Hydrogel array PCR chip, its preparation method and application

Abstract

The application discloses a hydrogel array PCR chip and a preparation method and application thereof, relates to the technical field of hydrogel PCR, and discloses the hydrogel array PCR chip, which comprises a substrate, a hydrophobic region and an arrayed hydrophilic site in the hydrophobic region arranged on the surface of the substrate, a hydrogel array comprising a plurality of hydrogel microparticles fixed on the hydrophilic sites respectively, and a primer of a target to be detected connected to the hydrogel microparticles through a covalent bond. The hydrogel array PCR chip has high-throughput detection capacity, excellent detection lower limit and good linear response relationship, improves the efficiency of PCR reaction, simplifies the preparation process, reduces the preparation cost, has no cross interference between array units, and has good spatial isolation effect and specificity.

Description

Technical Field

[0001] This application relates to the field of hydrogel PCR technology, and in particular to a hydrogel array PCR chip, its preparation method and application. Background Technology

[0002] Hydrogels possess a hydrophilic three-dimensional network structure and good biocompatibility, enabling PCR amplification reactions within them. Immobilizing hydrogels onto glass chips to form hydrogel microparticles and binding specific primers to them allows for rapid detection of multiplex and even hypermultiplexed samples, overcoming the limitations of traditional tube-based qPCR, such as primer competition, limited detection multiples, and high cost. However, current hydrogel PCR technologies generally suffer from low amplification efficiency. Furthermore, silicon-based chip fabrication is costly, and photolithography is time-consuming and expensive, making it difficult to meet high-throughput detection demands. Although lower detection limits can be obtained in laboratory studies, they are insufficient for routine testing or clinical applications. Therefore, existing technologies require further improvement. Summary of the Invention

[0003] The main purpose of this application is to propose a hydrogel array PCR chip, its preparation method and application, which aims to solve or at least partially alleviate the problems of low amplification efficiency, complex preparation process and high cost of existing hydrogel PCR chips.

[0004] To achieve the above objectives, in a first aspect, this application proposes a hydrogel array PCR chip, comprising: A substrate having hydrophobic regions on its surface and arrayed hydrophilic sites located within the hydrophobic regions; A hydrogel array comprising a plurality of hydrogel microparticles respectively fixed on each of the hydrophilic sites; The hydrogel microparticles contain primers for the target being tested, which are covalently linked inside.

[0005] In some embodiments, the hydrogel microparticles are columnar, crown-shaped, or hemispherical; and / or, The solid-liquid contact angle of the hydrogel microparticles on the substrate is greater than 90 degrees.

[0006] In some embodiments, the hydrogel microparticles are formed by polymer crosslinking of a hydrogel prepolymer solution comprising polyethylene glycol diacrylate and polyethylene glycol. In some embodiments, the volume ratio of polyethylene glycol diacrylate to polyethylene glycol in the hydrogel prepolymer solution is 1:(0.66~1.5).

[0007] In some embodiments, the primers include forward primers and / or reverse primers of the target to be tested; The primer is covalently linked to the polymer backbone of the hydrogel via its 5' end acrylamide group.

[0008] In some embodiments, the number of hydrogel microparticles is 1 to 1000, the diameter is 0.03 to 1 mm, and the spacing between adjacent hydrogel microparticles is 0.03 to 1 mm.

[0009] In some embodiments, the hydrogel array PCR chip further includes a cover plate disposed above the substrate, forming a reaction chamber between the substrate and the cover plate, the cover plate having at least one sample application well, and the reaction chamber having a length of 5-30 mm, a width of 5-30 mm, and a height of 0.1-5 mm.

[0010] Secondly, this application also proposes a method for preparing the hydrogel array PCR chip proposed in the first aspect of this application, comprising: S1. A hydrophobic layer is formed on the surface of the substrate, and an array of sites is etched on the hydrophobic layer; S2. The arrayed sites are modified with alkenyl groups by silanization. S3. Spot the hydrogel prepolymer solution containing acrylamide-modified primers to each of the arrayed sites; S4. Free radical polymerization is initiated by ultraviolet light to solidify the hydrogel prepolymer solution into hydrogel microparticles. At the same time, the primers are covalently anchored inside the hydrogel microparticles through their alkenyl groups participating in the polymerization reaction, and the alkenyl groups introduced by the silanization modification of the hydrogel microparticles are chemically bonded to the substrate surface.

[0011] In some embodiments, the hydrophobic layer is formed by vapor deposition of at least one of (1H,1H,2H,2H-heptadecyl)silane or 1H,1H,2H,2H-perfluorooctyltriethoxysilane. In some embodiments, the alkenyl-containing silanization modification is performed using 3-(isobutenoyloxy)propyltrimethoxysilane.

[0012] In some embodiments, the hydrogel prepolymer solution comprises polyethylene glycol diacrylate, polyethylene glycol, a photoinitiator, a buffer solution, and Tween-20; The volume ratio of polyethylene glycol diacrylate to polyethylene glycol is 1:(0.66~1.5).

[0013] Thirdly, this application also proposes the application of the hydrogel array PCR chip proposed in the first aspect of this application and the hydrogel array PCR chip prepared by the preparation method proposed in the second aspect of this application in the detection of intestinal flora. The primers include forward primers or reverse primers in a primer set for detecting at least one of Bifidobacterium, Lactobacillus, Akkermansia, Faecalibacterium, Bacteroides, Prevotella, Trichophyton, Veillonella, Enterococcus, and Streptococcus. The primer sequences are shown in any of SEQ ID NO.1 to 20.

[0014] The hydrogel array PCR chip proposed in this application includes a substrate with hydrophobic regions and arrayed hydrophilic sites, and hydrogel microparticles covalently linked to the target primers inside. Since all hydrogel microparticles are exposed in the same reaction chamber, the nucleic acid of the sample can freely flow through each microparticle, undergoing matched amplification with its internally immobilized primers. However, the primers are firmly anchored to the hydrogel backbone by covalent bonds, preventing crosstalk caused by liquid flow. Compared to traditional multi-chamber physical separation schemes, there is no need to distribute trace amounts of sample to different reaction units, thus avoiding target loss or decreased sensitivity due to uneven distribution, making it particularly suitable for parallel detection of low-abundance targets. Simultaneously, the hydrophobic-hydrophilic patterned design of the substrate surface allows hydrogel prepolymer droplets to be positioned at hydrophilic sites using a microelectronic printer, eliminating the need for complex microchannels or physical dikes to separate reaction areas. This not only reduces the precision requirements for chip manufacturing but also simplifies sample loading operations. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram of the structure of the hydrogel array PCR chip provided in this application; Figure 2 This is a schematic flowchart of the preparation method of the hydrogel array PCR chip provided in this application; Figure 3 Amplification curves of real-time PCR performed on the hydrogel array PCR chip provided in this application: (a) Hydrogel array PCR chip prepared in Example 1; (b) Hydrogel array PCR chip prepared in Example 2; (c) Hydrogel array PCR chip prepared in Example 3; (d) Hydrogel array PCR chip prepared in Example 4; (e) Hydrogel array PCR chip prepared in Example 5; (f) Solution control group; Figure 4 Melting curve of the hydrogel array PCR chip provided in the embodiments of this application for real-time PCR: (a) Hydrogel array PCR chip prepared in Example 1; (b) Hydrogel array PCR chip prepared in Example 2; (c) Hydrogel array PCR chip prepared in Example 3; (d) Hydrogel array PCR chip prepared in Example 4; (e) Hydrogel array PCR chip prepared in Example 5; (f) Solution control group; Figure 5 The hydrogel array PCR chip amplification efficiency test graph provided in the embodiments of this application: A bar chart comparing the Ct values ​​of the PCR amplification curves: (a) A bar chart comparing the Ct values ​​of the PCR amplification curves of Example 2 and the solution control group; (b) A standard PCR curve of the hydrogel array PCR chip prepared in Example 2; Figure 6 Physical images of hydrogel PCR chips of different specifications provided in the embodiments of this application: (a) A hydrogel array PCR chip prepared in Example 6; (b) A hydrogel array PCR chip prepared in Example 7; (c) A hydrogel array PCR chip prepared in Example 8; (d) A hydrogel array PCR chip prepared in Example 9; Figure 7 This is a fluorescence image of a hydrogel quadrupole sample after PCR reaction provided in an embodiment of this application. Figure 8 The fitting curve for the PCR amplification curve of the hydrogel array PCR chip provided in the embodiments of this application; Figure 9 The standard curve is a linear fit between the Ct value and the sample concentration for PCR amplification of samples of different concentrations provided in the embodiments of this application using the hydrogel array PCR chip.

[0017] Icon labels: 100. Hydrogel array PCR chip; 1. Substrate; 11. Hydrophobic region; 12. Hydrophilic site; 2. Hydrogel array; 21. Hydrogel microparticles; 3. Cover plate; 31. Sample well; 32. Pore.

[0018] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0020] It should be noted that if the embodiments of this application involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0021] Furthermore, if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed in this application.

[0022] Hydrogels are a class of three-dimensional network structures formed by chemical or physical cross-linking of hydrophilic polymers. They possess a three-dimensional network structure with adjustable pores and good compatibility with a water-like environment. These unique properties enable PCR amplification reactions to be performed within hydrogels. Utilizing this characteristic, hydrogels are immobilized on glass chips to form hydrogel microparticles. By binding sample-specific primers to these microparticles, rapid detection of multiplex or even hypermultiplexed samples on hydrogel PCR chips can be achieved. This overcomes the limitations of existing tube-based qPCR multiplex detection methods, such as primer competition, limited detection multiples, and high detection costs.

[0023] Currently, hydrogel multiplex PCR is mainly achieved through two strategies: First, hydrogel multiplex PCR can be achieved using a physically isolated hydrogel microparticle or microcolumn strategy. This involves spatially separating the hydrogel to enable multiplex target detection. Currently, physical isolation strategies for hydrogel multiplex PCR primarily rely on inkjet printing and template methods. Inkjet printing technology has significant advantages in the localization of functional components in hydrogel arrays and particles; however, the shear force of piezoelectric inkjet printing and the localized high temperature of thermal inkjet printing can lead to the inactivation of hydrogel materials. Template methods can be further divided into photomask methods, PDMS template methods, and glass template methods. Both photomask methods and soft lithography require the pre-preparation of photomasks. Yeom et al. successfully achieved five-fold detection of Alzheimer's disease-related miRNAs using the photomask method; however, the photocuring of hydrogels with five different primers requires repeated "infusion-step exposure-rinsing," making this method unsuitable for preparing hydrogels with more multiple targets. Alternatively, PDMS templates can be prepared using soft lithography, or glass templates can be prepared using laser etching. The hydrogel prepolymer solution is then infused into the template and cured to form hydrogel microspheres or microcolumns. However, this method typically requires significant time and economic costs in template preparation, and the process steps are relatively complex. Overall, while the template method offers advantages such as strong structural controllability and good repeatability, its high cost and low efficiency limit its large-scale application to some extent.

[0024] Second, signal differentiation strategies based on material coding. Hydrogel materials possess excellent identifiable coding capabilities, enabling the differentiation of different targets. Currently, the coding patterning of hydrogels mainly relies on soft lithography, 3D printing, and DMPP technologies. Soft lithography can fabricate structures at the micrometer and nanometer scales and is applicable to various linear, circular, flexible, and soft substrates. By dropping a few nanoliters to a few microliters of prepolymer solution onto a pre-patterned polydimethylsiloxane (PDMS) plate located on a hydrophobic plate, and then subjecting the microdroplet array to short-term UV irradiation to solidification, the hydrogel released from the hydrophobic plate acquires a unique and identifiable coded pattern. Soft lithography for hydrogel patterning requires a pre-customized photomask, which limits the size of the hydrogel. Furthermore, this method requires significant investment in cost, manpower, and time, further restricting its flexibility and reproducibility. 3D printing technology has a natural advantage in fabricating complex, multi-material, microscale hydrogel patterns; however, the long fabrication time of 3D printing hydrogel particles often leads to the inactivation of PCR reaction system components at room temperature. DMPP (digital micromirror device-based modulating projection printing) is a projection-based photopolymerization method based on digital dynamic masks. Unlike soft lithography, which relies on physical molds, DMPP modulates light patterns in real time using digital micromirror devices to locally expose and solidify hydrogel prepolymer solutions, thereby enabling the rapid, flexible, and reproducible construction of 2D or 3D hydrogel microstructures.

[0025] While existing hydrogel PCR technology can utilize the spatial separation and coding properties of hydrogels, its amplification efficiency is often low. Furthermore, the fabrication cost of currently used silicon-based chips is high, and the time and money involved in photolithography preparation are also significant, failing to meet the demands of high-throughput detection. Although high detection limits can be achieved in laboratory studies, current hydrogel PCR platforms clearly cannot meet the requirements for routine testing or clinical applications.

[0026] Based on the above issues, please refer to Figure 1 In a first aspect, embodiments of this application propose a hydrogel array PCR chip. The hydrogel array PCR chip 100 generally includes: a substrate 1, the surface of which is provided with hydrophobic regions 11 and arrayed hydrophilic sites 12 located within the hydrophobic regions 11; and a hydrogel array 2, comprising a plurality of hydrogel microparticles 21 respectively immobilized on each hydrophilic site 12. The hydrogel microparticles 21 are covalently linked to primers for the target to be tested.

[0027] The hydrogel PCR chip proposed in this application uses a substrate 1 with hydrophobic regions 11 and arrayed hydrophilic sites 12, and hydrogel microparticles 21 covalently linked to the target primers inside. All hydrogel microparticles 21 are exposed in the same reaction chamber, allowing the nucleic acid of the sample to flow freely through each microparticle and undergo matched amplification with its internally immobilized primers. However, the primers are firmly anchored to the hydrogel backbone by covalent bonds, preventing crosstalk caused by liquid flow. Compared to traditional multi-chamber physical separation schemes, it eliminates the need to distribute trace amounts of sample to different reaction units, thus avoiding target loss or decreased sensitivity due to uneven distribution. This is particularly suitable for parallel detection of low-abundance targets.

[0028] Furthermore, the hydrophobic-hydrophilic patterned design on the surface of substrate 1 allows hydrogel prepolymer droplets to be positioned at hydrophilic sites 12 via a microelectronic printer, eliminating the need for complex microchannels or physical dikes to separate the reaction areas. This not only reduces the precision requirements for chip manufacturing but also simplifies sample loading—simply injecting the reaction solution into the chamber is sufficient to simultaneously wet all detection sites.

[0029] In some embodiments, the hydrogel microparticles 21 are columnar, spherical, or hemispherical; and / or, the solid-liquid contact angle of the hydrogel microparticles 21 on the substrate 1 is greater than 90 degrees.

[0030] When hydrogel prepolymer droplets have a high contact angle (>90°) at their hydrophilic sites, the droplets are constrained by the surrounding hydrophobic regions and shrink in the height direction, forming a three-dimensional spherical crown structure after solidification. Compared to the flat, disc-shaped gel formed when the contact angle is smaller, the spherical crown-shaped particles have a significantly higher specific surface area (surface area to volume ratio). During PCR, target nucleic acids, DNA polymerase, dNTPs, and fluorescent dyes in the reaction solution need to diffuse from the bulk liquid phase into the hydrogel to participate in the reaction. A higher specific surface area means a larger mass transfer interface and a shorter diffusion path, thereby accelerating the exchange rate of reactants inside and outside the hydrogel, reducing the negative impact of diffusion limitation on amplification efficiency, and ultimately resulting in earlier Ct values ​​and stronger fluorescence signal intensity. In addition, the three-dimensional spherical crown morphology allows each hydrogel particle to have a larger fluorescence emission volume in the vertical direction, which is beneficial for effective signal acquisition and grayscale analysis during fluorescence imaging.

[0031] In some embodiments, the hydrogel microparticles 21 are formed by polymer crosslinking of a prepolymer solution containing polyethylene glycol diacrylate (PEGDA) and polyethylene glycol (PEG).

[0032] Polyethylene glycol diacrylate (PEG) acts as a crosslinking agent, rapidly forming a stable three-dimensional network framework through UV-initiated free radical polymerization. PEG, acting as a porogen, embeds itself into the network structure during polymerization and binds a large number of water molecules through hydrogen bonding, thus creating abundant nanoscale pores within the gel. This material combination endows the polymerized and crosslinked hydrogel with both mechanical stability and high permeability, providing a suitable microenvironment for PCR reactions: on the one hand, the covalently crosslinked network ensures that the hydrogel particles 21 maintain structural integrity under repeated thermal cycling and liquid scouring; on the other hand, the hydrophilicity and porosity imparted by PEG guarantee efficient mass transfer of biomolecules (such as Taq DNA polymerase) within the hydrogel.

[0033] In some embodiments, the volume ratio of polyethylene glycol diacrylate to polyethylene glycol in the hydrogel prepolymer solution is 1:(0.66~1.5). For example, the volume ratio of polyethylene glycol diacrylate to polyethylene glycol is 4:6, 4.5:5.5, 5:5, 5.5:4.5, or 6:4, etc.

[0034] By adjusting the relative ratio of polyethylene glycol diacrylate (PEG) to polyethylene glycol (PEG), the cross-linking density and pore size of the hydrogel can be precisely controlled. When the PEG content is too low, the cross-linking network is too dense, restricting the entry of macromolecules such as polymerases, leading to a decrease in amplification efficiency. When the PEG content is too high, the gel's mechanical strength is insufficient and the pore size is too large, potentially causing leakage of immobilized primers or escape of amplification products. Preferably, when the volume ratio of PEG diacrylate to PEG is 4.5:5.5, the hydrogel exhibits an amplification curve most closely resembling that of solution PCR. This range balances high mass transfer efficiency with good structural integrity, ensuring that the detection sensitivity, linear range, and amplification efficiency of the hydrogel PCR chip meet practical application requirements.

[0035] In some embodiments, the primers include forward and / or reverse primers for the target to be tested. Further, the primers are covalently linked to the polymeric backbone of the hydrogel via an acrylamide group at their 5' end.

[0036] Primers fixed by traditional physical adsorption or embedding methods are prone to desorption and migration under high-temperature denaturation or prolonged immersion, leading to signal crosstalk between adjacent detection sites. By utilizing the acrylamide double bond modified at the 5' end of the primer, it can be directly incorporated into the hydrogel backbone as a comonomer during UV-initiated polymerization of polyethylene glycol diacrylate. This chemical bond makes the primer an integral part of the hydrogel backbone, ensuring its firm confinement within the target hydrogel particles even after dozens of thermal cycles and continuous liquid immersion. Furthermore, during subsequent use, the DNA product generated by the amplification reaction is physically confined within the particles because the 5' end of one strand is anchored to the gel backbone via the primer. The fluorescence signal is then enriched at a specific spatial location, fundamentally solving the problems of primer dimer cross-interference and non-specific amplification in traditional multiplex PCR, significantly improving detection specificity.

[0037] In some implementations, the 5' end of the primer is modified with Acrydite.

[0038] In some embodiments, the number of hydrogel microparticles 21 is 1 to 1000, the diameter is 0.03 to 1 mm, and the spacing between adjacent hydrogel microparticles is 0.03 to 1 mm.

[0039] If the diameter of the hydrogel particles 21 is too small (less than 0.03 mm), the total fluorescence signal may be insufficient, affecting the detection signal-to-noise ratio; if it is too large (greater than 1 mm), it may increase the diffusion distance and reduce amplification efficiency. By limiting the diameter and spacing of the hydrogel particles 21 within the above-mentioned range, a high-density array layout can be achieved as much as possible while ensuring that the fluorescence signals at adjacent hydrogel particles 21 do not experience optical crosstalk during detection. It is understood that those skilled in the art can customize the array size according to the actual number of detection targets, thereby meeting the needs of parallel detection from a few layers to dozens or even hundreds of layers.

[0040] In some implementations, such as Figure 1 As shown, the hydrogel array PCR chip 100 also includes a cover plate 3 disposed above the substrate 1, forming a reaction chamber between the substrate 1 and the cover plate 3. The cover plate 3 has at least one sample application well 31. The cover plate can be made of materials such as glass chips, transparent rigid polymer materials, silicone-based materials, transparent silicone rubber materials, and transparent polymer soft films, with a thickness of 0.1–10 mm. The diameter of the sample application well 31 is 0.2–2 mm. The reaction chamber has a length of 5–30 mm, a width of 5–30 mm, and a height of 0.1–5 mm.

[0041] In some embodiments, the cover plate 3 is also provided with at least one vent 32. The vent 32 facilitates the closing of the cover plate 3 and the substrate 1, and the gas in the reaction chamber can be discharged through the vent 32 during the closing process.

[0042] Please see Figure 2 This application also proposes a method for preparing a hydrogel array PCR chip as described above, comprising: S1. A hydrophobic layer is formed on the substrate surface, and an array of sites is etched on the hydrophobic layer; S2. The arrayed sites are modified with alkenyl groups by silanization. S3. Spot the hydrogel prepolymer solution containing acrylamide-modified primers to each arraying site; S4. Free radical polymerization is initiated by ultraviolet light to solidify the hydrogel prepolymer solution into hydrogel microparticles. At the same time, the primers are covalently anchored inside the hydrogel microparticles through their alkenyl groups participating in the polymerization reaction, and the alkenyl groups introduced by the hydrogel microparticles through silanization modification are chemically bonded to the substrate surface.

[0043] Under ultraviolet light, polyethylene glycol diacrylate prepolymer polymerizes to form a hydrogel network. Simultaneously, acrylamide-modified primers participate in copolymerization via double bonds and are immobilized within the hydrogel. Furthermore, the double bonds at the bottom of the hydrogel covalently couple with the alkenyl groups introduced by silanization modification on the substrate surface, firmly welding the hydrogel particles to the hydrophilic sites. Compared to soft lithography relying on photomasks or template methods requiring precise molds, this laser-based direct-write etching combined with surface chemical modification eliminates the need for physical molds, significantly shortening the chip fabrication cycle and reducing equipment and consumable investment.

[0044] In some embodiments, in S1, the hydrophobic layer is formed by vapor deposition of at least one of (1H,1H,2H,2H-heptadecyl)silane or 1H,1H,2H,2H-perfluorooctyltriethoxysilane.

[0045] In some embodiments, in S1, the etching is performed using infrared laser etching with a laser power of 11~15 W.

[0046] In some embodiments, in S2, the arrayed sites are modified with 3-(isobutenoyloxy)propyltrimethoxysilane.

[0047] 3-(isobutenoyloxy)propyltrimethoxysilane has a trimethoxysilane end, which can undergo hydrolysis and condense with the silanol groups on the glass surface to form a stable Si-O-Si covalent bond; the other end is methacryloxy, containing a carbon-carbon double bond that can participate in free radical polymerization. This bifunctional structure makes it a "molecular bridge" connecting the inorganic glass substrate and the organic hydrogel. During the UV curing stage, the methacryloxy group copolymerizes with polyethylene glycol diacrylate monomer, thereby firmly anchoring the hydrogel particles to the chip surface through chemical bonds, significantly improving the mechanical stability and lifespan of the chip under repeated thermal cycling and liquid rinsing conditions.

[0048] In some embodiments, the hydrogel prepolymer solution comprises polyethylene glycol diacrylate, polyethylene glycol, a photoinitiator, a buffer solution, Tween-20, and target primers. The buffer solution is a primer buffer. The volume ratio of polyethylene glycol diacrylate to polyethylene glycol is 1:(0.66~1.5). The amount of photoinitiator added is 4%~6% of the hydrogel prepolymer solution. For example, the amount of photoinitiator added is 4%, 4.5%, 5%, 5.5%, or 6% of the hydrogel prepolymer solution.

[0049] In some embodiments, the method for preparing the hydrogel prepolymer solution includes: Polyethylene glycol diacrylate (PEGDA) and low molecular weight polyethylene glycol (PEG) were mixed to obtain a preliminary mixed solution. Subsequently, a photoinitiator, buffer solution and primer solution were added to the preliminary mixed solution to obtain a hydrogel prepolymer solution.

[0050] Preferably, the preparation method of the hydrogel and the specific solution is as follows: Mix 23.4% v / v polyethylene glycol diacrylate, 28.6% v / v polyethylene glycol, 28% v / v 3×Tris-EDTA buffer, 0.05% v / v Tween-20 and 6% v / v 2-hydroxy-2-methyl-1-phenyl-1-propanone, and then add 18% v / v 100 μM forward primer to form a hydrogel prepolymer solution.

[0051] In some embodiments, the alkenyl-modified primers are 5' end-modified with Acrydite, and the volume ratio of the primer solution to the hydrogel prepolymer solution is (1~5):9. For example, the volume ratio of the primer solution to the hydrogel prepolymer solution is 1:9, 2:9, 3:9, 4:9, or 5:9, etc. The primer concentration is 80~120 μM. For example, the primer concentration is 80 μM, 90 μM, 100 μM, 110 μM, or 120 μM, etc.

[0052] In some embodiments, in S4, the wavelength of the ultraviolet light is 340~380 nm. For example, the wavelength of the ultraviolet light is 340 nm, 350 nm, 360 nm, 370 nm, or 380 nm, etc. The intensity range of the ultraviolet light is 0.1~50 mJ / cm². 2 For example, the intensity of ultraviolet light is 0.1 mJ / cm². 2 1 mJ / cm 2 10 mJ / cm 2 25 mJ / cm 2 Or 50 mJ / cm 2Irradiation time is 5 to 15 minutes. For example, irradiation time is 5 minutes, 10 minutes, or 15 minutes, etc.

[0053] This application also proposes the application of a hydrogel array PCR chip as described above, and a hydrogel array PCR chip prepared by the preparation method described above, in the detection of intestinal flora. The primers include forward or reverse primers from a primer set for detecting at least one of Bifidobacterium, Lactobacillus, Akkermansia, Faecalibacterium, Bacteroides, Prevotella, Trichophyton, Veillonella, Enterococcus, and Streptococcus.

[0054] In some embodiments, the primer sequences are as shown in any of SEQ ID NO. 1 to 20, as detailed in Table 1. Those skilled in the art will understand that in standard DNA sequence representation, Y is not a specific base, but a degenerate (fuzzy) symbol representing a pyrimidine base, i.e., C (cytosine) or T (thymine). This is one of the nucleic acid coding rules defined by the International Union of Pure and Applied Chemistry (IUPAC), and is commonly used to represent situations where multiple bases may exist at a site (e.g., sequence alignment, primer design, or unknown mutations).

[0055] Table 1. Primer sequences

[0056] In some embodiments, the method for detecting gut microbiota using a hydrogel array PCR chip as described above includes: S21. Add the PCR reaction solution containing the nucleic acid of the sample to be tested to the chip, so that the reaction solution flows through all the hydrogel particles; S22. Perform PCR thermal cycling amplification; S23. By detecting the fluorescence signal of each of the hydrogel microparticle sites, the presence of at least one of Bifidobacterium, Lactobacillus, Akkermansia, Faecalibacterium, Bacteroides, Prevotella, Trichophyton, Veillonella, Enterococcus, and Streptococcus in the sample to be tested is simultaneously determined.

[0057] In some implementations, the PCR reaction solution includes SYBR Green I fluorescent dye, reverse primers, DNA polymerase, and dNTPs.

[0058] Specifically, the PCR reaction system is as follows: 2× Super Master Mix is ​​1×, forward and reverse primers are 0.1~0.3 μM, the DNA to be tested is 1~3 μL, and enzyme-free water is added to a final volume of 16 μL.

[0059] In some implementations, the amplification program for quantitative real-time PCR is as follows: 94–96°C, 2–4 min; pre-amplification stage: 93–96°C, 8–12 s; 52–60°C, 40–50 s; 12–18 cycles. PCR cycling stage: 93–96°C, 8–12 s; 52–60°C, 20–40 s; 25–38 cycles.

[0060] Preferably, the amplification program for real-time PCR is as follows: 95℃, 3 min; pre-amplification stage: 95℃, 10 s; 55℃, 44 s; 15 cycles. PCR cycling stage: 95℃, 10 s; 55℃, 30 s; 30 cycles.

[0061] In some implementations, the hydrogel microparticles are irradiated with 470 nm excitation light during PCR amplification, and a 520 nm fluorescence signal is collected for imaging. The amplification curve is obtained by analyzing the gray value of each hydrogel microparticle.

[0062] The following specific examples provide further details.

[0063] Example 1 (1) Preparation of hydrogel prepolymer solution: Mix 20.8 μL of polyethylene glycol diacrylate, 31.2 μL of polyethylene glycol, 28 μL of 3×Tris-EDTA buffer, 0.05 μL of Tween-20 and 6 μL of 2-hydroxy-2-methyl-1-phenyl-1-propanone, and then add 18 μL of 100 μM forward primer (as shown in Table 1) to form hydrogel prepolymer solution.

[0064] (2) Preparation of hydrogel array PCR chip: Hydrophobic chemical modification of (1H,1H,2H,2H-heptadecyl)silane was performed on the chip substrate. Each chip was hydrophobically deposited using 25 μL of (1H,1H,2H,2H-heptadecyl)silane. Then, the array area of ​​the chip was laser etched using an infrared laser etching machine (power of 13W). The etched array was 8×8. Finally, the hydrophilic array area was chemically modified with 8% 3-(isobutenoyloxy)propyltrimethoxysilane solution. The hydrogel prepolymer solution prepared in (1) was dispensed onto the chemically modified array area. The hydrogel was then sequentially sprayed at a wavelength of 360 nm and an intensity of 25 mJ / cm. 2 The hydrogel array PCR chip was obtained by curing under ultraviolet light for 10 min and washing with 0.05% Tween-20 1×TE solution.

[0065] Example 2 The preparation method of this embodiment is the same as that of Example 1, except that the hydrogel prepolymer solution is prepared as follows: 23.4 μL of polyethylene glycol diacrylate, 28.6 μL of polyethylene glycol, 28 μL of 3×Tris-EDTA buffer, 0.05 μL of Tween-20 and 6 μL of 2-hydroxy-2-methyl-1-phenyl-1-propanone are mixed, and then 18 μL of 100 μM forward primer is added to prepare the hydrogel prepolymer solution.

[0066] Example 3 The preparation method of this embodiment is the same as that of Example 1, except that the hydrogel prepolymer solution is prepared as follows: 26 μL of polyethylene glycol diacrylate, 26 μL of polyethylene glycol, 28 μL of 3×Tris-EDTA buffer, 0.05 μL of Tween-20 and 6 μL of 2-hydroxy-2-methyl-1-phenyl-1-propanone are mixed, and then 18 μL of 100 μM forward primer is added to prepare the hydrogel prepolymer solution.

[0067] Example 4 The preparation method of this embodiment is the same as that of Example 1, except that the hydrogel prepolymer solution is prepared as follows: 28.6 μL of polyethylene glycol diacrylate, 23.4 μL of polyethylene glycol, 28 μL of 3×Tris-EDTA buffer, 0.05 μL of Tween-20 and 6 μL of 2-hydroxy-2-methyl-1-phenyl-1-propanone are mixed, and then 18 μL of 100 μM forward primer is added to prepare the hydrogel prepolymer solution.

[0068] Example 5 The preparation method of this embodiment is the same as that of Example 1, except that the hydrogel prepolymer solution is prepared as follows: 31.2 μL of polyethylene glycol diacrylate, 20.8 μL of polyethylene glycol, 28 μL of 3×Tris-EDTA buffer, 0.05 μL of Tween-20 and 6 μL of 2-hydroxy-2-methyl-1-phenyl-1-propanone are mixed, and then 18 μL of 100 μM forward primer is added to prepare the hydrogel prepolymer solution.

[0069] Examples 6-9 The specific steps of Examples 6 to 9 are the same as those of Example 1, except that the etched arrays in step (2) are 4×4, 6×6 and 10×10 in sequence.

[0070] The etched substrate was observed, and the results are as follows: Figure 6 As shown in (a), (b), (c) and (d), from Figure 6As can be seen, Examples 1, 6-9 successfully obtained arrays composed of 64, 16, 36 and 100 hydrogel microparticles, respectively, which shows that the method provided in the embodiments of this application can effectively realize the dot matrix fixation of hydrogel microparticles on the chip surface.

[0071] Example 10 The preparation steps in this embodiment are similar to those in Example 8, the difference being that different primers are added to different hydrogel microparticles. Specifically: No primers were added in rows 1, 3, 5, and 7 for the preparation of hydrogel particles, serving as a blank negative control. For row 2, forward primers for *Bifidobacterium*, *Lactobacillus*, *Ackermania*, and *Femtobacter* were added. For row 4, forward primers for *Ackermania*, *Femtobacter*, *Bacteroides*, and *Prevotella* were added. For row 6, forward primers for *Bacteroides*, *Prevotella*, *Trichophyton*, and *Veillonella* were added. For row 8, forward primers for *Trichophyton*, *Veillonella*, *Enterococcus*, and *Streptococcus* were added. All primers were the same as those in Table 1.

[0072] To evaluate the amplification efficiency of hydrogel PCR, the hydrogel prepolymer solutions prepared in Examples 1-5 were tested. The specific steps are as follows: (1) Preparation of PCR reaction systems for viral plasmid sample solutions of different concentrations: 50 μL of SuperStar Universal SYBR Master Mix (containing SYBR Green I fluorescent dye) was mixed with 20 μL of 1 μM reverse primer, 17.5 μL of ultrapure water, and 12.5 μL of viral plasmid samples of different concentrations (viral plasmid sample concentration was 10 copies / μL, 1×10... 2 copies / μL, 1×10 3 copies / μL, 1×10 4 copies / μL, 1×10 5 The samples (copies / μL) were mixed thoroughly to form the PCR reaction system, with a total reaction volume of 320 μL, yielding C1, C2, C3, C4, and C5, respectively. Simultaneously, a control group (NTC) without the added viral plasmid was prepared.

[0073] (2) The prepared PCR reaction system was added to the hydrogel prepolymer solutions prepared in Examples 1-5. Tube-type hydrogel PCR amplification was performed using a real-time PCR instrument for a total of 45 cycles, with fluorescence data recorded starting from the 16th cycle. The amplification efficiency of hydrogel groups with different pore sizes was compared with that of the solution control group. The results are as follows: Figure 3 As shown.

[0074] observe Figure 3 (a) The Ct values ​​corresponding to the amplification curves of real-time PCR using the hydrogel array PCR chip prepared in Example 1 for concentrations C1, C2, C3, C4, and C5 were 8, 11, 14, 19, and 23, respectively; observation Figure 3 (b) The Ct values ​​corresponding to the amplification curves of real-time PCR using the hydrogel array PCR chip prepared in Example 2 for concentrations C1, C2, C3, C4, and C5 were 6, 9, 12, 16, and 19, respectively; observation Figure 3 (c) The Ct values ​​corresponding to the amplification curves of real-time PCR using the hydrogel array PCR chip prepared in Example 3 were 8, 11, 14, 18, and 21 for concentrations C1, C2, C3, C4, and C5, respectively; observation Figure 3 (d) The Ct values ​​corresponding to the amplification curves of real-time PCR using the hydrogel array PCR chip prepared in Example 4 for concentrations C1, C2, C3, C4, and C5 were 9, 12, 15, 18, and 24, respectively; observation Figure 3 (e) The Ct values ​​corresponding to the concentrations C1, C2, C3, C4 and C5 of the amplification curves of real-time PCR using the hydrogel array PCR chip prepared in Example 5 are 10, 13, 17, 20 and 24, respectively. Figure 3 (f) represents the solution control group, with Ct values ​​corresponding to amplification curve concentrations C1, C2, C3, C4, and C5 being 3, 6, 9, 12, and 16, respectively. Comparing the Ct values ​​of the amplification curves of the hydrogel prepolymer solutions prepared in Examples 1-5 for quantitative real-time PCR, the hydrogel prepolymer solution prepared in Example 2 exhibited the highest amplification efficiency for quantitative real-time PCR. In contrast, the Ct values ​​of the hydrogel prepolymer solutions prepared in the other examples were significantly higher at the same sample concentration, and the amplification signal of low-concentration samples was significantly weakened, even approaching the detection limit. This demonstrates that by adjusting the relative ratio of polyethylene glycol diacrylate to polyethylene glycol, this application can precisely regulate the crosslinking density and pore size of the hydrogel, balancing high mass transfer efficiency with good structural integrity. This allows the detection sensitivity, linear range, and amplification efficiency of the hydrogel prepolymer solution to meet practical requirements, making it suitable for preparing hydrogel array PCR chips.

[0075] After PCR amplification using the hydrogel prepolymer solutions prepared in Examples 1-5, the temperature was slowly increased from a lower value while the fluorescence signal was continuously monitored. The specificity of the amplified product was evaluated by analyzing the changes in fluorescence signal with temperature and its derivative peak. The program was as follows: heating at 94℃ for 5 minutes, then reducing to 37℃ and heating for 5 minutes, followed by a gradual increase from 37℃ at a rate of 0.02℃ / s. The melting curves of the corresponding PCR reactions are shown below. Figure 4 As shown. Among them, Figure 4 (a) shows the melting curve of PCR amplification using the hydrogel prepolymer solution prepared in Example 1. Figure 4 (b) shows the melting curve of PCR amplification using the hydrogel prepolymer solution prepared in Example 2. Figure 4 (c) shows the melting curve of PCR amplification using the hydrogel prepolymer solution prepared in Example 3. Figure 4 In the middle (d), the melting curve of the PCR amplification using the hydrogel prepolymer solution prepared in Example 4 is shown. Figure 4 (e) shows the melting curve of PCR amplification using the hydrogel prepolymer solution prepared in Example 5. Figure 4 In the middle (f), the melting curve of PCR amplification by conventional liquid chromatography is shown.

[0076] from Figure 4 As can be seen, the hydrogel prepolymer solutions prepared in Examples 1-5 all showed a single and sharp melting peak in the range of approximately 82-86℃, while the NTC group did not show any obvious characteristic peaks and only maintained a low baseline signal, indicating that no obvious non-specific amplification or primer dimer formation occurred in the system, and the amplification products had good specificity.

[0077] Furthermore, the PCR amplification efficiency of the hydrogel prepolymer solution prepared in Example 2 was compared with that of a conventional liquid-phase PCR reaction system, and the results are as follows: Figure 5 As shown. From Figure 5 As can be seen in (a), the Ct value distribution (approximately 6–19 cycles) of the hydrogel prepolymer solution prepared in Example 2 is closest to that of the liquid-phase PCR reaction system, and the ΔCt remains at approximately 3 cycles between each concentration gradient, indicating that the hydrogel prepolymer solution prepared in Example 2 has good detection sensitivity from high to low concentrations. Furthermore, samples from high concentration C1 to low concentration C5 can be stably detected, demonstrating good detection sensitivity and dynamic range for samples with different initial concentrations.

[0078] The amplification efficiency of the hydrogel group was calculated using the PCR amplification efficiency formula, as follows: ; Where Slope is the slope and E is the amplification efficiency.

[0079] To evaluate the amplification performance of the hydrogel prepolymer solution prepared in Example 2, different concentrations of template (10¹~10¹⁰) were tested. 5 Amplification was performed using copies / μL, and a standard curve was plotted between the Ct value and the logarithm of the template concentration. Figure 5 As shown in Figure (b), the Ct value exhibits a good linear relationship with the logarithm of the template concentration, and its linear regression equation is y = 22.48. The correlation coefficient was 3.3x, and the correlation coefficient was R² = 0.99863, indicating that the system has a high linear correlation and good quantitative ability.

[0080] The sample to be tested was added to the hydrogel array PCR chip prepared in Example 10. The reaction system of the sample to be tested consisted of: 50 μL SuperStar Universal SYBR Master Mix (containing SYBR Green I fluorescent dye), 20 μL of 1 μM reverse primers (a combination of reverse primers for Bifidobacterium, Lactobacillus, Akkermansia, and Faecalibacterium), 17.5 μL of ultrapure water, and 12.5 μL of the viral plasmid sample to be tested (the viral plasmid sample concentration was 10 copies / μL, 1×10⁻⁶). 2 copies / μL, 1×10 3 copies / μL, 1×10 4 copies / μL, 1×10 5 (copies / μL), where the added primers are the reverse primers shown in Table 1.

[0081] PCR amplification was performed, with 45 cycles of detection, starting from the first cycle to detect fluorescence. Results are as follows: Figures 7 to 9 As shown. From Figure 7 As can be seen, the positive group (rows 2, 4, 6, and 8) emitted significant fluorescence, while the negative group (rows 1, 3, 5, and 7) did not show any obvious fluorescence signal. This indicates that the positive group had specific amplification of the corresponding target sample, and the negative group did not show obvious contamination.

[0082] from Figure 8 and Figure 9 It can be seen that the Ct values ​​corresponding to different sample concentrations are: C1 = 18, C2 = 21, C3 = 25, C4 = 28, and C5 = 33. Linear fitting of the Ct values ​​with the sample concentrations yields the standard curve equation y = 36.1 - 3.7x. Based on the amplification efficiency calculation formula, the amplification efficiency of this system is approximately 86.3%, indicating that the constructed hydrogel PCR chip has good amplification performance.

[0083] The above description is merely an exemplary embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the technical concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.

Claims

1. A hydrogel array PCR chip, characterized in that, include: A substrate having hydrophobic regions on its surface and arrayed hydrophilic sites located within the hydrophobic regions; A hydrogel array comprising a plurality of hydrogel microparticles respectively fixed on each of the hydrophilic sites; The hydrogel microparticles contain primers for the target being tested, which are covalently linked inside.

2. The hydrogel array PCR chip as described in claim 1, characterized in that, The hydrogel particles are columnar, crown-shaped, or hemispherical; and / or, The solid-liquid contact angle of the hydrogel microparticles on the substrate is greater than 90 degrees.

3. The hydrogel array PCR chip as described in claim 1, characterized in that, The hydrogel microparticles are formed by polymerization and crosslinking of a hydrogel prepolymer solution containing polyethylene glycol diacrylate and polyethylene glycol.

4. The hydrogel array PCR chip as described in claim 3, characterized in that, The volume ratio of polyethylene glycol diacrylate to polyethylene glycol in the hydrogel prepolymer solution is 1:(0.66~1.5).

5. The hydrogel array PCR chip as described in claim 1, characterized in that, The primers include the forward primer and / or reverse primer of the target to be tested; The primer is covalently linked to the polymer backbone of the hydrogel via its 5' end acrylamide group.

6. The hydrogel array PCR chip as described in claim 1, characterized in that, The number of hydrogel microparticles is 1 to 1000, the diameter is 0.03 to 1 mm, and the spacing between adjacent hydrogel microparticles is 0.03 to 1 mm.

7. The hydrogel array PCR chip as described in claim 1, characterized in that, The hydrogel array PCR chip also includes a cover plate disposed above the substrate, forming a reaction chamber between the substrate and the cover plate. The cover plate is provided with at least one sample application well. The reaction chamber has a length of 5-30 mm, a width of 5-30 mm, and a height of 0.1-5 mm.

8. A method for preparing a hydrogel array PCR chip according to any one of claims 1 to 7, characterized in that, include: S1. A hydrophobic layer is formed on the surface of the substrate, and an array of sites is etched on the hydrophobic layer; S2. The arrayed sites are modified with alkenyl groups by silanization. S3. Spot the hydrogel prepolymer solution containing acrylamide-modified primers to each of the arrayed sites; S4. Free radical polymerization is initiated by ultraviolet light to solidify the hydrogel prepolymer solution into hydrogel microparticles. At the same time, the primers are covalently anchored inside the hydrogel microparticles through their alkenyl groups participating in the polymerization reaction, and the alkenyl groups introduced by the silanization modification of the hydrogel microparticles are chemically bonded to the substrate surface.

9. The preparation method according to claim 8, characterized in that, The hydrophobic layer is formed by vapor deposition of at least one of (1H,1H,2H,2H-heptadecyl)silane or 1H,1H,2H,2H-perfluorooctyltriethoxysilane.

10. The preparation method according to claim 8, characterized in that, The silanization modification with alkenyl groups uses 3-(isobutenoyloxy)propyltrimethoxysilane.

11. The preparation method according to claim 8, characterized in that, The hydrogel prepolymer solution comprises polyethylene glycol diacrylate, polyethylene glycol, a photoinitiator, a buffer solution, and Tween-20; The volume ratio of polyethylene glycol diacrylate to polyethylene glycol is 1:(0.66~1.5).

12. The application of the hydrogel array PCR chip according to any one of claims 1 to 7, and the hydrogel array PCR chip prepared by the preparation method according to any one of claims 8 to 11, in the detection of intestinal flora, characterized in that, The primers include forward or reverse primers from a primer set for detecting at least one of Bifidobacterium, Lactobacillus, Akkermania, Faecalibacterium, Bacteroides, Prevotella, Trichophyton, Veillonella, Enterococcus, and Streptococcus. The primer sequences are shown in any of SEQ ID NO.1 to 20.

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