Photonic crystal hydrogel for visualizing uric acid detection and preparation method and application thereof

Abstract

The application discloses a photonic crystal hydrogel for visualizing uric acid detection and a preparation method and application thereof, and relates to the technical field of medical detection. The photonic crystal hydrogel is obtained by uniformly filling a hydrogel pre-polymer solution in monodisperse microspheres with a particle size of 150nm-300nm to form a uniform hierarchical structure, and then washing and drying the obtained hierarchical structure; the hydrogel pre-polymer solution is obtained by dissolving a uric acid recognition monomer, a swelling functional monomer and a gel-forming skeleton monomer in water or a PBS phosphate buffer solution as a composite hydrogel matrix, adding a glutaraldehyde aqueous solution and uniformly stirring; the uric acid recognition monomer is a uricase produced by Prosthecochloris aminophilus, Escherichia coli or Candida utilis. Different response substance concentration ranges correspond to different colors, and the effect of visualizing uric acid detection is achieved; the operation is simple, the photonic crystal hydrogel is easy to carry, is not easy to be polluted, has a short detection time, and does not need precise instrument detection and professional detection personnel, and therefore, the photonic crystal hydrogel can be used for daily monitoring of patients with abnormal uric acid levels and achieves the purpose of preliminary diagnosis.

Description

Technical Field

[0001] This invention relates to visual uric acid detection technology, specifically to a photonic crystal hydrogel for visual uric acid detection, its preparation method, and its application. Background Technology

[0002] Uric acid is an important product of purine metabolism in the human body. In clinical diagnosis, the uric acid content in blood and urine is closely related to the body's metabolic status. Abnormal uric acid levels are closely associated with various diseases, such as gout, essential hypertension, and metabolic syndrome, which pose significant health risks. Therefore, for individuals with abnormal uric acid levels, regular monitoring of their uric acid levels and timely medical treatment based on their individual circumstances are crucial. However, clinical blood tests are time-consuming for patients and do not provide immediate results. Furthermore, while conventional uric acid detection techniques, such as fluorescence spectroscopy, colorimetry, and liquid chromatography, offer good accuracy, they are limited by time consumption, the need for sophisticated equipment, and high costs. Sensors developed based on electrochemical methods can achieve short-term detection to some extent, but the electrodes are susceptible to contamination during use, and repeated testing data fluctuates significantly. These limitations prevent these methods from meeting the needs of patients for daily self-monitoring at home.

[0003] Responsive photonic crystal hydrogels can undergo a volume phase transition and change their optical color in response to external responsive substances, providing a clear indication of the concentration range of the responsive substance. Therefore, applying photonic crystal hydrogels to the visualization of biomolecules can facilitate self-monitoring by patients and provide intuitive results without relying on additional detection instruments. However, due to the current lack of a suitable hydrogel matrix that can directly crosslink with uric acid to induce a volume phase transition, no research has yet combined uric acid detection with photonic crystal hydrogels.

[0004] Chinese invention patent CN111690090B discloses a glycosylated photonic crystal hydrogel and its application in influenza virus detection. It uses lactobionic acid as a raw material and performs terminal amination treatment to introduce unsaturated double bonds to obtain a galactose-functionalized monomer. Acrylamide and GAC are used as functional monomers, and methylenebisacrylamide is used as a crosslinking agent. The monomers are then polymerized on the surface of a two-dimensional photonic crystal to obtain the glycosylated photonic crystal hydrogel. This technology utilizes the swelling of the two-dimensional photonic crystal hydrogel and Debye ring measurement for preliminary detection of influenza virus. However, it still requires laser light for Debye ring measurement, which is not intuitive or convenient enough for routine patient monitoring.

[0005] Chinese invention patent CN107462531B discloses an enzyme-free colorimetric detection method for uric acid. This technology first prepares an integrated CoP / NF monolithic peroxidase-like enzyme. CoP / NF acts as the peroxidase-like enzyme, catalyzing the redox reaction of TMB and hydrogen peroxide solution to produce a chromogenic substance, TMBox. Because TMBox can be selectively reduced to colorless TMB by uric acid, the absorbance of the original colorimetric reaction decreases with increasing uric acid concentration. By measuring the absorbance of the final chromogenic substance, a curve showing the decrease in absorbance with increasing uric acid concentration is obtained. The uric acid content of the analyte is determined based on this standard curve. However, this technology still requires the use of a UV-Vis spectrophotometer for absorbance measurement. This instrument is not cost-effective for everyday home use, and patients without professional testing knowledge may find it difficult to perform the necessary procedures. These limitations make it inconvenient for patients to perform daily monitoring.

[0006] Chinese invention patent application CN114935572A discloses a visual uric acid detection method based on nanomaterials. It utilizes positively charged gold nanoparticles as peroxidase and 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic agent, allowing for rapid detection and analysis of uric acid content based on color intensity. However, since TMB is a chemical chromogenic agent, it can only produce a single-color chromogenic substance during its reaction with generated hydrogen peroxide. Therefore, uric acid concentration can only be determined by the intensity of the color. Without the aid of absorbance or colorimetric instruments, the single color can lead to errors in visual observation, which is also unfavorable for routine monitoring. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention aims to provide a photonic crystal hydrogel for visual uric acid detection, and its preparation method thereof, which is simple to operate, portable, not easily contaminated, has a short detection time, good visualization effect, does not rely on precision instruments or professional testing personnel, and can meet the needs of patients for home self-testing.

[0008] Another objective of this invention is to provide a method for applying photonic crystal hydrogels for visualizing uric acid detection in uric acid detection.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A photonic crystal hydrogel for visualizing uric acid detection: It is obtained by uniformly filling a hydrogel prepolymer solution into monodisperse microspheres with a particle size of 150nm-300nm to form a uniform hierarchical structure, followed by washing and drying. The hydrogel prepolymer solution is prepared by dissolving uric acid recognition monomers, swelling functional monomers, and gelling backbone monomers in water or PBS phosphate buffer solution as a composite hydrogel matrix, adding glutaraldehyde aqueous solution, and vortexing to homogenize. The uric acid recognition monomer is uricase produced by *Protozoa protozoa*, *Escherichia coli*, or *Candida utilis*. The swelling functional monomer is horseradish peroxidase (HRP), lignin peroxidase (LIP), or soybean peroxidase (SBP). The gelling backbone monomer is keratin, silk fibroin, or bovine serum albumin (BSA).

[0011] To further achieve the objectives of this invention, preferably, the monodisperse microspheres are any one of polystyrene microspheres, silica microspheres, polyacrylamide microspheres, and polymethyl methacrylate microspheres; the activity of uric acid in recognizing monomers... The activity of swelling functional monomers .

[0012] Preferably, the formation of a uniform hierarchical structure of the monodisperse microspheres is achieved by vertical deposition or drop-coating self-assembly.

[0013] Preferably, the drop-coating self-assembly method involves adding monodisperse microspheres to a solvent to prepare a dispersion, ultrasonically dispersing them evenly, and then dropping them onto a hydrophilic glass slide to form a photonic crystal template. Another glass slide is then placed on top of the photonic crystal template to form a sandwich structure of glass slide-photonic crystal-glass slide.

[0014] Preferably, the hydrophilic treatment involves treating the glass slide with a piranha solution or a saturated sodium hydroxide isopropanol solution; the solvent is water.

[0015] Preferably, the composite hydrogel matrix contains 0.1-10 mg / mL of uric acid recognition monomer, 25-75 mg / mL of swelling functional monomer, and 0.15-0.20 g / mL of gel-forming backbone monomer.

[0016] Preferably, the PBS phosphate buffer is prepared from disodium hydrogen phosphate and potassium dihydrogen phosphate, wherein the molar ratio of disodium hydrogen phosphate to potassium dihydrogen phosphate is 3:1.5-3:4.5, the concentration is 0.01M-1M, and the pH is 7-8.

[0017] Preferably, the mass fraction of the crosslinking agent glutaraldehyde aqueous solution is 3%-8%, and the volume ratio of glutaraldehyde solution to composite hydrogel matrix is ​​1:3-1:6.

[0018] The uniform filling of the hydrogel prepolymer is achieved by dripping the hydrogel prepolymer onto the monodisperse microsphere hierarchical structure and using capillary force to introduce the hydrogel prepolymer into the entire monodisperse microsphere structure, and then crosslinking it at a temperature of 25℃-35℃ for 4-6 hours.

[0019] The washing refers to washing the photonic crystal hydrogel in water or 0.01M-1M PBS solution for 12-24 hours after cross-linking; the drying is carried out at 15℃-30℃.

[0020] The rotational speed of the vortex is 2000-3000 rpm, and the time is 5-10 seconds.

[0021] The method for preparing the photonic crystal hydrogel for visualizing uric acid detection includes the following steps:

[0022] (1) The uric acid recognition monomer, swelling functional monomer, and gelling backbone monomer are dissolved in water or PBS phosphate buffer solution to form a composite hydrogel matrix. Glutaraldehyde is added, and the mixture is vortexed to obtain a hydrogel prepolymer solution. The uric acid recognition monomer is uricase produced by Protosporum protosporum, Escherichia coli, or Candida utilis. The swelling functional monomer is horseradish peroxidase (HRP), lignin peroxidase (LIP), or soybean peroxidase (SBP). The gelling backbone monomer is keratin, silk fibroin, or bovine serum albumin (BSA).

[0023] (2) The hydrogel prepolymer is uniformly filled into monodisperse microspheres with a particle size of 150nm-300nm to form a uniform hierarchical structure;

[0024] (3) The uniform hierarchical structure is washed and dried to obtain a photonic crystal hydrogel for visual uric acid detection.

[0025] The method for applying the photonic crystal hydrogel for visualizing uric acid detection in uric acid detection is as follows: place the photonic crystal hydrogel for visualizing uric acid detection in the test solution for 15-90 minutes, observe the color of the photonic crystal hydrogel, and determine the concentration of uric acid in the test solution based on the color.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1. This invention is the first in the field of photonic crystal hydrogels to achieve a response to uric acid, which is difficult to respond to directly, through an indirect conversion method. First, uric acid is catalyzed by a uric acid recognition monomer to generate hydrogen peroxide. Then, hydrogen peroxide induces the deactivation of the swelling functional monomer, causing the hydrogel system to swell. This results in changes in the interparticle spacing of the photonic crystal and a significant change in structural color, thereby achieving the purpose of visual detection of uric acid.

[0028] 2. This invention provides a novel method for uric acid detection. Simply placing the prepared photonic crystal hydrogel in the test solution for a certain period allows the uric acid concentration range to be determined through color changes. It eliminates the need for conventional instruments such as ultraviolet spectrophotometers or fluorometers, as well as specialized operators. The operation is simple, the results are intuitive, and it is convenient for various patients. Furthermore, the simple preparation method facilitates large-scale, standardized preparation.

[0029] 3. This invention exhibits excellent optical color change and corresponding reflection wavelength within the 0-1mM range, and can complete detection within 90 minutes. It is highly efficient and convenient, with high color change, meeting the needs of patients for routine preliminary testing. When used with a colorimetric card similar to pH test strips, it can determine the concentration range of uric acid. This invention can control the color change range within a specific range by changing the content of the template and hydrogel matrix. For specific ranges, it can be controlled within easily identifiable color intervals, such as a bright orange-red at the critical state, orange below the critical value, and red or light red above the critical value, making the color distinction between different concentration ranges more obvious. Furthermore, due to the characteristics of its gel, it is not reusable and can be used as a disposable test strip, making it less susceptible to contamination. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the preparation and swelling mechanism of the photonic crystal hydrogel for visualizing uric acid detection according to the present invention.

[0031] Figure 2a This is a particle size distribution diagram of polystyrene microspheres used in Example 1 of the present invention for preparing three-dimensional photonic crystals.

[0032] Figure 2b This is an electron microscope image of the three-dimensional photonic crystal template prepared in Example 1 of the present invention.

[0033] Figure 3a The image shows the normalized curves of the maximum reflection wavelength of the photonic crystal hydrogel used for visual hydrogen peroxide detection in Example 1 of this invention after being treated with hydrogen peroxide solutions of different concentrations for 45 minutes.

[0034] Figure 3b The average maximum reflection wavelength and corresponding optical color of the photonic crystal hydrogel for visualizing hydrogen peroxide detection in Example 1 of this invention after being treated in hydrogen peroxide solutions of different concentrations for 45 minutes are shown.

[0035] Figure 4 This is a scanning electron microscope image of the photonic crystal hydrogel for visualizing uric acid detection prepared in Example 2 of the present invention.

[0036] Figure 5The relationship between the maximum reflection wavelength and uric acid concentration of the photonic crystal hydrogel for visual uric acid detection prepared in Example 2 of the present invention in uric acid solutions of different concentrations.

[0037] Figure 6 The images show scanning electron microscope (SEM) images of the photonic crystal hydrogel for visualizing uric acid detection prepared in Example 2 of this invention, obtained after treatment with uric acid solution and subsequent re-drying. Figures af and f represent the SEM images of the photonic crystal hydrogel for visualizing uric acid detection after treatment with uric acid solutions of concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1 mM for 45 min, followed by re-drying.

[0038] Figure 7 The maximum reflection wavelength of the photonic crystal hydrogel for visual uric acid detection prepared in Example 3 of this invention after different treatment times is shown.

[0039] Figure 8 This is a comparison graph showing the relationship between the average maximum reflection wavelength and uric acid concentration after treatment in different uric acid concentrations for 45 minutes in Example 2 of the present invention and the relationship between the average maximum reflection wavelength and solution concentration after treatment in different concentrations of uric acid solution or hydrogen peroxide solution for 45 minutes in Example 4 of the present invention.

[0040] Figure 9 This is a comparison of the maximum reflection wavelengths after treatment with uric acid solutions of concentrations of 0 mM, 0.4 mM, and 0.8 mM for 45 minutes in Example 5 of the present invention.

[0041] Figure 10 This is a comparison of the maximum reflection wavelengths after treatment with uric acid solutions of concentrations of 0mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, and 1mM for 45 minutes, as shown in Example 6 of the present invention. Detailed Implementation

[0042] To better understand the present invention, the invention is further described below with reference to the accompanying drawings and embodiments. However, the embodiments do not constitute a limitation on the scope of protection of the claims of the present invention. Other embodiments obtained by those skilled in the art based on the embodiments without creative effort are all within the scope of protection of the present invention.

[0043] Figure 1This paper presents a method for preparing a photonic crystal hydrogel for visualizing uric acid detection and its response mechanism. The method uses uricase (derived from *Protozoa protozoa*) as the uric acid recognition monomer, horseradish peroxidase (HRP) as the swelling functional monomer, and bovine serum albumin (BSA) as the gelling backbone monomer. A uniform hierarchical structure of monodisperse microspheres with a particle size of 150 nm-300 nm is formed on a glass slide to create a three-dimensional photonic crystal template. A glass slide is then placed on top, forming a sandwich structure. A hydrogel prepolymer is introduced into the sandwich structure using capillary forces, allowing for full cross-linking to form the photonic crystal hydrogel. After removal, cleaning, and drying, the visualized photonic crystal hydrogel for uric acid detection is obtained. Under treatment with uric acid solutions of different concentrations, uricase catalyzes the production of hydrogen peroxide, which gradually inactivates horseradish peroxidase, causing structural changes. This leads to swelling changes in the hydrogel network, altering the interparticle spacing and resulting in a change in structural color.

[0044] This invention utilizes a uric acid recognition monomer on a photonic crystal hydrogel to catalyze the formation of allantoin and hydrogen peroxide (H₂O₂) from uric acid. The swelling functional monomer gradually deactivates under the action of the catalyzed H₂O₂. This deactivation is due to the attack of the porphyrin structure by H₂O₂, leading to the loss of Fe in the heme groups and a transformation from a cyclic porphyrin structure to a chain-like tetrapyrrole structure. This transformation causes a volume change in the composite hydrogel system formed by the swelling functional monomer. The lattice spacing of the three-dimensional photonic crystal coated by the composite hydrogel changes, resulting in changes in the maximum reflection wavelength and macroscopic color. The gelling framework monomer co-crosslinks with the swelling functional monomer to form a mechanically stable gel system, ensuring the bioactivity of the loaded uric acid recognition monomer. In particular, the photonic crystal hydrogel of this invention swells in the test solution containing the responder uric acid. The higher the concentration of the responder, the greater the swelling change, resulting in a redshift of the photonic bandgap. The greater the swelling change, the greater the redshift in the color reflection wavelength. Different responder concentration ranges correspond to different colors. Photonic crystal hydrogels can detect uric acid and H2O2 in the range of 0-1 mM.

[0045] Example 1

[0046] A method for preparing a photonic crystal hydrogel for visualizing hydrogen peroxide detection includes the following steps:

[0047] (1) Select 0.1 mg uricase (produced from Escherichia coli, with high activity) ) as a recognition monomer, 75mg horseradish peroxidase (HRP, activity) Using 0.2 g bovine serum albumin (BSA) as a swelling functional monomer and 0.2 g bovine serum albumin (BSA) as a gelling backbone monomer and a carrier for maintaining enzyme activity, the above substances were dissolved in 1 mL of 0.1 M PBS phosphate buffer to form a composite hydrogel matrix. 5 wt% glutaraldehyde was added as a cross-linking agent, with a volume ratio of 5 wt% glutaraldehyde to composite hydrogel matrix solution of 1:4 (v:v). The mixed solution was vortexed at 3000 rpm for 10 s to obtain a homogenized hydrogel prepolymer.

[0048] (2) Monodisperse polystyrene microspheres were prepared using a soap-free emulsion method with styrene, methacrylic acid, and ammonium persulfate as an initiator. The monodisperse polystyrene microspheres were dispersed in deionized water to prepare a 10% polystyrene dispersion. Then, 200 ml of the dispersion was taken... A polystyrene dispersion was dropped onto a glass slide that had been hydrophilically treated with a saturated sodium hydroxide solution in isopropanol and spread evenly. After the dispersion dried, polystyrene microspheres formed a three-dimensional photonic crystal template on the slide. Another glass slide was then placed over the prepared photonic crystal template to assemble a sandwich structure. The hydrogel prepolymer prepared in step 1 was pipetted into the cavities between the sandwich structure layers. The hydrogel prepolymer gradually filled the entire cavity between the sandwich layers under capillary action. After introducing the prepolymer, it was allowed to spread evenly throughout the entire interlayer structure without leaving any air bubbles. The mixture was then allowed to fully crosslink at 25°C for 5 hours to form a photonic crystal hydrogel.

[0049] Figure 2a The image shows the particle size distribution of the polystyrene microspheres used in Example 1. It can be seen that the average particle size of the polystyrene microspheres is about 230 nm, and they have a bright structural color, which is a prerequisite for the subsequent photonic crystal hydrogel to have a good visualization effect. Figure 2b The image shows an electron microscope image of the uniform photonic crystal template prepared in Example 1. It can be seen that the microspheres of the prepared photonic crystal template have uniform particle size and good self-assembly effect, so they can reflect bright structural colors, which is the microstructure basis for its bright structural colors.

[0050] (3) Remove the glass slide on the surface of the photonic crystal hydrogel prepared in step 2, place the photonic crystal hydrogel in 0.01M PBS buffer for 24h to remove excess prepolymer and dry at 25°C to obtain the photonic crystal hydrogel for visual hydrogen peroxide detection.

[0051] Hydrogen peroxide solution was diluted with 0.01M PBS buffer to prepare hydrogen peroxide solutions with concentrations of 0mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM, 2.0mM, and 5.0mM, respectively. The photonic crystal hydrogel for visualizing hydrogen peroxide detection prepared in step (3) was placed in hydrogen peroxide solutions of different concentrations for 45 minutes, then removed. The optical color was observed and corresponding optical photographs were taken using UV-Vis technology. The maximum reflected wavelength of the hydrogel sensor was measured using a Vis spectrophotometer (Hitachi U-3900), and the test results are shown in Table 1. It should be noted that the color descriptions in Table 1 are approximate and do not strictly adhere to color naming conventions.

[0052] Table 1

[0053]

[0054] Figure 3a This is a normalized curve of the maximum reflection wavelength of the photonic crystal hydrogel used in Example 1 after treatment in hydrogen peroxide solutions of different concentrations for 45 min. It can be observed that as the hydrogen peroxide concentration increases, the maximum reflection wavelength of the photonic crystal hydrogel continuously redshifts, and the reflection wavelengths fall within different ranges. Figure 3b The figures show the average maximum reflection wavelength and corresponding optical color of the photonic crystal hydrogel used in Example 1 after treatment with hydrogen peroxide solutions of different concentrations for 45 minutes. It can be observed that the maximum reflection wavelength of the photonic crystal hydrogel changes from 556 nm initially in the untreated hydrogel to 696 nm after treatment with 5 mM hydrogen peroxide. The optical color of the photonic crystal hydrogel, as captured by the camera, also gradually changes from the initial green gel to yellow, then orange, orange-red, and finally red. This indicates that the prepared photonic crystal hydrogel exhibits different maximum reflection wavelengths and optical colors under different concentrations of hydrogen peroxide solution, and that the optical color varies within different ranges.

[0055] The application of Example 1 simply requires placing the photonic crystal hydrogel sensor in the test solution for 45 minutes to determine the initial concentration range of hydrogen peroxide by color, demonstrating its advantages of simple operation and short detection time. Since the color can be observed with the naked eye (orange-red or deeper red at concentrations above 0.4 mmol / L), it meets the purpose of visual detection, eliminating the need for instruments such as UV spectrophotometers or laser pointers. This makes it suitable for routine home testing by patients without requiring a trip to the hospital for blood tests. Furthermore, the sensor prepared in this example can be mounted on a 76×25 mm glass slide, demonstrating its portability. It can also be discarded directly after testing, minimizing sensor contamination issues caused by repeated measurements.

[0056] The photonic crystal hydrogel prepared in Chinese invention patent CN113956388B requires detection of hydrogen peroxide volume fraction at a high concentration. In comparison, the accuracy of this embodiment is higher, and it can respond to a lower range of hydrogen peroxide concentrations. The detection of hydrogen peroxide concentration in Example 1 is also the basis for the ability of subsequent embodiments to respond to low concentrations of uric acid.

[0057] Example 2

[0058] The preparation method of photonic crystal hydrogel for visualizing uric acid detection includes the following steps.

[0059] (1) Select 6mg uricase (produced from Protosporum protosporum, with high activity) ) as a recognition monomer, 75mg horseradish peroxidase (HRP, activity) Using 0.17 g bovine serum albumin (BSA) as a swelling functional monomer and 0.17 g bovine serum albumin (BSA) as a gelling backbone monomer and a carrier for maintaining enzyme activity, the above substances were dissolved in 1 mL of 0.1 M PBS phosphate buffer to form a composite hydrogel matrix. 5 wt% glutaraldehyde was added as a cross-linking agent, with a volume ratio of 5 wt% glutaraldehyde to composite hydrogel matrix solution of 1:3 (v:v). The mixed solution was vortexed at 3000 rpm for 10 s to obtain a homogenized hydrogel prepolymer.

[0060] (2) Monodisperse polystyrene microspheres were prepared using a soap-free emulsion method with styrene, methacrylic acid, and ammonium persulfate as an initiator. The monodisperse polystyrene microspheres were dispersed in deionized water to prepare an 8% polystyrene dispersion. Then, 200 ml of the dispersion was taken... Polystyrene dispersion was dropped onto a glass slide that had been hydrophilically treated with a saturated sodium hydroxide solution in isopropanol and spread evenly. After the dispersion dried, polystyrene microspheres formed a three-dimensional photonic crystal template on the glass slide. Another glass slide was then placed over the prepared photonic crystal template to assemble a sandwich structure. The hydrogel prepolymer prepared in step 1 was pipetted into the cavities between the sandwich structure layers. The hydrogel prepolymer gradually filled the entire cavity between the sandwich layers under capillary action. After introducing the prepolymer, it was allowed to spread evenly throughout the entire interlayer structure without leaving any air bubbles, and then fully crosslinked at 30°C for 6 hours.

[0061] (3) Remove the glass slide on the surface of the photonic crystal hydrogel prepared in step 2, place the photonic crystal hydrogel in 0.01M PBS buffer for 24h to remove excess prepolymer and dry at 25℃ to obtain the photonic crystal hydrogel for visual uric acid detection.

[0062] Uric acid solutions of concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM were prepared by diluting the uric acid solution with 0.01 M PBS buffer, and the pH value was controlled at 7-8. The photonic crystal hydrogel prepared in step (3) was placed in the solution for 45 min, then removed, and its optical color was observed and corresponding optical photographs were taken using UV-Vis technology. The maximum reflected wavelength of the hydrogel sensor was measured using a visspectrophotometer (Hitachi U-3900). Specific test results are shown in Table 2. The descriptions of the colors displayed in the images in the table are approximate and do not strictly adhere to color naming conventions.

[0063] Table 2

[0064]

[0065] Figure 4 The image shows a scanning electron microscope (SEM) image of the photonic crystal hydrogel for visualizing uric acid detection prepared in Example 2. The image reveals that the gaps in the photonic crystal template are filled with hydrogel. This is the structural basis for the change in structural color of the photonic crystal hydrogel sensor due to the change in lattice spacing caused by hydrogel swelling.

[0066] Figure 5The relationship between the maximum reflection wavelength and uric acid concentration of the photonic crystal hydrogel for visual uric acid detection prepared in Example 2 in uric acid solutions of different concentrations is shown in the figure. It can be observed from the figure that after treatment in different uric acid solutions for 45 minutes, the maximum reflection wavelength of the photonic crystal hydrogel for visual uric acid detection increases with increasing uric acid concentration, corresponding to the corresponding color change. Within the range of 0-1 mM (0 mM - yellow-orange, 0.2 mM - orange, 0.4 mM - orange-red, 0.6 mM - red, 0.8 mM - light red, 1 mM - pink), the prepared visual uric acid sensor exhibits good color change. This demonstrates that it achieves the purpose of visual detection by observing color changes with the naked eye. Simply placing the photonic crystal hydrogel in the test solution for 45 minutes allows the concentration range to be determined by color change, showcasing its advantages of simple operation and short detection time. Because the color can be observed with the naked eye (the color is orange-red or a deeper red when the uric acid level is above the critical level of 0.4 mmol / L), it meets the purpose of visual detection, eliminating the need for instruments such as ultraviolet spectrophotometers or laser pointers. This makes it suitable for daily home testing by patients without requiring a trip to the hospital for blood tests. Furthermore, the sensor prepared in this embodiment can be mounted on a 76×25mm glass slide, demonstrating its portability. It can also be discarded directly after testing, minimizing the risk of sensor contamination from repeated measurements.

[0067] Chinese patent CN111999275B provides a method for rapid quantitative determination of pH and / or uric acid; however, its detection of uric acid still requires fluorescence measurement by a fluorescence molecular instrument, which is an additional instrument required compared to Example 2.

[0068] Figure 6 The images (a, b) show scanning electron microscope (SEM) images of the photonic crystal hydrogel for visual uric acid detection prepared in Example 2, obtained after treatment with uric acid solutions of concentrations 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1 mM for 45 min, followed by re-drying. The images show that the higher the concentration of uric acid solution used for treatment, the more tightly the interparticle spaces are filled after drying. This is because a greater degree of swelling in the gel leads to greater compression of the gel particles after drying and shrinkage. Therefore, the SEM images demonstrate that when the photonic crystal hydrogel for visual uric acid detection is placed in the uric acid solution to be tested, the degree of swelling of the hydrogel increases with increasing uric acid concentration. This is the microscopic basis for visual uric acid detection.

[0069] Example 3

[0070] The preparation method of photonic crystal hydrogel for visualizing uric acid detection includes the following steps.

[0071] (1) Select 6mg uricase (produced from Protosporum protosporum, with high activity) ) as a recognition monomer, 75mg horseradish peroxidase (HRP, activity) Using 0.2 g bovine serum albumin (BSA) as a swelling functional monomer and 0.2 g bovine serum albumin (BSA) as a gelling backbone monomer and a carrier for maintaining enzyme activity, the above substances were dissolved in 1 mL of 0.1 M PBS phosphate buffer to form a composite hydrogel matrix. 5 wt% glutaraldehyde was added as a cross-linking agent, with a volume ratio of 5 wt% glutaraldehyde to composite hydrogel matrix solution of 1:3 (v:v). The mixed solution was vortexed at 3000 rpm for 10 s to obtain a homogenized hydrogel prepolymer.

[0072] (2) Monodisperse polystyrene microspheres were prepared using a soap-free emulsion method with styrene, methacrylic acid, and ammonium persulfate as an initiator. The monodisperse polystyrene microspheres were dispersed in deionized water to prepare a 10% polystyrene dispersion. Then, 200 ml of the dispersion was taken... A polystyrene dispersion was dropped onto a glass slide that had been hydrophilically treated with a saturated sodium hydroxide solution in isopropanol and spread evenly. After the dispersion dried, polystyrene microspheres formed a three-dimensional photonic crystal template on the glass slide. Another glass slide was then placed over the prepared photonic crystal template to assemble a sandwich structure. The hydrogel prepolymer prepared in step 1 was pipetted into the cavities between the sandwich structure layers. The hydrogel prepolymer gradually filled the entire cavity between the sandwich layers under capillary action. After introducing the prepolymer, it was allowed to spread evenly throughout the entire interlayer structure without leaving any air bubbles, and then fully crosslinked at 25°C for 5 hours.

[0073] (3) Remove the glass slide on the surface of the photonic crystal hydrogel prepared in step 2, place the photonic crystal hydrogel in 0.01M PBS buffer and wash for 12h to remove excess prepolymer and dry at 25°C to obtain the photonic crystal hydrogel for visual uric acid detection.

[0074] Uric acid solutions of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM concentrations were prepared by diluting the uric acid solution with 0.01 M PBS buffer, and the pH value was controlled at 7-8. The photonic crystal hydrogel prepared in step 3 was then immersed in the solution for 15, 30, 45, 60, 75, and 90 min respectively, and then removed. The optical color was observed and corresponding optical photographs were taken using UV-Vis technology. The visspectrophotometer (Hitachi U-3900) measures the maximum reflected wavelength of its hydrogel sensor.

[0075] Figure 7The image shows the maximum reflection wavelength of the photonic crystal hydrogel for visual uric acid detection prepared in Example 3 after different treatment times. It can be observed that in this example, the maximum reflection wavelength of the photonic crystal hydrogel sensor for visual uric acid detection gradually increases with increasing treatment time at the same uric acid concentration. Furthermore, within the 0-1 mM uric acid concentration range, the maximum reflection wavelength tends to stabilize within 90 minutes, meaning it can be used to measure uric acid content. This demonstrates the advantages of the photonic crystal hydrogel for visual uric acid detection: simple operation and short detection time.

[0076] Example 4

[0077] A method for preparing a photonic crystal hydrogel for visualizing uric acid detection includes the following steps:

[0078] (1) 75mg horseradish peroxidase (HRP, active) was selected. Using 0.15 g bovine serum albumin (BSA) as a swelling functional monomer and 0.15 g bovine serum albumin (BSA) as a gelling backbone monomer and a carrier for maintaining enzyme activity, the above substances were dissolved in 1 mL of 0.1 M PBS phosphate buffer to form a composite hydrogel matrix. 5 wt% glutaraldehyde was added as a cross-linking agent, with a volume ratio of 5 wt% glutaraldehyde to composite hydrogel matrix solution of 1:4 (v:v). The mixed solution was vortexed at 3000 rpm for 10 s to obtain a homogenized hydrogel prepolymer.

[0079] (2) Monodisperse polystyrene microspheres were prepared using a soap-free emulsion method with styrene, methacrylic acid, and ammonium persulfate as an initiator. The monodisperse polystyrene microspheres were dispersed in deionized water to prepare a 10% polystyrene dispersion. Then, 200 ml of the dispersion was taken... A polystyrene dispersion was dropped onto a glass slide that had been hydrophilically treated with a saturated sodium hydroxide solution in isopropanol and spread evenly. After the dispersion dried, polystyrene microspheres formed a three-dimensional photonic crystal template on the glass slide. Another glass slide was then placed over the prepared photonic crystal template to assemble a sandwich structure. The hydrogel prepolymer prepared in step 1 was pipetted into the cavities between the sandwich structure layers. The hydrogel prepolymer gradually filled the entire cavity between the sandwich layers under capillary action. After introducing the prepolymer, it was allowed to spread evenly throughout the entire interlayer structure without leaving any air bubbles, and then fully crosslinked at 25°C for 5 hours.

[0080] (3) Remove the glass slide on the surface of the photonic crystal hydrogel prepared in step 2, place the photonic crystal hydrogel in 0.01M PBS buffer and wash for 12h to remove excess prepolymer and dry at 30°C to obtain the photonic crystal hydrogel for visual hydrogen peroxide detection.

[0081] Hydrogen peroxide solutions with concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM were prepared by diluting hydrogen peroxide solution with 0.01 M PBS buffer. Similarly, uric acid solutions with concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM were prepared by diluting uric acid solution, maintaining the pH at 7-8. The photonic crystal hydrogel prepared in step 3 was then immersed in the solutions for 45 min, and its optical color was observed and corresponding optical photographs were taken using UV-Vis technology. The Vis spectrophotometer (Hitachi U-3900) measures the maximum reflected wavelength of its hydrogel sensor.

[0082] Figure 8 This is a comparison graph showing the relationship between the average maximum reflected wavelength and uric acid concentration after treatment in different uric acid concentrations for 45 minutes in Example 2 and the relationship between the average maximum reflected wavelength and solution concentration after treatment in different concentrations of uric acid solution or hydrogen peroxide solution for 45 minutes in Example 4. The graph shows that, comparing the maximum reflected wavelengths of Examples 2 and 4 at different uric acid or hydrogen peroxide concentrations, the photonic crystal hydrogel prepared in Example 4, without the addition of a uric acid recognition monomer, exhibits a gradient-like reflected wavelength in hydrogen peroxide solution, but not a significant gradient-like reflected wavelength in uric acid solution. This indicates that it does not have the function of visualizing the uric acid response. It can be concluded that the uric acid recognition monomer is the core response material of the photonic crystal hydrogel for visualizing uric acid detection. Adding the uric acid recognition monomer prevents the visualization of the uric acid response, while the swelling-functional monomer can exert its swelling response function in hydrogen peroxide.

[0083] Example 5

[0084] A method for preparing a photonic crystal hydrogel for visualizing uric acid detection includes the following steps:

[0085] (1) 3mg uricase (produced from Candida utilis, active) was selected. ) as a recognition monomer, 50 mg horseradish peroxidase (HRP, activity) Using 0.2 g bovine serum albumin (BSA) as a swelling functional monomer and 0.2 g bovine serum albumin (BSA) as a gelling backbone monomer and a carrier for maintaining enzyme activity, the above substances were dissolved in 1 mL of 0.1 M PBS phosphate buffer to form a composite hydrogel matrix. 5 wt% glutaraldehyde was added as a cross-linking agent, with a volume ratio of 5 wt% glutaraldehyde to composite hydrogel matrix solution of 1:3 (v:v). The mixed solution was vortexed at 3000 rpm for 10 s to obtain a homogenized hydrogel prepolymer.

[0086] (2) Monodisperse silica microspheres were prepared using the Stobber method. The monodisperse silica microspheres were dispersed in deionized water to prepare a 10% silica dispersion. Then, 200 ml of the dispersion was taken... A silica dispersion was dropped onto a glass slide that had been hydrophilically treated with a saturated sodium hydroxide solution in isopropanol and spread evenly. After the dispersion dried, silica microspheres formed a three-dimensional photonic crystal template on the slide. Another glass slide was then placed over the prepared photonic crystal template to assemble a sandwich structure. The hydrogel prepolymer prepared in step 1 was pipetted into the cavities between the sandwich structure layers. The hydrogel prepolymer gradually filled the entire cavity between the sandwich layers under capillary action. After introducing the prepolymer, it was allowed to spread evenly throughout the entire interlayer structure without leaving any air bubbles, and then fully crosslinked at 25°C for 5 hours.

[0087] (3) Remove the glass slide on the surface of the photonic crystal hydrogel prepared in step 2, place the photonic crystal hydrogel in 0.01M PBS buffer for 24h to remove excess prepolymer and dry at 25℃ to obtain the photonic crystal hydrogel for visual uric acid detection.

[0088] Uric acid solutions of 0 mM, 0.4 mM, and 0.8 mM concentrations were prepared by diluting the uric acid solution with 0.01 M PBS buffer, and the pH value was controlled at 7-8. The photonic crystal hydrogel prepared in step 3 was placed in the solution for 45 min, then removed and its optical color was observed and corresponding optical photographs were taken using UV-Vis technology. The vis spectrophotometer (Hitachi U-3900) measures the maximum reflected wavelength of its hydrogel sensor.

[0089] Appendix Figure 9 This is a comparison of the maximum reflection wavelengths after treatment with uric acid solutions of concentrations 0 mM, 0.4 mM, and 0.8 mM for 45 min, as shown in Example 5 of the present invention. As can be observed from the figure, the position of the maximum reflection peak of the photonic crystal hydrogel shifted from 595 nm at 0 mM to 616 nm (0.4 mM) and 631 nm (0.8 mM). The color at equilibrium changed from yellow to orange and then to orange-red, thus achieving a visual response detection of uric acid.

[0090] Example 6

[0091] A method for preparing a photonic crystal hydrogel for visualizing uric acid detection includes the following steps:

[0092] (1) Select 6mg uricase (produced from Escherichia coli, with high activity) ) as a recognition monomer, 70mg horseradish peroxidase (HRP, activity) Using 0.2 g bovine serum albumin (BSA) as a swelling functional monomer and 0.2 g bovine serum albumin (BSA) as a gelling backbone monomer and a carrier for maintaining enzyme activity, the above substances were dissolved in 1 mL of 0.1 M PBS phosphate buffer to form a composite hydrogel matrix. 5 wt% glutaraldehyde was added as a cross-linking agent, with a volume ratio of 5 wt% glutaraldehyde to composite hydrogel matrix solution of 1:3 (v:v). The mixed solution was vortexed at 3000 rpm for 10 s to obtain a homogenized hydrogel prepolymer.

[0093] (2) Monodisperse silica microspheres were prepared using the Stober method. The monodisperse silica microspheres were dispersed in deionized water to prepare an 8 wt% silica dispersion. Then, 200 ml of the dispersion was taken... A silica dispersion was dropped onto a glass slide that had been hydrophilically treated with a saturated sodium hydroxide solution in isopropanol and spread evenly. After the dispersion dried, silica microspheres formed a three-dimensional photonic crystal template on the slide. Another glass slide was then placed over the prepared photonic crystal template to assemble a sandwich structure. The hydrogel prepolymer prepared in step 1 was pipetted into the cavities between the sandwich structure layers. The hydrogel prepolymer gradually filled the entire cavity between the sandwich layers under capillary action. After introducing the prepolymer, it was allowed to spread evenly throughout the entire interlayer structure without leaving any air bubbles, and then fully crosslinked at 25°C for 5 hours.

[0094] (3) Remove the glass slide on the surface of the photonic crystal hydrogel prepared in step 2, place the photonic crystal hydrogel in 0.01M PBS buffer for 24h to remove excess prepolymer and dry at 25℃ to obtain the photonic crystal hydrogel for visual uric acid detection.

[0095] Uric acid solutions of concentrations 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM were prepared by diluting the uric acid solution with 0.01 M PBS buffer, and the pH value was controlled at 7-8. The photonic crystal hydrogel prepared in step 3 was placed in the solution for 45 min, then removed, and its optical color was observed and corresponding optical photographs were taken using UV-Vis technology. The Vis spectrophotometer (Hitachi U-3900) measures the maximum reflected wavelength of its hydrogel sensor.

[0096] Figure 10This is a comparison of the maximum reflection wavelengths after treatment with uric acid solutions of concentrations 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1 mM for 45 min, as shown in Example 6 of the present invention. The graph shows that the position of the maximum reflection peak of the photonic crystal hydrogel shifted from 608 nm at 0 mM to 621 nm (0.2 mM), 630 nm (0.4 mM), 640 nm (0.6 mM), 655 nm (0.8 mM), and 674 nm (1 mM). The color at equilibrium changed from orange to orange-red, red, light red, and pink, thus achieving a visual response detection of uric acid.

[0097] It should be noted that the present invention is not limited to the above embodiments. Various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention, and all such changes and modifications fall within the scope of protection of the present invention as claimed. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for preparing a photonic crystal hydrogel for visualizing uric acid detection, characterized in that... Includes the following steps: (1) The uric acid recognition monomer, swelling functional monomer, and gelling backbone monomer were dissolved in PBS phosphate buffer solution to form a composite hydrogel matrix. Glutaraldehyde aqueous solution was added, and the mixture was vortexed for 10 seconds at 3000 rpm to obtain a homogeneous hydrogel prepolymer. The uric acid recognition monomer was uricase produced by *Protozoa protozoa* with an activity ≥20 u / mg; the swelling functional monomer was horseradish peroxidase with an activity ≥300 u / mg; and the gelling backbone monomer was bovine serum albumin. The composite hydrogel matrix contained 6 mg / mL of uric acid recognition monomer, 75 mg / mL of swelling functional monomer, and 0.17 g / mL of gelling backbone monomer. The glutaraldehyde aqueous solution had a mass fraction of 5%, and the volume ratio of glutaraldehyde solution to the composite hydrogel matrix was 1:

3. (2) The hydrogel prepolymer was uniformly filled into a uniform hierarchical structure formed by monodisperse polystyrene microspheres with a particle size of 150nm-300nm, and crosslinked at 30℃ for 6h. (3) The cross-linked uniform hierarchical structure was washed and dried at 25°C to obtain a photonic crystal hydrogel for visual uric acid detection; The photonic crystal hydrogel was placed in the test solution for 45 minutes, and the color of the photonic crystal hydrogel was observed. The concentration of uric acid in the test solution was determined based on the color. The uric acid concentration was yellow-orange at 0 mM, orange at 0.2 mM, orange-red at 0.4 mM, red at 0.6 mM, light red at 0.8 mM, and pink at 1 mM.

2. The method for preparing the photonic crystal hydrogel for visualizing uric acid detection according to claim 1, characterized in that: The uniform hierarchical structure formed by uniformly filling the hydrogel prepolymer liquid into monodisperse polystyrene microspheres with a particle size of 150nm-300nm is achieved by vertical deposition or drop-coating self-assembly.

3. The method for preparing the photonic crystal hydrogel for visualizing uric acid detection according to claim 2, characterized in that: The drop-coating self-assembly method involves adding monodisperse microspheres to a solvent to prepare a dispersion, ultrasonically dispersing them evenly, and then dropping them onto a hydrophilic glass slide to form a photonic crystal template. Another glass slide is then placed on top of the photonic crystal template to form a sandwich structure of glass slide-photonic crystal-glass slide.

4. The method for preparing the photonic crystal hydrogel for visualizing uric acid detection according to claim 3, characterized in that: The hydrophilic treatment involves treating the glass slide with a piranha solution or a saturated sodium hydroxide isopropanol solution; the solvent is water.

5. The method for preparing the photonic crystal hydrogel for visualizing uric acid detection according to claim 1, characterized in that: The PBS phosphate buffer solution is prepared from disodium hydrogen phosphate and potassium dihydrogen phosphate, wherein the molar ratio of disodium hydrogen phosphate to potassium dihydrogen phosphate is 3:1.5-3:4.5, the concentration is 0.01M-1M, and the pH is 7-8.

6. The method for preparing the photonic crystal hydrogel for visualizing uric acid detection according to claim 1, characterized in that: The washing process refers to washing the photonic crystal hydrogel in water or PBS phosphate buffer solution for 12-24 hours after the hydrogel has been cross-linked.

Images