Permeable gel in the reaction vessel

Abstract

The present invention uses a gel layer to coat and / or separate one or more aqueous reaction mixtures in a reaction vessel. The gel layer can prevent reagents or reaction products from coming into contact with the atmosphere. The gel layer can also separate multiple aqueous reaction mixtures, preventing them from coming into contact with each other until the gel layer is broken. An oil layer can be inserted directly beneath the gel layer. A micropipette tip can be inserted to penetrate the gel layer and deliver a solution, such as a sample, beneath the gel layer. The gel layer can also be broken using a micropipette tip or an inert rod, mixing the aqueous reaction mixtures above and below the layer. The gel layer can also be broken by selective heat application or by the movement of magnetic beads. Multiple gel layers separating multiple aqueous reagent mixtures can be broken in a predetermined sequence for a series of bioprocess steps.

Description

Technical Field

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 466,982, filed on May 16, 2023, the entire content of which is incorporated herein by reference for all purposes.

Background Art

[0002] In amplification reactions such as polymerase chain reaction (PCR) and several commonly performed laboratory detection assays (e.g., immunoassays), multiple aqueous reaction mixtures are used and these mixtures are separated and held from each other until a predetermined step of the assay is performed. Separation can be done by individual containers containing stocks of each aqueous reaction mixture in a kit, and then the experimenter dispenses them into appropriate reaction vessels. However, in such a method, there is a risk that the stock aqueous reaction mixtures will be contaminated and there may be human errors in preparing individual assay reactions (e.g., forgetting to add the reaction mixture to the sample, contamination before, during, or after the reaction due to adding the mixture twice and disturbing the required ratio / concentration of the reagents). Even if no errors occur, preparing reactions for large-scale experiments, such as for a 96-well or 384-well PCR plate reaction, takes a significant amount of time and labor. Therefore, in PCR assays involving multiple reagent subsets (e.g., primer subsets and enzyme subsets) and multiple steps (e.g., RT-PCR), it is necessary to simplify the PCR assay procedure and reduce the involvement in the cold chain when transporting reagent products to end-users. For this reason, it is desirable that reagent products be provided in a ready-to-use or "just add sample" format and transported at ambient temperature. This is important in eliminating human procedural errors, improving the speed and accuracy of the assay, and reducing transportation costs without compromising the performance of the reagents.

[0003] False results due to laboratory contamination before, during, or after the reaction have always been a concern. To address this problem, several solutions have been proposed, particularly for PCR reactions. For example, Johnson et al. describe the use of lyophilized pellets of reagent materials (see, e.g., U.S. Patents 10,144,954 and 11,098,344). One problem inherent in lyophilization, or freeze-drying, is maintaining a sufficiently low moisture level in the lyophilized reagent to ensure functionality during reconstitution. Johnson reported coating or impregnating the lyophilized reagent component with a wax component for this purpose. However, the wax required heating to a specific temperature in a thermal cycler to release the lyophilized reagent into the aqueous component of the PCR reaction added by the experimenter. While this is acceptable in standard thermal cycler-based PCR methods with a high-temperature denaturation step, in newer isothermal PCR methods, the temperature may not be high enough to release the lyophilized reagent and allow for reconstitution. More recent attempts to optimize amplification reactions have involved selecting the best characteristics from conventional optimization methods and integrating them into a format that can be used immediately with only sample addition (see, for example, WO2022 / 026670). While this appears to alleviate the problems of PCR reactions, such as the extensive sample preparation and time-consuming, error-prone steps mentioned above, these methods ultimately rely on proprietary instruments to perform specific POC assays using specific reaction cartridges.

[0004] Previous attempts to optimize PCR reactions have also used waxes and other immiscible hydrophobic substances (mineral oil, silicone oil, various inert greases, etc.) primarily for two purposes: (1) to prevent evaporation of the PCR mixture during thermal cycling and ensure a constant reaction volume and consistency between reactions; and (2) to avoid nonspecific PCR products generated by mispriming (such as primer dimerization or oligomerization) that can occur at temperatures much lower than the thermal cycling temperature during the PCR reaction when mixing PCR reagents at ambient temperature (U.S. Patent No. 5,411,876). This patent reports a method for separating and containing two subsets of PCR reagents using grease or wax to extend the shelf life of PCR reagents and enhance protection against contamination of the laboratory environment by PCR products. The improvement to PCR is achieved by changing the method of mixing the PCR reagents: the reagent subsets separated by grease or wax are mixed for the first time in the initial heating step of PCR amplification. In this heating step, the grease or wax barrier separating the reagent subsets melts and liquefies. During the initial amplification cycle, when the grease or wax melts into a liquid lighter than water, the molten grease or wax and the aqueous layer are mutually replaced, the mixed aqueous reagent is convectively mixed, and then the amplification reaction occurs. However, even with improved wax layer formation using hydrophilic surface-treated tubes as described in Example 5 of U.S. Patent No. 5,411,876, it is not possible to produce PCR products that are comparable to those of the mineral oil control group in terms of target product yield and nonspecific primer dimer quantity: 1) In 3 of the 15 reactions using the wax form, the specific product was significantly less than in the other reactions, while all 6 oil control groups showed a slightly higher yield of specific product than the wax form. 2) In the case of wax, the primer dimer was slightly larger and the primer dimer yield was slightly higher than in oil.

[0005] U.S. Patent No. 5,576,197 reports on PCR containers in which wax is attached to the inner surface located at or above the estimated meniscus position of the PCR mixture. The wax melts at the PCR temperature and covers the surface of the PCR mixture, preventing evaporation during the thermal cycle. According to this patent, similar methods, such as small solid wax balls or microtubes containing solid wax at the bottom of the tube, have the following major drawbacks: In the former case, loading individual wax balls into each reaction tube is prone to contamination, redundant, and time-consuming. In the latter case, the wax and PCR mixture must be inverted by centrifugation, and incomplete inversion leads to problems that are difficult to resolve. Although U.S. Patent No. 5,576,197 attempts to address the above problems, it is not practical to mass-produce microtubes with wax precisely positioned inside the tube in good yield. This manufacturing challenge becomes even more serious when using small-volume microtubes, such as 0.1 mL. Furthermore, the resulting microtubes are also inconvenient to use. Filling reaction mixtures through the small lumen of wax tube containers is cumbersome because wax clumps can clog the pipette tip or detach from the tube wall due to inaccurate pipette positioning.

[0006] Furthermore, many of these methods simply replicated the shortcomings of standard PCR in various aspects of the reaction process. Instead of potentially contaminating the stock of reaction reagents, there was a risk of contaminating the inert oils or greases applied to multiple samples. Adding these components was time-consuming and required pipetting of oily substances. Additionally, these methods were unsuitable for isothermal amplification reactions, as they required an initial step involving high temperatures in a thermal cycler to release and mix the separated components.

[0007] The COVID-19 pandemic, which began in 2019, highlighted the bottlenecks in large-scale and frequent mass infection testing. To address this challenge, Batas et al. reported a wax-layered reagent system that maintains thermal stability during transport without requiring cold chain packaging, offering a concept close to "just add the sample" for immediate clinical testing (Batas et al. Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2, Viruses 2021, 13, 742). This wax reagent system uses a multilayer structure of paraffin wax and silicone wax to separate and package all RT-LAMP (reverse transcription loop-mediated isothermal amplification) reagent components into a single PCR tube. The resulting wax-layered reagent is stable under transport conditions at room temperature and has been confirmed to be usable for testing clinical samples of COVID-19. However, faint fluorescence was observed in some samples, and more importantly, the negative and positive predictive values ​​were only 60% and 74%, respectively, significantly lower than those of freshly prepared liquid reagents. As the authors point out, these underperformance results may be due to the lack of uniform mixing of all reagent components due to the non-pipette operation. Another notable drawback of this wax-layered system is that it requires several manual steps, including melting and stirring the wax layer offline, before it can be loaded into a thermal instrument for isothermal reaction.

[0008] As an alternative to physical barriers such as mineral oil or paraffin wax, one method to suppress carryover contamination in amplification reactions is to use uracil DNA glycosylase in PCR. In this method, uracil-containing DNA (U-DNA) is generated by substituting dTTP with dUTP. Before PCR amplification, the subsequent PCR reaction mixture is treated with uracil DNA glycosylase (UNG), and then the polynucleotides containing apyrimidine moieties are cleaved at high temperature (e.g., 95°C) under alkaline conditions (during the first denaturation step), thereby removing contaminated U-DNA from the sample. (Longo et al., Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene). In other words, the presence of dUTP in the DNA distinguishes PCR products (amplicons, etc.) from natural DNA templates. Since natural templates do not contain dUTP, the templates remain intact even in the presence of enzymes. However, amplicon DNA containing dUTP is more susceptible to enzymatic cleavage.

[0009] However, for the UNG method to be an effective strategy, all PCR reactions in the laboratory would need to be performed using dUTP instead of dTTP, which is not practical in a clinical setting. Furthermore, this method does not always achieve complete removal of contaminants, especially when the length of the PCR product is short (which is common in real-time PCR assays). In addition, UNG contamination can reduce amplification efficiency, potentially delaying or missing target detection, especially when the initial sample contains one or a few target molecules (Pierce and Wangh, Effectiveness and limitations of uracil-DNA glycosylases in sensitive real-time PCR assays, Biotechniques 2004 Jan;36(1):44-6, 48). Other drawbacks of this strategy compared to conventional PCR methods include the need for extra material costs and redundant assay development work when using this chemical-based method to suppress carryover contamination.

[0010] From the above perspective, there is a need for a compartmentalized reaction vessel that is ready for immediate use, has performance equivalent to conventional liquid-type reaction vessels using PCR tubes or ELISA microtiter plates, is thermally and mechanically stable to transport at or below ambient temperature, has a long shelf life under freezing temperature, ambient temperature, or below ambient temperature storage conditions, and can form a non-flowable seal inside the vessel to prevent carryover contamination by reaction products after the reaction. As will become clear from the following considerations, the present invention provides these features and other features individually or in combination. [Overview of the project]

[0011] The present invention provides a reaction vessel comprising a gel layer, the gel layer being pierceable by a micropipette tip, and the reaction vessel being located within the vessel. Optionally, the reaction vessel further comprises one or more additives within the gel layer and mineral oil beneath the gel layer. Optionally, the reaction vessel further comprises an aqueous reaction mixture above and / or below and / or inside the gel layer. Optionally, the aqueous reaction mixture is for the detection of an analyte. Optionally, the aqueous reaction mixture is located below the gel layer, the gel layer prevents the aqueous reaction mixture from dispersing across the gel layer, the gel layer is pierceable by a micropipette tip, and a solution is delivered to the reaction mixture to cause a reaction and detect the analyte. Optionally, the delivered solution is a sample containing the analyte. Optionally, the gel layer prevents the aqueous reaction mixture from dispersing across the gel layer, and the tip of a micropipette is pierceable within a temperature range of at least 18–35°C, optionally within a temperature range of 20–25°C. Optionally, the reaction vessel comprises a plurality of gel layers, a plurality of oil layers, and a plurality of aqueous reaction mixture layers, wherein the number of gel layers differs from the number of oil layers, and / or the number of gel layers differs from the number of aqueous reaction mixture layers. Optionally, the micropipette comprises a single tip or a bundle of multiple tips or capillaries.

[0012] Optionally, the reaction vessel comprises a first aqueous reaction mixture, a first gel layer covering the first aqueous reaction mixture and through which a micropipette tip can penetrate, a second aqueous reaction mixture above the first gel layer, and a second gel layer covering the second aqueous reaction mixture and through which a micropipette tip can penetrate, optionally further comprising a first mineral oil layer between the first aqueous reaction mixture and the first gel layer, and optionally further comprising a second mineral oil layer between the second aqueous reaction mixture and the second gel layer. Optionally, one of the first and second aqueous reaction mixtures is a sample, and the other comprises one or more reagents for producing a reaction to detect an analyte in the sample when the first and second aqueous reaction mixtures are mixed. Optionally, the first and second aqueous reaction mixtures are each a partial reaction mixture that produces a detection reaction when mixed with each other and with the sample. Optionally, a third aqueous reaction mixture is present above the second gel layer, and the plurality of aqueous reaction mixtures comprises reagents for carrying out ordered bioprocess steps. Optionally, the plurality of aqueous reaction mixtures are for nucleic acid sequencing library preparation, nucleic acid sample extraction, immunoassay, biochemistry, or nucleic acid amplification and detection. Optionally, the first aqueous reaction mixture and the second aqueous reaction mixture each contain polymerase and primers, or vice versa, for nucleic acid detection by PCR amplification, qPCR amplification, reverse transcriptase PCR reaction, digital PCR amplification, or isothermal amplification. Optionally, the first aqueous reaction mixture and the second aqueous reaction mixture each contain different reagents required for the detection reaction in combination with each other and with the sample.

[0013] Optionally, the combination of the first aqueous reaction mixture and the second aqueous reaction mixture is for performing immunoassay and detection, and optionally, a third aqueous solution is present above the second gel layer for sequential reactions. Optionally, the aqueous reaction mixture contains magnetic beads.

[0014] Optionally, the reaction vessel is coupled to a heater module that slides along the outside of the reaction vessel, and the heater melts the gel layer when it is adjacent to the gel layer. Optionally, the reaction vessel is coupled to a magnetic module that slides along the external or internal surface of the reaction vessel, and magnetic beads are moved from the aqueous reaction mixture across the gel layer beneath the aqueous reaction mixture. Optionally, the sliding magnetic module is slidable along the inside of the reaction vessel and is coupled to a rod for detecting the reaction within the reaction vessel.

[0015] Optionally, the aqueous reaction mixture comprises a rod for detecting the reaction in the reaction vessel. Optionally, the rod is an electrode. Optionally, the reaction vessel is a well in a multi-well plate (e.g., a PCR plate, deep-well plate, microtiter plate, or ELISA plate), where each well contains a gel layer. Optionally, the reaction vessel is a tube or tubing (e.g., PCR tubes) comprising multiple such vessels, either individually or arranged as a strip or matrix and interconnected. Optionally, the reaction vessel is made of plastic, glass, or metal and has one or more grooves or channels.

[0016] Optionally, the reaction vessel is a well on a chip or tubing (e.g., a glass slide or silicon wafer or a plastic slide or a metal slide), and optionally has a plurality of wells providing a plurality of reaction vessels, optionally the plurality of wells are connected by channels, and optionally the wells contain a membrane or fragments of glass fiber.

[0017] Optionally, the inner surface of the reaction vessel may be hydrophobic or hydrophilic.

[0018] Optionally, the gel layer may be made of a polymer gel matrix consisting of inorganic, organic, or a combination of inorganic and organic materials, or a mixture of wax and mineral oil or silicone oil in a weight ratio of 25:75 to 75:25, with or without surfactant additives.

[0019] The present invention further provides a method for carrying out a reaction, comprising the steps of providing a reaction vessel as defined above, and delivering a solution from the micropipette tip by penetrating the gel layer with the micropipette tip, thereby causing a reaction to occur. Optionally, the solution comprises a sample and optionally a reaction reagent. Optionally, the gel layer melts during and after the reaction and then re-solidifies. Optionally, the method further comprises the steps of rupturing the gel layer with an inert rod or a stoppered micropipette tip, and then delivering one or more aqueous reaction mixtures to the reaction vessel by penetrating the gel with the micropipette tip. Optionally, the stopper in the tip is wax or gel. Optionally, the reaction vessel comprises a first aqueous reaction mixture below a first gel layer, a second aqueous reaction mixture above the first gel layer, and a second gel layer above the second aqueous reaction mixture, and the method comprises the step of mixing the first aqueous reaction mixture and the second aqueous reaction mixture by penetrating the gel layers using an inert rod or micropipette tip, so that the reaction vessel contains a mixed reaction mixture covered by a third gel layer formed from the first and second gel layers, and the method further comprises the step of delivering the solution from the micropipette tip to the mixed reaction mixture by penetrating the third gel layer with the micropipette tip. Optionally, the method further comprises the step of centrifugating the reaction vessel to facilitate mixing of the reaction mixture between the gel layers. Optionally, the solution is an aqueous reaction mixture comprising all reaction reagents including a sample for a reaction to detect a target in a sample, or some reaction reagents for a reaction to detect a target in a sample. Optionally, the aqueous reaction mixture is delivered to an oil or gel layer to form emulsion droplets or other reaction compartments for a droplet digital PCR reaction or a droplet immunoassay PCR reaction.

[0020] The present invention further provides a method for carrying out a reaction, comprising the steps of providing a reaction vessel as defined in claim 1, and solutions disposed above and below the gel layer, and displacing or melting the gel layer, thereby mixing the solutions and causing a reaction to occur.

[0021] The present invention further provides a reaction vessel comprising a plurality of aqueous reaction mixtures separated from each other by a plurality of gel layers disposed within the reaction vessel, wherein the plurality of aqueous reaction mixtures are mixable by melting and / or breaking the gel layers in order to perform a series of processing steps in a predetermined sequence. Optionally, the reaction vessel further comprises a mineral oil layer beneath one or more of the gel layers. Optionally, the reaction vessel is connected to a heater slidable along the outer surface of the reaction vessel to melt the gel layer adjacent to the heater. Optionally, at least one aqueous reaction mixture contains magnetic beads, and the reaction vessel is connected to a magnet slidable along the outer surface of the reaction vessel to move the magnetic beads through the gel layer beneath the aqueous reaction mixture containing the magnetic beads. Optionally, the gel is a mixture of paraffin wax and mineral oil or silicone oil in a volume or weight ratio in the range of 99:1 to 25:75. Optionally, the aqueous reaction mixture contains reagents for performing predetermined steps of an integrated immunoassay. Optionally, the aqueous reaction mixture contains different reagents for a predetermined bioprocess step, and optionally contains different reagents for a predetermined step of an immunoassay. Optionally, each of the aqueous reaction mixtures contains one or more of the following for performing a predetermined step of an integrated immunoassay: magnetic beads, capture antibody, washing solution, detection antibody, enzyme, substrate, and stop solution. Optionally, each of the aqueous reaction mixtures contains one or more of the following for performing a predetermined step of integrated DNA / RNA purification: lysis buffer, DNA / RNA binding solution, magnetic beads, washing buffer, and elution buffer. Optionally, each of the aqueous reaction mixtures contains one or more of the following for performing a predetermined step of integrated NGS library preparation: fragmentation enzyme or end repair reagent, ligation reagent, and library amplification reagent.

[0022] The present invention further provides a method for carrying out a reaction, comprising the steps of providing the reaction vessel described above, and melting one of the gel layers, thereby mixing the aqueous reaction mixture above and below the gel layer to initiate the reaction. Optionally, the reaction vessel is connected to a heater that is slidable on the outer surface of the reaction vessel, and the heater is moved along the surface to a position where one of the gel layers can be melted. Optionally, the heater is part of a module. Optionally, the heater is a thermoelectric device. Optionally, the thermoelectric heating element is a semiconductor-based Peltier element. Optionally, the module further comprises a cooling element for re-solidifying the gel layer after melting. Optionally, the module is connected to a magnetic module for moving magnetic beads. Optionally, the magnet is an electromagnet.

[0023] The present invention further provides a method for carrying out a reaction, comprising the steps of providing the reaction vessel described above, connected to a slidable magnetic module located on the external or internal surface of the reaction vessel, wherein at least one of the aqueous reaction mixtures comprises magnetic beads, and the method further comprises moving the slidable magnetic module along the external or internal surface to a position where the beads can be moved. Optionally, the slidable magnetic module is connected to one or more modules from among mechanical, thermal, electrical, magnetic, and optical modules to assist the reaction in the reaction vessel and to detect the reaction in the reaction vessel in real time or after the reaction has finished. Optionally, the reaction vessel is connected to a device for processing a sample or reagent and for detecting chemical or physical changes resulting from the reaction in the reaction vessel, such as color, fluorescence, emission, chemiluminescence, electrochemistry, radiation, reflection, phase change, magnetoresistance, turbidity, mobility shift, pH, ionic strength, etc.

[0024] The present invention further provides a reaction vessel comprising a first gel layer containing a first aqueous reaction mixture, an optional second gel layer containing a second aqueous reaction mixture, and an optional third aqueous reaction mixture or sample above the second gel layer, wherein the gel is meltable by heating, thereby mixing the aqueous reaction mixtures. Optionally, the reaction vessel further comprises an oil layer having a density higher than that of water above the second gel layer.

Brief Description of Drawings

[0025] [Figure 1] It shows a single-layer reaction tube without reagents, pre-filled with a gel layer. 1: Tube, 2: Gel layer

[0026] [Figure 2] It shows a two-layer reaction tube without reagents, pre-filled with a gel layer. 1: Tube, 2: Gel layer, 3: Mineral oil

[0027] [Figure 3] It shows a six-layer reaction tube containing reagents, pre-filled with a gel and mineral oil. 1: Tube, 2: Gel layer, 3: Mineral oil, 4: Second aqueous reaction mixture, 5: Gel layer, 6: Mineral oil, 7: First aqueous reaction mixture

[0028] [Figure 4] It shows a three-layer reaction tube containing reagents, pre-filled with a gel layer. 1: Tube, 2: Gel layer, 3: Mineral oil, 4: Aqueous reaction mixture.

[0029] [Figure 5]This diagram shows an ELISA reaction vessel system using magnetic beads. The system includes reagents divided into multiple compartments separated by permeable gel layers, a movable external heating element, and magnetic pieces. The arrows indicate the direction of movement of the heating element and magnetic pieces during the assay. 1: Tube 2: First gel layer 3: Movable magnet 4: Movable heating element 5: Magnetic bead chamber 6: Magnetic beads coated with captured antibody 7: Second gel layer 8: Washing chamber I 9: Third gel layer 10: Detection antibody-antigen binding chamber 11: Fourth gel layer 12: Washing chamber II 13: Fifth gel layer 14: Reaction product detection chamber 15: Sixth gel layer 16: Stop solution chamber

[0030] [Figure 6] This diagram shows an integrated nucleic acid purification system based on magnetic beads. The system includes a multi-compartment reagent with a heat-sensitive gel layer, a movable external heating / cooling module, and a magnetic module. The arrows indicate the direction of movement of the heating and magnetic modules during the purification process. 1: Tube 2: First gel layer 3: First movable heating / cooling element 4: Movable magnet 5: Second movable heating / cooling element 6: Dissolution chamber 7: Second gel layer 8: Magnetic bead chamber 9: Magnetic beads 10: Third gel layer 11: Washing I chamber 12: Fourth gel layer 13: Washing II chamber 14: Fifth gel layer 15: Washing III chamber 16: Sixth gel layer 17: Elution chamber

[0031] [Figure 7] This diagram shows an integrated reaction vessel system containing multi-compartment reagents pre-filled for NGS library preparation. 1: Tube 2: First gel layer 3: Fragmentation chamber 4: First heating element 5: First heating / cooling element 6: Second gel layer 7: Ligation chamber 8: Third gel layer 9: PCR amplification chamber 10: Second heating / cooling element

[0032] [Figure 8] The following are representative PCR amplification curves for three reaction tubes: i) liquid form (――); ii) gel layer alone (- - -); iii) two-layer form with gel layer and mineral oil layer (―·―).

[0033] [Figure 9] Images of three types of reaction tubes before and after thermal cycling are shown.

[0034] [Figure 10A-C] The following shows the four-color amplification curves for three tube types: [Figure 10A] two-layer format, [Figure 10B] Gel-only form, [Figure 10C] liquid format.

[0035] [Figure 11] The amplification curve of a PCR reaction using multilayer reagents is shown.

[0036] [Figure 12] Images of multilayer reagent tube strips before and after thermal cycling are shown.

[0037] [Figure 13A-D] The four-color amplification curves for multilayer UTI reagent tubes (black) and liquid-type tubes (gray) are shown. [Figure 13A] FAM channel, [Figure 13B] HEX Channel, [Figure 13C] ROX channel, [Figure 13D] Cy5 Channel

[0038] [Figure 14] The images show multilayer reagent tube strips before (top) and after (bottom) isothermal amplification at 60°C for 90 minutes.

[0039] [Figure 15] The amplification curves (FAM channels) of the reaction using HPV31 reagent tube strips (black) and liquid tube strips (gray) are shown.

[0040] [Figure 16A-B]The results of a free-fall experiment with multilayer reagent tubes are shown. Twelve tube strips were dropped three times from a height of approximately 10 feet onto the laboratory floor. [Figure 16A] Before falling, [Figure 16B] After the fall.

[0041] [Figure 17A-B] The two-color amplification curves (gray: FAM channel, black: ROX channel) are shown for two types of multilayer tube strips after repeated freeze-thaw cycles and long-term transport at ambient temperature. [Figure 17A] A 5-layer tube with no mineral oil layer above the enzyme mix layer at the bottom. [Figure 17B] A 6-layer tube with a mineral oil layer on top of a bottom enzyme mix layer. Definition of terms

[0042] Unless otherwise defined, all technical and scientific terms have the same meaning as they are commonly understood in the art to which this invention relates. The following definitions supplement the definitions in the art and relate to this application, and do not belong to any related or unrelated cases, such as jointly owned patents or applications. Any methods and materials similar to or equivalent to those described herein may be used in carrying out the tests of the present invention, but preferred materials and methods are described herein. Accordingly, the terms used herein are intended solely to describe specific embodiments and are not intended to limit them. The terms "a" or "an" refer to one or more entities. For example, "a nucleic acid" refers to one or more nucleic acids. Accordingly, the terms "a" (or "an"), "one or more," and "at least one" are interchangeable herein.

[0043] The term "nucleic acid" encompasses any sequence of physical monomer units that can be associated with a sequence of nucleotides, including polymers of nucleotides (e.g., common DNA or RNA polymers), peptide nucleic acids (PNAs), and modified oligonucleotides (e.g., oligonucleotides containing bases not typical of biological RNA or DNA in solution, such as 2'-O-methylated oligonucleotides). Nucleic acids include DNA, RNA, and any combination thereof. Nucleic acids can be double-stranded or single-stranded. DNA can be genomic DNA, cDNA, methylated DNA, or synthetic DNA, etc. RNA can be mRNA, miRNA, tRNA, rRNA, hnRNA, or methylated RNA, etc.

[0044] The most common nucleotide bases are A, T or U, C, and G, with T being present in DNA and U in RNA. The nucleotides included in the target are usually natural nucleotides (deoxyribonucleotides or ribonucleotides). The same applies to the nucleotides that make up the primers.

[0045] Nucleic acid chain complementarity means that the chains form a stable double helix through hydrogen bonds between groups of nucleic acid bases. Complementary bases are A and T, C and G in DNA, and C and G, U and A in RNA. Nucleotides in each chain are complementary if, when the chains are maximally aligned, they form one of these (Watson-Crick base pairs). If the nucleotides do not form complementary base pairs when the chains are maximally aligned, they are mismatched. Chain complementarity can be complete or partial. Complete complementarity between two chains means that the two chains can form a double helix, and all bases in the double helix bond with complementary bases through Watson-Crick pairing. Partial complementarity means that, under hybridization conditions (e.g., salt concentration and temperature), most (but not all) of the bases in the chains form Watson-Crick base pairs, creating a stable hybrid complex. For example, some primers can form a double helix with the primer binding site even with one, two, or three mismatches, but these mismatches are not at the 3' end, and preferably not in its vicinity (e.g., within 4 nucleotides). Such conditions can be predicted using sequences and standard mathematical calculations to predict the Tm of the hybridized strands, or by empirically determining Tm using conventional methods. Tm refers to the temperature at which 50% of the total number of hybridization complexes formed between two nucleic acid strands denature. Below Tm, the formation of hybridization complexes is promoted, while above Tm, the melting or separation of strands in the hybridization complex is promoted. The Tm of nucleic acids with a known G+C content in a 1M NaCl aqueous solution can be estimated, for example, using Tm = 81.5 + 0.41 (%G+C) - 675 / N - % mismatches (N = total number of bases).

[0046] Hybridization or annealing conditions include the chemical components and concentrations (salts, chelating agents, formamide, etc.) of the aqueous or organic solution containing nucleic acids, and the temperature of the mixture in which one nucleic acid chain binds to a second nucleic acid chain through complementary chain interactions to form a hybridization complex.

[0047] The analyte is the molecule detected in a binding assay. Analytes can be various things, such as antigens, small molecules, and nucleic acids.

[0048] An "antigen" is a substance that, upon contact with a target or organism (for example, if present within the target or organism, or detected by the target or organism), elicits a detectable immune response from that target or organism. Antigens can be, for example, lipids, proteins, carbohydrates, nucleic acids, or combinations or variations thereof. For example, an "antigen peptide" refers to a peptide that, when present within the target or organism, or detected by the target or organism, elicits the expression of an immune response in that target or organism. Therefore, an antigen can be recognized by an antigen-binding protein, such as an antibody produced by the target or organism upon contact with the antigen.

[0049] The term "epitope" refers to a portion of an antigen recognized by an antigen-binding protein. A single antigen (such as an antigen polypeptide) may have multiple epitopes. Epitopes can be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and are defined as linear sequences of residues that directly contribute to the binding ability of the antigen-binding polypeptide to the antigen. Epitopes may be three-dimensional, i.e., composed of non-linear amino acids. Epitopes may contain determinants that are chemically active surface substrates of molecules, such as amino acids, sugar side chains, phosphorylation groups, and sulfonyl groups, and may have specific three-dimensional structural properties and / or specific charge properties. Epitopes formed from consecutive amino acids are usually retained by exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost by denaturing solvent treatment.

[0050] A sample is a composition that may contain one or more analytes and includes patient samples, plant or animal materials, waste, forensic analysis materials, environmental samples, circulating tumor cells (CTCs), free DNA, and liquid biopsies. Samples may include any tissue, cells, or extracts derived from living or deceased individuals that may contain target nucleic acids, such as peripheral blood, bone marrow, plasma, serum, biopsy tissue including lymph nodes, respiratory tissue or exudate, gastrointestinal tissue, urine, feces, semen, and other bodily fluids. Particularly important samples are tissue samples (including bodily fluids) from humans or animals with or suspected of having a disease or symptom, especially a viral infection. Other important samples include industrial samples used for water quality testing, food testing, and contamination control. The components of a sample may include target and non-target nucleic acids, target and non-target antigens, other analytes, and other substances such as salts, acids, bases, surfactants, proteins, carbohydrates, lipids, and other organic or inorganic substances. Samples may or may not be treated to purify the target nucleic acid or antigen before amplification, detection, or other analysis. Further processing of nucleic acids may include treatment with surfactants or denaturants to release nucleic acids from cells or viruses, removal or inactivation of non-nucleic acid components, and concentration of nucleic acids. Further processing of polypeptide antigens may include any of the appropriate methods identified in the “Strategies for Protein Purification and Characterization” collection (updated April 2022) of Current Protocols in Protein Science 1995, published by Wiley, the contents of which are fully incorporated herein by reference. Any of the above types of lipids, carbohydrates, or complex antigens may be processed by appropriate methods necessary to ensure the concentration and purity appropriate for the assay of interest.

[0051] "Target nucleic acid" refers to a group of nucleic acid molecules or related nucleic acid molecules that are present or may be present in a sample. Target nucleic acid may include a segment to be amplified, defined by a primer binding site. This segment may be the entire nucleic acid or any segment of a nucleic acid of amplified length. The target nucleic acid may be an entire chromosome, gene, or cDNA, and the target segment may be, for example, only 40 to 500 of these nucleotides. The target segment may be present on any strand of the structure (sense strand or antisense strand). Target nucleic acid may be RNA (e.g., viral RNA, microRNA, mRNA, cRNA, rRNA, hnRNA, cfRNA), DNA (genomic DNA, somatic DNA, cfDNA, cffDNA, cDNA), etc.

[0052] The target nucleic acids may be derived from pathogenic microorganisms such as viruses, bacteria, and fungi, or they may be endogenous to the patient. Viral nucleic acids (genomic RNA, mRNA, etc.) are useful targets for viral sequence analysis. Examples of detectable viruses include HIV, hepatitis (types A, B, and C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, Epstein-Barr virus), adenovirus, XMRV, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, cornovirus, coronavirus, RSV, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, MLV-associated virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, arbovirus encephalitis virus, and others. Examples of bacteria include Chlamydia, Rickettsia, Mycobacterium tuberculosis, Staphylococcus, Treptococcus, Streptococcus pneumoniae, Neisseria meningitidis, Conococcus, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Clostridium botulinum, Bacillus anthrax, Plague, Leptospirosis, Lyme disease, Streptococcus, Neisseria, and others. rRNA is a particularly useful target nucleic acid in bacterial typing. Detection of human or animal genes is useful in detecting the presence or susceptibility to disease. Examples of genes that can be detected include oncogene fusions, BRACA-1 or BRAC-2, p53, CFTR, and cytochrome P450. These genes are used for genotyping (forensic identification, paternity testing, heterozygous carriers of genes that act in homozygous individuals, HLA typing, etc.), determining the effectiveness of drugs on individuals (companion diagnostics, etc.), and other applications.

[0053] The term "dNTP" generally refers to a single or combination of deoxynucleotides containing phosphate, sugar, and triphosphate-type organic bases, providing precursors necessary for DNA synthesis by DNA polymerase. A dNTP mixture may contain naturally occurring deoxynucleotides (i.e., adenine (A), guanine (G), cytosine (C), uracil (U), and / or thymine (T)). In some embodiments, each of the naturally occurring dideoxynucleotides may be substituted or supplemented with synthetic analogs such as inosine, iso-G, iso-C, deaza-G, and deaza-A.

[0054] The primer binding site is the complete or partial site within the target nucleic acid that the primer hybridizes to.

[0055] A primer or probe is an oligonucleotide that is complementary to the primer or probe binding site, either in whole or in part, of the target nucleic acid. The primer or probe can be bound at its 5' end to another nucleic acid (also called a tail) that is not present in or complementary to the target nucleic acid.

[0056] Primers or probes are oligonucleotides. The term “oligonucleotide” encompasses both the singular and plural forms and refers to any polymer consisting of two or more nucleotides, nucleosides, nucleic acid bases, or related compounds used as reagents in the amplification method and subsequent detection method of the present invention. Oligonucleotides may be DNA, RNA, and / or their analogues, and / or DNA-RNA chimeras. The term oligonucleotide does not indicate a specific function of a reagent but is used to collectively encompass all such reagents described herein. Oligonucleotides can perform a variety of functions. For example, they may function as primers if they are hybridizable to a complementary strand and can be further extended in the presence of nucleic acid polymerase. They may function as promoters if they contain a sequence that is recognized by RNA polymerase and enables transcription. They may also contain detection reagents for signal generation / amplification, and, if properly positioned and / or modified, may function to inhibit hybridization or primer extension. Specific oligonucleotides of the present invention are described in more detail below. In this specification, oligonucleotides can be substantially any length and are limited only by their specific function in amplification reactions or in the detection of amplification products of amplification reactions. Oligonucleotides having a defined sequence and chemical structure can be produced by conventional techniques such as chemical or biochemical synthesis, and in vitro or in vivo expression from recombinant nucleic acid molecules such as bacterial or viral vectors. Oligonucleotides may be modified in any way, as long as the given modification is compatible with the desired function of the oligonucleotide to the extent that the modification can be easily determined. Modifications include base modifications, sugar modifications, or skeletal modifications. Examples of base modifications include, but are not limited to, the use of adenine, cytidine, guanosine, thymine, and uracil, as well as C-5 propine, 2-aminoadenine, 5-methylcytidine, inosine, and dP and dK bases.The sugar groups of the nucleoside subunits are ribose, deoxyribose, and their analogues, such as ribonucleosides having a 2'-O-methyl (2'-O-ME) substitution on the ribofuranosyl moiety. See "Method for Amplifying Target Nucleic Acids Using Modified Primers," (Becker, Majlessi, & Brentano, 2000, U.S. Patent No. 6,130,038). Other sugar modifications include, but are not limited to, 2'-amino, 2'-fluoro, (L)-α-treophranosyl, and pentopranosyl modifications. Nucleoside subunits may be linked by phosphodiester bonds, modification bonds, or non-nucleotide moieties that do not interfere with hybridization of the oligonucleotide with a complementary target nucleic acid sequence. Modification bonds include those in which a standard phosphodiester bond is replaced by a different bond, such as a phosphorothioate bond or a methylphosphonate bond. Nucleic acid base subunits may be linked, for example, by replacing the natural deoxyribose phosphate backbone of DNA with a pseudo-peptide backbone such as a 2-aminoethylglycine backbone, which links the nucleic acid base subunits to a central secondary amine via a carboxymethyl linker. (DNA analogs having a pseudo-peptide backbone are commonly called "peptide nucleic acids" or "PNAs," and are disclosed in Nielsen et al., "Peptide Nucleic Acids," (Nielsen, Buchardt, Egholm, & Berg, 1996, U.S. Patent No. 5,539,082). Other linkage modifications include, but are not limited to, morpholino linkages. Non-limiting examples of oligonucleotides or oligomers envisioned in the present invention include nucleic acid analogs, including bicyclic and tricyclic nucleosides and nucleotide analogs (LNAs).See Imanishi et al., "Bicyclonucleoside and Oligonucleotide Analogues," (Imanishi & Obika, 2001, U.S. Patent No. 6,268,490) and Wengel et al., "Oligonucleotide Analogues," (Wengel & Nielsen, 2003, U.S. Patent No. 6,670,461). In this invention, any nucleic acid analogue is envisioned as long as the modified oligonucleotide can perform its intended function, such as hybridizing to a target nucleic acid under harsh hybridization or amplification conditions, or interacting with DNA or RNA polymerase to elongate or initiate transcription. In the case of detection probes, the modified oligonucleotide must be able to preferentially hybridize to the target nucleic acid under harsh hybridization conditions. The 3' end of an oligonucleotide (or other nucleic acid) can be blocked in various ways using blocking modification groups, as described later. A "blocked" oligonucleotide will not be efficiently elongated even if a nucleotide is added to its 3' end by DNA or RNA-dependent DNA polymerase to generate complementary DNA. Therefore, "blocked" oligonucleotides cannot serve as "primers."

[0057] The term "degenerate primer" refers to a mixture of similar primers that have different bases at various positions (Mitsuhashi, J. Clin. Lab. Anal., 10(5): 285 93 (1996); von Eggeling et al., Cell. Mol. Biol., 41(5):653 70 (1995); (Zhang et al., Proc. Natl. Acad. Sci. USA, 89:5847 5851 (1992); Telenius et al., Genomics, 13(3):718 25 (1992)). Such primers may include inosine, as inosine can base-pair with adenosine, cytosine, guanine, or thymidine. Degenerate primers enable annealing and amplification to a variety of relevant target sequences. Degenerate primers that anneal to target DNA can function as priming sites for further amplification. A degenerate region is a region in which part of the primer changes, while the rest of the primer remains the same. A degenerate primer (or region) can refer to multiple primers and may be random. A random primer (or region) means that the sequence has not been selected and may be degenerate, but does not necessarily have to be. In some embodiments, the Tm of the 3' target-specific region is approximately 5°C to 50°C. In some embodiments, the Tm of the 15-mer is less than approximately 60°C.

[0058] A primer, "3' segment, 3' binding region, 3' binding site, or 3' hybridization region," can bind to genomic sequences or other nucleic acid sequences that appear at a specific frequency in the genome. In some embodiments, this frequency is approximately 0.01% to 2.0%, for example, approximately 0.05% to 0.1%, or approximately 0.1% to 0.5%. In some embodiments, the length of the primer's "binding site" depends primarily on the average length of the predicted PCR product based on bioinformatics calculations. This definition includes, but is not limited to, binding regions of approximately 4 to 12 nucleotides in length. In more specific embodiments, the length of the 3' binding region can be, for example, approximately 4 to 20 nucleotides, or approximately 8 to 15 nucleotides. Binding regions with a Tm value of approximately 10°C to 60°C are also included in this definition. As used herein, the term "primer binding segment" refers to a primer for a specific sequence.

[0059] Polymerases are enzymes capable of template-directed extension of primers hybridized to a template. They can be DNA polymerases, RNA polymerases, or reverse transcriptases. Examples of DNA polymerases include E. coli DNA polymerase I, Taq DNA polymerase, S. pneumoniae DNA polymerase I, Tfl DNA polymerase, D. radiodurans DNA polymerase I, Tth DNA polymerase, Tth XL DNA polymerase, M. tuberculosis DNA polymerase I, M. thermoautotrophicum DNA polymerase I, herpes simplex type 1 DNA polymerase, T4 DNA polymerase, thermosecunease, or wild-type or modified T7 DNA polymerase, Φ29 polymerase, Bst polymerase, Vent polymerase, 9°Nm polymerase, and Klenow fragments of DNA polymerase I. Examples of reverse transcriptases include AMV reverse transcriptase, MMLV reverse transcriptase, and HIV reverse transcriptase. Examples of RNA polymerases include T7 RNA polymerase or SP6 RNA polymerase, bacterial RNA polymerase, eukaryotic RNA polymerase, and recombinase.

[0060] Amplification refers to the generation of additional copies (multiple copies) of all or part of a target nucleic acid by template-induced primer extension (target amplification), or the amplification of a detection signal for qualitative / quantitative measurement (signal amplification), or both. Amplification can be performed under temperature cycling conditions, isothermal conditions, or a combination thereof. Amplification can be performed linearly or exponentially. The target nucleic acid can be conjugated with an antibody for immunoPCR or immunoisothermal amplification.

[0061] The terms "thermal cycling," "thermal cycle(plural)," or "thermal cycle(singular)" refer to a repeating cycle of temperature changes from the total denaturation temperature to the annealing (or hybridizing) temperature, the extension temperature, and back to the total denaturation temperature. These terms also refer to a repeating cycle of the denaturation temperature and the extension temperature, in which case the annealing temperature and the extension temperature are combined into a single temperature. At the total denaturation temperature, all double-stranded fragments unwind into single strands. At the annealing temperature, primers can hybridize (anneal) to the complementary sequence of the separated strand of the nucleic acid template. At the extension temperature, the nascent DNA strand of the amplicon is synthesized.

[0062] The term "amplification mixture" or "PCR mixture" refers to a mixture of components necessary to amplify at least one amplicon from a nucleic acid template. The mixture may include nucleotides (dNTPs), heat-stable polymerases, primers, and multiple nucleic acid templates. The mixture may further include Tris buffer, monovalent salts, and Mg 2+ It may contain the following. The concentrations of each component are well known in the art and can be further optimized.

[0063] The term "amplification product" or "amplicon" refers to a fragment of DNA that has been amplified by polymerase using a pair of primers in amplification methods such as PCR.

[0064] The term "phosphor" refers to a part of a material that absorbs light energy at a defined excitation wavelength and emits light energy at a different defined wavelength.

[0065] The term "quencher" includes any site that, when placed in close proximity to an excited fluorescent label, can absorb and dissipate its energy. Quenchers can be fluorescent quenchers or non-fluorescent quenchers (also called dark quenchers). The above-mentioned phosphors can act as quenchers when in close proximity to other phosphors, and FRET quenching or contact quenching may occur. It is preferable to use dark quenchers that do not emit any visible light. Examples of dark quenchers include, but are not limited to, DABCYL (4-(4'-dimethylaminophenylazo)benzoic acid) succinimidyl ester, diallylrhodamine carboxylic acid, succinimidyl ester (QSY-7), and 4',5'-dinitrofluorescein carboxylic acid, succinimidyl ester (QSY-33), quenchers, or Black Hole Quencher® (BHQ-1, BHQ-2, and BHQ-3), nucleotide analogs, nucleotide G residues, nanoparticles, and gold particles.

[0066] A "mutation" refers to a condition in which one or more nucleotides in a target nucleic acid sequence differ from the typical form of the target nucleic acid, known as the wild type. The wild type refers to the most common allele form of the sequence, the first form discovered, and / or the sequence form associated with normality (non-disease phenotype). Single nucleotide polymorphisms (SNPs) are a type of mutation.

[0067] The term "surface" refers to any solid surface on which nucleic acids can covalently bond, such as latex beads, dextran beads, polystyrene, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, and silicon wafers. Preferably, the solid support is a glass surface.

[0068] "Adhesion to a surface" refers to any chemical or non-chemical method of attachment (including chemically modifiable functional groups). "Attachment" refers to the immobilization of nucleic acids on a solid support by covalent bonding, irreversible passive adsorption, or intermolecular affinity (e.g., fixation to an avidin-coated surface by biotinylated molecules). The attachment must be strong enough that it cannot be removed by washing with water or aqueous buffer under DNA denaturation conditions.

[0069] A sticky end is a single-stranded end adjacent to a double-stranded segment of a nucleic acid. Nucleic acids with sticky ends that have complementary sequences can anneal via the sticky ends and undergo ligation reactions with each other.

[0070] A "genetic marker" refers to a polynucleotide sequence or modification of a polynucleotide sequence that exists in the genomic sequence of a reference chromosome with a known physical location that enables identification. Examples of genetic markers include, but are not limited to, different alleles that are distinguished from each other based on differences in polynucleotide sequences (e.g., polymorphisms, etc.) (e.g., alleles from two different individuals, such as fetal alleles and pregnant alleles), or the presence or absence of a sequence (e.g., a sequence present on the Y chromosome of a male fetus but not in the genome of a pregnant woman). In this context, a "methylation marker" located on a chromosome related to chromosomal aneuploidy refers to a genomic polynucleotide sequence on a chromosome with an abnormal number of markers. Furthermore, if there is an extra fragment on a chromosome, or if a part of a chromosome is missing, a "methylation marker" refers to the presence of the relevant chromosomal fragment or part. Differences in the methylation profiles of methylation markers allow for the distinction of corresponding methylation markers between two different individuals, such as a fetus and a pregnant woman.

[0071] The term "single nucleotide polymorphism" or "SNP" refers to a single-nucleotide polynucleotide sequence variation that exists between different alleles of the same gene. This can be between two copies of the same gene on the same chromosome in the same individual (e.g., two alleles from a fetus), or between two different individuals (e.g., a fetus and a pregnant woman). This variation can occur within the coding region of a gene, in a non-coding region (e.g., the promoter region or its vicinity, or an intron), or in an intergene region. By detecting one or more SNPs, it is possible to distinguish between different alleles of a single gene.

[0072] A "simple tandem repeat polymorphism" refers to a polynucleotide sequence variation that occurs in the number of tandem repeats (such as tandem repeats of one or more nucleotides) of a nucleotide sequence between different alleles of the same gene. This variation may occur in the same gene on two copies of the same chromosome in the same individual (e.g., a fetus), or in the same gene in two different individuals (e.g., a fetus and a pregnant woman). This variation often occurs within the non-coding region of the gene (promoter region or its vicinity, or introns, etc.) or within the inter-gene region. By detecting differences in the number of tandem repeats, it is possible to distinguish between different alleles of a single gene.

[0073] "Insertion / deletion polymorphisms" refer to polynucleotide sequence variations characterized by the presence or absence of short nucleotide sequences (1-3 nucleotides, etc.) between different alleles of the same gene. These mutations may occur in the same gene on two copies of the same chromosome in the same individual (e.g., a fetus), or in the same gene in two different individuals (e.g., a fetus and a pregnant woman). These mutations can occur both within the coding region and non-coding region of a gene (promoter region or its vicinity, or introns, etc.), or within intergenetic regions. By detecting the presence or absence of short nucleotide sequences, it is possible to distinguish between different alleles of a single gene.

[0074] The term "blood" refers to a blood sample. This term includes all parts of blood, such as whole blood, serum, free DNA in plasma, and conventionally defined plasma. Examples of blood samples include, but are not limited to, samples from pregnant women or women being tested for potential pregnancy, and samples from individuals with a disease or infection who are being monitored for potential disease or infection.

[0075] The term "bisulfite" refers to any type of bisulfite, such as sodium bisulfite. These can be used to selectively modify DNA sequences based on the methylation status of the DNA because they can chemically convert cytosine (C) to uracil (U) without chemically modifying methylated cytosine.

[0076] A "locus" refers to a DNA segment (genomic location or chromosomal location, etc.) defined by the start and end nucleotide positions on a chromosome in a reference genome assembly (e.g., the Human Genome March 2006 Assembly (hg18) in the UCSC Genome Browser). A locus may or may not overlap with the genomic location of a gene, CpG island, or transcription / translation product. For example, a locus typically includes, but is not limited to, a contiguous DNA segment identified by experimental data (e.g., MeDIP chip datasets) and subsequent data analysis (e.g., MAT, TAS). A locus may contain one or more CpG sites. A locus can be subdivided into shorter segments (genomic sequences, fragments, or regions containing CpGs) suitable for analysis (e.g., epityper assays, bisulfite sequencing, polynucleotide amplification and determination). A locus can be divided into one or more fetal epigenetic markers. Alternatively, a locus may refer to a contiguous DNA segment identified by specific bioinformatics criteria.

[0077] The term "molecular counting" refers to any method that can quantitatively measure the number of molecules or molecular complexes, often measured relative to other coexisting molecules or complexes with different properties. Various methods of molecular counting are described, for example, in Leaner et al., Analytical Chemistry 69:2115-2121, 1997; Hirano and Fukami, Nucleic Acids Symposium Series No. 44:157-158, 2000; Chiu et al., Trends in Genetics 25:324-331, 2009; and U.S. Patent No. 7,537,897.

[0078] A reaction refers to the interaction between at least two reagents. The reagents for the reaction may be present in the sample or added to the sample. The interaction can have chemical or physical properties. Polymerase catalytic elongation, antibody-antigen binding, and contact between beads and washing solution are all considered reactions.

[0079] An integration process refers to a sequence of steps in which the product obtained in one step becomes the starting material for the next step. Each step in such a process is called a bioprocess step.

[0080] An aqueous reaction mixture contains water and at least one dissolved or suspended reagent for carrying out a reaction such as a bioprocess step, or a mixture of a complete set of sample and reaction reagent. While aqueous reaction mixtures are usually liquids, they may be gel-like at ambient temperature and become liquid above ambient temperature (e.g., reaction mixtures dissolved in agarose gel). An aqueous reaction mixture may contain a complete reaction mixture containing all the reagents necessary for sample analysis (not necessarily the sample itself), or it may contain a partial reaction mixture containing at least one reaction component (i.e., reagent) necessary for carrying out the reaction. Two or more partial reaction mixtures may be combined to form a complete reaction mixture.

[0081] Ambient temperature refers to room temperature, for example, 18°C ​​to 35°C, more preferably 20°C to 25°C. This is the temperature range that many people prefer indoors and is common in laboratory environments. It is the temperature at which one feels comfortable when wearing typical indoor clothing. Low temperature refers specifically to temperatures lower than room temperature in the atmosphere, for example, above 0°C and below 8°C (i.e., the temperatures common in refrigerators and cold storage rooms). The solidification temperature is the temperature at which a phase transition occurs, where a liquid changes into a solid, such as when the temperature of an aqueous solution falls below 0°C. Reaction reagents may be stored at 0°C to -20°C or 0°C to -50°C. According to internationally established definitions, solidification usually refers to the phase change in which a liquid or the liquid component of a substance solidifies due to cooling. For many substances, the melting point and the solidification point are the same temperature, but certain substances have different solid-liquid transition temperatures. For example, agar exhibits hysteresis in its melting and freezing points, melting at 85°C (185°F) and solidifying between 32°C and 40°C (89.6°F to 104°F).

[0082] When a permeable gel is described as permeable by a micropipette, it means that a micropipette equipped with a tip for delivering volumes in the range of 1 pL to 5000 μL, when applied manually or by an automated liquid processing device, can penetrate the gel at ambient temperature and deliver a desired volume of liquid contents beneath the gel layer stored in the reaction vessel. A permeable gel can be blocked by a pipette tip or inert rod before the micropipette tip delivers the liquid through the gel layer. The tip blocking the gel layer may or may not be stoppered. The stopper may be wax or gel. Stoppered tips may also be used to store reagents for any reaction inside the tip. Some gels may be permeable by a tip for delivering the lower limit of the above range but not by a tip for delivering the upper limit, and vice versa. On the other hand, other gels may be permeable by any tip within the above range. For example, some gels are permeable by 1 μL to 1 mL, 1 μL to 500 μL, or 1 μL to 100 μL tips.

[0083] A micropipette refers to a liquid handler or other device capable of transferring or delivering a liquid in a predetermined volume to a given reaction vessel. A micropipette may be fitted with one or more micropipette tips (e.g., 1 to 10,000). A tip is any device connected to a micropipette that delivers or transfers a predetermined volume of liquid to a given reaction vessel. The volume ranges from 1 pL to 5000 μL. The tip may be a standard industrial micropipette tip or one or more capillaries (such as a bundle of capillaries). A capillary is a hollow tube commonly used to deliver volumes of less than 1 μL. A micropipette tip can deliver liquid above, below, or to the center of a gel layer in a reaction vessel. In the case of an oil-containing reaction vessel, a micropipette tip can deliver liquid above, below, or to the center of an oil layer. Liquid delivered to the center of an oil or gel layer may form emulsion droplets or reaction compartments.

[0084] The present invention therefore further provides a micropipette method for delivering a reaction solution through a gel layer to an oil layer beneath the gel layer, thereby forming an emulsion droplet of oil and water. The number of emulsion droplets ranges from 1 to 1 million. The micropipette may be a multi-channel pipette. The tip for delivering the reaction solution may be a standard industrial micropipette tip or a capillary bundle. The reaction volume in the emulsion droplets may range from 0.0001 μL to 5000 μL.

[0085] A module is a device that performs at least one function. Multiple modules can be linked together in an integrated device to perform multiple interrelated functions, such as a magnetic module, a heating module, a reading module, and a liquid processing module. Heating and cooling may be performed in the same module or in different modules. A module, or an integrated device containing multiple modules, may also include connectors (e.g., for sliding along the surface of a vessel) for connecting the module or device to a reaction vessel. A module or a device containing modules may include a computer or its components, and may be adapted to receive signals from a connected or remote computer that controls the module or device.

[0086] A rod refers to a rod, stick, or elongated instrument made of glass, optical fiber, metal, plastic, or silicon. The surface of the rod may be chemically modified, unmodified, or partially modified. The rod may be conductive or nonconductive. The rod may be used for mixing solutions, stirring gel layers, or as an electrode. The rod may also be used to bind targets in a sample or to deliver reagents or targets into a reaction solution. The rod may be used as a sensor to detect chemical or physical changes in a reaction vessel. The rod may be reactive or nonreactive in aqueous solution.

[0087] Where this disclosure indicates a range of values, it should be understood that, unless otherwise required by context, it provides additional disclosure of the range of numbers within the range and the range of the portion formed by those numbers within the range.

[0088] Where this disclosure describes a composition, product, or method comprising (or including) a particular component or step, it should be understood that, unless otherwise required by context, this disclosure provides additional disclosure of a composition, product, or method consisting of, or essentially consisting of, that particular component or step. Detailed Description of the Invention

[0089] I. Overview The present invention utilizes a gel layer to coat and / or separate one or more aqueous reaction mixtures within a reaction vessel. The gel layer prevents the reagents or reaction products from coming into contact with the atmosphere, thereby serving as an alternative to freeze-drying during storage or transport. The gel layer can also separate multiple aqueous reaction mixtures, preventing them from coming into contact with each other until the gel layer is broken and the reaction mixtures come into contact with each other. The gel layer is permeable by inserting a micropipette tip, thereby allowing a solution (such as a sample) to be delivered to the reaction mixture beneath the gel layer. The gel layer can also be broken using a micropipette tip or an inert rod, resulting in the mixing of the aqueous reaction mixtures above and below the layer. The gel layer can also be broken by selectively applying heat to a particular layer to melt the gel, resulting in the mixing of the aqueous reaction mixtures above and below the layer. The reaction vessel may have multiple gel layers to separate multiple aqueous reagent mixtures, in which case the gel layers are broken in a predetermined order, such as from top to bottom of the reaction vessel, allowing a series of bioprocess steps to be executed in a sequential manner. Optionally, an additional oil layer may be inserted directly beneath any or all of the gel layers.

[0090] II. Gel Composition and Properties A gel is a semi-solid in which a liquid phase is confined within a three-dimensional matrix of solid phases. The matrix can be formed by cross-linking polymerization and / or self-assembly (e.g., hydrogen bonding between particles forming the matrix). For example, a gel can be formed by simultaneously heating wax and oil, confining the liquid oil phase between wax particles. Gels can be characterized by their viscosity, which is usually measured in centipoise units. Viscosity is a measure of the gel's resistance to flow. The viscosity of gels is intermediate between that of oil and wax. Unlike liquids, gels typically maintain their shape or remain in place unless acted upon by forces other than gravity. In other words, while the liquid phase may diffuse within a gel, the gel does not exhibit flow in a steady state. Gels can conform to shape and flow under pressure. The shape, hardness, and tackiness of a gel are determined by the matrix within the gel. Viscosity can be measured as the gel's resistance to objects passing through it. The viscosity of a gel can be controlled by the ratio of wax to oil. The ratio of wax to oil can be adjusted by weight or volume from 99:1 to 25:75, and can be adjusted to 25:75 to 75:25, or 40:60 to 60:40, 45:55 to 55:45, or approximately 50:50. In a reaction vessel containing multiple gel layers, each gel layer may have the same or different composition. By using gels with different compositions and melting points, the layers can be selectively melted by temperature control.

[0091] The gel may contain other chemicals or additives. For example, these chemicals or additives may include surfactants, which may have a hydrophilic "head" and a hydrophobic "tail." The "head" of a surfactant is polar and may or may not be electrically charged. The "tails" of many surfactants have very similar structures, containing hydrocarbon chains such as branched, straight, or aromatic chains. Fluorine-based surfactants have fluorocarbon chains, and siloxane-based surfactants have siloxane chains.

[0092] Many important surfactants contain polyether chains with highly polar anionic groups at their ends. These polyether groups often have ethoxylated (similar to polyethylene oxide) sequences inserted to enhance the surfactant's hydrophilicity. Conversely, polypropylene oxide can be inserted to increase the surfactant's lipophilicity. Surfactant molecules have one or two tails; those with two tails are referred to as double-chain.

[0093] Surfactants are typically organic compounds that are close to amphiphilic. That is, the molecule has a dual action, containing a hydrophilic "water-seeking" group (head) and a hydrophobic "water-avoiding" group (tail). As a result, surfactants contain both water-soluble and water-insoluble components. Surfactants diffuse into water and are adsorbed at the air-water interface, or, in the case of a mixture of water and oil, at the oil-water interface. The water-insoluble hydrophobic group can spread from the bulk aqueous phase to non-aqueous phases such as air and oil, while the water-soluble head remains bound to the aqueous phase.

[0094] Hydrophobic tails can be either lipophilic ("seeking oil") or oleophobic ("avoiding oil") depending on their chemical properties. Hydrocarbon groups are usually lipophilic and are used in soaps and detergents. On the other hand, fluorine groups are oleophobic and are used to repel dirt and reduce surface tension.

[0095] Waxes are diverse compositions with similar composition and physical properties to beeswax. Waxes can be produced from petroleum, oil shale, animals, and plants, with or without modification. Paraffin waxes contain hydrocarbons and mixtures of alkanes from the same group, typically consisting of chains of about 12 to 40 carbon atoms. Paraffin waxes may also contain mixtures of saturated n- and iso-alkanes, naphthenes, alkyl and naphthene-substituted aromatic compounds. The chemical composition of a typical alkane paraffin wax is hentriacincontane (C 31 H 64 ) General formula C n H 2n+2It contains hydrocarbons. The chain may or may not be branched. Another type of wax is a simple lipid that is a long-chain (12-32 carbon atoms) ester. Wax is solid at ambient temperature and melts above approximately 37°C. Wax cannot be pierced by a micropipette at ambient temperature without deforming or clogging the tip.

[0096] A liquid is a fluid that has a constant volume but not a constant shape, is nearly incompressible, conforms to the shape of the container, but maintains a (nearly) constant volume regardless of pressure. Liquids are fluid and can take the shape of a container. Water and oil are the most common liquids on Earth. Mineral oil is sometimes simply called oil. Mineral oil or oil as referred to in this invention is interchangeable. Oils in this invention include mineral oils obtained from petroleum distillation, synthetic oils, silicone oils, algal oils, and vegetable oils such as olive oil, palm oil, soybean oil, canola oil, corn oil, and peanut oil. Mineral oil, also called white oil, paraffin oil, liquid paraffin, liquefied paraffin (Latin), or liquid petroleum, is a liquid byproduct of crude oil refining, a colorless and transparent oil, mainly composed of alkanes and cycloalkanes, and is similar to petrolatum. Oil may also be a mixture of different types of oils and may or may not contain surfactants. Adding a surfactant to an oil mixture promotes emulsion droplet formation in digital PCR amplification or inhibits emulsion droplet formation in qPCR detection. Different types of oils and mixtures thereof can have different densities, such as being lower than, the same as, or higher than water.

[0097] The gel used in this invention has sufficient viscosity at ambient temperature to form a layer that covers the aqueous mixture in the reaction vessel, preventing or at least inhibiting the diffusion of the aqueous mixture throughout the tube, maintaining the gel itself and the aqueous reaction mixture beneath it in place during storage and transport of the tube before use, and preventing the aqueous mixture from coming into contact with the atmosphere. However, the gel described in this invention is not so viscous as to prevent a micropipette tip from penetrating the gel at ambient temperature to deliver a solution such as a sample and / or reagent(s) to the reaction mixture beneath the gel in the reaction vessel. The gel described in this invention preferably withstands shrinkage below ambient temperature (such as cold or solidification temperature) and prevents multiple aqueous reaction mixtures separated by the gel layer from mixing during storage or transport. The gel can melt at temperatures above ambient temperature and reform as a gel when returned to ambient temperature.

[0098] In contrast to the gel used in this invention, pure paraffin wax has the following drawbacks: With conventional pipettes, the wax stopper can get stuck in the tip hole, making it difficult to pass the sample through. Also, because the volume expansion rate when changing from solid to liquid is typically 15%, it can cause undesirable expansion / contraction effects of the barrier, or cross-leakage or mixing of separated component reagents in the reagent container, and may even form a perforated defective layer barrier (U.S. Patent No. 5,411,876). Furthermore, if the wax barrier is melted by heating to mix the separated component reagents in the container, the reaction may be delayed due to the slow melting of the wax barrier.

[0099] Semi-solid gels possess physical properties that overcome the drawbacks seen when using pure paraffin wax as a layering barrier. The gels can have customized hardness and can be easily penetrated and separated by pipette tips, needle-shaped plastic picks, or rods. Their volume expansion rate is 3-5% or less, reducing the risk of uncontrolled mixing and fusion of separated reagents within the reagent container, and the risk of perforated, defective wax barriers forming during reagent container manufacturing.

[0100] Similar to pure paraffin wax, which is a temperature-dependent phase-change material (PCM), the gel used in this invention changes from a semi-solid that does not flow due to gravity at ambient temperature (e.g., 23°C) to a fluid liquid at high temperatures (e.g., 50°C). When the container returns to ambient temperature, the liquefied gel re-solidifies into a semi-solid gel, i.e., a non-fluid substance. Since the molten semi-solid gel has a lower density than water or mineral oil, which is lighter than the aqueous phase, the semi-solid gel mixture floats to the top of the liquid phase during the heating reaction and forms a non-fluid layer on top of the liquid contents in the container after the reaction.

[0101] III. Layered reaction mixtures A gel layer can serve multiple roles in a reaction vessel. A single gel layer covering an aqueous reaction mixture can retain the reaction mixture at the bottom of the vessel rather than diffusing it throughout, forming a barrier between the aqueous reaction mixture and the atmosphere, protecting the reaction mixture from degradation due to interaction with the atmosphere (such as oxygenation and moisture absorption), and / or reducing environmental pollution by the reaction products. A gel layer can be used to absorb contaminants from a sample during the reaction, reducing reaction inhibition by contaminants. A gel layer can be used to control the release of components within the gel layer into the reaction mixture, or to absorb components from the reaction mixture.

[0102] A single gel layer covering an aqueous solution can be prepared by adding the gel to a reaction vessel containing the aqueous solution, or by adding the aqueous solution to a vessel already containing the gel, heating the reaction vessel, melting the gel layer, and allowing it to float and cover the aqueous solution.

[0103] An oil layer placed between the reaction mixture and the gel layer can enhance protection from the atmosphere and, if present, is retained by the gel layer. The gel layer (and the oil layer, if present) can be penetrated by a micropipette tip, thereby allowing liquid to be delivered to the aqueous layer. The delivered liquid may be one or more additional reagents not yet present in the aqueous reaction mixture, or a sample to be mixed with the aqueous reaction mixture, or both. Sequential addition of reagents and / or samples can also be achieved by multiple penetrations with a micropipette tip. Typically, the aqueous reaction mixture contains reagents for processing the sample by amplification or immunoassay, etc., and the sample is delivered to the aqueous reaction mixture using a pipette tip that penetrates the gel layer.

[0104] Gel layers can also be used to separate multiple aqueous reaction mixtures, for example, when the reaction mixture contains different components that need to be combined to perform a sample assay. For example, a reaction vessel may contain two, three, four, five, six, seven, eight, nine, or ten or more layers of aqueous reaction mixture separated from each other by gel layers and optionally oil layers. Thus, for example, in an amplification assay, polymerase can be separated from primers, probes, and / or dNTPs until use to reduce artifacts in amplification. Similarly, in an immunoassay, capture reagents and detection reagents can be separated before use. In this form, a gel layer can be used to cover a first reaction mixture, separating the first reaction mixture from a second reaction mixture, and a second gel layer can be used to cover the second reaction mixture. Both reaction mixtures are thus held in place within the reaction vessel and protected from decomposition by the atmosphere. If necessary, an oil layer can be included between the gel layer and the reaction mixture beneath it, similar to the description for a single gel layer. Also, if the reaction vessel contains multiple aqueous solutions, an oil layer may be included between two gel layers. The same principle can be applied to other reaction mixtures and gel layers within the same container.

[0105] The use of multilayer reaction mixtures requires mixing the mixture and / or delivering further reagents and / or samples through the gel layers using a micropipette. The reaction mixture layers separated by the gel layers can be mixed by inserting a rigid rod or micropipette tip into the gel layers and stirring the contents of the reaction vessel. The initially separated reaction mixtures are combined into a single layer at the bottom of the reaction vessel, and the initially separated gel layers are combined into a single layer on top of the combined reaction mixture. The aqueous mixture separated by the gel layers can be thoroughly mixed by centrifugation after breaking the gel layers with a micropipette tip or inert rod. Micropipette tips can also be used for delivering reagents or samples. Such delivery can occur when the separated reaction layers and gel layers penetrate the reformed gel layer, resulting in a single reaction mixture and gel layer. Alternatively, if the micropipette tip is used to stir the gel layers, the reagents or samples can be delivered by stirring. The integration of the reaction mixture layers can also occur by heating the gel with a heater to melt it. When the density of oil is greater than that of water, the high-density oil layer in the reaction vessel facilitates the mixing of two aqueous reaction mixtures. For example, the aqueous reaction mixture at the bottom is covered by a first gel layer. Above the first gel layer is an oil mixture that is denser than water. Above the oil layer is a second aqueous reaction mixture. Above the second aqueous reaction mixture is a second gel layer. When the reaction vessel is heated, the first gel layer melts, the oil falls to the bottom of the reaction vessel, pushing up the first aqueous reaction mixture and mixing it with the second aqueous reaction mixture. Optionally, the first and second aqueous mixtures may be gel-like in the reaction vessel at ambient temperature. When the reaction vessel is heated, both gels become fluid and are mixed by the oil layer between them, which is denser than water. The layers of reaction mixtures can also be mixed by using magnets on the outside or inside of the reaction vessel to move magnetic beads present in one aqueous reaction mixture through the gel layer to the other reaction mixture.Optionally, the heater or magnet used in this manner may be connected to a reaction vessel and slide along the outer or inner surface of the reaction vessel so as to be adjacent to a gel layer melted by the heater or adjacent to a gel layer destroyed by the movement of magnetic beads induced by the magnet. Optionally, the reaction vessel may be connected to an apparatus comprising both a heater module and a magnetic module.

[0106] After the reaction occurs, the gel layer can be removed.

[0107] Alternatively, the gel layers may have different compositions and melting points, and as a result, by controlling the temperature of the entire reaction vessel (for example, by immersing it in a heating bath), the layers may be destroyed in an order defined according to their melting points.

[0108] A device including a reader may be used to assist or accelerate the processing of sample solutions, handling of reagents, and detection reactions carried out within a reaction vessel. Once all reactions are prepared, the reaction vessel is placed in a suitable reader (such as a thermal cycler for conventional or real-time PCR, or a hot block / water bath for isothermal amplification) to complete and monitor the reaction. A device including a reader module may include one or more combinations of mechanical, heating, electrical, magnetic, liquid handling, and optical modules to assist the reaction within the reaction vessel and to detect the reaction within the vessel in real time or after the reaction has completed. The reader may detect chemical or physical changes in the reaction within the reaction vessel, such as color, fluorescence, emission, chemiluminescence, electrochemistry, radiation, reflection, phase change, magnetoresistance, turbidity, mobility shift, pH, or ionic intensity.

[0109] A reaction vessel containing multilayer aqueous reagents separated by gel layers can be used to carry out an integrated bioprocess involving a series of ordered steps. By selectively disrupting the gel layers, only the aqueous reaction mixtures above and below the gel layers can be mixed at once. After mixing, reactions occur between the reagents in the mixture, and one or more products of the reaction serve as starting materials to be mixed with another aqueous reaction mixture by disrupting further gel layers. The products of this further reaction may be mixed with yet another aqueous reaction mixture by disrupting yet another gel layer. Typically, the gel layers are continuously disrupted downwards, although the top gel layer may be left intact to continuously separate the reagents and reaction products from the atmosphere. The gel layers may be disrupted using a micropipette tip (which can also be used for introducing samples and other reagents), by selectively heating the gel layers using an inert rod to melt them, by moving magnetic beads across the gel layers in a magnet-induced aqueous layer, or by any combination of these techniques.

[0110] Other reaction vessels contain aqueous reaction mixtures, gels, and, if necessary, oil layers in a random order between the vessels.

[0111] The reaction vessel may be fitted with a device equipped with a module for heating / cooling and / or magnetic field application, which is slidable along the outer surface of the vessel and can directly apply heating or cooling to a specific gel layer or apply a magnetic field to magnetic beads in a specific aqueous reaction mixture. The heater may be a thermoelectric device such as a semiconductor-based Peltier element. The magnet may be an electromagnet.

[0112] IV. Types of reaction vessels Reaction vessels are used to hold gels and contain reaction reactions or their components. Examples include microtubes of various sizes, from microliters to milliliters, commonly used in laboratories. Reaction vessels may or may not have lids such as snaps, screws, plastic wrap, or metal foil. They may or may not be suitable for use in a microcentrifuge. Reaction vessels may be disposable or reusable. Reaction vessels may be channels, trenches, or hollow tubes, or they may have planes. Reaction vessels may be individual containers or may be connected to each other as strips or matrix containers to facilitate parallel processing. Reaction vessels may be in the form of multi-well plates (96, 384, or 1536 wells, etc.), or chips or microchips. In this case, each well of the plate or chip can be considered a reaction vessel, and the wells in the chip may be connected via channels.

[0113] The reaction vessel may be made of plastic (such as polypropylene), glass, metal, or other material suitable for the desired reaction (such as a material that is impermeable to or inert to the aqueous reaction mixture, gel, and oil components inside the vessel), depending on the application. The reaction vessel may have inherent hydrophobic or hydrophilic properties depending on its material composition, and may be treated to achieve the hydrophobic / hydrophilic properties required for the desired reaction.

[0114] Suitable containers for PCR include single PCR tubes, PCR tube strips, PCR plates, and cartridges, which allow for the simultaneous reaction of one or more samples. Suitable reaction containers for ELISA assays include commercially available ELISA plates, deep-well plates, microtiter plates, and cartridges, which also allow for the simultaneous reaction of one or more samples.

[0115] The reaction vessels are sealed during storage or transport. They may be sealed with a film such as aluminum foil and stored in a heat-sealable metal storage bag. They may be transported at ambient temperature or in a polystyrene box filled with blue ice or dry ice. They may be stored at a desired temperature.

[0116] V. Reactions suitable for reaction vessels Gel layers can be used to cover solutions before, during, or after a reaction. Gel layers can be used to separate reaction mixtures in a wide variety of reactions, including various chemical reactions, immunoassays, nucleic acid detection assays, sequencing, and / or purification protocols. Any assay method involving multiple aqueous reaction mixtures held individually until the assay is performed is adaptable to the strategies of this disclosure, including using more or fewer reaction layers than those shown in the representative examples and figures.

[0117] Gel layers can be used to inhibit interactions between reagents before the desired reaction occurs, such as nonspecific amplification or primer dimer interactions in nucleic acid amplification. Gel layers can be used in sequential reactions, such as performing a reverse transcription (RT) reaction on the gel layer, followed by mixing the PCR reagent with the RT product for amplification and detection. Another example is performing Nicking amplification around the gel layer, and then dissolving the gel layer to perform the PCR reaction. Another application is nested PCR. The first stage of amplification can be performed on the gel layer. The second stage of amplification can be performed after the gel has dissolved. In other words, multi-stage amplifications such as NASBA, T7, CRISPR, RPA, and EXPAR can be processed sequentially on different gel layers. Another example is a reaction vessel with a solution beneath the gel layer. This solution contains proteinase K. The sample is added to the reaction vessel, and the vessel is heated at 95°C for 10 minutes. The processed sample may be used in other reactions, or PCR reagent may be added to the reaction vessel for amplification and detection.

[0118] Another example is immunoPCR or immunoisothermal amplification. An immune reaction occurs on a gel layer through proximity interactions, and the analyte is then mixed with other reagents for PCR or isothermal amplification to detect the analyte.

[0119] 1. Amplification reaction: Amplification reactions are used to detect target nucleic acids, gene markers, single nucleotide polymorphisms, insertions, deletions, insertions, inversions, translocations, translocation versions, deletions, indels, microsatellite repeats, minisatellite repeats, short tandem repeats, transposable elements, large structural chromosomal mutations, methylation, and combinations thereof. Amplification is also used in applications such as nucleic acid cloning protocols and sequencing.

[0120] Many known methods of nucleic acid target amplification require thermal cycling to alternate between denaturation of double-stranded nucleic acids and hybridization of primers, while other known nucleic acid amplification methods are isothermal. Polymerase chain reaction (PCR), commonly known as PCR (Mullis, 1987, U.S. Patent No. 4,683,202; Saiki et al., 1985, Science (New York, NY), 230(4732), 1350-1354), exponentially increases the copy number of the target sequence by repeating denaturation, annealing of the primer pair to the opposite strand, and primer extension multiple times. In a variant called RT-PCR, reverse transcriptase (RT) is used to create complementary DNA (cDNA) from mRNA, and then the cDNA is amplified by PCR to generate multiple copies of the DNA (Gelfand et al., "Reverse Transcription with Thermostable DNA Polymerases - High Temperature Reverse Transcription," (Gelfand, 1994, U.S. Patent No. 5,322,770; Gelfand & Myers, 1994, U.S. Patent No. 5,310,652). Another method for amplifying nucleic acids is called LCR (ligase chain reaction, Laffler, Carrino, & Marshall, 1993, Annales De Biologie Clinique, 51(9), 821-826). Methods 821-826) are based on a reaction in which two adjacent probes hybridize to a target sequence and are linked together by a ligase. If the target nucleotide sequence is not present, the two probes will not be linked, so the presence of the linked product serves as an indicator of the target nucleotide sequence. In the LCR method, temperature control is required to separate the complementary strand from the template.Another method is the strand substitution amplification method (George T. Walker, Little, & Nadeau, 1993, U.S. Patent No. 5,270,184; George T. Walker, 1995, U.S. Patent No. 5,455,166; GT Walker et al., 1992, Nucleic Acids Research, 20(7), 1691-1696, 1992, Proceedings of the National Academy of Sciences of the United States of America, 89(1), 392-396). This method, commonly referred to as SDA, utilizes the following cycle: First, a pair of primer sequences is annealed to the opposite strand of the target sequence, and the primers are extended in the presence of dNTPs to produce a hemiphosphorothioate-treated double-stranded primer extension product. Next, the hemi-modified restriction endonuclease recognition site is nicked with an endonuclease, and the polymerase extends a primer from the 3' end of the nick, replacing the existing chain and synthesizing the chain for the next cycle of primer annealing, nicking, and chain replacement. This ultimately achieves exponential amplification of the product. Thermophilic SDA (tSDA) is essentially the same method, but uses a thermophilic endonuclease and polymerase at high temperatures (Fraiser, Spargo, Van, Walker, & Wright, 2002, European Patent No. 0684315).Other amplification methods include nucleic acid sequence-based amplification (Compton, 1991, Nature, 350(6313), 91-92, Malek, Davey, Henderson, & Sooknanan, 1992), commonly known as NASBA; methods that amplify the probe molecule itself using RNA replicases (Lizardi, Guerra, Lomeli, Tussie-Luna, & Russell Kramer, 1988, Nature Biotechnology, 6(10), 1197-1202), commonly known as Qβ replicases; transcription-based amplification (Kwoh et al., 1989, Proceedings of the National Academy of Sciences of the United States of America, 86(4), 1173-1177); and self-persistent sequence replication (3SR) (Guatelli et al., 1990, Proceedings of the National Academy of Sciences of the United States of America). Examples include 87(5), 1874-1878; Landgren (1993) Trends in Genetics 9, 199-202; Lee, H. et al., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES (1997)); and transcription-mediated amplification (Kwoh et al., 1989, Proceedings of the National Academy of Sciences of the United States of America, 86(4), 1173-1177; Kacian & Fultz, 1995, US Pat. No. 5,480,784; Kacian & Fultz, 1996, US Pat. No. 5,399,491) (commonly referred to as TMA).For further discussion of known amplification methods, see Persing, David H., 1993, "In Vitro Nucleic Acid Amplification Techniques" in Diagnostic Medical Microbiology: Principles and Applications (edited by Persing et al.), pp. 51-87 (American Society for Microbiology, Washington, D.C.).Other exemplary amplification methods suitable for use in accordance with the present invention include rolling circle amplification (RCA) (Fire & Xu, 1995, Proceedings of the National Academy of Sciences, 92(10), 4641-4645; Lizardi, 1998, U.S. Patent No. 5,854,033), nucleic acid amplification with nicking agents (Van Ness, Galas, & Van Ness, 2006, U.S. Patent No. 7,112,423), nicking and extension amplification reaction (NEAR) (Maples et al., 2009, U.S. 2009-0017453 A1), helicase amplification (Hyper-Speedings and Extension Amplification Reaction), and dependent amplification (HDA) (Kong, Vincent, & Xu, 2004, U.S. 2004-0058378 A1; Kong, Vincent, & Xu, 2007 US pat. US2007 / 0254304 A1); Loop-mediated isothermal amplification (LAMP) (Notomi & Hase, 2002, US Patent No. 6,410,278); Quadruple-chain priming amplification (Analyst, 2014, 139, 1644-1652), Expar amplification (PNAS April 15, 2003, 100, 4504-4509), Cross-priming amplification (Sci Rep. 2012; 2: 246), SMAP amplification (Nature Methods 04 / 2007; 4(3):257-62), Multiple substitution amplification (MDA, Proceedings of the National Academy of Sciences, 2005, 102(48): 17332-6), Recombinase polymerase amplification (RPA) (Journal of Clinical Virology 54(4): Examples include the single-primer isothermal amplification method (SPIA) (clinical chemistry, 2005 vol. 51 no. 10, 1973-1981), and the hybridization chain reaction (HCR) (Chem Soc Rev. 2017 Jul 17; 46(14):4281-4298).

[0121] The primers and probes used in the above method may be primers of a limited composition in which at least one of the four standard nucleotide types is located at two or fewer internal positions and / or at the 5' end, as described in WO2019 / 033065 and WO2016172632.

[0122] Another aspect of amplification is signal amplification. When a sufficient amount of the nucleic acid to be detected is present, there is an advantage to directly detecting the target sequence rather than increasing the number of copies of the target sequence (such as in PCR or LCR). Conventional direct detection methods such as Northern blotting, Southern blotting, and RNase protection assays typically require the use of radioactivity and are not suitable for automation. Other techniques have attempted to eliminate the use of radioactivity and / or improve sensitivity in an automated form. Cycling probe reactions (CPR) (Duck, Alvarado-Urbina, Burdick, & Collier, 1990b, BioTechniques, 9(2), 142-148) use long chimeric oligonucleotides composed of RNA in the middle and DNA at both ends. The probe is hybridized to the target DNA and exposed to heat-stable RNase H, which degrades the RNA portion. This destabilizes the remaining DNA portion of the double helix, releasing the rest of the probe from the target DNA, allowing another probe molecule to repeat the process. Branched DNA (bDNA), reported in Urdea et al., 1987, Gene, 61(3), 253-264, contains branched oligonucleotides, each capable of carrying 35-40 labels (such as alkaline phosphatase enzymes). This enhances signals from hybridization events, while also enhancing signals from nonspecific binding.Other signal amplification methods include invasive nucleic acid cleavage (Prudent, Hall, Lyamichev, Brow, & Dahlberg, 2006, U.S. Patent No. 7,011,944), hybridization chain reaction (HCR) (RM Dirks & Pierce, 2004, Proceedings of the National Academy of Sciences of the United States of America, 101(43), 15275-15278, R. Dirks & Pierce, 2012, U.S. Patent No. 8,105,778), guanine quadruplex deoxyribozyme-based colorimetric detection, CHA amplification (J. Am. Chem. Soc., 2013, 135 (20), pp 7430-7433), and SMART signal amplification (Biotechniques 2002 Mar; 32(3):604-6). Examples include 608-11.)

[0123] The amplification product can be detected qualitatively (e.g., a positive signal relative to a control) or quantitatively (e.g., signal intensity related to the absolute or relative amount of the analyte producing the amplification product). Detection may include, but is not required, further analysis such as sequencing of the amplification product. The methods provided by the present invention may also include the direct detection of specific nucleic acids in the capture reaction product or amplification reaction product, such as a specific target amplicon or amplicon set. Accordingly, the mixture of the present invention may include special probe sets such as TAQMAN® (Livak, Flood, & Marmaro, 1996, U.S. Patent No. 5,538,848), which uses a hydrolysis probe containing a detectable reporter and quencher moiety that can be released by a DNA polymerase having 5'→3' exonuclease activity; Molecular Beacon (Tyagi, Kramer, Lizardi, 1999, U.S. Patent No. 5,925,517), which uses a hairpin probe with a reporter moiety and a quenching moiety at opposite ends; Fluorescence Resonance Energy Transfer (FRET) primers (Wittwer, Ririe, Rasmussen, 2001, U.S. Patent No. 6,174,670), which uses a pair of adjacent primers having a fluorescent donor moiety and an acceptor moiety, respectively; and LIGHTUP® (Kubista & Svanvik, 2001, U.S. Patent No. 6,329,144), which is a single short probe that fluoresces only when bound to a target. Similarly, SCORPION® (Whitcombe, Theaker, Gibson, & Little, 2001, U.S. Patent No. 6,326,145) and SIMPLEPROBES® (Wittwer et al., 2003, U.S. Patent No. 6,635,427) use a single reporter / dye probe. The amplification product detection probe may be designed according to the specific detection mode used and as described in the above patents.Other detection methods include gel electrophoresis, mass spectrometry, capillary electrophoresis, melting curves, detection of amplification products using nucleic acid-based fluorescent chelating dyes such as SYBR® Green, or fluorescent labeling and soluble quenchers (Will, Gupta, & Geyer, 2014, U.S. Patent No. 8,658,366).

[0124] The term "multiplex amplification" refers to the amplification of multiple nucleic acids. For example, it can refer to the amplification of multiple sequences from the same sample, or the amplification of one sequence from among multiple sequences in a sample. This is explained, for example, in George T. Walker, Nadeau, & Little, 1995, U.S. Patent No. 5,422,252, and George T. Walker, Nadeau, Spears, et al., 1995, U.S. Patent No. 5,470,723, which provide examples of multiplex strand substitution amplification. The term also refers to the simultaneous or stepwise amplification of one or more sequences present in multiple samples.

[0125] The term "digital polymerase chain reaction" or "dPCR" refers to an improved version of the conventional polymerase chain reaction (PCR) method that directly quantifies and clonally amplifies nucleic acids such as DNA, cDNA, and RNA, allowing for the direct quantitative measurement of the amount of target nucleic acids. Digital PCR achieves direct quantitative measurement by dividing the sample into multiple separatory liquids within a number of independent reaction chambers, where the individual target nucleic acid molecules present in the sample can be localized and concentrated to detectable levels of amplification products. Preferably, the sample is divided so that most of the separatory liquids (e.g., at least 50%, 75%, 90%, 95%, or 99%) contain either 0 or 1 molecule of each target nucleic acid to be detected. If the reaction vessel contains oil beneath the gel layer, a micropipette can be used to inject the required amount of reaction mixture into the oil layer, forming the required number of oil-water emulsion droplets. The reaction volume can range from picoliters to microliters. The number of emulsion droplets can range from 1 to 1 million. The micropipette is a multi-channel pipette. The tip delivering the reaction mixture may be a bundle of capillaries. Optionally, the reaction mixture may be delivered directly to a gel layer to form multiple reaction compartments. The gel is heated to a desired temperature to initiate the reaction in each compartment. After PCR amplification, the presence of a signal in any droplet or compartment indicates the presence of the target nucleic acid, and the number of droplets or compartments containing the final PCR product is a direct measurement of the absolute amount of the target nucleic acid. Capture or isolation of individual nucleic acid molecules is generally performed by dilution and may be done on a capillary, microemulsion, small chamber array, or nucleic acid binding surface. Basic techniques for digital PCR are described, for example, in Sykes et al., Biotechniques 13 (3): 444-449, 1992, and Vogelstein and Kinzler, PNAS 1999; 96:9236-41. Other amplification forms described herein, such as transcription-mediated amplification, can also be performed digitally. The digital PCR method described in the present invention can also be applied to immunoassays using nucleic acid tags (such as digital immunoassays described in U.S. Patent No. 9,896,717) and other assays for the absolute quantification of one or more targets in a sample to be analyzed.

[0126] The term "real-time amplification" refers to an amplification reaction in which the amount of reaction products (such as amplicons) is monitored as the reaction progresses. The form of real-time amplification differs mainly depending on the detection mechanism used to monitor the reaction products. Detection methods are outlined in Mackay, Arden, & Nitsche, 2002, Nucleic Acids Research, 30(6), 1292-1305, which is incorporated as a reference in this text.

[0127] The term "detection label" refers to any atom or molecule that can be attached to a nucleic acid or protein and can be used to provide or assist in providing a detectable (preferably quantifiable) signal. Labels may provide a detectable signal by fluorescence, radioactivity, colorimetric analysis, gravimetric analysis, magnetism, enzymatic activity, etc. Detection labels can be incorporated in a variety of ways: (1) Primers include labels attached to, for example, a base, ribose, phosphate, or a similar structure of a nucleic acid analog. (2) A nucleotide triphosphate is modified with a label at either a base or ribose (or a similar structure of a nucleic acid analog). The label-modified nucleotide is then incorporated into a newly synthesized chain by an elongation enzyme such as polymerase. (3) Modified nucleotides containing functional groups that can be used (after the enzymatic reaction) to attach a detectable label are used. (4) Similarly, modified primers containing functional groups that can be used to attach a detectable label are used. (5) Labeled probes that are directly labeled and hybridize to a portion of the amplicon may be used. (6) Labels that can be incorporated into amplification products, (7) Labels that can react with by-products of amplification reactions.

[0128] The techniques, apparatus, and methods described herein offer advantages over the prior art in providing reaction vessels. For example, conventionally, PCR strips or plates, or immunoassay ELISA plates, are sold as empty wells. The present invention provides a reaction vessel having a pre-filled gel layer through which a micropipette can penetrate, optionally containing oil beneath the gel layer. The pre-filled gel reaction vessel allows for the partial or complete pre-filling of the reaction mixture within the vessel, extending the shelf life of the reagents and preventing carryover contamination into the laboratory environment. Thus, the reaction vessel is configured to contain a pre-filled reaction mixture, separated by the gel layer within the vessel. After the addition of the sample, the reaction can be initiated beneath the gel layer to prevent the reaction mixture from coming into contact with the atmosphere. A heat-induced reaction generates a temperature higher than the ambient temperature, followed by the gel changing from a semi-solid to a liquid lighter than water, with the liquefied gel moving to the top of the liquid phase of the reaction mixture. Once the reaction is complete, the reaction vessel is cooled to ambient temperature, and the gel solidifies, forming a non-flowable barrier over the aqueous reactants. This prevents the amplification product from leaking and contaminating the laboratory environment, even if the container is not properly sealed after the reaction or is handled improperly.

[0129] Referring to Figure 1 as one embodiment, a reaction tube without reagents is configured to contain a pre-filled gel layer. For example, after adding 20 μL of reaction master mix and, for example, 5 μL of sample to the reaction tube, during the first thermal cycle of the PCR reaction, for example, 25 μL of the reaction mixture passes through the gel layer and settles to the bottom of the tube. This is due to the density difference between the molten gel layer and the aqueous phase, and the surface tension of the gel layer surface. In this embodiment, a gel layer with a low melting point, an aqueous phase containing salt, and a large volume ratio of gel layer to aqueous phase are preferred. This is to promote the settling of the added aqueous layer to the bottom of the tube during the first thermal cycle of the PCR reaction, or as early as possible in the isothermal reaction. The molten gel layer cools and solidifies, forming a solid barrier over the liquid contents in the tube after the reaction.

[0130] When using only the gel layer, the equal volume of the aqueous layer may not sink to the bottom of the tube during the PCR reaction, or may not even sink below the molten gel layer. To ensure that the aqueous layer sinks below the molten gel layer during the PCR reaction, the volume ratio of the gel layer to the aqueous layer should be 2:1 or greater, for example, 40 μL:20 μL. However, a larger volume ratio means that the reaction solution added before the PCR reaction moves further away from the bottom of the tube, increasing the heat transfer time between the heat source and the reaction solution, ultimately delaying the amplification reaction. Furthermore, an increase in the amount of gel in the tube takes longer to melt, delaying the reaction. Therefore, gel reaction tubes pre-filled with pure wax may experience a decrease in reaction kinetics and a delay in Ct. Figure 2 shows an improved example configured to add a certain amount of mineral oil below the gel layer. To use this two-layer reaction tube, first break the gel layer using a micropipette tip or a needle-shaped plastic pick, and then add the constituent reagents (e.g., 20 μL) and / or sample (5 μL) through the opening of the broken gel layer. To mimic the heat transfer conditions of conventional liquid PCR, where the constituent reagents and sample are at the bottom of the reaction tube before the first thermal cycle of the amplification cycle, the tube containing mineral oil and the tube containing the sample are centrifuged in a microcentrifuge to facilitate the settling of the aqueous phase to the bottom of the tube before the reaction. Similar to the embodiment shown in Figure 1, in the two-layer reaction tube, a non-flowing gel covering is formed on the top of the tube after the reaction.

[0131] The embodiments shown in Figures 1 and 2 provide a reaction tube that can universally accommodate any reagents for contamination-free PCR, isothermal amplification, or any biochemical reaction. However, for users who desire reduced human error, rapid acquisition of assay results, and improved laboratory work efficiency, a reaction tube that requires less reagent preparation and pipetting, or one that is "sample-only," is desirable. The embodiment shown in Figure 3 is configured to contain a complete set of reagents or a subset of complementary reagents within the reaction vessel, with the subset of complementary reagents separated into an isolated compartment using mineral oil and a gel layer. It is undesirable to pre-mix a subset of PCR reagents or isothermal amplification reagents at ambient temperature before thermal cycling or steady-state heating. This is because primer depletion can delay the reaction, leading to the formation of nonspecific primer dimers and other byproducts, resulting in delayed target product yield and reduced performance. In this multilayer configuration, one subset of PCR reagent or isothermal reagent (e.g., 10 μL volume) is packed into the bottom of, for example, a 0.1 mL tube, which may be either a primer mix or an enzyme mix, followed by a fixed amount of mineral oil, a fixed amount of gel layer mixture, another fixed amount of mineral oil (e.g., 10 μL of a complementary reagent subset), and another fixed amount of gel layer mixture, which are then packed in sequence.

[0132] To use this embodiment for PCR or isothermal reactions, the two gel layers are first broken using a pipette tip or needle-shaped plastic pick, similar to the procedure described in the embodiment shown in Figure 2, and then, for example, 5 μL of sample is added through the broken gel layers. An additional layer of mineral oil is filled between the two gel layers to completely capture the small amount of sample (e.g., 1.0 μL) into the liquid phase of the reagent tube. Finally, the tube containing the sample is capped and briefly centrifuged in a microcentrifuge. This collects all aqueous components to the bottom of the tube, allowing for the most efficient thermal reaction during the first thermal cycle of the PCR reaction or isothermal amplification. The upper gel layer prevents the liquid contents from flowing into any part of the inner wall of the reagent tube during product manufacturing, storage, transport, or analytical handling. Similar to the embodiment shown in Figure 2, in the 6-layer reaction vessel, a non-flowing gel layer is formed at the top of the reaction vessel after or during the reaction.

[0133] In the embodiment shown in Figure 4, the reaction tube is configured to contain only one subset of the constituent reagents, with a layer of mineral oil on top of it, and a layer of gel on top of that. This embodiment is advantageous over embodiments containing multiple subsets of reagents when the analytical procedure is carried out in an open area in the countryside where the reagents are exposed to temperatures exceeding the ambient temperature, such as 40°C, for an extended period, such as several hours or more. In this case, the gel layer may melt and liquefy at high temperatures. However, even if the gel layer melts, it does not adversely affect the performance of the constituent reagents pre-filled in the tube, because the gel layer is thermally stable at temperatures much lower than the temperature at which it chemically decomposes.

[0134] 2. Nucleic acid detection assay Target nucleic acids, or one or more gene loci within target nucleic acids, can be detected using various binding assays with nucleic acid primers and probes. For example, target nucleic acids can be detected by oligonucleotides immobilized on a support. Alternatively, target nucleic acids can also be detected by capture probes in solution that bind to the target nucleic acid and the immobilized probe. In either form, the bound target nucleic acid can be detected using a labeled detection probe. Target nucleic acids can also be detected by primers using target-specific extension assays or single-nucleotide extension assays. Nucleic acids can also be analyzed using various sequencing methods, such as the Sangerdideoxy synthesis method and the Maxam-Gilbert chemical decomposition method. Furthermore, newer techniques such as 454 pyrosequencing (Siqueira et al., J Oral Microbiol. 2012; :10.3402 / jom.v4i0.10743. doi:10.3402 / jom.v4i0.10743), ion torrents (see Hu et al., Human Immunology 82, 801-811 (2021)), and Illumina bridge amplification sequencing (see, for example, Slatko et al., Curr. Protoc. Mol. Biol. 122(1), e59 (2018)) are also examples of sequencing methods. Nucleic acids can also be analyzed using similar techniques such as bisulfite sequencing, epigenetic analysis, and molecular counting.

[0135] 3. Immunoassay Immunoassays are bioanalytical methods that quantify analytes by utilizing the reaction between an analyte and an antibody (see Darwish, Int J Biomed Sci. 2006 Sep; 2(3): 217-235). These reagents include antibodies and signal-generating labels, and methods that generate detectable signals using oligonucleotides, such as PCR (immunoPCR), sequencing (NGS, next-generation sequencing), immunoisothermal amplification, and separation matrix methods. Oligonucleotides generate detectable signals through proximity reactions. Antibodies may be polyclonal or monoclonal antibodies. Immunoassays can be applied to analyze a single analyte or two or more analytes (at least 1, 5, 10, 20, or 100) within the same sample in parallel, employing various approaches.

[0136] Signal-generating labels in immunoassays include radioactive atoms (primarily 125I, 3H, and 14C). While the use of radioactive labels enables highly sensitive and accurate testing, it has several drawbacks, including health risks, special handling of reagents, staff training, the short half-lives of isotopes, and the high cost of equipment for radioactivity measurement. Therefore, non-radioactive labels such as enzymes, fluorescent probes, chemiluminescent materials, metals and metal chelates, and liposomes have been introduced as alternatives. Enzyme labeling can amplify the signal, potentially improving test sensitivity.

[0137] Matrix materials used to separate immune complexes formed as a result of immunoassay reactions include activated carbon, polyethylene glycol, secondary antibodies, microbeads containing magnetic beads, or multi-well plates in which one component of the reaction analyte or antibody is coated on the bottom surface of the plate wells, allowing the immune complexes to form on the well surface. The use of these plates simplifies washing procedures and reagent pipetting, and facilitates automation.

[0138] In a typical radioimmunoassay, a known amount of target is radioactive. This is usually done by labeling it with a gamma-ray radioactive isotope such as iodine-125-I, or tritium bound to tyrosine. When this radiolabeled target is mixed with a known amount of antibody against that target, the two bind specifically. Next, a sample containing an unknown amount of the same target is added. The unlabeled target in the sample competes with the radiolabeled antigen for antibody binding sites. As the concentration of unlabeled target increases, more unlabeled target binds to the antibody, replacing the radiolabeled variant. This reduces the ratio of antibody-bound radiolabeled target to free radiolabeled target. The bound target is then separated, and the radioactivity of the free (unbound) target remaining in the supernatant is measured using a gamma counter.

[0139] Enzyme-mediated immunoassay (EIA) is similar to radioisotope assay (RIA), except that the label is an enzyme rather than a radioisotope. The basic approach to using an enzyme as a label in immunoassay involves binding an enzyme molecule to one of the immunoassay reagents (analyte or antibody) using appropriate chemical techniques, followed by the standard immunoassay reaction. After separation into bound and free components, the enzyme activity of either of the two is monitored. This is done by adding a substrate and then monitoring the conversion rate from substrate to product. The product must have a measurable physical or chemical difference from the substrate. For example, a colorless chromogenic substrate may be converted to a colored product by the action of the enzyme label. In this case, the colored product can be easily measured with a spectrophotometer. The measured signal is then correlated with the concentration of the analyte.

[0140] Enzyme-linked immunosorbent assay (ELISA) is a method for detecting and quantifying specific proteins in complex mixtures (Hayrapetyan et al. Methods Mol. Biol 2612:1-17 (2023)). This method allows for the analysis of protein samples immobilized on a solid support (such as magnetic beads) using a specific antibody. ELISA can be performed in a 96-well or 384-well polystyrene plate in which the antibody and protein are passively bound to magnetic beads. Immobilizing the ELISA reaction on magnetic beads facilitates the separation of bound and unbound substances during analysis.

[0141] Various forms of ELISA typically involve the following four basic elements: (1) Coating / Capture - Immobilizing the antigen directly or indirectly onto the surface of a solid support (e.g., wells in a multiwell plate, paper, spherical beads, rods, etc., as detailed below). (2) Blocking - Adding unrelated proteins or other molecules to cover all unsaturated surface binding sites in the reaction vessel. (3) Probing / Detection - Incubating with an antigen-specific antibody that affinity-bound to the target antigen. (4) Signal Measurement - Detecting the signal generated by a direct or secondary binding enzyme on the affinity-binding antibody, as detailed below.

[0142] In ELISA, multiple binding and washing steps are performed to ensure specific binding. Separating the reagents in a gel within a horizontal or vertical chamber allows for a stepwise reaction without the need for cumbersome liquid handling. The protein-coated beads pass through the gel-separated reagent layers for washing and binding before the final detection step.

[0143] ELISA can be performed in various forms, primarily plate-based, tip-based, bead-based, paper-based (PVDF, nitrocellulose, cellulose), and rod-based. The basic concepts for performing ELISA on these platforms are largely the same as those for conventional immunoassays described above. Plate-based, tip-based, bead-based, and / or rod-based ELISAs can be automated for high-throughput screening, while paper-based ELISAs are primarily used for on-site immediate testing. Rod-based ELISAs use analyte-binding molecules fixed to the lower end of a disposable rod. The rod may be a glass rod, fiber optic, plastic, metal rod, capillary, electrode, or magnetic bar, and may or may not have a cover. The rod may be used in combination with a cartridge for automated high-throughput immunoassays with the ability to simultaneously detect multiple analytes from a large number of samples, or conversely, with a cartridge for automated on-site immediate immunoassays. Optionally, the rod is made of a conductive magnetic material and is used for capturing the analyte for real-time detection by measuring a magnetic sensor or magnetoresistance (as described in US20130274131), and / or as an electrode for the ECL reaction. Real-time detection can be performed based on the rod with a magnetic sensor (US20130274131). In such cases, for example, the detection antibody is coated with magnetic bead labeling. When the beads adhere to the rod, the magnetoresistance of the rod changes (magnetic sensor). Alternatively, the rod may be used in combination with a magnetic bead-based immunoassay or nucleic acid signal amplification assay such as HCR. The rod may also assist in moving the beads between different gel layers or reaction chambers, and may also be used as an electrode for the ECL reaction. The cartridge is a reaction cuvette containing diffusion-bound and non-diffusion-bound reagents for performing an immunoassay, and in this invention, the reagents are separated by a permeable gel. A cartridge assembly tray can be used to fix multiple test cartridges and rods into an assembly and apply to an immunoassay instrument for high-throughput testing.

[0144] Ni 2+ Magnetic beads with various surface chemical properties, such as sulfo-SMCC, tosyl groups, and carboxyl groups, are used to immobilize proteins, antibodies, or molecules targeted in ELISA. The beads function as a molecular immobilization platform to initiate ELISA, after which subsequent steps of washing and binding are performed by moving the beads through a gel layer. Bead-based ELISA enables high-throughput sample processing through automation and reduces turnover time. Separating the washing solution and binding molecules into different gel chambers allows bead-based ELISA to be performed in a one-pot format. By guiding magnetic beads to different chambers or gel layers for specific reactions, continuous reactions are possible without repeated liquid handling.

[0145] ELISA subtypes can be classified into direct, indirect, and sandwich methods. The target antigen is either directly adsorbed onto assay beads or indirectly immobilized via a capture antibody bound to the beads. The analyte is then detected directly (using a labeled primary antibody) or indirectly (using a labeled secondary antibody specific to the primary antibody, or an amplification biotin-streptavidin complex, etc.). Specificity of the secondary antibody to the primary antibody can be achieved by using capture antibodies and primary antibodies derived from different host species (e.g., mouse IgG and rabbit IgG). In sandwich assays, cross-adsorbed secondary antibodies are effective in removing secondary antibodies that may have affinity for the capture antibody. In sandwich ELISA, the analyte to be measured is bound between two primary antibodies (capture antibody and detection antibody), each detecting a different epitope of the antigen.

[0146] The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP), or ECL immunoassay (described in Journal of Electroanalytical Chemistry Vol. 919, 15 August 2022, 116511). Other enzymes that can be used include β-galactosidase, acetylcholinesterase, and catalase. A wide variety of substrates are commercially available for ELISA using HRP or AP conjugates. The choice of substrate depends on the required assay sensitivity and the instrument available for signal detection (spectrophotometer, fluorometer, or luminometer).

[0147] Figure 5 shows a preferred embodiment of the present invention for performing an efficient integrated immunoassay using a reaction vessel containing pre-sealed or pre-loaded reagents. In this embodiment, a permeable gel layer mixture is used as a compartmentalizing barrier to separate all individual reagents in a manner consistent with the workflow of a typical magnetic bead-based ELISA assay for measuring a target antigen. To facilitate the movement of magnetic beads through each gel layer in the vessel, a heating element is mounted in front of an external magnetic piece and may be configured as a 2-in-1 module. As the assay progresses to the stage of moving beads across the gel layers, the heating element is positioned to engage with the target gel layer and heat the gel layer to reduce the viscosity of the barrier, thereby allowing the beads to move between chambers or layers. To further facilitate such movement through one or more gel layers, a pair or multiple magnetic pieces may be introduced, thereby reducing the number of beads per magnet and consequently increasing the magnetic force on the beads.

[0148] The preparation of such a reaction vessel involves procedures similar to those described in the section on amplification reaction vessels of the present invention. Briefly, the following reagents and gel layers are sequentially added from the bottom upwards to a plastic container, for example, 1.5 mL or 2.0 mL with an inner diameter of 5.0 mm: for example, 100 μL of stop solution, 20 μL of gel layer, 100 μL of substrate solution, 20 μL of gel layer, second wash buffer, 20 μL of gel layer, 100 μL of detection antibody solution, 20 μL of gel layer, 100 μL of first wash buffer, 20 μL of gel layer, 50 μL of solution containing magnetic beads coated with capture antibody, and 20 μL of gel layer. After cooling, the container is sealed with a foil seal. Optionally, the separation of reagent components is enhanced by adding, for example, 10-20 μL of mineral oil to each aqueous layer. Optionally, the reaction vessel contains two aqueous reaction mixtures separated by gel layers. The lower aqueous reaction mixture contains a signal amplification element such as a substrate of HRP, AP, or ECL. The upper aqueous reaction mixture contains magnetic beads encoded with a capture antibody and a labeled detection antibody. After adding the sample to the upper aqueous solution and incubating at a constant temperature, the beads can be moved from the first gel layer to the first aqueous reaction mixture containing the enzyme substrate or ECL substrate, thereby generating a chemiluminescent or color reaction.

[0149] Before use, remove the foil seal from the container and pierce the upper gel layer with an inert rod. Next, add, for example, 100 μL of sample to the liquid phase of the bead chamber. To accelerate the immunoconjugation reaction, activate the device equipped with a thermal module and a magnetic module, and resuspend and mix the magnetic beads in the sample solution by alternately switching the electromagnetic field of the magnetic piece on and off. Next, place the heating element in the second gel layer and, after heating for a short time, move the activated magnetic module forward. This causes the beads to penetrate / pass through the gel layer and enter the first washing chamber. Next, place the magnetic module in the center of the target chamber and effectively wash the beads by programmed electromagnetic mixing. By following these similar operating procedures, the beads enter the detection chamber, where they emit a detectable color due to the enzymatic reaction. Optionally, to control the time-dependent color intensity of the detection chamber, place the heating module connected to the magnetic module in the lower gel layer and heat to melt / liquefy the gel layer. As a result, the colored reaction mixture falls through the liquefied gel layer and merges with the stop solution below. This allows for endpoint quantification of the target antigen while stabilizing the color of the reaction mixture. The intensity of the color observed or measured in the combined reaction mixture is proportional to the level of antigen molecules present in the sample.

[0150] 4. Integrated Bioprocesses The reaction vessel can be used for integrated, continuous reactions.

[0151] For example, DNA / RNA purification is the process of separating and extracting DNA or RNA from biological samples such as blood, tissue, cells, and microbial cultures. This purification process removes impurities and contaminants that may affect the quality and integrity of the genetic material.

[0152] DNA or RNA samples may contain various types of impurities and contaminants depending on their origin and processing steps. Samples of biological or environmental origin generally contain PCR inhibitors, which significantly inhibit DNA polymerase activity and reduce the sensitivity and efficiency of PCR reactions.

[0153] DNA polymerases exhibit varying resistance to PCR inhibitors. For example, DNA polymerases such as Taq polymerase, widely used in real-time and quantitative PCR, are completely inactive in the presence of small amounts of blood (0.004% to 0.2%). The problem of PCR inhibition due to DNA samples can be resolved by various methods, such as sample dilution and purification. However, this inhibition can still be a concern in many PCR-based human blood tests, as even after purifying DNA from blood, trace amounts of PCR inhibitors can lead to high false-negative rates. Another example is the testing of agricultural microorganisms, infectious disease pathogens, and bioterrorism-related pathogens in soil samples. Direct extraction of total DNA from soil samples also extracts humic acid, known as one of the strongest soil inhibitors against PCR analysis. Humic substances are mixtures of polyphenols, whose properties are partially elucidated, produced during the decomposition of organic matter. Other inhibitory components include fulvic acid, polysaccharides, and metal ions, which can be present in soil samples at various concentrations.

[0154] The most common impurities and contaminants include:

[0155] Proteins: Proteins can be purified along with DNA or RNA and may interfere with downstream applications such as PCR, sequencing, and gene expression analysis.

[0156] Salt: High salt concentrations can affect the quality of DNA or RNA samples and may inhibit enzymatic reactions such as PCR.

[0157] Organic solvents: Organic solvents such as phenol and chloroform, commonly used in DNA / RNA extraction protocols, can be purified along with the DNA or RNA and may affect downstream applications.

[0158] Residual cellular residue: Cellular residues such as lipids, polysaccharides, and other macromolecules are purified along with DNA or RNA and may affect the quality and quantity of the sample.

[0159] Nucleases: Nucleases are enzymes that can break down DNA or RNA, and may be present in the sample or introduced during processing.

[0160] Environmental contaminants: During sample collection or processing, contaminants such as dust, aerosols, and other microorganisms may enter the sample and affect the quality of the DNA or RNA.

[0161] Removing these impurities and contaminants from DNA or RNA samples ensures the accuracy and reliability of downstream applications. Purified DNA or RNA can then be used in a variety of downstream applications, including PCR, sequencing, cloning, and gene expression analysis.

[0162] There are several methods for DNA / RNA purification, and the choice of method depends on the type of sample and the downstream application. Below are some common DNA / RNA purification methods.

[0163] 1) Organic solvent extraction: This method uses the following organic solvents.

[0164] Phenol-chloroform, β-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), chloroform-isoamyl alcohol, isopropanol and salts, and TRIZOL®.

[0165] Nucleic acids can also be treated with proteinase K under a gel layer, for example, at 95°C for 10 minutes.

[0166] 2) Centrifugation

[0167] 3) Phase separation: After centrifugation, the sample separates into three distinct layers: an aqueous phase containing nucleic acids, a lower organic phase containing denatured proteins, and an intermediate phase containing other contaminants.

[0168] 4) Precipitation: The nucleic acids are then precipitated with ethanol or isopropanol, washed with a washing buffer, and then dissolved in a buffer for further analysis.

[0169] The present invention can be applied to organic solvent extraction by pre-arranging all necessary reagents in a single container. For example, from top to bottom, the layers are: gel layer 1, solvent, gel layer 2, organic solvent, gel layer 3, void, ethanol, gel layer 4, and elution buffer. When using, the sample is first added to gel layer 1. After melting gel layer 1, the sample is mixed with the solvent. After dissolution, gel layer 2 is melted, and the sample is mixed with the organic solvent. After stirring and centrifugation, gel layer 3 blocks the organic phase, allowing only the liquid phase containing nucleic acids to pass through. The obtained nucleic acids are precipitated by the ethanol layer. After centrifugation, the precipitated nucleic acids pass through gel layer 4, and the ethanol layer is blocked, so purified nucleic acids are obtained in the lower elution buffer layer. This embodiment of the present invention can also be combined with other layers mentioned elsewhere in this application. In this configuration, all reagents can be contained in a single container, thus protecting the user from exposure to toxic reagents. This all-in-one design simplifies the entire procedure and increases adaptability to automated equipment.

[0170] Column purification and size-selective purification: This method uses a column packed with a resin, membrane, or silica spin column that selectively binds to DNA or RNA. When the sample is packed into the column, nucleic acids are captured while impurities pass through. After washing the column, the purified DNA or RNA is eluted with buffer. DNA size-selective purification is the process of separating DNA fragments of a specific size range from a mixture of DNA fragments.

[0171] This technique is widely used in molecular biology experiments that require DNA fragments of specific sizes, such as cloning, PCR, and sequencing. One of the most commonly used methods is purification using a column. DNA fragments can be separated by size using a size exclusion column. A size exclusion column separates DNA fragments based on their size and charge.

[0172] After packing the sample into the column, the desired DNA fragment can be eluted using an appropriate buffer.

[0173] The present invention can be applied to column-based DNA separation by pre-arranging all necessary reagents in a single container. For example, from top to bottom, there may be gel layer 1, gel layer 2, gel layer 3... with a void at the end. Each gel layer has a different binding affinity for DNA / RNA and a different melting point due to the different wax-to-oil ratio. Therefore, the size range of DNA / RNA that binds to each layer is different. When in use, the sample is added on top of gel layer 1. The tube is then centrifuged until all the liquid phase has passed through the gel layers and reached the void region at the bottom. The DNA / RNA is extracted by melting the specific gel layer and transferring it with a pipette. For example, if the target DNA / RNA is bound to gel layer 2, first melt gel layer 1 and remove it with a pipette. Then melt gel layer 2 and transfer the DNA / RNA, which is contained in the gel layer, to a clean tube. This embodiment of the present invention can also be combined with other forms of gel layers described elsewhere in this application. The advantage of this embodiment is that all reagents are contained in a single container. This all-in-one design makes the entire process simpler and more streamlined, and also increases its adaptability to automated equipment.

[0174] Purification using magnetic beads: This method uses magnetic beads coated with ligands that selectively bind to DNA or RNA. Examples of ligands include -OH, -SH, -NH2, and -COOH. When the sample is mixed with the beads, the DNA or RNA binds to the magnetic beads and is captured by the magnetic field. After washing, the purified DNA or RNA is eluted. This technique can also be used to separate and purify DNA fragments of a specific size range from a mixture of DNA fragments using magnetic beads. This method is widely used in molecular biology experiments that require DNA fragments of a specific size, such as cloning, PCR, and sequencing. The basic procedure for purification using magnetic beads is as follows:

[0175] (a) Dissolution: First, the sample is dissolved to release nucleic acids from cells or tissues. The lysis buffer usually contains a surfactant that breaks down the cell membrane and releases nucleic acids. Examples of lysis reagents include sodium dodecyl sulfate (SDS), Triton X-100, guanidine thiocyanate or chloride, proteinase K, or other proteases that break down proteins, lysozyme, and combinations thereof.

[0176] (b) Binding DNA to magnetic beads: Add magnetic beads to the sample containing the target DNA fragment, and mix the beads to bind the DNA to the beads. Binding reagents include, but are not limited to, water, Tris-HCl buffer, PBS buffer, ethanol, isopropanol, propanol, butanol, other water-miscible alcohols, DMSO, THF, NaCl, and other salts.

[0177] (c) Bead washing: Wash the beads to remove contaminants and unbound DNA fragments. Washing reagents include, for example, water, Tris-HCl buffer, PBS buffer, ethanol, isopropanol, propanol, butanol, other water-miscible alcohols, DMSO, THF, NaCl, other salts, and any combination thereof.

[0178] (d) DNA elution: Elute the target DNA fragment from the beads using an appropriate buffer or solvent. Examples of elution reagents include water, Tris-HCl buffer, PBS buffer, NaCl or other salts, or combinations thereof.

[0179] The advantages of purification using magnetic beads include high yield and purity of DNA fragments, rapid and efficient purification, and ease of automation and scale-up, making it suitable for high-throughput applications. However, purification using magnetic beads also has potential drawbacks, such as higher costs compared to other purification methods, a limited size range of purifiable DNA fragments with some magnetic beads, and the possibility of loss of some DNA fragments due to non-specific binding of DNA fragments to the magnetic beads.

[0180] The present invention can be applied to the separation of DNA or RNA using magnetic beads by pre-distributing all necessary reagents into a single container. Figure 6 shows a preferred embodiment of a magnetic bead-based integrated DNA / RNA purification system. Architecturally, the reaction vessel of this integrated purification system is similar to that described in the section on integrated immunoassay systems. However, since many commercially available magnetic bead kits require heating, dissolution, and elution at high temperatures (e.g., 50-60°C) to improve yield and purity, preventing the breakdown of the heat-sensitive gel layer during the purification process is a challenge. Therefore, when constructing this integrated container system, cooling of the gel layer in direct contact with the dissolution or elution chamber is incorporated. Heating and cooling of the container can be controlled, for example, using a Peltier element that functions as a thermoelectric cooler or thermoelectric generator depending on the polarity of the DC current applied to the device. Furthermore, using a thicker gel layer connecting these two chambers reduces the risk of gel layer damage during processing.

[0181] To use the container shown in Figure 6, first remove the foil seal (not shown) on the top of the container and break the upper gel layer using an inert rod or pipette tip. Next, pipette, for example, 100-200 μL of the raw sample into a lysis chamber containing, for example, 100 μL of lysis buffer. After resealing the opening of the container with the lid, attach the heating / cooling module to the wall of the lysis chamber. The heating element is positioned around the liquid portion of the lysis chamber for dissolution, and the cooling element is positioned on the downstream gel layer for barrier protection. The dissolution process is carried out at, for example, 50-60°C, and barrier cooling is carried out at, for example, 15-20°C. After dissolution for, for example, 5-10 minutes, the module's operation is switched from cooling mode to heating mode by switching the polarity of the DC power supply. As a result, the affected gel layer is heated and partially melted. The lysis solution then falls through the gel layer and mixes with the DNA / RNA binding solution and magnetic beads in the bead chamber. To completely mix the aqueous solution and beads in the bead chamber, the electromagnet pair is switched on and off alternately for, for example, 5 to 10 minutes. Next, the heating element is placed in the next gel layer and a short warm-up is performed there to soften the gel barrier, allowing the beads to penetrate the gel layer with less resistance. The penetration and passage of the beads through the heated gel layer is facilitated by moving the externally driven electromagnet downstream. After the beads enter the first washing chamber, the electromagnet pair is placed in the central part of the washing chamber, where the electromagnet pair is started and switched on and off alternately for, for example, 1 to 2 minutes. This washing step washes away most of the impurities attached to the beads. This washing procedure is repeated in the next two washing steps in the subsequent washing chambers. Each step takes, for example, about 1 to 2 minutes to complete. Finally, the washed beads are transferred to the elution chamber, where they undergo a thermal elution treatment by the heating module and the electromagnetic module. Here, to prevent the upper gel layer from collapsing when the elution chamber is heated to, for example, 50-60°C, a pair of thermoelectric coolers are placed over the upper gel layer, and cooling conditions (e.g., 10-20°C) are maintained until elution is complete. After elution is complete (e.g., it takes about 3-5 minutes), the beads are returned to the third washing chamber, leaving behind a pure eluate containing purified DNA / RNA.This eluate is used for subsequent processing or reactions (such as integrated downstream amplification, etc.).

[0182] 5. Reaction Vessel for Integrated NGS Library Preparation Next-generation sequencing (NGS) is a technology that enables rapid and accurate sequencing of the entire genome or target regions of DNA or RNA. NGS employs massively parallel sequencing and can read multiple sequences simultaneously, thus realizing a much faster and more efficient process than conventional sequencing methods. DNA libraries are prepared from biological samples and used as starting materials for sequencing.

[0183] The preparation of libraries usually involves several steps. <Fragmentation of DNA

[0184] The preparation of NGS libraries begins with the fragmentation of DNA. In this step, long DNA strands are divided into small fragments that can be sequenced. There are mainly two methods for DNA fragmentation: enzymatic fragmentation and mechanical fragmentation. In enzymatic fragmentation, restriction enzymes or other enzymes are used to cut DNA into small fragments, and in certain enzymatic methods, end repair of the fragmented DNA is not necessary. On the other hand, in mechanical fragmentation, physical forces such as sonication or Covaris shearing are used to break DNA strands.

[0185] <End Repair After DNA fragmentation, the DNA fragments are often subjected to an end repair step. In this step, nucleotides are added to the ends of the fragments to repair possible damage that occurred during the fragmentation process. The repaired DNA ends are essential for adapter ligation and subsequent PCR amplification.

[0186] <Adapter Ligation The next step in NGS library preparation is adapter ligation. An adapter is a small synthetic DNA sequence that is added to the ends of DNA fragments, enabling attachment to the sequencing platform. Adapters can be ligated to DNA in various ways, such as using T4 DNA ligase, which catalyzes the formation of phosphodiester bonds between the adapter and the DNA fragment.

[0187] <PCR Amplification> Once the adapter is ligated (joined) to the DNA fragment, the library is PCR amplified. PCR amplification is used to create millions of copies of the DNA fragment to ensure there is sufficient material for sequencing. Since PCR can introduce bias and errors, it is important to optimize the reaction conditions to minimize these effects.

[0188] <Bead Cleanup> The final step in NGS library preparation is bead cleanup. In this step, unligated adapters, adapter dimers, and other impurities are removed from the library. Magnetic beads, which can bind to DNA fragments and wash away unwanted substances, are used for bead washing. Once the DNA fragment is purified, the library is ready for sequencing.

[0189] Commercially available kits can streamline the library preparation process and omit certain steps, such as bead cleanup after ligation. Enzymatic steps, such as enzymatic fragmentation, can be performed using heat-labile enzymes that can be inactivated by mild heat treatment. Mechanically sheared DNA requires end repair to ensure ends suitable for adapter ligation. Overall, NGS has revolutionized the field of genomics, enabling rapid and accurate sequencing of complex genomes and leading to significant advancements in fields such as medicine, agriculture, and environmental science.

[0190] The present invention provides a novel approach to preparing NGS libraries by utilizing a reaction vessel containing three aqueous reagent layers separated by three gel layers, as shown in Figure 7. This reaction vessel design allows the entire NGS library preparation process to be carried out in a single container, eliminating the need for multiple tubes and plates and reducing the risk of material contamination and loss.

[0191] Referring to Figure 7, the first aqueous reagent layer contains an enzymatic fragmentation reagent or end repair reagent to divide the DNA into smaller fragments and repair any damage that may occur during fragmentation. The enzyme used in this aqueous reagent layer is heat-unstable and can be inactivated with mild heat treatment, allowing for easy removal of the enzyme before the next step. The second aqueous reagent layer contains an adapter ligation reagent for attaching adapters to the ends of the DNA fragments. This reagent layer is separated from the first layer by a gel layer to prevent the two layers from mixing. The third aqueous reagent layer contains a library amplification reagent to provide enough DNA fragments for sequencing. This layer is separated from the second layer by another gel layer.

[0192] Overall, this reaction vessel design provides a simple and efficient method for NGS library preparation, allowing the entire process to be completed in a single vessel. The use of a gel layer prevents mixing of different reagent layers. This approach can simplify the library preparation process and improve the reproducibility and reliability of NGS results.

[0193] In some embodiments, intact DNA is added to the first layer of aqueous reagent containing the enzymatic fragmentation reagent via a micropipette tip that penetrates the upper gel layer. This method ensures that the DNA is uniformly dispersed throughout the reagent in the first layer. The reaction vessel is then incubated, for example, at 25°C for 30 minutes. During this time, the enzymatic fragmentation reagent breaks down the DNA into smaller fragments. After the completion of the enzymatic fragmentation step, the reaction vessel is incubated, for example, at 50°C for 10 minutes to inactivate the heat-unstable enzyme. This step ensures that the enzyme loses its activity and does not interfere with subsequent steps while maintaining the integrity of the second and third gel layers.

[0194] Next, the second layer of the gel is disrupted using a micropipette tip, and the fragmented DNA is mixed with the adapter ligation reagent contained in the second layer of the aqueous reagent. The reaction vessel is then incubated, for example, at 25°C for 30 minutes to ligate the adapter to the DNA fragments. After the adapter ligation step is complete, the third layer of the gel is disrupted using a micropipette tip, and the ligation product is mixed with the library amplification reagent contained in the third layer of the aqueous reagent. Finally, the reaction vessel is incubated with a PCR program to amplify the library and generate a sufficient amount of DNA fragments for sequencing.

[0195] Mechanically cut DNA refers to DNA that has been fragmented using mechanical means such as sonication or shearing, rather than enzymatic fragmentation. In this embodiment, the DNA is added to the first layer of aqueous reagent containing the end repair reagent through a micropipette tip that penetrates the upper gel layer. Next, the end repair reagent is reacted with the DNA for 30 minutes at, for example, 25°C. Then, the heat-unstable enzymes are inactivated by incubating the reaction vessel for 10 minutes at, for example, 50°C, while maintaining the integrity of the second and third gel layers. Next, the second gel layer is broken using the pipette tip and the end-repaired DNA is mixed with the adapter ligation reagent. Next, the adapter is ligated to the DNA fragments by incubating the reaction vessel for 30 minutes at, for example, 25°C. Then, the third gel layer is broken using the micropipette tip and the ligation product is mixed with the library amplification reagent. Finally, the reaction vessel is incubated with a PCR program to amplify the library. This yields a library of DNA fragments that can be sequenced using NGS technology.

[0196] In embodiments of ligation-mediated PCR, a type of PCR is performed using a reaction vessel to amplify a specific region of a DNA sequence. This process begins by adding intact DNA to a first layer of aqueous reagent containing either a restriction enzyme or an enzymatic fragmentation reagent that breaks down DNA into smaller fragments. This step is performed using a micropipette tip that penetrates the top layer of a permeable gel. Next, the reaction vessel is incubated for 30 minutes at, for example, 25°C to 37°C to allow the enzyme to act. Then, the reaction vessel is incubated for 10 minutes at, for example, 50°C to inactivate the heat-unstable enzyme while maintaining the integrity of the second and third gel layers. Next, the second gel layer is disrupted using a micropipette tip, and the fragmented DNA is mixed with an adapter ligation reagent that attaches adapters to the ends of the DNA fragments. Next, the reaction vessel is incubated for 30 minutes at, for example, 25°C, to allow the adapters to ligate the DNA fragments. Finally, the third gel layer is disrupted using a micropipette tip, and the ligation product is mixed with the PCR reagent. This initiates the ligation-mediated PCR process. Finally, the reaction vessel is incubated with a PCR program to amplify the DNA fragments, thereby generating a large quantity of the target DNA sequence.

[0197] In some embodiments, the process uses a reaction vessel having two distinct layers: a gel top layer and an aqueous reagent layer beneath it. The aqueous reagent layer contains one or more specific fragmentation enzymes, end repair reagents, ligation reagents, or library amplification reagents, depending on the desired reaction. The gel top layer allows for easy addition of a sample through a pipette tip that can penetrate the gel layer.

[0198] After adding the sample, the reaction vessel is incubated to induce a specific enzyme fragmentation, end repair, ligation, or library amplification reaction.

[0199] The incubation time may vary depending on the specific reaction and the desired outcome.

[0200] During the incubation period, the enzymatic reaction proceeds in the aqueous reagent layer, while the gel layer acts as a barrier to prevent contamination and ensure efficient reaction conditions.

[0201] In some embodiments of the described process, the reaction vessel contains two layers of aqueous reagents separated by a two-layer gel. This configuration allows two different enzymatic reactions to be carried out in the same vessel.

[0202] As one configuration, the first layer of aqueous reagent may contain a specific enzyme fragmentation reagent or end repair reagent, depending on the desired reaction. For example, in tagmentation library preparation on beads, using transposomes bound to the beads enables a more uniform tagmentation reaction compared to tagmentation reactions in solution. The chemical reaction of transposomes bound to beads integrates the steps of DNA extraction, fragmentation, library preparation, and library normalization. This reduces the number of workflow steps, the amount of sample required, and shortens both working time and turnaround time.

[0203] These reagents are designed to break down DNA molecules into smaller fragments or to repair DNA ends. The second layer of the aqueous reagent may contain a ligation reagent used to attach adapters to the ends of the DNA fragments.

[0204] Alternatively, the aqueous reagent may consist of a first layer containing a ligation reagent and a second layer containing a library amplification reagent. The ligation reagent in the first layer is used to attach adapters to the ends of the DNA fragments, which are then amplified using the library amplification reagent in the second layer. This allows for the creation of multiple copies of the target DNA fragment, which can be further analyzed or manipulated as needed.

[0205] In some embodiments, the PCR amplification library is purified by a bead cleanup method.

[0206] Magnetic beads and binding buffer are added to the completed library amplification reaction mixture. The binding buffer facilitates the binding of the DNA library to the bead surface. The bead-DNA complex is then separated from the liquid by dipping the magnetic rod into the reaction vessel and withdrawing the beads. Next, the beads are washed by dipping the magnetic rod with the beads twice in succession into 70% ethanol. This removes any impurities that may remain after the amplification reaction. Finally, the beads are eluted from the beads by dipping the magnetic rod with the beads into elution buffer.

[0207] All publications, patents, patent applications, accession numbers, websites, etc., described herein are incorporated herein by reference to the same extent as individual publications, patents, or patent applications are referenced individually. Where different content is associated with an accession number or other reference at different times, it refers to the content as of the effective filing date of this application. The effective filing date is the filing date of the earliest priority application disclosing the accession number. Unless otherwise apparent from the context, any element, embodiment, step, feature, or aspect of the Invention may be practiced in combination with other elements, embodiments, steps, features, or aspects.

[0208] <Examples> <Example 1: Gel Preparation> Eight paraffin wax blocks with a melting point of 50-52°C were placed in an aluminum wax melting container with internal dimensions of 100 x 140 x 50 mm. Each wax block weighed 32 g, and the total weight of the wax blocks was 256 g. The wax container was heated to a constant temperature of 75°C, and the wax blocks melted into a liquid. The molten wax reached a height of approximately 25 mm in the container. Next, mineral oil was added to the wax container until the liquid level reached 45 mm. The weight of the mineral oil added to the container was measured at 234.5 g using the container weight subtraction method. The resulting wax and mineral oil mixture was stirred with a spatula at 75°C to obtain a homogeneous and transparent liquid mixture. This mixture was suitable for gel distribution and the preparation of reaction vessels containing gel. The weight ratio of wax to mineral oil in the molten mixture was 256 g / 234.5 g or 1.09 (w / w). The volume ratio of wax to mineral oil in the molten mixture was (100 × 140 × 25 mm / 100 × 140 × 20 mm) or 1.25 (v / v).

[0209] The mixture, melted at 75°C, was dispensed into an 8-tube PCR tube strip using an 8-channel pipette equipped with an 8-channel aluminum nozzle adapter. The 8-tube adapter was heated to 75°C using a thermostat temperature controller.

[0210] SUPELCO® pure wax (melting point 42-44°C, EMD Millipore) was melted at 60°C using the same wax container as above, and dispensed into an 8-tube PCR tube strip using the same 8-channel heating pipette as above.

[0211] <Example 2: Reagent-free reaction tube pre-filled with a single layer of gel> Using four 0.1 mL 8-tube PCR strips, two different types of reaction tubes were prepared: (i) a gel-containing tube strip, and (ii) a two-layer tube strip containing gel and mineral oil. The amplification performance of the obtained tube strips was evaluated in comparison to that of liquid-type tube strips that did not contain any foreign matter in the PCR tubes.

[0212] To prepare the tubes containing the gel, 40 μL of liquefied paraffin wax was added to each of the two tube strips. First, 30 μL of mineral oil was added, followed by 30 μL of gel, which was a mixture of liquefied paraffin wax and mineral oil in a volume ratio of 1.25:1.0, as described in Example 1, to prepare a two-layer tube strip containing the gel. After preparing the above tube strips, 10 μL of master mix, 9 μL of monochromatic (FAM) primer mix, and 1 μL of DNA template were added to each tube strip, and also added to 12 empty tubes as liquid controls. The tube strips containing the reaction mixture were capped with lid strips and placed in a cold block, then processed in a Bio-Rad CFX96 thermal cycler. The thermal cycle was performed in two stages with the lid temperature set to 105°C. 1) 9 cycles of 0:15 minutes at 90°C and 0:30 minutes at 60°C were repeated. 2) The reaction was repeated 39 times, with 0:15 minutes at 95°C and 0:30 minutes at 65°C. Figure 8 shows representative amplification curves for the three types of tube reactions. Table 1 shows the results of Ct analysis for the amplification curves of the three tube types. As seen in the figure, the double-layer tube strip showed performance comparable to the liquid type, but the wax-only type showed a delay in the Ct value and a lower signal than the previous two. Figure 9 shows images of the three types of tubes before and after thermal cycling. This shows that a non-flowing solidified wax or gel covering is sealed over the liquid reaction mixture inside the tube.

[0213] Results of PCR amplification curves from three reaction tubes [Table 1]

[0214] The performance of these three tube configurations was also evaluated using isothermal amplification reagents. For example, 10 μL of a four-color HPV screening primer mix, 10 μL of an enzyme mix, and 5 μL of a positive control template were added to each of the three types of tube strips, and two strips were prepared for each tube configuration. After capping with a cap strip, the tube strips were placed in a cold block and then loaded into a Bio-Rad thermal cycler for an isothermal reaction at a constant temperature of 60°C for 72 cycles. Figures 10A-C show the amplification curves for the three tube configurations. Table 2 shows the results of Ct analysis for the amplification curves of the three reaction tubes. As can be seen in the figures, the three tube configurations showed comparable performance in terms of amplification rate and accuracy as determined by Ct analysis.

[0215] Results of Ct analysis on four-color amplification curves for three tube formats (two-layer format, gel-only format, and liquid format) [Table 2]

[0216] <Example 3> Multilayer reaction tube containing pre-filled reagents and a gel layer The preparation of tube strips containing a gel-mineral oil mixture and PCR reagents is described below. 10 μL of a single-color primer mixture was added to each tube of a 0.1 mL eight-tube PCR strip. Then, mineral oil was added to the second layer, a gel layer to the third layer, 10 μL of enzyme mix to the fourth layer, mineral oil to the fifth layer, and gel to the sixth layer. The two gel layers in this experiment have the same composition as those described in Example 2. The PCR reagents used in this experiment are the same as those described in Example 2.

[0217] To use the obtained reagent tubes for the PCR reaction, the foil seals were peeled off the tube strips, and the two gel layers were separated by piercing them with a needle-shaped plastic pick. Next, using a conventional pipette, 1 μL of PCR-positive control template was added to the liquid phase of the tube through the broken opening in the gel layer, and the lid of the tube strip containing the sample was closed. The closed tube strip was centrifuged in a microcentrifuge for several seconds. Finally, the centrifuged tube strip was placed in a cold block, and then the sample was measured in a Bio-Rad thermal cycler. The thermal cycling was performed in two stages with the lid temperature set to 105°C: 1) 9 cycles of 0:15 min at 90°C and 0:30 min at 60°C. 2) 39 cycles of 0:15 min at 95°C and 0:30 min at 65°C. This is the same thermal cycling setting as described in Example 2. Figure 11 shows the amplification curves of the reaction using two multilayer PCR reagent tube strips. Table 3 shows the results of the Ct analysis of the amplification curve shown in Figure 11, and it can be seen that the amplification rate and accuracy are equivalent to those shown in Table 1. Figure 12 shows images of the tube strip before and after the thermal cycle, and it shows that a non-flowing, solidified gel covering is sealed over the liquid reaction mixture inside the tube.

[0218] Results of Ct analysis on PCR amplification curves of multilayer reagent tubes [Table 3]

[0219] In parallel with the PCR reagent tubes described above, multilayer isothermal amplification reagent tubes were also prepared and their performance evaluated, as described below, in comparison to the liquid form. A 0.2 ml eight-tube multilayer UTI (urinary tract infection) test tube strip was prepared in the same manner as described above. The resulting multilayer reagent tube had a layer structure from bottom to top: 10 μL of enzyme mix, mineral oil, gel layer, four-color UTI primer mix, mineral oil, and gel layer. The completed tube strip was sealed with an adhesive foil strip. After removing the foil seal from the tube strip, the two layers of the wax-mineral oil gel barrier were punctured and separated using a needle-shaped plastic pick. Next, using a conventional pipette, 5 μL of UTI positive control template was added to the liquid phase of the tube through the broken opening in the wax-oil gel layer, and the lid of the sample-added tube strip was closed. The closed tube strip was centrifuged in a microcentrifuge for several seconds. Finally, the centrifuged tube strip was placed in a cold block for measurement in a thermal cycler. Liquid reaction tubes were prepared using empty 0.2 mL PCR tube strips by pipetting 10 μL of enzyme mix, 10 μL of 4-color UTI primer mix, and 5 μL of UTI positive control template into each tube of the strip. After short-term centrifugation in a microcentrifuge, the completed tube strips were placed in a Bio-Rad CFX96 thermal cycler along with the multilayer reagent tube strips and processed at a constant temperature of 60°C for 72 cycles or 90 minutes. Figures 13A-D show the 4-color amplification curves of the reaction in the multilayer UTI isothermal reagent tube strips and the control liquid form. Table 4 shows the Ct analysis results of the amplification curves shown in Figures 13A-D, demonstrating that the amplification rate and accuracy of the multilayer UTI reagent tubes are comparable to those of the liquid form. Figure 14 shows images of the multilayer reagent tube strips before (top) and after (bottom) isothermal amplification at 60°C for 90 minutes, showing the solidified gel cover that formed extensively over the liquid layer in the tubes after sample processing.

[0220] Results of Ct analysis on amplification curves of multilayer reagent tubes and liquid-type tubes [Table 4]

[0221] To evaluate the assay sensitivity of the multilayer reagent tubes, an HPV31 positive control template was added to an HPV31 reagent tube strip at a detection limit concentration of 7.5 copies per 25 μL of reaction solution, and the reaction was carried out in a Bio-Rad thermal cycler at 60°C for 90 minutes. The reaction with the liquid control was performed in parallel with the HPV31 reagent tube strip. Figure 15 shows the amplification curves (FAM channels) of the reactions using the HPV31 reagent tube strip and the liquid tube strip, demonstrating comparable assay sensitivity for both reagent formats.

[0222] <Example 4> Evaluation of multilayer reagent tubes equipped with HPV clinical samples Multilayer HPV screening reagent tube strips were evaluated using clinical HPV samples. HPV screening assays were performed on 78 HPV samples using two HPV reagent tube strips, namely HPV16 (FAM channel) and HPV18 (CY5 channel). These HPV samples were lysated swab samples collected from 78 patients. The HPV reagent tube strips for this evaluation study were prepared in the same manner as described in Example 2. The sample volume was 5 μl, and a total volume of 16 μl (4 × 4) was used to repeat the amplification reaction four times for each of the 78 samples. Isothermal sampling of these samples was performed using the same heating conditions as described in Example 2. Table 5 summarizes the results of the screening assays performed on the 78 HPV samples, showing good agreement between the multilayer reagent format and the conventional liquid format.

[0223] Results of HPV screening assays using 78 HPV samples (ND: Not detectable, HR: High risk) [Table 5]

[0224] <Example 5> Stability of multilayer reagent tubes The physical or mechanical stability of multilayer reagent tubes was evaluated by dropping reagent tube strips onto the floor from a height of approximately 10 feet. The objective of this study was to investigate the adhesion of two wax-mineral oil gel layers to the tube walls after being subjected to free-fall impact onto a hard surface. Prior to the drop impact experiment, 12 reagent tube strips in a plate holder were stored overnight at -20°C. The following day, the strip plate was removed from the -20°C state and allowed to equilibrate to ambient temperature for approximately 15 minutes. Next, the 12 strips were dropped onto the laboratory floor from a height of approximately 10 feet, and this procedure was repeated twice for these 12 strips. Figures 16A and 16B show images of the 12 tube strips before and after the drop impact. Visual inspection of the tube strips showed that the two wax-mineral oil layers remained well and stably adhered to the inner walls of the tubes even after three free-falls, suggesting good physical stability and feasibility of the tube strips during transport and handling under moderate impact conditions.

[0225] The thermal stress stability of multilayer reagent tubes was evaluated by simulating long-term transport conditions at ambient temperature and storing reagent tube strips in a commuter vehicle. For comparison, a 5-layer reagent tube strip was prepared by removing the mineral oil layer surrounding the reagent components at the bottom of the tube and compared with the standard 6-layer tube strip described in Example 2.

[0226] In this experiment, six strips of 6-layer 4-color HPV18 reagent tubes and six strips of 4-color 5-layer HPV18 reagent tubes were stored overnight at -20°C. The next day, the strips were removed and placed on a workbench at room temperature (20-25°C) for approximately 7 hours. The strips were then stored again overnight at -20°C. After repeating this freeze / thaw process three times, the strip plates were packed into a sealed styrofoam box without refrigerant and carried in the trunk of a commuter vehicle for four consecutive days. The temperature inside the styrofoam box, recorded by a temperature logger, was 15-30°C. Next, the tube strips were removed from their packaging and placed on a workbench for reaction preparation as described in Example 2. Briefly, 5 μL of HPV18 positive template was added to each of the two types of layered tube strips at a Lod (Limit of Detection) concentration of 15 copies per reaction. The sample-added tube strips were processed in a Bio-Rad thermal cycler at 60°C for 90 minutes. Figures 17A and 17B show the FAM and ROX amplification curves after repeated freeze-thaw cycles and long-term transport at ambient temperature for two types of tube strips (curves for HEX and Cy5 channels are not shown). As can be seen in the figures, the reagent stored in the 5-layer tubes showed a significantly increased background signal in the FAM channel and an increased Ct value compared to the reagent stored in the 6-layer tubes.

[0227] Considering the effectiveness of reagent separation through multilayer design, the thermal stability of these multilayer reaction tubes at high temperatures was evaluated. Table 6 shows the results of the thermal stability of 6-layer reagent tubes at ambient temperatures of 23–45°C. In the thermal stability tests, when the tube strip was exposed to a high temperature close to the melting point of the gel layer for a certain period of time (e.g., 39 minutes at 40°C), it was observed that a large portion of the gel layer thinned, eventually losing its adhesion to the inner wall of the tube, and the second aqueous layer penetrated the gel barrier and mixed with the first aqueous layer at the bottom of the tube. To improve the thermal stability of the 6-layer reaction tube, increasing the proportion of wax component in the wax-oil gel increases the tube breakage time, but at the cost of increased hardness of the gel layer against pipette tip penetration.

[0228] Thermal stability results of 6-layer reaction tubes at various temperatures [Table 6]

Claims

1. A reaction vessel, Equipped with a gel layer, The gel layer is a reaction vessel through which a micropipette tip can penetrate, and which is located inside the container.

2. A reaction vessel according to claim 1, A reaction vessel further comprising one or more additives within the gel layer and mineral oil beneath the gel layer.

3. A reaction vessel according to claim 1 or claim 2, The gel layer further optionally contains one or more aqueous reaction compounds above and / or below and / or inside the gel layer. The aqueous reaction mixture is a reaction vessel for detecting the analyte.

4. The reaction vessel according to claim 3, The aqueous reaction mixture is located below the gel layer. The gel layer prevents the aqueous reaction mixture from dispersing across the gel layer. The gel layer is permeable by a micropipette tip, and the reaction vessel delivers the solution to the reaction mixture to cause a reaction and detect the analyte.

5. The reaction vessel according to claim 3, A reaction vessel in which the solution to be delivered is a sample containing the analyte.

6. The reaction vessel according to claim 5, The gel layer prevents the aqueous reaction mixture from dispersing across the gel layer, and the reaction vessel is such that the tip of a micropipette can penetrate it at a temperature range of at least 18 to 35°C, and optionally at a temperature range of 20 to 25°C.

7. A reaction vessel according to any one of claims 1 to 6, The reaction vessel comprises a plurality of gel layers, a plurality of oil layers, and a plurality of aqueous reaction mixture layers. A reaction vessel in which the number of gel layers differs from the number of oil layers, and / or the number of gel layers differs from the number of aqueous reaction mixture layers.

8. A reaction vessel according to any one of claims 1 to 7, A reaction vessel in which the micropipette comprises a single tip, multiple tips, or a bundle of capillaries.

9. A reaction vessel according to any one of claims 1 to 8, The apparatus comprises a first aqueous reaction mixture, a first gel layer covering the first aqueous reaction mixture and through which a micropipette tip can penetrate, a second aqueous reaction mixture above the first gel layer, and a second gel layer covering the second aqueous reaction mixture and through which a micropipette tip can penetrate, Optionally, a first mineral oil layer may be further provided between the first aqueous reaction mixture and the first gel layer. A reaction vessel optionally further comprising a second mineral oil layer between the second aqueous reaction mixture and the second gel layer.

10. A reaction vessel according to claim 9, A reaction vessel in which one of the first and second aqueous reaction mixtures is a sample, and the other contains one or more reagents for producing a reaction to detect an analyte in the sample when the first and second aqueous reaction mixtures are mixed.

11. A reaction vessel according to claim 9, A reaction vessel in which the first aqueous reaction mixture and the second aqueous reaction mixture are each a partial reaction mixture that produces a detection reaction when mixed with each other and with a sample.

12. A reaction vessel according to claim 9, A reaction vessel in which a third aqueous reaction mixture is optionally present above the second gel layer, and the plurality of aqueous reaction mixtures contain reagents for carrying out ordered bioprocess steps.

13. A reaction vessel according to claim 12, A reaction vessel in which the plurality of aqueous reaction mixtures are used for preparing nucleic acid sequencing libraries, extracting nucleic acid samples, performing immunoassays, biochemistry, or nucleic acid amplification and detection.

14. A reaction vessel according to claim 12, A reaction vessel in which the first aqueous reaction mixture and the second aqueous reaction mixture each contain a polymerase and a primer, or vice versa, for nucleic acid detection by PCR amplification, qPCR amplification, reverse transcriptase PCR reaction, digital PCR amplification, or isothermal amplification.

15. A reaction vessel according to claim 12, A reaction vessel comprising the first aqueous reaction mixture and the second aqueous reaction mixture, each containing different reagents required for the detection reaction in combination with each other and with the sample.

16. A reaction vessel according to claim 15, A reaction vessel in which the mixing of the first aqueous reaction mixture and the second aqueous reaction mixture is for performing an immunoassay and detection, and optionally a third aqueous reaction mixture is present above the second gel layer for sequential reactions.

17. The reaction vessel according to claim 3, A reaction vessel containing the aqueous reaction mixture and magnetic beads.

18. A reaction vessel according to any one of claims 1 to 17, A reaction vessel coupled to a heater module that is slidable along the outside of the reaction vessel, and which melts the gel layer when the heater is adjacent to the gel layer.

19. A reaction vessel according to any one of claims 1 to 18, A reaction vessel coupled to a magnetic module that is slidable along the external or internal surface of the reaction vessel, for moving magnetic beads from the aqueous reaction mixture across the gel layer beneath the aqueous reaction mixture.

20. A reaction vessel according to claim 19, A reaction vessel in which the slidable magnetic module is slidable along the interior of the reaction vessel and connected to a rod for detecting a reaction within the reaction vessel.

21. The reaction vessel according to claim 3, A reaction vessel comprising the aqueous reaction mixture and a rod for detecting the reaction in the reaction vessel.

22. The reaction vessel according to claim 21, wherein the rod is an electrode.

23. A reaction vessel according to any one of claims 1 to 22, A reaction vessel in which wells in a multiwell plate (e.g., a PCR plate, deepwell plate, microtiter plate, or ELISA plate) each contain a gel layer.

24. A reaction vessel according to any one of claims 1 to 22, A reaction vessel comprising a plurality of such containers, which are tubes or pipes (e.g., PCR tubes), arranged individually or as strips or matrices, and connected to one another.

25. A reaction vessel according to any one of claims 1 to 22, A reaction vessel made of plastic, glass, or metal, having one or more grooves or channels.

26. A reaction vessel according to any one of claims 1 to 23, A reaction vessel having a well on a chip or on a pipe (e.g., a glass slide or silicon wafer or a plastic slide or a metal slide), which optionally provides a plurality of reaction vessels, wherein the plurality of wells are optionally connected by channels, and optionally the wells contain a membrane or fragments of glass fiber.

27. A reaction vessel according to any one of claims 1 to 26, A reaction vessel in which the inner surface of the reaction vessel is hydrophobic or hydrophilic.

28. A reaction vessel according to any one of claims 1 to 27, A reaction vessel in which the gel layer is made of a polymer gel matrix consisting of inorganic, organic, or a combination of inorganic and organic materials, or a mixture of wax and mineral oil or silicone oil in a weight ratio of 25:75 to 75:25, with or without surfactant additives.

29. A method for carrying out a reaction, A method comprising providing a reaction vessel as defined in claim 1, and delivering a solution from the micropipette tip by penetrating the gel layer with the micropipette tip, thereby causing a reaction.

30. The method according to claim 29, A method wherein the aforementioned solution contains a sample and optionally contains a reaction reagent.

31. The method according to claim 29 or 30, A method comprising melting the gel layer during and after the reaction, and then re-solidifying it.

32. A method according to any one of claims 29 to 31, A method further comprising the steps of disrupting the gel layer using an inert rod or a stoppered micropipette tip, and then using the micropipette tip to penetrate the gel and deliver one or more aqueous reaction mixtures to the reaction vessel.

33. A method according to claim 32, wherein the stopper in the tip is wax or gel.

34. A method according to any one of claims 29 to 33, The reaction vessel comprises a first aqueous reaction mixture below a first gel layer, a second aqueous reaction mixture above the first gel layer, and a second gel layer above the second aqueous reaction mixture. The method comprises a step of mixing the first aqueous reaction mixture and the second aqueous reaction mixture by penetrating the gel layer with an inert rod or micropipette tip, thereby the reaction vessel containing the mixed reaction mixture covered by a third gel layer formed from the first and second gel layers. The method further comprises the step of delivering the solution from the micropipette tip to the mixed reaction mixture by penetrating the third gel layer with the micropipette tip.

35. The method according to claim 34, A method further comprising the step of centrifuging the reaction vessel to facilitate mixing of the reaction mixture between the gel layers.

36. A method according to any one of claims 29 to 35, The method wherein the solution is an aqueous reaction mixture comprising all reaction reagents containing a sample for a reaction to detect a target in a sample, or some reaction reagents for a reaction to detect a target in a sample.

37. A method according to any one of claims 29 to 36, A method comprising delivering the aqueous reaction mixture onto an oil or gel layer to form emulsion droplets or other reaction compartments for a droplet digital PCR reaction or a droplet immunoassay PCR reaction.

38. A method for carrying out a reaction, A method comprising the steps of providing a reaction vessel as defined in claim 1, a solution disposed above and below the gel layer, and displacing or melting the gel layer, thereby mixing the solutions and causing a reaction.

39. A reaction vessel, The reaction vessel comprises a plurality of aqueous reaction mixtures separated from each other by a plurality of gel layers arranged within the reaction vessel, A reaction vessel in which the plurality of aqueous reaction mixtures can be mixed by melting and / or breaking the gel layer in order to carry out a series of processing steps.

40. A reaction vessel according to claim 39, A reaction vessel further comprising a mineral oil layer beneath one or more of the aforementioned gel layers.

41. A reaction vessel according to claim 39 or 40, It is connected to a heater that is slidable along the outer surface of the reaction vessel, A reaction vessel for melting the gel layer adjacent to the heater.

42. A reaction vessel according to any one of claims 39 to 41, A reaction vessel comprising at least one aqueous reaction mixture containing magnetic beads, wherein the reaction vessel is connected to a magnet that slides along the outer surface of the reaction vessel, and moves the magnetic beads through a gel layer beneath the aqueous reaction mixture containing the magnetic beads.

43. A reaction vessel according to any one of claims 39 to 42, A reaction vessel in which the gel is a mixture of paraffin wax and mineral oil or silicone oil in a volume or weight ratio in the range of 99:1 to 25:

75.

44. A reaction vessel according to any one of claims 39 to 43, A reaction vessel comprising the aqueous reaction mixture containing reagents for performing predetermined steps of an integrated immunoassay.

45. A reaction vessel according to any one of claims 39 to 43, A reaction vessel comprising the aqueous reaction mixture containing different reagents for a predetermined bioprocess step and optionally different reagents for a predetermined step of an immunoassay.

46. A reaction vessel according to any one of claims 39 to 45, A reaction vessel in which each of the aqueous reaction mixtures contains one or more of the following for performing a predetermined step of an integrated immunoassay: magnetic beads, capture antibody, washing solution, detection antibody, enzyme, substrate, and stop solution.

47. A reaction vessel according to any one of claims 39 to 46, A reaction vessel in which each of the aqueous reaction mixtures contains one or more of the following for performing a predetermined step of integrated DNA / RNA purification: lysis buffer, DNA / RNA binding solution, magnetic beads, wash buffer, and elution buffer.

48. A reaction vessel according to any one of claims 39 to 47, A reaction vessel in which each of the aqueous reaction mixtures contains one or more of the following for performing a predetermined step of integrated NGS library preparation: a fragmentation enzyme or end repair reagent, a ligation reagent, and a library amplification reagent.

49. A method for carrying out a reaction, A step of providing a reaction vessel according to any one of claims 37 to 46, A method comprising the step of melting one of the gel layers, thereby mixing the aqueous reaction mixture above and below the gel layer to initiate a reaction.

50. The method according to claim 49, A method comprising: connecting the reaction vessel to a heater that is slidable on the outer surface of the reaction vessel, and moving the heater along the surface to a position in which one of the gel layers can be melted.

51. A method according to claim 49 or 50, wherein the heater is part of a module.

52. A method according to claim 50 or 51, wherein the heater is a thermoelectric device.

53. A method according to any one of claims 50 to 52, A method wherein the thermoelectric heating element is a semiconductor-based Peltier element.

54. A method according to any one of claims 50 to 52, A method wherein the module further comprises a cooling element for re-solidifying the gel layer after it has been melted.

55. A method according to any one of claims 50 to 54, A method wherein the module is connected to a magnetic module for moving magnetic beads.

56. A method according to claim 55, wherein the magnet is an electromagnet.

57. A method for carrying out a reaction, The step of providing the reaction vessel according to claim 18 by connecting it to a slidable magnetic module located on the outer surface of the reaction vessel or on the inner surface of the reaction vessel, At least one of the aqueous reaction mixtures includes magnetic beads. A method comprising the step of moving the slidable magnetic module along the outer surface or inner surface to a position where the beads can be moved.

58. The method according to claim 57, A method wherein the slidable magnetic module is connected to one or more modules, including a mechanical module, a thermal module, an electrical module, a magnetic module, and an optical module, to assist the reaction in the reaction vessel and to detect the reaction in the reaction vessel in real time or after the reaction has finished.

59. The method according to claim 57, The reaction vessel is connected to the device, A device for processing a sample or reagent, and a method for detecting chemical or physical changes resulting from a reaction in the reaction vessel, such as color, fluorescence, emission, chemiluminescence, electrochemistry, radiation, reflection, phase change, magnetoresistance, turbidity, mobility shift, pH, and ionic strength.

60. A reaction vessel, A reaction vessel comprising a first gel layer containing a first aqueous reaction mixture, a second gel layer optionally containing a second aqueous reaction mixture, and optionally a third aqueous reaction mixture or sample on the second gel layer, wherein the gel is meltable by heating, thereby mixing the aqueous reaction mixtures.

61. A reaction vessel according to claim 60, A reaction vessel further comprising an oil layer denser than water on top of the second gel layer.

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