Dry-state hydrogel-based plasma-Fabry-Perot cavity biological detector as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological detection, in particular to a dry-state hydrogel-based plasma-Fabry-Perot cavity biological detector as well as a preparation method and application thereof. Compared with ELISA and other immunological detection methods depending on chemical tags and signal amplification, the method does not need a signal amplification process and shortens the detection time. The integrated plasma interlayer thickness sensing structure provided by the invention realizes direct signal reading after sample incubation is finished. According to the invention, the hydrogel is used as a sensing interlayer, and the hydrogel is used as a three-dimensional medium, so that a larger thickness change can be generated in a spatial scale. The biological detector can still keep relatively high specificity in a complex protein liquid phase environment (serum). The device has the structural characteristics of chip formation and miniaturization, and can be used for detecting various types of high-throughput proteins at the same time.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a dry hydrogel-based plasma-Faber cavity biodetector, its preparation method, and its application. Background Technology

[0002] Precise qualitative or quantitative analysis of proteins is of great significance in molecular biology research fields such as disease diagnosis, epidemic prevention, and pathological analysis. Currently, the gold standard method for protein quantification, recognized in clinical and research institutions, involves adding a specific labeling secondary antibody to amplify the signal of the protein to be detected, followed by signal reading using appropriate detection methods. Mainstream detection methods of this type include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), immunofluorescence assay (IFA), and immunochromatographic assay (LFIA). However, the complexity of the signal amplification process increases the actual cost and time cost of such detection methods, making them unsuitable for personal care applications. Therefore, label-free and signal-amplification-free immunosensors have become a current research hotspot. Among them, optical sensors based on surface plasmon resonance (SPR) are widely used in molecular biology because they can characterize subtle refractive index changes at the interface through the frequency and intensity of surface electron oscillations. These sensors, currently developed, possess high sensitivity, good specificity, and real-time signal output. However, this type of sensor is limited by its signal transduction mechanism, making high-throughput integration and visual optical analysis difficult. Current methods for detecting immunological proteins in clinical and research settings rely on large-scale instruments such as ELISA readers, dark-field microscopes, and fluorescence spectrometers for signal acquisition. This increases testing costs and is unsuitable for point-of-care testing at home. Therefore, there is an urgent need to develop an optically visualized immunosensor that combines label-free, high-throughput, multi-component analysis for application in point-of-care testing (POCT). Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a dry hydrogel-based plasma-Fabry-Perot cavity biodetector, its preparation method, and its application. The present invention provides a dry hydrogel-based plasma-Fabry-Perot cavity biodetector that requires no specialized instruments; the operator can obtain the detection results by naked-eye colorimetry or by analyzing photos taken with a smartphone. Furthermore, traditional detection methods, such as ELISA, require a series of signal amplification processes, including target labeling, which increases the cost and time consumption of the detection process. The present invention utilizes its structural optical properties to achieve highly sensitive and specific diagnosis without labeling.

[0004] This invention provides a dry hydrogel-based plasma-Faber cavity biodetector, comprising a hydrogel detection layer made of photocrosslinked gel, wherein biological ligand molecules binding to detection targets are immobilized in the hydrogel detection layer;

[0005] The bottom of the hydrogel detection layer is provided with a reflective substrate layer with specular reflection properties;

[0006] Metal nanoparticles are dispersed on the surface of the hydrogel detection layer.

[0007] In some embodiments, the photocrosslinking gel includes at least one of polyacrylamide-based hydrogel, polyvinyl alcohol-based hydrogel, and sodium alginate-based hydrogel;

[0008] The biological ligand molecules include at least one of antigens, antibodies, nucleic acid molecules, aptamers, enzymes, receptors, and lectins;

[0009] The reflective substrate layer includes at least one of a gold film, a silver film, an aluminum film, a liquid metal film, and a silicon wafer;

[0010] The metal nanoparticles include at least one of gold, silver, aluminum, and copper.

[0011] In some embodiments, the thickness of the hydrogel detection layer is 20~130 nm;

[0012] The metal nanoparticles have a particle size of 2-200 nm and a density of 20-200,000 particles / μm. 2 .

[0013] This invention provides a method for preparing the aforementioned dry hydrogel-based plasma-Fabry-Perot cavity biodetector, comprising the following steps:

[0014] Step 1: Acrylamide and acrylic acid are copolymerized, activated, and functionalized with allylamine to obtain an allyl-modified copolymer;

[0015] Step 2: Dissolve, mix, and filter the copolymer, photoinitiator, and light absorber to obtain a photocurable hydrogel precursor solution;

[0016] Step 3: Spin-coat the photocurable hydrogel precursor solution onto the aldehyde-modified reflective substrate to obtain a hydrogel precursor film. The hydrogel precursor film is then irradiated with ultraviolet light to obtain a composite of the hydrogel detection layer and the reflective substrate.

[0017] Step 4: Take a solid substrate with adsorbed metal nanoparticles and spin-coat it with polymethyl methacrylate solution to obtain a metal nanoparticle-polymer composite film.

[0018] Step 5: Take the gold nanoparticle-polymer composite film from Step 4 and transfer it to the surface of the hydrogel detection layer from Step 3 using a wet method. After dissolving polymethyl methacrylate in acetone, the dry hydrogel-based plasma-Faber cavity biodetector is obtained.

[0019] In some embodiments, in step 1, the mass-to-volume ratio of acrylamide to acrylic acid is (5~8) g:(1~2).

[0020] The activation includes activation using EDC / NHS.

[0021] In some embodiments, in step 2, the mass ratio of the light absorber, the photoinitiator, and the copolymer is (250~350):(30~50):(400~500), the light absorber includes N,N'-methylenebisacrylamide, and the photoinitiator includes photoinitiator 2959.

[0022] In some embodiments, in step 3,

[0023] The aldehyde-modified reflective substrate layer is obtained by sequentially treating the reflective substrate layer with 3-aminopropyltrimethoxysilane, cleaning, and glutaraldehyde solution.

[0024] The thickness of the hydrogel precursor film is 50~300nm, and the thickness of the hydrogel detection layer after ultraviolet irradiation is 20~130nm.

[0025] In some embodiments, in step 3, the 3-aminopropyltrimethoxysilane treatment includes treatment with 3-aminopropyltrimethoxysilane at 60-75°C for 0.5-1.5 hours, the cleaning includes sequential cleaning with ethanol and deionized water, and the concentration of the glutaraldehyde solution is 5 vol%-20 vol.

[0026] In some embodiments, in step 4, the solid substrate includes a quartz substrate, which is treated with a silane coupling agent to adsorb metal nanoparticles to obtain a solid substrate with adsorbed metal nanoparticles.

[0027] The present invention provides a dry hydrogel-based plasma-Faber cavity biodetector prepared by the aforementioned method.

[0028] This invention provides the application of the dry hydrogel-based plasma-Faber cavity biodetector and / or the dry hydrogel-based plasma-Faber cavity biodetector prepared by the aforementioned method in the fabrication of visualized high-throughput biodetection chips.

[0029] The present invention provides a visualization high-throughput biodetection chip, comprising the dry hydrogel-based plasma-Faber cavity biodetector and / or the dry hydrogel-based plasma-Faber cavity biodetector prepared by the preparation method and a physical isolation structure, wherein the physical isolation structure covers the dry hydrogel-based plasma-Faber cavity biodetector and forms at least one reaction chamber.

[0030] In some specific embodiments, the physical isolation structure is a PDMS fence.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. Compared with immunological detection methods such as ELISA that rely on chemical tags and signal amplification, this invention eliminates the need for signal amplification, thus shortening the detection time.

[0033] 2. As an integrated plasma sandwich thickness sensing structure, this invention enables direct signal reading after sample incubation.

[0034] 3. This invention uses hydrogel as a sensing interlayer. As a three-dimensional medium, hydrogel can produce greater thickness variations in space.

[0035] 4. This invention has extremely high naked-eye response capability for cavity (hydrogel interlayer) thickness, with a maximum grayscale sensitivity of 6.11 / nm (i.e., grayscale changes by 6.11 for every 1 nm change in thickness); in terms of protein binding quantification, it has a low detection limit (“Biotin / Streptavidin” system: 60.8 pg / mL), and the detection limit of this system using grayscale analysis is 2.04 ng / mL.

[0036] 5. This biodetector maintains high specificity even in complex protein liquid environments (serum).

[0037] 6. This invention features a chip-based and miniaturized structure, enabling simultaneous detection of multiple types of proteins at high throughput. Attached Figure Description

[0038] Figure 1 The diagram shows the structure of the dry hydrogel-based plasma-Faber cavity biodetector of the present invention, wherein, a is a schematic diagram of the sandwich sensing structure and b is a scanning electron microscope image;

[0039] Figure 2 A reference diagram showing the structure of a high-throughput biodetection chip;

[0040] Figure 3The detection results of the "S-peptide-S-peptide antibody" detection model in Example 2 are shown in Figure a. The reflectance detection results based on fiber optic spectrometer at different antibody concentrations are shown in Figure b. The standard curve based on reflectance is shown in Figure c. The microscope images obtained at different antibody concentrations are shown in Figure d. The standard curve obtained based on gray value detection results is shown in Figure d.

[0041] Figure 4 Example 3 illustrates the effects of different densities of metal nanoparticles (left figure) and different crosslinking agent concentrations (right figure) on the reflectance difference of a dry hydrogel-based plasma-Faber cavity biodetector.

[0042] Figure 5 Example 3 illustrates the effect of antigen grafting concentration (left figure) and incubation time (right figure) on the reflectance difference of a dry hydrogel-based plasma-Faber cavity biodetector.

[0043] Figure 6 The detection flowchart in Example 4 is shown;

[0044] Figure 7 The visualization high-throughput biodetection chip prepared in Example 4 was used to detect the results of negative and positive standard samples of the corresponding antigens.

[0045] Figure 8 The results of using a visualization high-throughput biodetection chip for clinical testing are shown. Figure a is a heatmap of the expression of COVID-19 antibody subtypes in serum obtained from a large number of clinical serum samples using the visualization high-throughput biodetection chip. Figure b is a microscopic image of some chip detection results. Figure c is the ROC curve for determining the detection results. Figure d is the correlation between the detection results of this application and the clinical CLIA calibration values. Detailed Implementation

[0046] This invention provides a dry hydrogel-based plasma-Fabry-Perot cavity biodetector, its preparation method, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0047] This invention constructs a stimulus-responsive plasma-Fabry-Perot cavity sandwich optical structure. The invention uses a stimulus-responsive hydrogel as the cavity material, and the cavity length can dynamically change according to the environment. Combined with the upper plasma structure, it outputs optical signals in the visible light region.

[0048] The gel type used in this invention can respond to a variety of signals, such as the water content, pH value, and protein concentration in the liquid environment.

[0049] This invention fabricates an integrated sandwich structure detector. Compared to traditional sandwich sensors that require the construction of a nano-sandwich structure after sample reaction to output a signal, the target protein in this invention can directly diffuse into the hydrogel sandwich and specifically bind inside the hydrogel, enabling immediate signal output and simplifying the testing process. Wherein:

[0050] 1. Regarding the selection of gel precursors, gel components with photocrosslinking properties can be used to replace the gel selection in this invention.

[0051] 2. Regarding the selection of the substrate, substrates with specular reflective properties, such as gold films, silver films, aluminum films, and liquid metal films, can all replace the substrate selection in this invention.

[0052] 3. The metal nanoparticles in the metal nanoparticle layer are spherical, rod-shaped, star-shaped, cubic, or irregular in shape. The metals in the layer include gold, silver, aluminum, and copper. The characteristic size of the metal nanoparticles is 2-200 nm, and their arrangement can be ordered or disordered. The average density of the metal nanoparticles is 20-200,000 / μm. 2

[0053] 4. Regarding the selection of capture units, the capture units are used to capture the target protein. The types of capture units include proteins, small molecules, polymers, and nucleic acid molecules. The capture units are fixed inside the patterned gel membrane obtained in step 3 by means of liquid incubation, liquid drying, manual spotting, printing with a microarray bioprinter, or introducing the capture molecule solution through microfluidic channels.

[0054] 5. Regarding the selection of microfluidic channels, the materials for microfluidic channels are polydimethylsiloxane, polymethyl methacrylate, cyclic olefin copolymers, or photocurable resins.

[0055] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0056] S-peptide: purchased from Abcam, catalog number ab273063;

[0057] S-peptide antibody: purchased from Abcam, catalog number ab272504;

[0058] Graft antigens (a) to (i) were all purchased from Sino Biological, as detailed below:

[0059] Alpha Spike RBD (40592-V08H82)

[0060] Gamma Spike RBD (40592-V08H86)

[0061] Delta Spike RBD (40592-V08H90)

[0062] B.1.1.529 (Omicron) Spike RBD (40592-V08H121)

[0063] Kappa Spike RBD (40592-V08H88)

[0064] XBB.1.5 (Omicron) Spike RBD (40592-V08H146)

[0065] BQ.1.1 (Omicron) Spike RBD (40592-V08H143)

[0066] BA.4 / BA.5 (Omicron) Spike RBD (40592-V08H130)

[0067] JN.1 (Omicron) Spike RBD (40592-V08H155).

[0068] The present invention will be further illustrated below with reference to the embodiments.

[0069] Example 1

[0070] The fabrication method of a dry hydrogel-based Fabry-Perot cavity coupled surface plasmon resonance biodetector (dry hydrogel-based plasmon-Fabry-Perot cavity biodetector) includes the following steps:

[0071] Step 1: Preparation of a hydrogel precursor with photocrosslinking properties: Dissolve 1.028 mL of acrylic acid, 6.04 g of acrylamide, and 1.14 g of ammonium persulfate in 100 mL of deionized water. Adjust the pH of the solution to 10 with sodium hydroxide solution. Purge the mixture with nitrogen gas for 30 minutes to remove oxygen, then heat at 50°C for two hours. After the reaction is complete, dialyze the mixture for seven days using a dialysis apparatus with a molecular weight cutoff of 12000. Freeze-dry to obtain the acrylamide-acrylic acid copolymer. Add 0.5 g of the prepolymer prepared above to 20 mL of deionized water, along with 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.3 g of N-hydroxysuccinimide (NHS), and stir thoroughly for 1 hour. Add 200 μL of allylamine to the mixture, react in a water bath at 50°C for 24 hours, dialyze using a dialysis apparatus with a molecular weight cutoff of 12000, and freeze-dry to obtain the allyl-functionalized acrylamide-acrylic acid copolymer. 180 mg of the above-mentioned functionalized copolymer was added to 3 mL of deionized water to prepare a 6% solution A. 4 mg of N,N'-methylenebisacrylamide (Bis) and 64 mg of photoinitiator 2959 were added to 2 mL of deionized water to prepare solution B. Solutions A and B were mixed at a volume ratio of 5:7, impurities were filtered out, and the mixture was sealed and stored in the dark as a hydrogel precursor solution.

[0072] Step 2: Preparation of functionalized substrates with photoinitiated chemical bonding: The substrate used here should have obvious specular reflective properties, including but not limited to silicon wafers, noble metal films such as gold films, and liquid metals. Subsequent descriptions will use silicon wafers as the substrate. The silicon wafer was sequentially placed in hexane, anhydrous ethanol, and deionized water, and sonicated for 5 minutes each. The substrate surface was then thoroughly cleaned with deionized water. Next, the substrate was placed in a piranha solution (a mixture of 98% concentrated sulfuric acid and 30% hydrogen peroxide in a 7:3 volume ratio) and heated at 140°C for 2 hours until no obvious bubbles were generated. The substrate was then thoroughly cleaned with deionized water and dried with nitrogen. Substrates not used immediately were placed in anhydrous ethanol for later use. The substrate was then placed in a vacuum desiccator, and 60 μL of 3-aminopropyltrimethoxysilane (APTMS) was added. The substrate was heated at 70°C for 1 hour. Remove the silicon wafer and wash it three times with ethanol and deionized water in sequence. Place it in PBS solution and add glutaraldehyde solution at a volume ratio of 1:10. Remove the wafer, wash it with deionized water, and dry it with nitrogen.

[0073] Step 3: Preparation of patterned ultrathin hydrogel films: After spin-coating a hydrogel precursor solution onto an aldehyde-modified silicon substrate, selective UV exposure is performed using a photomask combination. The substrate is then rinsed with deionized water and dried with nitrogen to obtain a patterned hydrogel ultrathin film chemically bonded to the substrate. The thickness of the hydrogel ultrathin film can be controlled between 20 nm and 130 nm, with control parameters including the crosslinking agent concentration in the precursor solution, spin-coating speed, and UV exposure time. The rinsed hydrogel ultrathin film is then soaked in deionized water overnight to remove unreacted components and dried with nitrogen for later use.

[0074] Step 4: Nanoparticle Film Preparation and Transfer: The quartz substrate cleaning process is the same as the silicon wafer cleaning process in Step 2. The substrate is placed in a vacuum desiccator, 3-aminopropyltrimethoxysilane is added, and the mixture is heated at 70°C for 1 hour. It is then soaked in water for 2 hours to reduce the amino density. Next, it is soaked in a solution of metal nanoparticles synthesized from sodium citrate-reduced chloroauric acid, dried with nitrogen, and spin-coated with a polymethyl methacrylate solution to form a gold nanoparticle-polymer composite film. The nanoparticle-polymer composite film is transferred to the dry hydrogel film obtained in Step 3. The polymethyl methacrylate is dissolved using acetone, leaving the gold nanoparticles on the hydrogel surface, thus constructing a dry hydrogel-based Fabry-Perot cavity coupled surface plasmon resonance biodetector chip. Its structural schematic diagram and scanning electron microscope image are shown below. Figure 1 a, Figure 1 As shown in b.

[0075] Step 5: Microfluidic Channel Fabrication: A glass plate with a chromium film and photoresist layer is exposed to ultraviolet light under a mask of a transparent microfluidic channel array. The substrate is then immersed in a developer (sodium hydroxide solution) for 15 seconds to obtain a patterned photoresist array glass plate with a chromium layer. The plate is then immersed in a chromium etching solution to remove the chromium, followed by immersion in a glass etching solution (a mixed solution of HF, HNO3, and NH4F) for 2 hours to obtain a glass microfluidic channel mold with a surface height of 60 μm. The glass microfluidic channel mold is then cleaned using an oxygen plasma cleaner and immersed in 0.1% trichloroisocyanuric acid (1H₂, 1H₂, 2H₂O) solution. The PDMS prepolymer was immersed in an acetone solution of 2H-tridecyl fluorooctyl)silane for 10 minutes, cleaned with ethanol and dried with nitrogen. The PDMS prepolymer and curing agent were mixed evenly at a mass ratio of 10:1. After vacuum degassing, the mixture was poured onto the surface of the glass mold and placed in an oven at 60°C for 4 hours to cure. The PDMS microfluidic microfluidic channels were then obtained by peeling it off.

[0076] Step 6: Coating with biological ligand molecules and protein detection: The structure obtained in Step 4 was activated in a carboxyl activation solution for 30 min (activation solution solvent was 0.1 M morpholine ethanesulfonic acid solution, pH=6.0, solute was 0.1 M EDC; 0.1 M NHS). After activation, it was rinsed with deionized water and dried with nitrogen. Immediately after activation, the PDMS microfluidic channels prepared in Step 5 were inverted onto the patterned lattice of the activated hydrogel membrane. The width of the channels was larger than the maximum width of the hydrogel pattern to ensure that the channels completely covered the hydrogel pattern. The solution of biological ligand molecules to be coated was introduced under negative pressure and reacted for 3 h. After the reaction, the PDMS channels were removed and immediately rinsed with plenty of deionized water. Unreacted active sites were blocked with 0.1 M glycine solution. After blocking, it was immediately rinsed with plenty of deionized water and dried with nitrogen. The resulting gel film coated with biological ligand molecules can be used immediately or stored in a sealed container under vacuum at -20℃ for long-term use. For the specific chip structure, please refer to [reference needed]. Figure 2 A PDMS fence was applied to a substrate coated with bioligand molecules to divide the chip into independent detection areas. 8 μL of the sample to be tested (antigen or antibody standard solution, human or other species serum or whole blood dilution) was added to each detection area, and the reaction was allowed to proceed for 1 hour. After the reaction, the chip was thoroughly washed five times with PBS, the fence was removed, and the chip was rinsed with deionized water and dried under nitrogen.

[0077] Step 7, the specific steps for calculating the concentration of biomarkers in the sample are as follows: observe with the naked eye or use equipment to detect the reflectance intensity of the gel point after the reaction, and establish the relationship curve between the concentration of biomarkers in the sample and the change in reflectance and grayscale.

[0078] Example 2

[0079] The “S-peptide-S-peptide antibody” detection model, as illustrated in this embodiment, demonstrates the protein-antibody interaction model of the present invention for basic antigen-antibody interactions. The steps are as follows:

[0080] 1. The photocurable hydrogel precursor solution is drop-coated onto the surface of a functionalized silicon wafer, and a hydrogel precursor film with a target thickness (about 90 nm) is obtained by spin-coating according to the corresponding parameters. The film is then irradiated with ultraviolet light to obtain a hydrogel protein detection film with a dry thickness of about 20 nm.

[0081] 2. The quartz substrate cleaned with piranha solution is aminated on the surface using a silane coupling agent (such as KH550). The resulting substrate is then immersed in a gold nanoparticle solution for adsorption. The adsorption density can be controlled by the concentration of the gold nanoparticle solution, the adsorption time, and the amyl grafting density. The synthesis of gold nanoparticles is based on the known one-pot method and seed growth method. After adsorption, the substrate is cleaned and dried, and then spin-coated with polymethyl acrylate solution to form a gold nanoparticle-polymer composite film.

[0082] 3. The nanoparticle-polymer composite film obtained in step 2 is transferred to the dry hydrogel film obtained in step 1 by wet transfer, and polymer dissolution treatment is performed to realize the construction of the dry hydrogel-based Fabry cavity coupled surface plasmon resonance biodetector chip.

[0083] 4. Different detection areas were demarcated on the substrate surface using PDMS fencing. The carboxyl functional groups of the hydrogel were activated into active NHS groups using an EDC / NHS reaction according to the area. PDMS microfluidic channels were then covered on the surface, and S-peptide antigen (0.2 mg / mL) was grafted into the hydrogel via liquid-phase incubation. Unreacted S-peptide molecules were washed with phosphate buffer, and unreacted grafted active sites were passivated with glycine solution (0.2M, phosphate buffer PBS) to prevent non-specific adsorption. After phosphate buffer washing, different concentrations of S-peptide antibody solution (0-200 ug / mL, 50 mg / mL BSA (PBS) solution) were added to each detection area. After incubation in liquid phase at room temperature for 1 h, the area was washed with PBST solution (tween 20, 0.05%, w / v, PBS) and dried with nitrogen.

[0084] 5. The chip obtained in step 4 was subjected to a portable fiber optic spectroscopy device to measure its reflectance spectrum, and an image was acquired using a complementary metal-oxide-semiconductor (CMOS) sensor. The image acquisition conditions were (gamma = 0.2, exposure time = 70 ms). The reflectance change at a specific wavelength (depending on the size and density of the gold nanoparticles used) was selected as the signal output to obtain the reflectance standard curve for the "S-peptide-S-peptide antibody" detection model. Figure 3 ab). After processing the photo into grayscale, the grayscale value is measured to obtain the grayscale standard curve of the "Biotin / Streptavidin" detection model. Figure 3 (cd). Results analysis using reflectance showed that the limit of detection for the dry hydrogel-based Fabry-Perot cavity coupled surface plasmon resonance biodetector in this model was 60.8 pg / mL.

[0085] Example 3

[0086] Based on Example 2, the effects of gold nanoparticle density, gel crosslinking degree, antigen grafting concentration, and sample incubation time on the effectiveness of the antigen / antibody detection model were investigated.

[0087] 1. Investigation into the density of gold nanoparticles

[0088] High-throughput sensor chips are fabricated using the following density grouping:

[0089] Particle-free: density is 0 particles / μm²;

[0090] High-density particles: density is 146 particles / μm²;

[0091] Medium-density particles: 78 particles / μm²;

[0092] Low-density particles: density is 41 particles / μm²;

[0093] Test results as follows Figure 4 As shown in the left-middle figure, the results demonstrate that optimal sensing performance is achieved under low-density gold nanoparticle parameters.

[0094] 2. Investigation of the degree of gel crosslinking

[0095] Crosslinking agents were set at 0.1%, 0.2%, 0.5%, and 1%, respectively, and corresponding high-throughput sensor chips were prepared. The detection results are as follows: Figure 4 As shown in the right-middle figure, the results indicate that the gel membrane prepared with a crosslinking agent concentration of 0.1% in the precursor solution exhibits the best sensing performance.

[0096] 3. Investigation into the grafting concentration of antigen

[0097] The ligand grafting concentrations were set to 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, respectively, and corresponding high-throughput sensing chips were fabricated. The detection results are as follows: Figure 5 As shown in the middle left figure, the results indicate that the high-throughput sensor chip prepared at a ligand concentration of 0.2 mg / mL exhibits the best sensing performance.

[0098] 4. Investigation into antigen incubation time

[0099] Antigen incubation times were set to 0, 2 min, 5 min, 10 min, 15 min, 30 min, 60 min, 120 min, and 240 min, respectively, and corresponding high-throughput sensor chips were prepared. The detection results are as follows: Figure 5 As shown in the right-middle figure, the results indicate that optimal sensing performance can be achieved under a sample incubation period of 2 hours.

[0100] Example 4

[0101] Serological detection of SARS-CoV-2 antibody subtypes was achieved using a dry hydrogel-based Fabry-Perot cavity coupled to surface plasmon resonance (SPR) biodetector chip. This embodiment demonstrates the application of the present invention in protein-based clinical diagnostics, and the steps are as follows:

[0102] 1. The operation steps are the same as steps 1-4 in Example 1. The concentration of the grafted antigen is 0.2 mg / mL. The types of grafted antigens include the following: (a) Alpha Spike RBD; (b) Gamma Spike RBD; (c) Delta Spike RBD; (d) B.1.1.529 (Omicron) Spike RBD; (e) Kappa Spike RBD; (f) XBB.1.5 (Omicron) Spike RBD; (g) BQ.1.1 (Omicron) Spike RBD; (h) BA.4 / BA.5 (Omicron) Spike RBD; (i) JN.1 (Omicron) Spike RBD. The test sample is human serum. The serum sample (n = 108) is directly added to the substrate fence, incubated at room temperature for 1 hour, and then washed.

[0103] 2. The chip obtained in step 1 is used to read visible light signals through various methods such as portable fiber optic spectroscopy equipment and microscope photography.

[0104] The specific detection process in this embodiment is as follows: Figure 6 As shown.

[0105] Before the experiment, the high-throughput sensor chip prepared in this embodiment was used to detect the negative and positive standard samples of the corresponding antigens (the positive standard is the antibody corresponding to the antigen). The specific results are as follows: Figure 7 As shown, it can determine the immune status (positive or negative) of a sample based on the color change of the sensor points after incubation.

[0106] like Figure 8 As shown in Figure a, the dry hydrogel-based Fabry-Perot cavity coupled surface plasmon resonance biodetector uses image grayscale values ​​as signal output to analyze and obtain the detection values ​​of COVID-19 antibody content in 108 human serum samples (92 cases in the viral nucleic acid positive group or PCR positive group, and 16 cases in the healthy group), which are displayed in the form of a heatmap. Figure 8 The diagram in Figure b illustrates a representative result obtained after using this structure to detect a large number of samples. The detection results show good consistency with the clinical judgment results of the actual samples. Figure 8 As shown in the CD image, using Alpha S1 antibody expression level as the research object, the statistical results show that this detector has 100% detection specificity and 98.9% detection sensitivity. The results are consistent with the clinical analysis method CLIA, with a consistency R = 0.877.

[0107] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A dry hydrogel-based plasma-Faber cavity biodetector, characterized in that, It includes a hydrogel detection layer made of photocrosslinked gel, wherein biological ligand molecules that bind to the detection target are immobilized in the hydrogel detection layer; The bottom of the hydrogel detection layer is provided with a reflective substrate layer with specular reflection properties; Metal nanoparticles are dispersed on the surface of the hydrogel detection layer.

2. The dry hydrogel-based plasma-Fabry-Perot cavity biodetector according to claim 1, characterized in that, The photocrosslinking gel includes at least one of polyacrylamide-based hydrogel, polyvinyl alcohol-based hydrogel, and sodium alginate-based hydrogel; The biological ligand molecules include at least one of antigens, antibodies, nucleic acid molecules, aptamers, enzymes, receptors, and lectins; The reflective substrate layer includes at least one of a gold film, a silver film, an aluminum film, a liquid metal film, and a silicon wafer; The metal nanoparticles include at least one of gold, silver, aluminum, and copper.

3. The dry hydrogel-based plasma-Fabry-Perot cavity biodetector according to claim 1 or 2, characterized in that, The thickness of the hydrogel detection layer is 20~130nm; The metal nanoparticles have a particle size of 2-200 nm and a density of 20-200,000 particles / μm. 2 .

4. The method for preparing the dry hydrogel-based plasma-Fabry-Perot cavity biodetector according to any one of claims 1 to 3, characterized in that, Includes the following steps: Step 1: Acrylamide and acrylic acid are copolymerized, activated, and functionalized with allylamine to obtain an allyl-modified copolymer; Step 2: Dissolve, mix, and filter the copolymer, photoinitiator, and crosslinking agent to obtain a photocurable hydrogel precursor solution; Step 3: Spin-coat the photocurable hydrogel precursor solution onto the aldehyde-modified reflective substrate to obtain a hydrogel precursor film. The hydrogel precursor film is then irradiated with ultraviolet light to obtain a composite of the hydrogel detection layer and the reflective substrate. Step 4: Take a solid substrate with adsorbed metal nanoparticles and spin-coat it with polymethyl methacrylate solution to obtain a metal nanoparticle-polymer composite film. Step 5: The gold nanoparticle-polymer composite film described in Step 4 is transferred to the surface of the hydrogel detection layer described in Step 3 by a wet method. After dissolving polymethyl methacrylate in acetone, the biological ligand molecules are fixed into the hydrogel detection layer by liquid phase incubation to obtain the dry hydrogel-based plasma-Faber cavity biological detector.

5. The preparation method according to claim 4, characterized in that, In step 1, the mass-to-volume ratio of acrylamide to acrylic acid is (5~8) g: (1~2). The activation includes activation using EDC / NHS; In step 2, the mass ratio of the crosslinking agent, the photoinitiator, and the copolymer is (250~350):(30~50):(400~500), the crosslinking agent includes N,N'-methylenebisacrylamide, and the photoinitiator includes photoinitiator 2959.

6. The preparation method according to claim 4, characterized in that, In step 3 The aldehyde-modified reflective substrate layer is obtained by sequentially treating the reflective substrate layer with 3-aminopropyltrimethoxysilane, cleaning, and glutaraldehyde solution. The thickness of the hydrogel precursor film is 50~300nm, and the thickness of the hydrogel detection layer after ultraviolet irradiation is 20~130nm.

7. The preparation method according to claim 4, characterized in that, In step 4, the solid substrate includes a quartz substrate, which is treated with a silane coupling agent to adsorb metal nanoparticles to obtain a solid substrate with adsorbed metal nanoparticles.

8. The preparation method according to claim 4, characterized in that, In step 5, during the liquid phase incubation, the concentration of the biological ligand molecule is 0.05~0.2 mg / mL, and the incubation time is 2~240 min.

9. The application of the dry hydrogel-based plasma-Faber cavity biodetector according to any one of claims 1 to 3 and / or the dry hydrogel-based plasma-Faber cavity biodetector prepared by the preparation method according to any one of claims 4 to 8 in the preparation of a visualized high-throughput biodetection chip.

10. A high-throughput visual biodetection chip, characterized in that, The invention includes the dry hydrogel-based plasma-Faber cavity biodetector as described in any one of claims 1 to 3 and / or the dry hydrogel-based plasma-Faber cavity biodetector and physical isolation structure prepared by the preparation method described in any one of claims 4 to 8, wherein the physical isolation structure covers the dry hydrogel-based plasma-Faber cavity biodetector and forms at least one reaction chamber.

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