Horseradish peroxidase hydrogel photonic crystal sensor and method
By crosslinking horseradish peroxidase (HRP) with acrylamide (AAm) to form a polymeric hydrogel photonic crystal sensor, the problems of high cost and poor stability of existing sensors are solved, and sensitive label-free detection of hydrogen peroxide is achieved, which is suitable for disease diagnosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIHANG UNIV
- Filing Date
- 2025-04-07
- Publication Date
- 2026-07-21
AI Technical Summary
Existing sensor methods for detecting hydrogen peroxide require trained personnel and large, expensive instruments, making them difficult to popularize in social production and daily life. Furthermore, the immobilization method for horseradish peroxidase is costly and its stability is affected by proteins.
Horseradish peroxidase (HRP) was used to double-bond and crosslink with acrylamide (AAm) to form a polymeric hydrogel, which was then integrated with a photonic crystal array. Label-free visualization detection was performed by utilizing the changes in gel volume caused by the specific reaction of HRP with hydrogen peroxide and the changes in diffraction signals of the photonic crystal array.
It achieves low-cost and easy-to-operate hydrogen peroxide detection. The sensor has abundant raw materials and good stability, and does not require expensive equipment or professional personnel, making it suitable for immediate disease diagnosis.
Smart Images
Figure CN120253762B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to hydrogen peroxide detection technology, and in particular to a horseradish peroxidase hydrogel photonic crystal sensor and method. Background Technology
[0002] Hydrogen peroxide, due to its excellent oxidation, disinfection, and bleaching properties, has been widely used in industries such as textiles, papermaking, clinics, pharmaceuticals, environmental protection, and food processing. Beyond its extensive industrial applications, hydrogen peroxide also plays a crucial role in living organisms. In organisms, hydrogen peroxide acts as a rich signaling molecule, regulating various physiological processes such as metabolism, immune responses, and vascular remodeling. As a common reactive oxygen species, hydrogen peroxide is associated with many diseases, including tumors, cancer, diabetes, osteoarthritis, rheumatoid arthritis, and neurodegenerative diseases, making it an important biomarker for diagnosis, treatment, and prognosis. Although many existing conventional analytical methods, such as spectroscopic techniques, electrochemical techniques, and colorimetric techniques, can achieve precise quantification of hydrogen peroxide, these methods typically require highly trained personnel and large, expensive instruments, hindering their widespread adoption in social production and daily life. Therefore, developing a low-cost, simple-to-prepare, and easy-to-operate sensor for hydrogen peroxide detection is of great significance in both medical diagnostics and clinical research.
[0003] Photonic crystals are ordered structural materials composed of two or more dielectric materials with different dielectric constants (refractive indices) arranged in a certain periodic order in space. When the periodic arrangement scale of a photonic crystal is comparable to the wavelength of visible light, its periodic structure can generate a "photonic bandgap" within a certain frequency range through Bragg diffraction. Electromagnetic waves with frequencies within the photonic bandgap will be completely reflected and thus cannot propagate within the photonic crystal. If this frequency falls within the visible light range, the photonic crystal will produce a bright, vivid structural color that can be perceived by the human eye. By rationally designing the constituent materials, effective refractive index, and lattice parameters of photonic crystals, we can artificially fabricate photonic crystals that meet our needs. Due to these excellent properties, photonic crystals have enormous application prospects in optoelectronics, information anti-counterfeiting, inkjet printing, and sensors. Photonic crystal hydrogels, obtained by embedding photonic crystals into stimulus-responsive hydrogel networks, are a very attractive class of smart polymer sensing materials. Under external stimuli (such as changes in temperature, humidity, electric field, magnetic field, pH, etc.), hydrogels undergo volume changes, which in turn cause changes in the lattice spacing of the embedded photonic crystal array. According to the Bragg diffraction equation, changes in the particle spacing of the photonic crystal alter the photonic bandgap, thus converting changes in the external environment into optically readable signals. Responsive photonic crystal hydrogel sensors have become a research hotspot in recent years due to their low cost, simple fabrication, easy operation, and high signal-to-noise ratio. Currently, researchers have developed various responsive photonic crystal hydrogel sensors, achieving effective detection of proteins, biomarkers, organophosphorus nerve agents, amino acids, and microorganisms.
[0004] Horseradish peroxidase (HRP) is an iron-containing glycoprotein derived from horseradish, widely used in biochemical analysis and clinical testing. This enzyme can catalyze the oxidation of various compounds in the presence of hydrogen peroxide, exhibiting stable properties and has been widely applied in the food industry, biomedicine, and wastewater treatment. HRP consists of 308 amino acids, including 6 lysines and 4 disulfide bonds, and can be effectively chemically modified. Cai et al. invented a method for immobilizing HRP using bovine serum albumin as a scaffold material (ZL 2021 1 1322536.1), achieving the detection of hydrogen peroxide. However, the stability of the sensor is affected by the stability of the protein, requiring stringent storage conditions and incurring relatively high costs. Immobilizing HRP using polymers is a cost-effective method, but it has not yet been reported. Summary of the Invention
[0005] This invention addresses the shortcomings of existing technologies by providing a horseradish peroxidase hydrogel photonic crystal sensor and method. The method involves double-bonding horseradish peroxidase (HRP), which serves as the recognition molecule. This allows the double-bonded HRP to crosslink with other monomers containing double bonds, such as acrylamide (AAm), to form a polymeric hydrogel that embeds colloidal particles within a photonic crystal array. This enables sensitive, label-free, and visual detection of hydrogen peroxide content in the analyte by utilizing the changes in gel volume (particle spacing) caused by the specific binding of HRP to hydrogen peroxide, and the resulting changes in the diameter of the Debye rings diffracted by the photonic crystal array.
[0006] The technical solution of the present invention is as follows:
[0007] A horseradish peroxidase hydrogel photonic crystal sensor is characterized by comprising a sheet-like hydrogel and a two-dimensional photonic crystal array formed by distributing colloidal particles in an interlocking manner on the surface of the hydrogel. The hydrogel contains double-bonded horseradish peroxidase Ac-HRP, which serves as a hydrogen peroxide recognition molecule, and a monomeric acrylamide AAm, which has double bonds, both participating in the hydrogel crosslinking reaction.
[0008] The Ac-HRP is prepared by reacting horseradish peroxidase (HRP) and N-hydroxysuccinimide acrylate (NAS) in phosphate-buffered saline (PBS).
[0009] The hydrogel has the following mass ratio: AAm:Ac-HRP = 2 to 8:1.
[0010] The colloidal particles in the two-dimensional photonic crystal array are polystyrene, polymethyl methacrylate, SiO2, ZnS, or TiO2. The two-dimensional photonic crystal array is formed by a two-dimensional photonic crystal colloidal particle array prepared by a tip-guided gas-liquid interface self-assembly method.
[0011] The Ac-HRP is deactivated to a corresponding degree in the hydrogen peroxide recognition reaction system according to the concentration of hydrogen peroxide, causing the hydrogel to swell accordingly. The swelling increases the spacing between colloidal particles in the photonic crystal array, and the increased spacing between colloidal particles reduces the diameter of the Debye rings diffracted by the photonic crystal array, thereby realizing qualitative or quantitative sensing for hydrogen peroxide detection.
[0012] The Ac-HRP has 6 lysine residues.
[0013] A method for preparing the above-mentioned horseradish peroxidase hydrogel photonic crystal sensor, characterized by comprising the following steps:
[0014] Step A: A two-dimensional photonic crystal array and double-bonded horseradish peroxidase Ac-HRP are prepared. The Ac-HRP is prepared by reacting horseradish peroxidase HRP and N-hydroxysuccinimide acrylate (NAS) dissolved in phosphate buffer PBS.
[0015] Step B involves mixing Ac-HRP, AAm, a crosslinking agent, an initiator, and an accelerator to form a hydrogel prepolymer solution. The hydrogel prepolymer solution is then spread on the two-dimensional photonic crystal array to undergo a crosslinking reaction, thereby forming a polymeric hydrogel. The colloidal particles in the two-dimensional photonic crystal array are all embedded in the polymeric hydrogel to form a colloidal particle distribution layer. Ac-HRP is a hydrogen peroxide recognition molecule.
[0016] Step C involves cutting the polymer hydrogel into sheets of specified dimensions to obtain the horseradish peroxidase hydrogel photonic crystal sensor.
[0017] The fabrication of the two-dimensional photonic crystal array in step A includes the following steps:
[0018] Step A1, Synthesis of polystyrene colloidal particles: Styrene, potassium persulfate and deoxygenated distilled water are mixed under a nitrogen atmosphere and heated in a water bath under stirring. After the reaction, polystyrene colloidal particles are prepared by centrifugation and washing.
[0019] Step A2, preparation of polystyrene two-dimensional photonic crystal array: Polystyrene colloidal particles are dispersed into an emulsion and mixed with n-propanol. After vortexing, the mixture is injected onto the water surface. The polystyrene colloidal particles are retrieved using a hydrophilically treated glass slide and dried in the air to obtain a polystyrene two-dimensional photonic crystal array. The hydrophilic treatment includes immersing the glass slide in a piranha solution, then washing the glass slide with distilled water and anhydrous ethanol in sequence, and finally drying it with nitrogen gas.
[0020] A method for detecting hydrogen peroxide, characterized by employing the aforementioned horseradish peroxidase hydrogel photonic crystal sensor, includes the following steps:
[0021] Step 1: Prepare at least two horseradish peroxidase hydrogel photonic crystal sensors. Place the first sensor in a phosphate buffer solution that does not contain hydrogen peroxide, and place the second sensor into the analyte.
[0022] Step 2: After allowing the Ac-HRP in the second sheet to undergo a specific reaction with the hydrogen peroxide in the analyte for a preset time, causing the hydrogel to swell, remove the second sheet and then remove the first sheet.
[0023] Step 3: Irradiate the first and second plates with a laser respectively, measure the diameter D of the Debye ring diffracted by the photonic crystal array on the first plate and the diameter D' of the Debye ring diffracted by the photonic crystal array on the second plate, and determine the hydrogen peroxide concentration in the analyte based on the difference between D and D'.
[0024] The technical effects of this invention are as follows: This invention provides a horseradish peroxidase hydrogel photonic crystal sensor and method, which addresses the challenge of preparing smart gel materials by immobilizing enzymes. By double-bonding horseradish peroxidase (HRP) as a recognition molecule, the double-bonded horseradish peroxidase (HRP) can cross-link with other monomers with double bonds, acrylamide (AAm), to form a polymer hydrogel, thereby preparing a two-dimensional photonic crystal sensor and a sensitive, label-free, and visual method for detecting hydrogen peroxide.
[0025] This invention discloses a horseradish peroxidase hydrogel photonic crystal sensor. Acrylamide is used as a scaffold material for immobilizing horseradish peroxidase, and a horseradish peroxidase / acrylamide smart hydrogel is prepared. After integration with a two-dimensional photonic crystal, a two-dimensional photonic crystal sensor is obtained. Based on the volume change of the smart hydrogel caused by the specific reaction of horseradish peroxidase with hydrogen peroxide and the unique Debye diffraction signal of the two-dimensional photonic crystal, label-free selective and sensitive detection of hydrogen peroxide is achieved. The sensor uses widely available and stable raw materials, and the detection process is easy to operate. It does not require expensive large-scale instruments or trained personnel, which is beneficial for the miniaturization and portability of the sensing device and holds promise for applications in the field of point-of-care diagnosis.
[0026] The present invention has the following characteristics: (1) The raw materials of the present invention are abundant and stable, and the sensor preparation process is simple. (2) The detection instrument sensor of the present invention is simple to operate and has good linear response. (3) The present invention has strong versatility; it can detect other targets by simply changing the specific enzyme. (4) The sensor of the present invention has excellent selectivity, good sensitivity and stability. (5) The sensor of the present invention is not reversible. (6) The present invention optimizes the HRP content of the sensor. By keeping the mass of AAM constant and adjusting the mass of HRP, it was found that the sensor performs optimally under the condition of HRP:AAM = 1:2 (w:w). (7) The present invention optimizes the optimal pH conditions of the sensor. Within the range of pH = 6.3 to 8.7, it was found that the sensor performs optimally under the condition of pH = 8.7. Attached Figure Description
[0027] Figure 1 This is a schematic diagram of the structure of a horseradish peroxidase hydrogel photonic crystal sensor that implements the present invention.
[0028] Figure 2This is a schematic diagram illustrating the principle of a horseradish peroxidase hydrogel photonic crystal sensor in hydrogen peroxide detection according to the present invention.
[0029] Figure 3 This is a schematic diagram illustrating the effect of different horseradish peroxidase contents on sensor performance.
[0030] Figure 4 This is a schematic diagram illustrating the effect of different pH values on the sensor's response performance.
[0031] Figure 5 This is a schematic diagram comparing the response performance of AAm / HRP-0.1 hydrogel and AAm hydrogel to hydrogen peroxide detection.
[0032] Figure 6 This is a schematic diagram comparing the response performance of the AAm / HRP-0.1 hydrogel two-dimensional photonic crystal sensor to eight different analytes. Detailed Implementation
[0033] The following is in conjunction with the attached diagram ( Figures 1-6 The invention will be described in the following sections and examples.
[0034] Figure 1 This is a schematic diagram of the structure of a horseradish peroxidase hydrogel photonic crystal sensor that implements the present invention. Figure 1 The device includes a horseradish peroxidase hydrogel 1 and a photonic crystal array 2. Hydrogel 1 has a sheet-like structure, and the colloidal particles in the photonic crystal array 2 are embedded on the surface of the sheet-like structure to form a colloidal particle distribution layer. Hydrogel 1 is a polymeric hydrogel formed by crosslinking double-bonded horseradish peroxidase (HRP) with acrylamide (AAm), a crosslinking agent, an initiator, and an accelerator. The horseradish peroxidase (HRP) in the polymeric hydrogel serves as the recognition molecule. Figure 2 This is a schematic diagram illustrating the principle of a horseradish peroxidase hydrogel photonic crystal sensor in hydrogen peroxide detection according to the present invention. Figure 2 The left side of the middle section indicates that the Debye rings diffracted after the laser passes through the photonic crystal array in the sensor have a large diameter D (i.e., the spacing between colloidal particles is small, and there is no hydrogen peroxide in the analyte). Figure 2 The right side of the image shows that the Debye rings diffracted after the laser passes through the photonic crystal array in the sensor have a smaller diameter, D' (i.e., a larger interparticle spacing, indicating the presence of hydrogen peroxide in the analyte). The change in the Debye ring diameter determines the change in the interparticle spacing, which in turn determines the hydrogen peroxide content in the analyte. Alternatively, the change in the Debye ring diameter can be used to directly determine the hydrogen peroxide content in the analyte. Figure 3 This is a schematic diagram illustrating the effect of different horseradish peroxidase contents on sensor performance. Figure 3The x-axis represents the concentration of hydrogen peroxide detection solution (H2O2 / mM, unit: millimoles / liter, solvent is phosphate buffer), and the scale values include 0-20-40-60-80-100. Figure 3 The vertical axis represents the change in particle spacing (increase, nm stands for nanometer), with scale values including 0-50-100-150-200-250. Figure 3 The sensor performance curves include three HRP contents, from bottom to top: AAm / HRP-0.1 (horseradish peroxidase HRP 0.1 g / ml), AAm / HRP-0.075 (horseradish peroxidase HRP 0.075 g / ml), and AAm / HRP-0.05 (horseradish peroxidase HRP 0.05 g / ml). Figure 4 This is a schematic diagram illustrating the effect of different pH values on the sensor's response performance. Figure 4 The x-axis represents the concentration of hydrogen peroxide detection solution (H2O2 / mM, unit: millimoles / liter, solvent is phosphate buffer), and the scale values include 0-20-40-60-80-100. Figure 4 The vertical axis represents the change in particle spacing (increase, nm stands for nanometer), with scale values including 0-50-100-150-200-250. Figure 4 It includes three pH performance curves, from bottom to top: pH 8.7, pH 7.4, and pH 6.3. Figure 5 This is a schematic diagram comparing the response performance of AAm / HRP-0.1 hydrogel and AAm hydrogel to hydrogen peroxide detection. The values represent the concentration of the hydrogen peroxide detection solution (H2O2 / mM, unit: millimoles / liter, solvent is phosphate buffer), with scale values including 0-20-40-60-80-100. Figure 5 The vertical axis represents the change in interparticle spacing (increase, nm stands for nanometer), with scale values including 0-50-100-150-200-250. Figure 6 This is a schematic diagram comparing the response performance of the AAm / HRP-0.1 hydrogel two-dimensional photonic crystal sensor to eight different analytes. Figure 6 The vertical axis represents the change in particle spacing (increase, nm stands for nanometer), with scale values including 0-50-100-150-200-250. Figure 6The results include the response performance of AAm / HRP-0.1 hydrogel to eight reagents, which are listed from left to right as blank, glucose, fructose, ascorbic acid, sodium chloride (NaCl), glycine, arginine, and hydrogen peroxide (H2O2).
[0035] refer to Figures 1 to 6 As shown, a horseradish peroxidase hydrogel photonic crystal sensor includes a sheet-like hydrogel 1 and a two-dimensional photonic crystal array 2 formed by distributing colloidal particles in an interlocking manner on the surface of the hydrogel. The hydrogel contains double-bonded horseradish peroxidase Ac-HRP, which serves as a hydrogen peroxide recognition molecule, and a monomeric acrylamide AAm, which has double bonds, both participating in the hydrogel crosslinking reaction.
[0036] The Ac-HRP is prepared by reacting horseradish peroxidase (HRP) and N-hydroxysuccinimide acrylate (NAS) dissolved in phosphate-buffered saline (PBS). The hydrogel has the following mass ratio: AAm:Ac-HRP = 2–8:1. The colloidal particles in the two-dimensional photonic crystal array are polystyrene, polymethyl methacrylate, SiO2, ZnS, or TiO2. The two-dimensional photonic crystal array is formed by a two-dimensional photonic crystal colloidal particle array prepared using a tip-guided gas-liquid interface self-assembly method.
[0037] The Ac-HRP in the hydrogen peroxide recognition reaction system undergoes corresponding inactivation with varying hydrogen peroxide concentrations, causing the hydrogel to swell. This swelling increases the interparticle spacing in the photonic crystal array, which in turn reduces the diameter of the Debye rings diffracted by the photonic crystal array, thereby enabling qualitative or quantitative sensing for hydrogen peroxide detection. The Ac-HRP contains six lysine residues.
[0038] A method for preparing the above-mentioned horseradish peroxidase hydrogel photonic crystal sensor includes the following steps: Step A, preparing a photonic crystal array and double-bonded horseradish peroxidase Ac-HRP, wherein Ac-HRP is prepared by reacting horseradish peroxidase HRP and N-hydroxysuccinimide acrylate (NAS) dissolved in phosphate buffered saline (PBS); Step B, mixing Ac-HRP, acrylamide (AAm), a crosslinking agent, an initiator, and an accelerator to form a hydrogel prepolymer, spreading the hydrogel prepolymer on the photonic crystal array for crosslinking reaction to form a polymeric hydrogel, wherein the colloidal particles in the photonic crystal array are embedded in the polymeric hydrogel to form a colloidal particle distribution layer, and Ac-HRP is a hydrogen peroxide recognition molecule; Step C, cutting the polymeric hydrogel into sheets of specified dimensions to obtain the horseradish peroxidase hydrogel photonic crystal sensor.
[0039] The preparation of the photonic crystal array in step A includes the following steps: Step A1, synthesizing polystyrene colloidal particles: under a nitrogen atmosphere, styrene, potassium persulfate, and deoxygenated distilled water are mixed and reacted in a water bath under stirring. After the reaction, the polystyrene colloidal particles are obtained by centrifugation and washing. Step A2, preparing a two-dimensional polystyrene photonic crystal array: the polystyrene colloidal particles are dispersed into an emulsion and mixed with n-propanol. After vortexing, the mixture is injected onto the water surface. The polystyrene colloidal particles are retrieved using a hydrophilically treated glass slide and dried in the air to obtain a two-dimensional polystyrene photonic crystal array. The hydrophilic treatment includes immersing the glass slide in a piranha solution, then washing the glass slide with distilled water and anhydrous ethanol in sequence, and finally drying it with nitrogen.
[0040] A method for detecting hydrogen peroxide, using the aforementioned horseradish peroxidase hydrogel photonic crystal sensor, includes the following steps: Step 1, prepare at least two horseradish peroxidase hydrogel photonic crystal sensors, placing the first sensor in a phosphate buffer solution free of hydrogen peroxide, and placing the second sensor in the analyte; Step 2, after allowing the Ac-HRP in the second sensor to undergo a specific reaction with the hydrogen peroxide in the analyte for a preset time, remove the second sensor and the first sensor; Step 3, irradiate the first sensor and the second sensor with a laser respectively, measure the diameter D of the Debye ring diffracted by the photonic crystal array on the first sensor and the diameter D' of the Debye ring diffracted by the photonic crystal array on the second sensor, and determine the hydrogen peroxide concentration in the analyte based on the difference between D and D'.
[0041] This invention relates to hydrogen peroxide detection technology, and in particular to a horseradish peroxidase hydrogel photonic crystal sensor and method. By double-bonding horseradish peroxidase (HRP), which serves as the recognition molecule, the double-bonded HRP can crosslink with other double-bonded monomers such as acrylamide (AAm) to form a polymeric hydrogel that integrates a photonic crystal. This allows for sensitive, label-free, and visual detection of the hydrogen peroxide content in the analyte by utilizing the changes in gel volume (particle spacing) caused by the specific binding of HRP to hydrogen peroxide, and the changes in the diameter of the Debye rings diffracted by the photonic crystal array due to these changes in particle spacing.
[0042] A horseradish peroxidase hydrogel photonic crystal sensor includes a hydrogel and a photonic crystal array. The hydrogel has a sheet-like structure, and colloidal particles in the photonic crystal array are embedded on the surface of the sheet-like structure to form a colloidal particle distribution layer. The hydrogel is a polymeric hydrogel formed by crosslinking double-bonded horseradish peroxidase (HRP) with acrylamide (AAm), a crosslinking agent, an initiator, and an accelerator. The horseradish peroxidase (HRP) is the recognition molecule. The photonic crystal array is a two-dimensional photonic crystal array. The horseradish peroxidase (HRP) is used to recognize hydrogen peroxide. In a specific reaction with hydrogen peroxide, the horseradish peroxidase (HRP) is inactivated to a corresponding degree according to the concentration of hydrogen peroxide in the reaction system. This inactivation causes the hydrogel to expand, and the spacing between the colloidal particles in the photonic crystal array increases with the volume change of the hydrogel. The increased spacing between the colloidal particles reduces the diameter of the Debye rings diffracted by the photonic crystal array, thereby achieving qualitative or quantitative sensing for hydrogen peroxide detection.
[0043] Contents not described in detail in this specification are prior art known to those skilled in the art. It is hereby indicated that the above description is intended to help those skilled in the art understand this invention, but does not limit the scope of protection of this invention. Any equivalent substitutions, modifications, improvements, and / or simplifications of the above descriptions that do not depart from the essential content of this invention fall within the scope of protection of this invention.
Claims
1. A horseradish peroxidase hydrogel photonic crystal sensor, characterized in that, The invention includes a sheet-like hydrogel and a two-dimensional photonic crystal array formed by distributing colloidal particles in an interlocking manner on the surface of the hydrogel. The sheet-like hydrogel contains double-bonded horseradish peroxidase Ac-HRP, which acts as a hydrogen peroxide recognition molecule, and a monomeric acrylamide AAm, which has a double bond, both of which participate in the hydrogel crosslinking reaction. The Ac-HRP has 6 lysine residues; The hydrogel has the following mass ratio: AAm: Ac-HRP = 2 to 8: 1; The preparation of the horseradish peroxidase hydrogel photonic crystal sensor includes the following steps: Step A: A two-dimensional photonic crystal array and double-bonded horseradish peroxidase Ac-HRP are prepared. The Ac-HRP is prepared by reacting horseradish peroxidase HRP and N-hydroxysuccinimide acrylate (NAS) dissolved in phosphate buffer PBS. Step B involves mixing Ac-HRP, AAm, a crosslinking agent, an initiator, and an accelerator to form a hydrogel prepolymer solution. The hydrogel prepolymer solution is then spread on the two-dimensional photonic crystal array to undergo a crosslinking reaction, thereby forming a polymeric hydrogel. The colloidal particles in the two-dimensional photonic crystal array are all embedded in the polymeric hydrogel to form a colloidal particle distribution layer. Ac-HRP is a hydrogen peroxide recognition molecule. Step C involves cutting the polymer hydrogel into sheets of specified dimensions to obtain the horseradish peroxidase hydrogel photonic crystal sensor.
2. The horseradish peroxidase hydrogel photonic crystal sensor according to claim 1, characterized in that, The colloidal particles in the two-dimensional photonic crystal array are polystyrene, polymethyl methacrylate, SiO2, ZnS, or TiO2. The two-dimensional photonic crystal array is formed by a two-dimensional photonic crystal colloidal particle array prepared by a tip-guided gas-liquid interface self-assembly method.
3. The horseradish peroxidase hydrogel photonic crystal sensor according to claim 1, characterized in that, The Ac-HRP is deactivated to a corresponding degree in the hydrogen peroxide recognition reaction system according to the concentration of hydrogen peroxide, causing the hydrogel to swell accordingly. This swelling increases the spacing between colloidal particles in the photonic crystal array, which in turn reduces the diameter of the Debye rings diffracted by the photonic crystal array, thereby achieving qualitative or quantitative sensing for hydrogen peroxide detection.
4. A method for preparing a horseradish peroxidase hydrogel photonic crystal sensor as described in any one of claims 1-3, characterized in that, Includes the following steps: Step A: A two-dimensional photonic crystal array and double-bonded horseradish peroxidase Ac-HRP are prepared. The Ac-HRP is prepared by reacting horseradish peroxidase HRP and N-hydroxysuccinimide acrylate (NAS) dissolved in phosphate buffer PBS. Step B involves mixing Ac-HRP, AAm, a crosslinking agent, an initiator, and an accelerator to form a hydrogel prepolymer solution. The hydrogel prepolymer solution is then spread on the two-dimensional photonic crystal array to undergo a crosslinking reaction, thereby forming a polymeric hydrogel. The colloidal particles in the two-dimensional photonic crystal array are all embedded in the polymeric hydrogel to form a colloidal particle distribution layer. Ac-HRP is a hydrogen peroxide recognition molecule. Step C involves cutting the polymer hydrogel into sheets of specified dimensions to obtain the horseradish peroxidase hydrogel photonic crystal sensor.
5. The method for preparing the horseradish peroxidase hydrogel photonic crystal sensor according to claim 4, characterized in that, The fabrication of the two-dimensional photonic crystal array in step A includes the following steps: Step A1, Synthesis of polystyrene colloidal particles: Styrene, potassium persulfate and deoxygenated distilled water are mixed under a nitrogen atmosphere and heated in a water bath under stirring. After the reaction, polystyrene colloidal particles are prepared by centrifugation and washing. Step A2, preparation of polystyrene two-dimensional photonic crystal array: Polystyrene colloidal particles are dispersed into an emulsion and mixed with n-propanol. After vortexing, the mixture is injected onto the water surface. The polystyrene colloidal particles are retrieved using a hydrophilically treated glass slide and dried in the air to obtain a polystyrene two-dimensional photonic crystal array. The hydrophilic treatment includes immersing the glass slide in a piranha solution, then washing the glass slide with distilled water and anhydrous ethanol in sequence, and finally drying it with nitrogen gas.
6. A method for detecting hydrogen peroxide, characterized in that, The horseradish peroxidase hydrogel photonic crystal sensor as described in any one of claims 1-3 includes the following steps: Step 1: Prepare at least two horseradish peroxidase hydrogel photonic crystal sensors. Place the first sensor in a phosphate buffer solution that does not contain hydrogen peroxide, and place the second sensor into the analyte. Step 2: After allowing the Ac-HRP in the second sheet to undergo a specific reaction with the hydrogen peroxide in the analyte for a preset time, causing the hydrogel to swell, remove the second sheet and then remove the first sheet. Step 3: Irradiate the first and second plates with a laser respectively, measure the diameter D of the Debye ring diffracted by the photonic crystal array on the first plate and the diameter D' of the Debye ring diffracted by the photonic crystal array on the second plate, and determine the hydrogen peroxide concentration in the analyte based on the difference between D and D'.