Penetrable gel in a reaction vessel

Abstract

The present invention covers and / or separates one or more aqueous reaction mixtures in a reaction vessel using a gel layer. The gel layer can protect a reagent or a reaction product from being in contact with the atmosphere. The gel layer may also separate a plurality of aqueous reaction mixtures such that they do not contact each other before the gel layer breaks. The oil layer can be directly inserted under the gel layer. By inserting the tip of the micropipettor, a solution (such as a sample) can be delivered below the gel layer through the gel layer. A micropipette tip or inert rod may also be used to break the gel layer resulting in mixing of the aqueous reaction mixture above and below the layer. The gel layer may also be ruptured by selectively applying heat or moving the magnetic beads. A plurality of gel layers separating a plurality of aqueous reagent mixtures may be ruptured in a prescribed order to perform a series of ordered biological treatment steps.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480032830.4 (22) Application Date 2024.05.15 (30) Priority Data 63 / 466,982 2023.05.16 US (85) PCT International Application Entering National Phase Date 2025.11.14 (86) PCT International Application Application Data PCT / US2024 / 029451 2024.05.15 (87) PCT International Application Publication Data WO2024 / 238645 EN 2024.11.21 (71) Applicant: Attila Biosystems, Inc. Address: Palo Alto, California, USA (72) Inventors: Shi Yining, Wang Youxiang, Wang Rong, Yang Zhijie, Chen Xin, Li Jialuo, Jiang Bo, Chen Shuqin, Soma Chodari, Chen Yifan, Li Bin (74) Patent Agency: Guangzhou Sanhuan Patent & Trademark Agency Co., Ltd., 44202 Patent Attorney: Hao Chuanxin (51) Int.Cl. B01L 3 / 00 (2006.01) C08J 7 / 04 (2020.01) C08L 91 / 06 (2006.01) (54) Invention Title: Permeable Gel in a Reaction Vessel (57) Abstract: This invention uses a gel layer to cover and / or separate one or more aqueous reaction mixtures in a reaction vessel. The gel layer can protect reagents or reaction products from contact with the atmosphere. The gel layer can also separate multiple aqueous reaction mixtures so that they do not come into contact with each other before the gel layer breaks. An oil layer can be inserted directly below the gel layer. A solution (such as a sample) can be delivered below the gel layer by inserting a pipette tip into a micropipette. A micropipette tip or inert rod can also be used to disrupt the gel layer, causing mixing of the aqueous reaction mixture above and below the layer. The gel layer can also be broken by selectively applying heat or moving magnetic beads. Multiple gel layers separating multiple aqueous reagent mixtures can be broken in a prescribed sequence for a series of ordered biological processing steps. Claims 4 pages, Description 39 pages, Drawings 17 pages, CN 121219073 A 2025.12.26 CN 1 21 21 90 73 A 1. A reaction vessel comprising a gel layer that can be penetrated by a micropipette tip disposed within the vessel. 2. The reaction vessel of claim 1, further comprising one or more additives in the gel layer and mineral oil below the gel layer. 3. The reaction vessel according to claim 1 or 2, further comprising a liquid aqueous reaction mixture above and / or below and / or inside the gel layer, optionally wherein one or more of the liquid aqueous reaction mixtures are used for detecting the analyte.4. The reaction vessel of claim 3, wherein the liquid aqueous reaction mixture is located below the gel layer, the gel layer preventing the liquid aqueous reaction mixture from diffusing through the gel layer, and the gel layer being permeable by a micropipette tip to deliver a solution to the reaction mixture for reaction to detect an analyte. 5. The reaction vessel of claim 3, wherein the delivered solution is a sample comprising the analyte. 6. The reaction vessel of claim 5, wherein the gel layer prevents the liquid aqueous reaction mixture from diffusing through the gel layer and is permeable by a micropipette tip at least in the temperature range of 18-35°C, optionally 20-25°C. 7. The reaction vessel of any one of claims 1 to 6, wherein the reaction vessel comprises a plurality of gel layers, a plurality of oil layers, and a plurality of liquid aqueous reaction mixture layers, the number of gel layers being different from the number of oil layers and / or the number of liquid aqueous reaction mixture layers. 8. The reaction vessel of any one of claims 1 to 7, wherein the micropipette comprises a single tip or a plurality of tips or a bundle of capillaries. 9. A reaction vessel according to any one of the preceding claims, comprising: a first aqueous liquid reaction mixture; a first gel layer permeable to a micropipette tip covering the first aqueous liquid reaction mixture; a second aqueous liquid reaction mixture above the first gel layer; and a second gel layer permeable to a micropipette tip covering the second aqueous liquid solution; and optionally, a first mineral oil layer between the first aqueous liquid reaction mixture and the first gel layer, and optionally, a second mineral oil layer between the second aqueous liquid reaction mixture and the second gel layer. 10. The reaction vessel of claim 9, wherein one of the first and second aqueous liquid reaction mixtures is a sample, and the other comprises one or more reagents for reacting when the first and second aqueous liquid reaction mixtures are combined to detect an analyte in the sample. 11. The reaction vessel of claim 9, wherein the first aqueous liquid reaction mixture and the second aqueous liquid reaction mixture are each a partial reaction mixture that, when combined with each other and the sample, produces a detection reaction. 12. The reaction vessel of claim 9, wherein optionally, a third aqueous liquid reaction mixture is present above the second gel layer, and the plurality of aqueous liquid reaction mixtures comprise reagents for performing ordered biological treatment steps. 13. The reaction vessel according to claim 12, wherein the plurality of liquid aqueous reaction mixtures are used for nucleic acid sequencing library preparation or nucleic acid sample extraction or immunoassay, or biochemical or nucleic acid amplification and detection.14. The reaction vessel of claim 12, wherein the first and second liquid aqueous reaction mixtures 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. 15. The reaction vessel of claim 12, wherein the first and second liquid aqueous reaction mixtures contain different reagents, which, when combined with each other and the sample, are necessary for the detection reaction. 16. The reaction vessel of claim 15, wherein the combination of the first and second liquid aqueous reaction mixtures is used for immunoassay and detection, optionally, a third liquid aqueous reaction mixture is located above the second gel layer for sequential reaction. 17. The reaction vessel of claim 3, wherein the liquid aqueous reaction mixture contains magnetic beads. 18. The reaction vessel of any of the preceding claims, connected to a heater module slidable along the exterior of the reaction vessel for melting the gel layer when the heater is near the gel layer. 19. A reaction vessel according to any one of the preceding claims, wherein a magnetic module is connected to a slidable magnetic module along an outer or inner surface of the reaction vessel for moving magnetic beads from a liquid aqueous reaction mixture through a gel layer beneath the liquid aqueous reaction mixture. 20. A reaction vessel according to claim 19, wherein the slidable magnetic module is slidable along the interior of the reaction vessel and connected to a rod for detecting a reaction in the reaction vessel. 21. A reaction vessel according to claim 3, wherein the liquid aqueous reaction mixture includes a rod for detecting a reaction in the reaction vessel. 22. A reaction vessel according to claim 21, wherein the rod is an electrode. 23. A reaction vessel according to any one of the preceding claims, wherein a well in a multi-well plate (e.g., a PCR plate, a deep-well plate, a microtiter plate, or an ELISA plate) contains a gel layer in each well. 24. A reaction vessel according to any one of claims 1 to 22, wherein a tube or tubing (e.g., a PCR tube) is individually arranged or arranged as strips or a matrix, comprising a plurality of interconnected vessels. 25. A reaction vessel according to any one of claims 1 to 22, wherein it is made of plastic, glass, or metal having one or more grooves or channels. 26. The reaction vessel according to any one of claims 1 to 23, wherein it is a hole on a chip or tube (e.g., a glass slide, silicon wafer, plastic slide, or metal slide), optionally having a plurality of holes, providing a plurality of reaction vessels, optionally the plurality of holes being connected by channels, optionally the holes comprising a membrane or glass fiber.27. The reaction vessel according to any one of the preceding claims, wherein the inner surface of the reaction vessel is hydrophobic or hydrophilic. 28. The reaction vessel according to any one of the preceding claims, wherein the gel layer is made of an inorganic or organic or a combination of inorganic and organic polymer gel matrices, 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 of carrying out a reaction comprising providing a reaction vessel as defined in claim 1 and penetrating the gel layer with a micropipette tip to deliver a solution from the micropipette tip, thereby causing the reaction to occur. 30. The method of claim 29, wherein the solution comprises a sample and optionally a reaction reagent. 31. The method of claim 29 or 30, wherein the gel layer melts during and after the reaction and then re-solidifies. 32. The method of any one of claims 29 to 31, further comprising breaking up the gel layer with an inert rod or a micropipette tip with a stopper thereon before penetrating the gel with the micropipette tip to deliver one or more liquid aqueous reaction mixtures to the reaction vessel. 33. The method of claim 32, wherein the stopper in the pipette tip is wax or gel. 34. The method of any one of claims 29 to 33, wherein the reaction vessel comprises a first liquid aqueous reaction mixture below a first gel layer and a second liquid aqueous reaction mixture above the first gel layer, and a second gel layer above the second liquid aqueous reaction mixture, and the method comprises penetrating the gel layer with an inert rod or micropipette tip to mix the first liquid aqueous reaction mixture and the second liquid aqueous reaction mixture, wherein the reaction vessel comprises a mixed reaction mixture covered by a third gel layer formed of the first and second gel layers, and the method further comprises penetrating the third gel layer with the micropipette tip to deliver the solution from the micropipette tip to the mixed reaction mixture. 35. The method of claim 34, further comprising centrifuging the reaction vessel to promote mixing of the reaction mixture between the gel layers. 36. The method of any one of claims 29 to 35, wherein the solution is a liquid aqueous reaction mixture comprising all reaction reagents, including a sample for detecting a reaction of a target in the sample or a portion of the reaction reagents for detecting a reaction of a target in the sample. 37. The method according to any one of claims 29 to 36, wherein the liquid aqueous reaction mixture is delivered into an oil layer or a gel layer to form emulsion droplets or other reaction compartments for droplet digital PCR reactions or droplet immunoassay PCR reactions.38. A method of carrying out a reaction, comprising providing a reaction vessel as defined in claim 1 and a solution disposed above and below the gel layer, and moving or melting the gel layer, whereby the solution mixes and reacts. 39. A reaction vessel comprising a plurality of liquid aqueous reaction mixtures separated from each other by a plurality of gel layers disposed within the reaction vessel, wherein the plurality of liquid aqueous reaction mixtures can be combined by melting and / or breaking the gel layers to sequentially perform a series of processing steps. 40. The reaction vessel of claim 39, further comprising a mineral oil layer located below one or more of the gel layers. 41. The reaction vessel of claim 39 or 40, connected to a heater slidable along an outer surface of the reaction vessel for melting a gel layer adjacent to the heater. 42. The reaction vessel of any one of claims 39 to 41, wherein at least one liquid aqueous reaction mixture comprises magnetic beads, and the reaction vessel is connected to a magnet slidable along an outer surface of the reaction vessel for moving the magnetic beads through a gel layer containing the magnetic beads. 43. The reaction vessel according to any one of claims 39 to 42, wherein the gel is a mixture of paraffin and mineral oil or silicone oil in a volume or weight ratio ranging from 99:1 to 25:75. 44. The reaction vessel according to any one of claims 39 to 43, wherein the liquid aqueous reaction mixture comprises reagents for performing ordered steps in an integrated immunoassay. 45. The reaction vessel according to any one of claims 39 to 43, wherein the liquid aqueous reaction mixture comprises various reagents for ordered bioprocessing steps, optionally for ordered steps in an immunoassay. 46. The reaction vessel according to any one of claims 39 to 45, wherein each of the liquid aqueous reaction mixtures comprises one or more of magnetic beads, capture antibodies, washing solutions, detection antibodies, enzymes, substrates, and termination solutions for performing ordered steps in an integrated immunoassay. 47. The reaction vessel according to any one of claims 39 to 46, wherein each of the liquid aqueous reaction mixtures comprises one or more of lysis buffer, DNA / RNA binding solution, magnetic beads, washing buffer, and elution buffer for performing ordered steps in an integrated DNA / RNA purification process. 48. The reaction vessel according to any one of claims 39 to 47, wherein each of the liquid aqueous reaction mixtures comprises one or more of a fragmentation enzyme or end-repair reagent, a ligation reagent, and a library amplification reagent, for performing ordered steps of integrated NGS library preparation.49. A method of carrying out a reaction, comprising providing a reaction vessel according to any one of claims 37 to 46; melting one of the gel layers to incorporate a liquid aqueous reaction mixture above and below the layer to initiate a reaction. 50. The method of claim 49, wherein the reaction vessel is connected to a heater slidable on an outer surface of the reaction vessel, and the heater is moved along the surface to a position where it can melt one of the gel layers. 51. The method of claim 49 or 50, wherein the heater is part of a module. 52. The method of claim 50 or 51, wherein the heater is a thermoelectric device. 53. The method of any one of claims 50 to 52, wherein the thermoelectric heating element is a semiconductor-based Peltier device. 54. The method of any one of claims 50 to 52, wherein the module further comprises a cooling element for re-solidifying the gel layer after melting. 55. The method of any one of claims 50 to 54, wherein the module is connected to a magnetic module for moving magnetic beads. 56. The method of claim 55, wherein the magnet is an electromagnet. 57. A method of carrying out a reaction, comprising providing a reaction vessel of claim 18, a slidable magnetic module connected to an outer surface or an inner surface of the reaction vessel, wherein at least one of the liquid aqueous reaction mixtures comprises magnetic beads, and moving the slidable magnetic module along the outer or inner surface to a position where it can move the magnetic beads. 58. The method of claim 57, wherein the slidable magnetic module is connected to one or more modules of the following: a mechanical module, a heating module, an electrical module, a magnetic module, 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 is completed. 59. The method of claim 57, wherein the reaction vessel is connected to a device for processing a sample or reagent and detecting a chemical or physical change caused by the reaction in the reaction vessel, such change being, for example, color, fluorescence, luminescence, chemiluminescence, electrochemistry, radiation, reflection, phase transition, magnetoresistance, turbidity, mobility shift, pH, or ionic strength. 60. A reaction vessel comprising a first gel layer, optionally a second gel layer, and optionally a third aqueous reaction mixture or sample on top of the second gel layer, the first gel layer comprising a first aqueous reaction mixture, the second gel layer comprising a second aqueous reaction mixture, the gel being meltable upon heating to mix the aqueous reaction mixture. 61. The reaction vessel of claim 60, further comprising an oil layer with a density greater than water above the second gel layer.Claims 4 / 4 Page 5 CN 121219073 A Cross-Reference to Related Applications for Permeable Gel in Reaction Vessel

[0001] This application claims the benefit of U.S. Application No. 63 / 466,982, filed May 16, 2023, which is incorporated herein by reference in its entirety for all purposes. Background Art

[0002] Amplification reactions, such as polymerase chain reaction (PCR), and some other commonly performed laboratory assays (such as immunoassays) involve multiple aqueous reaction mixtures that are separated from each other until the predetermined step of the assay is performed. Separation can be achieved by using separate containers containing stock of each aqueous reaction mixture in a kit, which are then dispensed by the experimenter into the appropriate reaction vessel. However, this approach carries the risk of contaminating the stock aqueous reaction mixtures and provides opportunities for human error in preparing individual assay reactions (e.g., forgetting to add the reaction mixture to the sample, adding the mixture twice, and compromising the necessary ratio / concentration of the reagents, causing contamination before, during, or after the reaction). Even without errors, these methods still require significant time and effort when preparing reactions for large-scale experiments, such as 96-well or 384-well PCR plate reactions. Therefore, when multiple reagent subsets (e.g., primer subsets and enzyme subsets) and / or multiple steps (e.g., RT-PCR) are involved, there is a need to simplify the procedures for performing PCR assays and reduce the cold chain involvement in the transport of reagent products to the end user. Therefore, reagent products provided in ready-to-use or “add-sample-only” form, and transported at ambient temperature, are ideal, thereby eliminating human procedural errors, improving assay speed and accuracy, and saving transportation costs without affecting reagent performance.

[0003] Erroneous results due to laboratory contamination before, during, or after the reaction have always been a concern. Several solutions have been proposed to address this issue, particularly for PCR reactions. For example, Johnson et al. described the use of lyophilized particles of reagent materials (see, for example, U.S. Patent Nos. 10,144,954 and 11,098,344). One of the inherent problems with lyophilization or freeze-drying is maintaining sufficiently low moisture levels in lyophilized reagents to ensure functionality during reconstitution. Johnson reports that, for this purpose, the lyophilized reagent components are coated or impregnated with a wax component. However, the wax needs to be heated to a certain temperature in a thermal cycler to release the lyophilized reagent into the aqueous components of the PCR reaction added by the experimenter. While acceptable for standard thermal cycler-based PCR methods with a high-temperature denaturation step, newer isothermal PCR methods may not reach sufficient temperatures to release the lyophilized reagent and facilitate its remodeling.Recent attempts to optimize amplification reactions have sought to select the best properties of existing optimization techniques and combine them into a ready-to-use form that only requires the addition of samples (see, for example, WO 2022 / 026670). While these approaches appear to reduce some problematic aspects of PCR reactions, including extensive sample preparation and time-consuming and error-prone steps, as described above, they ultimately rely on proprietary and dedicated equipment for specific point-of-care assays using defined reaction cassettes.

[0004] Early attempts to optimize PCR reactions also used waxes and other immiscible hydrophobic substances, such as mineral oil and silicone oil or various inert greases, primarily for two purposes: (1) to prevent PCR mixtures from evaporating during thermal cycling to ensure consistent reaction volumes and fidelity between reactions; and (2) to avoid nonspecific PCR products resulting from primer misbinding (e.g., primer dimers and oligomerization) when mixing PCR reagents at ambient temperatures, as such events can occur at temperatures far below the thermal cycling temperatures during PCR reactions (US Patent No. 5,411,876). This patent reports the use of grease or wax to contain two subsets of PCR reagents in a separated manner, thereby improving the shelf life of PCR reagents and increasing protection of the laboratory environment by preventing PCR product contamination. The improvement in PCR is achieved by changing the mixing method of the PCR reagents: Before a heating step in the PCR amplification specification (page 1 / 39, 6 CN 121219073 A) melts and liquefies the grease or wax barrier separating the reagent subsets, mixing of the reagent subsets separated by the grease or wax does not occur. When the grease or wax melts into a liquid lighter than water in the first amplification cycle, the melted grease or wax and the aqueous layer displace each other, resulting in convective mixing of the aqueous reagents that are now combined and subsequent amplification reactions. However, in terms of target product yield and the amount of nonspecific primer dimers, even with improved wax layering using tubes with hydrophilic surfaces, as described in Example 5 of US 5,411,876, PCR products comparable to those of the mineral oil control could not be produced: 1) three of the fifteen reactions using the wax form showed significantly less specific product than the other reactions, while all six oil controls showed slightly higher specific product yields than the wax form; and 2) the primer dimers produced by the wax were slightly larger than those produced by the oil, and the primer dimer yields were slightly higher than those produced by the oil.

[0005] US Patent 5,576,197 reports a PCR container preloaded with wax, which is attached to an inner surface located at or above the estimated meniscus position of the PCR mixture. The wax melts at PCR temperature and covers the surface of the PCR mixture, thereby preventing evaporation during thermal cycling.According to the patent, other similar methods, such as small solid wax balls and microtubes with solid wax at the bottom, have the following main drawbacks: for the former, loading individual wax balls into each reaction tube is prone to contamination, cumbersome, and time-consuming; for the latter, the wax and PCR mixture must be phased by centrifugation, and incomplete phase inversion is problematic and difficult to resolve. Although US 5,576,197 attempts to address the above problems, it is impractical to mass-produce microtubes containing wax precisely positioned within the microtube in good yield. This manufacturing challenge is further exacerbated when small volumes (e.g., 0.1 mL) of microtubes are involved. Furthermore, the resulting microtubes are user-unfriendly because loading the reaction mixture through the small lumen of the wax tube container is cumbersome, and wax clumps may clog pipette tips or detach from the inner wall of the tube due to inaccurate pipette positioning.

[0006] Moreover, many of these methods simply reintroduce the disadvantages of standard PCR in different aspects of the reaction process, namely, the risk of contamination from applying inert oil or grease to multiple samples is now present, rather than the risk of contaminating the stock of reagents in the reaction mixture. Adding these components is both time-consuming and cumbersome because it requires pipetting oily substances. Finally, these methods are also unsuitable for isothermal amplification reactions because they rely on a high-temperature initial step 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, frequent infection testing of the population. To address this challenge, Batas et al. described a wax-layered reagent system approaching the "just add sample" concept for point-of-care diagnostic testing, which is thermally stable during transport and requires no cold chain packaging (Batas et al. Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2, Viruses 2021, 13, 742). In this wax reagent system, multiple layers of paraffin and silicone wax are used to separate and package all RT-LAMP (reverse transcription loop-mediated isothermal amplification) reagent components in a single PCR tube. While the resulting wax-layered reagent was found to be stable under room temperature transport conditions and usable for testing clinical COVID-19 samples, weak fluorescence was observed in some samples. More importantly, the negative predictive value and positive predictive value were only 60% and 74%, respectively, significantly lower than those of freshly prepared liquid reagents. As the authors note, these poor performance findings may be attributed to the lack of uniform mixing of all reagent components due to the absence of pipetting. Another significant drawback of this wax-layered system is the requirement for multiple manual steps, including offline wax melting and vortexing, before it can be loaded onto a thermal instrument for isothermal reaction.

[0008] To control carryover contamination in amplification reactions, an alternative to physical barriers (such as mineral oil or paraffin) is the use of uracil DNA glycosylase in PCR, where dTTP is replaced by dUTP to produce uracil-containing DNA (U-DNA). Prior to PCR amplification, the subsequent PCR reaction mixture is treated with uracil DNA glycosylase (UNG), followed by cleavage of uracil-free polynucleotides at high temperatures (e.g., 95°C) under alkaline conditions (during the initial denaturation step), which removes contaminating U-DNA from the sample. (Longo et al., Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reaction. Specification 2 / 39, page 7, CN 121219073, A reaction.Gene). In other words, the presence of dUTP in the DNA distinguishes the PCR product (i.e., the amplicon) from the native DNA template. Because the native template lacks dUTP, it remains intact in the presence of the enzyme. However, any amplicon DNA containing dUTP is susceptible to enzymatic cleavage.

[0009] However, in order to be effective as a strategy, the UNG method requires all PCR reactions in the laboratory to be performed using dUTP instead of dTTP, which may be impractical in a clinical setting. In addition, this technique does not always completely eliminate contaminants, especially when the PCR product length is short, which is common in real-time PCR assays. Furthermore, especially when the initial sample contains only one or a few target molecules, the addition of UNG may reduce amplification efficiency, thereby delaying or preventing the detection of the target (Pierce and Wangh, Effectiveness and limitations of uracil-DNA glycosylases in sensitive real-time PCR assays, Biotechniques January 2004; 36(1): 44-6,48). Other disadvantages of this strategy compared to conventional PCR techniques may include the additional material costs and cumbersome assay development work involved in suppressing contaminants using this chemical-based method.

[0010] In view of the above, there is a need for a ready-to-use compartmentalized reaction vessel with performance comparable to that of liquid forms using conventional PCR tubes or ELISA microtiter plates, exhibiting thermal and mechanical stability during transport at ambient or sub-ambient temperatures, a long shelf life at freezing (or solidification) temperatures or at ambient or sub-ambient storage temperatures, and the ability to form a non-flowing seal within the vessel after the reaction to prevent carryover contamination of one or more reaction products. The present invention provides these and other features, individually or in combination, as will become apparent from the following description. Summary of the Invention

[0011] The present invention provides a reaction vessel comprising a gel layer permeable by a micropipette tip disposed within the vessel. Optionally, the reaction vessel further comprises one or more additives in the gel layer and mineral oil beneath the gel layer. Optionally, the reaction vessel further comprises a liquid aqueous reaction mixture above and / or below and / or inside the gel layer. Optionally, one or more liquid aqueous reaction mixtures are used for the detection of an analyte. Optionally, the liquid aqueous reaction mixture is located beneath the gel layer, which prevents the liquid aqueous reaction mixture from diffusing through the gel layer. The gel layer can be penetrated by a micropipette tip to deliver a solution to the reaction mixture for reaction to detect the analyte. Optionally, the delivered solution is a sample containing the analyte. Optionally, the gel layer prevents the liquid aqueous reaction mixture from diffusing through the gel layer and can be penetrated by a micropipette tip at least within a temperature range of 18–35°C (e.g., 20–35°C or 20–25°C). Optionally, the reaction vessel comprises multiple gel layers, multiple oil layers, and multiple layers of liquid aqueous reaction mixture, the number of gel layers differing from the number of oil layers and / or the number of liquid aqueous reaction mixture layers. Optionally, the micropipette comprises a single tip or multiple tips or a bundle of capillaries.

[0012] Optionally, the reaction vessel includes a first aqueous liquid reaction mixture, a first gel layer permeable to a micropipette tip covering the first aqueous liquid reaction mixture, a second aqueous liquid reaction mixture above the first gel layer, and a second gel layer permeable to a micropipette tip covering the second aqueous liquid solution, and optionally, a first mineral oil layer between the first aqueous liquid reaction mixture and the first gel layer, and optionally, a second mineral oil layer between the second aqueous liquid reaction mixture and the second gel layer. Optionally, one of the first and second aqueous liquid reaction mixtures is a sample, and the other contains one or more reagents for reacting when the first and second aqueous liquid reaction mixtures are combined to detect an analyte in the sample. Optionally, the first and second aqueous liquid reaction mixtures are each partial reaction mixtures that produce a detection reaction when combined with each other and the sample. Optionally, a third aqueous liquid reaction mixture is present above the second gel layer, and the plurality of aqueous liquid reaction mixtures contain reagents for performing ordered biological processing steps.Optionally, multiple liquid aqueous reaction mixtures are used for nucleic acid sequencing library preparation or nucleic acid sample extraction or immunoassay, or biochemical or nucleic acid amplification and detection. Optionally, the first liquid aqueous reaction mixture and the second liquid aqueous reaction mixture each contain polymerase and primers, or vice versa, for nucleic acid detection by PCR amplification, qPCR amplification, reverse transcriptase PCR reaction, or digital PCR amplification or isothermal amplification. Optionally, the first liquid aqueous reaction mixture and the second liquid aqueous reaction mixture contain different reagents, which are necessary for the detection reaction when combined with each other and the sample.

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

[0014] Optionally, the reaction vessel is connected to a heater module that slides along the outside of the reaction vessel for melting the gel layer when the heater is close to the gel layer. Optionally, the reaction vessel is connected to a magnetic module slidable along the outer or inner surface of the reaction vessel for moving magnetic beads from the liquid aqueous reaction mixture through a gel layer beneath the liquid aqueous reaction mixture. Optionally, the slidable magnetic module is slidable along the interior of the reaction vessel and connected to a rod for detecting the reaction in the reaction vessel.

[0015] Optionally, the liquid aqueous reaction mixture includes 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, a deep-well plate, a microtiter plate, or an ELISA plate), each well containing a gel layer. Optionally, the reaction vessel is a tube or tubing (e.g., a PCR tube) arranged individually or as strips or a matrix, comprising multiple interconnected containers. Optionally, the reaction vessel is made of plastic, glass, or metal having one or more grooves or channels.

[0016] Optionally, the reaction vessel is a hole in a chip or tube (e.g., a glass slide, silicon wafer, plastic substrate, or metal substrate), optionally having multiple holes, providing multiple reaction vessels, optionally the multiple holes are connected by channels, optionally these holes contain a membrane or glass fiber.

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

[0018] Optionally, the gel layer is made of an inorganic or organic or inorganic and organic polymer gel matrix, 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 also provides a method of carrying out a reaction comprising providing a reaction vessel as defined above and penetrating the gel layer with a micropipette tip to deliver a solution from the micropipette tip, thereby causing a reaction. 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 includes breaking the gel layer with an inert rod or a micropipette tip with a stopper thereon before penetrating the gel with a micropipette tip to deliver one or more liquid aqueous reaction mixtures to the reaction vessel. Optionally, the stopper in the tip is wax or gel. Optionally, the reaction vessel includes a first liquid aqueous reaction mixture below the first gel layer and a second liquid aqueous reaction mixture above the first gel layer, and a second gel layer above the second liquid aqueous reaction mixture, and the method includes penetrating the gel layer with an inert rod or a micropipette tip to mix the first liquid aqueous reaction mixture and the second liquid aqueous reaction mixture, whereby the reaction vessel includes a mixed reaction mixture covered by a third gel layer formed of the first and second gel layers, and the method further includes penetrating the third gel layer with a micropipette tip to deliver a solution from the micropipette tip to the mixed reaction mixture. Optionally, the method further includes centrifuging the reaction vessel to promote mixing of the reaction mixtures between the gel layers. Optionally, the solution is a liquid aqueous reaction mixture containing all reaction reagents, including a sample for detecting a target in a sample or a portion of the reaction reagents for detecting a target in a sample (i.e., the sample and a portion of the reaction reagents are used for the reaction, which is a reaction to detect a target in a sample). Optionally, the liquid aqueous reaction mixture is delivered into an oil layer or a gel layer to form an emulsion droplet or other reaction compartment for droplet digital PCR reaction or droplet immunoassay PCR reaction as described on page 4 / 39 of the specification, CN 121219073 A.

[0020] The present invention also provides a method of carrying out a reaction comprising providing a reaction vessel as defined in claim 1 and a solution placed above and below a gel layer, and moving or melting the gel layer, whereby the solution is mixed and a reaction occurs.

[0021] The present invention also provides a reaction vessel comprising a plurality of liquid aqueous reaction mixtures separated from each other by a plurality of gel layers disposed within the reaction vessel, wherein the plurality of liquid aqueous reaction mixtures may be combined by melting and / or breaking the gel layers to sequentially perform a series of processing steps. Optionally, the reaction vessel further comprises a mineral oil layer located below one or more gel layers. Optionally, the reaction vessel is connected to a heater that can slide along the outer surface of the reaction vessel for melting a gel layer adjacent to the heater. Optionally, at least one liquid aqueous reaction mixture includes magnetic beads, and the reaction vessel is connected to a magnet that can slide along the outer surface of the reaction vessel for moving the magnetic beads through a gel layer beneath the liquid 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 ranging from 99:1 to 25:75. Optionally, the liquid aqueous reaction mixture contains reagents for performing ordered steps of an integrated immunoassay.Optionally, the liquid aqueous reaction mixture contains different reagents for ordered biological processing steps, optionally for ordered steps in an immunoassay. Optionally, each liquid aqueous reaction mixture contains one or more of magnetic beads, capture antibodies, washing solutions, detection antibodies, enzymes, substrates, and termination solutions for performing ordered steps in an integrated immunoassay. Optionally, each liquid aqueous reaction mixture contains one or more of lysis buffer, DNA / RNA binding solution, magnetic beads, washing buffer, and elution buffer for performing ordered steps in an integrated DNA / RNA purification process. Optionally, each liquid aqueous reaction mixture contains one or more of fragmentation enzymes or end-repair reagents, ligation reagents, and library amplification reagents for performing ordered steps in an integrated NGS library preparation process.

[0022] The present invention also provides a method of carrying out a reaction comprising providing a reaction vessel as described above, melting one of the gel layers to incorporate liquid aqueous reaction mixtures above and below the layer to initiate a reaction. Optionally, the reaction vessel is connected to a heater that can slide on an outer surface of the reaction vessel, and the heater is moved along the surface to a position where it can melt one of the gel layers. 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 device. Optionally, the module further includes 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 also provides a method of carrying out a reaction comprising providing a reaction vessel as described above, the reaction vessel being connected to a slidable magnetic module on an outer surface or an inner surface of the reaction vessel, wherein at least one liquid aqueous reaction mixture contains magnetic beads, and moving the slidable magnetic module along the outer or inner surface to a position where it can move the magnetic beads. Optionally, the slidable magnetic module is connected to one or more of the following modules: a mechanical module, a heating module, an electrical module, a magnetic module, 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 is completed. Optionally, the reaction vessel is connected to a means for processing samples or reagents and detecting chemical or physical changes (e.g., color, fluorescence, luminescence, chemiluminescence, electrochemistry, radiation, reflection, phase transition, magnetoresistance, turbidity, mobility shift, pH, or ionic strength) caused by the reaction in the reaction vessel.

[0024] The present invention also provides a reaction vessel comprising a first gel layer, and optionally a second gel layer, and optionally a third aqueous reaction mixture or sample on top of the second gel layer, the first gel layer comprising a first aqueous reaction mixture, the second gel layer comprising a second aqueous reaction mixture, the gel being fusible upon heating to mix the aqueous reaction mixture. Optionally, the reaction vessel further includes an oil layer with a density greater than water above the second gel layer.

[0025] Figure 1: Reagent-free monolayer reaction tube with preloaded gel layer. 1. Tube, 2. Gel layer. Specification 5 / 39 pages 10 CN 121219073 A

[0026] Figure 2: Reagent-free bilayer reaction tube with preloaded gel layer. 1. Tube, 2. Gel layer, 3. Mineral oil.

[0027] Figure 3: Reagent-containing six-layer reaction tube with preloaded 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: Reagent-containing three-layer reaction tube with preloaded gel layer. 1. Tube, 2. Gel layer, 3. Mineral oil, 4. Aqueous reaction mixture.

[0029] Figure 5: Integrated ELISA reaction vessel system based on magnetic beads, comprising a multi-compartment reagent with a permeable gel layer as a separation barrier, and a movable external heating element and magnetic sheet. Arrows indicate the direction of heating and magnetic strip passage 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 capture antibodies 7. Second gel layer 8. Washing chamber I 9. Third gel layer 10. Chamber for detecting antibody-antigen binding 11. Fourth gel layer 12. Washing chamber II 13. Fifth gel layer 14. Reaction product detection chamber 15. Sixth gel layer 16. Termination solution chamber

[0030] Figure 6: Integrated nucleic acid purification system based on magnetic beads, comprising a multi-compartment reagent with a thermosensitive gel layer, and a movable external heating / cooling module and magnetic module. Arrows indicate the direction of passage of the heating module and magnetic module 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. Pyrolysis chamber 7. Second gel layer 8. Magnetic bead chamber 9. Magnetic bead 10. Third gel layer 11. Washing chamber I 12. Fourth gel layer Instruction manual 6 / 39 pages 11 CN 121219073 A 13. Washing chamber II 14. Fifth gel layer 15. Washing chamber III 16. Sixth gel layer 17. Elution chamber

[0031] Figure 7: Integrated reaction vessel system containing preloaded multi-compartment reagents 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. Connecting chamber 8. Third gel layer 9. PCR amplification chamber 10. Second heating / cooling element

[0032] Figure 8: Representative PCR amplification curves of three reaction tubes: i) liquid form (—); ii) single gel layer form (—--); and iii) bilayer form with gel and mineral oil layers (—·—).

[0033] Figure 9: Photographs of the three reaction tube types before and after thermal cycling.

[0034] Figures 10A-C: 4-color amplification curves for three tube formats: A) bilayer format; B) single gel format and C) liquid format.

[0035] Figure 11: Amplification curves of PCR reactions using multilayer reagents.

[0036] Figure 12: Photographs of multilayer reagent strips before and after thermal cycling.

[0037] Figures 13A-D: 4-color amplification curves of multilayer UTI reagent tubes (black) and liquid form tubes (gray), where (A) FAM channel, (B) HEX channel, (C) ROX channel and (D) Cy5 channel.

[0038] Figure 14: Photographs of multilayer reagent strips before (top) and after (bottom) isothermal amplification at 60°C for 90 minutes.

[0039] Figure 15: Amplification curves (FAM channel) of reactions using HPV31 reagent strips (black) and liquid strips (gray).

[0040] Figures 16A, B: Free fall experiments of multilayer reagent tubes. Twelve tubes were allowed to be dropped freely from a height of approximately 10 feet onto the laboratory floor three times. (A) Before the drop and (B) After the drop.

[0041] Figures 17A, B: Two-color amplification curves (gray-FAM channel; black-ROX channel) of two types of layered tubes after repeated freeze / thaw cycles and long-term transport at ambient temperature. (A) 5-layer tube with no mineral oil layer on top of the bottom enzyme mixture layer; (B) 6-layer tube with a mineral oil layer on top of the bottom enzyme mixture layer. Detailed Definitions

[0042] Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood in the art to which this invention pertains. The following definitions are supplementary to the definitions in the art and are specific to this application and should not be extrapolated to any related or unrelated circumstances, such as any co-owned patents or applications. Although any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. Belonging to an entity “a or an” means one or more of that entity; for example, “nucleic acid” means one or more nucleic acids. Therefore, the terms “a or an,” “one or more,” and “at least one” are used interchangeably herein.

[0043] The term “nucleic acid” encompasses any physical monomer unit string that may correspond to a nucleotide string, including nucleotide polymers (e.g., typical DNA or RNA polymers), peptide nucleic acids (PNAs), modified oligonucleotides (e.g., oligonucleotides containing atypical bases of biological RNA or DNA in solution, such as 2'-O-methylated oligonucleotides), etc. Nucleic acids include DNA, RNA, and any combination thereof. Nucleic acids can be double-stranded or single-stranded.DNA can be genomic, cDNA, methylated DNA, or synthetic DNA, etc. RNA can be mRNA, miRNA, tRNA, rRNA, hnRNA, or methylated RNA, etc.

[0044] The four common nucleotide bases are A, T / U, C, and G, where T is present in DNA and U is present in RNA. The nucleotides found in the target are usually natural nucleotides (deoxyribonucleotides or ribonucleotides). The same is true for the nucleotides that form primers.

[0045] The complementarity of nucleic acid strands means that these strands form a stable double helix due to hydrogen bonds between their nucleobases. The complementary bases in DNA are A and T and C and G, while in RNA they are C and G and U and A. When the strands are maximally aligned, the nucleotides in each strand form one of these (Watson-Crick pair), and they are complementary. When the respective strands are maximally aligned, the nucleotides do not form complementary pairs, and the nucleotides are mismatched. The complementarity of the strands can be complete or basic. Perfect complementarity between two strands means that the two strands can form a double helix, in which each base in the double helix binds to a complementary base via Watson-Crick pairing. Basic complementarity means that under a set of hybridization conditions (such as salt concentration and temperature), most, but not all, bases in the strands form Watson-Crick pairs, thus forming a stable hybridization complex. For example, despite the presence of up to one, two, or three mismatches, some primers can form a double helix with their primer-binding sites, provided that these mismatches are not at the 3' end, preferably not nearby (e.g., within four nucleotides). These conditions can be predicted by using sequence analysis and standard mathematical calculations, or by empirically determining Tm using conventional methods. Tm refers to the temperature at which the population of the hybridization complex formed between the two nucleic acid strands denatures by 50%. Temperatures below Tm favor the formation of the hybridization complex, while temperatures above Tm favor the melting or separation of the chains within the hybridization complex. For nucleic acids with known G+C content, in a 1M NaCl aqueous solution, Tm can be estimated by, for example, using Tm = 81.5 + 0.41(%G+C) - 675 / N - % mismatch rate, where N = total number of bases.

[0046] Hybridization or annealing conditions include the chemical composition and concentration of the aqueous or organic solution containing nucleic acids (e.g., salts, chelating agents, formamide), and the temperature of the mixture at which one nucleic acid chain binds to a second nucleic acid chain through complementary strand interactions to produce a hybridization complex.

[0047] An analyte is the molecule to be detected in the binding assay. An analyte can be an antigen, a small molecule, or a nucleic acid, etc.

[0048] An "antigen" is a substance that, when exposed to a subject or organism (e.g., when present in or detected by the subject or organism), causes a detectable immune response in the subject or organism. An antigen can be, for example, a lipid, protein, carbohydrate, nucleic acid, or a combination and variant thereof.For example, an "antigen peptide" refers to a peptide that, when present in or detected by a subject or organism, causes an immune response in the subject or organism. Therefore, an antigen can be recognized by antigen-binding proteins (e.g., antibodies produced by a subject or organism in contact with the antigen).

[0049] The term "epitope" refers to a portion of an antigen that is recognized by antigen-binding proteins. A single antigen (such as an antigenic polypeptide) may have multiple epitopes. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and are defined as those linear sequences of residues that directly contribute to the affinity between the antigen-binding polypeptide and the antigen. Epitopes can also be conformational, i.e., composed of non-linear amino acids. Epitopes may include determinants, which are chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and may have specific three-dimensional structural features and / or specific charge features. Epitopes formed from consecutive amino acids are typically preserved upon exposure to denaturing solvents, while epitopes formed from ternary folds are typically lost upon treatment with denaturing solvents.

[0050] A sample is a composition in which one or more analytes may be present, including patient samples, plant or animal material, waste, forensic analysis material, environmental samples, circulating tumor cells (CTCs), cell-free DNA (cell-free DNA), liquid biopsies, etc. Samples include any tissue, cell, or extract derived from a living or dead organism 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, or other bodily fluids. Samples of particular interest are tissue samples (including bodily fluids) from people or animals that have or are suspected of having a disease or condition, particularly a viral infection. Other samples of interest include industrial samples, such as those used for water testing, food testing, pollution control, etc. Sample components may include target and non-target acids, target and non-target antigens, and other analytes, as well as other materials such as salts, acids, bases, detergents, proteins, carbohydrates, lipids, and other organic or inorganic materials. Samples may or may not be treated to purify the target nucleic acid or antigen prior to amplification, detection, or other analysis. Further treatment of nucleic acids may include treatment with detergents or denaturants to release nucleic acids from cells or viruses, removal or inactivation of non-nucleic acid components, and concentration of nucleic acids. Further treatment of peptide antigens may include any such suitable methods identified in the Wiley Protein Science Current Protocol 1995, “Protein Purification and Characterization Strategies” (updated April 2022), the entire contents of which are incorporated herein by reference. Lipids, carbohydrates, or any combination of antigens of the types described above may be treated as needed by any such suitable means to ensure appropriate concentrations and purities for the intended assay.

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

[0052] Target nucleic acids can originate from pathogenic microorganisms, such as viruses, bacteria, or fungi, or they can be endogenous to the patient. Viral nucleic acids (such as genomes and mRNA) form useful targets for analyzing viral sequences. Some examples of viruses that can be detected include HIV, hepatitis A, B, or C, herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein-Barr virus), adenovirus, XMRV, influenza virus, flavivirus, echovirus, rhinovirus, Coxsackievirus, coronavirus, respiratory syncytial virus, 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, and arbovirus.Examples of such bacteria include chlamydia and rickettsia. Mycobacteria, Staphylococci, Streptococci, Pneumococci, Meningococci, and Gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria pathogens, Salmonella, Bacilli, cholera pathogens, tetanus pathogens, botulism pathogens, anthrax pathogens, plague pathogens, leptospirosis pathogens, and Lyme disease bacteria. bacteria), streptococci, or neisseria. rRNA is a particularly useful target nucleic acid for bacterial typing. Detection of human or animal genes can be used to detect the presence or susceptibility to disease. Examples of genes that can be tested include oncogene fusions, BRACA-1 or BRAC-2, p53, CFTR, cytochrome P450, for genotyping (e.g., forensic identification, paternity testing, heterozygous carriers of genes that function when homozygous, HLA typing), determining drug efficacy for an individual (e.g., companion diagnostics), and other uses.

[0053] The term “dNTP” generally refers to an individual or combination of deoxynucleotides containing phosphate, sugar, and organic base in triphosphate form, which provide precursors required for DNA polymerase to synthesize DNA. The dNTP mixture may include each naturally occurring deoxynucleotide (i.e., adenine (A), guanine (G), cytosine (C), uracil (U), and / or thymine (T)). In some embodiments, each naturally occurring dideoxynucleotide may be replaced or supplemented with synthetic analogs such as inosine, isoG, isoC, denitroG, denitroA, etc.

[0054] The primer binding site is a complete or partial site in the target nucleic acid that hybridizes with the primer.

[0055] The primer or probe is an oligonucleotide complementary to all or part of the primer or probe binding site contributed by the target nucleic acid. The primer or probe may be attached to another nucleic acid (sometimes called a tail) at its 5' end, which is not in the target nucleic acid or is complementary to the target nucleic acid.

[0056] Primers or probes are oligonucleotides. The term “oligonucleotide” encompasses both the singular and plural “oligonucleotide” and refers to any polymer of two or more nucleotides, nucleosides, nucleobases, or related compounds used as reagents in the amplification methods and subsequent detection methods of the present invention. Oligonucleotides can be DNA and / or RNA and / or their analogues and / or DNA-RNA chimeras. The term oligonucleotide does not indicate any particular function of the reagent; rather, it is generally used to encompass all such reagents described herein. Oligonucleotides can perform a variety of different functions; for example, they can serve as primers if they are able to hybridize with complementary strands and can be further extended in the presence of nucleic acid polymerases; they can provide promoters if they contain sequences recognized by RNA polymerases and allow transcription; they can contain detection reagents for signal generation / amplification; and, if the position and / or modification is appropriate, they can prevent hybridization or hinder primer extension. Specific oligonucleotides of the present invention are described in more detail below. As used herein, oligonucleotides can be of almost any length, limited only by their specific function in the amplification reaction or in detecting the amplification products of the amplification reaction. Oligonucleotides with defined sequences and chemical structures can be produced using conventional techniques, such as chemical or biochemical synthesis, and through in vitro or in vivo expression of recombinant nucleic acid molecules (e.g., bacterial or viral vectors). Oligonucleotides can be modified in any manner, as long as the given modification is compatible with the desired function of the given oligonucleotide, which can be readily determined. Modifications include base modifications, sugar modifications, or backbone modifications. In addition to adenine, cytidine, guanosine, thymine, and uracil, base modifications include, but are not limited to, the use of the following bases: C-5 propyne, 2-aminoadenine, 5-methylcytidine, inosine, and dP and dK bases. The glycosyl group of the nucleoside subunit can be ribose, deoxyribose, and analogues, including, for example, ribonucleosides with a 2′-O-methyl (2′-O-ME) substitution on the furanyl ribosyl 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-threonyl, and pentopyranyl modifications. Nucleoside subunits can be linked via phosphodiester bonds, modification bonds, or non-nucleotide moieties that do not prevent the oligonucleotide from hybridizing with its complementary target nucleic acid sequence. Modified linkages include those that replace the standard phosphodiester linkage with different linkages, such as thiophosphate linkages or methylphosphonate linkages.Nucleotide subunits can be linked, for example, by replacing the native deoxyribose phosphate backbone of DNA with a pseudopeptide backbone, such as a 2-aminoethylglycine backbone, which links the nucleotide subunit to a central secondary amine via a carboxymethyl linker. (DNA analogs with pseudopeptide backbones are commonly referred to as “peptide nucleic acids” or “PNAs” and are disclosed by Nielsen et al., “Peptide Nucleic Acids” (Nielsen, Buchardt, Egholm, & Berg, 1996, U.S. Patent No. 5,539,082). Other linking modifications include, but are not limited to, morpholino bonds. Non-limiting examples of oligonucleotides or oligomers contemplated in this invention include nucleic acid analogs containing bicyclic and tricyclic nucleosides and nucleotide analogs (LNAs). See Imanishi et al., “Bicyclonucleoside and 15 CN 121219073 A Oligonucleotide Analogues” (Imanishi & Obika, 2001, US Patent No. 6,268,490); and Wengel et al., “Oligonucleotide Analogues” (Wengel & Nielsen, 2003, US Patent No. 6,670,461). This invention contemplates any nucleic acid analogue, provided the modified oligonucleotide can perform its intended function, such as hybridizing with the target nucleic acid under stringent hybridization or amplification conditions, or interacting with DNA or RNA polymerases to initiate elongation or transcription. In the case of detection probes, the modified oligonucleotide must also be able to preferentially hybridize with the target nucleic acid under stringent hybridization conditions. The 3′ end of the oligonucleotide (or other nucleic acid) can be blocked in a variety of ways using a blocking motif, as described below. A “blocked” oligonucleotide cannot be effectively elongated by DNA or RNA-dependent DNA polymerases adding nucleotides to its 3′ end, thus failing to produce a complementary DNA strand. Therefore, "blocked" oligonucleotides cannot be "primers".

[0057] The term "degenerate primer" refers to a mixture of similar primers with different bases at different positions (Mitsuhashi, J. Clin. Lab. Anal., 10 (5): 28593 (1996); von Eggeling et al., Cell. Mol. Biol., 41 (5): 65370 (1995); Zhang et al., Proc. Natl. Acad. Sci. USA, 89: 58475851 (1992); Telenius et al., Genomics, 13 (3): 71825 (1992)).Such primers may include inosine, as inosine is capable of base pairing with adenosine, cytosine, guanine, or thymidine. Degenerate primers allow annealing and amplification with a variety of potentially relevant target sequences. Degenerate primers annealed to target DNA can serve as initiation sites for further amplification. A degenerate region is a variable region of the primer, while the rest of the primer may remain unchanged. A degenerate primer (or region) represents multiple primers, which may be random. A random primer (or region) represents a sequence that has not been selected; it may be degenerate, but it is not required to be. In some embodiments, the Tm of the 3' target-specific region is between about 5°C and 50°C. In some embodiments, the Tm of the 15-mer is less than about 60°C.

[0058] The primer “3' fragment or 3' binding region or 3' binding site or 3' hybridization region” is capable of binding to a genomic sequence (which occurs at a specific frequency in the genome) or other nucleic acid sequences. In some embodiments, the frequency is between about 0.01% and 2.0%, for example, between about 0.05% and 0.1% or between about 0.1% and 0.5%. In some embodiments, the length of the primer's "binding site" depends primarily on the average length of the predicted PCR product calculated based on bioinformatics. This definition includes, but is not limited to, "binding regions" of about 4 to 12 bases in length. In more specific embodiments, the length of the 3' binding region may, for example, be between about 4 to 20 bases or between about 8 to 15 bases. Binding regions with Tm between about 10°C and 60°C are also included in this definition. The term "primer-binding fragment" as used herein refers to a primer having a specific sequence.

[0059] A polymerase is an enzyme that can perform template-directed extension of a primer that hybridizes to a template. It may be a DNA polymerase, an RNA polymerase, or a reverse transcriptase. Examples of DNA polymerases include: *Escherichia coli* DNA polymerase I, Taq DNA polymerase, *Streptococcus pneumoniae* DNA polymerase I, Tfl DNA polymerase, *Radiatae migrans* DNA polymerase I, Tth DNA polymerase, TthXL DNA polymerase, *Mycobacterium tuberculosis* DNA polymerase I, *Methanobacter thermophilus* DNA polymerase I, *Herpes simplex-1* DNA polymerase, T4 DNA polymerase, hot sequencer or wild-type or modified T7 DNA polymerase, Φ29 polymerase, Bst polymerase, Vent polymerase, 9°Nm polymerase, and the Klenow fragment 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 polymerases and eukaryotic RNA polymerases, as well as recombinases.

[0060] Amplification refers to the generation of one or more additional copies of all or part of the target nucleic acid through template-guided primer extension (target amplification), and / or amplification of the detection signal for qualitative / quantitative measurement (signal amplification).Amplification can be performed under temperature cycling, isothermal conditions, or a combination of both. Amplification can be linear or exponential. The target nucleic acid can be conjugated with an antibody for immunoPCR or immunoisothermal amplification.

[0061] The term “thermally cycling” refers to a repeated cycle of temperature changes from the complete denaturation temperature to the annealing (or hybridization) temperature, then to the extension temperature, and back to the complete denaturation temperature. These terms also refer to repeated cycles of denaturation and extension temperatures, where the annealing and extension temperatures are combined into a single temperature. The complete denaturation temperature unwinds all double-stranded fragments into single strands. The annealing temperature allows the primers to hybridize or anneal to the complementary sequences of the separated strands of the nucleic acid template. The extension temperature allows the synthesis of the nascent DNA strands of the amplicon.

[0062] The term “amplification mixture” or “PCR mixture” refers to a mixture of components required to amplify at least one amplicon from a nucleic acid template. The mixture may contain nucleotides (dNTPs), thermostable polymerase, primers, and multiple nucleic acid templates. The mixture may also contain Tris buffer, monovalent salts, and Mg2+. The concentrations of each component are well known in the art and can be further optimized.

[0063] The term "amplification product" or "amplifier" refers to a DNA fragment amplified using a pair of primers in an amplification method such as PCR with a polymerase.

[0064] The term "fluorophore" refers to a portion that absorbs light energy at a defined excitation wavelength and emits light energy at different defined wavelengths.

[0065] The term "quencher" includes any portion that can absorb the energy of an excited fluorescent label when it is near the fluorescent label and can dissipate that energy. The quencher may be a fluorescent quencher or a non-fluorescent quencher (also called a dark quencher). Fluorophores listed above can act as quenchers when near another fluorophore, where FRET quenching or contact quenching can occur. Preferably, a dark quencher that does not emit any visible light is used. Examples of dark quenchers include, but are not limited to, DABCYL (4-(4′-dimethylaminophenylazo)benzoic acid) succinimide ester, diarylrhodamine carboxylic acid succinimide ester (QSY-7) and 4′,5′-dinitrofluorescein carboxylic acid succinimide ester (QSY-33), quencherl or BlackHole (BHQ-1, BHQ-2 and BHQ-3), nucleotide analogs, nucleotide G residues, nanoparticles and gold particles.

[0066] The term “mutation” refers to one or more nucleotides in the target nucleic acid sequence that differ from the prototype form of the target nucleic acid designated as wild-type.The sequence designated as wild-type is the most common allelic form of the sequence, the first-discovery sequence form, and / or the sequence form associated with a normal (non-disease) phenotype. A single nucleotide polymorphism (SNP) is a form of mutation.

[0067] The term “surface” refers to any solid surface that can be covalently attached to nucleic acids, 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] The term “attachment to a surface” refers to any chemical or non-chemical linking method, including chemically modifiable functional groups. “Attachment” involves immobilizing nucleic acids on a solid support by covalent attachment or by irreversible passive adsorption or by intermolecular affinity (e.g., immobilization on an avidin-coated surface by biotinylated molecules). The attachment must be strong enough to be washed away with water or an aqueous buffer under DNA denaturing conditions.

[0069] A sticky end is a single-stranded end of a nucleic acid adjacent to a double-stranded segment of the nucleic acid. Nucleic acids with sticky ends having complementary sequences can be annealed at the sticky ends and linked together.

[0070] A “genetic marker” refers to a polynucleotide sequence or modification of a polynucleotide sequence present in a genomic sequence of a reference chromosome, having a known physical location that allows for 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., polymorphism) or the presence or absence of sequences (e.g., sequences present on the Y chromosome of a male fetus but not in the genome of a pregnant woman) (e.g., alleles from two different individuals, such as an allele from the fetus and an allele from the pregnant woman). In this context, a “methylation marker” located on a chromosome and associated with chromosomal aneuploidy refers to a genomic polynucleotide sequence on a chromosome with an abnormal number; or, in the case of the presence of an extra segment of the chromosome or the deletion of a portion of the chromosome, a “methylation marker” is located within a segment or portion of the relevant chromosome. Differences in the methylation profiles of methylation markers allow for the distinction of corresponding methylation markers from two different individuals (e.g., a fetus and a pregnant woman). Instructions 12 / 39 pages 17 CN 121219073 A

[0071] The term “single nucleotide polymorphism” or “SNP” refers to a polynucleotide sequence variation on a single nucleotide residue between different alleles of the same gene. This could be the same gene located on two copies of the same chromosome from the same individual (e.g., two alleles from a fetus), or it could be the same gene from two different individuals (e.g., a fetus and a pregnant woman). Such variation can occur within coding or non-coding regions of a gene (e.g., promoter regions or their vicinity, or introns), or between gene regions. Detecting one or more SNPs allows for the differentiation of different alleles of a single gene.

[0072] The term "simple tandem repeat polymorphism" refers to the polynucleotide sequence variation exhibited by the difference in the number of tandem repeats (e.g., one or more nucleotides) of nucleotide sequences between different alleles of the same gene. This could be the same gene located on two copies of the same chromosome from the same individual (e.g., a fetus), or it could be the same gene from two different individuals (e.g., a fetus and a pregnant woman). Such variation typically occurs within non-coding regions of a gene (e.g., promoter regions or their vicinity, or introns) or between gene regions. Detecting differences in the number of tandem repeats allows for the differentiation of different alleles of a single gene.

[0073] The term "insertion-deletion polymorphism" refers to the polynucleotide sequence variation exhibited by the presence or absence of short nucleotide sequences (e.g., 1-3 nucleotides) between different alleles of the same gene. This could be the same gene located on two copies of the same chromosome from the same individual (e.g., a fetus), or it could be the same gene from two different individuals (e.g., a fetus and a pregnant woman). Such variation can occur within coding and non-coding regions of a gene (e.g., promoter regions or their vicinity, or introns), or between gene regions. Detecting the presence of short nucleotide sequences allows for the differentiation of different alleles of a single gene.

[0074] The term “blood” refers to a blood sample. This term encompasses whole blood or any part of blood, such as serum, cell-free DNA in plasma, and plasma as defined conventionally. Examples of blood samples include, but are not limited to, preparations from pregnant women or women being tested for possible pregnancy, preparations from people with a disease or infection or monitoring for possible disease or infection.

[0075] The term “bisulfite” refers to all types of bisulfites, such as sodium bisulfite, which are capable of chemically converting cytosine (C) to uracil (U) without chemically modifying methylated cytosine, and thus can be used for differential modification of DNA sequences based on the methylation state of DNA.

[0076] The term “locus” refers to a segment of DNA defined by the start nucleotide position to the end nucleotide position (i.e., genomic location or chromosomal location) on a chromosome assembled from a reference genome (e.g., the March 2006 assembly of the human genome on the UCSC Genome Browser (hg18)). A locus may or may not overlap with a gene's genomic location, CpG islands, or any transcription / translation product. For example, a locus can typically be, but is not limited to, a contiguous DNA fragment identified through experimental data (e.g., MeDIP microarray datasets) and subsequent data analysis (e.g., MAT, TAS) to contain varying levels of DNA methylation. A locus may contain one or more CpG sites.Loci can be subdivided into shorter segments (e.g., CpG-containing genomic sequences, fragments, or regions) that are easily analyzed (e.g., Epityper assays, bisulfite sequencing, polynucleotide amplification and assays). Loci can be classified into one or more fetal epigenetic markers. Loci can also refer to continuous DNA segments identified by certain bioinformatics standards.

[0077] The term “molecular counting” refers to any method that allows for the quantitative measurement of the number of molecules or molecular complexes, typically relative to other coexisting molecules or complexes with different characteristics. Various molecular counting methods 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 any interaction between at least two reagents. The reagents used in the reaction may be present in the sample (see page 13 / 39 of the specification, 18 CN 121219073 A) or added to the sample. The interaction may be chemical or physical in nature. Polymerase-catalyzed extensions, antibody-antigen binding, and contact between beads and washing solutions are all considered reactions.

[0079] An integrated approach refers to a series of ordered steps in which one or more products of one step become one or more starting materials for the next step. Steps in this approach may be referred to as bioprocessing steps.

[0080] An aqueous reaction mixture comprises water and at least one dissolving or suspending reagent for carrying out the reaction (such as a bioprocessing step), or a mixture of the sample and a complete set of reaction reagents. An aqueous reaction mixture is typically a liquid, but may be in gel form at ambient temperature and become liquid at temperatures above ambient temperature (e.g., a reaction mixture dissolved in an agarose gel). An aqueous reaction mixture may comprise a complete reaction mixture, i.e., all reagents required for sample analysis (but not necessarily the sample itself), or may be partial, comprising at least one reaction component (in other words, a reagent) required to carry 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, such as 18°C ​​to 35°C, more preferably 20°C to 25°C, which is the air temperature range that most people prefer for indoor environments and is typical of laboratory environments. It feels comfortable when wearing typical indoor clothing. Low temperature refers to a temperature below room temperature, especially in the atmosphere, such as above 0°C and below 8°C (i.e., the typical temperature of a refrigerator or freezer).Freezing temperature is a phase transition that occurs when the temperature of a liquid drops below its freezing point, at which point it solidifies. For example, this temperature is below 0°C for aqueous solutions. Reagents used in reactions are sometimes stored between -20°C and -0.50°C. According to the internationally recognized definition, freezing refers to the solidification phase transition of a liquid or substance's liquid contents, usually occurring due to cooling. For most substances, the melting point and freezing point are the same temperature; however, some substances have different solid-liquid transition temperatures. For example, agar exhibits hysteresis between its melting point and freezing point. It melts at 85°C (185°F) and solidifies between 32°C and 40°C (89.6°F to 104°F).

[0082] When it is mentioned that a permeable gel can be permeated by a micropipette, this means that when applied manually or by an automated liquid handling device, a tip-equipped micropipette for delivering a volume in the range of 1 picoliter to 5000 μL can permeate the gel at ambient temperature and deliver the desired volume of liquid contents below the gel layer stored in the reaction vessel. The permeable gel may be interrupted / disrupted by the pipette tip or inert rod before the micropipette tip delivers the liquid through the gel layer. The tip that interrupts / disrupts the gel layer may or may not have a stopper. The stopper may be wax or gel. Stoppered tips may also be used to store reagents for any reaction within the tip. Some gels can be permeated by a tip to deliver volumes at the lower limit of the aforementioned range rather than the upper limit, and vice versa, while other gels can be permeated by any tip within the aforementioned range. For example, some gels can be permeated by tips with cap sizes of 1 μL to 1 mL, 1 μL to 500 μL, or 1 μL to 100 μL.

[0083] A micropipette is a liquid processor or any device capable of transferring or delivering a desired volume of liquid to a desired reaction vessel. A micropipette may carry one or more micropipette tips, for example, 1 to 10,000 tips. A tip is any object attached to a micropipette for delivering or transferring liquid in a desired volume to a desired reaction vessel. The volume range is from 1 pL to 5000 μL. The tip can be a standard industrial micropipette tip or one or more capillaries (e.g., a bundle of capillaries). A capillary is a hollow tube typically used for delivering volumes <1 μL. Micropipette tips can deliver liquid above, below, or in the middle of a gel layer in a reaction vessel. For oil-containing reaction vessels, micropipette tips can deliver liquid above, below, or in the middle of an oil layer. Liquid delivered to the middle of an oil layer or gel layer can form an emulsion droplet or reaction compartment.

[0084] Therefore, the present invention also provides a method for micropipette delivery of a reaction solution through a gel layer to an oil layer below the gel layer, forming an oil-water emulsion droplet, the number of which is between 1 and 1 million. The micropipette may be a multichannel pipette (see specification 14 / 39 pages 19 CN 121219073 A). The pipette tip used to deliver the reaction solution may be a standard industrial micropipette tip or a bundle of capillaries. The reaction volume in the emulsion droplet ranges from 0.0001 μL to 5000 μL.

[0085] A module is a device that performs at least one function. Multiple modules may be connected together to form an integrated device that performs multiple related functions, such as a magnetic module, a heating module, a reading module, and a liquid handling module. Heating and cooling may be performed by the same or different modules. The module or integrated device containing multiple modules may also include a connector that allows the module or device to be connected to a reaction vessel (e.g., allows sliding along the surface of the vessel). A module or device including a module may include a computer or a component thereof, or may be adapted to receive signals from a link to or remotely connected to a control module or device.

[0086] A rod refers to a bar or stick, or an elongated tool made of glass, optical fiber, metal, plastic, or silicon. The surface of the rod may be chemically modified or unmodified or partially modified. It may be conductive or non-conductive. The rod may be used to mix solutions or stir gel layers or as an electrode. The rod may be used to bind a target in a sample or to deliver a reagent or target 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 non-reactive in aqueous solutions.

[0087] When this disclosure discloses a range of values, it should be understood, unless the context otherwise requires, that all values ​​within that range and subranges formed by all values ​​within that range are also disclosed.

[0088] When this disclosure discloses a composition, product, or method that comprises (or includes) a particular component or step, it should be understood, unless the context otherwise requires, that additional disclosure of a composition, product, or method consisting of or substantially consisting of the particular component and step is also disclosed. I. Overview

[0089] This invention uses a gel layer to cover and / or separate one or more aqueous reaction mixtures in a reaction vessel. The gel layer can protect reagents or reaction products from contact with the atmosphere, thus providing an alternative to lyophilization for storage or transport. The gel layer can also separate multiple aqueous reaction mixtures so that they do not come into contact with each other before the gel layer is broken and the reaction mixtures come into contact with each other. A solution (such as a sample) can be delivered to the reaction mixture below the gel layer by inserting a micropipette tip. A micropipette tip or inert rod can also be used to break the gel layer, resulting in mixing of the aqueous reaction mixtures above and below the layer. The gel layer can also be broken by selectively applying heat to the layer to melt the gel, also resulting in 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 may be broken in a prescribed order, for example from top to bottom of the reaction vessel, to achieve an ordered series of biological treatment steps. Optionally, another oil layer may be inserted directly beneath any or all of the gel layers. II. Gel Composition and Properties

[0090] A gel is a semi-solid form in which a liquid phase is embedded in a solid three-dimensional matrix. The matrix may be formed by cross-linking polymerization and / or self-assembly, for example by forming a matrix through hydrogen bonds between particles. For example, a gel may be formed by heating wax and oil together, causing the liquid oil phase to be trapped between the wax particles. Gels may be characterized by their viscosity, typically in centipoise. Viscosity is a measure of the resistance to gel flow. The viscosity of a gel is between that of oil and wax. Unlike liquids, gels generally retain their shape or remain in situ without forces other than gravity. In other words, gels do not flow in a steady state, although the liquid phase may diffuse within the gel. Gels may conform to shape or flow under pressure. The shape, stiffness, and adhesiveness (viscosity) of a gel are imparted by the matrix within the gel. Viscosity can be measured as the resistance of a gel to an object passing through it. The viscosity of a gel can be controlled by the ratio of wax to oil, which can range from 99:1 to 25:75 by weight or volume, specifically from 25:75 to 75:25, or from 40:60 to 60:40, 45:55 to 55:45, or about 50:50. In a reaction vessel comprising multiple gel layers, the gel layers can have the same or different compositions from each other. Gels with different compositions and different melting temperatures can be used in the specification, page 15 / 39, 20 CN 121219073 A, for temperature-controlled selective melting layers.

[0091] The gel may contain other chemicals or additives. For example, chemicals or additives include surfactants, which are compounds that may contain a hydrophilic “head” and a hydrophobic “tail.” The “head” of a surfactant is polar and may or may not carry a charge. The “tail” of most surfactants is quite similar and includes hydrocarbon chains that can be branched, linear, or aromatic. Fluorinated surfactants have fluorocarbon chains. Siloxane surfactants have siloxane chains.

[0092] Many important surfactants include polyether chains terminated in highly polar anionic groups. The polyether groups typically contain ethoxylated (polyethylene oxide-like) sequences that are inserted to increase the hydrophilicity of the surfactant. Conversely, polypropylene oxide can be inserted to increase the lipophilicity of the surfactant. Surfactant molecules have one or two tails; those with two tails are called double-chains.

[0093] Surfactants are generally amphiphilic organic compounds, meaning that such molecules, as dual agents, each contain a hydrophilic “water-seeking” group (head) and a hydrophobic “water-repelling” group (tail). Thus, surfactants comprise water-soluble and water-insoluble components.Surfactants diffuse in water and are adsorbed at the air-water interface or at the oil-water interface when water and oil are mixed. Water-insoluble hydrophobic groups can extend from the aqueous bulk into non-aqueous phases, such as air or oil, while water-soluble head groups remain bound in the aqueous phase.

[0094] Depending on their chemical properties, the hydrophobic tail may be lipophilic (“oil-seeking”) or lipophobic (“oil-repelling”). Hydrocarbon groups are typically lipophilic and used in soaps and detergents, while fluorocarbon groups are lipophobic and used to repel stains or reduce surface tension.

[0095] Waxes are a diverse class of compositions with similar composition and physical properties to beeswax. Waxes can be derived from petroleum, oil shale, animal or plant matter, and may or may not be modified. Paraffin waxes comprise hydrocarbons, typically a mixture of homologous alkanes with chain lengths of about 12-40 carbon atoms. Paraffin waxes may also comprise mixtures of saturated n-alkanes and isoalkanes, cycloalkanes, and aromatic compounds substituted with alkyl and cycloalkanes. Typical alkane paraffins have chemical compositions including hydrocarbons of the general formula CnH2n+2, such as hexadecane C31H64. The chains may or may not be branched. Another type of wax is a simple lipid, which is a long-chain (12-32 carbon atoms) ester. Waxes are solid at ambient temperature and melt above about 37°C. At ambient temperature, waxes will not be penetrated by micropipettes without causing pipette tip deformation or clogging.

[0096] A liquid is a state having a definite volume but no fixed shape. It is a nearly incompressible fluid that conforms to the shape of its container but maintains a (nearly) constant volume, independent of pressure. Liquids are capable of flowing and taking the shape of their container. Water and petroleum are the most common liquids on Earth. Mineral oil is sometimes simply referred to as oil. The mineral oil or oil mentioned in this invention are interchangeable. Oils in this invention include mineral oils distilled from petroleum, synthetic oils, silicone oils, algal oils, and oils from plant sources such as olive, palm, soybean, rapeseed, corn, and peanut. Mineral oil, also known as white oil, paraffin oil, liquid paraffin, liquid paraffin (Latin), and liquid petroleum, is a liquid byproduct of crude oil refining. It is a transparent, colorless oil mainly composed of alkanes and cycloalkanes, and is associated with petrolatum. The oil can also be a mixture of different types of oils, containing / not containing surfactants. Surfactants added to the oil mixture promote the formation of emulsion droplets for digital PCR amplification or reduce the formation of emulsion microdroplets for qPCR detection. Different types of oils and their mixtures can have different densities, less than, equal to, or greater than the density of water.

[0097] The gel used in this invention has sufficient viscosity at ambient temperature to form a layer that can cover the aqueous mixture in the reaction vessel, preventing or at least resisting the diffusion of the aqueous mixture throughout the tube, and holding itself and the aqueous reaction mixture below it in place throughout the storage and transport of the tube before use, and protecting the aqueous mixture from contact with the atmosphere.However, the gel described in this invention is not viscous enough to prevent micropipette tips from penetrating the gel at ambient temperature to deliver a solution (which may be a sample and / or one or more reagents) into the reaction mixture in the reaction vessel, as described on pages 16 / 39 of the specification 21 CN 121219073 A below the gel. The gel described in this invention is preferably resistant to shrinkage at temperatures below ambient temperature (e.g., at cryogenic or freezing / solidification temperatures) to prevent multiple aqueous reaction mixtures separated by the gel layer from mixing during storage or transport. The gel may melt at temperatures above ambient temperature and reform into a gel once it returns to ambient temperature.

[0098] Pure paraffin has the following disadvantages compared to the gel used in this invention. It is difficult to add samples by penetrating with a conventional pipette because the intruding wax plug blocks the tip orifice. Its typical volume expansion of 15% when changing from a solid to a liquid can lead to unwanted expansion / contraction effects on the barrier, or cross-leakage and mixing of the constituent reagents separated in the reagent vessel, and the risk of forming a defective layered barrier with voids (US Patent No. 5,411,876). Furthermore, if heating is used to melt the wax barrier to mix the separated constituent reagents in the container, the slow melting of the wax barrier may delay the reaction.

[0099] The semi-solid gel provides several physical properties that overcome the drawbacks observed in pure paraffin as a layered barrier. The gel can have a customized hardness that can be easily penetrated and broken with a pipette tip or a needle-shaped plastic pick or rod. It can have a volume expansion of 3%–5% or less, which helps to reduce the risk of uncontrolled mixing / merging of the separated constituent reagents in the reagent container, as well as the risk of forming a defective wax barrier with voids during the manufacturing process of the reagent container.

[0100] Similar to pure paraffin (a temperature-dependent phase change material, i.e., PCM), the gel used in this invention can transform from a non-flowable semi-solid under gravity at ambient temperature (e.g., 23°C) to a flowable liquid at high temperatures (e.g., 50°C); when the container returns to ambient temperature, the liquefied gel becomes a semi-solid gel, i.e., re-solidifies, which is a non-flowable substance. Because the density of molten semi-solid gels is typically lower than that of mineral oil, which is lighter than water or an aqueous phase, the semi-solid gel mixture floats to the top of the liquid phase during the heated reaction and forms a non-flowing capping layer on top of the liquid contents in the container after the reaction. III. Layered Reaction Composition

[0101] The gel layer can play several roles in the reaction vessel. A single gel layer covering the aqueous reaction mixture can be used to hold the reaction mixture in place at the bottom of the container rather than spreading throughout the container, and to form a barrier between the aqueous reaction mixture and the atmosphere, protecting the reaction mixture from degradation due to interaction with the atmosphere (e.g., oxidation or water absorption) and / or reducing environmental contamination by reaction products.The gel layer can be used to absorb contaminants from the sample during the reaction, reducing the inhibitory effect of contaminants on the reaction. The gel layer can be used to control the release of components in 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 a gel to a reaction vessel containing an aqueous solution, or by adding an aqueous solution to a vessel that already contains a gel, and heating the reaction vessel to melt the gel layer, which then floats above and covers the aqueous solution.

[0103] An oil layer disposed between the reaction mixture and the gel layer can increase protection against the atmosphere and, if present, can also be held in place by the gel layer. The gel layer (and the oil layer, if present) can be penetrated by a micropipette tip to deliver liquid to the aqueous layer. The delivered liquid can be one or more other reagents not already present in the aqueous reaction mixture, and / or can be a sample to be combined with the aqueous reaction mixture. Continuous addition of reagents and / or samples can also be performed by multiple penetrations of a micropipette tip. Typically, aqueous reaction mixtures contain reagents for processing samples, such as by amplification or immunoassay, and samples are delivered to the aqueous reaction mixture using pipette tips that penetrate the gel layer.

[0104] The gel layer can also be used to separate / divide (hereinafter also referred to as separation or partition) multiple aqueous reaction mixtures, for example, when the reaction mixture contains different components that need to be combined for sample assay. For example, a reaction vessel may contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more layers of aqueous reaction mixture, separated from each other by a gel layer and optionally an oil layer. Thus, for example, in amplification assays, one or more polymerases may be separated from primers, probes and / or dNTPs until use to reduce amplification artifacts. In immunoassays, capture and detection reagents may also be separated before use. In this form, a gel layer may be used to cover a first reaction mixture and separate the first reaction mixture from a second reaction mixture, and a second gel layer may be used to cover the second reaction mixture. Therefore, both reaction mixtures remain in their proper positions within the reaction vessel and are protected from atmospheric degradation. Optionally, for a single gel layer, an oil layer may be included between the gel layer and the reaction mixture below it, or, when the reaction vessel contains multiple aqueous solutions, an oil layer may be included between two gel layers. The same principle can be extended to other reaction mixtures and gel layers within the same vessel.

[0105] Using multilayer reaction mixtures involves combining mixtures and / or delivering another reagent and / or sample through the gel layer using a micropipette.The reaction mixture layers separated by the gel layer can be combined by penetrating the gel layer with a rigid pipette tip or micropipette tip and stirring the contents of the reaction vessel. The initially separated reaction mixtures combine into one layer at the bottom of the reaction vessel, and the initially separated gel layers combine into one layer on top of the mixed reaction mixture. After breaking the gel layer with a micropipette tip or inert rod, the aqueous reaction mixture separated by the gel layer can be well mixed by centrifugation. Micropipette tips can also be used to deliver reagents or samples. This delivery can occur after multiple reaction layers and gel layers have been separated into individual reaction mixtures and gel layers by penetrating the reformed gel layer. Alternatively, reagents or samples can be delivered during stirring if the gel layer is stirred with a micropipette tip. The reaction mixture layers can also be melted by applying heat to the gel using a heater to facilitate combination. When the density of oil is greater than that of water, a high-density oil layer in the reaction vessel can promote the mixing of two aqueous reaction mixtures. For example, the aqueous reaction mixture at the bottom is covered by a first gel layer. On top of the first gel layer is an oil mixture with a density greater than that of water. On top of the oil layer is a second aqueous reaction mixture. A second gel layer is placed on top of the second aqueous reaction mixture. After heating the reaction vessel, the first gel layer melts, and oil droplets fall to the bottom of the reaction vessel, pushing the first aqueous reaction mixture upwards and mixing with the second aqueous reaction reactant. Optionally, at ambient temperature, the first and second aqueous reaction mixtures can be gels in the reaction vessel. After heating the reaction vessel, both gels become fluid and gel mix because the density of the oil layer between them is higher than that of water. The reaction mixture layers can also be combined by moving magnetic beads present in one aqueous reaction mixture through the gel layer to the other reaction mixture using a magnet located outside or inside the reaction vessel. Optionally, the heater or magnet used in such methods can be attached to the reaction vessel such that the heater or magnet can slide along the outer or inner surface of the reaction vessel until it approaches the gel layer to be melted by the heater or destroyed by the movement of the magnetic beads caused by the magnet. Optionally, the reaction vessel can be attached to a device including a heater and a magnetic module.

[0106] The gel layer can be removed after the reaction has occurred.

[0107] Alternatively or additionally, the gel layer may have a different composition and melting temperature, thereby allowing the layer to be broken in a specified order according to its melting temperature by controlling the temperature of the entire reaction vessel (e.g., by immersion in a heating bath).

[0108] Devices including readers can be used to assist or facilitate the handling of sample liquids of reagents and the detection reactions occurring in the reaction vessel. When all reactions are ready, the reaction vessel is placed in an appropriate reader (e.g., a thermal cycler for conventional PCR or real-time PCR or a hot block / water bath for isothermal amplification) to complete and monitor the reaction.Devices including a reader module may also include combinations of one or more of the following modules: mechanical modules, heating modules, electrical modules, magnetic modules, liquid handling modules, 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 is completed. The reader can detect chemical or physical changes in the reaction in the reaction vessel, such as color, fluorescence, luminescence, chemiluminescence, electrochemistry, radiation, reflection, phase transition, magnetoresistance, turbidity, mobility shift, pH, or ionic strength.

[0109] A reaction vessel comprising a multilayer of aqueous reagents separated by gel layers can be used to perform an integrated biological process involving a series of ordered steps, as described on pages 18 / 39 of CN 121219073 A. The steps can be performed by selectively breaking the gel layers, such that only the aqueous reaction mixture above and below a certain gel layer is mixed at a time. After mixing, the reagents in the mixture react with each other, and one or more products of the reaction then become one or more starting materials, which are combined with another aqueous reaction mixture by breaking another gel layer. Then, by breaking another gel layer, one or more products of this further reaction can be combined with yet another aqueous reaction mixture. Typically, the gel layer is gradually broken down downwards, except for the top gel layer, which may remain in place, thus continuously separating the reagents and products of the reaction from the atmosphere. The gel layer can be broken down by selectively applying heat to the layer with a micropipette tip (which can also be used to introduce samples or other reagents), with an inert rod, or by causing magnetic beads present in the aqueous layer to move through the gel layer by a magnet, or any combination of these techniques.

[0110] Other reaction vessels include layers of aqueous reaction mixture, gel layer, and optionally oil layer arranged in a random order within the vessel.

[0111] The reaction vessel may be fitted with a device including modules for heating / cooling and / or applying a magnetic field, which can slide along the outer surface of the vessel to allow heat or cooling to be directed onto a particular gel layer, or a magnetic field to be applied to magnetic beads in a particular aqueous reaction mixture. The heater may be a thermoelectric device, such as a semiconductor-based Peltier device. The magnet may be an electromagnet. IV. Reaction Vessel Forms

[0112] The reaction vessel is used to contain a gel and contains a reaction mixture or any component thereof. Examples include microtubes of various sizes, ranging from microliters to milliliters, commonly used in laboratories. Reaction vessels may or may not have lids, such as snaps, screws, plastic wrap, or foil, and may or may not be suitable for microcentrifuges. Reaction vessels can be single-use or reusable. They can be channels or trenches or hollow tubes, or include flat surfaces. Reaction vessels can be individual containers or interconnected as strips or matrices to facilitate parallel processing.The reaction vessel may also be in the form of a multi-well plate (e.g., 96, 384, or 1536 wells), a pipette tip, or a chip, in which case each well of such a plate or chip can be considered a reaction vessel, and the wells in the chip can be connected by channels.

[0113] Depending on the desired application, the reaction vessel may be plastic (e.g., polypropylene), glass, metal, or any other material suitable for the intended reaction (e.g., impermeable to or inert to the intended aqueous reaction mixture, gel, and oil contents of the vessel). Depending on its material composition, the reaction vessel may be inherently hydrophobic or hydrophilic, or may be treated to achieve the hydrophobic / hydrophilic properties required for the intended reaction.

[0114] Suitable vessels for PCR include single PCR tubes, PCR strips, and / or PCR plates or detection kits, etc., which can react one or more samples simultaneously. Reaction vessels for ELISA assays include commercially available ELISA plates, deep-well plates, and / or microtiter plates, and / or detection kits, etc., which can react one or more samples simultaneously.

[0115] The reaction vessel is sealed during storage or transport. The reaction vessel can be sealed with a film such as aluminum foil and then stored in a heat-sealed metal storage bag. The reaction vessel can be transported at ambient temperature or in a polystyrene foam box containing blue ice or dry ice. The reaction vessel can be stored at the desired temperature. V. Reaction compatible with the reaction vessel

[0116] The gel layer can be used to cover the solution before, during or after the reaction. The gel layer can be used to separate the reaction mixtures for various reactions, including various chemical reactions, immunoassays, nucleic acid detection assays, sequencing and / or purification protocols. Any assay method that contains multiple aqueous reaction mixtures stored separately until the assay can be adapted to the disclosed strategy, including using more or fewer reaction layers than shown in the representative examples and figures.

[0117] The gel layer can be used to inhibit the interaction between reagents before the desired reaction occurs, for example, to inhibit nonspecific amplification of nucleic acid or primer dimers. The gel layer can be used for sequential reactions, such as reverse transcription reaction instructions on page 19 / 39 of CN 121219073 A (RT) above the gel layer, and then PCR reagents are mixed with the RT product for amplification and detection. Another example is that amplification can be performed first by nicking around the gel layer, and then the gel layer can be melted for PCR. Another application is nested PCR. The first stage of amplification can be performed above the gel layer. The second stage of amplification can be performed after the gel melts. In summary, multi-stage amplification, such as NASBA, T7, CRISPR, RPA, and EXPAR, can be performed sequentially in different gel layers. Another example is a reaction vessel containing a solution below the gel layer. The solution contains proteinase K. The sample is added to the reaction vessel, and the reaction vessel is heated at 95°C for 10 minutes.The treated sample can be used for other reactions, or PCR reagents can be added to the reaction vessel for amplification and detection.

[0118] Another example is for immunoPCR or immunoisothermal amplification. The immunoreaction occurs above the gel layer due to proximity interactions, and then is mixed with another reagent for PCR or isothermal amplification to detect the analyte. 1. Amplification reaction:

[0119] Amplification reactions are used to detect target nucleic acids, genetic markers, single nucleotide polymorphisms, insertions, deletions, inversions, rearrangements, transversions, deletions, insertions and deletions, microsatellite repeats, small satellite repeats, short tandem repeats, transposable elements, large-scale structural chromosomal variations, methylation, and combinations thereof. Amplification is also used in applications such as nucleic acid cloning protocols and sequencing.

[0120] Many well-known nucleic acid target amplification methods require thermal cycling to alternately denature double-stranded nucleic acids and hybridization primers; however, other well-known nucleic acid amplification methods are isothermal. Polymerase chain reaction, commonly known as PCR (Mullis, 1987, US Patent No. 4,683,202; Saiki et al., 1985, Science (New York, N.Y.), 230(4732), 1350–1354), uses multiple cycles of denaturation, primer pair and reverse strand annealing and primer extension to exponentially increase the copy number of the target sequence. In a variant called RT-PCR, a complementary DNA (cDNA) is produced from mRNA using reverse transcriptase (RT), and then the cDNA is amplified by PCR to produce multiple copies of DNA (Gelfand et al., "Reverse Transcription with Thermostable DNA Polymerases—High Temperature Reverse Transcription," (Gelfand, 1994, US Patent No. 5,322,770; Gelfand & Myers, 1994, US Patent No. 5,310,652). Another method for amplifying nucleic acids is called the LCR method (ligase chain reaction, Laffler, Carrino, & Marshall, 1993, Annales De Biologie Clinique, 51(9), 821–826). LCR (Laffler et al., 1993, Annales De Biologie Clinique, 51(9)). LCR (821–826) is a reaction based on the hybridization of two adjacent probes to a target sequence and their ligation by an enzyme. In the absence of a target nucleotide sequence, the two probes cannot ligate; therefore, the presence of the ligation product indicates the target nucleotide sequence. The LCR method also requires temperature control to separate the template and complementary strand.Another method is chain substitution amplification (George T. Walker, Little, & Nadeau, 1993, US Patent No. 5,270,184; George T. Walker, 1995, US Patent No. 5,455,166; G.T. 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), commonly known as SDA, uses multiple cycles of primer sequence pair annealing to the opposite strand of the target sequence, primer extension in the presence of dNTPs to produce double-stranded hemithiophosphate primer extension products, endonuclease-mediated nick formation at the recognition site of a semi-modified restriction endonuclease, and polymerase-mediated primer extension from the 3′ end of the nick to displace the existing strand and generate a new strand, which is used for the next round of primer annealing, nick formation, and strand displacement, resulting in geometric amplification of the product. Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same manner (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; a method using RNA replicase to amplify the probe molecule itself (Lizardi, Guerra, Lomeli, Tussie-Luna, & Russell Kramer, 1988, Nature Biotechnology, 6 (10), 1197–1202), commonly known as Qβ replicase; transcription-based amplification methods (Kwoh et al., 1989, Proceedings of the National Academy of Sciences of the United States of America, 86 (4), 1173–1177); and self-sustaining sequence replication (3SR), (Guatelli et al., 1990). ,Proceedings of the National Academy of Sciences of the United States of America, 87(5), 1874–1878; Landgren (1993) Trends in Genetics 9, 199–202; and 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 Patent No. 5,480,784; Kacian & Fultz, 1996, US Patent 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, DC). Other illustrative amplification methods applicable to the present invention include rolling circle amplification (RCA) (Fire & Xu, 1995, Proceedings of the National Academy of Sciences, 92(10), 4641–4645; Lizardi, 1998, US Patent No. 5,854,033); nucleic acid amplification using a nicking agent (VanNess, Galas, & VanNess, 2006, US Patent No. 7,112,423); nick and extension amplification reaction (NEAR) (Maples et al., 2009, US2009-0017453A1); helicase-dependent amplification (HDA) (Kong, Vincent, & Xu, 2004, US2004-0058378A1; Kong Vincent, & Xu, 2007 (US Patent US2007 / 0254304A1); and loop-mediated isothermal amplification (LAMP) (Notomi & Hase, 2002, US Patent No. 6, 410,278) and quadruple primer amplification (Analyst, 2014, 139, 1644–1652). Expar amplification (PNAS April 15, 2003, 100, 4504–4509). Cross primer 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): 308–12).Single primer isothermal amplification (SPIA) (clinical chemistry, 2005, Vol. 51, No. 10, 1973-1981); hybridization chain reaction (HCR) (Chem Soc Rev. July 17, 2017; 46(14): 4281-4298).

[0121] The primers and probes used in the above methods can be primers with limited composition, wherein at least one of the four standard nucleotide types is present at no more than two internal positions and / or the 5' end, as described in WO2019 / 033065 and WO2016172632.

[0122] Another aspect of amplification is signal amplification. When the amount of detectable nucleic acid is sufficient, it is advantageous to directly detect the sequence rather than to make more copies of the target (e.g., in PCR and LCR). Traditional direct detection methods (including Northern and Southern blotting and RNase protection assays) usually require the use of radioactive materials and are not suitable for automation. Other techniques attempt to eliminate the use of radioactive materials and / or improve the sensitivity of automated forms. Cyclic probe reactions (CPR) (Duck, Alvarado-Urbina, Burdick, & Collier, 1990b, BioTechniques, 9(2), 142–148) use a long chimeric oligonucleotide in which the central portion is composed of RNA and the two ends are composed of DNA. Hybridization of the probe with the target DNA and exposure to thermostable RNase H results in the digestion of the RNA portion. This destabilizes the remaining DNA portion in the double helix, releasing the remaining portion of the probe from the target DNA and allowing another probe molecule to repeat the process. Branched DNA (bDNA), described in Urdea et al., 1987, Gene, 61(3), 253–264, involves oligonucleotides with a branched structure, each carrying 35 to 40 tags (e.g., alkaline phosphatase). While this enhances the signal of the hybridization event, the signal of nonspecific binding is also increased.Other signal amplification methods include: invasive cleavage of nucleic acids (Prudent, Hall, Lyamichev, Brow, & Dahlberg, 2006, US Patent No. 7,011,944); hybridization chain reaction (HCR) specification 21 / 39 pages 26 CN 121219073 A (RMDirks & Pierce, 2004, Proceedings of the National Academy of Sciences of the United States of America, 101(43), 15275–15278, R.Dirks & Pierce, 2012, US Patent No. 8,105,778); and colorimetric detection based on G quadruplex DNase. CHA amplification (J. Am. Chem. Soc., 2013, 135(20), pp. 7430–7433). SMART signal amplification (Biotechniques March 2002; 32(3):604-6,608-11).

[0123] The amplification product can be detected qualitatively (i.e., a positive signal relative to a control) or quantitatively (signal intensity is related to the absolute or relative amount of the analyte that produced the amplification product). Detection may include, but does not require, further analysis, such as sequencing of the amplification product. The method provided by the present invention may also include direct detection of specific nucleic acids, such as specific target amplicons or a set of amplicons, in the capture reaction product or amplification reaction product. Therefore, the mixtures of the present invention can contain specialized probe sets, including TAQMAN™, which uses a hydrolyzable probe containing a detectable reporter and quencher motif, which can be released by a DNA polymerase with 5'->3' exonuclease activity (Livak, Flood, & Marmaro, 1996, U.S. Patent No. 5,538,848); molecular beacons, which use hairpin probes having a reporter and quencher motif at opposite ends (Tyagi, Kramer, & Lizardi, 1999, U.S. Patent No. 5,925,517); fluorescence resonance energy transfer (FRET) primers, which use a pair of adjacent primers having a fluorescent donor and acceptor motif, respectively (Wittwer, Ririe, & Rasmussen, 2001, U.S. Patent No. 6,174,670); and LIGHTUP™, a single short probe that fluoresces only upon binding to a target (Kubista & Svanvik, (2001, US Patent No. 6,329,144).Similarly, SCORPION™ (Whitcombe, Theaker, Gibson, & Little, 2001, U.S. Patent No. 6,326,145) and SIMPLEPROBEST™ (Wittwer et al., 2003, U.S. Patent No. 6,635,427) use a single reporter / dye probe. Amplicon detection probes can be designed according to the specific detection method used, and as discussed in the aforementioned patents. Other detection methods include gel electrophoresis, mass spectrometry or capillary electrophoresis, melting curves, nucleic acid-based fluorescent chelating dyes such as SYBR™ Green, or detection of amplified products using 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 of interest. For example, it can refer to the amplification of multiple sequences from the same sample or the amplification of one of several sequences in a sample, as described in George T. Walker, Nadeau, & Little, U.S. Patent No. 5,422,252, 1995; and George T. Walker, Nadeau, Spears, et al., U.S. Patent No. 5,470,723, 1995, which provides examples of multiple 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 for the direct quantification and clonal amplification of nucleic acids, including DNA, cDNA, or RNA, thereby allowing direct quantification of the amount of target nucleic acid. Digital PCR achieves this direct quantification by distributing a single target nucleic acid molecule present in a sample into multiple aliquots in many separate reaction chambers, which enable the amplification products to be localized and concentrated to detectable levels. Preferably, the sample is dispensed such that most aliquots (e.g., at least 50%, 75%, 90%, 95%, or 99%) receive zero or one of each target nucleic acid molecule to be detected. When the reaction vessel contains oil beneath the gel layer, a micropipette can deliver a desired volume of one or more reaction mixtures to the oil layer, forming the desired number of oil-water emulsion droplets. The reaction volume ranges from picoliters to microliters. The number of emulsion droplets ranges from one to one million. The micropipette is a multichannel pipette. The pipette tip for delivering the reaction mixture can be a bundle of capillaries. Optionally, the reaction mixture can be delivered directly into the gel layer to form multiple reaction compartments. The gel is warmed to the desired temperature to initiate the reaction in the compartments.Following PCR amplification, the presence of a signal in any droplet or compartment indicates the presence of the target nucleic acid, and the count of droplets or compartments containing the final PCR product is a direct measure of the absolute amount of the target nucleic acid. Individual nucleic acid molecules are typically captured or isolated by dilution, which can be performed in capillaries, microemulsions, miniaturized chamber arrays, or on nucleic acid-binding surfaces. The basic methods of digital PCR are described, for example, in Sykes et al., Biotechniques 13(3):444-449, 1992; and Vogelstein and Kinzler, Specification 22 / 39 pages 27 CN 121219073 A PNAS1999; 96:9236-41. Other forms of amplification described herein (such as transcription-mediated amplification) can be similarly performed digitally. The digital PCR methods described in this invention can also be used for immunoassays using nucleic acid tags (digital immunoassays, as described in US Patent 9,896,717), or for other assays for absolute quantification of a single target in a sample or multiple 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 (i.e., amplicons) is monitored during the reaction process. The forms of real-time amplification differ primarily in the detection mechanisms used to monitor the reaction products. Detection methods are reviewed in Mackay, Arden, & Nitsche, 2002, Nucleic Acids Research, 30(6), 1292–1305, which are incorporated herein by reference.

[0127] The term “detection label” refers to any atom or molecule that can be used to provide or assist in providing a detectable (preferably quantifiable) signal and can be attached to nucleic acids or proteins. Labels can provide signals that can be detected by fluorescence, radioactivity, colorimetry, gravimetric analysis, magnetism, enzyme activity, etc. The detection label can be incorporated in a variety of ways: (1) the primer contains one or more labels, for example, attached to a similar structure in a base, ribose, phosphate or nucleic acid analog; (2) the triphosphate nucleotide is modified with one or more labels on a base or ribose (or a similar structure in a nucleic acid analog); then the labeled nucleotide is incorporated into the newly synthesized chain by an elongation enzyme such as a polymerase; (3) the modified nucleotide contains a functional group that can be used to add a detectable label (after enzymatic reaction); (4) the modified primer contains a functional group that can be used to add a detectable label in a similar manner; (5) the labeled probe can be used, which is directly labeled and hybridizes with a portion of the amplicon; (6) the label can be incorporated into the amplification product; (7) the label can react with the byproducts of the amplification reaction.

[0128] The techniques, apparatus and methods described herein are improvements over the prior art in providing reaction containers. For example, PCR strips or plates or immunoassay ELISA plates are conventionally sold as empty wells.The present invention provides a reaction vessel having a preloaded gel layer permeable to a micropipette, optionally including oil beneath the gel layer. The preloaded gel reaction vessel allows for the preloading of some or all of the reaction mixture into the reaction vessel, thereby extending the shelf life of the reagents and preventing carryover contamination of the laboratory environment. Specifically, the reaction vessel is configured to contain the preloaded reaction mixture and is isolated using the gel layer within the vessel. After the sample is added, the reaction can begin beneath the gel layer to prevent the reaction mixture from contacting the atmosphere. The thermally induced reaction causes the temperature to rise above ambient temperature, subsequently changing the gel from a semi-solid to a liquid lighter than water, with the liquefied gel migrating to the top of the liquid phase of the reaction mixture. When the reaction is complete, the reaction vessel cools to ambient temperature, causing the gel to solidify and forming a non-flowing barrier covering the top of the aqueous reaction, thereby preventing leakage of amplified products and contamination of the laboratory environment due to improper sealing or handling of the vessel after the reaction.

[0129] Referring to FIG1 as an example, a reagent-free reaction tube is configured to contain the preloaded gel layer. After adding, for example, 20 μL of the master reaction mixture 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 will fall through the gel layer and settle to the bottom of the tube due to the density difference between the molten gel layer and the aqueous phase, as well as the surface tension of the gel layer surface. In this embodiment, the gel layer with a lower melting point, the salt-containing aqueous phase, and the larger volume ratio of the gel layer to the aqueous phase are beneficial for the added water layer to settle to the bottom of the tube during the first thermal cycle of the PCR reaction or the earliest possible time of the isothermal reaction. After the reaction, the molten gel layer cools and solidifies, forming a solid barrier on top of the liquid contents in the tube.

[0130] When only the gel layer is used, an equal volume of water layer may not settle to the bottom of the tube during the PCR run, or even below the level of the molten gel layer. To ensure that the water layer settles below the molten gel layer during the PCR run, a gel layer to water layer volume ratio of 2:1 or greater can be used, for example, 40 μL: 20 μL. However, with increasing volume ratios, the added reaction mixture moves further away from the bottom of the tube before PCR run, leading to prolonged heat transfer between the heat source and the reaction mixture, ultimately delaying the amplification reaction. Furthermore, the increased gel content in the tube requires longer melting time, further delaying the reaction. Therefore, pre-loaded pure wax gel reaction tubes may result in decreased reaction kinetics or a delayed Ct. Figure 2 shows an improved configuration containing a certain amount of mineral oil beneath the gel layer. To use this bilayer reaction tube, the user first breaks the gel layer with a micropipette tip or a needle-like plastic pick, then adds the constituent reagents (e.g., 20 μl) and / or the sample (5 μl) through the pores of the broken gel layer.To simulate the heat transfer conditions of traditional liquid PCR (where the constituent reagents and samples are at the bottom of the reaction tube before the first thermal cycle of the amplification cycle), centrifugation of the tube containing mineral oil and added sample using a microcentrifuge helps to bring the aqueous phase down to the bottom of the tube before the reaction. As in the embodiment shown in Figure 1, the bilayer reaction tube forms a non-flowing gel capping layer on top of the tube after the reaction.

[0131] Although the embodiments shown in Figures 1 and 2 provide reaction tubes capable of universally accommodating any reagents for contamination-protected PCR or isothermal amplification or any biochemical reaction, the reaction tubes involve less reagent preparation and pipetting operations, or are provided in a “sample-only” manner, which is desirable for users who want to reduce human procedural errors, speed up assay results, and improve laboratory efficiency. The embodiment shown in Figure 3 is configured to contain the complete constituent reagents or complementary subsets of reagents in the reaction vessel, wherein the complementary subsets of reagents are isolated in separate compartments using mineral oil and a gel layer. Premixing a subset of PCR reagents or isothermal amplification reagents at ambient temperature before thermal cycling or constant heating is undesirable because it can delay the reaction and delay the production of the target product or reduce performance by depleting primers, leading to nonspecific primer dimers or other byproducts. In this multilayer configuration, a subset of PCR or isothermal reagents, such as a 10 μL volume, is loaded at the bottom of a 0.1 mL test tube, which may be a primer mixture or enzyme mixture, followed by a volume of mineral oil, a volume of gel layer mixture, another volume of mineral oil, such as a 10 μL complementary subset of reagents, and another volume of gel layer mixture.

[0132] To use this embodiment for PCR or isothermal reactions, following the procedure described in the embodiment shown in FIG2, the user first breaks up the two gel layers using a pipette tip or a needle-like plastic pick, and then adds, for example, 5 μL of sample through the broken gel layer. To facilitate complete capture of small volumes of sample, such as 1.0 μL, in the liquid phase of the test tube, an additional layer of mineral oil is loaded between the two gel layers. Finally, the capped and sample-added tubes are briefly centrifuged using a microcentrifuge to collect all aqueous components at the bottom of the tubes for the most efficient thermal reaction during the first thermal cycle or isothermal amplification of the PCR reaction. The top gel layer is used to prevent liquid contents from flowing to all locations on the inner wall of the reagent tube during product manufacturing, storage, transportation, and / or assay processing. As in the embodiment shown in Figure 2, the six-layer reaction vessel forms a non-flowing gel layer on 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 a subset of the constituent reagents, which is covered with a layer of mineral oil and then topped with a layer of gel.This embodiment is superior to the embodiment containing multi-subset reagents when the assay procedure is performed in an open field at temperatures above ambient temperature (e.g., 40°C) for an extended period (e.g., several hours or more). Therefore, the gel layer may melt and liquefy at high temperatures. However, melting of this layer does not adversely affect the performance of the pre-loaded constituent reagents in the tube, as it is thermally stable at temperatures well below its chemical degradation temperature. 2. Nucleic Acid Detection Assay

[0134] The target nucleic acid or one or more loci therein can be detected using various binding assays involving nucleic acid primers and probes. For example, the target nucleic acid can be detected using oligonucleotides immobilized on a support. The target nucleic acid can also be detected using a capture probe in solution, which binds to both the target nucleic acid and the immobilized probe. In either form, a labeled detection probe can be used to detect the bound target nucleic acid. The target nucleic acid can also be detected by primers using target-specific extension or single-base extension assays. Nucleic acids can also be analyzed using a variety of sequencing methods, including Sanger dideoxy synthesis and Maxam-Gilbert chemical cleavage, as well as newer methods such as 454 pyrosequencing (Siqueira et al., J Oral Microbiol. 2012;:10.3402 / jom.v4i0.10743.doi:10.3402 / jom.v4i0.10743), Ion Torrent (see Hu et al., Human Immunology 82, 801-811 (2021)), and Illumina bridging amplification sequencing (see, for example, Slatko et al., Curr. Protoc. Mol. Biol. 122(1), e59 (2018)). Nucleic acids can also be analyzed using bisulfite sequencing and similar techniques used for epigenetic analysis and molecular counting. 3. Immunoassay

[0135] An immunoassay is a bioanalytical method in which the quantification of an analyte depends on the reaction between the analyte and an antibody (see Darwish, Int J Biomed Sci. 2006 Sep; 2(3):217–235). These reagents are antibodies, signal-generating markers, including the use of nucleic acid oligonucleotides to generate detectable signals, such as PCR methods (immunoPCR), sequencing (NGS, next-generation sequencing), or immunoisothermal amplification and separation of the matrix. Nucleic acid oligonucleotides can generate detectable signals through proximity reactions. Antibodies can be polyclonal or monoclonal. Immunoassay methods can be used to analyze a single analyte, or multiple analytes in parallel (e.g., at least 1, 5, 10, 20, or 100) in the same sample, by employing different methods.

[0136] Signal-generating markers in immunoassays include radioactive atoms (primarily 125I, 3H, and 14C). The use of radioactive markers provides extremely sensitive and fairly accurate assays; however, they also have drawbacks (e.g., health hazards, special considerations for reagent handling, staff training, short isotope half-lives, and expensive instruments for radioactive counting). Therefore, alternative non-radioactive markers have been introduced, such as enzymes, fluorescent probes, chemiluminescent substances, metals and metal chelates, and liposomes. Enzyme labeling can amplify the signal, potentially increasing the sensitivity of the method.

[0137] Matrix materials used for separating immune complexes formed by immunoassay reactions include charcoal, polyethylene glycol, secondary antibodies, microbeads (including magnetic beads), or multiwell plates, wherein a component of the analyte or antibody is coated on the surface at the bottom of the wells, forming immune complexes on the surface of the wells. The use of these plates facilitates washing steps and reagent pipetting, thereby promoting automation.

[0138] In an exemplary radioimmunoassay, a known amount of a target is made radioactive, typically by labeling it with a gamma radioisotope of iodine (such as 125-I) or tritium bound to tyrosine. This radiolabeled target is then mixed with a known amount of antibody against that target, resulting in specific binding. A sample containing an unknown amount of the same target is then added. The unlabeled target in the sample competes with the radiolabeled antigen for antibody binding sites. As the concentration of the unlabeled target increases, more target binds to the antibody, replacing the radiolabeled variant and reducing the ratio of antibody-bound radiolabeled target to free radiolabeled target. The bound target is then separated, and the radioactivity of the remaining free (unbound) target in the supernatant is measured using a gamma counter.

[0139] Enzyme immunoassay (EIA) is similar to RIA, but the label is an enzyme instead of a radioisotope. The basic approach to using enzymes as immunoassay markers involves coupling enzyme molecules to one of the immunoassay reagents (analytes or antibodies) using appropriate chemical techniques, followed by a conventional immunoassay reaction. After separating the bound and free portions, enzyme activity in either portion is monitored. This is achieved by adding a substrate and subsequently monitoring the conversion of the substrate to a product. The product must exhibit a measurable physical or chemical difference compared to the substrate. For example, a colorless chromogenic substrate is converted to a colored product by enzyme labeling. The colored product can be easily measured using a spectrophotometer. The measured signal is then correlated with the analyte concentration.

[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 enables the analysis of protein samples immobilized on a solid support, such as magnetic beads, using specific antibodies.ELISA can be performed in 96-well or 384-well polystyrene plates, where antibodies and proteins are passively bound to magnetic beads. Immobilizing ELISA reactants on magnetic beads facilitates the separation of bound and unbound substances during the assay. Specification 25 / 39 pages 30 CN 121219073 A

[0141] Various forms of ELISA generally comprise four basic elements: (1) coating / capture – immobilizing antigens directly or indirectly onto the surface of a solid support (e.g., wells of a multi-well plate, paper, spherical beads, or rods, as described below); (2) blocking – adding irrelevant proteins or other molecules to cover all unsaturated surface binding sites of the reaction vessel; (3) probe / detect – incubating with antigen-specific antibodies that bind to the antigen of interest with affinity; and (4) signal measurement – ​​detecting the signal generated on the affinity-bound antibody by a direct or secondary coupling enzyme, as described in more detail below.

[0142] ELISA involves multiple rounds of binding and washing to ensure specific binding. Separating reagents at each step using gels in horizontal and / or vertical chambers allows for stepwise reactions without cumbersome liquid handling. Protein-coated beads can pass through gel-separated reagent layers for washing and binding prior to the final detection stage.

[0143] ELISA can be performed in various forms, primarily plate-based, chip-based, bead-based, paper-based (PVDF, nitrocellulose, cellulose), or rod-based. The general concept of performing ELISA on these platforms is essentially similar to the conventional immunoassays mentioned above. Plate-based, chip-based, bead-based, and / or rod-based ELISAs can be used for high-throughput screening through automation, while paper-based ELISAs are primarily used for point-of-care testing. Rod-based ELISAs are performed by anchoring analyte-binding molecules to the bottom tip of a disposable rod. The rod can be a glass rod, optical fiber, plastic, metal rod, capillary, electrode, or magnetic rod with / without a cap. The rod can be used in conjunction with a detection kit for automated, high-throughput immunoassays capable of simultaneously detecting multiple analytes in multiple samples, or alternatively, for automated, point-of-care immunoassays. Optionally, the rod is made of a conductive magnetic material and serves as an electrode for capturing analytes for real-time detection (as described in patent US20130274131 by measuring a magnetic sensor or magnetoresistance), and / or for ECL reactions. Real-time detection based on the rod can be performed using a magnetic sensor (US20130274131). In this case, for example, the detection antibody is coated with magnetic beads. The attachment of the beads to the rod causes a change in the rod's magnetoresistance (magnetic sensor). Alternatively, the rod can be used in conjunction with magnetic bead-based immunoassays or nucleic acid signal amplification assays (such as HCR). The rod can also assist in moving beads through different gel layers or reaction chambers and can be used as an electrode for ECL reactions.The test kit is a reaction dish containing diffusion-binding and non-diffusion-binding reagents for immunoassay, wherein the reagents are separated by a permeable gel in this invention. A test kit assembly tray can be used to hold multiple test kits and rods into the assembly for use in immunoassay devices for high-throughput testing.

[0144] Magnetic beads with different surface chemical compositions (including Ni2+, sulfonyl SMCC, toluenesulfonyl, carboxyl) are designed to immobilize proteins or antibodies or molecules of interest for ELISA. The beads serve as a platform for molecule immobilization to initiate the ELISA, followed by subsequent washing and binding steps by moving the beads through the gel layer. Bead-based ELISA allows for automated processing of high-throughput samples, thus reducing turnaround time. The gel chambers separate the washing solution and the binding molecules, allowing bead-based ELISA to be performed in a one-pot configuration. Guiding the magnetic beads through different chambers or gel layers for specific reactions enables continuous reactions without repeated liquid handling.

[0145] ELISA subtypes can also be classified as direct, indirect, and sandwich types. After immobilizing the antigen of interest, the analyte is detected directly (with a labeled primary antibody) or indirectly (e.g., a secondary antibody specifically labeled for the primary antibody, or a biotin-streptavidin complex for amplification) by adsorption directly onto the assay bead or indirectly by capture antibodies attached to the bead. Specificity of the secondary antibody to the primary antibody can be achieved using capture antibodies and primary antibodies from different host species (e.g., mouse IgG and rabbit IgG, respectively). For sandwich assays, it is advantageous to use cross-adsorbed secondary antibodies to remove any secondary antibodies that may have affinity for the capture antibody. In sandwich ELISA, the analyte binds between two primary antibodies, each detecting a different epitope of the antigen—the capture antibody and the detection antibody. The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP) or ECL immunoassays, as described in the Journal of Electroanalytical Chemistry, Vol. 919, August 15, 2022, 116511. Other usable enzymes include β-galactosidase, acetylcholinesterase, and catalase. A wide range of substrates are available commercially for use with HRP or AP conjugates in ELISA, as described on pages 26 / 39 of the instruction manual (CN 121219073 A). The choice of substrate depends on the required assay sensitivity and the instrument available for signal detection (spectrometer, fluorometer, or photometer).

[0146] Figure 5 illustrates a preferred embodiment of the invention for 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 compartment barrier to isolate all individual reagents in a manner consistent with a typical magnetic bead-based ELISA assay workflow for determining the target antigen. To facilitate the transport of beads through each gel layer in the container, a heating element is attached to the front side of an external magnetic sheet, which can be configured as a 2-in-1 module. As the assay continues to pass the beads through the gel layers, the heating element is engaged and positioned at the target gel layer, where heating helps reduce the viscosity of the barrier, thereby allowing the beads to be transported from compartment to compartment or from layer to layer. To further facilitate this traversal movement through one or more gel layers, pairs or multiple magnetic sheets can be introduced, reducing the number of beads per magnet and thus increasing the magnetic force on the beads.

[0147] The manufacture of this reaction container involves a similar manufacturing process as described in the amplification reaction container section of this invention. In short, add the following reagents and gel layers in the following order from bottom to top to a plastic container with an inner diameter of, for example, 1.5 or 2.0 mL and an inner diameter of, for example, 5.0 mm: 100 μL stop solution, 20 μL gel layer, 100 μL substrate solution, 20 μL gel layer, second wash buffer, 20 μL gel layer, 100 μL detection antibody solution, 20 μL gel layer, 100 μL first wash buffer, 20 μL gel layer, 50 μL solution containing magnetic beads coated with capture antibody, 20 μL layer. After cooling, seal the container with a foil seal. Optionally, for example, 10–20 μL of mineral oil is added to each aqueous layer to enhance the isolation of reagent components. Optionally, the reaction vessel contains two aqueous reaction mixtures separated by the gel layers. The bottom aqueous reaction mixture contains a substrate of a signal amplification element, such as HRP, AP, or ECL. The top aqueous reaction mixture contains magnetic beads coated with capture antibody and labeled detection antibody. After the sample is added to the top aqueous solution and incubated at a specific temperature, the beads can move through the first gel layer into a first aqueous reaction mixture containing an enzyme substrate or ECL substrate to produce a chemiluminescent or color reaction.

[0148] In use, the foil seal is peeled off from the container and the top gel layer is then pierced with an inert rod. Then, for example, 100 μL of sample is added to the liquid phase of the bead chamber. To accelerate the immunobinding reaction, a device comprising a thermal module and a magnetic module is used to resuspend and mix the magnetic beads in the sample solution by alternately switching the electromagnetic field of the magnetic sheets. The heating element is then placed on the second gel layer and briefly heated thereon, and the actuated magnetic module is moved forward, thereby dragging the beads through / through the gel layer and into the first washing chamber. Next, the magnetic module is placed in the central portion of the target chamber and programmed electromagnetic mixing is performed to effectively wash the beads. By following these similar operating procedures, the beads enter the detection chamber, where they produce a detectable color through an enzymatic reaction therein.Optionally, to control the color intensity of the detection chamber over time, a heating module connected to the magnet module is placed on the bottom gel layer and heated to melt and liquefy the gel layer. As a result, the colored reaction mixture falls through the liquefied gel layer and merges with the termination solution below, thereby allowing endpoint quantification of the target antigen with the stable color of the reaction mixture. The color intensity observed or measured in the merged reaction mixture is proportional to the level of antigen molecules present in the sample. 4. Integrated Bioprocessing

[0149] Reaction vessels can be used to integrate sequential reactions.

[0150] For example, DNA / RNA purification is the process of isolating and extracting DNA or RNA from biological samples such as blood, tissue, cells, or microbial cultures. The purification process involves removing impurities and contaminants that may affect the quality and integrity of the genetic material.

[0151] Various types of impurities or contaminants may be present in DNA or RNA samples, depending on the source and the processing steps involved. Biological and environmental samples often contain PCR inhibitors, which significantly inhibit the activity of DNA polymerase and reduce the sensitivity and efficiency of PCR reactions. Instructions for Use, Pages 27 / 39, 32 CN 121219073 A

[0152] DNA polymerases vary in their tolerance to PCR inhibitors. For example, DNA polymerases widely used in real-time and quantitative PCR (such as Taq polymerase) are completely inactivated in the presence of small amounts of blood (0.004% to 0.2%). The problem of PCR inhibition from DNA samples can be addressed in various ways, particularly through sample dilution and purification. However, this inhibition remains a problem for many PCR-based human blood assays because even after purifying DNA from blood, trace amounts of PCR inhibitors can produce high false-negative rates. Another example is the detection of agricultural-related microorganisms, infectious disease pathogens, and bioterrorism-related pathogens in soil samples. Direct extraction of total DNA from soil samples results in the co-extraction of humic acid, the most potent soil inhibitor in PCR analysis. Humic substances represent partially characterized (not yet fully characterized) polyphenols produced during the decomposition of organic matter. Other inhibitory components include fulvic acid, polysaccharides, and metal ions, which may be present in soil samples at varying concentrations.

[0153] Some of the most common impurities or contaminants include:

[0154] Proteins: Proteins can be co-purified with DNA or RNA and may interfere with downstream applications such as PCR, sequencing, and gene expression analysis.

[0155] Salts: High salt concentrations can affect the quality of DNA or RNA samples and can inhibit enzymatic reactions such as PCR.

[0156] Organic solvents: Organic solvents commonly used in DNA / RNA extraction protocols (such as phenol or chloroform) can be co-purified with DNA or RNA and may affect downstream applications.

[0157] Residual cell debris: Cell debris (such as lipids, polysaccharides, and other macromolecules) can be co-purified with DNA or RNA and may affect the quality and quantity of the sample.

[0158] Nucleases: Nucleases are enzymes that can degrade DNA or RNA and may be present in the sample or introduced during processing.

[0159] Environmental contaminants: Contaminants (such as dust, aerosols, or other microorganisms) can enter the sample during collection or processing and may affect the quality of DNA or RNA.

[0160] Removing these impurities or contaminants from DNA or RNA samples ensures the accuracy and reliability of downstream applications. Purified DNA or RNA can be used for various downstream applications, such as PCR, sequencing, cloning, gene expression analysis, etc.

[0161] There are several methods for DNA / RNA purification, and the choice of method depends on the sample type and downstream application. Here are some common DNA / RNA purification methods:

[0162] 1) Organic solvent extraction: This method involves the use of organic solvents such as,

[0163] phenol-chloroform, β-mercaptoethanol and polyvinylpyrrolidone (PVPP-40), chloroform-isoamyl alcohol, isopropanol and salts, and TRIZOL™.

[0164] Nucleic acids can also be treated with proteinase K below the gel layer, for example, at 95°C for 10 minutes.

[0165] 2) Centrifugation

[0166] 3) Phase separation: After centrifugation, the sample is separated into three distinct layers: an aqueous phase containing nucleic acids, a lower organic phase containing denatured proteins, and an intermediate phase containing other contaminants.

[0167] 4) Precipitation: The nucleic acids are then precipitated with ethanol or isopropanol, washed with washing buffer, and then dissolved in buffer for further analysis.

[0168] This invention can be applied to organic solvent extraction by pre-distributing all the necessary reagents in one container: for example, from top to bottom, these layers are gel layer 1, lysis solution, gel layer 2, organic solvent, gel layer 3, voids, ethanol, gel layer 4, and elution buffer. In use, the sample is first added to the top of gel layer 1. After gel layer 1 melts, the sample is mixed with the lysis solution. After lysis, gel layer 2 melts, and the sample is mixed with the organic solvent. After vortexing 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 then precipitated through the ethanol layer. After centrifugation, the precipitated nucleic acids pass through gel layer 4, while the ethanol layer is blocked, resulting in purified nucleic acids in the bottom elution buffer layer. This embodiment of the invention can also be combined with other layers mentioned elsewhere in this application. This format can include all reagents in one container, protecting the user from exposure to toxic reagents. This integrated design also makes the entire procedure easier and simpler, which can be further adapted to automated instruments.

[0169] Column-based purification and size-selective purification: This method involves using columns filled with resin or membranes, or silica centrifuge columns, which selectively bind DNA or RNA. A sample is loaded onto the column, nucleic acids are captured, while impurities pass through. After washing the column, the purified DNA or RNA is eluted with a buffer. DNA size-selective purification is a process that allows the separation of DNA fragments within a specific size range from a mixture of DNA fragments.

[0170] This technique is commonly used in molecular biology experiments requiring specific DNA fragment sizes, such as cloning, PCR, and sequencing. One of the most common methods is column-based purification. DNA fragments can be separated by size using size exclusion columns, which separate DNA fragments based on their size and charge.

[0171] After loading a sample onto the column, the DNA fragment of interest can be eluted using an appropriate buffer.

[0172] The present invention can be applied to column-based DNA separation by pre-dispensing all the necessary reagents in a single container: for example, from top to bottom, these layers are gel layer 1, gel layer 2, gel layer 3… and finally, voids. The binding affinity and melting point of different gel layers for DNA / RNA vary due to different wax-oil ratios. Therefore, different layers will bind DNA / RNA of different size ranges. In use, the sample is added on top of gel layer 1. The tube is then centrifuged until all liquid phase has passed through the gel layer and reached the bottom void region. DNA / RNA is removed by melting a specific gel layer and then transferring it with a pipette. For example, the targeted DNA / RNA is bound in gel layer 2. Gel layer 1 is first melted and removed with a pipette. Then gel layer 2 is melted and transferred to a clean tube containing the DNA / RNA contained in the gel layer. This embodiment of the invention can also be combined with other gel layer forms described elsewhere in this application. The advantage of this embodiment is that it includes all reagents in one container. This integrated design makes the entire procedure easier and simpler, which can be further adapted to automated instruments.

[0173] Purification based on magnetic beads: This method involves the use of magnetic beads coated with ligands that can selectively bind DNA or RNA. Ligands include, for example, -OH, -SH, -NH2, and -COOH. When the sample is mixed with the beads, DNA or RNA binds to the magnetic beads, which are trapped by a 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 commonly used in molecular biology experiments that require specific DNA fragment sizes, such as cloning, PCR, and sequencing. The basic steps of magnetic bead-based purification are as follows:

[0174] (a) Lysis: The sample is first lysed to release nucleic acids from cells or tissues. Lysis buffers typically contain detergents that help break down cell membranes and release nucleic acids.Lysis reagents include, for example, sodium dodecyl sulfate (SDS), Triton X-100, guanidine thiocyanate or chloride salts, proteinase K or other proteases that degrade proteins, lysozyme, and combinations thereof.

[0175] (b) Binding DNA to magnetic beads: Magnetic beads are added to a sample containing the DNA fragment of interest, and the beads are mixed 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 or other water-miscible alcohols, DMSO, THF, NaCl or other salts.

[0176] (c) Washing magnetic beads: The beads are washed to remove any contaminants or unbound DNA fragments. Washing reagents may include, for example, water, Tris-HCl buffer, PBS buffer, ethanol, isopropanol, propanol, butanol or other water-miscible alcohols, DMSO, THF, NaCl or other salts, and any combination thereof.

[0177] (d) Eluting DNA: The DNA fragment of interest is eluted from the beads using a suitable buffer or solvent. Elution reagents include, for example, water, Tris-HCl buffer, PBS buffer, NaCl or other salts and any combination thereof.

[0178] Some advantages of magnetic bead-based purification include high yield and purity of DNA fragments; rapid and efficient purification; ease of automation and expansion for high-throughput applications. However, magnetic bead-based purification also has some potential disadvantages, including: higher cost compared to other purification methods; a limited range of DNA fragment sizes that can be purified using certain types of magnetic beads; and the possibility of nonspecific binding of DNA fragments to magnetic beads, resulting in the loss of some DNA fragments.

[0179] By pre-dispensing all the necessary reagents into a single container, the present invention can be applied to magnetic bead-based DNA or RNA isolation. Figure 6 is a preferred embodiment of an integrated magnetic bead-based DNA / RNA purification system. In terms of configuration, the reaction vessel of this integrated purification system is similar to those described in the Integrated Immunoassay System section. However, since most commercially available magnetic bead reagent kits require high-temperature (e.g., 50–60°C) heating and elution procedures to improve yield and purity, preventing the collapse of the heat-sensitive gel layer during purification is a challenge. Therefore, when configuring such an integrated container system, a cooling operation is incorporated for the gel layer directly contacting the lysis or elution chamber. The heating and cooling of the container portion is controlled using, for example, a Peltier device, which can function as a thermoelectric cooler or thermoelectric generator depending on the polarity of the DC current applied to the device. Furthermore, a thicker gel layer can be used as the interface between the two chambers to reduce the risk of gel layer failure during processing.

[0180] To use the container shown in Figure 6, the user peels off the foil seal (not shown) at the top of the container and breaks up the top gel layer using an inert rod or pipette tip. The user then transfers 100–200 μL of the original sample into the lysis chamber, which contains, for example, 100 μL of lysis buffer. After resealing the container opening with the cap, the heating and cooling modules are engaged to contact the wall portion of the lysis chamber. The heating element wraps around the liquid portion of the chamber for lysis, and the cooling element is located on the downstream gel layer for barrier protection. The lysis process occurs, for example, at 50–60°C, while barrier cooling occurs, for example, at 15–20°C. After, for example, 5–10 minutes of lysis, the operation of the modules is switched from cooling mode to heating mode by switching the polarity of the applied DC power supply. As a result, the affected gel layer is heated and partially melted, causing the lysis solution to fall through the gel layer and merge with the DNA / RNA binding solution and magnetic beads in the bead chamber. To ensure thorough mixing of the aqueous solution with the beads in the bead chamber, a pair of electromagnets is allowed to be actuated in an alternating switching manner, for example, for 5–10 minutes. Next, a heating element is placed on the next gel layer, where a brief period of heating is applied to soften the gel barrier, allowing the beads to penetrate and pass through with less resistance. The beads are driven to penetrate and pass through the heated gel layer by moving an externally actuated electromagnet downstream. After the beads enter the first washing chamber, the pair of electromagnets is positioned in the center of the chamber, where actuation is initiated and operated in an alternating switching manner, for example, for 1–2 minutes. This washing step removes most of the impurities carried on the beads. In subsequent washing chambers, this washing procedure is repeated for the next two washing steps, each taking approximately 1–2 minutes to complete. Finally, the washed beads are conveyed to the elution chamber for thermal elution via a heating and electromagnetic module. Similarly, to prevent the upper gel layer from collapsing when heating the elution chamber at, for example, 50–60°C, a pair of thermoelectric coolers are placed on the upper gel layer and maintained at cooling conditions (e.g., 10–20°C) until elution is complete. When elution is complete, after approximately 3-5 minutes, the beads are transferred back to the third washing chamber, leaving a pure elution buffer containing purified DNA / RNA for subsequent processing or reactions, such as integration of downstream amplification. 5. Reaction vessel for integration of NGS library preparation

[0181] Next-generation sequencing (NGS) is a technology that allows for rapid and accurate sequencing of the entire genome or targeted regions of DNA or RNA. NGS utilizes massively parallel sequencing, which means that many sequences can be read simultaneously, making the process faster and more efficient than traditional sequencing methods. DNA libraries are prepared from biological samples and used as starting material for sequencing. Library preparation typically involves several steps: DNA fragmentation:

[0182] NGS library preparation begins with DNA fragmentation. This step breaks long DNA strands into smaller fragments that can be sequenced.There are two main methods for DNA fragmentation: enzymatic fragmentation and mechanical fragmentation. Enzymatic fragmentation involves using restriction enzymes or other enzymes to cut DNA into smaller fragments. Some enzymatic methods do not require end repair of the fragmented DNA. On the other hand, mechanical fragmentation uses physical forces (such as ultrasound or Covaris shearing) to break the DNA strands. End Repair:

[0183] After fragmentation, DNA fragments typically undergo an end repair step. This step involves adding nucleotides to the ends of the fragments to repair any damage that may have occurred during fragmentation. Repaired ends of DNA are necessary for adapter ligation and subsequent PCR amplification. Adapter Ligation:

[0184] The next step in NGS library preparation is adapter ligation. Adapters are small synthetic DNA sequences added to the ends of DNA fragments, enabling them to bind to sequencing platforms. Adapters can be ligated to DNA using various methods, including T4 DNA ligase, which catalyzes the formation of phosphodiester bonds between the adapter and the DNA fragment. PCR Amplification:

[0185] Once the adapters are ligated to the DNA fragments, the library is subjected to PCR amplification. PCR amplification is used to generate millions of copies of DNA fragments to provide sufficient material for sequencing. PCR can also introduce bias or errors, so optimizing reaction conditions to minimize these effects is important. Bead Clean-Up:

[0186] The final step in NGS library preparation is bead clean-up. This step removes any unligated adapters, adapter dimers, or other impurities from the library. Bead clean-up involves using magnetic beads that bind to the DNA fragments and allow unwanted substances to be washed away. Once the DNA fragments are purified, the library is ready for sequencing.

[0187] Commercial kits can simplify the library preparation process and omit certain steps, such as post-ligation bead clean-up. 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 that the DNA ends are compatible with adapter ligation. Overall, NGS has revolutionized the field of genomics, enabling rapid and accurate sequencing of complex genomes and achieving breakthroughs in fields such as medicine, agriculture, and environmental science.

[0188] This invention provides a novel method for preparing NGS libraries by utilizing a reaction vessel comprising 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 vessel, eliminating the need for multiple tubes or plates and reducing the risk of contamination or material loss.

[0189] Referring to Figure 7, the first aqueous reagent layer contains enzymatic fragmentation reagents or end-repair reagents, which are used to cleave DNA into smaller fragments and repair any damage that may occur during fragmentation.The enzymes used in this aqueous layer are thermally unstable, meaning they can be inactivated by mild heat treatment, making them easily removed before the next step. The second aqueous layer contains adapter ligation reagents for attaching adapters to the ends of DNA fragments. This layer is separated from the first layer by a gel layer to prevent mixing of the two layers. The third aqueous layer contains library amplification reagents to provide sufficient DNA fragments for sequencing. This layer is separated from the second layer by another gel layer.

[0190] Overall, this reaction vessel design provides a convenient and efficient method for NGS library preparation, allowing the entire process to be completed in a single vessel. The use of gel layers helps prevent mixing of different reagent layers. This approach has the potential to simplify the library preparation process and improve the reproducibility and reliability of NGS results.

[0191] In some embodiments, intact DNA is added to the first aqueous layer containing enzymatic fragmentation reagents using a micropipette tip that penetrates the top gel layer. This method ensures that the DNA is uniformly dispersed in the first reagent layer. The reaction vessel is then incubated, for example, at 25°C for 30 minutes, during which time the enzymatic fragmentation reagents cleave the DNA into smaller fragments. Once the enzymatic fragmentation step is complete, the reaction vessel is incubated at, for example, 50°C for 10 minutes to inactivate the heat-labile enzyme. This step ensures that the enzyme is no longer active and will not interfere with subsequent steps, while maintaining the integrity of the second and third gel layers.

[0192] The second gel layer is then broken using a micropipette tip to mix the fragmented DNA with the adapter ligation reagent in the second aqueous reagent layer. The reaction vessel is then incubated at, for example, 25°C for 30 minutes to ligate the adapters to the DNA fragments. After the adapter ligation step is complete, the third gel layer is broken using a micropipette tip to mix the ligation product with the library amplification reagent in the third aqueous reagent layer. Finally, the reaction vessel is incubated using a PCR program to amplify the library, producing sufficient DNA fragments for sequencing.

[0193] Mechanically sheared DNA refers to DNA fragmented using mechanical means (such as ultrasound or forced shearing) rather than enzymatic fragmentation. In this embodiment, DNA is added to a first layer of aqueous reagent containing end-repair reagent using a micropipette tip that penetrates the top gel layer. The end-repair reagent is then allowed to act on the DNA at, for example, 25°C for 30 minutes. After this, the heat-labile enzyme is inactivated by incubating the reaction vessel at, for example, 50°C for 10 minutes, while maintaining the integrity of the second and third gel layers. The second gel layer is then disrupted using a pipette tip, allowing the end-repaired DNA to mix with the adapter ligation reagent. The reaction vessel is then incubated at, for example, 25°C for 30 minutes to ligate the adapter to the DNA fragment.Following this, the third gel layer is disrupted using a micropipette tip, allowing the ligation product to mix with the library amplification reagent. Finally, the reaction vessel is incubated using a PCR program to amplify the library, resulting in a DNA fragment library that can be sequenced using NGS technology.

[0194] In the ligation-mediated PCR embodiment, the reaction vessel is used for a variant of the PCR technique, namely, amplifying specific regions of a DNA sequence. The process begins by adding intact DNA to a first layer of aqueous reagent containing restriction enzymes or enzymatic fragmentation reagents that cleave the DNA into smaller fragments. This step is performed using a micropipette tip that penetrates the top permeable gel. The reaction vessel is then incubated, for example, at 25°C–37°C for 30 minutes to allow the enzymes to activate. Next, the reaction vessel is incubated, for example, at 50°C for 10 minutes to inactivate the heat-labile enzymes while maintaining the integrity of the second and third gel layers. The second gel layer is then disrupted using a micropipette tip, allowing the fragmented DNA to mix with the adapter ligation reagent, thereby attaching the adapter to the ends of the DNA fragments. The reaction vessel is then incubated, for example, at 25°C for 30 minutes to allow the adapter to ligate to the DNA fragment. The third gel layer is then disrupted using a micropipette tip to mix the ligation product with PCR reagents, thereby initiating a ligation-mediated PCR process. Finally, the reaction vessel is incubated with a PCR program to amplify the DNA fragment, generating a large amount of the target DNA sequence.

[0195] In some embodiments, the process involves a reaction vessel with two distinct layers: a top gel layer and a lower aqueous reagent layer. 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 top gel layer allows for convenient addition of the sample using a pipette tip that can penetrate the gel layer.

[0196] Once the sample has been added, the reaction vessel is incubated for a specific enzyme fragmentation, end-repair, ligation, or library amplification reaction.

[0197] The incubation period may vary depending on the specific reaction and desired outcome.

[0198] During this incubation period, the enzymatic reaction takes place in the aqueous reagent layer, while the gel layer acts as a barrier to prevent contamination and ensure effective reaction conditions.

[0199] In some embodiments of the process, the reaction vessel comprises two layers of aqueous reagent separated by two layers of gel. This configuration allows two different enzymatic reactions to be carried out in the same vessel.

[0200] In one possible configuration, the first aqueous reagent layer may contain a specific enzymatic fragmentation reagent or an end-repair reagent, depending on the desired reaction.For example, compared to reactions in solution, on-bead tagmentation library prep uses bead-linked transposomes for a more uniform tagmentation reaction (see specification 32 / 39, page 37, CN 121219073 A). Bead-linked transposome chemistry integrates DNA extraction, fragmentation, library preparation, and library normalization steps. This reduces the number of workflow steps, requires a small sample input, and reduces hands-on and turnaround time.

[0201] These reagents are designed to break down DNA molecules into smaller fragments or repair DNA ends. The second aqueous layer may contain a ligation reagent for adding adapters to the ends of DNA fragments.

[0202] In another possible configuration, the first aqueous layer contains a ligation reagent, and the second layer may contain a library amplification reagent. The ligation reagent in the first layer is used to add adapters to the ends of DNA fragments, which are then amplified using the library amplification reagent in the second layer. This allows the creation of multiple copies of the desired DNA fragment, which can be further analyzed or manipulated as needed.

[0203] In some embodiments, the PCR-amplified library is purified by bead purification.

[0204] Magnetic beads and binding buffer are added to the completed library amplification reaction system. The binding buffer promotes the binding of the DNA library to the bead surface. The bead-DNA complex is then separated from the liquid using a magnetic rod, which is immersed in the reaction vessel to pull out the beads. Next, the magnetic rod with beads is immersed twice in 70% ethanol to wash the beads and remove any impurities that may remain after the amplification reaction. Finally, the magnetic rod with beads is immersed in elution buffer to elute the DNA library from the beads.

[0205] All publications, patents and patent applications, accession numbers, websites, etc., mentioned in this specification are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is explicitly cited separately. If different content is associated with accession numbers or other references at different times, the content that is valid at the effective filing date of this application shall prevail. The effective filing date is the date of the earliest priority application disclosing the accession number in question. Unless otherwise apparent from the context, any element, embodiment, step, feature, or aspect of the invention may be performed in combination with any other element, embodiment, step, feature, or aspect. Example 1: Preparation of Gel

[0206] Eight pieces of paraffin wax with a melting point of 50-52°C were added to an aluminum wax melting reservoir with internal dimensions of 100×140×50mm. Each wax block weighed 32g, and the total weight of the wax blocks was 256g. The wax reservoir was kept at a constant temperature and heated to 75°C to melt the wax blocks into a liquid. The height of the molten wax in the reservoir was approximately 25mm.Then, mineral oil was added to the wax reservoir until the connector liquid level reached 45 mm. The weight of the mineral oil added to the reservoir was determined to be 234.5 g using the bottle weight subtraction method. The resulting mixture of wax and mineral oil was stirred with a spatula at 75°C until a homogeneous and transparent liquid mixture was obtained, which could be used for gel dispensing and preparation of gel-containing reaction vessels. 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).

[0207] The molten mixture at 75°C was dispensed into 8-tube PCR strips using an 8-channel pipette equipped with an 8-aluminum nozzle adapter. The 8-nozzle adapter was heated at 75°C using a thermostat temperature controller.

[0208] Pure wax of EMD Millipore with a melting point of 42-44°C was melted at 60°C using the same wax reservoir as described above, and dispensed into 8-tube PCR strips using the same 8-channel heated pipette as described above. Example 2: Reagent-free reaction tubes containing pre-loaded monolayer gel

[0209] Two different types of reaction tubes were prepared using four 0.1 mL 8-tube PCR strips: i) strips containing gel; ii) gel and mineral oil bilayer strips. The performance of the resulting strips for amplification was evaluated compared to the liquid form in which the PCR tubes contained no foreign substances. Specification 33 / 39 pages 38 CN 121219073 A

[0210] To prepare the gel-containing tubes, 40 μL of liquefied paraffin was added to each of the two strips. As described in Example 1, 30 μL of mineral oil was first added, followed by 30 μL of a gel capping layer, a mixture of liquefied paraffin and mineral oil at a volume ratio of 1.25:1.0, to prepare gel-containing bilayer strips. After preparing the strips, 10 μL of the master mixture, 9 μL of a single-color (FAM) primer mixture, and 1 μL of DNA template were added to each strip, along with 12 empty tubes as liquid controls. The strips containing the reaction mixture were capped and placed in a cold block, then run on a Bio-Rad CFX96 thermal cycler. The thermal cycling was performed in a two-stage setting with the cap temperature set to 105 °C: 1) 90 °C for 0 min 15 s, 60 °C for 0 min 30 s, repeated for 9 cycles; 2) 95 °C for 0 min 15 s, 65 °C for 0 min 30 s, repeated for 39 cycles. Figure 8 shows representative amplification curves for the three types of tube reactions. Table 1 shows the Ct analysis results of the amplification curves for the three types of tubes. It can be seen that the performance of the double-layer tubing is comparable to that of the liquid form, while the single wax form shows an observable delay in Ct value and a lower signal than the other two forms.Figure 9 shows photographic images of the three tube types before and after thermal cycling, demonstrating the non-flowable cured wax or gel coating sealed on top of the liquid reaction mixture within the tube. Pore-only gel-mineral oil liquid 1 20.82 20.02 19.58 2 20.94 20.17 19.41 3 20.38 20.21 19.24 4 20.74 19.3 19.45 5 20.26 20 19.11 6 20.85 19.65 19.55 7 21.31 19.96 19.31 8 21.94 19.64 19.18 9 20.78 20.39 19.37 10 20.77 19.6 19.44 11 20.73 20.03 19.19 12 20.81 19.56 19.51 13 20.95 19.16 14 20.32 18.8 15 20.91 20.04 16 20.43 19.35 Mean 20.83 19.77 19.36 Standard Deviation 0.41 0.43 0.16 Coefficient of Variation 1.94% 2.20% 0.80% Table 1: PCR amplification curve results for the three reaction tubes.

[0211] The performance of these three tube formats was also evaluated using isothermal amplification reagents. For example, 10 μL of a 4-color HPV screening primer mixture, 10 μL of an enzyme mixture, and 5 μL of a positive control template were added to each of the three types of tubes, with two tubes per format. After being capped, the tubes were placed in a cold block and then loaded into a Bio-Rad thermal cycler for 72 cycles of isothermal reaction at a constant temperature of 60°C. Figures 10A-C show the amplification curves for the three tube formats. Table 2 shows the Ct analysis results of the amplification curves for the three reaction tubes. It can be seen that the three tube formats are comparable in terms of amplification speed and accuracy by Ct analysis. Instruction manual 34 / 39 pages 39 CN 121219073 A Table 2: Ct analysis results of 4-color amplification curves for three tube formats (double-layer format, gel-only format, and liquid format). Example 3: Multilayer reaction tubes containing pre-loaded reagents and gel layers

[0212] The preparation of the strips containing gel-mineral oil mixture and PCR reagents is as follows. Add 10 μL of monochromatic primer mixture to each tube of 0.1 mL 8-tube PCR strip, then add mineral oil as the second layer, the gel layer as the third layer, 10 μL of enzyme mixture as the fourth layer, mineral oil as the fifth layer, and the gel as the sixth layer. The composition of the two gel layers in this experiment is the same as that described in Example 2. The PCR reagents used in this experiment are the same as those described in Example 2.

[0213] To use the obtained reagent tubes for PCR reactions, the foil seal was peeled off the strips, and the two gel layers were pierced and broken using a needle-shaped plastic pick. Then, 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 sample-added strip was capped. The capped strip was centrifuged for a few seconds using a microcentrifuge. Finally, the centrifuged strip was placed in a cold block and then placed in a Bio-Rad thermal cycler for sample running. The thermal cycling was performed in a two-stage setting with the cap temperature set at 105°C: 1) 90°C for 0 min 15 s, 60°C for 0 min 30 s, repeated 9 times; 2) 95°C for 0 min 15 s, 65°C for 0 min 30 s, repeated 39 times, the same thermal cycling setting as described in Example 2. Figure 11 shows the amplification curves of the reactions of the two multilayer PCR reagent strips. Table 3 shows the Ct analysis results of the amplification curves shown in Figure 11, indicating that the amplification speed and accuracy are comparable to those shown in Table 1. Figure 12 shows photographic images of the tube strip before and after thermal cycling, demonstrating the non-flowable, cured gel coating sealed on top of the liquid reaction mixture within the tube. Multilayer Reagent 1 19.82 2 18.48 3 18.63 Instruction Manual 35 / 39 Page 40 CN 121219073 A 4 18.88 5 19.39 6 18.24 7 19.35 8 18.62 9 19.21 10 18.62 11 19.46 12 18.5 13 18.62 14 19 15 19.26 16 19.17 Mean 18.94 Coefficient of Variation 2.34% Table 3: Ct analysis results of PCR amplification curves for multilayer reagent tubes.

[0214] In parallel with the PCR test tubes described above, multilayer isothermal amplification test tubes were also prepared and their performance was compared with that of the liquid form, as described below. 0.2 mL 8-tube multilayer UTI (urinary tract infection) detection strips were prepared in the same manner as described above. The resulting multilayer test tubes had the following layer configuration in sequence (from bottom to top): 10 μL enzyme mixture, mineral oil, gel layer, 10 μL 4-color UTI primer mixture, mineral oil, and gel layer. The completed strips were sealed with adhesive foil. After peeling the foil seal off the strips, the two wax-mineral oil gel barriers were pierced and broken using a needle-shaped plastic pick. Then, 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 using a conventional pipette, and the strip with the added sample was then capped. The capped strips were centrifuged for a few seconds using a microcentrifuge. Finally, the centrifuged strips were placed in a cold block, ready for operation on a thermal cycler.Liquid reaction tubes were prepared using empty 0.2 mL PCR strips by pipetting 10 μL of enzyme mixture, 10 μL of 4-color UTI primer mixture, and 5 μL of UTI positive control template into each tube of the strip. After brief centrifugation, the completed strips were placed side-by-side with multilayer reagent strips in a Bio-Rad CFX96 thermal cycler and run 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 using the multilayer UTI isothermal reagent strips and the control liquid form. Table 4 shows the Ct analysis results of the amplification curves shown in Figures 13A-D, indicating that the amplification speed and accuracy of the multilayer UTI reagent strips are comparable to those of the liquid form. Figure 14 shows photographic images of the multilayer reagent strips before (top) and after (bottom) isothermal amplification at 60 °C for 90 minutes, illustrating the formation of a widely solidified gel overlay on top of the liquid layer in the tube after sample run. Instructions for Use, Pages 36 / 39, 41, CN 121219073 A, Table 4: Ct analysis results of amplification curves for multilayer test tubes and liquid form tubes.

[0215] To evaluate the performance of multilayer test tubes in terms of assay sensitivity, HPV31 positive control template at a detection limit concentration of 7.5 copies per 25 μL was added to HPV31 test tube strips and run at 60°C for 90 minutes on a Bio-Rad thermal cycler. The liquid control run was performed alongside the HPV31 test tube strips. Figure 15 shows the amplification curves (FAM channel) of the reactions using HPV31 test tube strips and liquid tube strips, indicating that the assay sensitivity of the two reagent forms is comparable. Example 4: Evaluation of multilayer test tubes using clinical HPV samples

[0216] Multilayer HPV screening test tube strips were evaluated using clinical HPV samples. HPV screening assays were performed on 78 HPV samples using two types of HPV test tube strips, namely HPV16 (FAM channel) and HPV18 (Cy5 channel). These HPV samples were lysed swab specimens collected from 78 patients. The HPV reagent strips used in this evaluation study were prepared in the same manner as described in Example 2. Sample volumes were 5 μL, with a total volume of 16 μL (4 × 4), used for four replicate amplification reactions for each of the 78 samples. Isothermal sample runs for these samples were performed using the same heating conditions as described in Example 2. Table 5 summarizes the results of screening assays performed on the 78 HPV samples, demonstrating good consistency between the multilayer reagent form and the conventional liquid form. (Instructions for Use 37 / 39, Page 42, CN 121219073 A) Table 5: Results of HPV Screening Assays on 78 HPV Samples (ND - Undetectable; HR - High Risk)Example 5: Stability of Multilayer Reagent Tubes

[0217] The physical or mechanical stability of multilayer reagent tubes was evaluated by dropping strips of reagent tubes from a height of approximately 10 feet above the ground. The aim of this study was to examine the adhesion of the two wax-mineral oil gel layers to the tube wall after experiencing a free-fall impact on a hard surface. Prior to this drop impact test, 12 strips of reagent tubes were placed on a rack and stored overnight at -20 °C. The next day, the rack was removed from the -20 °C condition and allowed to equilibrate to the ambient temperature for approximately 15 minutes. Then, the 12 strips were dropped from a height of approximately 10 feet onto the laboratory floor, and this was repeated twice. Figures 16A and 16B show photographic images of the 12 strips before and after the drop impact, on pages 38 / 39 of the instruction manual, 43 CN 121219073 A. Visual inspection of the tubing showed that after three free falls, the two wax-mineral oil layers adhered well and stably to the inner wall of the tubing, indicating that the tubing has good physical stability and feasibility for transport and handling under moderate impact conditions.

[0218] The thermal stress stability of the multilayer reagent tubing was evaluated by simulating long-term transport conditions at ambient temperature and storing the reagent tubing in a commuter vehicle. For comparison, a 5-layer reagent tubing without a mineral oil layer encapsulating the reagent components at the bottom of the tube was prepared and compared with the conventional 6-layer tubing described in Example 2.

[0219] In this experiment, six 6-layer 4-color HPV18 reagent tubings and six 4-color 5-layer HPV18 reagent tubings were stored overnight at -20°C. The next day, the strips were removed and placed on a laboratory workbench at room temperature (20 to 25°C) for about 7 hours. Then, the strips were stored overnight again at -20°C. After repeating the freeze / thaw process three times, the strips were placed in a sealed foam box without coolant and stored in the trunk of a commuter vehicle for four consecutive days of daily commutes. The temperature inside the foam box was recorded using a temperature recorder, ranging from 15 to 30°C. The strips were then removed from the packaging and placed on a lab bench for reaction preparation, as described in Example 2. Briefly, 5 μL of HPV18 positive template was added to each strip of both types of layered form, with a Lod (limit of detection) concentration of 15 copies per reaction. The strips with added samples were run on a Bio-Rad thermal cycler at 60°C for 90 minutes. Figures 17A and 17B show the FAM and ROX amplification curves (HEX and Cy5 channels are not shown) of the two types of strips after repeated freeze / thaw cycles and long-term transport at ambient temperature. It can be seen that the reagent stored in the 5-layer tubes showed a significantly increased background signal and increased Ct value in the FAM channel compared to the reagent stored in the 6-layer tubes.

[0220] In view of the effectiveness of reagent isolation achieved by the multilayer design, the thermal stability of these multilayer reaction tubes at high temperatures was evaluated.Table 6 shows the thermal stability results of the 6-layer reagent tubes at ambient temperatures ranging from 23 to 45°C. In the thermal stability experiments, it was observed that when the tube strips were exposed to temperatures close to the melting point of the gel layer for a period of time, such as 40°C for 39 minutes, most of the gel layer thinned and eventually lost adhesion to the inner wall of the tube, causing the second water layer to fall through the gel barrier and merge with the first water layer at the bottom of the tube. To improve the thermal stability of these 6-layer reaction tubes, increasing the proportion of wax component in the wax-oil gel prolongs the tube failure time, but at the cost of increasing the hardness of the resulting gel layer, making it more difficult for pipette tips to penetrate. Ambient Temperature | Tube Failure Time 23°C | >7 months 35°C | 2 days 40°C | 39 min 45°C | 2-3 min Table 6: Thermal stability results of 6-layer reaction tubes at different temperatures.Instruction manual 39 / 39 pages 44 CN 121219073 A Figure 1 Figure 2 Instruction manual Figure 1 / 17 pages 45 CN 121219073 A Figure 3 Figure 4 Instruction manual Figure 2 / 17 pages 46 CN 121219073 A Figure 5 Instruction manual Figure 3 / 17 pages 47 CN 121219073 A Figure 6 Instruction manual Figure 4 / 17 pages 48 CN 121219073 A Figure 7 Instruction manual Figure 5 / 17 pages 49 CN 121219073 A Figure 8 Instruction manual Figure 6 / 17 pages 50 CN 121219073 A Figure 9 Instruction manual Figure 7 / 17 pages 51 CN 121219073 A Figure 10A Instruction manual Figure 8 / 17 pages 52 CN 121219073 A Figure 10B Figure 10C Instruction manual Figure 9 / 17 pages 53 CN 121219073 A Figure 11 Figure 12 Appendix to the Instruction Manual Page 10 / 17 54 CN 121219073 A Figure 13A Appendix to the Instruction Manual Page 11 / 17 55 CN 121219073 A Figure 13B Appendix to the Instruction Manual Page 12 / 17 56 CN 121219073 A Figure 13C Appendix to the Instruction Manual Page 13 / 17 57 CN 121219073 A Figure 13D Figure 14 Appendix to the Instruction Manual Page 14 / 17 58 CN 121219073 A Figure 15 Figure 16A Appendix to the Instruction Manual Page 15 / 17 59 CN 121219073 A Figure 16B Figure 17A Appendix to the Instruction Manual Page 16 / 17 60 CN 121219073 A Figure 17B Appendix to the Instruction Manual Page 17 / 17 61 CN 121219073 A.

Claims

1. A reaction vessel comprising a gel layer that can be penetrated by a micropipette tip disposed within the vessel.

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

3. The reaction vessel according to claim 1 or 2, further comprising a liquid aqueous reaction mixture above and / or below and / or inside the gel layer, optionally wherein one or more of the liquid aqueous reaction mixtures are used for detecting the analyte.

4. The reaction vessel of claim 3, wherein the liquid aqueous reaction mixture is located below the gel layer, the gel layer preventing the liquid aqueous reaction mixture from diffusing through the gel layer, the gel layer being permeable by a micropipette tip to deliver a solution to the reaction mixture for reaction to detect an analyte.

5. The reaction vessel of claim 3, wherein the delivered solution is a sample comprising the analyte.

6. The reaction vessel of claim 5, wherein the gel layer prevents the liquid aqueous reaction mixture from diffusing through the gel layer and is permeable by a micropipette tip at least in the temperature range of 18-35°C, optionally 20-25°C.

7. The reaction vessel according to any one of claims 1 to 6, wherein the reaction vessel comprises a plurality of gel layers, a plurality of oil layers and a plurality of liquid aqueous reaction mixture layers, the number of gel layers being different from the number of oil layers and / or the number of liquid aqueous reaction mixture layers.

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

9. The reaction vessel according to any one of the preceding claims, comprising: A first aqueous liquid reaction mixture, a first gel layer covering the first aqueous liquid reaction mixture and permeable by a micropipette tip, and a second aqueous liquid reaction mixture above the first gel layer; and A second gel layer covering the second liquid aqueous solution, which can be penetrated by a micropipette tip; as well as Optionally, a first mineral oil layer between the first liquid aqueous reaction mixture and the first gel layer, and optionally, a second mineral oil layer between the second liquid aqueous reaction mixture and the second gel layer.

10. The reaction vessel of claim 9, wherein one of the first and second liquid aqueous reaction mixtures is a sample, and the other comprises one or more reagents for reacting when the first and second liquid aqueous reaction mixtures are combined to detect an analyte in the sample.

11. The reaction vessel of claim 9, wherein the first liquid aqueous reaction mixture and the second liquid aqueous reaction mixture are each a partial reaction mixture, which, when combined with each other and the sample, produce a detection reaction.

12. The reaction vessel of claim 9, wherein optionally, a third liquid aqueous reaction mixture is present above the second gel layer, and the plurality of liquid aqueous reaction mixtures contain reagents for performing ordered biological treatment steps.

13. The reaction vessel according to claim 12, wherein the plurality of liquid aqueous reaction mixtures are used for nucleic acid sequencing library preparation or nucleic acid sample extraction or immunoassay, or biochemical or nucleic acid amplification and detection.

14. The reaction vessel according to claim 12, wherein the first liquid aqueous reaction mixture and the second liquid 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.

15. The reaction vessel of claim 12, wherein the first liquid aqueous reaction mixture and the second liquid aqueous reaction mixture contain different reagents, which, together with each other and the sample, are necessary for the detection of the reaction.

16. The reaction vessel of claim 15, wherein the combination of the first liquid aqueous reaction mixture and the second liquid aqueous reaction mixture is used for immunoassay and detection, optionally, the third liquid aqueous reaction mixture is located above the second gel layer for sequential reaction.

17. The reaction vessel of claim 3, wherein the liquid aqueous reaction mixture comprises magnetic beads.

18. The reaction vessel according to any one of the preceding claims, wherein a heater module is slidably connected along the outside of the reaction vessel for melting the gel layer when the heater is near the gel layer.

19. The reaction vessel according to any one of the preceding claims, wherein a magnetic module is connected to a magnetically movable module slidable along the outer or inner surface of the reaction vessel for moving magnetic beads from the liquid aqueous reaction mixture through a gel layer beneath the liquid aqueous reaction mixture.

20. The reaction vessel of claim 19, wherein the slidable magnetic module is slidable along the interior of the reaction vessel and connected to a rod to detect the reaction in the reaction vessel.

21. The reaction vessel of claim 3, wherein the liquid aqueous reaction mixture includes a rod for detecting the reaction in the reaction vessel.

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

23. The reaction vessel according to any one of the preceding claims, wherein each well of a multi-well plate (e.g., a PCR plate, a deep-well plate, a microtiter plate, or an ELISA plate) contains a gel layer.

24. The reaction vessel according to any one of claims 1 to 22, which is a tube or tubing (e.g., a PCR tube) arranged individually or as strips or a matrix, comprising a plurality of said vessels interconnected.

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

26. The reaction vessel according to any one of claims 1 to 23, wherein it is a hole on a chip or tube (e.g., a glass slide, silicon wafer, plastic slide, or metal slide), optionally having a plurality of holes, providing a plurality of reaction vessels, optionally the plurality of holes being connected by channels, optionally the holes comprising a membrane or glass fiber.

27. The reaction vessel according to any one of the preceding claims, wherein the inner surface of the reaction vessel is hydrophobic or hydrophilic.

28. The reaction vessel according to any one of the preceding claims, wherein the gel layer is made of a combination of an inorganic or organic or inorganic and organic polymer gel matrix, 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 of carrying out a reaction, comprising providing a reaction vessel as defined in claim 1 and penetrating the gel layer with a micropipette tip to deliver a solution from the micropipette tip, thereby causing the reaction to occur.

30. The method of claim 29, wherein the solution comprises a sample and optionally a reaction reagent.

31. The method of claim 29 or 30, wherein the gel layer melts during and after the reaction and then re-solidifies.

32. The method according to any one of claims 29 to 31, further comprising breaking the gel layer with an inert rod or a micropipette tip with a stopper thereon before penetrating the gel with the micropipette tip to deliver one or more liquid aqueous reaction mixtures to the reaction vessel.

33. The method of claim 32, wherein the stopper in the suction tip is wax or gel.

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

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

36. The method according to any one of claims 29 to 35, wherein the solution is a liquid aqueous reaction mixture comprising all reaction reagents, including a sample for detecting the reaction of the target in the sample or a portion of the reaction reagents for detecting the reaction of the target in the sample.

37. The method according to any one of claims 29 to 36, wherein the liquid aqueous reaction mixture is delivered into an oil layer or a gel layer to form emulsion droplets or other reaction compartments for droplet digital PCR reactions or droplet immunoassay PCR reactions.

38. A method of carrying out a reaction, comprising providing a reaction vessel as defined in claim 1 and a solution placed above and below the gel layer, and moving or melting the gel layer, whereby the solution mixes and reacts.

39. A reaction vessel comprising a plurality of liquid aqueous reaction mixtures separated from each other by a plurality of gel layers disposed within the reaction vessel, wherein the plurality of liquid aqueous reaction mixtures may be combined by melting and / or breaking the gel layers to sequentially perform a series of processing steps.

40. The reaction vessel of claim 39, further comprising a mineral oil layer located beneath one or more of the gel layers.

41. The reaction vessel according to claim 39 or 40, wherein the reaction vessel is connected to a heater that can slide along the outer surface of the reaction vessel for melting a gel layer near the heater.

42. The reaction vessel according to any one of claims 39 to 41, wherein at least one liquid aqueous reaction mixture comprises magnetic beads, and the reaction vessel is connected to a magnet slidable along an outer surface of the reaction vessel for moving the magnetic beads through a gel layer beneath the liquid aqueous reaction mixture containing the magnetic beads.

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

75.

44. The reaction vessel according to any one of claims 39 to 43, wherein the liquid aqueous reaction mixture comprises reagents for performing ordered steps of an integrated immunoassay.

45. The reaction vessel according to any one of claims 39 to 43, wherein the liquid aqueous reaction mixture comprises different reagents for ordered biological processing steps, optionally for ordered steps in immunoassays.

46. ​​The reaction vessel according to any one of claims 39 to 45, wherein each of the liquid aqueous reaction mixtures comprises one or more of magnetic beads, capture antibodies, washing solutions, detection antibodies, enzymes, substrates, and termination solutions for performing ordered steps of an integrated immunoassay.

47. The reaction vessel according to any one of claims 39 to 46, wherein each of the liquid aqueous reaction mixtures comprises one or more of a lysis buffer, a DNA / RNA binding solution, magnetic beads, a washing buffer, and an elution buffer for performing ordered steps of integrated DNA / RNA purification.

48. The reaction vessel according to any one of claims 39 to 47, wherein each of the liquid aqueous reaction mixtures comprises one or more of a fragmentation enzyme or end-repair reagent, a ligation reagent, and a library amplification reagent for performing ordered steps of integrated NGS library preparation.

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

50. The method of claim 49, wherein the reaction vessel is connected to a heater that can slide on the outer surface of the reaction vessel, and the heater is moved along the surface to a position where it can melt one of the gel layers.

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

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

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

54. The method according to any one of claims 50 to 52, wherein the module further comprises a cooling element for re-solidifying the gel layer after melting.

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

56. The method of claim 55, wherein the magnet is an electromagnet.

57. A method of carrying out a reaction, comprising providing a reaction vessel of claim 18, a slidable magnetic module attached to an outer surface or an inner surface of the reaction vessel, wherein at least one of the liquid aqueous reaction mixtures comprises magnetic beads, and moving the slidable magnetic module along the outer or inner surface to a position where it can move the magnetic beads.

58. The method of claim 57, wherein the slidable magnetic module is connected to one or more of the following modules: a mechanical module, a heating 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 is completed.

59. The method of claim 57, wherein the reaction vessel is connected to a means for processing a sample or reagent and detecting a chemical or physical change caused by a reaction in the reaction vessel, such change being, for example, color, fluorescence, luminescence, chemiluminescence, electrochemistry, radiation, reflection, phase transition, magnetoresistance, turbidity, mobility shift, pH, or ionic strength.

60. A reaction vessel comprising a first gel layer, and optionally a second gel layer, and optionally a third aqueous reaction mixture or sample on top of the second gel layer, the first gel layer comprising a first aqueous reaction mixture, the second gel layer comprising a second aqueous reaction mixture, the gel being fusible upon heating to mix the aqueous reaction mixture.

61. The reaction vessel of claim 60, further comprising an oil layer with a density greater than that of water above the second gel layer.

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