An integrated biosensing platform based on microneedles and hydrogels and its application in detecting monkeypox virus

Abstract

The present invention discloses an integrated biosensor platform based on microneedles and hydrogels and its application in detecting monkeypox virus, wherein the platform comprises a three-electrode microneedle, a hydrogel, a burning needle and a micro-electrochemical workstation. The microneedle electrode of the working electrode is combined with a nanoenzyme and an A29 protein antibody, and the monkeypox virus in the sample is extracted by the specific binding of the antibody with the monkeypox virus; the burning needle is used to connect the three-electrode microneedle and the micro-electrochemical workstation and transmit electrochemical signals. After the three-electrode microneedle is inserted into the hydrogel, the nanoenzyme catalyzes the TMB contained in the hydrogel and generates a blue oxidation product, thereby generating an electrochemical signal, which is collected by the micro-electrochemical workstation through the burning needle, and the electrochemical signal is collected through Bluetooth and a hydrogel colorimetric image is obtained, thereby realizing the detection of monkeypox virus. The integrated biosensor platform of the present invention has a low detection limit and has broad application prospects in the rapid on-site detection of monkeypox virus.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, and in particular to an integrated biosensor platform based on microneedles and hydrogels and an application thereof in detecting monkeypox virus. Background Art

[0002] Monkeypox virus is a double-stranded DNA virus belonging to the genus Orthopoxvirus of the family Poxviridae. It has been confirmed to cause monkeypox infection in humans and animals. Monkeypox virus is highly contagious, and symptoms of infection include fever, muscle pain, and characteristic rash. Since 2022, monkeypox virus has spread rapidly to many countries and regions around the world, resulting in more than 80,000 confirmed cases of monkeypox. Currently, monkeypox virus nucleic acid detection is a common method for its diagnosis. However, genetic testing usually requires professional equipment, cumbersome operation, and long detection time, which limits its effectiveness in rapid and on-site testing. Therefore, there is an urgent need for a simple, portable, and time-efficient analytical tool for sensitive detection of monkeypox virus under field conditions, which is of great significance for early diagnosis, isolation, and treatment.

[0003] Portable devices that integrate extraction and diagnosis are a promising solution that provides significant opportunities for the development of personalized medical devices. Microneedles, as micrometer-scale needles, have attracted widespread attention for their ability to achieve minimally invasive transdermal drug delivery in a safe, effective, and painless manner. In recent years, research on the extraction of biomolecules using microneedles has emerged, and microneedle-based biosensing strategies have been reported to monitor various endogenous biomarkers, metabolites, and proteins, including cholesterol, glucose, exosomes, interleukin-4, C-reactive protein, procalcitonin, etc.

[0004] The most commonly used detection principles for microneedle biosensors are electrochemistry and colorimetry, because these two methods have the characteristics of fast detection speed and simple operation. Therefore, by combining electrochemistry and colorimetry, microneedles can achieve instant diagnosis capabilities to detect glucose, uric acid, cholesterol, cysteine ​​C, epidermal growth factor receptor, etc. At present, there is no microneedle biosensor for detecting monkeypox virus. Summary of the invention

[0005] In order to overcome the above-mentioned defects and shortcomings in the prior art, the present invention provides an integrated biosensor platform based on microneedles and hydrogels and its application in detecting monkeypox virus.

[0006] The first object of the present invention is to provide a biosensing platform.

[0007] The second object of the present invention is to provide the application of the above-mentioned biosensor platform in the preparation of monkeypox virus detection products.

[0008] The third object of the present invention is to provide a method for using the above-mentioned biosensor platform.

[0009] The fourth object of the present invention is to provide a method for detecting monkeypox virus using the above-mentioned biosensor platform.

[0010] In order to achieve the above object, the present invention is implemented by the following scheme: The integrated biosensor platform of the present invention comprises a three-electrode microneedle (a microneedle electrode of a counter electrode, a reference electrode and a working electrode), a hydrogel, a burning needle and a micro-electrochemical workstation. The microneedle electrode of the working electrode is combined with MnO2-Au nanozyme and A29 protein antibody, and the monkeypox virus in the sample is extracted through the specific binding of the A29 protein antibody with the monkeypox virus; the burning needle is used to connect the three-electrode microneedle and the micro-electrochemical workstation and transmit electrochemical signals.

[0011] After the counter electrode, reference electrode and working electrode microneedle electrodes are inserted into the hydrogel together, the MnO2-Au nanozyme catalyzes the TMB contained in the hydrogel and generates a blue oxidation product, thereby generating an electrochemical signal. The signal is collected by the micro-electrochemical workstation through the burning needle and transmitted to the smartphone. At the same time, the hydrogel is photographed using the smartphone to obtain a colorimetric image, thereby realizing the detection of monkeypox virus.

[0012] Therefore, the present invention claims the following: A biosensor platform, comprising a collection module, a detection module, a connection module and an information transmission module; The collection module is divided into a test area and a control area, the test area is used to collect virus-infected samples, and the control area is used to collect normal samples; The test area and the control area both contain a counter electrode, a reference electrode and a working electrode, and the counter electrode, the reference electrode and the working electrode are all provided with microneedle electrodes; The microneedle electrode of the working electrode contains an antibody that specifically binds to the monkeypox virus and an enzyme for catalyzing 3,3',5,5'-tetramethylbenzidine to generate an oxidation product; The detection module is a hydrogel, and the hydrogel contains 3,3',5,5'-tetramethylbenzidine and electrolyte; The connection module is a circuit connector; The information transmission module is two electrochemical sensing units, which are detachably connected to the electrodes of the test area and the control area through connection modules respectively. Each electrochemical sensing unit is provided with an electrochemical signal acquisition device and an electrochemical signal transmission device.

[0013] Preferably, the electrochemical signal transmission device is a Bluetooth transmission device.

[0014] The two electrochemical sensing units constitute a micro electrochemical workstation, and the two electrochemical sensing units are used to obtain and transmit to the smart device the electrochemical signals generated in the test area and the control area, respectively. The electrochemical signals are generated after the microneedle electrodes of the counter electrode, reference electrode and working electrode in the test area and the control area penetrate into the hydrogel.

[0015] As an practicable manner, the microneedle electrode of the counter electrode is obtained by modifying multi-walled carbon nanotubes on the microneedle, the microneedle electrode of the reference electrode is obtained by modifying silver chloride on the microneedle, and the microneedle electrode of the working electrode is obtained by modifying multi-walled carbon nanotubes on the microneedle.

[0016] Since the A29 protein is a highly conserved surface envelope protein of the monkeypox virus and can be used as a specific antigen for detecting the monkeypox virus, preferably, the antibody is an A29 protein antibody.

[0017] Compared with natural enzymes, nanozymes overcome limitations such as high cost, low stability and sensitivity to environmental factors. Therefore, preferably, the enzyme is MnO2-Au nanozyme. The preparation method of the MnO2-Au nanozyme is: add HAuCl4 aqueous solution to the aqueous dispersion of MnO2 nanoflowers, heat to boiling, and then add sodium citrate aqueous solution to the boiling solution for sufficient reaction. After the reaction is completed, the product is collected and purified.

[0018] Preferably, the particle size of the MnO2 nanoflowers is 690-710 nm.

[0019] More preferably, the synthesis steps of the MnO2 nanoflowers are: fully mixing KMnO4 and MnSO4·H2O in water, transferring the mixture into an autoclave, heating for full reaction, and then cooling to room temperature, collecting the product and purifying it.

[0020] Further preferably, the mass volume ratio of KMnO4, MnSO4·H2O and water is (0.8-1.2) g: (0.2-0.6) g: 30 mL.

[0021] More preferably, the mass volume ratio of KMnO4, MnSO4·H2O and water is 1 g:0.4 g:30 mL.

[0022] More preferably, the heating temperature is 135-145° C., and the sufficient reaction time is 0.8-1.2 h.

[0023] More preferably, the heating temperature is 140° C. and the sufficient reaction time is 1 h.

[0024] More preferably, the mass ratio of the MnO2 nanoflowers, HAuCl4 and sodium citrate is (48-52) mg: (6.2-6.6) mg: (8-12) mg.

[0025] Further preferably, the mass ratio of the MnO2 nanoflowers, HAuCl4 and sodium citrate is 50 mg:6.4 mg:10 mg.

[0026] More preferably, the time for adding the sodium citrate aqueous solution to fully react is 28 to 32 minutes.

[0027] More preferably, the time for adding the sodium citrate aqueous solution to fully react is 30 min.

[0028] As an practicable manner, the hydrogel is a sodium alginate hydrogel.

[0029] As an practicable manner, the electrolyte is acetic acid-sodium acetate buffer.

[0030] Preferably, the circuit connector is 6 wires and 6 burning pins, the 6 burning pins are detachably connected to the information transmission module, and the counter electrode, reference electrode and working electrode of the test area and the control area are respectively connected to the corresponding burning pins through wires.

[0031] Further preferably, the information transmission module comprises a plurality of interfaces, and the other end of the burning needle is detachably connected to the interface, thereby being connected to the information transmission module.

[0032] Preferably, the biosensor platform further comprises a container for carrying the detection module, wherein the container is provided with a separated hydrogel region and a circuit connector region, the circuit connector is embedded in the circuit connector region, and the hydrogel is embedded in the hydrogel region.

[0033] Preferably, the biosensor platform also includes a container carrying an information transmission module, the acquisition module, the detection module, the container carrying the detection module, the information transmission module and the container carrying the information transmission module are stacked and assembled in sequence, and the circuit connector is embedded in the container carrying the detection module to connect the acquisition module and the information transmission module.

[0034] Use of any of the above-mentioned biosensor platforms in the preparation of a monkeypox virus detection product.

[0035] A method for using any of the above-mentioned biosensor platforms comprises the following steps: S11. Insert the counter electrode, reference electrode and working electrode of the test area into the sample to be tested, and insert the counter electrode, reference electrode and working electrode of the control area into the normal sample, and let stand for 8 to 15 min; S12. All the microneedle electrodes are inserted into the hydrogel together, and each electrode is connected to the information transmission module through the connection module, and the electrodes are left standing for 180 to 220 seconds; the electrochemical signals of the test area and the control area transmitted by the information transmission module are collected respectively, and the ratio of the electrochemical signals of the test sample and the normal sample is calculated; S13. Take out the microneedle electrode, obtain a photo of the hydrogel, and analyze the colorimetric image to obtain the colorimetric signal ratio of the sample to be tested and the normal sample.

[0036] Preferably, in step S11, the mixture is allowed to stand for 10 min.

[0037] Preferably, in step S12, the mixture is allowed to stand for 200 s.

[0038] Preferably, a smart device is used to collect the electrochemical signals of the test area and the control area transmitted by the information transmission module, the smart device contains a Bluetooth receiver, and the information transmission module transmits the electrochemical signals of the test area and the control area through a Bluetooth transmission device; The smart device also has a shooting function, and the hydrogel photo is taken by the smart device; The smart device contains a program or APP software for analyzing electrochemical signals and colorimetric signals.

[0039] A method for detecting monkeypox virus using any of the above-mentioned biosensor platforms comprises the following steps: S21. The test sample and the normal sample are tested according to the above method to obtain the electrochemical signal ratio and the colorimetric signal ratio of the test sample and the normal sample; S22. The standard samples and normal samples of different concentrations were tested according to the above method to obtain the electrochemical signal ratio and colorimetric signal ratio of the standard samples and normal samples of different concentrations, and a standard curve was drawn; S23. Obtaining the concentration of monkeypox virus in the sample to be tested according to the standard curve; The method is aimed at non-disease treatment and diagnosis.

[0040] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses an integrated biosensor platform based on microneedles and hydrogels and its application in detecting monkeypox virus. The integrated biosensor platform of the present invention has a low detection limit and good specificity, stability, accuracy and reliability.

[0041] Furthermore, a control area is also designed in the integrated biosensor platform of the present invention for collecting blank samples from normal skin, thereby realizing ratio analysis and quality control functions. In addition, the integrated biosensor platform of the present invention is also multifunctional and flexible, and the modular design enables rapid switching of analysis targets by replacing components. The preparation process of electrochemical microneedles and colorimetric hydrogels is simple and cost-effective, making large-scale production possible. Due to the maximum simplification of the entire operation process, the detection process is user-friendly. Therefore, the integrated biosensor platform of the present invention has broad application prospects in the rapid on-site detection of monkeypox virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the composition and working principle of the integrated biosensing platform based on microneedles and hydrogels; A: Schematic diagram of the microneedles extracting monkeypox virus from the skin and the composition of the microneedle patch, B: The principle of the integrated biosensing platform based on microneedles and hydrogels detecting monkeypox virus through electrochemical and colorimetric methods, C: Front view of the microelectrochemical workstation, D: Front view of the integrated biosensing platform based on microneedles and hydrogels.

[0043] Figure 2 Schematic diagram of the fabrication process of epoxy resin microneedles.

[0044] Figure 3 The effects of different factors on the catalytic activity of MnO2-Au nanozyme in microneedle patches, A: Typical absorption spectra and corresponding color changes of different reaction systems of MnO2-Au nanozyme, TMB, and TMB+MnO2-Au nanozyme, B: pH value, C: MnO2-Au nanozyme concentration, D: TMB concentration, E: Effect of reaction time on the activity of MnO2-Au nanozyme, each error bar represents the standard deviation of three independent measurements.

[0045] Figure 4 Scanning electron microscope (SEM) image of MnO2 nanoflowers.

[0046] Figure 5 SEM images; A: SEM image of MnO2-Au nanozyme, B: SEM-EDS element distribution map of MnO2-Au nanozyme, C: SEM image of unmodified microneedles, D: SEM image of sodium alginate hydrogel.

[0047] Figure 6 SEM images of the microneedle patch; A: Photo of the epoxy resin microneedle array, B: SEM image of the working electrode, C: SEM image of the counter electrode, D: SEM image of the reference electrode.

[0048] Figure 7Electrochemical characterization results of the working electrode in the microneedle patch; A: The working electrode of the microneedle patch was exposed to the probe solution (5 mM [Fe(CN)6] 3- / 4- and 0.1 M KCl), with the scan rate ranging from 10 mV / s to 200 mV / s. B: The linear relationship between the redox peak current and the square root of the scan rate.

[0049] Figure 8 The performance of the integrated biosensing platform based on microneedles and hydrogels; A: Square wave voltammetry (SWV) curves of A29 protein with different concentrations recorded on the integrated biosensing platform based on microneedles and hydrogels; B: The relationship between the concentration of A29 protein and the current ratio I C / I T Calibration curve of , C: Alginate hydrogel images of different A29 protein concentrations recorded by a smartphone, D: A29 protein concentration and grayscale value ratio G T / G C Calibration curve of , E: Specificity test of the integrated biosensing platform based on microneedles and hydrogels for A29 protein (100 ng / mL) and interfering substances (100 ng / mL), F: Reproducibility of 5 independently prepared integrated biosensing platforms based on microneedles and hydrogels for A29 protein, G: Current ratio I of the integrated biosensing platform based on microneedles and hydrogels at different storage times C / I T Gray value ratio G T / G C , Error bars represent the standard deviation of three replicate measurements.

[0050] Fig. 9 The detection results of A29 protein in samples based on the integrated biosensing platform of microneedles and hydrogels, A: simulated body fluid, B: artificial saliva sample, the error bars represent the standard deviation of three repeated measurements. DETAILED DESCRIPTION

[0051] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0052] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0053] Example 1 Preparation of an integrated biosensing platform based on microneedles and hydrogels The integrated biosensing platform based on microneedles and hydrogels consists of five components: a microneedle patch, a hydrogel, a hydrogel container, a microelectrochemical workstation and a microelectrochemical workstation container.

[0054] The microneedles in the microneedle patch are made of commercially available liquid epoxy resin and are used to pierce the skin and extract monkeypox virus. A test area and a control area are designed in the microneedle patch to collect samples from diseased skin and normal skin, respectively. A typical three-electrode system is configured in both the test area and the control area. Both areas include a reference electrode, a counter electrode, and a working electrode. The reference electrode contains 4 microneedle electrodes, the counter electrode contains 4 microneedle electrodes, and the working electrode contains 8 microneedle electrodes. One end of each electrode is connected to the corresponding wire ( Figure 1 A in ).

[0055] The hydrogel was cross-linked by sodium alginate and loaded with 3,3',5,5'-tetramethylbenzidine (TMB) and acetic acid-sodium acetate buffer, providing a sensing environment for electrochemical and colorimetric analysis.

[0056] The micro electrochemical workstation contains an interface for the burning needle and integrates two electrochemical sensor units, which are used to collect electrochemical signals from the test area and the control area respectively, and transmit data via Bluetooth ( Figure 1 C in.

[0057] Six burning needles are embedded in the hydrogel patch container. The burning needles are used to connect the three electrodes in the test area and the control area and the micro electrochemical workstation and transmit electrochemical signals. The six burning needles are detachably connected to the micro electrochemical workstation. The counter electrode, reference electrode and working electrode in the test area and the control area are connected to the corresponding burning needles through wires.

[0058] Since the hydrogel contains electrolytes, the counter electrode, reference electrode and working electrode on the microneedle patch penetrate the hydrogel to form an electrolytic cell and generate electrochemical signals. At this time, one end of the burning needle is connected to the electrode wire, and the other end is connected to the interface on the micro electrochemical workstation, thereby transmitting the electrochemical signal to the micro electrochemical workstation ( Figure 1 D in.

[0059] 1. Experimental Methods 1. Preparation of microneedle patches (1) Preparation of epoxy resin microneedles Schematic diagram of the preparation process of epoxy resin microneedles Figure 2 shown.

[0060] ① Two commercial stainless steel microneedle arrays (about 3000 μm in length) were placed vertically in a custom-designed 3D-printed container; ② Mix the epoxy resin substrate and the curing agent in a ratio of 3:1 (w / w) and degas to obtain a mixture, pour the mixture into a 3D printing container, and immerse the bottom of the microneedle; ③ Place the 3D printed container at 50°C for curing to fix the stainless steel microneedles as the final microneedle master mold; ④ Mix the polydimethylsiloxane base material and the curing agent in a ratio of 10:1 (w / w), and then degas to obtain a polydimethylsiloxane mixture. Pour the polydimethylsiloxane mixture into the master mold and cure it at 60°C, and then peel the formed polydimethylsiloxane mold from the master mold; ⑤ The microneedle substrate is made of commercial liquid epoxy resin. The epoxy resin substrate and the curing agent are mixed in a ratio of 3:1 (w / w) and degassed to obtain a mixture. Subsequently, 1.5 mL of the mixture is poured into the polydimethylsiloxane mold obtained in step ④, vacuumed at room temperature for 5 min to fill the mold cavity, and then cured at 60°C; ⑥ Peel off the epoxy resin microneedle array from the polydimethylsiloxane mold, and remove the needles in the second and fourth rows of the epoxy resin microneedle array.

[0061] (2) Synthesis of MnO2-Au nanozyme MnO2 nanoflowers were synthesized by hydrothermal method: 1.0 g KMnO4 and 0.4 g MnSO4·H2O were dissolved in 30 mL distilled water and stirred for 30 min to obtain a mixed solution. The mixed solution was transferred to a 50 mL Teflon-lined stainless steel autoclave, which was heated to 140°C for 1 h and then slowly cooled to room temperature. The product was collected by centrifugation at 8000 rpm for 5 min and washed three times with ethanol and distilled water to obtain MnO2 nanoflowers, which were dried in a vacuum oven at 60°C overnight.

[0062] 50 mg of MnO2 nanoflowers were ultrasonically dispersed in 50 mL of distilled water, and then 64 μL of 10% HAuCl4 aqueous solution (w / v) was added while stirring and heated to boiling. Then 100 μL of 10% sodium citrate solution (w / v) was slowly added to the boiling solution, and the reaction lasted for 30 min. After the reaction, the product was collected by centrifugation at 8000 rpm for 5 min, and washed three times with ethanol and distilled water in turn to obtain MnO2-Au nanozyme, which was dried overnight under vacuum at 60°C and sealed for further use.

[0063] The present invention also explored the parameters that affect the catalytic activity of MnO2-Au nanozymes. The results showed that the maximum catalytic activity of MnO2-Au nanozymes for TMB was obtained at pH 4.0 and a reaction time of 200 s. In addition, as the concentration of MnO2-Au nanozymes increased, the absorbance intensity also increased, indicating that the reaction rate was significantly improved ( Figure 3 ).

[0064] (3) Modification of epoxy resin microneedle circuit The epoxy resin microneedle substrate is patterned with silver paste to form a circuit. The specific steps are as follows: ① Cover the surface of the epoxy resin microneedle substrate with a laser-etched polyethylene terephthalate mask, then spin-coat the uncovered area with silver paste at 3000 rpm for 1 min and dry at 50°C; ② Microneedle electrode for the counter electrode: Use a glass capillary with a diameter of 1.0±0.1 mm to modify the multi-walled carbon nanotube aqueous slurry to the counter electrode area of ​​the epoxy resin microneedle; Microneedle electrode for reference electrode: Use a glass capillary with a diameter of 1.0±0.1 mm to modify the silver chloride paste to the reference electrode area of ​​the epoxy resin microneedle; Microneedle electrode of the working electrode: Use glass capillaries with a diameter of 1.0±0.1 mm to modify the working electrode area of ​​the microneedle with a multi-walled carbon nanotube aqueous slurry and a MnO2-Au nanozyme dispersion (2 mg / mL) in sequence, and dry them at 50°C.

[0065] ③ Use expansion resin to modify the epoxy resin microneedles: prepare a mixed aqueous solution containing 5 wt% poly(methyl vinyl ether-co-maleic acid), 5 wt% polyvinyl alcohol 8000, 3 wt% sodium carbonate and 0.2 wt% sodium polyacrylate, and stir it at 60 °C for 2 h. Then use a glass capillary with a diameter of about 1.0 mm to modify the mixed aqueous solution to the surface of the epoxy resin microneedles and dry it at 50 °C.

[0066] ④ Remove the mask and cover the epoxy resin microneedle surface with another layer of film to provide insulation.

[0067] (4) Processing of the microneedle electrode of the working electrode The microneedle electrode of the working electrode obtained in step (2) was immersed in a 4 mg / L A29 protein antibody (brand: Sino Biological, catalog number: 40891-V08E) solution, incubated at 4°C for 8 h, and then washed 3 times with PBS (0.01 M, pH 7.4). The A29 protein antibody was mainly connected to the MnO2-Au nanozyme on the microneedle electrode of the working electrode through electrostatic adsorption. Subsequently, the microneedle electrode of the working electrode was incubated in a 1% BSA solution (w / v) for 1 h to block the nonspecific recognition site, and then washed 3 times with PBS (0.01 M, pH 7.4).

[0068] Scanning electron microscopy (SEM) was used to characterize the morphology of the prepared MnO2 nanoflowers, MnO2-Au nanozymes and the counter electrode, reference electrode and working electrode on the microneedle patch, and energy dispersive spectroscopy was used to analyze the elemental composition of MnO2-Au nanozymes.

[0069] Cyclic voltammetry was performed in a probe solution (5 mM [Fe(CN)6] 3- / 4- The electrochemical properties of the microneedle electrode as the working electrode were investigated in 0.5 M KCl and 0.1 M KCl) with a voltage range of -0.5 to 0.4 V, a scan rate of 50 mV / s, and 3 scans.

[0070] 2. Preparation of Hydrogel 0.2 g of sodium alginate was dissolved in 15 mL of sodium acetate solution (0.2 mol / L, pH 6.0) by stirring to obtain a sodium alginate-sodium acetate solution. 5 mL of 20 mmol / L TMB was added to the sodium alginate-sodium acetate solution and stirred for 3 h to obtain a mixed solution. Subsequently, 2.0 mL of the mixed solution was transferred to the 3D printed mold and allowed to stand for 10 min. The mold was immersed in a 2% calcium chloride solution (w / v) for 2 h to obtain a sodium alginate hydrogel.

[0071] Scanning electron microscopy (SEM) was used to characterize the morphology of the prepared ammonium alginate hydrogel.

[0072] 2. Experimental Results MnO2 nanoflowers were synthesized by hydrothermal method. SEM images showed that they had three-dimensional flower-like nanosphere morphology with a size of about 700 nm ( Figure 4 ).

[0073] Gold nanoparticles were modified on the surface of MnO2 to obtain MnO2-Au nanozymes. SEM images showed that the average diameter of gold nanoparticles was 22 nm and they were distributed on the surface and folds of MnO2 nanoflowers ( Figure 5 Energy dispersive spectroscopy (EDS) analysis showed that MnO2-Au nanozyme is composed of three elements (Mn, O and Au) ( Figure 5 B in the figure).

[0074] The microneedles were prepared in a homemade mold using commercial epoxy resin and were in the shape of triangular prisms with a length of approximately 3000 μm, a base width of approximately 400 μm, and a thickness of approximately 200 μm ( Figure 5 The distance between each row of adjacent microneedles is 3000 μm ( Figure 6The distance between adjacent microneedles in the same row is 5000 μm. The SEM images of the microneedle electrodes of the working electrode, counter electrode, and reference electrode in the microneedle patch are shown in Figure 1. Figure 6 As shown in B, C and D.

[0075] SEM characterization of the cross-sectional morphology of sodium alginate hydrogel Figure 5 As shown in D in Figure , the sodium alginate hydrogel exhibits a dense structure with distributed cavities.

[0076] The electrochemical performance results of the microneedle electrode of the working electrode are as follows Figure 7 As shown in Figure 2, as the scan rate increases from 10 mV / s to 200 mV / s, the anodic and cathodic peak currents (I pa and I pc ) increases ( Figure 7 At the same time, the current value is proportional to the square root of the scan rate ( Figure 7 B in the figure indicates that there is a characteristic diffusion-controlled process on the surface of the microneedle electrode of the working electrode. Based on the Randles-Sevcik equation, the effective surface area of ​​the microneedle electrode of the working electrode is calculated to be 1.03 cm 2 .

[0077] Example 2 In vitro detection of monkeypox virus A29 protein is a highly conserved surface envelope protein in monkeypox virus. As a specific antigen for monkeypox virus detection, A29 protein was selected as the target analyte. In the integrated biosensing platform based on microneedles and hydrogels, the microneedle electrode of the working electrode is combined with MnO2-Au nanozyme and A29 protein antibody for specific recognition and extraction of A29 protein.

[0078] After extraction, the microneedle electrodes of the counter electrode, reference electrode and working electrode on the microneedle patch were inserted into the hydrogel, and the bifunctional signal probe TMB in the hydrogel could be catalyzed by the MnO2-Au nanozyme to produce blue oxidation products and electrochemical signals.

[0079] Since the hydrogel contains electrolytes, the counter electrode, reference electrode and working electrode on the microneedle patch form an electrolytic cell after piercing the hydrogel. At this time, one end of the burning needle is connected to the wire of each electrode, and the other end is connected to the interface on the micro electrochemical workstation, thereby transmitting the electrochemical signal to the micro electrochemical workstation. The electrochemical signal is collected by the micro electrochemical workstation and transmitted to the smartphone via Bluetooth. The colorimetric image of the TMB oxidation product can be captured by the smartphone camera and analyzed with Image J software to obtain the grayscale value result. The obtained colorimetric image and electrochemical results verify each other, which improves the reliability of the detection. At the same time, the integrated biosensing platform based on microneedles and hydrogels is also designed with a control area, which can collect electrochemical signals and colorimetric images under normal skin, thereby realizing ratio analysis and quality control functions ( Figure 1 B in the figure).

[0080] 1. Experimental Methods A29 protein solutions of different concentrations were prepared using PBS (0.01 M, pH 7.4). The counter electrode, reference electrode, and working electrode microneedle electrodes on the microneedle patch were immersed in the A29 protein solution for 10 min and then washed three times with PBS (0.01 M, pH 7.4). Subsequently, the counter electrode, reference electrode, and working electrode microneedle electrodes were simultaneously inserted into the sodium alginate hydrogel and allowed to stand for 200 s for electrochemical determination. The electrochemical signals were recorded by square wave voltammetry (SWV). The potential range of SWV was +0.1 V to +0.5 V, the amplitude was 25 mV, and the frequency was 15 Hz. The electrochemical signals were collected by a micro-electrochemical workstation and transmitted to a smartphone via Bluetooth. Finally, the microneedles were removed, and a colorimetric image of the hydrogel was taken using a smartphone. The grayscale value of the image was analyzed using Image J.

[0081] 2. Experimental Results The results are as follows Figure 8 As shown in the figure, with the increase of A29 protein concentration, the oxidation peak current of TMB gradually decreased. At the same time, the blue color of the hydrogel gradually became lighter ( Figure 8 C in TMB C / I T and G T / G C There was a good linear relationship between the logarithm of A29 protein concentration (lgC) in the range of 10 to 1000 ng / mL ( Figure 8 B and D). By electrochemical detection of I C / I T and G in colorimetric detection T / G CThe detection limits obtained were 0.1632 ng / mL and 0.3017 ng / mL, respectively. This suggests that setting up a control zone in the microneedle patch can achieve lower detection limits and help improve the validity of the measurement results.

[0082] Example 3 Specificity Analysis 1. Experimental Methods PBS (0.01 M, pH 7.4) was used to prepare 100 ng / mL A29 protein solution, α1-acid glycoprotein (AAG) solution, human serum albumin (HSA) solution, smallpox virus A27L protein solution, and Na + Solution, K + solution, glucose solution, and A29 protein, α1-acid glycoprotein (AAG), human serum albumin (HSA), smallpox virus A27L protein, Na + , K + The mixed sample solution after mixing with glucose, the concentration of each substance in the mixed sample solution is 100 ng / mL.

[0083] The assay was performed according to the method of Example 2 to evaluate the specificity of the integrated biosensing platform based on microneedles and hydrogels.

[0084] 2. Experimental Results The results are as follows Figure 8 As shown in E, all interfering substances had little interference with the determination of A29 protein. The ratio (I C / I T and G T / G C There was no significant difference in I between pure A29 protein sample and mixed sample (p>0.05). C / I T and G T / G C The differences were negligible (p>0.05). However, significant differences were found between the A29 protein sample and these interfering substances. C / I T and G T / G C The results showed significant differences (*p<0.05), indicating that the integrated biosensing platform based on microneedles and hydrogels is specific for A29 protein.

[0085] Example 4 Reproducibility and stability analysis 1. Experimental Methods Reproducibility analysis: Five integrated biosensor platforms based on microneedles and hydrogels were prepared according to the method of Example 1, and the A29 protein was measured according to the method of Example 2.

[0086] Stability analysis: The integrated biosensor platform based on microneedles and hydrogels was placed at 4°C for 1 d, 7 d, 14 d, and 21 d, respectively, and then the A29 protein was measured according to the method of Example 2.

[0087] 2. Experimental Results Figure 8 The F results in Figure 3 showed that the relative standard deviations (RSDs) of the electrochemical and colorimetric signals were 1.14% and 3.76%, respectively, demonstrating the remarkable reproducibility of the integrated biosensing platform based on microneedles and hydrogels.

[0088] Figure 8 The results in G showed that after storage at 4°C for 1 d, 7 d, 14 d, and 21 d, the electrochemical and colorimetric signals did not change significantly (RSD < 1.5%, n = 4), indicating that the integrated biosensing platform based on microneedles and hydrogels has good stability.

[0089] Example 5 Accuracy and reliability analysis 1. Experimental Methods 10, 50 and 100 ng / mL of A29 protein solution were added to simulated body fluid and artificial saliva samples, and the A29 protein was determined according to the method of Example 2.

[0090] At the same time, the above samples were measured using a commercially available A29 protein ELISA kit.

[0091] 2. Experimental Results The results of the integrated biosensing platform based on microneedles and hydrogels are shown in Fig. 9 As shown, the recovery rate of A29 protein in the samples was 89.0-110.9%, and there was no significant difference between the results obtained by electrochemical signals and colorimetric signals (p>0.05), indicating that the integrated biosensing platform based on microneedles and hydrogels has good accuracy and reliability.

[0092] The results of the measurement of A29 protein at different concentrations by the integrated biosensor platform based on microneedles and hydrogels and the commercially available A29 protein ELISA kit are shown in Table 1. The results show that there is no significant difference between the two, indicating that the integrated biosensor platform based on microneedles and hydrogels and the commercially available A29 protein ELISA kit have good consistency in detection content.

[0093] Table 1 Comparison of detection effects between the biosensor platform and the commercially available A29 protein ELISA kit

[0094] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A biosensing platform, characterized in that: The biosensor platform includes a collection module, a detection module, a connection module and an information transmission module; The collection module is divided into a test area and a control area, the test area is used to collect virus-infected samples, and the control area is used to collect normal samples; The test area and the control area both contain a counter electrode, a reference electrode and a working electrode, and the counter electrode, the reference electrode and the working electrode are all provided with microneedle electrodes; The microneedle electrode of the working electrode contains an antibody that specifically binds to the monkeypox virus and an enzyme for catalyzing 3,3',5,5'-tetramethylbenzidine to generate an oxidation product; The detection module is a hydrogel, and the hydrogel contains 3,3',5,5'-tetramethylbenzidine and electrolyte; The connection module is a circuit connector; The information transmission module is two electrochemical sensing units, which are detachably connected to the electrodes of the test area and the control area through connection modules respectively. Each electrochemical sensing unit is provided with an electrochemical signal acquisition device and an electrochemical signal transmission device.

2. The biosensor platform according to claim 1, characterized in that: The microneedle electrode of the counter electrode is obtained by modifying multi-walled carbon nanotubes on the microneedle, the microneedle electrode of the reference electrode is obtained by modifying silver chloride on the microneedle, and the microneedle electrode of the working electrode is obtained by modifying multi-walled carbon nanotubes on the microneedle.

3. The biosensor platform according to claim 1, characterized in that: The antibody is an A29 protein antibody.

4. The biosensor platform according to claim 1, characterized in that: The enzyme is MnO2-Au nanozyme, and the preparation method of the MnO2-Au nanozyme is as follows: adding HAuCl4 aqueous solution to the aqueous dispersion of MnO2 nanoflowers, heating to boiling, and then adding sodium citrate aqueous solution to the boiling solution for sufficient reaction, collecting the product after the reaction is completed and purifying it to obtain the product.

5. The biosensor platform according to claim 1, characterized in that: The hydrogel is sodium alginate hydrogel.

6. The biosensor platform according to claim 1, characterized in that: The circuit connector is composed of 6 wires and 6 burning pins, the 6 burning pins are detachably connected to the information transmission module, and the counter electrode, reference electrode and working electrode of the test area and the control area are respectively connected to the corresponding burning pins through wires.

7. The biosensor platform according to claim 1, characterized in that: The biosensor platform further comprises a container for carrying the detection module, wherein the container is provided with a separated hydrogel region and a circuit connector region, the circuit connector is embedded in the circuit connector region, and the hydrogel is embedded in the hydrogel region.

8. Use of the biosensor platform according to any one of claims 1 to 7 in the preparation of a product for detecting monkeypox virus.

9. The method for using the biosensor platform according to any one of claims 1 to 7, characterized in that: The following steps are involved: S11. Insert the counter electrode, reference electrode and working electrode of the test area into the sample to be tested, and insert the counter electrode, reference electrode and working electrode of the control area into the normal sample, and let stand for 8 to 15 min; S12. All the microneedle electrodes are inserted into the hydrogel together, and each electrode is connected to the information transmission module through the connection module, and the electrodes are left standing for 180 to 220 seconds; the electrochemical signals of the test area and the control area transmitted by the information transmission module are collected respectively, and the ratio of the electrochemical signals of the test sample and the normal sample is calculated; S13. Take out the microneedle electrode, obtain a photo of the hydrogel, and analyze the colorimetric image to obtain the colorimetric signal ratio of the sample to be tested and the normal sample.

10. A method for detecting monkeypox virus using the biosensor platform according to any one of claims 1 to 7, characterized in that: The following steps are involved: S21. The method of using according to claim 9 is used to detect the test sample and the normal sample to obtain the electrochemical signal ratio and the colorimetric signal ratio of the test sample and the normal sample; S22. The method of using the standard sample and the normal sample of different concentrations is detected according to claim 9 to obtain the electrochemical signal ratio and the colorimetric signal ratio of the standard sample and the normal sample of different concentrations, and to draw a standard curve; S23. Obtaining the concentration of monkeypox virus in the sample to be tested according to the standard curve; The method is aimed at non-disease treatment and diagnosis.

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