Immune response hydrogel, immune sensing detection device and preparation method

Abstract

The application discloses an immune response hydrogel, an immune sensing detection device and a preparation method. The preparation steps of the immune response hydrogel of the application include vinyl modification of an antigen protein, preparation and antibody coupling of gold nanoparticles, polymerization of the vinyl modified antigen protein and immune cross-linking of the antibody coupled gold nanoparticles, and finally the immune response hydrogel with the immune response function is obtained. The wireless immune detection device includes an immune response hydrogel resonance sensor, a readout coil and the like. The device can realize wireless readout of multiple sensing signals through extension of parallel intermediate coils, and meanwhile, the immune hydrogel resonance sensor and the intermediate coil are assembled in an article such as a mask, so that wearable detection of antigen proteins can be realized. The technology provides a rapid, sensitive, accurate and instant detection platform for applications such as virus antigen protein monitoring and wearable immune biochemical analysis.

Description

Technical Field

[0001] This invention relates to a novel immunosensing detection technology, particularly a method for preparing an immunoresponsive hydrogel, and an immunoresonance sensing detection device based on the immunoresponsive hydrogel and its applications. Background Technology

[0002] The ongoing evolution of COVID-19 and the seasonal outbreaks of infectious respiratory viruses such as influenza and respiratory syncytial virus (RSV) have placed a heavy burden on public health. Developing rapid and accurate diagnostic tools remains a significant need in the face of this evolving respiratory viral threat. Immunoassays are considered a complementary approach to nucleic acid testing, providing rapid but preliminary results. Recent research has made substantial progress in immunoassays by pursuing rapid and sensitive antigen or antibody detection through the fabrication of nanomaterials / nanostructures and the development of efficient transduction techniques. However, cumbersome pretreatment and sample collection, insufficient sensitivity, and a lack of clinical sample validation hinder the application of immunoassay technologies. Therefore, there is still a need to develop a rapid and accurate immunoassay technology, ideally with a wireless and battery-free configuration for compact integration into everyday wearable devices, enabling point-of-care immunoassay of viral aerosols. Summary of the Invention

[0003] This invention addresses the problems of poor specificity, insufficient detection limit, and bulky and complex detection equipment in traditional immunoassay methods by providing a wireless immunoassay technology based on immunoresponsive hydrogels, which can be integrated into wearable devices to achieve rapid, sensitive, and specific instant detection of antigen proteins.

[0004] To solve the above-mentioned technical problems, the present invention specifically adopts the following technical solution:

[0005] A method for preparing an immunoresponsive hydrogel involves first modifying an antigen protein with N-acryloyloxysuccinimide, incubating at 36°C, and centrifuging to obtain vinylated antigen; then preparing gold nanoparticles using a sodium citrate reduction method, and incubating them with an antibody overnight to obtain antibody-coupled gold nanoparticles; finally, employing a two-step method, the vinylated antigen is first mixed with a hydrogel monomer, an initiator, and a reaction catalyst to react and obtain a polymerized antigen protein; subsequently, antibody-coupled gold nanoparticles, a covalent crosslinking agent, and an initiator are added to perform covalent crosslinking and immunocrosslinking to form a precursor solution, ultimately yielding the immunoresponsive hydrogel. Details are as follows:

[0006] Vinylated antigens were obtained by modifying antigen proteins with N-acryloyloxysuccinimide.

[0007] The specific antibody corresponding to the antigen protein was added to a solution containing gold nanoparticles, and the mixture was incubated and stirred overnight to obtain antibody-coupled gold nanoparticles; wherein the molar ratio of the specific antibody to the gold nanoparticles was 10000:1 to 1:10.

[0008] Vinylated antigen, hydrogel monomer, initiator, and reaction catalyst were mixed in water at a mass ratio of 0.8:30000:456.4:278.88 and reacted at room temperature to obtain a polymeric antigen protein. Subsequently, antibody-coupled gold nanoparticles, covalent cross-linking agent, and initiator were added at a mass ratio of (0.9–4.5):40:456.4 to obtain a mixed solution. Covalent cross-linking and immunocross-linking were performed to form a precursor solution, and finally an immunoresponsive hydrogel was obtained. The mass fraction of hydrogel monomer in the mixed solution was 10%–20%.

[0009] Or include:

[0010] An antigen protein is added to a solution containing gold nanoparticles and incubated and stirred overnight to obtain antigen-coupled gold nanoparticles; wherein the molar ratio of the antigen protein to the gold nanoparticles is 1:10 to 10000:1.

[0011] Vinylated antibodies were obtained by modifying the specific antibodies corresponding to the antigen protein with N-acryloyloxysuccinimide.

[0012] Vinylated antibodies, hydrogel monomers, initiators, and reaction catalysts were mixed in water at a molar ratio of 0.8:30000:456.4:278.88 and reacted at room temperature to obtain polymeric antibodies. Subsequently, antigen-coupled gold nanoparticles, covalent cross-linking agents, and initiators were added at a mass ratio of (0.9–4.5):40:456.4 to obtain a mixed solution. Covalent cross-linking and immunocross-linking were then performed to form a precursor solution, ultimately yielding an immunoresponsive hydrogel. The mass fraction of hydrogel monomers in the mixed solution was 10%–20%.

[0013] Furthermore, the molar ratio of N-acryloyloxysuccinimide to the antigen protein is 15:1.

[0014] Furthermore, the antigen proteins include, but are not limited to, the SARS-CoV-2 nucleocapsid protein (N protein), the H1N1 influenza virus lectin (HA protein), and the respiratory syncytial virus fusion protein (FP protein).

[0015] Furthermore, the antibody is a specific antibody against the corresponding antigen protein.

[0016] Furthermore, the average particle size of the gold nanoparticles used is 8–30 nm, preferably 15–20 nm.

[0017] Furthermore, gold nanoparticles are prepared using the sodium citrate reduction method. The gold nanoparticles can be concentrated 2 to 10 times before incubation with antibodies, preferably 2 times.

[0018] Furthermore, the hydrogel monomer is acrylamide.

[0019] Furthermore, the concentration of acrylamide is preferably 10% by mass.

[0020] Furthermore, the initiator is ammonium persulfate.

[0021] Furthermore, the catalyst is tetramethylethylenediamine.

[0022] Furthermore, the reaction time to obtain the polymerized antigen protein is 1 to 10 minutes, preferably 1 minute.

[0023] Furthermore, the covalent crosslinking agent is N,N-methylenebisacrylamide.

[0024] Furthermore, the final concentration of the added antibody-coupled gold nanoparticles is preferably 4 ng / mL.

[0025] Furthermore, under otherwise unchanged conditions, N-acryloyloxysuccinimide can be used to modify the antibody, and gold nanoparticles can be incubated with the antigen protein to prepare an immunoresponsive hydrogel.

[0026] Meanwhile, the present invention also discloses a wireless immune sensing detection device, which includes:

[0027] One or more immunoresonant hydrogel sensors, the immunoresonant hydrogel sensors comprising the immunoresponsive hydrogel and a bottom resonator and a top resonator assembled on both sides of the immunoresponsive hydrogel; used to capture antigen proteins in a sample and generate a resonant frequency shift;

[0028] A readout coil is connected to a vector network analyzer. The readout coil reads the electrical signal of the immunohydrogel resonant sensor through an intermediate coil or radio frequency method and transmits it to the vector network analyzer.

[0029] Furthermore, the connection method via the intermediate coil is as follows: the immunohydrogel resonant sensor is fixed on the intermediate coil, and under mechanical deformation, the intermediate coil ensures a good electromagnetic coupling path between the immunohydrogel resonant sensor and the readout coil; the readout coil is connected to a vector network analyzer, and the resonant frequency shift generated by the immunohydrogel resonant sensor is detected through mutual inductance. By expanding the parallel intermediate coil, multiple sensor signals can be wirelessly read out without battery power, and it can be integrated into wearable devices such as masks to achieve wearable real-time immunoassay analysis of viral aerosols.

[0030] Furthermore, the resonator is obtained by laser cutting a flexible substrate covered with a metal film.

[0031] Furthermore, the flexible substrate includes, but is not limited to, polyimide and polyethylene terephthalate films.

[0032] Furthermore, the resonator can be a split-ring resonator or a spiral coil resonator.

[0033] Furthermore, an insulating layer is provided on the surface of the resonator, which is obtained by spin coating insulating adhesive onto the surface of the resonator.

[0034] Furthermore, it also includes using oxygen plasma to hydrophilize the resonator.

[0035] Furthermore, the resonator obtained above is placed at the bottom of the mold as the bottom resonator, and a precursor solution is injected into the mold. A resonator with the same processing parameters is placed on the surface of the precursor solution, which is symmetrical to the bottom resonator as the top resonator. After the immune-responsive hydrogel is fully formed, it is demolded and soaked in deionized water to remove unreacted reagents.

[0036] Furthermore, the intermediate coil is fabricated using flexible printed circuit board technology.

[0037] Furthermore, the readout coil is fabricated using printed circuit board technology.

[0038] Furthermore, the vector network analyzer is connected to a terminal to analyze the resonant frequency shift using corresponding Java software.

[0039] Furthermore, the parallel intermediate coils are extended, with an independent immunohydrogel resonant sensor fixed on each intermediate coil, enabling wireless readout of multiple sensing signals.

[0040] Furthermore, by assembling the immunohydrogel resonant sensor and intermediate coil in wearable devices such as masks, wearable real-time immunodetection and analysis of viral aerosols can be achieved.

[0041] Compared with traditional immunosensing technology, the present invention has the following advantages:

[0042] (1) The immunoresponsive hydrogel prepared by the method proposed in this invention has a nanoscale porous network structure, which can quickly prepare an immunoresponsive hydrogel material within 5 minutes. Combined with the wireless immunosensing detection device proposed in this invention, it can achieve sensitive and specific detection of trace antigen proteins within 10 minutes, with a detection limit as low as fg / L.

[0043] (2) The wireless immune sensing detection device proposed in this invention can realize wireless reading of sensing signals through radio frequency communication. The entire device does not require battery power and can realize synchronous reading of multiple sensing signals by extending parallel intermediate coils.

[0044] (3) The immune hydrogel resonant sensor and intermediate coil proposed in this invention are assembled in wearable devices such as masks to realize wearable instant immune detection of viral aerosols. Attached Figure Description

[0045] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0046] Figure 1 This is a flowchart of the preparation process of the immune-responsive hydrogel provided in the embodiments of the present invention;

[0047] Figure 2 This is a scanning electron microscope image of an immune-responsive hydrogel provided in an embodiment of the present invention, with a scale bar of 100 micrometers;

[0048] Figure 3 This is a scanning electron microscope image of an immune-responsive hydrogel provided in an embodiment of the present invention, with a scale bar of 500 nanometers;

[0049] Figure 4 This is a graph showing the optimized acrylamide concentration in an immune-responsive hydrocoagulation, as provided in an embodiment of the present invention.

[0050] Figure 5 This is a graph showing the optimized concentration of antibody-coupled gold nanoparticles in an immune-responsive hydrocoagulation, as provided in an embodiment of the present invention.

[0051] Figure 6 This is a schematic diagram of the wireless immune sensing detection device and signal readout provided in an embodiment of the present invention;

[0052] Figure 7 This is a flowchart illustrating the fabrication process of the immunohydrogel resonant sensor provided in this embodiment of the invention;

[0053] Figure 8 This is a photograph of the actual immunohydrogel resonant sensor provided in the embodiment of the present invention (using a split-ring resonator), with a scale bar of 5 mm;

[0054] Figure 9 This is a photograph of the actual immunohydrogel resonant sensor provided in the embodiment of the present invention (using a spiral coil resonator), with a scale bar of 5 mm;

[0055] Figure 10 This is a diagram showing the detection results of the SARS-CoV-2 nucleocapsid protein provided in an embodiment of the present invention;

[0056] Figure 11 This is a graph showing the H1N1 influenza virus agglutinin detection results provided in an embodiment of the present invention;

[0057] Figure 12 This is a diagram showing the detection results of respiratory syncytial virus fusion protein provided in an embodiment of the present invention;

[0058] Figure 13 This is a graph showing the rapid detection results of H1N1 influenza virus agglutinin within 10 minutes provided in this embodiment of the invention;

[0059] Figure 14 These are selective detection results of three types of immunohydrogel resonant sensors provided in the embodiments of the present invention;

[0060] Figure 15 This is a schematic diagram of wireless readout of multiple sensing signals via parallel intermediate coils provided in an embodiment of the present invention;

[0061] Figure 16 This is a schematic diagram illustrating the application of the wireless immunosensing detection technology based on immunoresponsive hydrogel provided in this embodiment of the invention to wearable point-of-care immunodetection.

[0062] In the figure: 1. Immunohydrogel resonant sensor; 2. Intermediate coil; 3. Readout coil; 4. Vector network analyzer; 5. Terminal; 11. Top layer resonator; 12. Immunoresponsive hydrogel; 13. Bottom layer resonator. Detailed Implementation

[0063] The embodiments, features, and aspects of this disclosure will be described in detail below with reference to the accompanying drawings, but this is not intended to limit the invention. Based on any embodiment extended from the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The same reference numerals in the drawings denote the same or similar functional elements. Although various aspects of the embodiments are shown in the drawings, they are not necessarily drawn to scale unless specifically indicated. Furthermore, numerous specific details are set forth in the following detailed description for better illustration of this disclosure. Those skilled in the art should understand that this disclosure can be practiced without certain specific details. Some methods and means well known to those in the art, and the use of components, are not described in detail to highlight the main points of this disclosure.

[0064] Example 1: Preparation of immune-responsive hydrogels

[0065] like Figure 1 As shown, the preparation method of immune-responsive hydrogel 12 includes the following steps:

[0066] Step 1: Add N-acryloyloxysuccinimide and antigen protein to deionized water at a molar ratio of 15:1, wherein the concentration of antigen protein is 0.02 μM, and heat in a water bath at 36°C for 1 hour.

[0067] Step 2: Add the above solution to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, centrifuge at 14000×g for 15 minutes at 4°C to obtain purified vinylized antigen protein, and prepare a solution with a concentration of 0.05 mg / mL for later use.

[0068] Step 3: Add 100 mL of 1 mM chloroauric acid to the flask and boil.

[0069] Step 4: Add 10 mL of 38.8 mM sodium citrate to boiling chloroauric acid, continue boiling for 20 min and then cool to obtain a gold nanoparticle solution.

[0070] Step 5: Centrifuge the cooled gold nanoparticle solution at 12000 rpm for 15 min, and resuspend the precipitate in an appropriate amount of supernatant to obtain a 2-fold concentrated gold nanoparticle solution with a specific concentration of 50 μg / mL.

[0071] Step 6: Add 16 ng of the specific antibody corresponding to the antigen protein to 1 mL of 2-fold concentrated gold nanoparticle solution and incubate overnight at 4°C to obtain antibody-coupled gold nanoparticles.

[0072] Step 7: Add acrylamide, 0.8 μg vinylized antigen protein, 3 μL of 0.8 M tetramethylethylenediamine solution, and 1 μL of 2 M ammonium persulfate solution to 124 μL of deionized water and react at room temperature for 1 min.

[0073] Step 8: Add 2 μL of 20 mg / mL N,N-methylenebisacrylamide, an appropriate amount of antibody-coupled gold nanoparticle solution, and 1 μL of 2 M ammonium persulfate solution to the above solution to obtain a mixed solution. The mass fraction of acrylamide in the mixed solution is 10%–20%, and the concentration of the antibody-coupled gold nanoparticle solution is 1–5 ng / mL. Perform covalent cross-linking and immunocross-linking to form a precursor solution, and completely gel within 5 min to form an immunoresponsive hydrogel 12.

[0074] The antigen protein used in step 1 of this embodiment is not limited. For ease of explanation, it can be one of the following: SARS-CoV-2 nucleocapsid protein, H1N1 influenza virus agglutinin, or respiratory syncytial virus fusion protein.

[0075] The specific antibody used in step 6 of this embodiment is not limited. For ease of explanation, it can be one of the following: SARS-CoV-2 nucleocapsid protein monoclonal antibody, H1N1 influenza virus lectin monoclonal antibody, or respiratory syncytial virus fusion protein monoclonal antibody.

[0076] like Figure 2 As shown, an immunoresponsive hydrogel 12, prepared from a mixed solution containing 10% acrylamide and 4 ng / mL antibody-coupled gold nanoparticles, was imaged using a scanning electron microscope after lyophilization, exhibiting a typical hydrogel network structure at the micrometer scale. Figure 3 As shown, further observation at the nanoscale revealed that the immunoresponsive hydrogel 12 possesses a nanoscale porous network structure. This structure is mainly caused by the steric hindrance of the gold nanoparticles, which facilitates the diffusion of antigen proteins and antibody binding. Simultaneously, the antibody-coupled gold nanoparticles constrain all the immune cross-linking networks around the gold nanoparticles, achieving the concentration of strain energy. When a small amount of antigen-antibody binding occurs, the fixation of the antibody-coupled gold nanoparticles in the hydrogel network can be disrupted, leading to a rapid and intense release of strain energy, ultimately improving detection sensitivity and detection speed.

[0077] In this embodiment, an immunoresponsive hydrogel 12 prepared by mixing a solution containing 10%-20% acrylamide and an antibody-coupled gold nanoparticle solution at a concentration of 4 ng / mL was incubated in 1 ng / mL antigen protein (the same antigen protein used to prepare the hydrogel, namely the SARS-CoV-2 N protein) for 10 min, and then the antigen protein immune response was tested. Figure 4 As shown, the immunoresponsive hydrogel 12 exhibits good immunoresponsiveness when the final concentration of acrylamide in the hydrogel is 10%–20% by mass, and the immunoresponsive hydrogel 12 has the optimal immunoresponsiveness when the acrylamide concentration is 10% by mass.

[0078] In this embodiment, an immunoresponsive hydrogel 12 prepared from a mixed solution containing 10% acrylamide and an antibody-coupled gold nanoparticle solution with a concentration of 1-5 ng / mL was incubated in 1 ng / mL antigen protein (the same antigen protein used to prepare the hydrogel, namely the SARS-CoV-2 N protein) for 10 min, and then the antigen protein immune response was tested. Figure 5 As shown, the immunoresponsive hydrogel 12 exhibits good immunoresponsiveness when the concentration of the antibody-coupled gold nanoparticles immobilized in it is between 1 ng / mL and 5 ng / mL, and the immunoresponsive hydrogel 12 exhibits the optimal immunoresponsiveness when the concentration of the immobilized antibody-coupled gold nanoparticles is 4 ng / mL.

[0079] Example 2: Construction of a Wireless Immunosensing Detection Device

[0080] like Figure 6As shown, this invention also discloses a wireless immunosensing detection device and its application. The device includes: an immunohydrogel resonant sensor 1, an intermediate coil 2, and a readout coil 3. The immunohydrogel resonant sensor 1 is fixed to the intermediate coil 2, and the intermediate coil 2 ensures a good electromagnetic coupling path between the immunohydrogel resonant sensor 1 and the readout coil 3 under mechanical deformation. The readout coil 3 is connected to a vector network analyzer 4, and the vector network analyzer 4 is connected to a terminal 5 (such as a laptop computer with Java software) to detect the resonant frequency shift.

[0081] In this process, the immunoresponsive hydrogel competitively binds to the antigen protein in the test sample, causing the hydrogel to expand in volume. This expansion further alters the capacitance parameters of the immunohydrogel resonant sensor 1. The capacitance change in the immunohydrogel resonant sensor 1 is detected by the vector network analyzer 4 through electromagnetic coupling between the intermediate coil 2 and the readout coil 3, and is reflected as a resonant frequency shift, which is displayed on the terminal 5.

[0082] The immunoresonant hydrogel sensor 1 includes the immunoresponsive hydrogel 12, a bottom resonator 13 assembled on both sides of the immunoresponsive hydrogel, and a top resonator 11. Figure 7 The diagram shows a method for constructing the immunohydrogel resonant sensor 1, which includes the following steps:

[0083] Step 1: Deposit a metal layer onto a flexible substrate using magnetron sputtering. The flexible substrate can be one or more of polyimide and polyethylene terephthalate films, and the metal layer can be one or more of copper, titanium, gold, and nickel.

[0084] Step 2: The resonator pattern is processed on the flexible substrate covered with a metal layer by laser cutting. The geometric parameters of the resonator are obtained by designing using electromagnetic simulation software.

[0085] Step 3: Peel off the laser-cut resonator, clean it with deionized water and isopropanol for 2 minutes each, and store it for later use.

[0086] Step 4: Spin-coat a layer of insulating adhesive, SU-8, onto the surface of the resonator.

[0087] Step 5: Treat the resonator obtained in the previous step with oxygen plasma for 120 seconds to give it a hydrophilic surface and enhance its adhesion to the immune-responsive hydrogel.

[0088] Step 6: Fix the resonator obtained in the previous step to the bottom of the mold as the bottom resonator 13, and fill the mold with the precursor solution just prepared in step 8 of Example 1.

[0089] Step 7: Take another resonator obtained in Step 5 of this embodiment and gently place it on the precursor solution in a manner symmetrical to the bottom resonator 13 as the top resonator 11. After standing for 5 minutes, the precursor solution completely gels to form an immunoresponsive hydrogel 12.

[0090] Step 8: Gently remove the assembled immunohydrogel resonant sensor 1 from the mold.

[0091] Step 9: Immerse the immunohydrogel resonant sensor 1 obtained in the previous step in deionized water for 15-30 minutes to remove unreacted reagents.

[0092] The resonator processed in step 2 of this embodiment is not limited and can be a split resonator. Figure 8 It can also be a helical coil resonator. Figure 9 For ease of explanation, typical parameters for a crack resonator can be a side length of 10mm, a crack length of 2mm, and a line width of 2mm.

[0093] Example 3: Application of Wireless Immunosensing Detection Technology Based on Immunoreactive Hydrogels

[0094] Based on Examples 1 and 2, this example constructs three types of immunohydrogel resonant sensors for three viral biomarkers: SARS-CoV-2 nucleocapsid protein (N protein), H1N1 influenza virus lectin (HA protein), and respiratory syncytial virus fusion protein (FP protein), respectively denoted as N protein sensor, HA protein sensor, and FP protein sensor.

[0095] By configuring standard solutions and using a medical-grade nebulizer, this embodiment prepared viral biomarker aerosols with various concentration gradients for sensor testing. During testing, the readout coil and intermediate coil were first coupled and matched to generate a clear resonant signal. Then, the viral biomarker aerosol generated by the medical-grade nebulizer was introduced into the test chamber containing the immunohydrogel resonant sensor through a silicone conduit. After a reaction time of 1–10 minutes, a vector network analyzer was used to scan and detect the resonant frequency shift of the immunohydrogel resonant sensor. Figures 10-12 As shown, through nonlinear fitting calculations, the detection limit of the N protein sensor is 19.7 fg / L, the detection limit of the HA protein sensor is 0.11 fg / L, and the detection limit of the FP protein sensor is 3.2 fg / L.

[0096] Furthermore, taking the HA protein sensor as an example, this embodiment tested the application of wireless immunosensing detection technology based on immunoresponsive hydrogels in rapid virus detection. Figure 13As shown, the HA protein sensor can clearly distinguish between negative control aerosol and HA protein aerosol with a concentration of 71.4 fg / L within 1, 5 and 10 minutes, indicating that the sensor can achieve rapid virus detection.

[0097] like Figure 14 As shown, this embodiment also tested the selectivity of three immunohydrogel resonant sensors. The concentration of the viral marker aerosol used was 71.4 pg / mL, and the test time was 10 minutes. All sensors showed significant selectivity for the target antigen protein, while the response to other non-target proteins was similar to that of the negative control.

[0098] Based on the above results, this embodiment can achieve wireless readout of multiple sensor signals by extending the parallel intermediate coil, such as... Figure 15 As shown. By adjusting the resonator geometry parameters in step 2 of Example 2 and the precursor solution filling amount in step 6 of Example 2 (e.g., 300–1000 μL) to control the hydrogel thickness, various immunohydrogel resonant sensors 1 with different resonant frequencies can be prepared. An independent immunohydrogel resonant sensor is fixed to each intermediate coil. By connecting the intermediate coils in parallel and coupling them to the readout coil, multiple signal readouts can be achieved in a single scan.

[0099] In addition to the intermediate coil, this invention can also achieve wireless signal transmission by reading the electrical signals of the immunohydrogel resonant sensor via radio frequency, specifically, as shown in... Figure 16 As shown, by assembling an immune hydrogel resonant sensor and an intermediate coil in wearable devices such as masks and connecting them to the readout coil via radio frequency communication, wearable wireless immune sensing detection can be performed by a vector network analyzer installed at monitoring points such as gates, enabling on-site real-time immune detection of targets such as viral aerosols.

[0100] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made without creative effort should be included within the protection scope of the present invention.

Claims

1. A method of preparing an immune-responsive hydrogel, characterized in that, include: Vinylated antigens were obtained by modifying antigen proteins with N-acryloyloxysuccinimide. The specific antibody corresponding to the antigen protein was added to a solution containing gold nanoparticles, and the mixture was incubated and stirred overnight to obtain antibody-coupled gold nanoparticles; wherein the molar ratio of the specific antibody to the gold nanoparticles was 10000:1 to 1:

10. Vinylated antigen, hydrogel monomer, initiator, and reaction catalyst were mixed in water at a mass ratio of 0.8:30000:456.4:278.88 and reacted at room temperature to obtain a polymeric antigen protein. Subsequently, antibody-coupled gold nanoparticles, covalent cross-linking agent, and initiator were added at a mass ratio of (0.9–4.5):40:456.4 to obtain a mixed solution. Covalent cross-linking and immunocross-linking were performed to form a precursor solution, and finally an immunoresponsive hydrogel was obtained. The mass fraction of hydrogel monomer in the mixed solution was 10%–20%. Or include: An antigen protein is added to a solution containing gold nanoparticles and incubated and stirred overnight to obtain antigen-coupled gold nanoparticles; wherein the molar ratio of the antigen protein to the gold nanoparticles is 1:10 to 10000:

1. Vinylated antibodies were obtained by modifying the specific antibodies corresponding to the antigen protein with N-acryloyloxysuccinimide. Vinylated antibodies, hydrogel monomers, initiators, and reaction catalysts were mixed in water at a molar ratio of 0.8:30000:456.4:278.88 and reacted at room temperature to obtain polymeric antibodies. Subsequently, antigen-coupled gold nanoparticles, covalent cross-linking agents, and initiators were added at a mass ratio of (0.9–4.5):40:456.4 to obtain a mixed solution. Covalent cross-linking and immunocross-linking were then performed to form a precursor solution, ultimately yielding an immunoresponsive hydrogel. The mass fraction of hydrogel monomers in the mixed solution was 10%–20%.

2. The method of claim 1, wherein, The molar ratio of N-acryloyloxysuccinimide to antigen protein is 15:

1.

3. The method of claim 1, wherein, The antigen proteins include one of the following: the nucleocapsid protein of the novel coronavirus (N protein), the lectin of the H1N1 influenza virus (HA protein), and the fusion protein of the respiratory syncytial virus (FP protein).

4. The method of claim 1, wherein, The average particle size of the gold nanoparticles used is 8–30 nm.

5. The method of claim 1, wherein, The hydrogel monomer is acrylamide.

6. The method of claim 1, wherein, The initiator is ammonium persulfate; the catalyst is tetramethylethylenediamine; the reaction time to obtain polymeric antigen protein or polymeric antibody is 1-10 minutes; the covalent crosslinking agent is N,N-methylenebisacrylamide.

7. An immunoresponsive hydrogel prepared by a method according to any one of claims 1-6.

8. An immunosensing device based on the immunoresponsive hydrogel of claim 7, characterized in that, include: One or more immunoresonant hydrogel sensors, the immunoresonant hydrogel sensors comprising the immunoresponsive hydrogel of claim 7 and a bottom resonator and a top resonator assembled on both sides of the immunoresponsive hydrogel; A readout coil is connected to a vector network analyzer. The readout coil reads the electrical signal of the immunohydrogel resonant sensor through an intermediate coil or radio frequency method and transmits it to the vector network analyzer.

9. The immune sensing detection device according to claim 8, characterized in that, The multiple immune hydrogel resonant sensors have different resonant frequencies. The resonant frequency of the immune hydrogel resonant sensors can be adjusted by adjusting the geometric parameters of the bottom resonator and the top resonator or the thickness of the immune-responsive hydrogel.

10. The immune sensing detection device according to claim 8, characterized in that, The resonator is either a split-ring resonator or a spiral coil resonator.

11. The immune sensing detection device according to claim 8, characterized in that, The immune hydrogel resonant sensor is assembled in a wearable device to enable wearable real-time immune detection and analysis of viral aerosols.

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