A gel precursor solution for digital nucleic acid detection, a post-gelation reaction solution, its preparation method, and its application.

Abstract

This invention provides a gel precursor solution, a post-gelatinization reaction solution, and their preparation methods and applications for digital nucleic acid detection. The gel precursor solution comprises a first polymeric component, a second polymeric component, a photoinitiator, and purified water; the first polymeric component is polyethylene glycol diacrylate; the second polymeric component is gelatin acrylamide (GelA); and the photoinitiator is preferably lithium phenyl (2,4,6-trimethylbenzoyl)phosphate. The post-gelatinization reaction solution prepared from this gel precursor solution maintains good fluidity before nucleic acid amplification and during droplet preparation, stably generating droplets at room temperature without additional heating or temperature control. It undergoes a post-gelatinization controllable transition after the nucleic acid amplification reaction, thereby achieving effective fixation, protection, and transport of the amplified products, while supporting the subsequent selective release of the amplified products.

Description

Technical Field

[0001] This invention belongs to the field of digital nucleic acid detection technology, specifically relating to a gel precursor solution for digital nucleic acid detection, a post-gelation reaction solution, its preparation method, and its application. Background Technology

[0002] Nucleic acid detection technology, as an important technique in molecular biology and in vitro diagnostics, is widely used in clinical diagnosis, infectious disease surveillance, food safety, and environmental monitoring. In recent years, digital nucleic acid detection technologies (such as digital PCR and digital isothermal amplification) have achieved absolute quantification of nucleic acid molecules by dividing the reaction system into a large number of independent microreaction units, demonstrating significant advantages in sensitivity and quantitative accuracy. In digital nucleic acid detection, the reaction solution containing nucleic acid templates, primers, enzymes, and buffer systems is typically prepared into a large number of uniform microdroplets, micropores, or microcavities to ensure that a single or limited number of nucleic acid templates independently complete the amplification reaction in each reaction unit. To meet the requirements of the amplification reaction, existing reaction solution systems are usually designed as low-viscosity aqueous phase systems to adapt to the droplet formation process and ensure the reaction kinetics conditions required for nucleic acid amplification.

[0003] However, traditional digital nucleic acid detection solutions remain liquid or weakly structured after the amplification reaction, making it difficult to effectively physically immobilize or encapsulate the amplified products. This characteristic makes the amplified products susceptible to mechanical disturbances, temperature changes, or transport processes during subsequent operations, posing risks of diffusion, leakage, cross-contamination, and nucleic acid degradation, which is detrimental to the protection, transportation, and subsequent analysis of the amplified products. To address these issues, existing technologies have attempted to introduce gelation systems. By adding thermally responsive or chemically cross-linked polymers to the reaction system, the reaction solution forms a gel structure under certain conditions, thereby achieving the immobilization and protection of the amplified products. However, such gelation systems typically have the following shortcomings: Firstly, existing gelation schemes often rely on temperature changes (e.g., heating or cooling) to trigger gel formation. During the droplet preparation stage, to ensure the reaction system remains in a flowable state, it is often necessary to operate at higher temperatures to prevent premature gel formation. This not only increases the reliance on temperature control equipment for droplet generation, raising system complexity and cost, but also, under conditions containing nucleic acid amplification enzymes and primers, heating operations can easily induce unexpected enzyme activity or pre-amplification reactions, thus affecting the quantitative accuracy and reliability of digital nucleic acid detection. On the other hand, some gel systems require the introduction of high concentrations of polymeric materials or cross-linking components before the reaction begins, which can easily inhibit the nucleic acid amplification reaction, affecting enzyme activity, amplification efficiency, and reaction uniformity, and exhibiting poor compatibility between different amplification systems. Furthermore, the release of amplification products after amplification in these systems typically relies on destructive treatment, which is complex and makes selective or controllable release difficult to achieve.

[0004] In summary, existing reaction solution systems for digital nucleic acid detection still have significant shortcomings in simultaneously ensuring stable droplet formation at room temperature, avoiding equipment dependence and pre-amplification risks associated with heating operations, and achieving in-situ gelation after amplification to protect, transport, and support the subsequent release of amplified products. Therefore, there is an urgent need to provide a new solution composition system for digital nucleic acid detection that can maintain good fluidity during droplet preparation and support droplet formation at room temperature, and achieve controllable gelation transition after amplification without interfering with the nucleic acid amplification reaction, thereby overcoming the aforementioned deficiencies in existing technologies. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a gel precursor solution, a post-gelatinization reaction solution, its preparation method, and its application for digital nucleic acid detection. The post-gelatinization reaction solution maintains good fluidity before the nucleic acid amplification reaction and during the droplet preparation stage, can stably generate droplets at room temperature without additional heating or temperature control, and undergoes a post-controllable gelation transition after the nucleic acid amplification reaction is completed, thereby achieving effective fixation, protection, and transport of the amplification products, while supporting the subsequent selective release of the amplification products.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] On one hand, the present invention provides a gel precursor solution for digital nucleic acid detection, the gel precursor solution comprising a first polymeric component, a second polymeric component, a photoinitiator, and purified water.

[0008] The first polymeric component is polyethylene glycol diacrylate;

[0009] Preferably, the polyethylene glycol diacrylate is a polyethylene glycol diacrylate with a molecular weight greater than or equal to 2000;

[0010] More preferably, the polyethylene glycol diacrylate is PEGDA 2000, PEGDA 10000 or PEGDA20000.

[0011] More preferably, the polyethylene glycol diacrylate is PEGDA10000.

[0012] The second polymeric component is gelatin acrylamide product GelA.

[0013] The photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0014] The mass concentration of the first polymeric component in the gel precursor solution is 2%-10%; preferably, the mass concentration of the first polymeric component in the gel precursor solution is 5%-9%; and even more preferably, the mass concentration of the first polymeric component in the gel precursor solution is 6%-8%.

[0015] More preferably, the mass concentration of the first polymeric component in the gel precursor solution is any value or range between 4% and 10%, specifically selected from: 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any two of these ranges.

[0016] The second polymeric component has a mass concentration of 4%-10% in the gel precursor solution; preferably, the mass concentration of the second polymeric component in the gel precursor solution is 5%-9%; and even more preferably, the mass concentration of the second polymeric component in the gel precursor solution is 6%-8%.

[0017] More preferably, the mass concentration of the second polymeric component in the gel precursor solution is any value or range between 4% and 10%, specifically selected from: 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any two of them.

[0018] The photoinitiator has a mass concentration of 0.1%-0.4% in the gel precursor solution; preferably, the photoinitiator has a mass concentration of 0.2%-0.3% in the gel precursor solution.

[0019] More preferably, the mass concentration of the photoinitiator in the gel precursor solution is any value or range between 0.1% and 0.4%, specifically selected from: 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, or any range between two of them.

[0020] On the other hand, the present invention also provides a method for preparing the above-mentioned gel precursor solution, which is to mix the first polymer component, the second polymer component, the photoinitiator and purified water.

[0021] In another aspect, the present invention also provides the application of the above-mentioned gel precursor solution in the preparation of a post-gelatinizable reaction solution for digital nucleic acid detection.

[0022] In another aspect, the present invention also provides a post-gelatinizable reaction solution for digital nucleic acid detection, wherein the post-gelatinizable reaction solution comprises the above-mentioned gel precursor solution and reagents required for nucleic acid amplification.

[0023] The digital nucleic acid detection described in this invention is a LAMP detection; the reagents required for nucleic acid amplification are common LAMP (loop-mediated isothermal amplification) reagents.

[0024] Furthermore, the present invention also provides a method for preparing the above-mentioned post-gelation reaction solution, which is obtained by adding the gel precursor solution to the reagents required for nucleic acid amplification.

[0025] The mass concentration of the first polymeric component in the post-gelatinizable reaction solution is 1%-5%; preferably, the mass concentration of the first polymeric component is 3%-4%; and even more preferably, the mass concentration of the first polymeric component is 3%.

[0026] More preferably, the mass concentration of the first polymeric component is any value or range between 2% and 5%, specifically selected from: 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or any two of them.

[0027] When the mass concentration of the first polymeric component in the post-gellable reaction solution is 2%-5%, it can maintain liquid properties over a wide temperature range.

[0028] The mass concentration of the second polymeric component in the post-gelatinization reaction solution is 2%-5%; preferably, the mass concentration of the second polymeric component is 3%-4%; and even more preferably, the mass concentration of the second polymeric component is 3%.

[0029] More preferably, the mass concentration of the second polymeric component is any value or range between 2% and 5%, specifically selected from: 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or any two of them.

[0030] The mass concentration of the second polymer component in the post-gellable reaction solution is 2%-5%, which allows the reaction solution to remain in a liquid state at room temperature, thereby ensuring that the reaction solution has good fluidity during the droplet preparation stage and can generate droplets at room temperature.

[0031] Tests showed that PEGDA10000 and GelA did not significantly inhibit nucleic acid amplification reactions at concentrations of 2%-5%, and were well compatible with the enzymes, primers, nucleotides, and buffer systems required for nucleic acid amplification.

[0032] The mass concentration of the photoinitiator in the post-gelling reaction solution is 0.05%-0.2%; preferably, the mass concentration of the photoinitiator is 0.1%-0.2%; and even more preferably, the mass concentration of the photoinitiator is 0.2%.

[0033] More preferably, the mass concentration of the photoinitiator in the post-gellable reaction solution is any value or range between 0.05% and 0.2%, specifically selected from: 0.05%, 0.08%, 0.1%, 0.12%, 0.15%, 0.2%, or any range between two of them.

[0034] When the mass concentration of the photoinitiator in the post-gellable reaction solution is 0.05%-0.2%, it can initiate a free radical polymerization reaction between PEGDA10000 and GelA under specific wavelength light irradiation conditions.

[0035] After the nucleic acid amplification reaction is completed, the reaction solution is irradiated with light of approximately 405 nm to activate the photoinitiator, thereby initiating a cross-linking polymerization reaction between PEGDA10000 and GelA. When the content and ratio of PEGDA10000 and GelA reach predetermined conditions, the polymerization reaction can transform the reaction solution from a liquid state to a gel state, achieving post-amplification.

[0036] In one embodiment of the invention, the reaction solution is applied to a loop-mediated isothermal amplification (LAMP) system. Experimental studies have shown that, in the LAMP system, the reaction solution containing PEGDA10000, GelA, and a photoinitiator can support the formation of stable digital droplets via microfluidics.

[0037] The droplets maintain structural stability during LAMP amplification without fusion or rupture. After the amplification reaction is completed, photo-initiated polymerization causes the internal reaction system of the droplets to gel, thereby achieving fixation and protection of the amplification products.

[0038] This invention achieves controllable gelation of digital nucleic acid detection reaction solutions after amplification without introducing a droplet preparation heating step, taking into account the needs of room temperature droplet generation, nucleic acid amplification compatibility, and protection and subsequent processing of amplification products.

[0039] Furthermore, the present invention also provides the application of the above-mentioned post-gelatinizable reaction solution in the preparation of digital nucleic acid detection products.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] (1) Supports droplet generation under ambient temperature conditions, reducing system complexity:

[0042] The reaction solution of this invention maintains good fluidity and low viscosity during the droplet preparation stage, and can stably generate uniform droplets at room temperature without heating or additional temperature control of the reaction system. This significantly reduces the dependence of the droplet generation process on temperature control equipment, simplifies the detection system structure, reduces equipment costs, and improves system integration and feasibility for field application.

[0043] (2) Avoid the risk of pre-amplification during droplet preparation and improve quantitative accuracy:

[0044] Since this invention does not rely on heating during the droplet generation stage, it can effectively avoid unexpected enzyme activity activation or premature template amplification under conditions containing nucleic acid amplification enzymes and primers. This reduces the probability of pre-amplification from the source, which is beneficial to maintaining the authenticity of the initial template distribution in each reaction unit, thereby improving the quantitative accuracy and repeatability of digital nucleic acid detection results.

[0045] (3) Achieve post-amplification to improve the physical stability of the amplification products:

[0046] The reaction solution of the present invention can undergo in-situ gelation after the nucleic acid amplification reaction is completed, so that the amplification product is fixed or embedded in the gel structure, effectively limiting the diffusion and leakage of the amplification product, and significantly improving the structural stability and reliability of the reaction unit in storage, transportation and subsequent processing.

[0047] (4) Compatible with nucleic acid amplification reactions, without affecting amplification efficiency and reaction uniformity:

[0048] This invention, through the rational design of solution components and their state transition characteristics, enables the reaction solution to maintain a suitable reaction environment for nucleic acid amplification during the amplification reaction stage, without significantly inhibiting amplification enzyme activity or altering the amplification kinetics. Thus, while achieving the post-gelation function, it still ensures the amplification efficiency and reaction uniformity required for digital nucleic acid detection.

[0049] (5) It is beneficial for the protection, transportation and long-term preservation of amplification products:

[0050] The gel structure formed after amplification can provide a physical barrier and stable microenvironment for nucleic acid amplification products, reducing the impact of external temperature changes, mechanical disturbances and contaminants on the amplification products. It is suitable for application scenarios where amplification products need to be transported, centrally analyzed or stored in stages, thus expanding the application scope of digital nucleic acid detection in multiple scenarios.

[0051] (6) Improve the overall reliability and application value of digital nucleic acid testing:

[0052] By integrating room-temperature droplet generation and post-amplification gelation into the same reaction solution system, this invention systematically solves the compatibility issues between droplet preparation, amplification process control, and post-processing of amplification products without increasing operational complexity, effectively improving the stability, reliability, and industrial application potential of the digital nucleic acid detection process. Attached Figure Description

[0053] Figure 1 Real-time fluorescence amplification curves of nucleic acid amplification by different concentrations of LAP initiator;

[0054] Figure 2 Real-time fluorescence amplification curves of nucleic acid amplification for PEGDA of different molecular weights;

[0055] Figure 3 A schematic diagram characterizing the storage modulus of gel formation at different concentrations of GelA;

[0056] Figure 4 A schematic diagram characterizing the storage modulus of gels formed by different concentrations of PEGDA;

[0057] Figure 5 A statistical chart of digital droplet size distribution;

[0058] Figure 6 This is a diagram showing the effect of digital droplet amplification.

[0059] Figure 7 This is a diagram showing the photopolymerization effect of digital droplets. Detailed Implementation

[0060] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0061] The experimental materials and instruments are shown in Table 1.

[0062] Table 1

[0063]

[0064] Example 1: A gel precursor solution and application for digital nucleic acid detection

[0065] Example 1-1: A gel precursor solution for digital nucleic acid detection

[0066] The components include: a photoinitiator of 0.2% by mass of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate and the balance being purified water.

[0067] Application Example 1-1: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0068] Add the gel precursor solution from Example 1-1 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelatinization reaction solution. The mass concentration of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in the post-gelatinization reaction solution is 0.1%.

[0069] Examples 1-2: A gel precursor solution for digital nucleic acid detection

[0070] The components include: a photoinitiator of 0.4% by mass of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate and the balance being purified water.

[0071] Application Example 1-2: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0072] Add the gel precursor solutions from Examples 1-2 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelatinization reaction solution. The mass concentration of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in the post-gelatinization reaction solution is 0.2%.

[0073] Comparative Example 1-1: A gel precursor solution for digital nucleic acid detection

[0074] The components include: a photoinitiator of 0.8% by mass of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate and the balance being purified water.

[0075] Comparative Example 1-1: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0076] Add the gel precursor solution of Comparative Example 1-1 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelation reaction solution. The mass concentration of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in the post-gelation reaction solution is 0.4%.

[0077] Effect test:

[0078] The fluorescence change curves over time during the amplification process of the reaction reagents were detected using a real-time fluorescence nucleic acid amplification instrument for the reaction solutions prepared in Application Example 1-1, Application Example 1-2, and Comparative Example 1-1, respectively. Figure 1 As shown.

[0079] according to Figure 1The results show that when the mass concentration of the photoinitiator is 0.1% and 0.2%, the fluorescence curve increases significantly during the LAMP reaction, indicating that a nucleic acid amplification reaction has occurred. This suggests that a photoinitiator concentration of 0.05%-0.2% is sufficient. However, when the mass concentration of the photoinitiator is 0.4%, the fluorescence curve does not increase, indicating that excessively high concentrations of LAP inhibit the nucleic acid amplification reaction.

[0080] Example 2: A gel precursor solution for digital nucleic acid detection and its application

[0081] Example 2-1: A gel precursor solution for digital nucleic acid detection

[0082] The components include: the first polymer component is PEGDA 2000 with a mass concentration of 2%, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate with a mass concentration of 0.4%, and the balance is purified water.

[0083] Application Example 2-1: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0084] Add the gel precursor solution from Example 2-1 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelation reaction solution. The mass concentration of PEGDA 2000 in the post-gelation reaction solution is 1%, and the mass concentration of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate is 0.2%.

[0085] Example 2-2: A gel precursor solution for digital nucleic acid detection

[0086] The components include: the first polymeric component is PEGDA 10000 at a mass concentration of 2%, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate at a mass concentration of 0.4%, and the balance is purified water.

[0087] Application Example 2-2: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0088] Add the gel precursor solution from Example 2-2 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelation reaction solution. The mass concentration of PEGDA 10000 in the post-gelation reaction solution is 1%, and the mass concentration of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate is 0.2%.

[0089] Examples 2-3: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0090] The components include: the first polymeric component is PEGDA 20000 at a mass concentration of 2%, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate at a mass concentration of 0.4%, and the balance is purified water.

[0091] Application Example 2-3: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0092] Add the gel precursor solutions from Examples 2-3 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelation reaction solution. The mass concentration of PEGDA 20000 in the post-gelation reaction solution is 1%, and the mass concentration of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate is 0.2%.

[0093] Comparative Example 2-1: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0094] The difference from Example 2-2 is that PEGDA 20000 is replaced with PEGDA 400. Otherwise, it is the same as Example 2-2 and Application Example 2-2.

[0095] Effect test:

[0096] The fluorescence change curves over time during the amplification process of the reaction reagents were detected using a real-time fluorescence nucleic acid amplification instrument using the post-gelatinization reaction solutions prepared in Application Example 2-1, Application Example 2-2, Application Example 2-3, and Comparative Example 2-1, respectively. Figure 2 As shown.

[0097] according to Figure 2 The results show that the real-time fluorescence amplification curve indicates that PEGDA with a molecular weight of 2000 or higher, as an additive, shows an increase in fluorescence over time, suggesting that LAMP nucleic acid amplification occurs normally. However, when PEGDA400 is used as an additive, the fluorescence curve does not show an upward step, indicating that it inhibits the LAMP reaction. This suggests that selecting PEGDA with a molecular weight of 2000 or higher as the polymer component is sufficient to meet the requirements.

[0098] Example 3: A gel precursor solution for digital nucleic acid detection and its application

[0099] Example 3-1: A gel precursor solution for digital nucleic acid detection

[0100] The composition, by mass concentration, includes: PEGDA 10000 2%, GelA 6%, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate 0.4%, with the balance being purified water.

[0101] Application Example 3-1: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0102] Add the precursor solution from Example 3-1 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelation reaction solution. The post-gelation reaction solution contains 5% PEGDA 10000, 3% GelA, and 0.2% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0103] Example 3-2: A gel precursor solution for digital nucleic acid detection

[0104] The product comprises, by mass concentration: PEGDA 10000 2%, GelA 10%, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate 0.4%, with the balance being purified water.

[0105] Application Example 3-1: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0106] Add the precursor solution from Example 3-2 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelation reaction solution. The post-gelation reaction solution contains 5% PEGDA 10000, 5% GelA, and 0.2% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0107] Comparative Example 3-1: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0108] The product comprises, by mass concentration: PEGDA 10000 2%, GelA 20%, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate 0.4%, with the balance being purified water.

[0109] Comparative Example 3-1: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0110] Add the precursor solution of Comparative Example 3-1 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelation reaction solution. The post-gelation reaction solution contains 5% PEGDA 10000, 10% GelA, and 0.2% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0111] Effect test:

[0112] The post-gelatinizable reaction solutions prepared in Application Example 3-1, Application Example 3-2, and Comparative Example 3-1 were used for nucleic acid amplification, and photoinitiated polymerization was performed. The mechanical strength of the resulting gels was tested as follows: Figure 3 As shown.

[0113] according to Figure 3 The results show that the concentration of GelA has little effect on photoinitiated gel formation, and the gel strength increases with increasing addition. However, considering the high cost of GelA in practical applications, and the fact that higher concentrations of GelA have higher viscosity at room temperature, a concentration of 5% or lower is preferable.

[0114] Example 4: A gel precursor solution for digital nucleic acid detection and its application

[0115] Example 4-1: A gel precursor solution for digital nucleic acid detection

[0116] The composition, by mass concentration, includes: PEGDA 10000 2%, GelA 6%, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate 0.4%, with the balance being purified water.

[0117] Application Example 4-1: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0118] Add the precursor solution from Example 4-1 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelation reaction solution. The post-gelation reaction solution contains 1% PEGDA 10000, 3% GelA, and 0.2% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0119] Example 4-2: A gel precursor solution for digital nucleic acid detection

[0120] The product comprises, by mass concentration: 6% PEGDA 10000, 6% GelA, 0.4% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, and the balance being purified water.

[0121] Application Example 4-2: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0122] Add the precursor solution from Example 4-2 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelation reaction solution. The post-gelation reaction solution contains 3% PEGDA 10000, 3% GelA, and 0.2% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0123] Example 4-3: A gel precursor solution for digital nucleic acid detection

[0124] The composition, by mass concentration, includes: PEGDA 10000 10%, GelA 6%, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate 0.4%, with the balance being purified water.

[0125] Application Examples 4-3: A method for preparing a post-gelatinizable reaction solution for digital nucleic acid detection

[0126] Add the precursor solution from Examples 4-3 to the LAMP reagent required for nucleic acid amplification to obtain a post-gelation reaction solution. The post-gelation reaction solution contains 5% PEGDA 10000, 3% GelA, and 0.2% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0127] Effect test:

[0128] The post-gelatinizable reaction solutions prepared in Application Examples 4-1, 4-2, and 4-3 were used for nucleic acid amplification, and photoinitiated polymerization was performed. The mechanical strength of the resulting gels was tested as follows: Figure 4 As shown.

[0129] according to Figure 4 As shown, increasing the concentration of PEGDA can increase gel strength, but the effect of further increasing gel strength at higher concentrations is limited. Therefore, a mass concentration of 5% or less is selected.

[0130] Effect detection:

[0131] Example 1: Droplet formation and droplet stability verification under room temperature conditions

[0132] The post-gelatinizable reaction solution prepared in Application Example 4-2 was introduced into a microfluidic droplet generator at room temperature. Droplets with a diameter of approximately 51.5 micrometers and extremely low dispersion were formed through the shearing action between the continuous and dispersed phases. No heating or temperature control was applied to the reaction solution during droplet formation. Experimental results show that the reaction solution can stably form uniformly sized droplets at room temperature. The droplet formation process is continuous and controllable, and no difficulties in droplet formation due to excessively high solution viscosity were observed (see [link to example 4-2]). Figure 5 ).

[0133] The generated droplets maintained good structural integrity in subsequent operations, and no obvious droplet fusion, rupture, or leakage of contents was observed, indicating that the reaction solution is suitable for forming independent reaction units in the form of droplets, micropores, or microcavities.

[0134] The above results demonstrate that the reaction solution described in this invention can achieve stable droplet generation without heating during the droplet preparation stage, thereby avoiding equipment dependence and potential pre-amplification risks associated with heating.

[0135] Example 2: Digital LAMP amplification and droplet stability during the amplification process

[0136] The droplets obtained in Example 1 were used as independent reaction units for LAMP reactions. The droplets contained a target nucleic acid template, a LAMP primer set, an amplification enzyme, and the post-gelatinizable reaction solution prepared in Example 1. During LAMP amplification, the droplets were placed under isothermal conditions suitable for the LAMP reaction. Experimental observations showed that the droplets maintained structural stability during the amplification reaction, without significant fusion or breakage.

[0137] The amplification results showed that the reaction solution system containing PEGDA10000 and GelA could support the normal progress of the LAMP amplification reaction. (See attached image) Figure 6 The left image is a bright-field image of densely packed digital droplets, showing that the droplets are neatly arranged and uniform in size, which meets the requirements of digital nucleic acid detection. The right image is a fluorescent image of digital droplets obtained after the amplification step of digital nucleic acid detection. Positive droplets show green fluorescence, indicating successful amplification. The nucleic acid amplification stage of this solution does not significantly affect the amplification efficiency and reaction uniformity, and can support the application requirements of digital nucleic acid detection.

[0138] Example 3: Photoinitiated gelation and immobilization of amplification products after amplification

[0139] After nucleic acid amplification was completed in Example 2, light with a wavelength of approximately 405 nm was applied to the droplet system to activate the photoinitiator in the reaction solution. Under light irradiation, PEGDA10000 inside the droplet underwent a cross-linking polymerization reaction with GelA, and the reaction system inside the droplet changed from a liquid state to a gel state.

[0140] Experimental observations showed that the gelled droplet structure was more stable, and the amplification product was immobilized within the gel structure without significant diffusion or leakage. The gelled droplets maintained good integrity during transport and short-term storage, demonstrating a protective effect on the amplification product. (See [link to related information]). Figure 7 The left image is a bright-field digital photograph of droplets in 7500 fluorinated oil, at which point the droplets have not yet solidified; the right image is a bright-field photograph of droplets gelling after photo-initiated polymerization, forming uniformly sized microgels. It can be seen that after the 7500 is evaporated, the boundaries of the microgels are distinct and no fusion occurs, indicating that the gel was already in a microgel state before the 7500 was evaporated.

[0141] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A gel precursor solution for digital nucleic acid detection, characterized in that: The gel precursor solution comprises a first polymeric component, a second polymeric component, a photoinitiator, and purified water; the first polymeric component is polyethylene glycol diacrylate with a molecular weight of 2000, 10000, or 20000; the second polymeric component is gelatin acrylamide product GelA; the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate; the mass concentration of the first polymeric component in the gel precursor solution is 2%-10%; the mass concentration of the second polymeric component in the gel precursor solution is 4%-10%; and the mass concentration of the photoinitiator in the gel precursor solution is 0.1%-0.4%.

2. The method of making a gel precursor solution of claim 1, characterized by: The first polymer component, the second polymer component, the photoinitiator, and purified water are mixed to obtain the final product.

3. The use of the gel precursor solution according to claim 1 in the preparation of a post-gelatinizable reaction solution for digital nucleic acid detection.

4. A post-gelable reaction solution for digital nucleic acid detection, characterized by: Includes the gel precursor solution as described in claim 1 and reagents required for nucleic acid amplification.

5. The method for preparing the post-gelable reaction solution according to claim 4, characterized by: The gel precursor solution is prepared by mixing it with the reagents required for nucleic acid amplification.

6. The post-gelable reaction solution of claim 4, wherein: The mass concentration of the first polymeric component in the post-gellable reaction solution is 1%-5%; the mass concentration of the second polymeric component is 2%-5%; and the mass concentration of the photoinitiator is 0.05%-0.2%.

7. The use of the post-gelatinizable reaction solution as described in claim 4 or 6 in the preparation of digital nucleic acid detection products.

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