to a first temperature of 101° to 160° Celsius.

[0018] In one aspect, the present disclosure provides a system for real-time polymerase chain reaction (PCR), comprising: (a) a rotatable disk comprising a loading chamber, a plurality of reaction chambers, and channels connecting the loading chamber to the plurality of reaction chambers, wherein the loading chamber comprises a chelating agent, a single-stranded nucleic acid binding protein, and a reducing agent; and (b) an analysis device comprising: (i) a sample holder for receiving the rotatable disk in a substantially horizontal plane and configured to rotate the rotatable disk within the substantially horizontal plane; (ii) a first heating element in contact with the reaction chambers and configured to heat the plurality of reaction chambers to a first temperature; and (iii) a second heating element in contact with the reaction chambers and configured to heat the plurality of reaction chambers to a second temperature. In some embodiments, the analysis device further comprises a light source oriented to generate excitation light of a first wavelength within a horizontal plane occupied by the plurality of reaction chambers of the rotatable disk. In some embodiments, the analysis device further comprises a photodetector oriented to detect emission light of a second wavelength emitted from the plurality of reaction chambers of the rotatable disk.

[0019] In one aspect, the present disclosure provides a method for processing a target nucleic acid, comprising: (a) loading a sample comprising the target nucleic acid into a loading chamber on a rotatable disk, the rotatable disk further comprising reaction chambers and channels connecting the loading chamber to the reaction chambers; (b) heating the sample comprising the target nucleic acid in the loading chamber to a temperature above 100° Celsius under high pressure conditions to produce a processed sample; (c) rotating the rotatable disk such that the processed sample flows through the channels into the reaction chambers; and (d) amplifying the target nucleic acid in the processed sample within the reaction chambers.

[0020] In one aspect, the present disclosure provides a method for processing a target nucleic acid, comprising: (a) loading a sample comprising the target nucleic acid into a loading chamber on a rotatable disk, the rotatable disk further comprising a reaction chamber and a channel connecting the loading chamber to the reaction chamber; (b) heating the sample comprising the target nucleic acid in the loading chamber under high pressure conditions to a temperature above 100°C to produce a processed sample; (c) rotating the rotatable disk so that the processed sample flows into the reaction chamber through the channel; and (d) contacting the rotatable disk with a sealer to seal the channel after the processed sample has flowed into the reaction chamber. In some embodiments, the heating step includes heating the sample to a temperature of 101°C to 160°C. In some embodiments, the heating step occurs with a temperature gradient rate of 5°C / sec to 50°C / sec. In some embodiments, the sample comprising the target nucleic acid further comprises one or more reagents selected from the group consisting of chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, and stabilizers. In some embodiments, the loading chamber is pre-loaded with one or more reagents selected from the group consisting of chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, and stabilizers, and the sample is mixed with one or more reagents in the loading chamber. In some embodiments, the sample comprising the target nucleic acid comprises a body sample selected from the group consisting of blood samples, tear samples, saliva samples, mucus samples, sputum samples, fecal samples, cerebrospinal fluid samples, and urine samples. In some embodiments, the target nucleic acid is not extracted, isolated, or otherwise purified from the body sample prior to heating. In some embodiments, the sample comprising the target nucleic acid comprises multiple molecular amplification inhibitors, and the heating step inactivates the molecular amplification inhibitors of the multiple molecular amplification inhibitors in the sample. In some embodiments, the heating step inactivates at least 70% of the multiple molecular amplification inhibitors, resulting in a processed sample.

[0021] (Integrated by reference) All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is incorporated by specific and individual reference. [Brief explanation of the drawing]

[0022] Novel features of the present invention are described in detail in the appended claims. A deeper understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings, which describe illustrative embodiments in which the principles of the present invention are utilized.

[0023] [Figure 1] Figure 1 illustrates a top view of a rotatable disk as described herein, in the context of the analytical device described herein.

[0024] [Figure 2] Figure 2 illustrates a top view of the rotatable disk described herein.

[0025] [Figure 3] Figure 3 illustrates a diagram representing the main elements required for thermal circulation on the analytical device described herein.

[0026] [Figure 4] Figure 4 illustrates a top view of the rotatable disk described herein during the steps of the method for RT-PCR described herein.

[0027] [Figure 5A]Figures 5A–5C illustrate several diagrams of the rotatable disk described herein. Figure 5A illustrates two components used to manufacture several embodiments of the rotatable disk described herein. Figure 5B illustrates an enlarged embodiment of the reaction chamber provided on the rotatable disk described herein. Figure 5C illustrates an additional diagram of a manufactured rotatable disk with multiple reaction chambers designed for RT-PCR. [Figure 5B] Figures 5A–5C illustrate several diagrams of the rotatable disk described herein. Figure 5A illustrates two components used to manufacture several embodiments of the rotatable disk described herein. Figure 5B illustrates an enlarged embodiment of the reaction chamber provided on the rotatable disk described herein. Figure 5C illustrates an additional diagram of a manufactured rotatable disk with multiple reaction chambers designed for RT-PCR. [Figure 5C] Figures 5A–5C illustrate several diagrams of the rotatable disk described herein. Figure 5A illustrates two components used to manufacture several embodiments of the rotatable disk described herein. Figure 5B illustrates an enlarged embodiment of the reaction chamber provided on the rotatable disk described herein. Figure 5C illustrates an additional diagram of a manufactured rotatable disk with multiple reaction chambers designed for RT-PCR.

[0028] [Figure 6] Figure 6 illustrates components used to manufacture some embodiments of the rotatable disk described herein.

[0029] [Figure 7] Figure 7 illustrates a schematic diagram of an analytical device for processing target nucleic acids.

[0030] [Figure 8A]Figures 8A and 8B illustrate schematic diagrams of analytical devices for processing target nucleic acids. Figure 8A shows a front view of the analytical device. Figure 8B shows a side view of the analytical device. [Figure 8B] Figures 8A and 8B illustrate schematic diagrams of analytical devices for processing target nucleic acids. Figure 8A shows a front view of the analytical device. Figure 8B shows a side view of the analytical device.

[0031] [Figure 9A] Figures 9A-9D illustrate schematic diagrams of analytical devices for processing target nucleic acids, showing thermal sealer elements for sealing channels on a rotatable disk. Figure 9A shows an overview of the thermal sealer housed within the analytical device above the channels on the rotatable disk. Figure 9B shows a magnified view of the thermal sealer housed within the analytical device above the channels on the rotatable disk. Figure 9C shows an overview of the thermal sealer housed within the analytical device in contact with the channels on the rotatable disk. Figure 9D shows a magnified view of the thermal sealer housed within the analytical device in contact with the channels on the rotatable disk. [Figure 9B] Figures 9A-9D illustrate schematic diagrams of analytical devices for processing target nucleic acids, showing thermal sealer elements for sealing channels on a rotatable disk. Figure 9A shows an overview of the thermal sealer housed within the analytical device above the channels on the rotatable disk. Figure 9B shows a magnified view of the thermal sealer housed within the analytical device above the channels on the rotatable disk. Figure 9C shows an overview of the thermal sealer housed within the analytical device in contact with the channels on the rotatable disk. Figure 9D shows a magnified view of the thermal sealer housed within the analytical device in contact with the channels on the rotatable disk. [Figure 9C]Figures 9A-9D illustrate schematic diagrams of analytical devices for processing target nucleic acids, showing thermal sealer elements for sealing channels on a rotatable disk. Figure 9A shows an overview of the thermal sealer housed within the analytical device above the channels on the rotatable disk. Figure 9B shows a magnified view of the thermal sealer housed within the analytical device above the channels on the rotatable disk. Figure 9C shows an overview of the thermal sealer housed within the analytical device in contact with the channels on the rotatable disk. Figure 9D shows a magnified view of the thermal sealer housed within the analytical device in contact with the channels on the rotatable disk. [Figure 9D] Figures 9A-9D illustrate schematic diagrams of analytical devices for processing target nucleic acids, showing thermal sealer elements for sealing channels on a rotatable disk. Figure 9A shows an overview of the thermal sealer housed within the analytical device above the channels on the rotatable disk. Figure 9B shows a magnified view of the thermal sealer housed within the analytical device above the channels on the rotatable disk. Figure 9C shows an overview of the thermal sealer housed within the analytical device in contact with the channels on the rotatable disk. Figure 9D shows a magnified view of the thermal sealer housed within the analytical device in contact with the channels on the rotatable disk.

[0032] [Figure 10A] Figures 10A-10D illustrate schematic front views of the analytical device, where the disclosed heating block elements clamp and unclamp the disclosed rotatable disk. A comparison of Figure 10A and Figure 10B illustrates how the first pair of heating blocks clamp or contact the rotatable disk. A comparison of Figure 10B and Figure 10C shows how the heating blocks are unclamped from the rotatable disk, allowing the rotatable disk to rotate to the second pair of heating blocks. A comparison of Figure 10C and Figure 10D illustrates how the second pair of heating blocks clamp or contact the rotatable disk. [Figure 10B]Figures 10A-10D illustrate schematic front views of the analytical device, where the disclosed heating block elements clamp and unclamp the disclosed rotatable disk. A comparison of Figure 10A and Figure 10B illustrates how the first pair of heating blocks clamp or contact the rotatable disk. A comparison of Figure 10B and Figure 10C shows how the heating blocks are unclamped from the rotatable disk, allowing the rotatable disk to rotate to the second pair of heating blocks. A comparison of Figure 10C and Figure 10D illustrates how the second pair of heating blocks clamp or contact the rotatable disk. [Figure 10C] Figures 10A-10D illustrate schematic front views of the analytical device, where the disclosed heating block elements clamp and unclamp the disclosed rotatable disk. A comparison of Figure 10A and Figure 10B illustrates how the first pair of heating blocks clamp or contact the rotatable disk. A comparison of Figure 10B and Figure 10C shows how the heating blocks are unclamped from the rotatable disk, allowing the rotatable disk to rotate to the second pair of heating blocks. A comparison of Figure 10C and Figure 10D illustrates how the second pair of heating blocks clamp or contact the rotatable disk. [Figure 10D] Figures 10A-10D illustrate schematic front views of the analytical device, where the disclosed heating block elements clamp and unclamp the disclosed rotatable disk. A comparison of Figure 10A and Figure 10B illustrates how the first pair of heating blocks clamp or contact the rotatable disk. A comparison of Figure 10B and Figure 10C shows how the heating blocks are unclamped from the rotatable disk, allowing the rotatable disk to rotate to the second pair of heating blocks. A comparison of Figure 10C and Figure 10D illustrates how the second pair of heating blocks clamp or contact the rotatable disk.

[0033] [Figure 11A] Figures 11A-11B illustrate enlarged views of the heating elements of the analytical devices provided herein. [Figure 11B]Figures 11A-11B illustrate enlarged views of the heating elements of the analytical devices provided herein.

[0034] [Figure 12A] Figures 12A-12B illustrate schematic diagrams of a rotatable disk that rotates from one heating block (Figure 12A) to a second heating block (Figure 12B). [Figure 12B] Figures 12A-12B illustrate schematic diagrams of a rotatable disk that rotates from one heating block (Figure 12A) to a second heating block (Figure 12B).

[0035] [Figure 13A] Figures 13A–13C provide photographs of examples of the disclosed rotatable disk and the disclosed analytical device. Figure 13A shows an example of a fabricated rotatable disk. Figure 13B shows an example of a magnified view of a sealed channel on the rotatable disk. Figure 13C shows an example of an analytical device used to process a target nucleic acid (e.g., RT-PCR). [Figure 13B] Figures 13A–13C provide photographs of examples of the disclosed rotatable disk and the disclosed analytical device. Figure 13A shows an example of a fabricated rotatable disk. Figure 13B shows an example of a magnified view of a sealed channel on the rotatable disk. Figure 13C shows an example of an analytical device used to process a target nucleic acid (e.g., RT-PCR). [Figure 13C] Figures 13A–13C provide photographs of examples of the disclosed rotatable disk and the disclosed analytical device. Figure 13A shows an example of a fabricated rotatable disk. Figure 13B shows an example of a magnified view of a sealed channel on the rotatable disk. Figure 13C shows an example of an analytical device used to process a target nucleic acid (e.g., RT-PCR).

[0036] [Figure 14A]Figures 14A and 14B illustrate data showing fluorescence readout values ​​from the device provided herein. Figure 14A illustrates signal detection with one color channel implemented in the device. Figure 14B illustrates signal detection with two color channels implemented in the device. [Figure 14B] Figures 14A and 14B illustrate data showing fluorescence readout values ​​from the device provided herein. Figure 14A illustrates signal detection with one color channel implemented in the device. Figure 14B illustrates signal detection with two color channels implemented in the device.

[0037] [Figure 15A] Figures 15A-15C illustrate multiple views of the high-pressure heating vessel. Figure 15A shows a side view of the high-pressure heating vessel, Figure 15B shows a top view, and Figure 15C shows a bottom view. [Figure 15B] Figures 15A-15C illustrate multiple views of the high-pressure heating vessel. Figure 15A shows a side view of the high-pressure heating vessel, Figure 15B shows a top view, and Figure 15C shows a bottom view. [Figure 15C] Figures 15A-15C illustrate multiple views of the high-pressure heating vessel. Figure 15A shows a side view of the high-pressure heating vessel, Figure 15B shows a top view, and Figure 15C shows a bottom view.

[0038] [Figure 16A] Figures 16A and 16B illustrate the high-pressure heating vessel, with a duckbill valve as the sample inlet. Figure 16A shows a side view of the high-pressure heating vessel and duckbill valve, and Figure 16B shows a cross-sectional view. [Figure 16B] Figures 16A and 16B illustrate the high-pressure heating vessel, with a duckbill valve as the sample inlet. Figure 16A shows a side view of the high-pressure heating vessel and duckbill valve, and Figure 16B shows a cross-sectional view.

[0039] [Figure 17] Figure 17 provides a photograph of an induction heating platform.

[0040] [Figure 18] Figure 18 illustrates a closed high-pressure heating chamber, which is housed within a high-pressure heating vessel and includes an outlet channel.

[0041] [Figure 19] Figure 19 provides a photograph of the bottom view of the disclosed high-pressure heating vessel and illustrates a laser valve that seals the disclosed outlet channel.

[0042] [Figure 20A] Figures 20A and 20B illustrate the movement of the sample and magnified views of the laser valve during sample injection and sample release. Figure 20A illustrates the movement of the sample during sample injection and provides a magnified view of the outlet channel and the closed laser valve. Figure 20B illustrates the movement of the sample during sample release and provides a magnified view of the sample movement through the outlet channel and the open laser valve. [Figure 20B] Figures 20A and 20B illustrate the movement of the sample and magnified views of the laser valve during sample injection and sample release. Figure 20A illustrates the movement of the sample during sample injection and provides a magnified view of the outlet channel and the closed laser valve. Figure 20B illustrates the movement of the sample during sample release and provides a magnified view of the sample movement through the outlet channel and the open laser valve.

[0043] [Figure 21] Figure 21 provides a gel image showing PCR products from a PCR reaction with a HeLa Genomic DNA sample performed on an analytical device provided herein.

[0044] [Figure 22] Figure 22 shows individual fluorescence traces collected from RT-PCR experiments involving two respiratory infectious pathogens, performed on an analytical device provided herein.

[0045] [Figure 23A]Figures 23A–23D show individual fluorescence traces collected from RT-PCR experiments with respiratory infectious pathogens, treated using the high-pressure heating method described herein and performed on the analytical device provided herein, demonstrating that the PCR product was detectable after 35–50 cycles for the PCR reaction. Figure 23A shows individual fluorescence traces collected for the PCR reaction using primer set PS3.3. Figure 23B shows individual fluorescence traces collected for the PCR reaction using primer set PS3.4. Figure 23C shows individual fluorescence traces collected for the PCR reaction using primer set PS3.5. Figure 23D shows individual fluorescence traces collected for the PCR reaction using primer set PS3.6. [Figure 23B] Figures 23A–23D show individual fluorescence traces collected from RT-PCR experiments with respiratory infectious pathogens, treated using the high-pressure heating method described herein and performed on the analytical device provided herein, demonstrating that the PCR product was detectable after 35–50 cycles for the PCR reaction. Figure 23A shows individual fluorescence traces collected for the PCR reaction using primer set PS3.3. Figure 23B shows individual fluorescence traces collected for the PCR reaction using primer set PS3.4. Figure 23C shows individual fluorescence traces collected for the PCR reaction using primer set PS3.5. Figure 23D shows individual fluorescence traces collected for the PCR reaction using primer set PS3.6. [Figure 23C]Figures 23A–23D show individual fluorescence traces collected from RT-PCR experiments with respiratory infectious pathogens, treated using the high-pressure heating method described herein and performed on the analytical device provided herein, demonstrating that the PCR product was detectable after 35–50 cycles for the PCR reaction. Figure 23A shows individual fluorescence traces collected for the PCR reaction using primer set PS3.3. Figure 23B shows individual fluorescence traces collected for the PCR reaction using primer set PS3.4. Figure 23C shows individual fluorescence traces collected for the PCR reaction using primer set PS3.5. Figure 23D shows individual fluorescence traces collected for the PCR reaction using primer set PS3.6. [Figure 23D] Figures 23A–23D show individual fluorescence traces collected from RT-PCR experiments with respiratory infectious pathogens, treated using the high-pressure heating method described herein and performed on the analytical device provided herein, demonstrating that the PCR product was detectable after 35–50 cycles for the PCR reaction. Figure 23A shows individual fluorescence traces collected for the PCR reaction using primer set PS3.3. Figure 23B shows individual fluorescence traces collected for the PCR reaction using primer set PS3.4. Figure 23C shows individual fluorescence traces collected for the PCR reaction using primer set PS3.5. Figure 23D shows individual fluorescence traces collected for the PCR reaction using primer set PS3.6.

[0046] [Figure 24A]Figures 24A-24B show results from RT-PCR experiments with two respiratory infectious pathogens performed on the analytical device provided herein, demonstrating that nasal swab influenza samples, treated with high-pressure heating and processed via rapid PCR thermal circulation conditions (approximately 7.5 minutes), maintain high sensitivity with an average cycle threshold of approximately 33–36. Figure 24A shows the cycle threshold for influenza A samples. Figure 24B shows the cycle threshold for influenza B samples. [Figure 24B] Figures 24A-24B show results from RT-PCR experiments with two respiratory infectious pathogens performed on the analytical device provided herein, demonstrating that nasal swab influenza samples, treated with high-pressure heating and processed via rapid PCR thermal circulation conditions (approximately 7.5 minutes), maintain high sensitivity with an average cycle threshold of approximately 33–36. Figure 24A shows the cycle threshold for influenza A samples. Figure 24B shows the cycle threshold for influenza B samples.

[0047] [Figure 25] Figure 25 shows a schematic diagram of the trade-off between speed and sensitivity in modern nucleic acid sample preparation techniques.

[0048] [Figure 26A] Figures 26A–26D show schematic diagrams and data relating to the high-pressure heating device provided herein. Figure 26A shows a schematic diagram of the steps of the high-pressure heating workflow. Figure 26B shows results from a time-series experiment in which a sample in a high-pressure heating sample container was heated using magnetic induction. Figure 26C shows an experiment in which ribonuclease activity in nasal matrix collected with a cotton swab was measured by fluorescence acquisition after 10 minutes of incubation with a fluorescent RNA probe. Figure 26D shows PCR cycle quantification to increase the volume of the high-pressure heating treated sample. [Figure 26B]Figures 26A–26D show schematic diagrams and data relating to the high-pressure heating device provided herein. Figure 26A shows a schematic diagram of the steps of the high-pressure heating workflow. Figure 26B shows results from a time-series experiment in which a sample in a high-pressure heating sample container was heated using magnetic induction. Figure 26C shows an experiment in which ribonuclease activity in nasal matrix collected with a cotton swab was measured by fluorescence acquisition after 10 minutes of incubation with a fluorescent RNA probe. Figure 26D shows PCR cycle quantification to increase the volume of the high-pressure heating treated sample. [Figure 26C] Figures 26A–26D show schematic diagrams and data relating to the high-pressure heating device provided herein. Figure 26A shows a schematic diagram of the steps of the high-pressure heating workflow. Figure 26B shows results from a time-series experiment in which a sample in a high-pressure heating sample container was heated using magnetic induction. Figure 26C shows an experiment in which ribonuclease activity in nasal matrix collected with a cotton swab was measured by fluorescence acquisition after 10 minutes of incubation with a fluorescent RNA probe. Figure 26D shows PCR cycle quantification to increase the volume of the high-pressure heating treated sample. [Figure 26D] Figures 26A–26D show schematic diagrams and data relating to the high-pressure heating device provided herein. Figure 26A shows a schematic diagram of the steps of the high-pressure heating workflow. Figure 26B shows results from a time-series experiment in which a sample in a high-pressure heating sample container was heated using magnetic induction. Figure 26C shows an experiment in which ribonuclease activity in nasal matrix collected with a cotton swab was measured by fluorescence acquisition after 10 minutes of incubation with a fluorescent RNA probe. Figure 26D shows PCR cycle quantification to increase the volume of the high-pressure heating treated sample.

[0049] [Figure 27A]Figures 27A–27C demonstrate that high-pressure heating enables highly sensitive PCR detection of multiple organisms across diverse biotypes. Figure 27A shows PCR cycle quantification in multiple levels of artificial SARS-CoV-2 samples in nasal samples for both high-pressure heated samples and Qiagen Viral RNA extracts. Figure 27B shows PCR cycle quantification for two different bacterial organisms, B. subtilis and B. cereus spores. Figure 27C shows PCR cycle quantification for S. cerevisiae. Figure 27D shows PCR cycle quantification for C. albicans. [Figure 27B] Figures 27A–27C demonstrate that high-pressure heating enables highly sensitive PCR detection of multiple organisms across diverse biotypes. Figure 27A shows PCR cycle quantification in multiple levels of artificial SARS-CoV-2 samples in nasal samples for both high-pressure heated samples and Qiagen Viral RNA extracts. Figure 27B shows PCR cycle quantification for two different bacterial organisms, B. subtilis and B. cereus spores. Figure 27C shows PCR cycle quantification for S. cerevisiae. Figure 27D shows PCR cycle quantification for C. albicans. [Figure 27C] Figures 27A–27C demonstrate that high-pressure heating enables highly sensitive PCR detection of multiple organisms across diverse biotypes. Figure 27A shows PCR cycle quantification in multiple levels of artificial SARS-CoV-2 samples in nasal samples for both high-pressure heated samples and Qiagen Viral RNA extracts. Figure 27B shows PCR cycle quantification for two different bacterial organisms, B. subtilis and B. cereus spores. Figure 27C shows PCR cycle quantification for S. cerevisiae. Figure 27D shows PCR cycle quantification for C. albicans. [Figure 27D]Figures 27A–27C demonstrate that high-pressure heating enables highly sensitive PCR detection of multiple organisms across diverse biotypes. Figure 27A shows PCR cycle quantification in multiple levels of artificial SARS-CoV-2 samples in nasal samples for both high-pressure heated samples and Qiagen Viral RNA extracts. Figure 27B shows PCR cycle quantification for two different bacterial organisms, B. subtilis and B. cereus spores. Figure 27C shows PCR cycle quantification for S. cerevisiae. Figure 27D shows PCR cycle quantification for C. albicans.

[0050] [Figure 28] Figure 28 shows the cycle threshold results for SARS-CoV-2 and RNase P controls with respect to a fully integrated high-pressure heating and PCR system as provided.

[0051] [Figure 29] Figure 29 shows a schematic diagram of one embodiment, which includes two heating blocks.

[0052] [Figure 30] Figure 30 shows a schematic diagram of the structure of one embodiment, which has three blocks instead of two.

[0053] [Figure 31] Figure 31 shows a graph demonstrating that the cooling rate of the PCR disk is accelerated using a heat sink block.

[0054] [Figure 32] Figure 32 shows an embodiment in which a hole is punctured within the heating block, allowing it to cool more rapidly.

[0055] [Figure 33] Figure 33 shows a method in one embodiment in which airflow can be directed through an air duct.

[0056] [Figure 34] Figure 34 displays a simulation graph demonstrating that the air duct equipment shown in Figure 33 allows the Swiss cheese-shaped semi-heated block to return to its original temperature within 5 seconds when set to 40°C and the room temperature is 30°C. [Modes for carrying out the invention]

[0057] Detailed explanation This disclosure provides a method and system enabling ultrafast real-time PCR for highly multiplexed assays from a volume of approximately 20–30 μL. The disclosed method can be readily integrated with a centripetal microfluidic device for sample preparation, enabling a complete integrated sample processing / analysis response system. The system may consist of a rotatable disk comprising PCR reaction chambers (e.g., cuvettes) located at the same radius from the rotation center of the rotatable disk. These reaction chambers may be designed to enable ultrafast heat transfer from the heating block of the analytical device to the PCR reagent.

[0058] Two or three separate heat blocks, for example, heating blocks, set to a constant temperature of approximately 50°C to 98°C, may be arranged in a ring shape directly above and below the reaction chamber of the rotatable disk. In one embodiment, the analytical device may have an optical head positioned at the same radius as the reaction chamber, capable of detecting one to six different wavelengths in the time it takes for the reaction chamber to pass the sensor. The analytical device may have two distinctly different optical heads, each capable of detecting two different wavelengths in the time it takes for the reaction chamber to pass the sensor.

[0059] The disclosure further relates to a method for using the device, wherein after filling the reaction chambers via centripetal force, each reaction chamber may be sealed by contact or non-contact means to avoid evaporation and escape of the heated liquid. Heat circulation is achieved by sequentially contacting a pair of heating blocks with the reaction chambers, starting with the denaturing block, then the annealing block, and optionally, then the extension block.

[0060] definition Unless otherwise defined, all technical terms, expressions, and other technical and scientific terms or technical terms used herein are intended to have the same meaning as those generally understood by those skilled in the art to which the claimed subject matter relates. Where there is a generally understood meaning, terms are defined herein for clarity and / or for easy reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is generally understood in the art.

[0061] Throughout this application, various embodiments may be presented in scope form. It should be understood that the scope descriptions are for convenience and simplification only and should not be interpreted as inflexible limitations on the scope of this disclosure. Therefore, scope descriptions should be considered to have all conceivable sub-scopes and individual numerical values ​​within those scopes, as specifically disclosed. For example, a scope description such as 1-6 should be considered to have all specifically disclosed sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numerical values ​​within those scopes, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the scope.

[0062] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless otherwise clearly determined by the context. For example, the term "sample" includes multiple samples, as well as mixtures thereof.

[0063] The terms “determine,” “measure,” “evaluate,” “assess,” “assay,” and “analyze” are often used synonymously in this specification to refer to forms of measurement. These terms include determining whether an element is present or not (e.g., detection). These terms may include quantitative, qualitative, or both quantitative and qualitative determinations. Assessments may be relative or absolute. “Detecting the presence of” may, depending on the context, include determining the quantity of something that is present, in addition to determining whether it is present or not.

[0064] The terms “subject,” “individual,” or “patient” are often used synonymously herein. “Subject” may be a biological entity containing genetic material. A biological entity may be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject may be a tissue, cell, or fragment thereof, derived from a biological entity obtained from within a living organism or cultured outside a living organism. A subject may be a mammal, for example, a human. A subject may be diagnosed or suspected of being at high risk for a particular disease. In some cases, a subject may not necessarily be diagnosed or suspected of being at high risk for that disease.

[0065] As used herein, the term “approximately” a number refers to a number within ±10% of that number. The term “approximately” range refers to a range from -10% of its lowest value to +10% of its highest value.

[0066] As used herein, the term “molecular amplification” refers to an assay, method, or test used to detect nucleic acids in a sample. This can be used, for example, in experimental research, clinical drug development, infectious disease diagnosis, gene cloning, and industrial quality control. Molecular amplification can also be used as part of a diagnostic test. As used herein, the term “molecular amplification” is intended to encompass any method designed to amplify (e.g., replicate, copy, etc.) nucleic acids, thereby generating more copies of the nucleic acid in a sample. Molecular amplification comprises nucleic acid amplification, enzyme amplification (e.g., PCR), isothermal amplification, and / or other alternative amplification methods developed within this art.

[0067] As used herein, the term “polymerase chain reaction” or “PCR” refers to a type of molecular or nucleic acid amplification that amplifies or generates more copies of a nucleic acid template. PCR methods can be used in conjunction with methods for detecting, identifying, and / or quantifying nucleic acids. As used herein, the term “PCR” is intended to encompass all different types of PCR, including, for example, sequential PCR and real-time PCR or reverse transcription PCR (RT-PCR).

[0068] As used herein, the term “biological sample” means a sample containing nucleic acids / biological agents such as clinical samples (e.g., cell fractions, mucous membranes, nasal swab samples, whole blood, plasma, serum, urine, tissues, cells, etc.), agricultural samples, environmental samples (e.g., soil, mud, minerals, water, air), food samples, forensic samples, or any other biological samples. A sample may contain infectious agents such as viral, bacterial, or parasitic infectious agents. “Whole blood” means blood, such as that collected by venous sampling, and contains, for example, white and red blood cells, platelets, plasma, and any infectious agents that may be present. Clinical samples may be of human or animal origin. Samples to be analyzed may be solid or liquid by nature. When solid materials are used, it is obvious that they are first dissolved in a suitable solution as known in the art.

[0069] As used herein, the term “nucleic acid” refers to DNA molecules, e.g., cDNA or genomic DNA, RNA molecules, e.g., mRNA, DNA-RNA hybrids, and analogues of DNA or RNA produced using nucleotide analogues. Nucleic acid molecules include nucleotides, oligonucleotides, double-stranded DNA, single-stranded DNA, multi-stranded DNA, complementary DNA, genomic DNA, non-coding DNA, messenger RNA (mRNA), single-stranded RNA, microRNA (miRNA), and nuclear small molecules. These may be RNA (snoRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), short interfering RNA (siRNA), heteronuclear RNA (hnRNA), or short hairpin RNA (shRNA).

[0070] overview Centripetal microfluidics can be used for sample preparation and for fluorescence acquisition in multiple cuvettes during real-time PCR amplification. Therefore, centripetal systems are used both for integrated sample processing / analysis response systems such as GenePOC, Spindiag, or Simplexa, and for standalone real-time thermal circulation systems such as Rotorgene, LightCycler, or MIC. However, spinning the entire consumable can present other challenges, particularly with respect to ultrafast thermal circulation. In microfluidic systems, the liquid may need to be kept in place after being pumped into the reaction chamber to avoid evaporation during the heating step of PCR. In microfluidic systems controlled to a steady pressure, this can be achieved by applying air pressure exceeding the vapor pressure that occurs during the denaturation step.

[0071] One advantage of centripetal microfluidics may be that centrifugal pressure can be applied to the fluid without the need for any direct connection to the equipment, which can help prevent contamination. Applying air pressure to this system during heat circulation may be difficult. Therefore, the liquid may need to be kept in place during PCR circulation by continuously spinning the consumables. However, ultrafast heat transfer for ultrafast heat circulation may be difficult because non-contact heating and cooling strategies must be used when the consumables are continuously spinning. Non-contact cooling and heating systems may be suboptimal.

[0072] For example, pulsed high and low temperature air is used in GenePOC and Rotorgene to achieve thermal circulation. However, due to the poor heat capacity of air, thermal circulation can be slow in these techniques. Mic and Simplexa use contact between the consumables and a metal holder heated via a non-contact method during spinning. Mic uses induction heating, while Simplexa uses infrared LAMP. Such methods still lack an efficient way to rapidly cool PCR reagents. Spindiag uses a fully electrified rotor, allowing heating and cooling of consumables during spinning. This approach can make the instrument extremely complex. However, the circulation time is too long and cannot be considered ultrafast.

[0073] Another challenge in enabling ultrafast heating and cooling strategies in centripetal devices may be maintaining the PCR reagent in place during heating. Another challenge may be preventing evaporation within the PCR reactor. This disclosure provides a method that allows all reactive chambers (e.g., cuvettes) to be filled in less than 30 seconds, while each cuvette is heat-sealed after being filled with amplification reagent.

[0074] Channels for dispersing liquids within a reaction chamber, formed from thermoformed thin films and sealed by a heat-sealing film, can be permanently closed by pressing the channel ceiling against the bottom sealing film. Once applied, the seal can prevent any liquid or water vapor from escaping. In various cases, the U-shape of the channel can be crushed, bringing the ceiling closer to the bottom film and enabling a tight seal.

[0075] This disclosure relates to an analytical device, a rotatable disk, a method for preparing and processing a sample, and a method for performing sample analysis, such as nucleic acid amplification, including polymerase chain reaction (PCR). In some embodiments, the analytical device comprises a thermal circulation device comprising one or more reaction chambers. A nucleic acid-containing sample can be processed and / or analyzed in one or more reaction chambers, for example, in a PCR reaction chamber. In some embodiments, the analytical device further comprises a heating chamber for sample preparation. The sample may be heated in this heating chamber prior to further processing and analysis in the plurality of reaction chambers. In some embodiments, the method for preparing and processing a sample may include the step of heating the sample under high-pressure heating conditions to inactivate one or more nucleic acid amplification inhibitors prior to nucleic acid amplification. In some embodiments, the method for preparing and processing a sample may include the step of amplifying and analyzing nucleic acids using a thermal circulation device as described herein.

[0076] This disclosure provides an ultrafast real-time polymerase chain reaction (PCR) method for detecting the presence or absence of a target nucleic acid in a biological sample. In one aspect, the method includes (a) loading a sample into a loading chamber on a rotatable disk, the rotatable disk further comprising a plurality of reaction chambers and channels that fluidly connect the loading chamber to the plurality of reaction chambers, the reaction chambers being loaded with a PCR reagent mixture comprising primers and a fluorescent probe, the sample flowing through the channels into the plurality of reaction chambers, thereby filling the reaction chambers following the loading of the sample; (b) bringing the channels in the rotatable disk into contact with a sealer, thereby sealing the channels after filling and preventing fluid communication between the plurality of reaction chambers; and (c) rotating the rotatable disk and heating the plurality of reaction chambers to a first temperature. The method includes (d) placing the reaction chambers adjacent to an element, thereby denaturing the target nucleic acid in the sample, if present, thereby producing a denatured target nucleic acid; (e) rotating a rotatable disk to place the plurality of reaction chambers adjacent to a second heating element maintained at a second temperature, thereby annealing the primers to the denatured target nucleic acid and replicating the denatured target nucleic acid; (f) exposing the plurality of reaction chambers to excitation light of a first wavelength, thereby exciting a phosphor probe; and (g) repeating steps (c)-(e) over a plurality of cycles, measuring the emission light of a second wavelength from the plurality of reaction chambers, wherein if emission light of a second wavelength is detected, the sample comprises the target nucleic acid.

[0077] In some embodiments, the primer comprises an oligonucleotide sequence that is complementary to at least a portion of the target nucleic acid.

[0078] In some embodiments, the disclosure may include an analytical device comprising a pair of heating blocks (Figures 1, 104, 105, and 106) arranged radially with a plurality of reaction chambers (e.g., cuvettes) (Figure 1, 103) on a rotatable disk. In some embodiments, the rotatable disk may include a cuvette insert comprising a plurality of reaction chambers (e.g., cuvettes). In some embodiments, the plurality of reaction chambers (e.g., cuvettes) (Figure 1, 103) rotate between each heating block and stop at the location of each block for a residence time required for the corresponding PCR denaturation, annealing, or extension step.

[0079] In some embodiments, each pair of heating blocks may be symmetrically positioned directly opposite the plane of the reaction chamber. Each pair of heating blocks may move perpendicular to the plane of the reaction chamber. The heating blocks may have a speed ranging from about 10 mm to about 200 mm / second. In some embodiments, the speed may be about 50 mm to about 80 mm / second. In some embodiments, each heating block in a pair of heating blocks may be operated separately. In some other embodiments, each heating block in a pair of heating blocks may be operated simultaneously.

[0080] In some embodiments, each pair of heating blocks may be set to a fixed temperature at the start of the reaction. Setting each pair of heating blocks to a fixed temperature may thereby allow for rapid heat circulation. The reaction chamber may move between heating blocks, which are held at a variable temperature. This can therefore avoid inertia that would change the temperature of the heating blocks themselves. In some embodiments, to minimize the required heating and cooling times, the heating blocks may be designed to have a thermal mass at least about four times greater than the thermal mass of the reaction chamber.

[0081] In some embodiments, the pair of heating blocks may include a first heating block (Figure 1 104, Figure 7 704, Figure 8A 802). The first heating block may be set to a temperature compatible with the PCR denaturation step. In some embodiments, the first heating block may be set to a fixed temperature of about 90°C to about 99°C. In some embodiments, the pair of heating blocks may include a second heating block (Figure 1 105, Figure 7 705, Figure 8A 803). The second heating block may be set to a temperature compatible with the PCR annealing step. In some embodiments, the second heating block may be set to a fixed temperature of about 50°C to about 72°C. In some embodiments, the pair of heating blocks may include a third heating block (Figure 1 106). The third heating block may be set to a temperature compatible with the PCR extension step. In some embodiments, the third heating block may be set to a fixed temperature of about 70°C to about 75°C. In some embodiments, the cooling step between a first heating block (set to a denaturation temperature) and a second heating block (set to an annealing temperature) may be provided by bringing the blocks into contact with the reaction chamber, which are set to an annealing temperature (e.g., 65°C). In some embodiments, the greater thermal mass of the blocks may absorb heat from the reaction chamber.

[0082] In one other embodiment, the pair of heating blocks comprises a first heating block (Figure 7, 704) and a second heating block (Figure 7, 705) located at the same radius. In some embodiments, the PCR annealing and extension steps are performed by the same heating block (e.g., the second heating block). In some embodiments, the second heating block may be fixed at a temperature of about 50°C to about 74°C. In some embodiments, the second heating block may be fixed at a temperature of about 65°C to about 70°C.

[0083] In some embodiments, each pair of heating blocks (e.g., a first heating block, a second heating block, or a third heating block) may be in contact with the reaction chamber or multiple reaction chambers from the top and bottom (Figure 10B 1005 and 1002). The pair of heating blocks may be in contact with the reaction chamber or multiple reaction chambers using a force of about 1 N to about 100 N. The pair of heating blocks may be in contact with the reaction chamber or multiple reaction chambers for a period of about 0.1 seconds to about 10 seconds, thereby allowing the gradient and residence time necessary for optimal PCR amplification.

[0084] In some embodiments, reaction chambers (e.g., cuvettes) on a rotatable disk may be designed and manufactured to be filled and sealed before thermal circulation. In some embodiments, the rotatable disk may comprise a cuvette insert, which may comprise a reaction chamber (e.g., a cuvette). In some embodiments, the ultrafast thermal circulation of the Disclosure may be carried out after the reaction chamber or a plurality of reaction chambers have been reliably sealed from the atmosphere. The reaction chambers may be openly connected to an upper chamber via a microfluidic connection before sealing. The reaction chambers may be filled via this microfluidic connection.

[0085] In some embodiments, the reaction chamber may be formed by sealing two thin polymer films together (Figures 5A and 5B). For example, one film (Figure 5A 501) may be thermoformed to define the shape and size of the reaction chamber (Figure 5B 503) and the channel (Figure 5B 504). For example, the second film may be a flat film (Figure 5A 502) which may be used to laminate and seal the thermoformed film. In some embodiments, the thermoformed reaction chamber and channel have an aspect ratio of at least about 10:1. In some embodiments, the aspect ratio is at least about 20:1. In some embodiments, the depth of the reaction chamber is at most about 1 mm. In some embodiments, the depth of the reaction chamber is at most about 250 μm. In some embodiments, the depth of the channel is at most about 200 μm. In some embodiments, the depth of the channel is at most about 100 μm.

[0086] In some embodiments, the channel is formed by a thermoformed film. This thermoformed film can be heated to seal the channel. The channel may be heat-sealed by compressing the thermoformed film forming the ceiling of the channel against a sealing film forming the floor of the channel. In some embodiments, the thermoformed film has a thickness of about 50 μm to about 500 μm. In some embodiments, the thermoformed film has a thickness of about 100 μm to about 300 μm. In some embodiments, the channel has a width of about 1 mm to about 5 mm. In some embodiments, the channel has a width of about 0.05 mm to about 5 mm. In some embodiments, the channel has a width of about 2 mm to about 4 mm. In some embodiments, the channel has a width of about 0.25 mm to about 4 mm. In some embodiments, the width of the channel, divided by the thickness of the thermoformed film, is greater than 2, thereby allowing the channel to be easily compressed, obtain a strong heat seal, and completely close the channel.

[0087] In some embodiments, the sealing film has a thickness of about 10 μm to about 500 μm. In some embodiments, the sealing film has a thickness of about 40 μm to about 100 μm. In some embodiments, the sealing film and the thermoformed film comprise a thermoplastic resin. In some embodiments, the sealing film and the thermoformed film comprise the same thermoplastic resin. In some other embodiments, the sealing film and the thermoformed film both comprise a thermoplastic resin that can be heat-sealed. In some embodiments, the sealing film or thermoformed film comprises a single polymer. In some embodiments, the sealing film or thermoformed film comprises a thermoplastic resin that is co-extruded and further comprises an inner layer polymer having a lower glass transition temperature (Tg) than the outer layer polymer. In some embodiments, the resin is selected from polyolefins, polycarbonates, polystyrene, PMMA, polyethylene, and polypropylene.

[0088] In one aspect of this disclosure, three heating blocks (e.g., a first heating block (Figure 1 104), a second heating block (Figure 1 105), and a third heating block (Figure 1 104)) are set to three different temperatures (e.g., a first temperature, a second temperature, and a third temperature) corresponding to a denaturation temperature (e.g., about 90°C to about 99°C), an annealing temperature (e.g., about 50°C to about 70°C), and an extension temperature (e.g., about 70°C to about 75°C). In some embodiments, the three heating blocks have a radial length, and the radial length is designed to correspond to a preferred time ratio for the denaturation, annealing, and extension steps. The analytical device may thereby perform the PCR amplification process while a rotatable disk rotates at a constant speed above a fixed heating block, maintaining physical contact with the block throughout the entire process. In some embodiments, the disclosure comprises two heating blocks (e.g., a first heating block (Figure 7704) and a second heating block (Figure 7705)) set to two different temperatures (e.g., a first temperature and a second temperature) corresponding to a denaturation temperature (e.g., about 90°C to about 99°C) and an annealing / extension temperature (e.g., about 50°C to about 75°C). In these embodiments, the analytical device may thereby perform the annealing and extension PCR steps at the same temperature using the same heating block.

[0089] In some embodiments, a first heating block, set to a denaturation temperature (e.g., a first temperature), has a radial length (e.g., a first radial length) designed to move in parallel for each reaction chamber, resulting in a contact time of about 500 milliseconds to about 2 seconds, while the rotatable disk rotates at a speed of about 4 RPM to about 16 RPM above the first heating block. In some embodiments, a second heating block, set to an annealing temperature (e.g., a second temperature), has a radial length (e.g., a second radial length) about 6 to about 7 times the radial length of the first heating block. In some embodiments, a second heating block, set to an annealing / extension temperature (e.g., a second temperature), has a radial length (e.g., a second radial length) about 8 to about 9 times the radial length of the first heating block. In some embodiments, a third heating block, set to an extension temperature (e.g., a third temperature), has a radial length approximately 2 to 3 times the radial length of the first heating block.

[0090] In some embodiments, the heating blocks (e.g., a first heating block, a second heating block, or a third heating block) are made of a material having a low coefficient of friction (e.g., Teflon® or boroaluminum magnesium). For example, but not limited to, the heating blocks may be made of or coated with a material having a low coefficient of friction. In some embodiments, contact between the rotatable disk and the heating block allows for rapid heat transfer to the reaction chamber. In some embodiments, contact between the rotatable disk and the heating block allows the rotatable disk to rotate at a constant speed. In some embodiments, the speed of the rotatable disk is about 4 RPM to about 16 RPM.

[0091] In some embodiments, the heating block (e.g., a first heating block, a second heating block, or a third heating block) comprises one heating block, with the rotatable disk positioned above the heating block. In another embodiment, the heating block comprises one heating block, with the rotatable disk positioned below the heating block. In yet another embodiment, the heating block comprises a pair of heating blocks (Figures 10A, 1002; Figures 10A, 1003) arranged symmetrically from the plane of the rotatable disk, which narrows the rotatable disk (Figures 10A, 1005).

[0092] In some embodiments, the rotatable disk is maintained in each heating block, and the bottom and top blocks are installed in mechanical contact with the rotatable disk. For example, the disk can be clamped between a pair of heating blocks, as shown by the comparison of element 1002 in Figures 10A and 10B. In some embodiments, the rotatable disk moves from one heating block to another after the rotatable disk has been mechanically released from the heating block, as shown by the comparison of element 1005 in Figures 10A and 10C.

[0093] In some embodiments, the analytical device further includes an optical head (Figures 1, 107; 7, 706, 8A, 804) for exciting and detecting fluorescence inside the reaction chamber while it is rotating. In some embodiments, the excitation source and detector are located on the same side of the rotatable disk. In some embodiments, the excitation source and detector are located on opposite sides of the rotatable disk. In some embodiments, the excitation source is a light source (e.g., a laser or LED) encompassing the wavelength of the phosphor of interest. In some embodiments, the phosphor of interest is selected from FAM (e.g., 495 nm / 520 nm), SUN (e.g., 538 nm / 554 nm), TEX615 (e.g., 596 nm / 613 nm), or Cy5 (e.g., 648 nm / 667 nm).

[0094] In some embodiments, the rotatable disk comprises reaction chambers (e.g., cuvettes) or a plurality of reaction chambers (e.g., cuvettes), as shown by element 209 in Figure 2. In some embodiments, the rotatable disk comprises a cuvette insert 211 comprising a plurality of reaction chambers 209. In some embodiments, the reaction chambers (e.g., cuvettes) are located at the edge of the rotatable disk (Figure 2, 201). In some embodiments, the plurality of reaction chambers comprises 1 to about 100 reaction chambers. In some embodiments, the plurality of reaction chambers comprises about 4 to about 10 reaction chambers. In one embodiment, but not limited to, there may be eight reaction chambers equally spaced apart from one another. In some embodiments, the plurality of reaction chambers are equally diffused over 360 degrees of the rotatable disk. In some embodiments, the plurality of reaction chambers (e.g., cuvettes) occupy a section of the rotatable disk, e.g., an arc of about 10 degrees to an arc of about 100 degrees.

[0095] In some embodiments, multiple reaction chambers (Figure 2, 209) are each connected to an upstream mixing chamber (Figure 2, 207) and an adjacent reaction chamber (Figure 2, 209) via channels (Figure 2, 208). In some embodiments, the channels comprise a thin plastic film (e.g., a sealing film) sealed onto a rotatable disk. In some embodiments, the thin plastic film (e.g., a sealing film) is in direct contact with the material of the rotatable disk (e.g., a rigid plastic disk). For example, the channels may not have any depth, and the sample liquid may be pushed through them by applying a high centripetal force to the rotatable disk. In some embodiments, the thin plastic film (e.g., a sealing film or thermoplastic material) comprises a material (e.g., a resin) selected from polycarbonate, polypropylene, PEET, and COC. In some embodiments, the thin plastic film (e.g., a sealing film or thermoplastic material) comprises a material (e.g., a resin) with a Tg of at least about 100°C. In some embodiments, the thin film (e.g., sealing film) has a thickness of about 10 μm to about 400 μm. In some embodiments, the rotatable disk comprises a thermoplastic (e.g., thermoformed film) with a Tg of at least about 100°C. In some embodiments, the rotatable disk comprises a thermoplastic (e.g., thermoformed film) polycarbonate. In some embodiments, the channel seal is achieved via heat and pressure, via laser welding, or via ultrasonic welding. In some embodiments, the channel seal is achieved via heat and pressure, but not limited to, by a thermal sealer, etc.

[0096] In some embodiments, each of the reaction chambers (e.g., cuvettes) has a volume capacity of about 10 μl to about 100 μl. In some embodiments, each of the reaction chambers has a depth of about 0.1 mm to about 1 mm. In some embodiments, each of the reaction chambers has a depth of about 0.2 mm to about 0.7 mm.

[0097] In some embodiments, the rotatable disk is linked by channels (e.g., channels with fluid transport paths) (Figure 1, 102) and comprises different chambers (e.g., loading chamber Figure 2, 203, measurement chamber Figure 2, 205, or mixing chamber Figure 2, 207, also referred herein to as heating chambers) for performing measurement and PCR reagent mixing after sample heating (e.g., high-pressure heating). In some embodiments, but not limited to, after extraction, nucleic acids may be introduced into multiple reaction chambers (e.g., cuvettes) (Figure 1, 103; Figure 2, 209) via channels (Figure 1, 108; Figure 2, 208). In some embodiments, RT-PCR reagents are stored in the mixing chamber in lyophilized form. The sample may be treated via heating in the loading chamber 203, measured in the measurement chamber 205, mixed with the PCR lyophilized reagent inside chamber 207, and then flowed through channel 208 into the reaction chamber (e.g., cuvette) 209. In some embodiments, PCR primers and fluorescent probes are stored in a dry form within multiple reaction chambers 209 and reconstituted when a solution containing the nucleic acid of interest is pushed through a channel and fills the multiple reaction chambers (e.g., cuvettes).

[0098] In some embodiments, channels are sealed between each of the reaction chambers after each reaction chamber has been filled with liquid, thereby preventing evaporation and escape of the liquid when the reaction chambers are heated to high temperatures. In some embodiments, channels are sealed using an electromagnetic source, such as a laser beam, which is mounted on a rail, but is not limited to this configuration. In some embodiments, channels are sealed by applying pressure and heat to the channels, for example, by a thermal sealer (Figures 9B, 901-9D, 901). In some aspects, this disclosure relates to a method, and the analytical device described herein is used to rapidly circulate the temperature of PCR or RT-PCR reagent in a volume of about 10 to about 100 μl within each reaction chamber. In the method described herein, up to about six phosphors per reaction chamber may be monitored simultaneously. In some embodiments, the method includes the step of filling the reaction chamber with PCR or RT-PCR reagent through channels using centripetal force generated by rotating a rotatable disk. In some embodiments, the reaction chamber is filled by spinning the rotatable disk at about 1,000 RPM to about 15,000 RPM. In some embodiments, after the reaction chamber is filled, the method further includes the step of sealing the reaction chamber by applying a heat source across the channels. In some embodiments, the heat source may be applied by contact or, but not limited to, via an electromagnetic source such as a laser. In some embodiments, the sealing film is sealed across the channel by pressing two blocks against a rotatable disk, resulting in a thermoformed film (see comparison between elements 901 in Figures 9B and 9D). In some embodiments, a laser mounted on a rail seals the channel through a window between the heating blocks.

[0099] In some embodiments, the analytical device further includes a heating chamber for sample preparation of nucleic acid-containing samples prior to processing and analysis by PCR. In some embodiments, the heating chamber is part of, or connected to, a rotatable disk comprising a reaction chamber. In some embodiments, the heating chamber is connected to a microfluidic network that can transfer the heat-treated sample to a mixing chamber, where the heat-treated sample can be mixed with PCR reagents and further transferred to one or more reaction chambers where a nucleic acid amplification reaction is carried out.

[0100] The methods and compositions provided herein can achieve high-speed nucleic acid amplification. For example, in some cases, the time from sample loading to acquisition of a signal output indicating the presence or absence of a target can be equal to or less than about 15 minutes, less than about 14 minutes, less than about 13 minutes, less than about 12 minutes, less than about 11 minutes, less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, or less than about 3 minutes. Methods for sample heating or sample preparation

[0101] This disclosure provides methods for sample preparation for use with biological samples comprising nucleic acids. Examples of biological samples comprising nucleic acids may be body samples (e.g., saliva or mucus) or cells (e.g., bacterial cells, fungal cells, or mammalian cells). This disclosure provides methods for sample preparation of nucleic acids in body samples without extraction, isolation, or other forms of purification of nucleic acids from body samples. This disclosure provides methods for heating nucleic acid samples under high-pressure heating conditions, thereby preparing them for further processing or analysis. In some embodiments, the heat-treated samples may undergo further sample processing or analysis. This disclosure provides methods for inactivating molecular amplification inhibitors (e.g., RNAses present in saliva or mucus) in a sample, thereby preparing the sample for further processing. The heat-treated or inactivated samples may be used for nucleic acid detection and / or analysis using PCR analysis or biological (e.g., diagnostic) assays or any of the nucleic acid analysis methods disclosed elsewhere herein.

[0102] This disclosure further provides a method for analyzing nucleic acids in a biological sample, comprising the steps of preparing the sample using the method of this disclosure and subsequently analyzing the nucleic acid. The sample preparation method described herein may prepare the sample for molecular amplification of nucleic acids in the sample. In some embodiments, the method described herein may improve the efficiency of nucleic acid analysis by improving the efficiency of molecular amplification. The method described herein may reduce the degree of nucleic acid degradation and improve the ability to detect nucleic acids or their molecularly amplified products for analysis.

[0103] The step of analyzing nucleic acids can be carried out using any number of techniques known in the art (e.g., sequencing of nucleic acids (e.g., sequencing by synthesis, sequencing by hybridization, nanopore sequencing, etc.), genotyping of amino acids (e.g., genotyping by hybridization, genotyping by sequencing, etc.), or detection of nucleic acids (e.g., detection by hybridization, antibody binding, fluorescence, radioisotope detection, etc.)). For example, in some embodiments, the step of detecting nucleic acids includes hybridizing a nucleic acid with a fluorescently labeled nucleic acid having a sequence complementary to at least a portion of the nucleic acid, using a method known in the art.

[0104] In some embodiments, the step of analyzing nucleic acids includes one or more of the following steps: detecting nucleic acids, sequencing nucleic acids, and genotyping nucleic acids. In some embodiments, the step of analyzing nucleic acids includes detecting nucleic acids. In some embodiments, the step of analyzing nucleic acids includes sequencing nucleic acids. In some embodiments, the step of analyzing nucleic acids includes genotyping nucleic acids.

[0105] In some embodiments, the step of analyzing nucleic acids includes the step of analyzing the molecular amplification product of the nucleic acid (for example, a DNA copy of RNA produced during PCR amplification of RNA in a sample using reverse transcriptase). For example, in some embodiments, the step of detecting nucleic acids may include the step of detecting the molecular amplification product of the nucleic acid. In some embodiments, the step of sequencing nucleic acids includes the step of sequencing the molecular amplification product of the nucleic acid. In some embodiments, the step of genotyping nucleic acids includes the step of genotyping the molecular amplification product of the nucleic acid. Those skilled in the art will understand that many means for analyzing nucleic acids are known in the art, and all of them are compatible with the methods of this disclosure and are discussed herein.

[0106] In some embodiments, molecular amplification comprises enzyme amplification. In some embodiments, molecular amplification comprises isothermal amplification. In some embodiments, nucleic acid amplification comprises polymerase chain reaction ("PCR"), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-persistent sequence replication (3SR), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling cycle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification (HAD), branched amplification method (RAM), transcription-mediated assay (TMA), Nicking enzyme amplification reaction (NEAR), recombinase polymerase amplification (RPA), or whole-genome amplification (WGA).

[0107] In some embodiments, molecular amplification comprises polymerase chain reaction ("PCR"). In some embodiments, PCR comprises reverse transcription polymerase chain reaction (RT-PCR), reverse transcription quantitative PCR (RT-qPCR), quantitative real-time PCR (qPCR), digital PCR (dPCR), digital droplet PCR (ddPCR), microfluidic PCR, multiplex PCR, variable number tandem repeat (VNTR) PCR, asymmetric PCR, nested PCR, quantitative PCR, hot-start PCR, touchdown PCR, assembly PCR, colony PCR, suicide PCR, cold-temperature co-amplification PCR (COLD-PCR), rapid amplification of cDNA ends (RACE) PCR, two-tail PCR, ligation-mediated PCR, methylation-specific PCR (MSP), intersequence-specific PCR (or ISSR-PCR), RNase H-dependent PCR (rhPCR), or vectoret PCR.

[0108] In some embodiments, nucleic acids can be analyzed after molecular amplification (e.g., PCR). In some embodiments, nucleic acids are analyzed as part of a downstream application. In some embodiments, the downstream application comprises molecular amplification or sequencing. In some embodiments, the downstream application comprises probe hybridization and / or probe detection.

[0109] In some aspects, the methods of the present disclosure include the step of heating the treatment sample. “Treatment sample” is a sample (e.g., an aqueous solution or suspension) containing at least nucleic acids and optionally a reagent such as an enzyme or chelating agent, as referred to herein. In some embodiments, the treatment sample may be a biological sample of nucleic acids collected from a subject and optionally diluted with water or a buffer containing one or more reagents. The biological sample may be a body sample such as saliva or mucus. The biological sample may comprise cells (e.g., unlysed cells). In some embodiments, the nucleic acid sample collected from a subject may be isolated or purified, e.g., from cells, proteins, or other biological preparations in the biological sample prior to dilution and / or addition of reagents. In some embodiments, the nucleic acid sample collected from a subject may not be isolated or purified, e.g., from cells, proteins, or other biological preparations in the biological sample prior to dilution and / or addition of reagents.

[0110] In some aspects, the method includes the step of heating a sample to be treated in a closed heating chamber under high-pressure heating conditions to a temperature above 100 degrees Celsius. For example, the closed heating chamber may substantially prevent air and vapor from entering or leaving the chamber. In some cases, there is a negligible airflow inside and outside the closed heating chamber. The closed heating chamber may remain closed during high-pressure heating. In some embodiments, “high-pressure heating conditions” as referred herein are conditions in which heating generates a higher pressure inside the closed heating chamber than outside the closed heating chamber. In some embodiments, the temperature as referred herein is the average temperature over the duration of the sample heating step or any part thereof. In some embodiments, the temperature as referred herein is the temperature inside the closed container. In some embodiments, the temperature as referred herein is the temperature of the heating device used to heat the sample under high pressure. In some cases, the temperature is estimated using an internal temperature sensor inside the closed container. In some cases, the internal temperature is correlated via an external temperature infrared sensor that assesses the external temperature of a closed container. In some aspects, the heating of the treatment sample occurs with a temperature gradient rate, for example, 6 degrees Celsius / second to 20 degrees Celsius / second. In some aspects, the method of the present disclosure includes the step of heating the treatment sample from a first temperature to a second temperature above 100 degrees Celsius over a gradient time.

[0111] In some aspects, the methods of the present disclosure include the step of analyzing nucleic acids in a body sample selected from blood, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, and urine. In one aspect, the methods include the step of providing a body sample and heating a treatment sample comprising the body sample. In some embodiments, nucleic acids are not extracted, isolated, or otherwise purified from the body sample.

[0112] In some aspects, the methods of the present disclosure include the step of heating a treatment sample comprising unlysynthesized cells comprising nucleic acids to produce a heat-treated sample. In some embodiments, the methods further include the step of analyzing nucleic acids. In some embodiments, nucleic acids are not extracted, isolated, or otherwise purified from the heat-treated sample prior to the analysis of nucleic acids.

[0113] In some aspects, the treatment sample, comprising nucleic acids, further comprises one or more reagents selected from chelating agents, single-stranded nucleic acid-binding proteins, and reducing agents. In some embodiments, the treatment sample comprises a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent.

[0114] In some aspects, the disclosure provides a method for inactivating molecular amplification inhibitors in a treatment sample comprising nucleic acids. In some aspects, the method includes the step of heating a treatment sample comprising i) nucleic acids and ii) a plurality of molecular amplification inhibitors. In one aspect, the method includes the step of heating the treatment sample in a closed heating chamber to a temperature above 100 degrees Celsius under high-pressure heating conditions, thereby inactivating the molecular amplification inhibitors of the plurality of amplification inhibitors and producing a heat-treated sample. In another aspect, the method includes the step of heating the treatment sample in a closed heating chamber under high-pressure heating conditions from a first temperature to a second temperature above 100 degrees Celsius over a gradient time, thereby inactivating the molecular amplification inhibitors of the plurality of amplification inhibitors and producing a heat-treated sample in which the nucleic acid is substantially undegraded. In some embodiments, the method further includes the step of detecting the nucleic acid.

[0115] In some aspects, the methods described herein include the step of heating a treatment sample in a closed heating chamber under high pressure to a temperature above 100 degrees Celsius. In some embodiments, the method includes the step of heating the treatment sample under high pressure to a temperature above 100 degrees Celsius. The temperature may be above the boiling point of water. In some embodiments, the treatment sample does not boil at temperatures above 100 degrees Celsius under high pressure. In some embodiments, the method includes the step of heating the treatment sample under high pressure to a temperature between 100 and 160 degrees Celsius. In some embodiments, the temperature is between 101 and 160 degrees Celsius. In some embodiments, the temperature is between 105 and 160 degrees Celsius. In some embodiments, the temperature is between 110 and 160 degrees Celsius. In some embodiments, the temperature is between 120 and 160 degrees Celsius. In some embodiments, the temperature is between 130 and 160 degrees Celsius. In some embodiments, the temperature is between 140 and 160 degrees Celsius. In some embodiments, the temperature is between 150 and 160 degrees Celsius. In some embodiments, the temperature is between 100 and 140 degrees Celsius. In some embodiments, the temperature is between 110 and 140 degrees Celsius. In some embodiments, the temperature is between 120 and 140 degrees Celsius. In some embodiments, the temperature is between 130 and 140 degrees Celsius. In some embodiments, the temperature is approximately 110 degrees Celsius. In some embodiments, the temperature is approximately 120 degrees Celsius. In some embodiments, the temperature is approximately 130 degrees Celsius. In some embodiments, the temperature is approximately 140 degrees Celsius. In some embodiments, the temperature is approximately 150 degrees Celsius. In some embodiments, the temperature is approximately 160 degrees Celsius.

[0116] In some aspects, heating of the treatment sample occurs over a first period. In some embodiments, the first period is, for example, about 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 110 seconds, 120 seconds, 130 seconds, 140 seconds, 150 seconds, 160 seconds, 170 seconds, 180 seconds, 190 seconds, 200 seconds, or 300 seconds. In some embodiments, the first period is, for example, 1 to 300 seconds, 5 to 300 seconds, 30 to 300 seconds, 40 to 300 seconds, 50 to 300 seconds, 60 to 300 seconds, 70 to 300 seconds, 80 to 300 seconds, 90 to 300 seconds, 100 to 300 seconds, 110 to 300 seconds, 120 to 300 seconds, or 200 to 300 seconds. In some embodiments, the first period is, for example, 10 to 180 seconds, 20 to 180 seconds, 30 to 180 seconds, 40 to 180 seconds, 50 to 180 seconds, 60 to 180 seconds, 70 to 180 seconds, 80 to 180 seconds, 90 to 180 seconds, 100 to 180 seconds, 110 to 180 seconds, or 120 to 180 seconds. In some embodiments, the first period is, for example, 10 to 120 seconds, 20 to 120 seconds, 30 to 120 seconds, 40 to 120 seconds, 50 to 120 seconds, 60 to 120 seconds, 70 to 120 seconds, 80 to 120 seconds, 90 to 120 seconds, 100 to 120 seconds, or 110 to 120 seconds. In some embodiments, the first period is, for example, 30 to 90 seconds, 40 to 90 seconds, 50 to 90 seconds, 60 to 90 seconds, 70 to 90 seconds, or 80 to 90 seconds. In some embodiments, the first period is, for example, 10 to 60 seconds, 20 to 60 seconds, 30 to 60 seconds, 40 to 60 seconds, or 50 to 60 seconds.

[0117] In some embodiments, the first period is, for example, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes. In some embodiments, the first period is, for example, 1 to 2 minutes, 1 to 3 minutes, 1 to 4 minutes, 1 to 5 minutes, 1 to 6 minutes, 1 to 7 minutes, 1 to 8 minutes, 1 to 9 minutes, or 1 to 10 minutes. In some embodiments, the first period is, for example, 1 to 5 minutes, 2 to 5 minutes, 3 to 5 minutes, or 4 to 5 minutes.

[0118] In some aspects, the method further includes the step of heating the treatment sample to a temperature above 100 degrees Celsius, and then maintaining the treatment sample at that temperature for a certain maintenance time prior to the cooling time. In some embodiments, the maintenance time is 0 to 300 seconds, 0 to 250 seconds, 0 to 200 seconds, 0 to 150 seconds, 0 to 120 seconds, 0 to 100 seconds, 0 to 50 seconds, 0 to 40 seconds, 0 to 30 seconds, 0 to 20 seconds, or 1 to 10 seconds. In some embodiments, the maintenance time is 5 to 300 seconds, 5 to 250 seconds, 5 to 200 seconds, 5 to 150 seconds, 5 to 100 seconds, 5 to 50 seconds, 5 to 40 seconds, 5 to 30 seconds, 5 to 20 seconds, or 5 to 10 seconds.

[0119] During the maintenance period, the closed heating chamber may remain closed (for example, substantially preventing air and steam from entering or leaving the chamber).

[0120] In some embodiments, the cooling time is 5 to 300 seconds. In some embodiments, the cooling time is 30 to 300 seconds. In some embodiments, the cooling time is 60 to 300 seconds. In some embodiments, the cooling time is 90 to 300 seconds. In some embodiments, the cooling time is 120 to 300 seconds. In some embodiments, the cooling time is 150 to 300 seconds. In some embodiments, the cooling time is 180 to 300 seconds. In some embodiments, the cooling time is 240 to 300 seconds. In some embodiments, the cooling time is 270 to 300 seconds.

[0121] In some embodiments, the cooling time is 10 to 120 seconds. In some embodiments, the cooling time is 30 to 120 seconds. In some embodiments, the cooling time is 60 to 120 seconds. In some embodiments, the cooling time is 90 to 120 seconds. During the cooling time, the closed heating chamber may remain closed (for example, substantially preventing air and vapor from entering or leaving the chamber). In some embodiments, nucleic acids are substantially not decomposed after heating.

[0122] In some aspects, the step of heating the treatment sample comprising the nucleic acid occurs with respect to a temperature gradient. In some embodiments, the temperature gradient is calculated as the time derivative of temperature. In some embodiments, the temperature gradient is calculated as the average rate of change of temperature with respect to time. In some embodiments, the temperature gradient is at least 0.5 degrees Celsius / second, at least 1 degree Celsius / second, at least 2 degrees Celsius / second, at least 3 degrees Celsius / second, at least 4 degrees Celsius / second, at least 5 degrees Celsius / second, at least 6 degrees Celsius / second, at least 7 degrees Celsius / second, at least 8 degrees Celsius / second, at least 9 degrees Celsius / second, at least 10 degrees Celsius / second, at least 11 degrees Celsius / second, at least 12 degrees Celsius / second, at least 13 degrees Celsius / second, at least 14 degrees Celsius / second, at least 15 degrees Celsius / second, and less At least 16 degrees Celsius, at least 17 degrees Celsius, at least 18 degrees Celsius, at least 19 degrees Celsius, at least 20 degrees Celsius, at least 25 degrees Celsius, at least 27.5 degrees Celsius, at least 30 degrees Celsius, at least 32.5 degrees Celsius, at least 35 degrees Celsius, at least 37.5 degrees Celsius, at least 40 degrees Celsius, at least 42.5 degrees Celsius, at least 45 degrees Celsius, at least 47.5 degrees Celsius, or at least 50 degrees Celsius.

[0123] In some embodiments, the temperature gradient ratio is 0.5°C / sec to 50°C / sec, 0.5°C / sec to 40°C / sec, 0.5°C / sec to 35°C / sec, 0.5°C / sec to 30°C / sec, 0.5°C / sec to 25°C / sec, 0.5°C / sec to 20°C / sec, or 0.5°C / sec to 15°C / sec.

[0124] In some embodiments, the temperature gradient ratio is 2 degrees Celsius to 50 degrees Celsius, 2 degrees Celsius to 40 degrees Celsius, 2 degrees Celsius to 35 degrees Celsius, 2 degrees Celsius to 30 degrees Celsius, 2 degrees Celsius to 25 degrees Celsius, 2 degrees Celsius to 20 degrees Celsius, or 2 degrees Celsius to 15 degrees Celsius.

[0125] In some embodiments, the temperature gradient is 5°C / sec to 50°C / sec, 5°C / sec to 40°C / sec, 5°C / sec to 35°C / sec, 5°C / sec to 30°C / sec, 5°C / sec to 25°C / sec, 5°C / sec to 20°C / sec, or 5°C / sec to 15°C / sec. In some embodiments, the nucleic acids are not substantially decomposed after heating.

[0126] In some aspects, the methods described herein include the step of heating a treatment sample comprising the nucleic acid in a closed heating chamber under high pressure conditions from a first temperature to a second temperature above 100 degrees Celsius over a gradient time, thereby producing a heat-treated sample. The closed heating chamber may remain closed during heating from the first temperature to the second temperature (e.g., substantially preventing air from entering or leaving the chamber). In some embodiments, the treatment sample does not boil at the second temperature above 100 degrees Celsius under high pressure conditions. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius and the second temperature is 100 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius and the second temperature is 101 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius to 40 degrees Celsius and the second temperature is 105 degrees Celsius to 160 degrees Celsius. In some embodiments, the first temperature is between 4 and 40 degrees Celsius, and the second temperature is between 110 and 160 degrees Celsius. In some embodiments, the first temperature is between 4 and 40 degrees Celsius, and the second temperature is between 120 and 160 degrees Celsius. In some embodiments, the first temperature is between 4 and 40 degrees Celsius, and the second temperature is between 130 and 160 degrees Celsius. In some embodiments, the first temperature is between 4 and 40 degrees Celsius, and the second temperature is between 140 and 160 degrees Celsius.

[0127] In some embodiments, the first temperature is 20 to 30 degrees Celsius, and the second temperature is 100 to 160 degrees Celsius. In some embodiments, the first temperature is 20 to 30 degrees Celsius, and the second temperature is 101 to 160 degrees Celsius. In some embodiments, the first temperature is 20 to 30 degrees Celsius, and the second temperature is 105 to 160 degrees Celsius. In some embodiments, the first temperature is 20 to 30 degrees Celsius, and the second temperature is 110 to 160 degrees Celsius. In some embodiments, the first temperature is 20 to 30 degrees Celsius, and the second temperature is 120 to 160 degrees Celsius. In some embodiments, the first temperature is 20 to 30 degrees Celsius, and the second temperature is 130 to 160 degrees Celsius. In some embodiments, the first temperature is 20 to 30 degrees Celsius, and the second temperature is 140 to 160 degrees Celsius.

[0128] In some embodiments, the first temperature is 4 degrees Celsius and 40 degrees Celsius, and the second temperature is at least 100 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius and 40 degrees Celsius, and the second temperature is at least 101 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius and 40 degrees Celsius, and the second temperature is at least 105 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius and 40 degrees Celsius, and the second temperature is at least 110 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius and 40 degrees Celsius, and the second temperature is at least 120 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius and 40 degrees Celsius, and the second temperature is at least 130 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius and 40 degrees Celsius, and the second temperature is at least 140 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius and 40 degrees Celsius, and the second temperature is at least 150 degrees Celsius. In some embodiments, the first temperature is 4 degrees Celsius and 40 degrees Celsius, and the second temperature is at least 160 degrees Celsius.

[0129] In some embodiments, the gradient time is 3-100 seconds, 6-100 seconds, 7-100 seconds, 8-100 seconds, 9-100 seconds, or 10-100 seconds. In some embodiments, the gradient time is 3-50 seconds, 6-50 seconds, 7-50 seconds, 8-50 seconds, 9-50 seconds, or 10-50 seconds.

[0130] In some embodiments, the method for analyzing nucleic acids further includes (a) maintaining the treatment sample at a second temperature for a certain maintenance time, after and prior to a cooling time. In some embodiments, the maintenance time is 0 to 300 seconds. In some embodiments, the maintenance time is 5 to 300 seconds. In some embodiments, the maintenance time is 30 to 300 seconds. In some embodiments, the maintenance time is 60 to 300 seconds. In some embodiments, the maintenance time is 90 to 300 seconds. In some embodiments, the maintenance time is 120 to 300 seconds. In some embodiments, the maintenance time is 150 to 300 seconds. In some embodiments, the maintenance time is 180 to 300 seconds. In some embodiments, the maintenance time is 240 to 300 seconds. In some embodiments, the maintenance time is 270 to 300 seconds.

[0131] In some embodiments, the maintenance time is 0 to 120 seconds. In some embodiments, the maintenance time is 10 to 120 seconds. In some embodiments, the maintenance time is 30 to 120 seconds. In some embodiments, the maintenance time is 60 to 120 seconds. In some embodiments, the maintenance time is 90 to 120 seconds. During the maintenance time, the closed heating chamber may remain closed (for example, substantially preventing air and vapor from entering or leaving the chamber).

[0132] In some embodiments, the cooling time is 0 to 300 seconds. In some embodiments, the cooling time is 5 to 300 seconds. In some embodiments, the cooling time is 30 to 300 seconds. In some embodiments, the cooling time is 60 to 300 seconds. In some embodiments, the cooling time is 90 to 300 seconds. In some embodiments, the cooling time is 120 to 300 seconds. In some embodiments, the cooling time is 150 to 300 seconds. In some embodiments, the cooling time is 180 to 300 seconds. In some embodiments, the cooling time is 240 to 300 seconds. In some embodiments, the cooling time is 270 to 300 seconds.

[0133] In some embodiments, the cooling time is 10 to 120 seconds. In some embodiments, the cooling time is 30 to 120 seconds. In some embodiments, the cooling time is 60 to 120 seconds. In some embodiments, the cooling time is 90 to 120 seconds. During the cooling time, the closed heating chamber may remain closed (for example, substantially preventing air and vapor from entering or leaving the chamber).

[0134] In some aspects, the step of heating the treatment sample in a closed chamber generates pressure inside the chamber. In some embodiments, the pressure inside the chamber is, for example, 1-200 PSI, 10-200 PSI, 20-200 PSI, 30-200 PSI, 40-200 PSI, 50-200 PSI, 10-100 PSI, 20-100 PSI, 30-100 PSI, 40-100 PSI, 50-100 PSI, 60-100 PSI, 70-100 PSI, 80-100 PSI, or 90-100 PSI, each exceeding 1 atm. In some embodiments, the pressure inside the chamber is, for example, 10-100 PSI, 10-90 PSI, 10-80 PSI, 10-70 PSI, 10-60 PSI, 10-50 PSI, 10-40 PSI, 10-30 PSI, 10-20 PSI, 20-100 PSI, 20-90 PSI, 20-80 PSI, 20-70 PSI, 20-60 PSI, 20-50 PSI, 20-40 PSI, 20-30 PSI, 30-100 PSI, 30-90 PSI, 30-80 PSI, 30-70 PSI, 30-60 PSI, 30-50 PSI, or 30-40 PSI, each exceeding 1 atm. In some embodiments, the pressure inside the chamber is 40-60 PSI, exceeding 1 atm. In some embodiments, the pressure inside the chamber is greater than 1 atm and is 40-50 PSI. In some embodiments, the nucleic acids are substantially undegraded after heating.

[0135] Any means for heating the sample may be used, but are not limited to, contact heating in a dry heating block, induction heating, microwave heating, nanophotonic heating, or any other heating means known in the art. In some aspects, the treatment sample is heated using a heat source. In some embodiments, the heat source comprises an induction heater, a heating element, or microwaves. For example, in some embodiments, a heating block or heat bath may be used by filling it with, for example, preheated aluminum beads. In some embodiments, heating of the biological sample is high-pressure heating. In some embodiments, the treatment sample is heated under high pressure using a heat source.

[0136] For example, in some embodiments, the treatment sample is heated using a heating block or heat bath that is preheated to a temperature ranging from 100 to 300 degrees Celsius, or more preferably, 100 to 150 degrees Celsius, and the high-pressure heating vessel is left standing for, for example, 1 second to 10 minutes after reaching 100 degrees Celsius. In another embodiment, the vessel is heated under high pressure for 20 to 40 seconds after reaching 100 degrees Celsius or a higher temperature.

[0137] In another embodiment of the present disclosure, heat can be applied to the sample via induction heating. In some embodiments, the container can be made of a paramagnetic material, or the paramagnetic material can be placed inside a heating container made of a highly magnetically heat-resistant material such as a plastic polymer, in direct contact with the sample. This provides an extreme temperature gradient that allows the sample to be brought above 100 degrees Celsius in less than 30 seconds, which allows complete sample preparation to be performed in one minute or less.

[0138] In some embodiments, a laser can be used to heat the treatment sample inside the container to a temperature above 100 degrees Celsius (e.g., high-pressure heating). Nanoparticles can be added to the heating system to capture the laser energy and convert it directly into the sample. This technique provides a very high heating gradient, which is suitable for ultraheated sample preparation. In some embodiments, the heat source is a laser. In some embodiments, nanoparticles are added to the laser.

[0139] In some aspects, this disclosure provides a method for heating a treatment sample comprising a nucleic acid. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is derived from a eukaryotic or prokaryotic cell. In some embodiments, the nucleic acid is derived from a virus. In some embodiments, the nucleic acid is selected from viral nucleic acids, bacterial nucleic acids, protist nucleic acids, eukaryotic nucleic acids, and fungal nucleic acids. In some embodiments, the nucleic acid is viral nucleic acid.

[0140] In some embodiments, the treatment sample, comprising a body sample containing nucleic acids, is heated under high-pressure conditions in a closed heating chamber. In some embodiments, the body sample comprises a substance selected from blood, plasma, serum, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, lymphatic fluid, bile, synovial fluid, cystic fluid, ascites, pleural fluid, ocular fluid, interstitial fluid, cervical fluid, and urine. In some embodiments, the body sample comprises mucus. In some embodiments, the body sample comprises a body fluid sample, tissue, or cells of the subject. In some embodiments, nucleic acids are not extracted, isolated, or otherwise purified from the body sample.

[0141] In some aspects, a body sample may be collected from a subject (e.g., a human). The body sample may be collected from the subject via nasopharyngeal swab collection, cervical swab collection, or nasal swab collection. In some cases, the body sample may comprise a pathogen or a part thereof. In some embodiments, the pathogen or a part thereof is selected from viruses or a part thereof, bacteria or a part thereof, protozoa or a part thereof, yeast or a part thereof, and fungi or a part thereof. In some embodiments, the pathogen is a bloodborne pathogen or a part thereof. In some embodiments, the pathogen is a respiratory pathogen or a part thereof.

[0142] In some embodiments, a treatment sample comprising a body sample containing nucleic acids is heated in a closed heating chamber under high pressure to a temperature exceeding 100 degrees Celsius, thereby producing a heat-treated sample. In some aspects, the nucleic acids are analyzed after high-pressure heating. In some cases, the nucleic acids are not extracted, isolated, or otherwise purified from the body sample. For example, in some cases, the nucleic acids are not extracted by phenol-chloroform extract, not purified via a commercial nucleic acid purification kit, or purified via column chromatography.

[0143] In some embodiments, the treatment sample, comprising nucleic acids and cells, is heated under high-pressure conditions in a closed heating chamber. The cells may be unlysed cells. In some embodiments, the cells are embedded in a biological matrix such as nasal mucus, cerebrospinal fluid, feces, vaginal mucus, urine, or saliva. In some embodiments, the cells are embedded in nasal mucus. In some embodiments, the cells are bacterial cells, e.g., B. subtilis, E. coli, or S. Pyogenes. In some embodiments, the cells are fungal cells, e.g., C. albicans. In some embodiments, the cells are yeast cells, e.g., S. cerevisiae. In some embodiments, the cells are mammalian cells, e.g., Chinese hamster ovary cells, BHK cells, or mouse C127 cells. In some embodiments, the cells are human cells, e.g., HeLa cells. In some embodiments, the treatment sample comprises bacterial spores, e.g., B. cereus bacterial spores.

[0144] In some embodiments, a treatment sample comprising unlysynthesized cells containing nucleic acids is heated in a closed heating chamber under high pressure to a temperature exceeding 100 degrees Celsius, thereby producing a heat-treated sample. In some embodiments, the nucleic acids are analyzed after high-pressure heating. In some cases, the nucleic acids are not extracted, isolated, or otherwise purified from the heat-treated sample prior to the analysis of the nucleic acids.

[0145] In some respects, this disclosure provides a method for inactivating a molecular amplification inhibitor in a treatment sample comprising a nucleic acid. In some cases, the molecular amplification inhibitor may be an active agent that binds to the nucleic acid. In some cases, the molecular amplification inhibitor may be an active agent that degrades the nucleic acid. In some cases, the molecular amplification inhibitor may be a nuclease. In some cases, the molecular amplification inhibitor is a DNase. In other cases, the molecular amplification inhibitor is an RNase.

[0146] In some aspects, the method includes the step of heating a treatment sample comprising i) nucleic acid and ii) a plurality of molecular amplification inhibitors. In one aspect, the method includes the step of heating the treatment sample in a closed heating chamber to a temperature above 100 degrees Celsius under high-pressure heating conditions, thereby inactivating the molecular amplification inhibitors of the plurality of amplification inhibitors and producing a heat-treated sample. In another aspect, the method includes the step of heating the treatment sample in a closed heating chamber under high-pressure heating conditions over a gradient time from a first temperature to a second temperature above 100 degrees Celsius, thereby inactivating the molecular amplification inhibitors of the plurality of amplification inhibitors and producing a heat-treated sample in which the nucleic acid is substantially undegraded. In some embodiments, the nucleic acid is degraded by, for example, 20%, 15%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the method further includes the step of amplifying the nucleic acid using molecular amplification. In some embodiments, the method further includes the step of detecting nucleic acids.

[0147] In some aspects, this method inactivates at least one molecular amplification inhibitor in the treatment sample. In some embodiments, this method inactivates at least 60% of multiple molecular amplification inhibitors. In some embodiments, this method inactivates at least 70% of multiple molecular amplification inhibitors. In some embodiments, this method inactivates at least 80% of multiple molecular amplification inhibitors. In some embodiments, this method inactivates at least 90% of multiple molecular amplification inhibitors.

[0148] In some aspects, nucleic acids are not substantially degraded after heating, such as under high pressure. In some embodiments, nucleic acids are degraded by, for example, 20% or less, 15% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less after heating under high pressure. In some embodiments, nucleic acids are intact by, for example, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, at least 99.5% after heating under high pressure.

[0149] In some aspects, nucleic acids are analyzed after high-pressure heating. In some aspects, a method for analyzing nucleic acids includes a step of amplifying the nucleic acid by molecular amplification (e.g., by polymerase) after high-pressure heating using any of the methods disclosed elsewhere herein. Molecular amplification may occur during a real-time polymerase chain reaction (real-time PCR), transcription-mediated amplification (TMA), or loop-mediated isothermal amplification (LAMP). Molecular amplification of nucleic acids can produce molecular amplification products. In some embodiments, a method for analyzing nucleic acids further includes a step of amplifying the nucleic acid by polymerase chain reaction (PCR) after heating, thereby producing a PCR product. In some embodiments, the method further includes a step of detecting the PCR product. In some cases, the PCR product is detected via a fluorescent signal released during amplification.

[0150] In some embodiments, molecular amplification products (e.g., PCR products) are detectable following fewer molecular amplification cycles than would be required in the absence of high-pressure heating of the treatment sample. In some embodiments, molecular amplification products (e.g., PCR products) are detectable following fewer molecular amplification cycles than would be required compared to the step of heating the treatment sample at a temperature below 100 degrees Celsius. In some embodiments, nucleic acids are detectable following fewer molecular amplification cycles than would be required compared to the step of heating the treatment sample at a temperature below the boiling point of the sample over the same period of time.

[0151] In some embodiments, molecular amplification products (e.g., PCR products) are detectable following 10–50 molecular amplification cycles, 15–50 molecular amplification cycles, 20–50 molecular amplification cycles, 25–50 molecular amplification cycles, 26–50 molecular amplification cycles, 27–50 molecular amplification cycles, 28–50 molecular amplification cycles, 29–50 molecular amplification cycles, or 30–50 molecular amplification cycles.

[0152] In some embodiments, molecular amplification products (e.g., PCR products) are detectable following 10-40 molecular amplification cycles, 20-40 molecular amplification cycles, 25-40 molecular amplification cycles, 26-40 molecular amplification cycles, 27-40 molecular amplification cycles, 28-40 molecular amplification cycles, 29-40 molecular amplification cycles, or 30-40 molecular amplification cycles.

[0153] In some embodiments, molecular amplification products (e.g., PCR products) are detectable following 10–35 molecular amplification cycles, 20–35 molecular amplification cycles, 25–35 molecular amplification cycles, 26–35 molecular amplification cycles, 27–35 molecular amplification cycles, 28–35 molecular amplification cycles, 29–35 molecular amplification cycles, or 30–35 molecular amplification cycles. In some embodiments, molecular amplification products (e.g., PCR products) are detectable following 28–35 molecular amplification cycles. In some embodiments, nucleic acids are detectable following, for example, 25–35 molecular amplification cycles, 25–34 molecular amplification cycles, 25–33 molecular amplification cycles, 25–32 molecular amplification cycles, 25–31 molecular amplification cycles, 25–30 molecular amplification cycles, 25–29 molecular amplification cycles, or 25–28 molecular amplification cycles.

[0154] In some embodiments, molecular amplification products (e.g., PCR products) are detectable following 25 or more molecular amplification cycles. In some embodiments, nucleic acids are detectable following, for example, 25 or more molecular amplification cycles, 26 or more molecular amplification cycles, 27 or more molecular amplification cycles, 28 or more molecular amplification cycles, 29 or more molecular amplification cycles, 30 or more molecular amplification cycles, 31 or more molecular amplification cycles, 32 or more molecular amplification cycles, 33 or more molecular amplification cycles, 34 or more molecular amplification cycles, or 35 or more molecular amplification cycles.

[0155] In some embodiments, molecular amplification products (e.g., PCR products) are detected after the nucleic acid has been amplified, using 10–40 molecular amplification cycles, 10–35 molecular amplification cycles, 20–35 molecular amplification cycles, 25–35 molecular amplification cycles, 26–35 molecular amplification cycles, 27–35 molecular amplification cycles, 28–35 molecular amplification cycles, 29–35 molecular amplification cycles, or 30–35 molecular amplification cycles.

[0156] In some embodiments, nucleic acid detection occurs simultaneously with the molecular amplification of nucleic acids. In some embodiments, nucleic acid detection and molecular amplification of nucleic acids occur sequentially. In some embodiments, nucleic acid detection occurs after each cycle of molecular amplification.

[0157] In some embodiments, the method for analyzing nucleic acids further includes at least one of the following steps: detecting the nucleic acid, sequencing the nucleic acid, and genotyping the nucleic acid. In some embodiments, the method for analyzing nucleic acids further includes at least two of the following steps: detecting the nucleic acid, sequencing the nucleic acid, and genotyping the nucleic acid. In some embodiments, the method for analyzing nucleic acids further includes detecting the nucleic acid, sequencing the nucleic acid, and genotyping the nucleic acid. In some embodiments, the method for analyzing nucleic acids includes detecting the nucleic acid via fluorescence detection.

[0158] As described herein, the heated treatment sample contains at least a nucleic acid and optionally a reagent such as an enzyme or chelating agent. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is derived from a eukaryotic or prokaryotic cell. In some embodiments, the nucleic acid is derived from a virus. In some embodiments, the nucleic acid is selected from viral nucleic acids, bacterial nucleic acids, protist nucleic acids, eukaryotic nucleic acids, and fungal nucleic acids. In some embodiments, the nucleic acid is viral nucleic acid.

[0159] In some embodiments, nucleic acids are extracted from organisms selected from prokaryotes. In some embodiments, nucleic acids are extracted from organisms selected from eukaryotes. In some embodiments, nucleic acids are extracted from parasites. In some embodiments, nucleic acids are extracted from viruses, bacteria, fungi, animals, or plants.

[0160] In some embodiments, nucleic acids are extracted from viruses. In some embodiments, nucleic acids are extracted from respiratory viruses. In some embodiments, the viruses are selected from influenza viruses, rhinoviruses, coronaviruses, metapneumoviruses, adenoviruses, cynthiaviruses, bocaviruses, and parainfluenza viruses.

[0161] In some embodiments, nucleic acids are extracted from bacteria. In some embodiments, nucleic acids are extracted from Gram-negative bacteria. In some embodiments, nucleic acids are extracted from Gram-positive bacteria. In some embodiments, nucleic acids are extracted from fungi. In some embodiments, nucleic acids are extracted from yeast. In some embodiments, nucleic acids are extracted from animals. In some embodiments, nucleic acids are extracted from plants.

[0162] In some cases, nucleic acids are present in the body sample. In some embodiments, nucleic acids are not extracted, isolated, or otherwise purified from the body sample. The body sample may be selected from blood, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, and urine. In some embodiments, the body sample comprises a substance selected from blood, plasma, serum, tears, saliva, mucus, sputum, feces, cerebrospinal fluid, lymphatic fluid, bile, synovial fluid, cystic fluid, ascites, pleural fluid, ocular fluid, interstitial fluid, cervical fluid, and urine. In some embodiments, the body sample comprises mucus. In some embodiments, the body sample comprises a body fluid sample, tissue, or cells of the subject.

[0163] In some aspects, body samples may be collected from subjects. In some embodiments, subjects are mammals. In some embodiments, mammals are non-human primates (marmosets, macaques, chimpanzees, etc.), rodents (mice, rats, gerbils, zillo's, glovefish mice, hamsters, cottonmouths, naked mole-rats, etc.), rabbits, domestic mammals (goats, sheep, pigs, dairy cows, cattle, horses, camels, etc.), pets (dogs, cats, etc.), or zoo mammals. In some embodiments, subjects are humans.

[0164] Body samples may be collected via nasopharyngeal swab collection, cervical swab collection, or nasal swab collection from the subject. In some cases, the body sample may comprise a pathogen or a portion thereof. In some embodiments, the pathogen or a portion thereof is selected from viruses or a portion thereof, bacteria or a portion thereof, protozoa or a portion thereof, yeast or a portion thereof, and fungi or a portion thereof. In some embodiments, the pathogen is a bloodborne pathogen or a portion thereof. In some embodiments, the pathogen is a respiratory pathogen or a portion thereof. In some embodiments, the respiratory pathogen comprises a bacterial or fungal pathogen. In some embodiments, the respiratory pathogen is Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pyogenes, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis, Klebsiella pneumoniae, Staphylococcus aureus, or Aspergillus sp. In some embodiments, the pathogen is a virus or a part thereof. In some embodiments, the virus is a respiratory virus. In some embodiments, the virus is SARS-CoV-2, influenza A virus (-H1N1 and other subtypes), influenza B virus, human respiratory syncytial virus (HRSV), human parainfluenza virus type I (HPIV-1), II (HPIV-2), III (HPIV-3), IV (HPIV-4), rhinovirus / enterovirus (RV / EV), adenovirus (ADV), human metapneumovirus (hMPV), human coronavirus (HCoV)-229E, HCoV-HKU1, HCoV-NL63, or HCoV-OC43. In some embodiments, the virus is SARS-CoV-2.

[0165] In some embodiments, the virus is selected from influenza virus, rhinovirus, coronavirus, metapneumovirus, adenovirus, cynthial virus, bocavirus, and parainfluenza virus. In some embodiments, the virus is influenza virus. In some embodiments, the virus is rhinovirus. In some embodiments, the virus is coronavirus. In some embodiments, the virus is metapneumovirus. In some embodiments, the virus is adenovirus. In some embodiments, the virus is cynthial virus. In some embodiments, the virus is bocavirus. In some embodiments, the virus is parainfluenza virus.

[0166] In some aspects, the treatment sample, consisting of a physical sample, is heated in a closed heating chamber under high pressure to a temperature exceeding 100 degrees Celsius, thereby producing a heat-treated sample.

[0167] In some aspects, the treatment sample comprises cells comprising nucleic acids. The cells may be unlysed cells. The cells may be eukaryotic or prokaryotic cells. In some embodiments, the cells are bacterial cells, e.g., B. subtilis, E. coli, or S. Pyogenes. In some embodiments, the cells are fungal cells, e.g., C. albicans. In some embodiments, the cells are yeast cells, e.g., S. cerevisiae. In some embodiments, the cells are mammalian cells, e.g., Chinese hamster ovary cells, BHK cells, or mouse C127 cells. In some embodiments, the cells are human cells, e.g., HeLa cells. In some embodiments, the cells are bacterial spores, e.g., B. cereus bacterial spores.

[0168] In some embodiments, the cells may be present in a body sample. In some embodiments, the cells are embedded in a biological matrix such as nasal mucus, cerebrospinal fluid, feces, vaginal mucus, urine, or saliva. In some embodiments, the cells are embedded in nasal mucus.

[0169] The treatment sample may be an aqueous solution or a suspension. In some embodiments, the treatment sample comprises isolated nucleic acids diluted in the collection buffer. In other embodiments, the treatment sample comprises a body sample (e.g., saliva or mucus) comprising nucleic acids diluted in the collection buffer. In further embodiments, the treatment sample comprises an undiluted body sample comprising nucleic acids. In some embodiments, the treatment sample comprises cells suspended in the collection buffer. The collection solution or buffer may be prepared from pure water or be a mixture of low-buffering buffers. Examples of such buffers can be obtained based on Tris HCl buffer ranging from 1 mm to 50 mM, with or without EDTA, at concentrations ranging from 0.5 mm to 1 mM.

[0170] In some embodiments, the treatment sample has a pH of about 8.0 to about 12.0. In some embodiments, the treatment sample has a pH of about 9.0 to about 12.0. In some embodiments, the treatment sample has a pH of about 10.0 to about 12.0. In some embodiments, the treatment sample has a pH of about 11.0 to about 12.0. In some embodiments, the treatment sample has a pH of about 8.0 to about 11.0. In some embodiments, the treatment sample has a pH of about 9.0 to about 11.0. In some embodiments, the treatment sample has a pH of about 10.0 to about 11.0. In some embodiments, the treatment sample has a pH of about 8.0 to about 10.0. In some embodiments, the treatment sample has a pH of about 9.0 to about 10.0.

[0171] In some embodiments, the treatment sample has a pH greater than 7.0. In some embodiments, the treatment sample has a pH greater than 8.0. In some embodiments, the treatment sample has a pH greater than 8.5. In some embodiments, the treatment sample has a pH greater than 9.0. In some embodiments, the treatment sample has a pH greater than 9.5. In some embodiments, the treatment sample has a pH greater than 10.0. In some embodiments, the treatment sample has a pH greater than 10.5. In some embodiments, the treatment sample has a pH greater than 11.0.

[0172] In some embodiments, the treatment sample has a pH of approximately 7.0. In some embodiments, the treatment sample has a pH of approximately 8.0. In some embodiments, the treatment sample has a pH of approximately 9.0. In some embodiments, the treatment sample has a pH of approximately 10.0. In some embodiments, the treatment sample has a pH of approximately 11.0. In some embodiments, the treatment sample has a pH of approximately 12.0.

[0173] In some embodiments, the treatment sample has a pH of about 8.0 to 11.0. In some embodiments, the treatment sample has a pH of about 9.0 to 11.0. In some embodiments, the treatment sample has a pH of about 10.0 to 11.0.

[0174] In some embodiments, the treatment sample has a pH of about 8.0 to 12.0. In some embodiments, the treatment sample has a pH of about 9.0 to 12.0. In some embodiments, the treatment sample has a pH of about 10.0 to 12.0. In some embodiments, the treatment sample has a pH of about 11.0 to 12.0.

[0175] In some embodiments, the treatment sample has a pH of about 4.0 to about 7.0. In some embodiments, the treatment sample has a pH of about 5.0 to about 7.0. In some embodiments, the treatment sample has a pH of about 6.0 to about 7.0. In some embodiments, the treatment sample has a pH of about 4.0 to about 6.0. In some embodiments, the treatment sample has a pH of about 5.0 to about 6.0. In some embodiments, the treatment sample has a pH of about 4.0 to about 5.0.

[0176] In some embodiments, the treatment sample has a pH of approximately 3.0. In some embodiments, the treatment sample has a pH of approximately 4.0. In some embodiments, the treatment sample has a pH of approximately 5.0. In some embodiments, the treatment sample has a pH of approximately 6.0. In some embodiments, the treatment sample has a pH of approximately 7.0.

[0177] In some embodiments, the treatment sample has a pH below about 3.0. In some embodiments, the treatment sample has a pH below about 4.0. In some embodiments, the treatment sample has a pH below about 5.0. In some embodiments, the treatment sample has a pH below about 6.0. In some embodiments, the treatment sample has a pH below about 7.0.

[0178] In some embodiments, the volume of the treatment sample is the total volume of the biological sample, the collection buffer, and the mixture of one or more additives prior to high-pressure heating. In some embodiments, the volume of the treatment sample is from 100 μl to 5 mL. In some embodiments, the volume of the treatment sample is from 200 μl to 5 mL. In some embodiments, the volume of the treatment sample is from 300 μl to 5 mL. In some embodiments, the volume of the treatment sample is from 400 μl to 5 mL. In some embodiments, the volume of the treatment sample is from 500 μl to 5 mL. In some embodiments, the volume of the treatment sample is from 1 ml to 5 mL. In some embodiments, the volume of the treatment sample is from 1.5 ml to 5 mL. In some embodiments, the volume of the treatment sample is from 2 ml to 5 mL. In some embodiments, the volume of the treatment sample is from 2.5 ml to 5 mL. In some embodiments, the volume of the treatment sample is from 3 ml to 5 mL. In some embodiments, the volume of the treatment sample is from 3.5 ml to 5 mL. In some embodiments, the volume of the treatment sample is from 4 ml to 5 mL. In some embodiments, the volume of the treatment sample is from 4.5 ml to 5 mL.

[0179] In some embodiments, the treatment sample comprises nucleic acid at 10 copies / mL to 10 9 copies / mL. In some embodiments, the treatment sample comprises nucleic acid at 50 copies / mL to 10 9 copies / mL. In some embodiments, the treatment sample comprises nucleic acid at 100 copies / mL to 10 9 copies / mL. In some embodiments, the treatment sample comprises nucleic acid at 10 3 copies / mL to 10 9 copies / mL. In some embodiments, the treatment sample comprises nucleic acid at 10 4 copies / mL to 10 9 copies / mL. In some embodiments, the treatment sample comprises nucleic acid at 10 5 copies / mL to 10 9 copies / mL. In some embodiments, the treatment sample comprises nucleic acid at 10 6Copy / mL~10 9 It contains nucleic acids at a rate of copies / mL.

[0180] In some embodiments, the treatment sample is 10 copies / mL~10 8 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 50 copies / mL to 10 8 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 100 copies / mL to 10 8 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 10 3 Copy / mL~10 8 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 10 4 Copy / mL~10 8 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 10 5 Copy / mL~10 8 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 10 6 Copy / mL~10 8 It contains nucleic acids at a rate of copies / mL.

[0181] In some embodiments, the treatment sample is 10 copies / mL~10 7 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 50 copies / mL to 10 7 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 100 copies / mL to 10 7 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 10 3 Copy / mL~10 7 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 10 4 Copy / mL~10 7 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 10 5 Copy / mL~10 7 It contains nucleic acids at copies / mL. In some embodiments, the treatment sample is 10 6Copy / mL~10 7 It contains nucleic acids at a rate of copies / mL.

[0182] In some respects, the treatment sample comprises one or more additives. These one or more additives may include chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, proteases, nuclease inhibitors, or combinations thereof.

[0183] In some embodiments, the treatment sample comprises a chelating agent. In some embodiments, the chelating agent is an insoluble chelating agent. In some embodiments, the insoluble chelating agent comprises a chelating resin. The chelating resin may be a polymer or copolymer. The chelating resin may be a cation binder or a metal ion binder. The chelating resin may be in the form of microbeads. In some embodiments, the chelating agent comprises crosslinked polystyrene. The chelating agent may comprise one or more functional groups. One or more functional groups may comprise a sulfonic acid or sulfonic acid base, a quaternary amino group (e.g., trimethylammonium), a primary, secondary, and / or tertiary amino group (e.g., polyethyleneamine), or a carboxylic acid or carboxylic acid base. In some embodiments, the insoluble chelating agent comprises a styrenedivinylbenzene copolymer. In some embodiments, the insoluble chelating agent comprises a Chelex resin or Chelex. In some embodiments, Chelex is stored in the collection buffer.

[0184] In some embodiments, the chelating agent is a soluble chelating agent. In some embodiments, the soluble chelating agent comprises ethylenediaminetetraacetic acid (EDTA). In some embodiments, the chelating agent comprises EDTA, nitrilotriacetic acid, n-hydroxyethylethylenediaminetriacetate (HEDTA), ethylenediamine, dimercaprol, porfin, heme, hemoglobin, or chlorophyll. In some embodiments, the chelating agent comprises a simple organic acid such as oxalic acid, malic acid, rubeanoic acid, or citric acid.

[0185] In some embodiments, the chelating agent is added prior to heating at a final concentration defined as the weight / volume percentage (w / v%) of the chelating agent relative to the volume of the treatment sample (e.g., a mixture comprising a biological sample, collection buffer, chelating agent, single-stranded nucleic acid-binding protein, and reducing agent). In some embodiments, the final concentration of the chelating agent in the treatment sample prior to heating is 1%–40%, 2.5%–35%, 5%–25%, 7.5%–20%, or 10%–15% as the weight / volume percentage (w / v%) of the chelating agent relative to the volume of the treatment sample. In some embodiments, the final concentration of the chelating agent in the treatment sample prior to heating is about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, or about 20% as the weight / volume percentage (w / v%) of the chelating agent relative to the volume of the treatment sample.

[0186] In some embodiments, the treatment sample further comprises a single-stranded nucleic acid-binding (SSB) protein. In some embodiments, the single-stranded nucleic acid-binding protein is thermally stable. In some embodiments, the SSB protein is thermally stable at temperatures of 4°C to 170°C, 4°C to 160°C, 4°C to 150°C, 4°C to 140°C, 4°C to 130°C, or 4°C to 120°C. In some embodiments, the SSB protein is thermally stable at temperatures of 90°C to 170°C, 90°C to 160°C, 90°C to 150°C, 90°C to 140°C, 90°C to 130°C, 90°C to 120°C, or 90°C to 110°C.

[0187] In some embodiments, prior to heating, the final molar concentration of SSB protein in the treatment sample (e.g., a mixture comprising a biological sample, collection buffer, chelating agent, SSB protein, and reducing agent) is one of the following concentrations: 0.1 μm to 5 μm, 0.1 μm to 4 μm, 0.1 μm to 3 μm, 0.1 μm to 2 μm, 0.1 μm to 1 μm, 0.2 μm to 0.9 μm, 0.3 μm to 0.7 μm, or 0.4 μm to 0.6 μm.

[0188] In some embodiments, prior to heating, the final molar concentration of SSB protein in the treatment sample (e.g., a mixture comprising a biological sample, collection buffer, chelating agent, SSB protein, and reducing agent) is approximately 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, or 5 μm.

[0189] In some embodiments, the single-stranded nucleic acid-binding protein is derived from a thermophilic organism. In some embodiments, the thermophilic organism is a thermophilic microorganism or thermophilic bacterium. In some embodiments, the single-stranded nucleic acid-binding protein is derived from an organism selected from Thermomotoga maritima (TmaSSB), Thermomotoga neapolitana (TneSSB), Thermococcus kodakarensis (KOD), and Thermomus thermophilus (TthSSB). In some embodiments, the single-stranded nucleic acid-binding protein is derived from, for example, Thermomus aquaticus (TaqSSB) or Thermococcus kodakarensis (KOD). In some embodiments, the single-stranded nucleic acid-binding protein is derived from an organism selected from Thermomotoga maritima (TmaSSB), Thermomotoga neapolitana (TneSSB), and Thermomus thermophilus (TthSSB). In some embodiments, the single-stranded nucleic acid-binding protein is derived from Thermomotoga maritima (TmaSSB). In some embodiments, the single-stranded nucleic acid-binding protein is derived from Thermotoga neapolitana (TneSSB). In some embodiments, the single-stranded nucleic acid-binding protein is derived from Thermococcus kodakarensis (KOD). In some embodiments, the single-stranded nucleic acid-binding protein is derived from Thermomus thermophilus (TthSSB). In some embodiments, the single-stranded nucleic acid-binding protein is selected from ET SSB, E. coli SSB, KOD SSB, TthSSB, TneSSB, TmaSSB, and TaqSSB.

[0190] In some embodiments, the treatment sample comprises a reducing agent. In some embodiments, the reducing agent is added to the treatment sample prior to heating. The reducing agent may be 2-mercaptoethanol, 2-mercaptoethylamine-HCl, TCEP, cysteine-HCl, dithiothreitol (DTT), TCEP-HCl, a thiol-based reducing agent, guanidine-HCl, or urea.

[0191] In some embodiments, prior to heating, the final molar concentration of the reducing agent in the treatment sample (e.g., a mixture comprising a biological sample, collection buffer, chelating agent, SSB protein, and reducing agent) is a concentration of 0.1 mm to 10 mM, 0.1 mm to 9 mM, 0.1 mm to 8 mM, 0.1 mm to 7 mM, 0.1 mm to 6 mM, 0.1 mm to 5 mM, 0.1 mm to 4 mM, 0.1 mm to 3 mM, 0.1 mm to 2 mM, 0.2 mm to 1.8 mM, 0.4 mm to 1.6 mM, 0.6 mm to 1.4 mM, or 0.8 mm to 1.2 mM. In some embodiments, prior to heating, the final molar concentration of the reducing agent in the treatment sample (e.g., a mixture comprising a biological sample, a collection buffer, a chelating agent, an SSB protein, and a reducing agent) is about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 1.2 mM, about 1.4 mM, about 1.6 mM, about 1.8 mM, about 2 mM, about 2.5 mM, about 5 mM, about 7.5 mM, or about 10 mM.

[0192] In some embodiments, the treatment sample comprises one or more reagents selected from a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent. In some embodiments, the treatment sample comprises at least two reagents selected from a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent. In some embodiments, the treatment sample comprises a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent. In some embodiments, the concentration of the chelating agent is 2.5% to 35% as w / v% of the volume of the treatment sample, the concentration of the single-stranded nucleic acid-binding protein in the treatment sample is 0.1 μm to 5 μm, and the concentration of the reducing agent in the treatment sample is 0.1 mm to 5 mM. In some embodiments, the concentration of the chelating agent is 5% to 25% as w / v% of the volume of the treatment sample, the concentration of the single-stranded nucleic acid-binding protein in the treatment sample is 0.1 μm to 2 μm, and the concentration of the reducing agent in the treatment sample is 0.1 mm to 2 mM. In some embodiments, the concentration of the chelating agent is 10% to 15% of the volume of the treatment sample as w / v%, the concentration of single-stranded nucleic acid-binding proteins in the treatment sample is 0.3 μm to 0.7 μm, and the concentration of the reducing agent in the treatment sample is 0.6 μm to 1.4 mM.

[0193] In some embodiments, the treatment sample comprises a stabilizer. In some embodiments, the treatment sample is mixed with the stabilizer prior to heating. In some cases, the stabilizer may help prevent the degradation of nucleic acids. In some cases, the stabilizer may help inactivate molecular amplification inhibitors during high-pressure heating. In some embodiments, the stabilizer may stabilize proteins. In some embodiments, the stabilizer may stabilize nucleic acids. In some embodiments, the stabilizer may stabilize nucleic acids and proteins. The stabilizer may improve the viscosity of the treatment sample. The stabilizer may help prevent or reduce aggregation between one or more proteins. The stabilizer may improve the solubility of proteins. The stabilizer may reduce nonspecific binding between one or more components (e.g., proteins). The stabilizer may be a blocking agent. The stabilizer may be a component that stabilizes one or more components in the treatment sample. In some embodiments, the stabilizer may stabilize nucleic acids. In some embodiments, the stabilizer may stabilize chelating agents. In some embodiments, the stabilizer may stabilize single-stranded nucleic acid-binding proteins. For example, the stabilizer may be bovine serum albumin (BSA). The stabilizer may also be gelatin. In some embodiments, the stabilizer has concentrations of 100 ng / mL to 15 mg / mL, 200 ng / mL to 15 mg / mL, 300 ng / mL to 15 mg / mL, 400 ng / mL to 15 mg / mL, 500 ng / mL to 15 mg / mL, or 1 mg / mL to 15 mg / mL in the treatment sample. In some embodiments, the stabilizer has concentrations of 100 ng / mL to 10 mg / mL, 200 ng / mL to 10 mg / mL, 300 ng / mL to 10 mg / mL, 400 ng / mL to 10 mg / mL, 500 ng / mL to 10 mg / mL, or 1 mg / mL to 10 mg / mL in the treatment sample.In some embodiments, the stabilizer has concentrations of 500 ng / mL to 2 mg / mL, 500 ng / mL to 3 mg / mL, 500 ng / mL to 4 mg / mL, 500 ng / mL to 5 mg / mL, 500 ng / mL to 6 mg / mL, 500 ng / mL to 7 mg / mL, 500 ng / mL to 8 mg / mL, 500 ng / mL to 9 mg / mL, or 500 ng / mL to 10 mg / mL in the treatment sample.

[0194] In some embodiments, the treatment sample comprises one or more reagents selected from chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, and stabilizers. In some embodiments, the treatment sample comprises at least two reagents selected from chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, and stabilizers. In some embodiments, the treatment sample comprises at least three reagents selected from chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, and stabilizers. In some embodiments, the treatment sample comprises a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent. In some embodiments, the concentration of the chelating agent is 2.5% to 35% by weight / volume percentage (w / v%) of the volume of the treatment sample, the concentration of the single-stranded nucleic acid-binding protein in the treatment sample is 0.1 μm to 5 μm, the concentration of the reducing agent in the treatment sample is 0.1 mm to 5 mM, and the concentration of the stabilizer is 500 ng / mL to 10 mg / mL. In some embodiments, the concentration of the chelating agent is 5% to 25% as w / v% of the volume of the treatment sample, the concentration of single-stranded nucleic acid-binding proteins in the treatment sample is 0.1 μm to 2 μm, the concentration of the reducing agent in the treatment sample is 0.1 mm to 2 mM, and the concentration of the stabilizer is 500 ng / mL to 10 mg / mL. In some embodiments, the concentration of the chelating agent is 10% to 15% as w / v% of the volume of the treatment sample, the concentration of single-stranded nucleic acid-binding proteins in the treatment sample is 0.3 μm to 0.7 μm, the concentration of the reducing agent in the treatment sample is 0.6 mm to 1.4 mM, and the concentration of the stabilizer is 500 ng / mL to 10 mg / mL.

[0195] In some embodiments, the treatment sample comprises a protease. In some embodiments, the treatment sample is mixed with the protease prior to heating. In some embodiments, the protease is, for example, protease K. In some embodiments, the biological sample is mixed with a reducing agent and a protease prior to heating. In some embodiments, prior to heating, the final concentration of the protease in the treatment sample (a mixture comprising, for example, a biological sample, collection buffer, chelating agent, SSB protein, reducing agent, and protease) is a concentration of 0.01 mg / mL to 5 mg / mL, 0.02 mg / mL to 4 mg / mL, 0.03 mg / mL to 3 mg / mL, 0.04 mg / mL to 2 mg / mL, 0.05 mg / mL to 1 mg / mL, 0.075 mg / mL to 0.75 mg / mL, or 0.1 mg / mL to 0.5 mg / mL. In some embodiments, prior to heating, the final concentration of protease in the treatment sample (e.g., a mixture comprising a biological sample, collection buffer, chelating agent, SSB protein, reducing agent, and protease) is about 0.05 mg / mL, about 0.075 mg / mL, about 0.1 mg / mL, about 0.25 mg / mL, about 0.5 mg / mL, about 0.75 mg / mL, or about 1 mg / mL.

[0196] In some embodiments, the treatment sample comprises a nuclease inhibitor. In some embodiments, the treatment sample comprises an RNAse inhibitor. In some embodiments, the amount of RNAse inhibitor added prior to heating is, for example, about 1 U, about 10 U, about 50 U, about 100 U, about 150 U, about 200 U, about 250 U, about 300 U, about 350 U, about 400 U, about 450 U, or about 500 U.

[0197] In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 1 U to about 500 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 10 U to about 500 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 50 U to about 500 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 100 U to about 500 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 150 U to about 500 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 200 U to about 500 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 250 U to about 500 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 300 U to about 500 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 350 U to about 500 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 400 U to about 500 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is approximately 450 U to approximately 500 U.

[0198] In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 1 U to about 200 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 10 U to about 200 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 50 U to about 200 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 100 U to about 200 U. In some embodiments, the amount of RNAse inhibitor in the treatment sample is about 150 U to about 200 U.

[0199] In some embodiments, the treatment sample further comprises a molecular amplification inhibitor. In some cases, the molecular amplification inhibitor may be an active agent that binds to nucleic acids. In some cases, the molecular amplification inhibitor may be an active agent that degrades nucleic acids. In some cases, the molecular amplification inhibitor may be a nuclease. In some cases, the molecular amplification inhibitor is a DNase. In other cases, the molecular amplification inhibitor is an RNase. In some embodiments, the treatment sample comprises one or more molecular amplification inhibitors that can be inactivated after heating.

[0200] Additional details and examples of the steps for processing the sample using high-pressure conditions are disclosed in International Application No. PCT / US23 / 67879 (the entire contents of which are incorporated herein by reference).

[0201] Methods for sample processing or analysis This disclosure provides methods for sample processing and / or analysis. For example, the method can be used to expose a sample comprising nucleic acid to nucleic acid amplification, such as a real-time polymerase chain reaction (PCR). In some embodiments, a centripetal microfluidic is applied to the fluid within the PCR device. One advantage of the centripetal microfluidic is that, for example, centrifugal pressure is applied to the fluid without the need for any direct connection to the instrument, which helps to prevent contamination. In some embodiments, this disclosure provides a method for amplifying nucleic acid within a PCR device comprising a rotatable disk. In some embodiments, the method includes the step of maintaining one or more reaction samples in a fixed position within one or more reaction chambers during heating, substantially preventing evaporation within one or more reaction chambers. In some embodiments, the method includes the step of transferring a sample comprising a target nucleic acid to be analyzed to one or more reaction chambers within 30 seconds or less, and subsequently sealing one or more reaction chambers. In some embodiments, the step of sealing the reaction chambers substantially prevents any liquid or water vapor from escaping from the reaction chambers.

[0202] In some embodiments, the method includes loading a sample (e.g., a body sample, a treatment sample, or a heat-treated sample) comprising a target nucleic acid into a loading chamber on a rotatable disk. In some cases, the sample is heated in a separate heating chamber (e.g., under high-pressure heating conditions) prior to being loaded into the loading chamber. In some cases, the loading chamber is or comprises a heating chamber, and the method includes heating the sample, comprising the target nucleic acid in the loading chamber, under high-pressure conditions to produce a treated sample (e.g., a heat-treated sample), as described elsewhere in this specification. In some cases, the sample is heated under high-pressure conditions to a temperature above 100 degrees Celsius. In some cases, the sample is heated under high-pressure conditions to a temperature between 101 degrees Celsius and 160 degrees Celsius. In some cases, the heating step occurs with a temperature gradient rate of 5 degrees Celsius / second to 50 degrees Celsius / second.

[0203] In some embodiments, the sample to be loaded is a treatment sample (e.g., a nucleic acid-containing sample prior to treatment by high-pressure heating) that is heated directly within the loading chamber, as described elsewhere in this specification. In some cases, the sample to be heated, comprising a target nucleic acid, comprises a body sample selected from the group consisting of blood samples, tear samples, saliva samples, mucus samples, sputum samples, fecal samples, cerebrospinal fluid samples, and urine samples. In some cases, the target nucleic acid is not extracted, isolated, or otherwise purified from the body sample prior to heating. In some cases, the sample comprising a target nucleic acid comprises a plurality of molecular amplification inhibitors, as described elsewhere in this specification, and the heating step inactivates the molecular amplification inhibitors of the plurality of molecular amplification inhibitors in the sample. In some cases, the heating step inactivates at least 70% of the plurality of molecular amplification inhibitors, resulting in a treated sample.

[0204] In some cases, the sample further comprises one or more reagents selected from the group consisting of chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, and stabilizers. For example, as described anywhere in this specification, a body sample (e.g., saliva) comprising nucleic acids can be pre-mixed with additives (e.g., reducing agents, chelating agents, single-stranded nucleic acid-binding proteins, stabilizers, or a combination thereof) to produce a treatment sample, which is then loaded into a loading chamber where the sample is heated. Alternatively, a body sample can be loaded into a loading chamber pre-loaded with additives (e.g., reducing agents, chelating agents, single-stranded nucleic acid-binding proteins, stabilizers, or a combination thereof), the sample is mixed with one or more reagents, and then heated in the loading chamber.

[0205] During sample heating, the sample may be in a sealed or closed heating chamber. In some embodiments, following heating, an outflow valve is opened to release the sample from the loading chamber (e.g., heating chamber). The outflow valve may be opened by electromagnetic means. For example, the outflow valve may be a laser valve, which is opened using a laser. In some embodiments, the method includes the step of rotating a rotatable disk so that the processed sample flows out of the loading chamber to the outflow valve via centrifugal force. In some cases, the centrifugal force is generated using a spin speed of 3,000 RPM to 6,000 RPM. In some cases, the rotatable disk further comprises a reaction chamber and a channel connecting the loading chamber to the reaction chamber. In some cases, the rotatable disk is rotated to transfer the processed sample from the loading chamber to the reaction chamber via the channel. In some cases, the processed sample flows from the loading chamber to one or more intermediate chambers before flowing into the reaction chamber via the channel.

[0206] For example, the step of rotating a rotatable disk may cause the processed sample to flow from the loading chamber through a channel into a collection chamber (e.g., a measurement chamber) located directly downstream of the loading chamber. In some embodiments, the method for sample processing and / or analysis further includes the step of measuring the specific volume of the sample to be processed or analyzed (e.g., a nucleic acid-containing sample, a body sample, a treatment sample, or a sample after high-pressure heating treatment). In some embodiments, the sample measurement occurs after the sample has been loaded into the loading chamber on the rotatable disk. In some embodiments, the sample measurement occurs after initial sample preparation (e.g., treatment by high-pressure heating). The sample measurement may occur in a measurement chamber, which can be the loading chamber into which the sample is loaded, or a separate collection chamber into which the sample flows after initial sample preparation (e.g., high-pressure heating in a heating chamber). In some cases, the volume is predetermined depending on the requirements of the sample processing and / or analytical assay. In some cases, multiple fractions of the sample are measured for multiplexed sample processing and / or analysis. In some embodiments, following sample measurement, the measured volume is flowed towards a reagent mixing chamber. In some embodiments, the flow of the measured volume into the reagent mixing chamber is controlled by a valve (e.g., a laser valve). In some embodiments, any excess volume of sample exceeding the measured volume flows into an overflow chamber.

[0207] In some embodiments, the method for sample processing and / or analysis further includes the step of mixing the sample to be processed or analyzed with a PCR reagent mixture (e.g., an RT-PCR reagent mixture). The PCR reagent mixture may comprise polymerase or reverse transcriptase or a combination thereof. The PCR reagent mixture may comprise a buffer and a plurality of nucleotides (e.g., dNTPs). The PCR reagent mixture may comprise BSA. In some embodiments, the PCR reagent mixture comprises primers comprising a sequence complementary to at least a portion of the target nucleic acid. In some embodiments, the PCR reagent mixture comprises probes comprising a sequence complementary to at least a portion of the target nucleic acid. In some embodiments, the PCR reagent mixture comprises polymerase, reverse transcriptase, magnesium, buffer solution, BSA, and a plurality of nucleotides. In some embodiments, the PCR reagent mixture is provided as a lyophilized powder. In some embodiments, one or more of the PCR reagents in the PCR reagent mixture are provided as lyophilized reagent beads. In some embodiments, the PCR reagent mixture is provided in a reagent mixing chamber, and the sample is mixed with the PCR reagent mixture in the reagent mixing chamber. In some embodiments, the reagent mixing chamber is a loading chamber or connected thereto. In some embodiments, a reagent mixing chamber is connected to a measurement chamber and receives a measured volume of a sample comprising the target nucleic acid. The sample, comprising the target nucleic acid and a PCR reagent mixture, can be mixed by Euler force to produce a combined target nucleic acid and PCR reagent mixture.

[0208] In some embodiments, a method for sample processing and / or analysis includes the step of transferring a target nucleic acid into a reaction chamber (e.g., a cuvette) or multiple reaction chambers (e.g., multiple cuvettes) on a rotatable disk. In some cases, a combined target nucleic acid and PCR reagent mixture is transferred from a reagent mixing chamber to one or more reaction chambers. The release of the combined mixture from the reagent mixing chamber can be controlled by a valve (e.g., a laser valve). In some embodiments, the nucleic acid and PCR reagent mixture is transferred from the reagent mixing chamber to the reaction chambers or multiple reaction chambers via centrifugal force. In some cases, the centrifugal force is generated using a spin rate of 1,000 RPM to 3,000 RPM.

[0209] In some embodiments, one or more reaction chambers (e.g., cuvettes) are pre-loaded with one or more PCR reagents. For example, one or more reaction chambers may be pre-loaded with one or more primers complementary to at least a portion of the target nucleic acid. One or more primers may be supplied as lyophilized powder. In some embodiments, one or more reaction chambers are pre-loaded with probes complementary to at least a portion of the target nucleic acid. Probes may be supplied as lyophilized powder. In another embodiment, the primers or probes are dried inside one or more reaction chambers (e.g., cuvettes). In some embodiments, a sample comprising the target nucleic acid is mixed with one or more PCR reagents in a PCR reagent mixing chamber and then mixed with one or more additional PCR reagents in a reaction chamber. For example, a sample comprising the target nucleic acid may be mixed in a PCR reagent mixing chamber with a PCR reagent mixture comprising polymerase or reverse transcriptase, multiple nucleotides, BSA, and reaction buffer, and then mixed with primers and probes in a reaction chamber.

[0210] In some embodiments, the method for sample processing and / or analysis further includes the step of transferring a mixture comprising the target nucleic acid into one or more reaction chambers, and then sealing one or more reaction chambers. In some embodiments, the step of sealing the reaction chambers substantially prevents the mixture from flowing out of the chambers. In some embodiments, a rotatable disk comprises a cuvette insert comprising one or more reaction chambers and cuvette channels through which a sample containing the target nucleic acid flows into a plurality of reaction chambers (e.g., cuvettes). One or more reaction chambers (e.g., cuvettes) can be sealed using a thermal sealer. In some embodiments, the rotatable disk is in contact with a sealer, thereby sealing the channels after the processed sample has flowed into one or more reaction chambers. In some embodiments, the channels are thermal sealed at a temperature of 200 to 300 degrees Celsius. In some embodiments, the channels are sealed by compressing or crushing them using a thermal sealer. In some embodiments, the step of sealing the cuvette channels substantially prevents fluid communication between the plurality of reaction chambers.

[0211] In some embodiments, the method for sample processing and / or analysis further includes a step of amplifying a target nucleic acid in one or more reaction chambers. In some embodiments, the nucleic acid amplification reaction is carried out after the one or more reaction chambers are heat-sealed. In some embodiments, a cuvette insert comprising one or more reaction chambers is first pressed between 95°C temperature blocks for 5 to 30 seconds to activate DNA polymerase. In some embodiments, the cuvette insert is moved between different heating elements (e.g., temperature blocks) to carry out different thermal circulation steps. In some embodiments, the thermal circulation includes a step of rotating a rotatable disk to bring the reaction chamber (e.g., cuvette) adjacent to a first heating element maintained at a first temperature, thereby denaturing the target nucleic acid in the sample. In some embodiments, the thermal circulation further includes a step of rotating the rotatable disk to bring the reaction chamber adjacent to a second heating element maintained at a second temperature, thereby annealing primers to the denatured nucleic acid and replicating the target nucleic acid. In some embodiments, the thermal circulation further includes the step of rotating a rotatable disk to bring a plurality of reaction chambers (e.g., cuvettes) adjacent to a third heating element maintained at a third temperature, thereby replicating the target nucleic acid.

[0212] In some respects, this disclosure provides a method for sample analysis. The method may include (a) loading a sample comprising a target nucleic acid into a loading chamber on a rotatable disk. The rotatable disk may further comprise a plurality of reaction chambers and channels connecting the loading chamber to the plurality of reaction chambers. Following the loading of the sample, the sample may flow into the plurality of reaction chambers through the channels. The method may further include (b) bringing the rotatable disk into contact with a sealer. The sealer can seal the channels after loading and prevent fluid communication between the plurality of reaction chambers. In some embodiments, the sealer is incorporated into a PCR device. The method may further include (c) rotating the rotatable disk to bring the plurality of reaction chambers adjacent to a first heating element maintained at a first temperature. The first heating element may be used to denature the target nucleic acid in the sample. The method may further include (d) rotating the rotatable disk to bring the plurality of reaction chambers adjacent to a second heating element maintained at a second temperature. A second heating element can be used to anneal the primer to the denatured target nucleic acid and to replicate the denatured target nucleic acid. The method may further include (e) a step of exposing a plurality of reaction chambers to excitation light of a first wavelength. The method may further include (f) a step of measuring the light emitted from the plurality of reaction chambers at a second wavelength. In some cases, some of the steps described herein, for example, steps (c)-(f), can be repeated until the emitted light is measured at an intensity indicating the presence of the target nucleic acid.

[0213] Another aspect of the present disclosure relates to a method for multiplexed real-time amplification and detection of multiple different target nucleic acids using a rotatable disk comprising multiple reaction chambers (e.g., cuvettes). The method may include (a) loading a sample comprising multiple different target nucleic acids into the rotatable disk. The sample may then be transferred to the multiple reaction chambers. The sample may be mixed with multiple primers, each of which is complementary to a corresponding portion of the multiple different target nucleic acids. The multiple primers may be pre-loaded into the multiple reaction chambers. The method may further include (b) rotating the rotatable disk to bring the multiple reaction chambers adjacent to a first heating element maintained at a first temperature. The first heating element may be used to denature each of the multiple target nucleic acids in the sample. The method may further include (c) rotating the rotatable disk to bring the multiple chambers adjacent to a second heating element maintained at a second temperature. A second heating element may be used to anneal multiple primers to the corresponding target nucleic acids denatured in (b) and to replicate multiple different target nucleic acids. The method may further include (d) a step of exposing multiple reaction chambers (e.g., cuvettes) to excitation light having multiple excitation wavelengths. The method may further include a step of detecting a signal from a sample. For example, the method may further include (e) a step of measuring the emitted light from multiple reaction chambers (e.g., cuvettes) having multiple emission wavelengths, each of which corresponds to the presence of each of multiple different target nucleic acids. In some cases, some of the steps, e.g., steps (b)-(e), may be repeated until the emitted light is measured at an intensity indicating the presence of multiple different target nucleic acids. In some embodiments, a rotatable disk further comprises a loading chamber that fluidly communicates with the multiple reaction chambers through channels.In some embodiments, prior to (b), the method further includes bringing a rotatable disk into contact with a sealer, thereby sealing the channel and preventing fluid communication between multiple reaction chambers (e.g., cuvettes).

[0214] In some embodiments, the sealer is housed within an analytical device for real-time PCR, thereby enabling a fully integrated sample processing / analysis response system by integrating a microfluidic sample preparation system and a nucleic acid processing system. In some embodiments, each reaction chamber (e.g., cuvette) is sealed by contact or non-contact means to avoid evaporation and escape of the heated liquid. In some embodiments, channel sealing is achieved via heat and pressure, via laser welding, or via ultrasonic welding. In some embodiments, channel sealing is achieved via heat and pressure, by a thermal sealer, etc., but not limited to thermal sealers. In some embodiments, the sealer is a thermal sealer. In some embodiments, the sealer comprises a first element configured to provide thermal energy and pressure onto a rotatable disk, and a second element configured to provide a reaction force to the pressure. In some embodiments, the step of bringing the rotatable disk and the sealer into contact includes the step of applying pressure onto the rotatable disk so that the channel deforms.

[0215] In some embodiments, prior to (b), the method further includes the step of rotating a rotatable disk to generate sufficient centripetal force on the sample to allow the sample to flow through the channels into multiple reaction chambers. In some embodiments, sufficient centripetal force is generated by spinning the rotatable disk at about 500 RPM to about 15,000 RPM.

[0216] In some embodiments, the channel is defined by two plastic sheets welded together by two parallel lines. In some embodiments, the channel comprises a thin plastic film with a diameter ranging from about 1 μm to about 1 mm. In some embodiments, the channel defines a lumen with a diameter ranging from about 1 μm to about 1 mm. In some embodiments, the channel comprises a thermoplastic material that seals the channel when heated to a target temperature. In some embodiments, the target temperature ranges from about 110°C to about 300°C.

[0217] In some embodiments, the reaction chamber (Figure 5 503) is defined by a thermoplastic film (Figure 5 501) sealed to a sealing film (Figure 5 502).

[0218] In another embodiment, the reaction chamber is defined by two films, a film (Figure 6 607), a thermoformed film (Figure 6 601), and a sealing film (Figure 6 602), with both sides sealed by two films. In this embodiment, the depth of the reaction chamber is defined not by thermoformation, but by the thickness of the film (Figure 6 607), which is 0.1 to 1 mm.

[0219] In some embodiments, the excitation light or emission light each comprises one wavelength. In some embodiments, the excitation light or emission light each comprises two or more wavelengths. In some embodiments, the excitation light or emission light each comprises 1 to 10 wavelengths. In some embodiments, the excitation light or emission light each comprises 1 to 6 wavelengths. In some embodiments, the excitation light or emission light each comprises 1 to 4 wavelengths. In some embodiments, the excitation light and emission light are independently selected between approximately 400 nm and approximately 750 nm for each generation. In some embodiments, the excitation light and emission light are a pair of wavelengths associated with excitation and emission from a phosphor probe. For example, the first and second wavelengths are compatible with FAM (e.g., 495 nm / 520 nm), SUN (e.g., 538 nm / 554 nm), 5'TEX615 (e.g., 596 nm / 613 nm), or Cy5 (e.g., 648 nm / 667 nm) detection. For example, the first wavelength can be independently selected from approximately 495 nm, 538 nm, 596 nm, and 648 nm, although this is not an exhaustive list. For example, the second wavelength can be independently selected from approximately 520 nm, 554 nm, 613 nm, and 667 nm, although this is not an exhaustive list.

[0220] In some embodiments, the sample is mixed with primers that are complementary to at least a portion of the target nucleic acid, which are pre-loaded into a plurality of reaction chambers. In some embodiments, the plurality of reaction chambers further comprises probes, and the sample is further mixed with the probes. In some embodiments, the plurality of primers are stored in the plurality of reaction chambers. In some embodiments, the plurality of primers are provided as lyophilized powders. In some embodiments, the probes are provided as lyophilized powders. In another embodiment, the primers and probes are dried inside each reaction chamber (e.g., cuvette).

[0221] In some embodiments, the sample is mixed with a PCR reagent mixture prior to being transferred to multiple reaction chambers. In some embodiments, the PCR reagent mixture comprises polymerase, reverse transcriptase, magnesium, buffer solution, BSA, and multiple nucleotides. In some embodiments, the components of the PCR reagent mixture are pre-mixed before contacting the sample or loading it into multiple reaction chambers. In some embodiments, the PCR reagent mixture is provided in a reagent mixing chamber, and the sample is mixed with the PCR reagent mixture within the reagent mixing chamber. In some embodiments, the PCR reagent mixture is provided as a lyophilized powder. In some embodiments, the sample is mixed with the PCR reagent mixture prior to being transferred into multiple reaction chambers.

[0222] In some embodiments, the reaction chambers are aligned at the same radial distance from the center of the rotatable disk and are equally spaced apart from one another. In some embodiments, the reaction chambers (e.g., cuvettes) occupy a 360-degree arc on the rotatable disk. In some embodiments, the reaction chambers occupy a segment of the arc on the rotatable disk ranging from approximately 10 to 100 degrees. In some embodiments, the reaction chambers (e.g., cuvettes) comprise 1 to 100 reaction chambers. In some embodiments, each reaction chamber has a volume ranging from approximately 5 μL to approximately 100 μL and a depth ranging from approximately 0.1 mm to approximately 1.0 mm. In some embodiments, the reaction chambers have a depth ranging from approximately 0.1 to approximately 0.7 mm. In some embodiments, the reaction chambers have a maximum depth of approximately 0.25 mm.

[0223] In some embodiments, the method of the present disclosure further includes the step of rotating a rotatable disk to position a plurality of reaction chambers (e.g., cuvettes) adjacent to a second heating element, followed by the step of rotating the rotatable disk to position a plurality of reaction chambers (e.g., cuvettes) adjacent to a third heating element maintained at a third temperature. In some embodiments, the first temperature is maintained over a first period, and the second temperature is maintained over a second period. In some embodiments, the third temperature is maintained over a third period. In some embodiments, the first period is selected from about 500 milliseconds to about 2 seconds, and the second period is selected from about 2 seconds to about 18 seconds. In some embodiments, the third period is selected from about 1 second to about 6 seconds. In some embodiments, the first temperature is selected from about 90°C to about 99°C, and the second temperature is selected from about 50°C to about 74°C. In some embodiments, the third temperature is selected from about 65°C to about 75°C. In some embodiments, the first heating element has a first radial length, and the second heating element has a second radial length. In some embodiments, the second radial length is about 6 to 9 times the first radial length. In some embodiments, the third heating element has a third radial length. In some embodiments, the third radial length is about 2 to 3 times the first radial length. In some embodiments, all blocks have the same radial length.

[0224] In some embodiments, the method further includes stabilizing the rotatable disk for a first period at a first temperature, for a second period at a second temperature, and / or for a third period at a third temperature. In some embodiments, the stabilization step includes clamping the rotatable disk between the first heating block and the second heating block of the first heating element, the second heating element, or the third heating element. In some embodiments, following the step of clamping the rotatable disk, the method further includes unclamping the rotatable disk from the first heating block and the second heating block of the first, second, or third heating element.

[0225] In some embodiments, excitation light is supplied by an optical head or a plurality of optical heads. In some embodiments, the optical head or a plurality of optical heads further comprises one or more wavelength filters to vary a first wavelength or a plurality of wavelengths for the excitation light. For example, all excitation wavelengths are activated sequentially over an excitation period. In some embodiments, the excitation period is about 200 milliseconds to about 500 milliseconds. In some embodiments, the excitation period is about 350 milliseconds. In other embodiments, all wavelengths are activated simultaneously by the optical head. In some embodiments, the emitted light is measured on a first fluorescence detector or a plurality of fluorescence detectors. In some embodiments, the first detector or a plurality of fluorescence detectors comprises one or more wavelength channels to measure the emission intensity. For example, all wavelengths are detected or measured sequentially over a measurement period. In some embodiments, the measurement period is about 200 milliseconds to about 500 milliseconds. In some embodiments, the measurement period is about 350 milliseconds. In other embodiments, all wavelengths are measured simultaneously on the fluorescence detector.

[0226] Another aspect of the present disclosure relates to a method for multiplexed real-time amplification and detection of multiple target sequences in a rotatable disk comprising a reaction chamber, the method comprising: (a) loading a sample comprising multiple target sequences into a reaction chamber (e.g., a cuvette), the sample being mixed with primers and / or probes stored in the reaction chamber; (b) thermally circulating the sample to be mixed with a PCR reaction mixture by rotating a rotatable disk, thereby sequentially bringing the reaction chamber closer to a plurality of heating elements positioned adjacent to the rotatable disk, each of which is maintained at a denaturation temperature, an annealing temperature, or an extension temperature; and (c) detecting a combination of multiple fluorescence signals from the sample and a first PCR reaction mixture.

[0227] In some embodiments, the rotatable disk further comprises a loading chamber that is in fluid communication with the reaction chamber through a channel. In some embodiments, the method further includes the step of loading a sample into the loading chamber. In some embodiments, the channel is defined by two plastic sheets welded together by two parallel lines. In some embodiments, the channel comprises a thin plastic film with a z dimension of about 1 μm to about 1 mm.

[0228] In some embodiments, the reaction chamber (e.g., cuvette) comprises one or more reaction chambers. In some embodiments, each reaction chamber has a volume of about 5 μL to about 100 μL and a depth of about 0.1 mm to about 1.0 mm. In some embodiments, the reaction chamber has a maximum depth of about 0.25 mm.

[0229] In some embodiments, the step of loading a sample into the reaction chamber includes rotating a rotatable disk to generate sufficient centripetal force on the sample to flow the sample through the channel into the reaction chamber. In some embodiments, sufficient centripetal force is generated by spinning the rotatable disk at about 500 RPM to about 15,000 RPM.

[0230] In some embodiments, prior to the step of thermally circulating the sample, the method further includes the step of bringing a rotatable disk and a sealer into contact, thereby sealing the channel and preventing fluid communication with the reaction chamber (e.g., cuvette). In some embodiments, the sealer is housed within an analytical device for real-time PCR, thereby enabling a fully integrated sample processing / analysis response system by integrating a microfluidic sample preparation system and a nucleic acid processing system. In some embodiments, each reaction chamber is sealed by contact or non-contact means to avoid evaporation and escape of the heated liquid. In some embodiments, sealing of the channel is achieved via heat and pressure, via laser welding, or via ultrasonic welding. In some embodiments, sealing of the channel is achieved via heat and pressure, by a thermal sealer, etc., but not limited to these. In some embodiments, the sealer is a thermal sealer. In some embodiments, the sealer comprises a first element configured to provide thermal energy and pressure onto the rotatable disk, and a second element configured to provide a reaction force to the pressure. In some embodiments, the step of bringing the rotatable disk into contact with the sealer includes the step of applying pressure to the rotatable disk so that the channel deforms.

[0231] In some embodiments, the step of thermally circulating the mixed sample includes sequentially maintaining the reaction chamber at the denaturation temperature for about 500 milliseconds to about 2 seconds, at the annealing temperature for about 3 seconds to about 18 seconds, and at the extension temperature for about 1 second to about 6 seconds. In some embodiments, the denaturation temperature is selected from about 90°C to about 99°C, the annealing temperature is selected from about 50°C to about 74°C, and the extension temperature is selected from about 65°C to about 75°C.

[0232] In some embodiments, prior to the step of detecting multiple fluorescence wavelengths, the method further includes the step of exposing the reaction chamber to multiple excitation wavelengths. In some embodiments, each of the multiple fluorescence signals corresponds to one target sequence of multiple target sequences. In some embodiments, each of the multiple excitation wavelengths and multiple fluorescence signals comprises one wavelength. In some embodiments, each of the multiple excitation wavelengths and multiple fluorescence signals comprises two or more wavelengths. In some embodiments, each of the multiple excitation wavelengths and multiple fluorescence signals comprises 1 to 10 wavelengths. In some embodiments, each of the multiple excitation wavelengths and multiple fluorescence signals comprises 1 to 6 wavelengths. In some embodiments, each of the multiple excitation wavelengths and multiple fluorescence signals comprises 1 to 4 wavelengths. In some embodiments, the multiple excitation wavelengths and multiple fluorescence signals are independently selected between approximately 400 nm and approximately 750 nm for each generation. In some embodiments, the multiple excitation wavelengths and multiple fluorescence signals are paired wavelengths associated with excitation and emission from the phosphor probe. For example, but not limited to, multiple excitation wavelengths and multiple fluorescence signals can be compatible with FAM (e.g., 495nm / 520nm), SUN (e.g., 538nm / 554nm), TEX615 (e.g., 596nm / 613nm), or Cy5 (e.g., 648nm / 667nm) detection. For example, but not limited to, multiple excitation wavelengths can be independently selected from approximately 495nm, approximately 538nm, approximately 596nm, and approximately 648nm. For example, but not limited to, multiple fluorescence signals can be independently selected from approximately 520nm, approximately 554nm, approximately 613nm, and approximately 667nm.

[0233] In some embodiments, the first PCR reagent mixture comprises a plurality of primers complementary to at least a portion of a plurality of target sequences, and a plurality of fluorescent probes complementary to each of the plurality of target sequences. In some embodiments, the primers or probes are provided as lyophilized powders.

[0234] In some embodiments, prior to the step of loading the sample into the reaction chamber, the method further includes the step of contacting the sample with the PCR reagent mixture. In some embodiments, the PCR reaction mixture comprises polymerase and a plurality of nucleotides. In some embodiments, the PCR reagent mixture is provided in a mixing chamber on a rotatable disk, and the sample is mixed with the PCR reagent mixture in the mixing chamber. In some embodiments, the PCR reagent mixture is provided in the mixing chamber as a lyophilized powder.

[0235] This disclosure provides a method for amplifying a target nucleic acid. The method may include (a) providing a sample comprising the target nucleic acid into a loading chamber on a rotatable disk. The rotatable disk may further comprise a plurality of reaction chambers (e.g., cuvettes) that are in fluid communication with the loading chamber through channels. The method may further include (b) sealing the channels by bringing the rotatable disk into contact with a sealer in the housing of an analytical device. Fluid communication between the plurality of reaction chambers (e.g., cuvettes) can be prevented after sealing. The method may further include (c) exposing the rotatable disk to thermal circulation within the housing of an analytical device, thereby amplifying the target nucleic acid.

[0236] In some embodiments, the thermal circulation includes the step of rotating a rotatable disk to bring a reaction chamber (e.g., a cuvette) adjacent to a first heating element maintained at a first temperature, thereby denaturing a target nucleic acid in the sample. In some embodiments, the thermal circulation further includes the step of rotating the rotatable disk to bring a reaction chamber adjacent to a second heating element maintained at a second temperature, thereby annealing a primer to the denatured nucleic acid and replicating the target nucleic acid. In some embodiments, the thermal circulation further includes the step of rotating a rotatable disk to bring a plurality of reaction chambers (e.g., cuvettes) adjacent to a third heating element maintained at a third temperature, thereby replicating the target nucleic acid. In some embodiments, the first temperature is maintained over a first period. The second temperature is maintained over a second period, and the third temperature is maintained over a third period. In some embodiments, the first temperature is selected from about 90°C to about 99°C, the second temperature is selected from about 50°C to about 74°C, and the third temperature is selected from about 65°C to about 75°C. In some embodiments, the first period is selected from about 500 milliseconds to about 2 seconds, the second period is selected from about 3 seconds to about 18 seconds, and the third period is selected from about 1 second to about 6 seconds. In some embodiments, the first heating element has a first radial length, the second heating element has a second radial length, and the third heating element has a third radial length. In some embodiments, the second radial length is about 6 to about 9 times the first radial length, and the third radial length is about 2 to about 3 times the first radial length. In some embodiments, the method further includes independently stabilizing the rotatable disk for a first period at a first temperature, for a second period at a second temperature, and for a third period at a third temperature. In some embodiments, the stabilization step includes clamping the rotatable disk between a first heating element, a second heating element, or a first heating block and a second heating block of a third heating element. In some embodiments, the method further includes, following the stabilization step, unclamping the rotatable disk from the first, second, or third heating element.

[0237] In some embodiments, following thermal circulation within the housing of the device, the method further includes the step of exposing the reaction chamber to excitation light of a first wavelength. In some embodiments, the method further includes the step of measuring the light emitted from the reaction chamber at a second wavelength. In some embodiments, the target nucleic acid comprises a plurality of different target nucleic acids. In some embodiments, the method further includes the step of exposing the plurality of different target nucleic acids in the reaction chamber to excitation light of a plurality of wavelengths. In some embodiments, the excitation light of a plurality of wavelengths comprises about 400 nm to about 750 nm. In some embodiments, the method further includes the step of detecting emitted light of a plurality of wavelengths from the plurality of different target nucleic acids. In some embodiments, the emitted light of a plurality of wavelengths comprises about 400 nm to about 750 nm. In some embodiments, the plurality of excitation wavelengths and the plurality of fluorescence signals each comprise one wavelength. In some embodiments, the plurality of excitation wavelengths and the plurality of fluorescence signals each comprise two or more wavelengths. In some embodiments, the plurality of excitation wavelengths and the plurality of fluorescence signals each comprise 1 to 10 wavelengths. In some embodiments, the plurality of excitation wavelengths and the plurality of fluorescence signals each comprise 1 to 6 wavelengths. In some embodiments, the multiple excitation wavelengths and multiple fluorescence signals each comprise one to four wavelengths. In some embodiments, the multiple excitation wavelengths and multiple fluorescence signals are independently selected between approximately 400 nm and approximately 750 nm for each generation. In some embodiments, the multiple excitation wavelengths and multiple fluorescence signals are paired wavelengths associated with excitation and emission from the phosphor probe. For example, but are not limited, the multiple excitation wavelengths and multiple fluorescence signals may be compatible with FAM (e.g., 495 nm / 520 nm), SUN (e.g., 538 nm / 554 nm), TEX615 (e.g., 596 nm / 613 nm), or Cy5 (e.g., 648 nm / 667 nm) detection. For example, but are not limited, the multiple excitation wavelengths may be independently selected from approximately 495 nm, approximately 538 nm, approximately 596 nm, and approximately 648 nm. For example, although not limited to them, multiple fluorescence signals can be independently selected from approximately 520 nm, 554 nm, 613 nm, and 667 nm.

[0238] In some embodiments, the excitation light is supplied by an optical head or a plurality of optical heads. In some embodiments, the optical head or a plurality of optical heads further comprises one or more wavelength filters to vary the first or a plurality of wavelengths for the excitation light. For example, all excitation wavelengths are activated sequentially over the excitation period. In some embodiments, the excitation period is about 200 milliseconds to about 500 milliseconds. In some embodiments, the excitation period is about 350 milliseconds. In another embodiment, all wavelengths are activated simultaneously by the optical head. In some embodiments, two or more excitation wavelengths selected from about 495 nm, about 538 nm, about 596 nm, and about 648 nm are used simultaneously to illuminate the reaction chamber (e.g., cuvette). In some embodiments, three or more excitation wavelengths selected from about 495 nm, about 538 nm, about 596 nm, and about 648 nm are used simultaneously to illuminate the reaction chamber (e.g., cuvette). In some embodiments, a total of four excitation wavelengths, selected from approximately 495 nm, 538 nm, 596 nm, and 648 nm, are used simultaneously to illuminate the reaction chamber (e.g., cuvette). In some embodiments, one or more filters are used to prevent crosstalk between different wavelengths.

[0239] In some embodiments, the emitted light is measured on a first fluorescence detector or a plurality of fluorescence detectors. In some embodiments, the first detector or the plurality of fluorescence detectors have one or more wavelength channels and measure the emission intensity. For example, all fluorescence emission wavelengths may be detected or measured sequentially over a measurement period. In some embodiments, the measurement period is about 200 milliseconds to about 500 milliseconds. In some embodiments, the measurement period is about 350 milliseconds. In some embodiments, substantially crosstalking fluorescence emission wavelengths of a phosphor are measured sequentially. In some embodiments, substantially overlapping fluorescence emission wavelengths are measured sequentially. In some embodiments, substantially crosstalking, or substantially overlapping, fluorescence emission wavelengths of a phosphor are measured in separate passages through a reaction chamber with multiple heating elements or in separate cycles of heat circulation. In some embodiments, substantially non-crosstalking fluorescence emission wavelengths of a phosphor are measured simultaneously. In some embodiments, two or more substantially non-overlapping fluorescence emission wavelengths may be measured simultaneously. In some embodiments, fluorescence emission wavelengths of a phosphor that are substantially free from crosstalk, or substantially non-overlapping, are measured over a single pass through a reaction chamber with multiple heating elements, or over a single cycle of thermal circulation.

[0240] In some embodiments, the fluorescence emission wavelength is measured over cycles exceeding one of the thermal circulations. In some embodiments, the fluorescence emission wavelength is measured over passes over one of the reaction chambers passing through multiple heating elements. For example, fluorescence signals from FAM (e.g., at 520 nm) and TEX615 (e.g., at 613 nm) can be measured during a first pass (e.g., cycle) through the reaction chamber passing through multiple heating elements, while fluorescence signals from Cy5 (e.g., at 667 nm) and SUN (e.g., at 554 nm) can be measured during a second pass (e.g., cycle) through the reaction chamber passing through multiple heating elements.

[0241] In another embodiment, without limitation, all fluorescence emission wavelengths are measured simultaneously on a fluorescence detector. In some embodiments, all fluorescence emission wavelengths are measured in a single pass during one cycle of thermal cycling. In some embodiments, all wavelengths are measured in a single pass from when one or more reaction chambers are transferred from a heating element maintained at an annealing temperature to when one or more reaction chambers are transferred to a heating element maintained at a denaturation temperature.

[0242] In some embodiments, the channel comprises a thermoplastic material that seals the channel when heated to a target temperature. In some embodiments, the target temperature ranges from 110 to 300 °C.

[0243] In some embodiments, the plurality of reaction chambers contain a PCR reaction mixture. In some embodiments, the PCR reaction mixture comprises at least one of the following: a primer, a polymerase, and a plurality of nucleotides.

[0244] In some embodiments, the plurality of reaction chambers are each aligned at the same radial distance from the center of a rotatable disk and are equally spaced from each other. In some embodiments, the plurality of reaction chambers occupy a 360-degree arc on the rotatable disk. In some embodiments, the plurality of reaction chambers occupy a segment of an arc of about 10 degrees to 100 degrees on the rotatable disk. In some embodiments, the plurality of reaction chambers (e.g., cuvettes) comprise from 1 to 100 reaction chambers. In some embodiments, the plurality of reaction chambers each have a volume of from about 10 μL to about 100 μL. In some embodiments, the plurality of reaction chambers (e.g., cuvettes) each have a depth of from about 0.1 mm to about 1.0 mm. In some embodiments, the plurality of reaction chambers (e.g., cuvettes) have a maximum depth of about 0.25 mm.

[0245] In one aspect, the present disclosure is directed to a method for real-time polymerase chain reaction (PCR), the method comprising (a) A step of loading a sample comprising a target nucleic acid into a loading chamber on a rotatable disk, the rotatable disk further comprising a plurality of reaction chambers (e.g., cuvettes) and channels connecting the loading chamber to the plurality of reaction chambers (e.g., cuvettes), wherein the sample flows through the channels into the plurality of reaction chambers (e.g., cuvettes) following the loading of the sample. (b) The step of bringing a rotatable disk into contact with a sealer, thereby sealing the channel and preventing fluid communication between multiple reaction chambers (e.g., cuvettes) after filling, (c) Rotating a rotatable disk to position multiple reaction chambers (e.g., cuvettes) adjacent to a first heating element maintained at a first temperature, thereby denaturing the target nucleic acid in the sample, (d) Rotating a rotatable disk to position multiple reaction chambers (e.g., cuvettes) adjacent to a second heating element maintained at a second temperature, thereby annealing the primers to the denatured target nucleic acid and replicating the denatured target nucleic acid, (e) Rotating a rotatable disk to position multiple reaction chambers (e.g., cuvettes) adjacent to a first heating element, while simultaneously exposing the multiple reaction chambers (e.g., cuvettes) to excitation light of a first wavelength, a second wavelength, a third wavelength, and a fourth wavelength; (f) The step of measuring the light emitted at first, second, third, and fourth wavelengths from a plurality of reaction chambers (e.g., cuvettes), (h) Repeat steps (c)-(f) until the emitted light is measured at an intensity that indicates the presence of the target nucleic acid, Includes.

[0246] In another aspect, this disclosure relates to a method for multiplexed real-time amplification and detection of multiple different target nucleic acids, using a rotatable disk comprising multiple reaction chambers (e.g., cuvettes), the method (a) A step of loading a sample comprising multiple different target nucleic acids into a rotatable disk, thereby transferring the sample to multiple reaction chambers (e.g., cuvettes), wherein the sample is mixed with multiple primers and probes, each of which primers and probes is complementary to a corresponding portion of the multiple different target nucleic acids. (b) Rotating a rotatable disk to position multiple reaction chambers (e.g., cuvettes) adjacent to a first heating element maintained at a first temperature, thereby denaturing each of the multiple target nucleic acids in the sample, (c) Rotating a rotatable disk and positioning multiple chambers adjacent to a second heating element maintained at a second temperature, thereby annealing multiple primers to the corresponding target nucleic acids denatured in (b), thereby replicating multiple different target nucleic acids, (d) Exposing multiple reaction chambers (e.g., cuvettes) to excitation light comprising multiple excitation wavelengths, (e) A step of measuring light emitted from a plurality of reaction chambers (e.g., cuvettes), wherein each of the emission wavelengths corresponds to the presence of each of a plurality of different target nucleic acids. (f) Repeat steps (b)-(e) until the emitted light is measured at an intensity that indicates the presence of multiple different target nucleic acids, Includes.

[0247] This disclosure provides a method for multiplexed real-time amplification and detection of multiple different target nucleic acids in a biological sample. In one aspect, the method includes the steps of (a) loading a sample into a loading chamber on a rotatable disk, the rotatable disk further comprising a plurality of reaction chambers and channels that fluidly connect the loading chamber to the plurality of reaction chambers, the reaction chambers being loaded with a PCR reagent mixture comprising a plurality of primers and a plurality of phosphor probes, the sample being mixed with the plurality of primers, each of which is complementary to a corresponding portion of the plurality of different target nucleic acids, the sample flowing through the channels into the plurality of reaction chambers, thereby filling the reaction chambers following the loading of the sample; (b) bringing the channels in the rotatable disk into contact with a sealer, thereby sealing the channels after filling and preventing fluid communication between the plurality of reaction chambers; and (c) rotating the rotatable disk and placing the plurality of reaction chambers on a first heating element maintained at a first temperature. (d) (c Devices for sample preparation, processing, or analysis

[0248] This disclosure provides analytical devices (Figure 7) for preparing, processing, and / or analyzing target nucleic acids. In some embodiments, the analytical device comprises a heating chamber for sample preparation. In some embodiments, the heating chamber is connected to a downstream microfluidic network, and the heat-treated sample can be transferred after heating. In some embodiments, the analytical device comprises a measurement chamber, a mixing chamber, one or more reaction chambers, or a combination thereof, and the heat-treated sample undergoes further processing and / or analysis. The analytical device is or may comprise a thermal circulation device. For example, the analytical device may comprise one or more heating elements. In some embodiments, one or more reaction chambers are housed in a rotatable disk that moves the reaction chambers from one heating element to a second heating element, thereby enabling thermal circulation of the reaction chambers between different temperatures. In some embodiments, the analytical device comprises heating elements that thermally seal one or more reaction chambers after they are loaded with reaction mixtures, thereby substantially preventing evaporation or escape of the reaction mixture.

[0249] In some embodiments, the Disclosure provides a heating chamber for sample preparation. The heating chamber may be a closed heating chamber. A closed heating chamber may be used to heat a sample to be treated, as described anywhere in this Spec. A closed heating chamber can substantially prevent air and vapor from entering or leaving the chamber. In some cases, there is a negligible airflow inside and outside the closed heating chamber. A closed heating chamber may remain closed during high-pressure heating. In some embodiments, the closed heating chamber is an inner chamber of a heating vessel.

[0250] In some embodiments, a closed chamber is opened at some point to release the sample from the heating vessel. In some embodiments, the sample may be released through an outlet, which may be part of an outlet channel 214 in the heating vessel (Figures 2, 18, 19). In some embodiments, the sample is released into a collection chamber (e.g., a measurement chamber) 205 (Figures 2, 18). In some embodiments, the collection chamber is part of the heating vessel. In some embodiments, the collection chamber and the heating vessel are part of a fluid network. In some cases, the measurement chamber may be designed to measure multiple fractions of the sample for multiplexed sample analysis.

[0251] The heating container and / or enclosed heating chamber may be made of different materials. In some embodiments, the heating container and / or enclosed heating chamber comprises glass. In some embodiments, the heating container and / or enclosed heating chamber comprises high-temperature polycarbonate. In some embodiments, the heating container and / or enclosed heating chamber comprises a thermally conductive material. In some embodiments, the heating container and / or enclosed heating chamber comprises metal. In some embodiments, the heating container and / or enclosed heating chamber may comprise zinc, stainless steel, copper, copper alloy, gold, silver, aluminum, aluminum nitride, iron, nickel, nickel alloy, cobalt, carbon fiber, platinum, brass, tungsten, silicon, silicon carbide, diamond, or graphite. In some embodiments, the heating container and / or enclosed heating chamber comprises a conductive material. In some embodiments, the heating container and / or enclosed heating chamber comprises a ferromagnetic material such as iron, nickel, or cobalt. For example, the heating container and / or enclosed heating chamber may be a glass ampoule, a plastic container, or a metal container, or comprise one thereof. In some embodiments, the heating vessel and / or closed heating chamber comprises an induction susceptor 1503 (e.g., a metal cup) (Figures 15A-15C, 16A-16B, 18) which may be used to heat a treatment sample by induction heating through contact with an induction platform 1701 (Figure 17).

[0252] In some embodiments, the heating vessel comprises a heating chamber 203 containing the treatment sample during high-pressure heating (Figures 16A-16B). The material of the vessel and / or heating chamber may be selected so that the heating chamber can have a temperature gradient of 2°C to 50°C, 2°C to 40°C, 2°C to 35°C, 2°C to 30°C, 2°C to 25°C, 2°C to 20°C, or 2°C to 15°C. The material of the container and / or heating chamber may be selected so that the heating chamber can have a temperature gradient of 5°C / sec to 60°C / sec, 5°C / sec to 50°C / sec, 5°C / sec to 40°C / sec, 5°C / sec to 35°C / sec, 5°C / sec to 30°C / sec, 5°C / sec to 25°C / sec, 5°C / sec to 20°C / sec, or 5°C / sec to 15°C / sec.

[0253] In some embodiments, the heating chamber is closed during high-pressure heating to prevent air and vapor from entering or leaving the chamber. In some embodiments, the closed heating chamber remains closed under high-pressure conditions, and the pressure inside the chamber is higher than the pressure outside the chamber. The material of the container or heating chamber may be selected so that the closed heating chamber can remain closed during high-pressure heating, for example, at room temperature to 160 degrees Celsius or when the pressure inside the heating chamber is greater than 1 atm and between 1 and 200 PSI.

[0254] In some embodiments, the closed heating chamber is or may comprise a glass ampoule. The glass ampoule may have a fused sample inlet. In other embodiments, the closed heating chamber is or may comprise a plastic container. The plastic container may comprise a plastic resin or a heat-resistant plastic, such as polycarbonate, high-density polypropylene, PEEK, or PEI. For example, the plastic container may be part of a cryogenic tube or microfluidic cartridge. In further embodiments, the closed heating chamber is or may comprise a metal container. The metal container may comprise an inductive susceptor (e.g., a metal cup).

[0255] In some embodiments, a closed heating chamber is created by sealing an open heating chamber. The sealing operation may include a step of fusing the open sample inlet. The sealing operation may include a step of using a cap, lid, plug, or valve. The manner in which the heating chamber is sealed may be selected so that the closed heating chamber can remain closed during high-pressure heating.

[0256] In some embodiments, the heating vessel includes a sample inlet 202 through which a sample can flow into a heating chamber 203, as depicted in Figures 2, 15A-C, 16A-16B, 18, and 20A-20B. The sample inlet may be sealed, creating a closed heating chamber within the heating vessel. In some embodiments, the sample inlet is sealed using a cap, lid, plug, or valve. In some embodiments, the sample inlet may be equipped with a valve. The valve may be a one-way valve 1601 (e.g., a duckbill valve) (Figures 16A, 16B) that allows the sample to flow into the heating chamber and prevents air and / or the sample from flowing out of the heating chamber. In some cases, the sample is introduced through the one-way valve 1601 (Figures 16A, 16B). The sample may be introduced manually using a pipette or injection needle. In other cases, the sample may be introduced through an automated system. The heating vessel may be part of a fluid system, and the one-way valve may be in contact with an upstream component of the fluid system. In some embodiments, the one-way valve can further prevent air and steam from flowing out of the heating chamber. The one-way valve provides a seal 1602 for the closed chamber during high-pressure heating (Figures 16A, 16B).

[0257] In some embodiments, a closed chamber is opened at some point to release the sample from the heating vessel. The closed chamber may be opened after high-pressure heating. In some embodiments, the heating chamber can be opened using mechanical force. In some embodiments, the heating chamber is opened by removing a cap, lid, plug, or valve from the outlet. In some embodiments, the heating chamber is opened by opening the outlet valve 204 at the outlet. For example, the outlet valve 204 may be a laser valve that is initially closed and can be opened by shining a laser beam onto the valve (Figure 19, 20A-20B). In some cases, the outlet may be the same as or in the same location as the sample inlet. In other cases, the outlet may be in a location separate from the sample inlet.

[0258] In some embodiments, the sample may be released through an outlet. The outlet may be part of an outlet channel 214 in the heating vessel (Figures 2, 18, 19). The outlet channel 214 may be equipped with or in contact with an outlet valve 204 (Figures 2, 18, 19). The outlet channel 214 may initially be sealed by the outlet valve 204, for example, during sample injection and high-pressure heating in operation 1101 in Figure 20A. The outlet channel 214 may then be unsealed by opening the outlet valve 204 for sample release in operation 1102 in Figure 20B. Once the outlet valve 204 is open, the sample may flow out of the heating chamber through the outlet channel 214. The sample may be propelled out of the heating chamber through the outlet channel 214 using a force, such as centrifugal force. In some embodiments, the sample is released into a collection chamber (e.g., a measurement chamber) 205 (Figure 2, 18). In some embodiments, the collection chamber is part of the heating vessel. In some embodiments, the collection chamber and heating vessel are part of a fluid network. The collection chamber may be a measuring chamber 205, designed to measure the precise volume of the sample depending on the requirements of subsequent sample analysis. In some cases, the measuring chamber is designed to measure different volumes for different sample analysis assays. In other cases, the measuring chamber may be designed to measure multiple fractional amounts of the sample for multiplexed sample analysis.

[0259] In some embodiments, the present disclosure provides an analysis device (FIG. 7) for processing and / or analyzing a target nucleic acid. In some embodiments, the analysis device is a PCR device. In some embodiments, the analysis device comprises a rotatable disk (FIG. 2) having a plurality of reaction chambers (e.g., cuvettes) (FIG. 22). The analysis device provided herein can comprise a housing. The housing can comprise a sample holder configured to hold and rotate the rotatable disk (FIG. 7 701). The device provided herein can further comprise a sealer (FIG. 7 703) configured to seal the reaction chambers while the rotatable disk is on the sample holder. The device can further comprise a first heating element (FIG. 7 704) maintained at a first temperature and configured to heat the rotatable disk to the first temperature. The device can further comprise a second heating element (FIG. 7 705) maintained at a second temperature and configured to heat the rotatable disk to the second temperature.

[0260] In some embodiments, the analysis device further comprises a rotatable platform capable of spinning at a maximum speed of 15,000 RPM. In some embodiments, the analysis device comprises a rotatable disk held within the sample holder. In some embodiments, the rotatable disk further comprises a loading chamber (e.g., a heating chamber) (FIG. 2 203) and a channel (FIG. 2 208) connecting the loading chamber to a plurality of reaction chambers (e.g., cuvettes) (FIG. 2 209). In some embodiments, the loading chamber is or comprises a heating chamber.

[0261] In some embodiments, the sealer is housed within an analytical device for real-time PCR, thereby enabling a fully integrated sample processing / analysis response system by integrating a microfluidic sample preparation system and a nucleic acid processing system. In some embodiments, each reaction chamber is sealed by contact or non-contact means to avoid evaporation and escape of the heated liquid. In some embodiments, channel sealing is achieved via heat and pressure, via laser welding, or via ultrasonic welding. In some embodiments, channel sealing is achieved via heat and pressure, by a thermal sealer, etc., but not limited to thermal sealers. In some embodiments, the sealer is a thermal sealer. In some embodiments, the sealer comprises a first element configured to provide thermal energy and pressure onto a rotatable disk, and a second element configured to provide a reaction force to the pressure. In some embodiments, the step of bringing the rotatable disk and the sealer into contact includes the step of applying pressure onto the rotatable disk so that the channel deforms.

[0262] In some embodiments, a plurality of reaction chambers (e.g., cuvettes) contain a first PCR reaction mixture comprising primers and a fluorescent probe. In some embodiments, a mixing chamber contains a second PCR reaction mixture comprising polymerase and a plurality of nucleotides.

[0263] In some embodiments, the multiple reaction chambers (e.g., cuvettes) have a volume of about 10 μL to about 100 μL and a depth of about 0.1 mm to about 1.0 mm. In some embodiments, each of the multiple reaction chambers (e.g., cuvettes) has a maximum depth of 0.25 mm. In some embodiments, the multiple reaction chambers (e.g., cuvettes) comprises 1 to 100 reaction chambers (e.g., cuvettes). In some embodiments, the channel has a z dimension of about 1 μm to about 1 mm and a width of about 1 mm to about 5 mm. In some embodiments, the aspect ratio of each of the multiple reaction chambers (e.g., cuvettes) to the channel is at least 10:1.

[0264] In some embodiments, each of the multiple reaction chambers (e.g., cuvettes) has an interior and an exterior. In some embodiments, each of the multiple reaction chambers (e.g., cuvettes) is suitable for transmitting excitation light of multiple wavelengths from the outside of the multiple reaction chambers (e.g., cuvettes), and is suitable for transmitting emitted light of multiple wavelengths from the inside of the multiple reaction chambers (e.g., cuvettes).

[0265] In some embodiments, the channel comprises a thermoplastic material that seals the channel when heated to a temperature of 110–300°C. In some embodiments, the rotatable disk comprises a thermoformed film and a sealing film that combine together to form a plurality of reaction chambers (e.g., cuvettes). In some embodiments, the thermoformed film forms the shape and size for the plurality of reaction chambers (e.g., cuvettes). In some embodiments, the thermoformed film has a Tg higher than about 100°C. In some embodiments, the sealing film and the thermoformed film comprises a resin. In some embodiments, the resin comprises a single polymer. In some embodiments, the resin comprises an inner polymer and an outer polymer, the inner polymer having a lower Tg than the outer polymer. In some embodiments, the single polymer, inner polymer, and outer polymer are each independently selected from polyolefins, polycarbonates, polystyrene, polymethylmethylacrylates, polyethylene, and polypropylene. In some embodiments, the resin for the sealing film and the resin for the thermoformed film are heat-sealed together. In some embodiments, the resin for the sealing film and the resin for the thermoformed film are identical.

[0266] In some embodiments, the first heating element has a first radial length, and the second heating element has a second radial length, the second radial length being about 6 to about 9 times the radial length of the first radial length. In some embodiments, the analytical device further comprises a third heating element configured to heat the rotatable disk to a third temperature and maintain it at a third temperature. In some embodiments, the third radial length is about 2 to about 3 times the radial length of the first radial length. In some embodiments, the first heating element, the second heating element, and the third heating element each independently comprise a pair of heating blocks designed to clamp the rotatable disk between them.

[0267] In some embodiments, the analytical device (Figure 7) further comprises multiple optical heads (Figure 7-706). In some embodiments, the multiple optical heads provide excitation light sources of one or more wavelengths. In some embodiments, each excitation light source comprises one wavelength. In some embodiments, each excitation light source comprises two or more wavelengths. In some embodiments, each excitation light source comprises 1 to 10 wavelengths. In some embodiments, each excitation light source comprises 1 to 6 wavelengths. In some embodiments, each excitation light source comprises 1 to 4 wavelengths. In some embodiments, the excitation light sources are independently selected between approximately 400 nm and approximately 750 nm for each generation. In some embodiments, the excitation light sources are associated with excitation for a phosphor probe. For example, but not limited to, the excitation light source can be compatible with FAM (e.g., 495nm / 520nm), SUN (e.g., 538nm / 554nm), TEX615 (e.g., 596nm / 613nm), or Cy5 (e.g., 648nm / 667nm) detection. For example, but not limited to, the excitation light source can be independently selected from approximately 495nm, approximately 538nm, approximately 596nm, and approximately 648nm.

[0268] In some embodiments, the optical head further comprises multiple filters to add one or more wavelengths to the excitation light source. In some embodiments, the analytical device further comprises a fluorescence detector to measure the emission intensity for one or more wavelengths. In some embodiments, the fluorescence detector can simultaneously measure the emission intensity for more than one wavelength.

[0269] In another aspect, this disclosure relates to an analytical device for processing target nucleic acids, comprising a housing, A sample holder configured to hold and rotate a rotatable disk comprising multiple reaction chambers (e.g., cuvettes), A sealer, configured to seal the channel, connects to the reaction chamber while the rotatable disk is on the sample holder, A first heating element configured to heat a rotatable disk to a first temperature and maintain it at a first temperature, A second heating element configured to heat a rotatable disk to a second temperature and maintain it at a second temperature, It is equipped with.

[0270] Rotatable disk for sample processing or analysis This disclosure also provides a rotatable disk (Figure 2) for sample processing or analysis. The rotatable disk may comprise a loading chamber 203 (e.g., a high-pressure heating chamber). The loading chamber may be a heating chamber (e.g., configured for high-pressure heating of a sample) as described elsewhere herein. The rotatable disk may comprise one or more reaction chambers (e.g., cuvettes) 209. In some embodiments, each reaction chamber is aligned at the same radial distance from the center of the rotatable disk. In some embodiments, multiple reaction chambers are aligned at different radial distances from the center of the rotatable disk. The rotatable disk may further comprise channels 208 connecting the loading chamber (e.g., a high-pressure heating chamber) to the multiple reaction chambers (e.g., cuvettes). In some embodiments, the rotatable disk further comprises one or more intermediate chambers (e.g., measuring chambers or mixing chambers) between the loading chamber and one or more reaction chambers, the one or more intermediate chambers being in fluid communication with the loading chamber and one or more reaction chambers. In some cases, channels connect the loading chamber, one or more intermediate chambers, and one or more reaction chambers.

[0271] In some cases, the rotatable disk comprises a loading chamber configured to seal a sample under high-pressure conditions to a temperature above 100 degrees Celsius, as described elsewhere in this specification. In some cases, the loading chamber is configured to seal a sample under high-pressure conditions to a temperature between 101 degrees Celsius and 160 degrees Celsius. The loading chamber may be configured to be heated by induction heating, as described elsewhere in this specification. In some cases, the loading chamber comprises one or more additives (e.g., chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, stabilizers, or combinations thereof), as described elsewhere in this specification. In some cases, the loading chamber comprises a one-way valve (e.g., a duckbill valve), as described elsewhere in this specification. The loading chamber is a closed chamber during high-pressure heating that can substantially prevent air from flowing out of the chamber. The loading chamber may comprise an outflow valve (e.g., a laser valve) configured to be opened after high-pressure heating. In some cases, the loading chamber is equipped with or connected to a channel through which the sample can be discharged in response to the opening of the discharge valve.

[0272] In some cases, the rotatable disk comprises a measurement chamber 205 (Figure 2). In some cases, the measurement chamber is located downstream of the loading chamber (e.g., a heating chamber). In some cases, the measurement chamber is connected to the loading chamber via a channel. A valve is located between the loading chamber and the measurement chamber and can control the flow of the sample (e.g., a heat-treated sample) from the loading chamber to the measurement chamber. The measurement chamber may be configured to measure a specific volume of the sample into one or more downstream chambers that are in fluid communication with the measurement chamber. In some cases, the volume is configured according to the requirements of the sample processing and / or analytical assay. In some cases, the measurement chamber is connected to one or more reaction chambers via a channel and is configured to measure a specific volume of the sample into one or more reaction chambers. In some cases, the measurement chamber is connected to an intermediate chamber such as a mixing chamber and is configured to measure a specific volume of the sample into the intermediate chamber. In some embodiments, the rotatable disk further comprises an overflow chamber 213 (Figure 2) that is in fluid communication with the measurement chamber and is configured to receive an excess volume of the sample that is not measured and is not transferred to the reaction chamber. In some embodiments, the rotatable disk includes a mixing chamber 207 (Figure 2) that is in fluid communication with the loading chamber and one or more reaction chambers. In some cases, the mixing chamber is further in fluid communication with the measurement chamber. In some cases, the mixing chamber is located downstream of the loading chamber and / or the measurement chamber and upstream of one or more reaction chambers. In some cases, a channel connects the mixing chamber to the loading chamber and one or more reaction chambers. In some cases, a channel further connects the mixing chamber to the measurement chamber.

[0273] In some embodiments, one or more reaction chambers (e.g., cuvettes) have a volume of about 5 μL to about 100 μL. In some embodiments, one or more reaction chambers (e.g., cuvettes) have a depth of about 0.1 mm to about 1.0 mm. In some embodiments, one or more reaction chambers (e.g., cuvettes) have a depth of about 0.2 to about 0.7 mm. In some embodiments, one or more reaction chambers (e.g., cuvettes) have a maximum depth of 0.25 mm. In some embodiments, one or more reaction chambers (e.g., cuvettes) are equally spaced apart from each other. In some embodiments, the multiple reaction chambers (e.g., cuvettes) occupy a 360-degree arc on a rotatable disk. In some embodiments, the multiple reaction chambers (e.g., cuvettes) occupy a segment of an arc of about 10 to 100 degrees on a rotatable disk. In some embodiments, the multiple reaction chambers (e.g., cuvettes) comprise 1 to 100 reaction chambers (e.g., cuvettes). In some embodiments, each of the multiple reaction chambers (e.g., cuvettes) has an interior and an exterior. In some embodiments, each of the multiple reaction chambers (e.g., cuvettes) is suitable for transmitting excitation light of multiple wavelengths from the outside of the multiple reaction chambers (e.g., cuvettes), and is suitable for transmitting emitted light of multiple wavelengths from the inside of the multiple reaction chambers (e.g., cuvettes).

[0274] In some embodiments, the rotatable disk comprises a thermoformed film and a sealing film, which are joined together to form a plurality of reaction chambers (e.g., cuvettes). In some embodiments, the thermoformed film forms the shape and size for the plurality of reaction chambers (e.g., cuvettes) and the shape and size for the channels. In some embodiments, the sealing film is sealed to the thermoformed film. In some embodiments, the sealing film is sealed to the thermoformed film via a framework 607, and two sides are co-extruded with a heat-sealed compliant material. In some embodiments, the thermoformed film has a thickness of about 50 μm to about 500 μm. In some embodiments, the ratio of the channel width to the thickness of the thermoformed film is greater than 2. In some embodiments, the sealing film has a thickness of about 10 μm to about 500 μm. In some embodiments, the thermoformed film has a Tg higher than about 100°C.

[0275] In some embodiments, the sealing film and the thermoformed film comprise a resin. In some embodiments, the resin comprises a single polymer. In some embodiments, the resin comprises an inner polymer and an outer polymer, the inner polymer having a lower Tg than the outer polymer. In some embodiments, the single polymer, the inner polymer, and the outer polymer are each independently selected from polyolefins, polycarbonates, polystyrene, polymethylmethylacrylates, polyethylene, and polypropylene. In some embodiments, the resin for the sealing film and the resin for the thermoformed film can be heat-sealed together. In some embodiments, the resin for the sealing film and the thermoformed film are identical.

[0276] In some embodiments, the channel connects a loading chamber, measuring chamber, mixing chamber, or a combination thereof to one or more reaction chambers. In some embodiments, the channel has a z dimension of about 1 μm to about 1 mm, a width of about 1 mm to about 5 mm, and a depth of up to about 200 μm. In some embodiments, the channel has a width of about 2 mm to about 4 mm and a depth of up to about 100 μm. In some cases, the channel has a depth of 10 μm to 500 μm, 20 to 400 μm, 30 to 300 μm, 40 to 200 μm, or 50 to 100 μm. In some embodiments, the rotatable disk further has an aspect ratio of the channel to each of the multiple reaction chambers (e.g., cuvettes), where the aspect ratio is at least 10:1. In some embodiments, the rotatable disk further has an aspect ratio of the channel to each of the multiple reaction chambers (e.g., cuvettes), where the aspect ratio is at least 20:1.

[0277] In some embodiments, the channel comprises a thermoplastic material. In some embodiments, the thermoplastic material has a thickness of about 10 μm to about 400 μm. In some cases, the thermoplastic material seals the channel when heated to a temperature of 110 to 300 degrees Celsius, 120 to 280 degrees Celsius, or 130 to 260 degrees Celsius. In some embodiments, the thermoplastic material is selected from polycarbonate, polypropylene, polyethylene terephthalate, and cyclic olefin copolymers. In some cases, the channel comprises a thermoformed thermoplastic film that is sealed to a sealing thermoplastic film. In some cases, the channel is heat-sealed by compressing the thermoformed film on the top of the channel into contact with the sealing film on the bottom of the channel. In some cases, the channel comprises a co-extruded thermoplastic film with an inner thermoplastic film layer having a lower Tg than the outer thermoplastic film layer. In some cases, the inner layer is configured to seal the channel when heated to a temperature of 110 to 300 degrees Celsius, 120 to 280 degrees Celsius, or 130 to 260 degrees Celsius. In some cases, the inner layer is configured to seal the channel by compression of the inner thermoplastic film layer and the outer layer.

[0278] system This disclosure also provides a system for sample processing or analysis. For example, the system can be used to expose a sample containing a target nucleic acid molecule to nucleic acid amplification. The nucleic acid amplification may comprise a real-time polymerase chain reaction (PCR). The system may comprise (a) a rotatable disk comprising a loading chamber, a plurality of reaction chambers (e.g., cuvettes), and channels connecting the loading chamber to the plurality of reaction chambers. The system may comprise (b) a sample holder for holding the rotatable disk in a substantially horizontal plane. The system may comprise a device configured to rotate the rotatable disk in a substantially horizontal plane. The system may further comprise (c) a sealer. The sealer may be coupled to an actuator that moves the sealer, thereby changing the distance between the sealer and the rotatable disk. The system may comprise (d) a first heating element located in close proximity to a second portion of the rotatable disk and maintained at a first temperature. The system may comprise (e) a second heating element located in close proximity to a second portion of the rotatable disk and maintained at a second temperature. The system may include (f) a light source oriented to generate excitation light of a first wavelength in a horizontal plane occupied by the reaction chambers (e.g., cuvettes) of a rotatable disk. The system may also include (g) a photodetector oriented to detect emitted light of a second wavelength emitted from the reaction chambers (e.g., cuvettes) of a rotatable disk. In various embodiments, once a liquid sample comprising nucleic acid is loaded into a loading chamber, the liquid sample can flow through channels into multiple reaction chambers (e.g., cuvettes). The liquid sample can be mixed with a PCR reagent mixture comprising primers, polymerase, and multiple nucleotides. The rotatable disk can be in contact with a thermal sealer to seal the channels and prevent fluid communication between the multiple reaction chambers (e.g., cuvettes).The rotatable disk is rotated in a substantially horizontal plane, and the rotatable disk can be oriented such that (i) multiple reaction chambers (e.g., cuvettes) are adjacent to a first heating element, thereby denaturing nucleic acids in a sample; (ii) multiple reaction chambers (e.g., cuvettes) are adjacent to a second heating element, thereby annealing primers to the denatured nucleic acids and replicating the nucleic acids; and / or (iii) excitation light generated by a light source flows into the reaction chambers, thereby generating emitted light that is emitted from the reaction chambers and detected by a light chamber.

[0279] In another respect, this disclosure pertains to a system for real-time polymerase chain reaction (PCR), which is (a) A rotatable disk comprising a loading chamber, a plurality of reaction chambers (e.g., cuvettes), and channels connecting the loading chamber to the plurality of reaction chambers, (b) A sample holder for holding a rotatable disk in a substantially horizontal plane, wherein the device is configured to rotate the rotatable disk within a substantially horizontal plane, (c) A sealer is connected to an actuator that moves the sealer, thereby changing the distance between the sealer and the rotatable disk, (d) A first heating element located adjacent to the second portion of the rotatable disk, which is maintained at a first temperature, (e) A second heating element located adjacent to the second portion of the rotatable disk and maintained at a second temperature, (f) A light source oriented to generate excitation light of a first wavelength in a horizontal plane occupied by the reaction chamber of a rotatable disk, (g) A first photodetector oriented to detect emitted light of a second wavelength emitted from the reaction chamber of the rotatable disk, It is equipped with.

[0280] In another aspect, the present disclosure provides a system for real-time polymerase chain reaction (PCR), the system comprising: (a) a rotatable disk comprising a loading chamber, a plurality of reaction chambers, and channels connecting the loading chamber to the plurality of reaction chambers, wherein the loading chamber is configured to be heated by induction heating; and (b) an analytical device comprising: (i) a sample holder for receiving the rotatable disk in a substantially horizontal plane, the sample holder configured to rotate the rotatable disk in a substantially horizontal plane; and (ii) a first heating element in contact with the reaction chambers and configured to heat the plurality of reaction chambers to a first temperature; and (iii) a second heating element in contact with the reaction chambers and configured to heat the plurality of reaction chambers to a second temperature.

[0281] In one aspect, the present disclosure provides a system for real-time polymerase chain reaction (PCR) comprising: (a) a rotatable disk comprising a loading chamber, a plurality of reaction chambers, and channels connecting the loading chamber to the plurality of reaction chambers; and (b) an analytical device comprising: (i) a sample holder for receiving the rotatable disk in a substantially horizontal plane, configured to rotate the rotatable disk in a substantially horizontal plane; (ii) a first heating element configured to heat the loading chamber to a first temperature above 100 degrees Celsius; (iii) a second heating element in contact with the reaction chambers and configured to heat the plurality of reaction chambers to a second temperature; and (iv) a third heating element in contact with the reaction chambers and configured to heat the plurality of reaction chambers to a third temperature.

[0282] In some cases, the system includes a second photodetector oriented to detect emitted light of a third wavelength emitted from the reaction chamber of a rotatable disk. In other cases, the system includes a third photodetector oriented to detect emitted light of a fourth wavelength emitted from the reaction chamber of a rotatable disk. In some cases, the first, second, and third photodetectors are different photodetectors. In other cases, the first, second, and third photodetectors are the same photodetector.

[0283] In some cases, when a liquid sample containing nucleic acid flows from a loading chamber through a channel into a plurality of reaction chambers, the rotatable disk comes into contact with a thermal sealer, thereby sealing the channel and preventing fluid communication between the plurality of reaction chambers. In other cases, the rotatable disk is rotated in a substantially horizontal plane, (i) positioning the plurality of reaction chambers adjacent to a first heating element, thereby denaturing the nucleic acid in the sample; (ii) positioning the plurality of reaction chambers adjacent to a second heating element, thereby annealing primers to the denatured nucleic acid and replicating the nucleic acid; and (iii) oriented the rotatable disk so that excitation light generated by a light source flows into the reaction chambers, thereby generating emitted light that is emitted from the reaction chambers and detected by a photodetector.

[0284] In some cases, the system comprises one or more PCR reagents to be mixed with a liquid sample. In some cases, the system comprises one or more PCR reagents in a loading chamber. In some cases, the system further comprises a mixing chamber installed to provide fluid communication between the loading chamber and the reaction chamber, and the liquid sample from the loading chamber is mixed with one or more PCR reagents or a mixture of PCR reagents. In some cases, the system further comprises one or more PCR reagents pre-loaded in the reaction chamber, and one or more PCR reagents are mixed with the sample. One or more PCR reagents may comprise polymerase or multiple nucleotides, primers, probes, or a combination thereof. In some embodiments, the loading chamber or mixing chamber comprises a first PCR reaction mixture comprising polymerase and multiple nucleotides, and the reaction chamber comprises a second PCR reaction mixture comprising primers and probes. In some embodiments, the loading chamber or mixing chamber comprises a PCR reaction mixture comprising primers, polymerase, and multiple nucleotides, and the reaction chamber comprises probes.

[0285] In some aspects, this disclosure provides a system for real-time polymerase chain reaction (PCR) comprising a loading chamber, a plurality of reaction chambers, and a rotatable disk having channels connecting the loading chamber to the plurality of reaction chambers. In some cases, the loading chamber is located at the center of the rotatable disk. In some cases, the plurality of reaction chambers are located at the periphery of the rotatable disk. In some cases, the loading chamber is configured to be heated by induction heating, as described elsewhere herein. In some cases, the loading chamber comprises a chelating agent, a single-stranded nucleic acid-binding protein, a reducing agent, a stabilizer, or a combination thereof, as described elsewhere herein.

[0286] In some cases, the system further comprises an analytical device comprising a sample holder for receiving a rotatable disk in a substantially horizontal plane, and configured to rotate the rotatable disk in a substantially horizontal plane. In some cases, the analytical device comprises a first heating element configured to contact the reaction chambers and heat a plurality of reaction chambers to a first temperature, and a second heating element configured to contact the reaction chambers and heat a plurality of reaction chambers to a second temperature. In some cases, the analytical device further comprises a third heating element configured to heat a loading chamber to a temperature above 100 degrees Celsius. In some cases, the third heating element is configured to heat the loading chamber to a temperature between 101 degrees Celsius and 160 degrees Celsius. In some cases, the third heating element is 0.5°C / sec to 50°C / sec, 0.5°C / sec to 40°C / sec, 0.5°C / sec to 35°C / sec, 0.5°C / sec to 30°C / sec, 0.5°C / sec to 25°C / sec, 0.5°C / sec to 20°C / sec, 0.5°C / sec to 15°C / sec, 2°C / sec to 50°C / sec, 2°C / sec to 40°C / sec, 2°C / sec to 35°C / sec, 2°C / sec to 50°C / sec The loading chamber is configured to heat at a temperature gradient of 30 degrees / second, 2 degrees / second to 25 degrees / second, 2 degrees / second to 20 degrees / second, 2 degrees / second to 15 degrees / second, 5 degrees / second to 50 degrees / second, 5 degrees / second to 40 degrees / second, 5 degrees / second to 35 degrees / second, 5 degrees / second to 30 degrees / second, 5 degrees / second to 25 degrees / second, 5 degrees / second to 20 degrees / second, or 5 degrees / second to 15 degrees / second. In some cases, the analytical device further comprises a light source oriented to generate excitation light of a first wavelength in a horizontal plane occupied by the multiple reaction chambers of the rotatable disk. In some cases, the analytical device further comprises a photodetector oriented to detect emitted light of a second wavelength emitted from the multiple reaction chambers of the rotatable disk.

[0287] Additional methods and devices This disclosure provides a method for multiplexed real-time amplification and detection of multiple target sequences in a rotatable disk comprising a reaction chamber, the method comprising: (a) loading a sample comprising multiple target sequences into a reaction chamber, the sample being mixed with primers and / or probes stored in the reaction chamber; (b) thermally circulating the sample to be mixed with a PCR reaction mixture by rotating a rotatable disk, thereby sequentially bringing the reaction chamber closer to a plurality of heating elements positioned adjacent to the rotatable disk, each of which is maintained at a denaturation temperature, an annealing temperature, or an extension temperature; and (c) detecting a combination of multiple fluorescence signals from the sample and a first PCR reaction mixture.

[0288] In some embodiments, the reaction chamber comprises one or more reaction chambers. In some embodiments, the step of thermally circulating the sample includes, sequentially, maintaining the reaction chamber at the denaturation temperature for about 500 milliseconds to about 2 seconds, at the annealing temperature for about 3 seconds to about 18 seconds, and at the extension temperature for about 1 second to about 6 seconds. In some embodiments, the denaturation temperature is selected from about 90°C to about 99°C, the annealing temperature is selected from about 50°C to about 74°C, and the extension temperature is selected from about 65°C to about 75°C. In some embodiments, the rotatable disk further comprises a loading chamber that is in fluid communication with the reaction chamber through a channel. In some embodiments, the method further includes a step of loading the sample into the loading chamber prior to (a). In some embodiments, the step of loading the sample into the reaction chamber in (a) includes rotating the rotatable disk to generate sufficient centripetal force on the sample to flow the sample into the reaction chamber through the channel. In some embodiments, sufficient centripetal force is generated by spinning the rotatable disk at about 500 RPM to about 15,000 RPM. In some embodiments, the channel is defined by two plastic sheets welded together by two parallel lines. In some embodiments, the channel comprises a thin plastic film with a z dimension of about 1 μm to about 1 mm. In some embodiments, the method further includes, prior to (b), bringing the rotatable disk into contact with a sealer, thereby sealing the channel and preventing fluid communication with the reaction chamber. In some embodiments, the sealer is a thermal sealer. In some embodiments, each reaction chamber has a volume of about 5 μL to about 100 μL and further has a depth of about 0.1 mm to about 1.0 mm. In some embodiments, the reaction chamber has a maximum depth of about 0.25 mm. In some embodiments, the method further includes, prior to (c), exposing the reaction chamber to a plurality of excitation wavelengths.In some embodiments, each of the multiple excitation wavelengths and multiple fluorescence signals has wavelengths independently selected between approximately 400 nm and approximately 750 nm for each generation. In some embodiments, each of the multiple fluorescence signals corresponds to one target sequence of multiple target sequences. In some embodiments, the first PCR reagent mixture comprises multiple primers complementary to at least some of the multiple target sequences and multiple fluorescence probes complementary to each of the multiple target sequences. In some embodiments, the primers or probes are provided as lyophilized powders. In some embodiments, the method further includes the step of contacting the sample with the PCR reagent mixture prior to the step of loading the sample into the reaction chamber. In some embodiments, the PCR reaction mixture comprises polymerase and multiple nucleotides. In some embodiments, the PCR reagent mixture is provided in a mixing chamber on a rotatable disk, and the sample is mixed with the PCR reagent mixture in the mixing chamber. In some embodiments, the PCR reagent mixture is provided in a mixing chamber as lyophilized powders.

[0289] This disclosure provides a method for amplifying a target nucleic acid, the method comprising: (a) providing a sample comprising a target nucleic acid into a loading chamber on a rotatable disk, the rotatable disk further comprising a plurality of reaction chambers that are in fluid communication with the loading chamber through channels; (b) bringing the rotatable disk into contact with a sealer in the housing of an analytical device to seal the channels and thereby prevent fluid communication between the plurality of reaction chambers; and (c) exposing the rotatable disk to the thermal circulation in the housing of an analytical device to amplify the target nucleic acid.

[0290] In some embodiments, the thermal circulation in (c) includes the step of rotating a rotatable disk to bring the reaction chamber adjacent to a first heating element maintained at a first temperature, thereby denaturing the target nucleic acid in the sample. In some embodiments, the thermal circulation in (c) includes the step of rotating a rotatable disk to bring the reaction chamber adjacent to a second heating element maintained at a second temperature, thereby annealing the primer to the denatured nucleic acid and replicating the target nucleic acid. In some embodiments, the thermal circulation in (c) further includes the step of rotating a rotatable disk to bring a plurality of reaction chambers adjacent to a third heating element maintained at a third temperature, thereby replicating the target nucleic acid. In some embodiments, the method further includes, following (c), the step of exposing the reaction chamber to excitation light of a first wavelength. In some embodiments, the method further includes the step of measuring the light emitted from the reaction chamber at a second wavelength. In some embodiments, the target nucleic acid comprises a plurality of different target nucleic acids. In some embodiments, the method further includes the step of exposing multiple different target nucleic acids in a reaction chamber to excitation light of multiple wavelengths. In some embodiments, the excitation light of multiple wavelengths comprises approximately 400 nm to approximately 750 nm. In some embodiments, the method further includes the step of detecting emission light of multiple wavelengths from multiple different target nucleic acids. In some embodiments, the emission light of multiple wavelengths comprises approximately 400 nm to approximately 750 nm. In some embodiments, the channel comprises a thermoplastic material that seals the channel when heated to a target temperature. In some embodiments, the target temperature is 110 to 300°C. In some embodiments, the multiple reaction chambers contain a PCR reaction mixture. In some embodiments, the PCR reaction mixture comprises a PCR reagent selected from the group consisting of primers, polymerase, and multiple nucleotides. In some embodiments, each of the multiple reaction chambers is aligned at the same radial distance from the center of a rotatable disk and is equally spaced apart from one another. In some embodiments, the multiple reaction chambers occupy a 360-degree arc on the rotatable disk.In some embodiments, the multiple reaction chambers occupy a section of arc about 10 to 100 degrees on a rotatable disk. In some embodiments, the multiple reaction chambers comprise 1 to 100 reaction chambers. In some embodiments, each of the multiple reaction chambers has a volume of about 10 μL to about 100 μL. In some embodiments, each of the multiple reaction chambers has a depth of about 0.1 mm to about 1.0 mm. In some embodiments, the multiple reaction chambers have a maximum depth of about 0.25 mm. In some embodiments, a first temperature is maintained over a first period. A second temperature is maintained over a second period, and a third temperature is maintained over a third period. In some embodiments, the first temperature is selected from about 90°C to about 99°C, the second temperature is selected from about 50°C to about 74°C, and the third temperature is selected from about 65°C to about 75°C. In some embodiments, the first period is selected from about 500 milliseconds to about 2 seconds, the second period is selected from about 3 seconds to about 18 seconds, and the third period is selected from about 1 second to about 6 seconds. In some embodiments, the first heating element has a first radial length, the second heating element has a second radial length, and the third heating element has a third radial length. In some embodiments, the second radial length is about 6 to about 9 times the first radial length, and the third radial length is about 2 to about 3 times the first radial length. In some embodiments, the method further includes independently stabilizing the rotatable disk over a first period at a first temperature, over a second period at a second temperature, and over a third period at a third temperature. In some embodiments, the stabilization step includes clamping the rotatable disk between a first heating element, a second heating element, or a first heating block and a second heating block of a third heating element. In some embodiments, the method further includes, following the stabilization step, unclamping the rotatable disk from the first, second, or third heating element. In some embodiments, the excitation light is supplied by a plurality of optical heads. In some embodiments, the plurality of optical heads further comprises a plurality of filters to vary the excitation light over a plurality of wavelengths.In some embodiments, the emitted light is measured on a first detector. In some embodiments, the first detector comprises one or more fluorescence detectors that measure the emission intensity for emitted light at multiple wavelengths. In some embodiments, the sealer is a thermal sealer.

[0291] This disclosure provides an analytical device for processing target nucleic acids, comprising: a housing comprising a sample holder configured to hold and rotate a rotatable disk comprising a plurality of reaction chambers; a sealer configured to seal the reaction chambers while the rotatable disk is on the sample holder; a first heating element configured to heat the rotatable disk to a first temperature and maintained at a first temperature; and a second heating element configured to heat the rotatable disk to a second temperature and maintained at a second temperature.

[0292] In some embodiments, the analytical device further comprises a rotatable platform capable of spinning at a speed of at least 15,000 RPM. In some embodiments, the analytical device comprises a rotatable disk held within a sample holder. In some embodiments, the sealer is a thermal sealer. In some embodiments, the rotatable disk further comprises a loading chamber and a channel connecting the loading chamber to a plurality of reaction chambers. In some embodiments, the channel comprises a thermoplastic material that seals the channel when heated to a temperature of 110–300°C. In some embodiments, the plurality of reaction chambers comprises a first PCR reaction mixture comprising primers and a phosphor probe. In some embodiments, the rotatable disk further comprises a mixing chamber, which comprises a second PCR reaction mixture comprising polymerase and a plurality of nucleotides. In some embodiments, the plurality of reaction chambers comprises a volume of about 10 μL to about 100 μL and a depth of about 0.1 mm to about 1.0 mm. In some embodiments, each of the plurality of reaction chambers comprises a maximum depth of 0.25 mm. In some embodiments, the plurality of reaction chambers comprises 1 to 100 reaction chambers. In some embodiments, the channel has a z dimension of about 1 μm to about 1 mm and a width of about 1 mm to about 5 mm. In some embodiments, the aspect ratio of each of the multiple reaction chambers to the channel is at least 10:1. In some embodiments, each of the multiple reaction chambers has an interior and an exterior. In some embodiments, each of the multiple reaction chambers is suitable for transmitting excitation light of multiple wavelengths from the exterior of the multiple reaction chambers, and the multiple reaction chambers are suitable for transmitting emitted light of multiple wavelengths from the interior of the multiple reaction chambers. In some embodiments, the rotatable disk comprises a thermoformed film and a sealing film that combine together to form the multiple reaction chambers. In some embodiments, the thermoformed film forms the shape and size for the multiple reaction chambers. In some embodiments, the thermoformed film has a Tg higher than about 100°C.In some embodiments, the sealing film and the thermoformed film comprises a resin. In some embodiments, the resin comprises a single polymer. In some embodiments, the resin comprises an inner polymer and an outer polymer, the inner polymer having a lower Tg than the outer polymer. In some embodiments, the single polymer, the inner polymer, and the outer polymer are each independently selected from polyolefins, polycarbonates, polystyrene, polymethylmethylacrylates, polyethylene, and polypropylene. In some embodiments, the resin for the sealing film and the resin for the thermoformed film are both heat-sealed. In some embodiments, the resin for the sealing film and the thermoformed film are identical. In some embodiments, the sealer is designed to heat-seal the reaction chamber, thereby applying pressure and heat to a rotatable disk. In some embodiments, the first heating element has a first radial length, and the second heating element has a second radial length, the second radial length being about 6 to about 9 times the radial length of the first radial length. In some embodiments, the analytical device further comprises a third heating element configured to heat a rotatable disk to a third temperature and maintain it at that third temperature. In some embodiments, the third radial length is about 2 to 3 times the radial length of the first radial length. In some embodiments, the first heating element, the second heating element, and the third heating element each comprise a pair of heating blocks independently designed to clamp the rotatable disk between them. In some embodiments, the analytical device further comprises a plurality of optical heads. In some embodiments, the plurality of optical heads provide excitation light sources of one or more wavelengths. In some embodiments, the excitation light source wavelengths are selected from 400 nm to 750 nm. In some embodiments, the optical heads further comprise a plurality of filters that add one or more wavelengths to the excitation light source. In some embodiments, the analytical device further comprises a fluorescence detector that measures emission intensity for one or more wavelengths. In some embodiments, the fluorescence detector can simultaneously measure emission intensity for more than one wavelength.In some embodiments, the thermal sealer comprises a first element configured to provide thermal energy and pressure onto a rotatable disk, and a second element configured to provide a reaction force to the pressure.

[0293] Detailed explanation of the diagram Figure 1 illustrates a top view diagram of a rotatable disk as described herein. The rotatable disk (101) comprises a set of fluid transport channels (102) connecting different chambers (e.g., cuvettes), each containing, for example, PCR reagents and nucleic acid samples, to a reaction chamber or a plurality of reaction chambers (103) via channels (108). In addition, provided herein are elements of an analytical device for thermal circulation. These elements include, for example, a heating block (104) for denaturation during PCR, a heating block (105) for annealing primers to target nucleic acids, and a heating block (106) for extension of the target nucleic acid sequence by polymerase. The device further comprises an optical device or optical head (107) designed to excite one or more target phosphors and detect the resulting fluorescence intensity.

[0294] Figure 2 illustrates a top view of a rotatable disk as described herein in the context of the analytical device described herein. The rotatable disk (201) is inserted onto an analytical device designed to process nucleic acid samples, for example, by RT-PCR. For example, but not limited to, a sample (e.g., a liquid sample containing a target nucleic acid) is inserted into an inlet motif (202) on the device. The sample then flows into a loading chamber (e.g., a heating chamber) (203) for sample processing to extract the target nucleic acid. Subsequently, a measurement chamber laser valve (204) is activated to release the flow from the loading chamber (e.g., a heating chamber) (203) to the measurement chamber (205), and the measurement well controls the sample flow before mixing with the PCR reagent mixture (e.g., buffer, polymerase, and dNTP). The measured volume of the sample is then mixed with the PCR reagent mixture (e.g., buffer, polymerase, or dNTP) in a mixing chamber (207). The sample, mixed with the reconstituted reagent, then flows from the mixing chamber on the disk body (210) through channels (208) on the rotatable disk reaction chamber insert (e.g., cuvette insert) (211) into multiple reaction chambers (209). Additional PCR reagent mixtures (e.g., primers and phosphors) are stored in the multiple reaction chambers and mixed with the sample flow from channels (208) into the reaction chambers (209). In one non-limiting embodiment as shown herein, the reaction chamber insert and disk body are connected to an analytical device via a connection chamber (212).

[0295] Figure 3 illustrates a diagram representing the main elements required for thermal circulation on the analytical device described herein. For example, these elements of the analytical device (301) include a heating block (304) for denaturation during PCR, a heating block (302) for annealing primers to target nucleic acids, and a heating block (303) for extension of the target nucleic acid sequence by polymerase. The device further comprises an optical device or optical head (305) designed to excite one or more target phosphors and detect the resulting fluorescence intensity.

[0296] Figure 4 illustrates a top view of the rotatable disk described herein during a step of the method for RT-PCR described herein. The sample inside the loading chamber is heated to a temperature above 100°C for a period of 15 seconds (Figure 4, 401). After heating is complete, a valve (V1, Figure 4, 402), for example, made of a PMMA membrane, is punctured using electromagnetic means (e.g., a laser). Puncture of V1 causes the liquid to flow from inside the loading chamber (Figure 2, 202) to the measurement chamber (Figure 2, 205) via a transport path. The sample is transferred into the measurement chamber by centrifugation at a speed of 3,000–60,000 RPM. The measurement chamber is designed to measure different volumes depending on the targeted multiplexing number. A measurement well (Figure 2, 206) is punctured before or during the instrument is used to measure the precise volume required for the assay (Figure 4, 403). For example, if the assay requires only one reaction chamber, measurement well 1 will be punctured. If the assay requires three reaction chambers, measurement well 3 will be punctured. During measurement, any excess sample volume will flow into the overflow chamber (Figure 2 213). The measured volume is then pumped towards the mixing chamber after opening laser valve V2 (Figure 4 404). Inside the reagent mixing chamber, the RT-PCR reagent is stored, for example, in a lyophilized form. The RT-PCR reagent is mixed with the measured sample by Euler force (Figure 4 405). After opening laser valve V3 (Figure 4 406), the resuspended RT-PCR reagent, mixed with the sample, is pumped into the reaction chamber insert at, for example, 1,000-30,000 RPM to fill the reaction chamber (Figure 4 407). After filling, the reaction chambers are sealed from each other via a thermal sealer, for example, mounted inside the equipment. In some embodiments, the thermal sealer compresses the channels (Figure 4 408). In some embodiments, the thermal sealing is carried out using a temperature ranging from 200 to 300°C. For example, after sealing, the RT step is carried out by pressing a rotatable disk between the annealing blocks at 65°C for 15 to 60 seconds (Figure 4 409).A short (e.g., 5-30 seconds) activation step of the high-temperature initiated polymerase is achieved by inserting a rotatable disk between 95°C blocks (Figure 4, 410). After DNA polymerase activation, thermal circulation begins (Figure 4, 411).

[0297] Figure 5 illustrates several diagrams of a rotatable disk as described herein. Figure 5A illustrates two components used to manufacture several embodiments of a rotatable disk as described herein. For example, the rotatable disk is manufactured from a thermoformed film (501) (e.g., with a thickness of about 140 μm to about 300 μm). The thermoformed film is thermoformed to define the size and shape of several reaction chambers (503) and channels (504). The thermoformed film (501) is sealed to a thin plastic sealing film (502) (e.g., with a thickness of about 50 μm to about 150 μm). The rotatable disk further includes reinforcing bars (505) on the rotatable disk, which contribute to the stability of the rotatable disk due to the design and manufacture of the disk using two films. Figure 5B illustrates an enlarged embodiment of reaction chambers provided on a rotatable disk as described herein. In some embodiments, as shown herein, the reaction chamber (503) has a substantially dome shape produced by a thermoformed film (501) and a sealing film (502). Additional element ports depicted are, for example, reinforcing bars (505) that assist in the stability of the rotatable disk and the channels (504) that transport the sample into the reaction chamber or multiple reaction chambers (503). Figure 5C illustrates an additional diagram of a manufactured rotatable disk having multiple reaction chambers designed for RT-PCR. In some embodiments, as shown herein, the rotatable disk comprises multiple reaction chambers (e.g., cuvettes), (503), channels (504), and reinforcing bars (505), and the rotatable disk further comprises an additional dome (506) having a z dimension, found on the other side of the channels (504) from each reaction chamber (e.g., cuvette) (503).

[0298] Figure 6 illustrates components used to manufacture several embodiments of a rotatable disk described herein. The components provided herein are an embodiment of a reaction chamber insert for a rotatable disk, comprising a thermoformed film (601), a sealing film (602), a plurality of reaction chambers (603), a channel (604), reinforcement (605) for the stability of the thermoformed film, an inlet (606) for the rotatable disk insert, and a co-extruded material (607) or framework for manufacturing the disk using the thermoformed film and the sealing film.

[0299] Figure 7 illustrates a schematic diagram of an analytical device for processing target nucleic acids. For example, a rotatable disk (701) is inserted into the analytical device to process a nucleic acid sample (e.g., RT-PCR). An embodiment of the analytical device shown herein comprises a first heating block (704) set to a denaturation temperature and a second heating block (705) set to a temperature suitable for both annealing and extension during PCR. The device further comprises two optical heads (706) designed to excite multiple target phosphors and detect their fluorescence emission intensities. Finally, the analytical device comprises a thermal sealer (703) designed to traverse the channel of the rotatable disk and seal multiple reaction chambers from each other.

[0300] Figure 8 illustrates a schematic diagram of an analytical device for processing target nucleic acids. Figure 8A illustrates a front view of the analytical device, which comprises a first heating block (802) set to a denaturation temperature and a second heating block (803) set to a temperature suitable for both annealing and extension during PCR. These heating blocks are ultimately shown to narrow a rotatable disk (805) containing the sample to be processed after sample injection through an inlet and loading chamber (806) on the device. In this embodiment, the device further comprises two optical heads (804) designed to excite multiple target phosphors and detect their fluorescence emission intensities. Finally, in this embodiment, the analytical device comprises a thermal sealer (801) designed to seal across the channel of the rotatable disk and to seal multiple reaction chambers from each other. In one embodiment, the analytical device shown in Figure 8B illustrates a side view of the analytical device. The elements of the analytical device shown in this figure include a thermal sealer (801), a second heating block (803), an optical device (804), a rotatable disk (805), and an inlet / loading chamber (806) for sample insertion.

[0301] Figure 9 illustrates a schematic diagram of an analytical device for processing target nucleic acids, showing a thermal sealer element for sealing channels on a rotatable disk. Figure 9A shows an overview of the thermal sealer housed within the analytical device above the channels on the rotatable disk. Figure 9B shows a magnified view of the thermal sealer housed within the analytical device above the channels on the rotatable disk. Figure 9C shows an overview of the thermal sealer housed within the analytical device in contact with the channels on the rotatable disk. Figure 9D shows a magnified view of the thermal sealer housed within the analytical device in contact with the channels on the rotatable disk. In one embodiment of the rotatable disk and analytical device, the elements shown herein by Figures 9A-D include a thermal sealer (901), a rotatable disk (903) comprising two heating blocks (902) for heat circulation, and a plurality of reaction chambers (904) and channels (905), and a loading chamber / inlet (906) for inserting a sample into the device. The comparison between Figures 9A / B and 9C / D illustrates an embodiment of a method using the device for processing nucleic acid samples, each including the step of bringing a thermal sealer (901) and a rotatable disk (903) into contact across the entire width of a channel (905) connected to multiple reaction chambers (904), thereby thermally sealing the channel and reaction chambers from each other and from the rest of the disk.

[0302] Figure 10 illustrates a schematic front view of the analytical device, in which the disclosed heating block element clamps and unclamps the disclosed rotatable disk. A comparison of Figure 10A and Figure 10B illustrates how the first pair of heating blocks clamps or contacts the rotatable disk. In one embodiment of the method performed on the analytical device, the first heating block (1002) (set to a temperature suitable for, for example, a denaturation step in PCR) presses down and clamps the rotatable disk (1005) from above, as shown by comparing element 1002 in Figures 10A and 10B, thereby enabling heat transfer to the reaction chamber containing the sample for PCR amplification. A comparison of Figures 10B and 10C shows how the heating block is unclamped from the rotatable disk, allowing the rotatable disk to rotate to a second pair of heating blocks. In a further embodiment of the method performed on an analytical device, the first heating block (1002) is unclamped from the rotatable disk (1005), and the rotatable disk rotates toward the side of the analytical device, which is equipped with a second heating block (1003) (set to a temperature suitable for, for example, the annealing / extension step in PCR). This process is illustrated by a comparison of elements 1002 and 1005 in Figures 10B and 10C. A comparison of Figures 10C and 10D illustrates how the second pair of heating blocks clamps or contacts the rotatable disk. In a further embodiment of the method performed on an analytical device, the second heating block (1003) (set to a temperature suitable for, for example, the annealing / extension step in PCR) presses down on and clamps the rotatable disk (1005), as shown by a comparison with element 1003 in Figures 10C and 10D, thereby enabling heat transfer to the reaction chamber, which is equipped with a sample for PCR amplification.

[0303] Figure 12 illustrates a schematic diagram of a rotatable disk that rotates from one heating block (Figure 12A) to a second heating block (Figure 12B). For example, the rotatable disk (1202) rotates from a first heating block (1203) (e.g., set to a denaturation temperature of approximately 95 degrees Celsius) to a second heating block (1204) (e.g., set to an annealing / extension temperature of 65 degrees Celsius). Additional elements shown in this embodiment include two optical devices or two optical heads (1201) for exciting and measuring emissions related to multiple target phosphors.

[0304] Figure 13 provides photographs of embodiments of the disclosed rotatable disk and the disclosed analytical device. Figure 13A shows an embodiment of the fabricated rotatable disk. Figure 13B shows an embodiment of a magnified view of a sealed channel on the rotatable disk. Figure 13C shows an embodiment of an analytical device used to process a target nucleic acid (e.g., RT-PCR). The elements shown in Figures 13A / B include a rotatable disk (1301) having a plurality of reaction chambers (1302) connected to a channel (1303), the channel (1303) being sealed by a thermal sealer (1304). Figure 13D shows elements of the analytical device, comprising a thermal sealer (1305) used to seal the channel in Figure 13B, and two optical heads (1306).

[0305] Figures 14A and 14B illustrate data showing fluorescence readout values ​​from the device provided herein. Figure 14A illustrates signal detection with one color channel implemented in the device. Figure 14B illustrates signal detection with two color channels implemented in the device.

[0306] Figure 29 shows a schematic diagram of the structure of one embodiment, where one pair of blocks is set to approximately 95°C and the other pair to approximately 65°C (annealing / extension temperature). By using two blocks, the cooling step becomes the longest step, taking approximately 7 seconds.

[0307] Figure 30 shows a schematic diagram of the structure of one embodiment, which has three blocks instead of two. In this embodiment, the pair of blocks for annealing are divided into two distinctly different temperature zones, which can be set to two different temperatures independently of each other. For assays requiring four cuvettes or fewer, two distinctly different blocks may be set to two different temperatures, with the half-block closer to the 95°C block being used as a heat sink and set to a temperature well below the annealing temperature. For example, the half-block closer to the 95°C block may be set to 40°C. The remaining half-block may be set to the annealing / extension temperature (e.g., 65°C).

[0308] The disk is rotated from the 95°C block and first clamped by a heat sink half-block to cool the PCR insert as quickly as possible. This allows the disk to cool more rapidly than if it were moved linearly relative to the annealing temperature block. After the disk reaches the annealing temperature, it is moved onto the annealing temperature half-block. The time it takes for the disk to reach the annealing temperature from 95°C when it comes into contact with the heat sink half-block can be calculated.

[0309] Figure 31 demonstrates that the cooling rate of the PCR disk is accelerated by using a heat sink block. Without a heat sink block, the disk would approach the annealing temperature more slowly in an asymptotic manner as the disk temperature approaches the annealing temperature. This prolongs the time it takes to reach the annealing temperature. In this embodiment, using a heat sink half-block reduces the time from 7 seconds to 4 seconds compared to the two block embodiments.

[0310] To implement the three-block embodiment, the half-block acting as a heat sink needs to dissipate heat very quickly in order to remain at the same temperature after multiple cycles. To be efficient, the half-block acting as a heat sink needs to return to its target temperature in a time shorter than the required residence time at the annealing / extension temperature + time under the modified block + time required to move the insert and complete the cycle, or in a time consisting of approximately 3 to 6 seconds. The block, therefore, must be capable of cooling very quickly.

[0311] Conventional heating blocks cool very slowly and often cannot recover to their original temperature within the given time range. Figure 32 shows an embodiment in which holes are punctured within the heating block, allowing it to cool more rapidly (e.g., a Swiss cheese-like configuration).

[0312] Figure 33 shows a method in one embodiment in which airflow may be directed through air ducts. In this embodiment, two fans connected to the air at room temperature outside the box blow air through two air ducts, both directed to their respective half-blocks. 3201 shows the fans in this embodiment. 3202 shows the gripper. 3203 shows the air duct annealing half-block. 3204 shows the flexible heater. 3205 shows the annealing half-block. 3206 shows the heat sink half-block. 3207 shows the air duct heat sink half-block.

[0313] Figure 34 displays a simulation graph demonstrating that the air duct equipment shown in Figure 33 allows the Swiss cheese-shaped semi-heated block to recover its temperature within approximately 5 seconds when set to approximately 40°C and the room temperature is approximately 30°C. When tested, the system showed that the cooling gradient rate was at least 2.5 times faster than that in the simulation. This indicates that the heat sink semi-block could potentially recover its temperature within approximately 2 seconds.

[0314] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous modifications, alterations, and substitutions will be conjured upon those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. The following claims define the scope of the present invention, and methods and structures within the scope of these claims, as well as their equivalents, are intended to be covered thereby. [Examples]

[0315] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention. [Examples]

[0316] Ultrafast polymerase chain reaction with human genome DNA In this example, an ultrafast PCR reaction for amplifying human genomic DNA was initiated on a PCR device comprising a rotatable disk and heating blocks at 98°C and 65°C, as described herein. In this example, 45 cycles of PCR were performed within 7 minutes. The PCR products were analyzed after 45 cycles of ultrafast PCR, followed by analysis via gel electrophoresis.

[0317] The target nucleic acid sample was first prepared using 620 copies of HeLa Genomic DNA in Tris-EDTA buffer. A custom assay targeting the 158-base pair region of the HPV-18 gene was used to detect the HeLa DNA in the sample. Two ultrafast PCR reagent kits, namely Promega 2x GoTaq Rapid Master Mix and KAPA3G, were tested. The PCR reagent, comprising buffer / salt, polymerase, nucleotides, reverse transcriptase, primers, probes, and the target nucleic acid sample, was combined in appropriate ratios with the desired PCR reaction volume. For readout, a final concentration of 250 nM TaqMan Probe was used. The complete PCR reaction product was loaded into the custom PCR reaction vessel. Each reaction chamber was fully filled and separated into independent reactions. For thermal circulation, a heating device with gripper heating blocks at annealing (65°C) and denaturation temperature (98°C) was used to transfer heat to the PCR reaction vessel. During thermal circulation, the reaction vessel was moved between heating blocks via a motor. The following thermal circulation conditions were used: 60 seconds of polymerase activation (98°C), followed by 45 cycles of 2 seconds of denaturation (98°C) and 7 seconds of annealing / extension (65°C). Shunting between different blocks was repeated over 45 cycles.

[0318] Post-PCR samples were analyzed via endpoint gel electrophoresis. Post-PCR samples were extracted using a syringe, and each replica of the PCR product, along with a 50-base pair PCR ladder, was loaded into individual electrophoresis lanes on a 1% agarose gel. A voltage of 120V was applied to the gel for 15 minutes, and the gel was imaged using a transilluminator to visualize the PCR product DNA. A template-free control was tested to ensure there was no out-of-target amplification. Figure 21 shows the imaged gels. Lane 2101 shows the PCR reaction with the HeLa Genomic DNA sample and the Promega kit. Lanes 2102, 2103, 2106, and 2107 show the PCR reaction with the HeLa Genomic DNA sample and the KAPA3G kit. Lanes 2104, 2105, 2108, and 2109 show the PCR reaction with the template-free control and the KAPA3G kit. The gels showed the correct PCR product size for the targeted 158 base pair region of the HPV-18 gene for both of the ultrafast PCR reagent kits tested. No out-of-target amplification was observed. [Examples]

[0319] Ultrafast polymerase chain reaction with respiratory infectious pathogens In this example, an ultrafast PCR reaction to amplify the respiratory infectious pathogens influenza B and RSV was initiated on a PCR device comprising a rotatable disk and heating blocks at 55°C, 98°C, and 65°C, as described herein. In this example, 50 cycles of ultrafast PCR were initiated, and real-time optical data were collected at the end of each PCR cycle.

[0320] Target nucleic acid samples were initially prepared using samples co-infected with influenza B and respiratory cynthiavirus, both at 5,000 copies / mL in 1x Tris EDTA buffer with 2 mg / mL bovine serum albumin and 0.02% sodium azide. The samples were heated according to a high-pressure PCR sample preparation method and then loaded with PCR reagents in a custom reaction vessel. A dual TaqMan probe assay was utilized, targeting the 94-base pair region of the matrix gene in influenza B (FAM TaqMan Probe) and the 83-base pair region of the matrix gene in RSV (TEX615 TaqMan Probe). PCR reagents, comprising buffer / salt, polymerase, nucleotides, reverse transcriptase, primers, and probes, were prepared at 5x concentration and, when combined with the target nucleic acid samples, diluted to 0.2x to produce the final PCR reaction mixture. The PCR reagents and target nucleic acid samples were combined in the desired PCR reaction volume and appropriate ratios. The complete PCR reaction material was loaded into a custom PCR reaction vessel. Each reaction chamber was fully filled and separated into independent reactions. For thermal circulation, a heating device with gripper heating blocks at reverse transcription (55°C), annealing (65°C), and denaturation temperature (98°C) was used to transfer heat to the PCR reaction vessel. During thermal circulation, the reaction vessel was moved between temperature blocks via a motor. PCR was initiated in the custom reaction vessel using the following circulation conditions: 60 seconds of reverse transcription (55°C), 30 seconds of polymerase activation (98°C), followed by 50 cycles of 2 seconds of denaturation (98°C) and 7 seconds of annealing / extension (65°C). Shunting between different blocks was repeated over 50 cycles, and real-time optical data was collected at the end of each PCR cycle.

[0321] Individual fluorescence traces were collected for each co-reaction replicate, as shown in Figure 22. The fluorescence traces indicated that both influenza B and RSV PCR products were detectable between 33 and 50 cycles. [Examples]

[0322] Sample preparation by high-pressure heating and ultrafast polymerase chain reaction with influenza B. In this example, samples containing influenza B in a nasal swab collection matrix were prepared via high-pressure heating treatment and processed and analyzed by ultrafast polymerase chain reaction on a PCR device as described herein (Figures 7, 8A–8B, 9). The PCR device comprises a rotatable disk with cuvette inserts, comprising eight reaction chambers, and heating elements set to 55°C, 98°C, and 65°C. In this example, 50 cycles of ultrafast PCR were initiated, and real-time optical data were collected at the end of each PCR cycle. Different primer sets and probes were tested to amplify and detect influenza B.

[0323] A treatment sample comprising influenza B at a concentration of 10,000 copies / mL, 500 nM of thermostable Thermococcus kodakarensis (KOD), 1 mM DTT, and 10% Chelex was first prepared and heated under high-pressure heating conditions in a closed heating chamber equipped with an induction susceptor. The heating chamber was heated via induction heating at a frequency of 103.5 kHz for 15 seconds. During the 15 seconds of induction heating, the sample was heated from room temperature to a temperature of 120°C to 130°C. Following high-pressure heating, 19 μL of the treated sample was mixed with 6 μL of PCR reaction mixture comprising 10x KAPA Buffer, 0.2 mM dNTPs, Superscript IV (10 units), RNAsin Inhibitor (5 units), KAPA3G DNA Polymerase (2 units), and 4.5 mM MgSO4 per PCR reaction mixture. The primer sets and probes were added to the PCR reaction mixture at final concentrations of 1,000 nM primers and 350 nM probes. In this example, four sets of primers and probes were tested as shown in Table 1. [Table 1]

[0324] A total of eight PCR reaction mixtures were transferred to separate chambers (e.g., cuvettes) within a cuvette insert on a rotatable disk, as described herein (Figure 2). The separate chambers were heat-sealed, and the PCR reaction was carried out using the following cyclic conditions: 60 seconds of reverse transcription (55°C), 30 seconds of polymerase activation (98°C), followed by 50 cycles of 2 seconds of denaturation (98°C) and 7 seconds of annealing / extension (65°C). For thermal circulation, a heating device equipped with gripper heating blocks at the reverse transcription (55°C), annealing (65°C), and denaturation temperature (98°C) was used to transfer heat to the PCR reaction vessels. During thermal circulation, the reaction vessels were moved between heating blocks via a motor. Real-time optical data were collected at the end of each PCR cycle.

[0325] Individual fluorescence traces were collected for each cuvette, as shown in Figures 23A–23D. The results showed that for each primer set and probe tested, the influenza B PCR product was detectable in all eight cuvettes between 35 and 50 cycles for the PCR reaction. The results demonstrate that nasal swab samples, treated by high-pressure heating and processed and analyzed by ultrafast polymerase chain reaction on the PCR device described herein, maintain high sensitivity. [Examples]

[0326] Limitations of detecting influenza A and influenza B in pooled nasal swab collections following sample processing by high-pressure heating and PCR thermal circulation. In this example, samples comprising influenza A and influenza B in a pooled nasal swab collection matrix were prepared via treatment by high-pressure heating and processed and analyzed by an ultrafast polymerase chain reaction on a PCR device as described herein (Figures 7, 8A–8B, 9). In this example, different concentrations of influenza A and influenza B were tested to determine the limitations of detection of influenza A and influenza B in the pooled nasal swab collection matrix.

[0327] To determine the detection limits, influenza A samples were processed and analyzed by ultrafast polymerase chain reaction at the following concentrations: 0, 25, 60, 125, 250, 500, 1,000, and 5,000 copies / mL. Influenza B samples were processed and analyzed by ultrafast polymerase chain reaction at the following concentrations: 25, 60, 125, 250, 500, and 5,000 copies / mL.

[0328] Influenza A and influenza B samples were treated by high-pressure heating in a treatment sample containing 500 nM of heat-stable Thermococcus kodakarensis (KOD), 1 mM DTT, and 10% Chelex. The samples were heated under high-pressure conditions in a closed heating chamber equipped with an induction susceptor. The heating chamber was heated via induction heating at a frequency of 103.5 kHz for 15 seconds. During the 15 seconds of induction heating, the samples were heated from room temperature to a temperature of 120°C to 130°C.

[0329] The PCR reaction with the treated sample was then initiated on a PCR device as described herein (Figures 7, 8A–8B, 9). The treated sample was mixed with PCR reagents, primers, and probes, and the PCR reaction was carried out in a separate reaction chamber that was heat-sealed prior to thermal circulation. During thermal circulation, the reaction chamber was moved between heating blocks via a motor. The thermal circulation time was approximately 7.5 minutes.

[0330] The PCR results for influenza A shown in Figure 24A illustrate the cycle thresholds for influenza A samples at different concentrations, indicating a detection limit of 250 copies / mL, which resulted in 100% detection across screening and confirmatory replicates with an average cycle threshold of approximately 34. Influenza A samples at a concentration of 125 copies / mL resulted in 92.5% detection across screening and confirmatory replicates.

[0331] The results ...

Claims

1. A method for ultrafast real-time polymerase chain reaction (PCR) for detecting the presence or absence of a target nucleic acid in a biological sample, wherein the method is: (a) Loading a sample into a loading chamber on a rotatable disk, the rotatable disk further comprising a plurality of reaction chambers and channels that fluidly connect the loading chamber to the plurality of reaction chambers, the reaction chambers being loaded with a PCR reagent mixture comprising primers and a phosphor probe, the sample flowing into the plurality of reaction chambers through the channels, thereby filling the reaction chambers following the loading of the sample; (b) To bring the channel in the rotatable disk into contact with the sealer, thereby sealing the channel after filling and preventing fluid communication between the plurality of reaction chambers, (c) Rotating the rotatable disk to bring the plurality of reaction chambers adjacent to a first heating element maintained at a first temperature, thereby denaturing the target nucleic acid in the sample, if present, thereby producing denatured target nucleic acid, (d) Rotating the rotatable disk and positioning the plurality of reaction chambers adjacent to a second heating element maintained at a second temperature, thereby annealing the primers to the denatured target nucleic acid and replicating the denatured target nucleic acid, (e) Exposing the plurality of reaction chambers to excitation light of a first wavelength, thereby exciting the phosphor probe, (f) Repeating steps (c)–(e) over multiple cycles, measuring the emission light of a second wavelength from the multiple reaction chambers, wherein if emission light of a second wavelength is detected, the sample comprises the target nucleic acid. Methods that include...

2. The method according to claim 1, wherein the primer comprises an oligonucleotide sequence complementary to at least a portion of the target nucleic acid.

3. The aforementioned phosphor probe is fluoroceine amidite (FAM), SUN ＴＭ The method according to any one of claims 1 to 2, comprising a phosphor selected from the group consisting of , TEX615, and cyanine-5 (Cy5).

4. The method according to claim 1, wherein the sealant is stored in an analytical device for real-time PCR.

5. The method according to claim 1 or 4, wherein the sealant is a heat sealant.

6. The method according to any one of claims 1 to 5, further comprising, between steps (a) and (b), rotating the rotatable disk to generate a sufficient centripetal force on the sample to allow the sample to flow through the channel into the plurality of reaction chambers.

7. The method according to claim 6, wherein the sufficient centripetal force is generated by spinning the rotatable disk at approximately 500 RPM to approximately 15,000 RPM.

8. The method according to any one of claims 1 to 7, wherein the channel defines a lumen having a diameter ranging from about 1 μm to about 1 mm.

9. The method according to any one of claims 1 to 8, wherein the channel comprises a thermoplastic material, and the thermoplastic material is heated to a target temperature and compressed by the sealer to seal the channel.

10. The method according to claim 9, wherein the target temperature is approximately 110°C to approximately 300°C.

11. The method according to any one of claims 1 to 10, wherein the sealer comprises a first element configured to provide thermal energy and pressure onto the rotatable disk, and a second element configured to provide a reaction force to the pressure.

12. The method according to claim 11, wherein bringing the rotatable disk and the sealer into contact involves applying a pressure at one or more locations along the channel on the rotatable disk that is opposed to the reaction force, such that the channel deforms and creates the plurality of reaction chambers.

13. The method according to any one of claims 1 to 12, wherein the excitation light or the emitted light comprises two or more wavelengths.

14. The method according to any one of claims 1 to 13, wherein the first wavelength and the second wavelength are independently selected from about 400 nm to about 750 nm.

15. The method according to any one of claims 1 to 14, further comprising, between step (d) and step (e), rotating the rotatable disk to bring the plurality of reaction chambers adjacent to a third heating element maintained at a third temperature.

16. The method according to any one of claims 1 to 15, wherein the sample is mixed with a primer that is complementary to at least a portion of the target nucleic acid pre-loaded in the plurality of reaction chambers.

17. The method according to claim 16, wherein the plurality of reaction chambers further comprise probes, and the sample is further mixed with the probes.

18. A method for multiplexed real-time amplification and detection of multiple different target nucleic acids in a biological sample, wherein the method is (a) Loading a sample into a loading chamber on a rotatable disk, the rotatable disk further comprising a plurality of reaction chambers and channels that fluidly connect the loading chamber to the plurality of reaction chambers, the reaction chambers being loaded with a PCR reagent mixture comprising a plurality of primers and a plurality of phosphor probes, the sample being mixed with the plurality of primers, each of the plurality of primers being complementary to a portion of the corresponding target nucleic acids of the plurality of different target nucleic acids, and the sample flowing through the channels into the plurality of reaction chambers, thereby filling the reaction chambers following the loading of the sample. (b) To bring the channel in the rotatable disk into contact with the sealer, thereby sealing the channel after filling and preventing fluid communication between the plurality of reaction chambers, (c) Rotating the rotatable disk to bring the plurality of reaction chambers adjacent to a first heating element maintained at a first temperature, thereby denaturing each of the plurality of target nucleic acids in the sample, if present, thereby producing a plurality of denatured target nucleic acids, (d) Rotating the rotatable disk and positioning the plurality of chambers adjacent to a second heating element maintained at a second temperature, thereby annealing the plurality of primers to the corresponding plurality of denatured target nucleic acids and replicating the plurality of different target nucleic acids, (e) Exposing the plurality of reaction chambers to excitation light having a plurality of excitation wavelengths, thereby exciting the plurality of phosphor probes, (f) Repeating steps (c)–(e) over multiple cycles, measuring multiple emitted light of multiple emission wavelengths from the multiple reaction chambers, wherein each of the multiple emission wavelengths corresponds to the presence of each of the multiple different target nucleic acids, and if emitted light of an emission wavelength is detected, the sample comprises the corresponding target nucleic acid. Methods that include...

19. The method according to claim 18, wherein the sealant is a heat sealant.

20. The method according to claim 18 or 19, wherein the sealant is stored in an analytical device for multiplexed real-time amplification.

21. The method according to any one of claims 18-20, further comprising, between steps (a) and (b), rotating the rotatable disk to generate a sufficient centripetal force on the sample to allow the sample to flow through the channel into the plurality of reaction chambers.

22. The method according to claim 21, wherein the sufficient centripetal force is generated by spinning the rotatable disk at approximately 500 RPM to approximately 15,000 RPM.

23. The method according to any one of claims 18-22, wherein the channel defines a lumen having a diameter ranging from about 1 μm to about 1 mm.

24. The method according to any one of claims 18-23, wherein each of the plurality of excitation wavelengths and the plurality of emission wavelengths is independently selected from about 400 nm to about 750 nm.

25. The method according to any one of claims 18-24, further comprising, between step (d) and step (e), rotating the rotatable disk to bring the plurality of reaction chambers adjacent to a third heating element maintained at a third temperature.

26. The method according to claim 18, wherein each of the plurality of primers comprises an oligonucleotide sequence that is complementary to at least a portion of the target nucleic acid.

27. The method according to any one of claims 1 to 26, wherein the plurality of primers are stored in the plurality of reaction chambers.

28. The method according to any one of claims 1 to 27, wherein the plurality of primers are provided as freeze-dried powders.

29. The method according to any one of claims 1 to 28, wherein the sample comprises a physical sample selected from the group consisting of a blood sample, a tear sample, a saliva sample, a mucus sample, a sputum sample, a fecal sample, a cerebrospinal fluid sample, and a urine sample.

30. The phosphor probes in the aforementioned plurality of phosphor probes are fluorothane amidite (FAM), SUN ＴＭ The method according to any one of claims 1 to 29, comprising a phosphor selected from the group consisting of , TEX615, and cyanine-5 (Cy5).

31. The method according to any one of claims 1 to 30, wherein the first heating element, the second heating element, or the third heating element comprises a heating block.

32. The method according to any one of claims 17 or 21-28, wherein the probe is provided as a freeze-dried powder.

33. The method according to any one of claims 1 to 32, wherein the rotatable disk is made of hard plastic.

34. The method according to any one of claims 6-15 or 21-28, further comprising mixing the sample with a PCR reagent mixture prior to transferring the sample to the plurality of reaction chambers.

35. The method according to claim 34, wherein the PCR reagent mixture comprises a polymerase and a plurality of nucleotides.

36. The method according to claim 34, wherein the PCR reagent mixture comprises polymerase, a plurality of nucleotides, a buffer, reverse transcriptase, primers, and a probe.

37. The method according to claim 34 or 35, wherein the components of the PCR reagent mixture are pre-mixed before contacting the sample or loading it into the plurality of reaction chambers.

38. The method according to any one of claims 1 to 37, wherein the PCR reagent mixture is provided in a reagent mixing chamber, and the sample is mixed with a second PCR reagent mixture in the reagent mixing chamber.

39. The method according to any one of claims 1 to 38, wherein the PCR reagent mixture is provided as a lyophilized powder.

40. The method according to any one of claims 1 to 37, wherein the sample is mixed with the PCR reagent mixture prior to being transferred into the plurality of reaction chambers in (a).

41. The method according to any one of claims 1 to 40, wherein each of the plurality of reaction chambers is aligned to be at the same radial distance from the center of the rotatable disk and is equally spaced apart from one another.

42. The method according to any one of claims 1 to 40, wherein the plurality of reaction chambers occupy a 360-degree arc on the rotatable disk.

43. The method according to any one of claims 1 to 40, wherein the plurality of reaction chambers occupy an arc section of about 10 to 100 degrees on the rotatable disk.

44. The method according to any one of claims 1-43, wherein the plurality of reaction chambers comprises 1 to 100 reaction chambers.

45. The method according to any one of claims 1 to 44, wherein each of the plurality of reaction chambers has a volume of about 5 μL to about 100 μL and a depth of about 0.1 mm to about 1.0 mm.

46. The method according to any one of claims 1 to 45, wherein the plurality of reaction chambers have a depth of about 0.1 mm to about 0.7 mm.

47. The method according to any one of claims 1 to 46, wherein the plurality of reaction chambers have a maximum depth of about 0.25 mm.

48. The method according to any one of claims 1 to 47, wherein the first temperature is maintained over a first period of time, and the second temperature is maintained over a second period of time.

49. The method according to claim 48, wherein the first period is selected from about 500 milliseconds to about 2 seconds, and the second period is selected from about 3 seconds to about 18 seconds.

50. The method according to any one of claims 1 to 49, wherein the first temperature is selected from about 90°C to about 99°C, and the second temperature is selected from about 50°C to about 74°C.

51. The method according to any one of claims 1 to 50, wherein the first heating element has a first radial length, and the second heating element has a second radial length.

52. The method according to claim 51, wherein the second radial length is approximately 6 to approximately 9 times the first radial length.

53. The method according to any one of claims 15 or 25-52, wherein the third temperature is selected from about 65°C to about 75°C.

54. The method according to any one of claims 15 or 25-53, wherein the third temperature is maintained over a third period of time.

55. The method according to claim 54, wherein the third period is selected from about 1 second to about 6 seconds.

56. The method according to any one of claims 15 or 25-55, wherein the third heating element has a third radial length.

57. The method according to claim 56, wherein the third radial length is approximately 2 to approximately 3 times the first radial length.

58. The method according to any one of claims 1 to 57, further comprising stabilizing the rotatable disk at the first temperature for the first period, at the second temperature for the second period, and / or at the third temperature for the third period, thereby enabling thermal circulation on the sample.

59. The method according to claim 58, wherein stabilization includes clamping the rotatable disk between the first heating element, the second heating element, or the first heating block and the second heating block of the third heating element.

60. The method according to claim 59, further comprising clamping the rotatable disk, followed by unclamping the rotatable disk from the first heating block and the second heating block of the first, second, or third heating element.

61. The method according to any one of claims 1 to 60, wherein the excitation light is supplied by an optical head or a plurality of optical heads.

62. The method according to claim 61, wherein the optical head or a plurality of optical heads further comprises wavelength filters for changing the first wavelength or the plurality of wavelengths of the excitation light.

63. The method according to any one of claims 1 to 62, wherein the emitted light is measured on a first fluorescence detector or a plurality of fluorescence detectors.

64. The method according to claim 63, wherein the first detector or a plurality of fluorescence detectors comprises one or more wavelength channels for measuring the emission intensity of the second wavelength or the plurality of emission wavelengths.

65. A rotatable disk, Loading chamber and Multiple reaction chambers, The loading chamber has a channel that connects it to the plurality of reaction chambers. Equipped with, The channel is a rotatable disk comprising a thermoplastic material that seals the channel when heated to a temperature of 110 to 300°C.

66. The rotatable disk according to claim 65, wherein each of the plurality of reaction chambers is aligned to be at the same radial distance from the center of the rotatable disk.

67. The rotatable disk according to claim 65 or 66, wherein each of the plurality of reaction chambers contains a first PCR reaction mixture comprising primers and a phosphor probe.

68. The rotatable disk according to any one of claims 65-67, wherein the loading chamber comprises a second PCR reaction mixture comprising polymerase and a plurality of nucleotides.

69. A rotatable disk, Loading chamber and A plurality of reaction chambers, each aligned at approximately the same radial distance from the center of the rotatable disk, and each of the plurality of reaction chambers comprises a first PCR reaction mixture comprising primers, The loading chamber has a channel that connects it to the plurality of reaction chambers. A rotatable disc equipped with the following features.

70. The rotatable disk according to claim 69, wherein the loading chamber comprises a polymerase and a second PCR reaction mixture comprising a plurality of nucleotides.

71. The rotatable disk according to claim 69 or 70, wherein the channel is provided with a thermoplastic material that seals the channel when heated to a temperature of 110 to 300°C.

72. The rotatable disk according to any one of claims 65-71, wherein each of the plurality of reaction chambers has a volume of about 5 μL to about 100 μL.

73. The rotatable disk according to any one of claims 65-72, wherein each of the plurality of reaction chambers has a depth of about 0.1 mm to about 1.0 mm.

74. The rotatable disk according to any one of claims 65-73, wherein the plurality of reaction chambers have a depth of about 0.2 to about 0.7 mm.

75. The rotatable disk according to any one of claims 65-74, wherein each of the plurality of reaction chambers has a maximum depth of 0.25 mm.

76. The rotatable disk according to any one of claims 65-75, wherein each of the plurality of reaction chambers is equally spaced apart from one another.

77. The rotatable disk according to any one of claims 65-76, wherein the plurality of reaction chambers occupy a 360-degree arc on the rotatable disk.

78. The rotatable disk according to any one of claims 65-77, wherein the plurality of reaction chambers occupy an arc section of about 10 to 100 degrees on the rotatable disk.

79. The rotatable disk according to any one of claims 65-78, wherein the plurality of reaction chambers comprises 1 to 100 reaction chambers.

80. The rotatable disk according to any one of claims 65-79, wherein the thermoplastic material is selected from polycarbonate, polypropylene, polyethylene terephthalate, and cyclic olefin copolymer.

81. The rotatable disk according to any one of claims 65-80, wherein the thermoplastic material has a thickness of about 10 μm to about 400 μm.

82. The rotatable disk according to any one of claims 65-81, wherein the channel has a z dimension of about 1 μm to about 1 mm, a width of about 1 mm to about 5 mm, and a maximum depth of about 200 μm.

83. The rotatable disk according to any one of claims 65-82, wherein the channel has a width of about 2 mm to about 4 mm and a maximum depth of about 100 μm.

84. The rotatable disk according to any one of claims 65-83, further comprising the aspect ratio of each of the plurality of reaction chambers and the channel, wherein the aspect ratio is at least 10:

1.

85. The rotatable disk according to any one of claims 65-84, further comprising the aspect ratio of each of the plurality of reaction chambers and the channel, wherein the aspect ratio is at least 20:

1.

86. The rotatable disk according to any one of claims 65-85, wherein each of the plurality of reaction chambers has an interior and an exterior.

87. The rotatable disk according to claim 86, wherein each of the plurality of reaction chambers is suitable for transmitting excitation light of multiple wavelengths from outside the plurality of reaction chambers, and the plurality of reaction chambers are suitable for transmitting emitted light of multiple wavelengths from inside the plurality of reaction chambers.

88. The rotatable disk according to any one of claims 65-87, wherein the rotatable disk comprises a thermoformed film and a sealing film that are joined together to form the plurality of reaction chambers.

89. The rotatable disk according to claim 88, wherein the thermoformed film forms the shape and size for the plurality of reaction chambers and the shape and size for the channels.

90. The rotatable disk according to claim 88 or claim 89, wherein the sealing film is sealed to the thermoformed film.

91. The rotatable disk according to claim 90, wherein the sealing film is sealed to the thermoformed film via a framework, together with two surfaces co-extruded from a heat-sealed compliant material.

92. The rotatable disk according to any one of claims 88-91, wherein the thermoformed film has a thickness of about 50 μm to about 500 μm.

93. The rotatable disk according to any one of claims 88-92, wherein the ratio of the width of the channel to the thickness of the thermoformed film is greater than 2.

94. The rotatable disk according to any one of claims 88-93, wherein the sealing film has a thickness of about 10 μm to about 500 μm.

95. The rotatable disk according to any one of claims 88-94, wherein the thermoformed film has a glass transition temperature (Tg) higher than about 100°C.

96. The rotatable disk according to any one of claims 88-95, wherein the sealing film and the thermoformed film are made of resin.

97. The rotatable disk according to claim 96, wherein the resin comprises a single polymer.

98. The rotatable disk according to claim 96 or claim 97, wherein the resin comprises an inner polymer and an outer polymer, and the inner polymer has a lower Tg than the outer polymer.

99. The rotatable disc according to claim 98, wherein the single polymer, the inner polymer, and the outer polymer are each independently selected from polyolefin, polycarbonate, polystyrene, polymethylmethylacrylate, polyethylene, and polypropylene.

100. The rotatable disk according to any one of claims 96-99, wherein the resin for the sealing film and the resin for the thermoformed film can both be heat-sealed.

101. The rotatable disk according to any one of claims 96-100, wherein the resin for the sealing film and the thermoformed film is the same.

102. A rotatable disk, Loading chamber and A plurality of reaction chambers, each of which is aligned at the same radial distance from the center of the rotatable disk, The loading chamber has a channel that connects it to the plurality of reaction chambers. Equipped with, The channel comprises a thermoformed thermoplastic film sealed to a sealing thermoplastic film, and the channel is a rotatable disk having a depth of 10 micrometers to 500 micrometers.

103. A rotatable disk, Loading chamber and A plurality of reaction chambers, each of which is aligned at the same radial distance from the center of the rotatable disk, The loading chamber has a channel that connects it to the plurality of reaction chambers. Equipped with, The channel is a rotatable disk that is heat-sealed by compressing a thermoformed film on the ceiling of the channel into contact with a sealing film on the floor of the channel.

104. A rotatable disk, Loading chamber and A plurality of reaction chambers, each of which is aligned at the same radial distance from the center of the rotatable disk, The loading chamber has a channel that connects it to the plurality of reaction chambers. Equipped with, The channel comprises a co-extruded thermoplastic film with an inner thermoplastic film layer having a Tg lower than that of the outer thermoplastic film layer. A rotatable disk wherein the inner layer is configured to seal the channel when heated to a temperature of 110° to 300° Celsius.

105. A rotatable disk, A loading chamber configured to seal the sample under high pressure conditions to temperatures exceeding 100°C, A plurality of reaction chambers, each of which is aligned at the same radial distance from the center of the rotatable disk, The loading chamber has a channel that connects it to the plurality of reaction chambers. A rotatable disc equipped with the following features.

106. The rotatable disk according to claim 105, wherein the loading chamber is configured to seal a sample under high-pressure conditions at temperatures from 101° to 160° Celsius.

107. The rotatable disk according to claim 105 or 106, wherein the channel is heat-sealed by compressing a thermoformed film on the ceiling of the channel into contact with a sealing film on the floor of the channel.

108. The rotatable disk according to any one of claims 65-107, wherein the loading chamber is configured to be heated by induction heating.

109. The rotatable disk according to any one of claims 65-108, wherein the loading chamber comprises a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent.

110. The rotatable disk according to claim 109, wherein the loading chamber further comprises a stabilizer.

111. A system for real-time polymerase chain reaction (PCR), (a) A rotatable disk comprising a loading chamber, a plurality of reaction chambers, and channels connecting the loading chamber to the plurality of reaction chambers, (b) A sample holder for holding the rotatable disk in a substantially horizontal plane, wherein the device is configured to rotate the rotatable disk in the substantially horizontal plane, (c) A sealer coupled to an actuator that moves the sealer, thereby changing the distance between the sealer and the rotatable disk, (d) A first heating element located in close proximity to the second portion of the rotatable disk and maintained at a first temperature, (e) A second heating element located in close proximity to the second portion of the rotatable disk and maintained at a second temperature, (f) A light source oriented to generate excitation light of a first wavelength within the horizontal plane occupied by the reaction chamber of the rotatable disk, (g) A first photodetector oriented to detect emitted light of a second wavelength emitted from the reaction chamber of the rotatable disk and Equipped with, When a liquid sample containing nucleic acid flows from the loading chamber through the channel into the plurality of reaction chambers, the rotatable disk is brought into contact with the thermal sealer, thereby sealing the channel and preventing fluid communication between the plurality of reaction chambers. A system comprising: a rotatable disk that is rotated in the substantially horizontal plane, (i) positioning the plurality of reaction chambers adjacent to the first heating element, thereby denaturing the nucleic acids in the sample; (ii) positioning the plurality of reaction chambers adjacent to the second heating element, thereby annealing the primers to the denatured nucleic acids and replicating the nucleic acids; and (iii) oriented the rotatable disk so that the excitation light generated by the light source flows into the reaction chambers, thereby generating the emitted light that is emitted from the reaction chambers and detected by the photodetector.

112. The system according to claim 111, further comprising a mixing chamber installed to be in fluid communication between the loading chamber and the reaction chamber, wherein the liquid sample from the loading chamber is mixed in the mixing chamber with a PCR reagent mixture comprising polymerase and a plurality of nucleotides prior to flowing through the channel into the plurality of reaction chambers.

113. The system according to claim 111 or 112, wherein each of the plurality of reaction chambers is pre-loaded with a PCR reagent comprising primers and probes.

114. The system according to any one of claims 111-113, further comprising a second photodetector oriented to detect emitted light of a third wavelength emitted from the reaction chamber of the rotatable disk.

115. The system according to claim 114, further comprising a third photodetector oriented to detect emitted light of a fourth wavelength emitted from the reaction chamber of the rotatable disk.

116. A system for real-time polymerase chain reaction (PCR), (a) A rotatable disk comprising a loading chamber, a plurality of reaction chambers, and channels connecting the loading chamber to the plurality of reaction chambers, wherein the loading chamber is configured to be heated by induction heating, (b) an analytical device, (i) A sample holder for receiving the rotatable disk in a substantially horizontal plane, wherein the sample holder is configured to rotate the rotatable disk within the substantially horizontal plane, (ii) A first heating element configured to contact the reaction chambers and heat the plurality of reaction chambers to a first temperature, (iii) A second heating element configured to contact the reaction chambers and heat the plurality of reaction chambers to a second temperature An analytical device equipped with A system equipped with these features.

117. The system according to claim 116, wherein the loading chamber is located at the center of the rotatable disk.

118. The system according to claim 116 or 117, wherein the plurality of reaction chambers are located on the periphery of the rotatable disk.

119. A system for real-time polymerase chain reaction (PCR), (a) A rotatable disk comprising a loading chamber, a plurality of reaction chambers, and channels connecting the loading chamber to the plurality of reaction chambers, (b) an analytical device, (i) A sample holder for receiving the rotatable disk in a substantially horizontal plane, wherein the sample holder is configured to rotate the rotatable disk within the substantially horizontal plane, (ii) A first heating element configured to heat the loading chamber to a first temperature exceeding 100 degrees Celsius, (iii) A second heating element configured to contact the reaction chambers and heat the plurality of reaction chambers to a second temperature, (iv) A third heating element configured to contact the reaction chambers and heat the plurality of reaction chambers to a third temperature An analytical device equipped with A system equipped with these features.

120. The system according to claim 119, wherein the first heating element is configured to heat the loading chamber to a first temperature of 101° to 160° Celsius.

121. A system for real-time polymerase chain reaction (PCR), (a) A rotatable disk comprising a loading chamber, a plurality of reaction chambers, and channels connecting the loading chamber to the plurality of reaction chambers, wherein the loading chamber comprises a chelating agent, a single-stranded nucleic acid-binding protein, and a reducing agent, (b) an analytical device, (i) A sample holder for receiving the rotatable disk in a substantially horizontal plane, wherein the sample holder is configured to rotate the rotatable disk within the substantially horizontal plane, (ii) A first heating element configured to contact the reaction chambers and heat the plurality of reaction chambers to a first temperature, (iii) A second heating element configured to contact the reaction chambers and heat the plurality of reaction chambers to a second temperature An analytical device equipped with A system equipped with these features.

122. The system according to any one of claims 116-121, further comprising a light source oriented to generate excitation light of a first wavelength in the horizontal plane occupied by the plurality of reaction chambers of the rotatable disk.

123. The system according to any one of claims 116-122, wherein the analytical device further comprises a photodetector oriented to detect emitted light of a second wavelength emitted from the plurality of reaction chambers of the rotatable disk.

124. A method for processing a target nucleic acid, wherein the method is (a) Loading a sample comprising a target nucleic acid into a loading chamber on a rotatable disk, wherein the rotatable disk further comprises a reaction chamber and a channel connecting the loading chamber to the reaction chamber, (b) Heating the sample containing the target nucleic acid in the loading chamber under high pressure conditions to a temperature exceeding 100°C to produce a processed sample, (c) Rotating the rotatable disk so that the processed sample flows through the channel into the reaction chamber, (d) Amplifying the target nucleic acid in the processed sample within the reaction chamber. Methods that include...

125. A method for processing a target nucleic acid, wherein the method is (a) Loading a sample comprising a target nucleic acid into a loading chamber on a rotatable disk, wherein the rotatable disk further comprises a reaction chamber and a channel connecting the loading chamber to the reaction chamber, (b) Heating the sample containing the target nucleic acid in the loading chamber under high pressure conditions to a temperature exceeding 100°C to produce a processed sample, (c) Rotating the rotatable disk so that the processed sample flows into the reaction chamber through the channel, (d) Bringing the rotatable disk and the sealer into contact, thereby sealing the channel after the processed sample has flowed into the reaction chamber. Methods that include...

126. The method according to claim 124 or 125, wherein the heating includes heating the sample to a temperature of 101° to 160° Celsius.

127. The method according to claim 126, wherein the heating is performed with a temperature gradient of 5°C / second to 50°C / second.

128. The method according to any one of claims 124-127, wherein the sample comprising the target nucleic acid further comprises one or more reagents selected from the group consisting of chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, and stabilizers.

129. The method according to any one of claims 124-128, wherein the loading chamber is pre-loaded with one or more reagents selected from the group consisting of chelating agents, single-stranded nucleic acid-binding proteins, reducing agents, and stabilizers, and the sample is mixed with the one or more reagents in the loading chamber.

130. The method according to any one of claims 124-128, wherein the sample comprising the target nucleic acid comprises a physical sample selected from the group consisting of a blood sample, a tear sample, a saliva sample, a mucus sample, a sputum sample, a fecal sample, a cerebrospinal fluid sample, and a urine sample.

131. The method according to claim 130, wherein the target nucleic acid is not extracted, isolated, or otherwise purified from the body sample prior to heating.

132. The method according to claim 131, wherein the sample comprising the target nucleic acid comprises a plurality of molecular amplification inhibitors, and the heating inactivates the molecular amplification inhibitors of the plurality of molecular amplification inhibitors in the sample.

133. The method according to claim 132, wherein the heating inactivates at least 70% of the plurality of molecular amplification inhibitors, resulting in the treated sample.

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