A DAAO / HRP@MAF-7 hydrogel and its preparation method and application

Abstract

The present invention belongs to the field of biosensor instant detection technology, and relates to a DAAO / HRP@MAF-7 hydrogel and its preparation method and application. The hydrogel is formed by immobilizing horseradish peroxidase HRP and D-amino acid oxidase DAAO in a hydrophilic metal organic framework MAF-7, and then the prepared DAAO / HRP@MAF-7 biocomposite material and the color developer TMB are co-loaded in a PEG-DA hydrogel. The DAAO / HRP@MAF-7 hydrogel can be used as a biosensor to detect D-amino acids.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensor instant detection and relates to a DAAO / HRP@MAF-7 hydrogel and a preparation method and application thereof. Background Art

[0002] The detection of D-amino acids (D-AAs) has important value that cannot be ignored in many disciplines such as medicine, biology, food science and pharmaceutical engineering. In the field of disease diagnosis, D-AAs presents specific concentration changes in the course of various diseases such as gastric cancer. This change is closely related to the occurrence, development and prognosis of the disease, making it a highly potential biomarker, providing a key basis for early accurate diagnosis of the disease, dynamic monitoring of the disease and in-depth exploration of the pathogenesis. In addition, D-AAs participates in many physiological processes, such as neurotransmission and hormone synthesis. The fluctuation of its level can be used as a sensitive indicator of pathological state, which helps to reveal the inherent laws of physiological and pathological changes in the body. The content of D-AAs is also related to the quality characteristics of food, the production and processing process, and the microbial contamination status. It can be used as an important reference indicator for evaluating food quality and safety, and provides strong support for food quality control and safety supervision. In biopharmaceutical research, D-AAs is an important component or precursor of drug synthesis. The accurate detection of its concentration and purity is a key link to ensure the quality and efficacy of drug research and development, and also provides important clues for the development of new drugs and the study of their mechanism of action.

[0003] At present, a variety of effective technologies have been developed for the detection of D-AAs, such as liquid chromatography (LC), gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS) and capillary electrophoresis (CE). However, these methods are often severely limited by the complex pretreatment process, long time consumption, high reagent cost, bulky equipment and other problems. Optical sensing methods have become a powerful tool for chiral analysis. At present, many chiral optical sensors based on organic small molecules have been designed and used for AAs enantiomer analysis, but they have problems such as narrow substrate range and limited enantiomer distinguishability. Although these problems have been solved by many chiral fluorescence strategies based on nanomaterials, the complex synthesis process, harsh synthesis conditions and unclear recognition mechanism still limit their application. Therefore, it is very challenging to synthesize high-performance optical sensors for D-AAs detection under mild conditions.

[0004] D-amino acid oxidase (DAAO) is an enantioselective flavin enzyme that can specifically catalyze the stereospecific oxidative deamination of D-AAs to generate α-keto acids and hydrogen peroxide (H2O2). Therefore, DAAO is considered to be a powerful tool for D-AAs analysis. At the same time, related studies have shown that horseradish peroxidase (HRP) is a very significant signal transduction material that can effectively catalyze the colorless and non-fluorescent substrate TMB to form blue-green oxTMB in the presence of H2O2. Since one enzyme can continuously trigger the activation of another enzyme to generate a signal, the cascade catalytic reaction of the two enzymes can be used to achieve signal amplification. Therefore, it is attractive and reliable to combine DAAO with HRP to form a cascade catalytic bioreactor for effective D-AAs detection. However, the limited mass transfer efficiency between substrates and free enzymes, the inherent fragility and non-recyclability of enzymes have greatly hindered the practical application of bioreactors. Enzyme immobilization is an effective method to overcome the above shortcomings. In particular, when multiple enzymes are confined in the nanoscale range, the favorable distance between the cascade enzymes will increase the diffusion rate of reaction intermediates from one enzyme to another. Therefore, the cascade catalytic efficiency of multi-enzymes will be greatly improved. However, the research on constructing multi-enzyme immobilized bioreactors for chiral analysis has received only limited attention. Zhou et al. proposed that mesoporous hydrogen-bonded organic frameworks (MHOFs) can be used as scaffolds to realize the biocatalytic cascade reaction of DAAO and HRP to prepare MHOF-based nanoreactors for enantiospecific and visual detection of D-AAs in saliva (doi.org / 10.1016 / j.snb.2024.136716), but the problem of achieving instant and efficient detection of D-AAs in complex samples containing a large number of interfering compounds has not been solved. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a DAAO / HRP@MAF-7 hydrogel and a preparation method and application thereof.

[0006] The present invention is achieved through the following technical solutions:

[0007] A DAAO / HRP@MAF-7 hydrogel is formed by immobilizing DAAO and HRP in a hydrophilic metal organic framework MAF-7, and then co-loading the prepared DAAO / HRP@MAF-7 biocomposite with a color developer TMB in a PEG-DA hydrogel.

[0008] A method for preparing DAAO / HRP@MAF-7 hydrogel comprises the following steps:

[0009] (1) Synthesis of DAAO / HRP@MAF-7 biocomposites

[0010] Horseradish peroxidase (HRP) and D-amino acid oxidase (DAAO) were added to the Zn(NO3)2 solution, mixed evenly, and then mixed with 3-methyl-1,2,4-triazole (Hmtz) aqueous solution, stirred at room temperature for reaction, and the precipitate was collected by centrifugation, washed with deionized water for multiple times, and freeze-dried to obtain the DAAO / HRP@MAF-7 biocomposite material;

[0011] (2) Preparation of hydrogel

[0012] PEG-DA, synthesized DAAO / HRP@MAF-7 biocomposite and 200 μL 5 mM TMB ethanol solution were dissolved in 4 mL, pH = 7, 20 mM phosphate buffer (PBS) to obtain a precursor solution, 200 μL of photoinitiator 651 with a mass concentration of 10% w / w and 1-vinyl-2-pyrrolidone as solvent were added, and the DAAO / HRP@MAF-7 hydrogel was obtained by polymerization under ultraviolet light.

[0013] Furthermore, in step (1), the amount of Zn(NO3)2 and 3-methyl-1,2,4-triazole is 1:(4-10), preferably 1:6, to avoid instability or defects in the MOF structure caused by insufficient coordination.

[0014] Furthermore, in step (1), the stirring reaction time is 4-24 hours, preferably 20 hours.

[0015] Furthermore, the mass ratio of horseradish peroxidase (HRP) and D-amino acid oxidase (DAAO) is 1: (4-0.25); the concentration of horseradish peroxidase is 1.4 mg / mL, and the concentration of D-amino acid oxidase is 0.35 mg / mL. Preferably, it is 1:4. During the oxidation of D-amino acid, DAAO oxidizes D-amino acid to generate hydrogen peroxide, while HRP catalyzes the oxidation reaction of hydrogen peroxide (such as color development reaction). By setting the concentration of HRP to 4 times that of DAAO, it is ensured that hydrogen peroxide can be consumed quickly to avoid its accumulation. Hydrogen peroxide is a strong oxidant, which may inhibit the activity of the enzyme at high concentrations. A higher concentration of HRP can quickly consume hydrogen peroxide and protect the enzyme activity in the system. HRP is a key catalyst for the color development reaction, and its high concentration (1.4 mg / mL) ensures a rapid oxidation reaction of the color developer (such as TMB), thereby improving the sensitivity and signal intensity of the detection.

[0016] Furthermore, in step (2), the mass ratio of PEG-DA to DAAO / HRP@MAF-7 biocomposite is 8:1. The high ratio of PEG-DA helps to evenly disperse the DAAO / HRP@MAF-7 biocomposite, avoiding the reduction of catalytic efficiency due to excessive local concentration of the enzyme complex. PEG-DA plays a mechanical supporting role in the hydrogel, which can provide a stable three-dimensional network environment for DAAO / HRP@MAF-7 and prevent the loss or inactivation of the composite material.

[0017] Furthermore, the molecular weight of the PEG-DA is 400. PEG-DA with a molecular weight of 400 has a lower viscosity in liquid state, is easy to dissolve, mix and evenly coat, is easier to react with an initiator, has a faster curing speed, and can improve the efficiency of material preparation. A higher cross-linking density can be formed to generate a stronger and denser hydrogel structure with smaller and more uniform pores, which is suitable for controlling molecular diffusion. The hydrogel network generated by low molecular weight PEG-DA can maintain a higher hydration capacity due to tighter cross-linking, which is conducive to providing a water environment suitable for enzymes or active substances, thereby improving their catalytic activity and stability. It exhibits stronger acid and alkali resistance, solvent resistance and mechanical stability, and can be used for a long time in a complex environment. The dense network structure formed by PEG-DA with a molecular weight of 400 helps to immobilize enzymes (such as DAAO and HRP) or other bioactive molecules, reduce their loss, and improve the stability and service life of the composite material.

[0018] Furthermore, the conditions for photopolymerization are: irradiation with 365 nm ultraviolet light, an irradiance of 20 mW / cm², and polymerization for 5 minutes.

[0019] The present invention also provides the use of the DAAO / HRP@MAF-7 hydrogel as a biosensor in detecting D-amino acids.

[0020] Beneficial technical effects of the present invention:

[0021] (1) DAAO- has high specificity and can specifically catalyze D-AAs, but has no effect on L-AAs. The present invention utilizes DAAO (D-amino acid oxidase) to oxidize D-amino acids to generate α-keto acids and hydrogen peroxide. HRP (horseradish peroxidase) further utilizes the product hydrogen peroxide to catalyze the oxidation reaction of the color developer TMB. The two enzymes work synergistically to make the entire catalytic process fast and efficient, and can instantly convert the intermediate product hydrogen peroxide of the reaction, making the color development reaction usually very sensitive, and convenient for detecting the target substrate concentration through color changes. Therefore, the combination of DAAO and HRP can achieve highly selective and highly sensitive detection and analysis.

[0022] (2) DAAO and HRP were fixed in the MAF-7 framework by in situ synthesis, taking advantage of the high specific surface area, hydrophilicity, porous structure and easy functionalization of MAF-7 to improve the stability of the enzyme and the synergistic catalytic efficiency. The specific performance is as follows:

[0023] The synthesis conditions of MAF-7 of the present invention are mild. MAF-7 is a hydrophilic metal organic framework (MOF). The hydrophilicity of MAF-7 helps to maintain the hydration environment required by the enzyme and avoid the reduction of activity due to dehydration of the enzyme. In addition, MAF-7 has a high specific surface area and uniform porosity, which can provide sufficient space for enzyme molecules to perform physical adsorption or chemical binding. The porous structure of MAF-7 is conducive to the diffusion of substrates and products, and enhances the efficiency of enzyme catalytic reactions.

[0024] The immobilized enzyme molecules are fixed in the framework of MAF-7, which can significantly reduce the loss of enzymes in the flow system and increase the service life of the enzymes. In addition, the enzymes immobilized on MAF-7 have stronger thermal stability and resistance to denaturation, such as resistance to pH changes and organic solvents.

[0025] The porous structure of MAF-7 allows DAAO and HRP molecules to be fixed in close proximity, thereby enhancing the synergistic effect between the two enzymes. Hydrogen peroxide, as a product of DAAO, can be quickly utilized by the adjacent HRP, reducing byproduct diffusion and loss and improving the overall reaction efficiency. The rapid conversion of hydrogen peroxide reduces its cumulative concentration in the system, avoiding its potential inhibitory effect on the enzyme, while improving the stability of the reaction.

[0026] The porous structure and high permeability of MAF-7 ensure that substrates and products can efficiently enter and leave the position of the immobilized enzyme. This feature greatly improves the dynamic performance of the immobilized enzyme catalytic system, making it suitable for continuous reactions and high-throughput detection.

[0027] By in situ synthesis, DAAO and HRP are fixed in the MAF-7 framework, which can be further embedded in hydrogels such as PEG-DA to form a composite material. The solid structure of MAF-7 can enhance the mechanical stability of the hydrogel and make the active area of ​​the enzyme more concentrated, thereby improving the catalytic performance and detection sensitivity of the composite material.

[0028] (3) The hydrogel formed by PEG-DA (polyethylene glycol diacrylate) is highly hydrophilic and provides an ideal reaction environment for immobilized enzymes (such as DAAO and HRP) or other biomolecules, improving their activity and stability. PEG-DA-derived hydrogels have good chemical stability in a variety of environments and are not easily degraded or dissolved. The integrated catalytic or functional materials can maintain their activity under the protection of PEG-DA and are suitable for long-term stable operating environments. The good adhesion of PEG-DA hydrogels enables them to be integrated with other devices, improving the stability of detection sensors.

[0029] (4) The present invention combines porous hydrogel and MAF-7 to not only protect the catalytic activity of DAAO and HRP in complex media, but also activate the dual enzyme cascade catalysis of DAAO and HRP in the presence of D-AAs substrate. PEG-DA can prevent macromolecules or cells in complex components from entering the composite hydrogel matrix without affecting the diffusion of amino acid small molecules, thereby realizing the detection of D-AAs in complex samples and improving the sensitivity and specificity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the result of the optimization experiment of the mass ratio of DAAO / HRP;

[0031] Figure 2 This is the SEM image of the DAAO / HRP@MAF-7 biocomposite prepared in Example 1;

[0032] Figure 3 This is the SEM image of the DAAO / HRP@MAF-7 biocomposite prepared in Example 2;

[0033] Figure 4 This is the SEM image of the DAAO / HRP@MAF-7 biocomposite prepared in Example 3;

[0034] Figure 5 This is the SEM image of the DAAO / HRP@MAF-7 biocomposite prepared in Example 4;

[0035] Figure 6 TEM image of the DAAO / HRP@MAF-7 biocomposite prepared in Example 1;

[0036] Figure 7 Energy dispersive spectra (EDS) of the DAAO / HRP@MAF-7 biocomposite prepared in Example 1;

[0037] Figure 8 The encapsulation efficiency results of Example 1 and Examples 5-9 are shown in FIG.

[0038] Fig. 9 Schematic diagram of the preparation of the hydrogel in Example 10;

[0039] Fig.10 This is the SEM characterization image of the DAAO / HRP@MAF-7 hydrogel prepared in Example 10;

[0040] Fig.11 Diagram of the detection process of functionalized hydrogel;

[0041] Fig.12 Schematic diagram of D-AAs detection;

[0042] Fig.13 The signal response diagram of DAAO / HRP@MAF-7 hydrogel prepared in Example 10 to 14 common AAs;

[0043] Fig.14 This is a diagram showing the optimization of pH conditions for D-Ala detection of the DAAO / HRP@MAF-7 hydrogel prepared in Example 10;

[0044] Fig.15 This is a diagram showing the optimized reaction time conditions for D-Ala detection of the DAAO / HRP@MAF-7 hydrogel prepared in Example 10;

[0045] Fig.16 This is the standard curve diagram for D-Ala detection;

[0046] Fig.17 This is a stability test diagram for detecting D-Ala in functionalized hydrogel. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with examples, but the present invention is not limited to the following examples.

[0048] The levels of D-alanine (D-Ala) are significantly increased in the biological fluids of gastric cancer patients, suggesting that they may be potential biomarkers for cancer. Therefore, the experiment used D-Ala as the target molecule and TMB as the chromogenic substrate. The UV absorption intensity of the product oxTMB at 652nm was used as a signal to optimize the addition ratio of D-amino acid oxidase (DAAO) / horseradish peroxidase (HRP). The mass ratios of DAAO / HRP were selected as 1:4, 2:3, 3:2, and 4:1. The results are shown in the figure below. Figure 1 As shown, it can be seen that when the mass ratio of DAAO / HRP is 1:4, the absorbance of the reaction system is the highest and the HRP concentration is high, which can ensure that hydrogen peroxide can be consumed quickly. Therefore, in the subsequent material synthesis experiments, the concentration ratio of DAAO / HRP is selected as 1:4 to prepare DAAO / HRP@MAF-7 biocomposites.

[0049] Example 1 DAAO / HRP@MAF-7 biocomposite material

[0050] Horseradish peroxidase (HRP) (1.4 mg / mL) and D-amino acid oxidase (DAAO) (0.35 mg / mL) were added to 4 mL of 0.05 M Zn(NO3)2 solution. After mixing evenly, the mixture was mixed with 4 mL of 0.3 M 3-methyl-1,2,4-triazole (Hmtz) aqueous solution. After stirring at room temperature for 20 h, the reaction was stopped. After centrifugation at 10000 rpm for 10 min, the precipitate was collected, washed with deionized water several times, and then freeze-dried to obtain the DAAO / HRP@MAF-7 biocomposite material.

[0051] Example 2 DAAO / HRP@MAF-7 biocomposite material

[0052] The difference between Example 2 and Example 1 is that the molar ratio of Zn(NO3)2 to 3-methyl-1,2,4-triazole (Hmtz) is 1:4, and the other conditions remain unchanged.

[0053] Example 3 DAAO / HRP@MAF-7 biocomposite material

[0054] The difference between Example 3 and Example 1 is that the molar ratio of Zn(NO3)2 to 3-methyl-1,2,4-triazole (Hmtz) is 1:8, and the other conditions remain unchanged.

[0055] Example 4 DAAO / HRP@MAF-7 biocomposite material

[0056] The difference between Example 4 and Example 1 is that the molar ratio of Zn(NO3)2 to 3-methyl-1,2,4-triazole (Hmtz) is 1:10, and the other conditions remain unchanged.

[0057] The electron microscopy images of the DAAO / HRP@MAF-7 biocomposite obtained in Example 1-4 are as follows: Figure 2-5 As shown in the figure, it can be seen that with the increase of concentration ratio, the morphology of MAF-7 gradually tends to be a regular octahedron. When the concentration ratio reaches 1:6, the morphology of MAF-7 has no obvious change. DAAO / HRP@MAF-7 crystals show a uniform morphology with an average particle size of about 150nm. 3-Methyl-1,2,4-triazole is alkaline. Considering that the enzyme will be denatured and inactivated under extremely alkaline conditions, it is finally determined that Zn 2+ The molar ratio of 3-methyl-1,2,4-triazole is 1:6.

[0058] DAAO is an enantioselective flavoenzyme, and its non-covalently bound cofactor (flavin adenine dinucleotide (FAD)) contains S and P elements. HRP is a heme protein, and its iron porphyrin contains Fe elements. The DAAO / HRP@MAF-7 biocomposite prepared in Example 1 was tested by energy dispersive spectroscopy (EDS) under TEM, and the results are shown in Figure 6-7 ,In the DAAO / HRP@MAF-7 biocomposite, Zn, Fe, S, and P elements coexist and are distributed relatively evenly, which also proves that the enzyme molecules are successfully encapsulated into MAF-7.

[0059] Example 5

[0060] The difference between Example 5 and Example 1 is that the stirring reaction time is 4 hours, and the other conditions are exactly the same.

[0061] Example 6

[0062] The difference between Example 6 and Example 1 is that the stirring reaction time is 8 hours, and the other conditions are exactly the same.

[0063] Example 7

[0064] The difference between Example 7 and Example 1 is that the stirring reaction time is 12 hours, and the other conditions are exactly the same.

[0065] Example 8

[0066] The difference between Example 8 and Example 1 is that the stirring reaction time is 16 hours, and the other conditions are exactly the same.

[0067] Example 9

[0068] The difference between Example 9 and Example 1 is that the stirring reaction time is 24 hours, and the other conditions are exactly the same.

[0069] The reaction solutions of Examples 1 and 5-9 and the washing solutions washed with deionized water were combined, and the enzyme concentration of the combined solution was determined using a BCA detection kit, and the encapsulation efficiency was calculated. The results obtained are as follows: Figure 8 The encapsulation efficiency of DAAO / HRP increased with the increase of reaction time and stabilized after 20 h. This result indicates that 20 h is sufficient to complete the growth of DAAO / HRP@MAF-7 composites.

[0070] Encapsulation efficiency: The ratio of the amount of DAAO / HRP encapsulated in the DAAO / HRP@MAF-7 biocomposite to the initial amount of DAAO / HRP. The calculation formula is as follows:

[0071]

[0072] inC 1 (mg / mL) is the initial DAAO / HRP concentration, V 1 (mL) is the initial enzyme volume, C 2 (mg / mL) is the enzyme concentration in the combined solution, V 2 (mL) is the volume of the combined solution.

[0073] Example 10 Preparation of hydrogel sensor

[0074] 0.4 g PEG-DA (molecular weight: 400), 50 mg DAAO / HRP@MAF-7 biocomposite material synthesized in Example 1 and 200 μL TMB ethanol solution (5 mM) were dissolved in 4 mL phosphate buffer (PBS, 20 mM, pH = 7) to obtain a precursor solution, and photoinitiator 651 was selected to crosslink and cure the hydrogel. After adding 200 μL photoinitiator 651 (2,2-dimethoxy-2-phenylacetophenone) (10% w / w, solvent is 1-vinyl-2-pyrrolidone) to the precursor solution, an appropriate amount of the mixed solution was loaded onto the lid of a 1.5 mL centrifuge tube, and then irradiated with 365 nm ultraviolet light (20 mW / cm²) for 5 minutes to obtain a DAAO / HRP@MAF-7 hydrogel. The SEM image of the obtained hydrogel is shown in FIG. Fig.10 As shown, it can be seen that the gel has multi-level pores at the micron level, which are expected to reach the nanometer level after swelling with water.

[0075] The schematic diagram of the preparation of the hydrogel in Example 1 of the present invention is as follows Fig. 9 As shown, the prepared functionalized hydrogel was then fixed on the cap of a centrifuge tube, which could be easily operated in the centrifuge tube and reliably quantified by a handheld colorimeter, see Fig.11 .

[0076] The detection principle of this embodiment is as follows Fig.12 As shown in the figure, DAAO can catalyze the oxidation of D-AAs to keto acids and H2O2 under the consumption of oxygen, but has no reaction to its enantiomer L-AAs. The generated H2O2 then uses HRP as a catalyst to rapidly oxidize the substrate TMB to blue-green oxTMB. The acquisition of RGB values ​​by a handheld colorimeter enables efficient and sensitive detection of D-AAs.

[0077] 1 mL of 0.3 mM 14 common AAs solutions were added to the centrifuge tube, and the cap of the centrifuge tube with the hydrogel fixed was fastened. The centrifuge tube was inverted and turned over after 30 minutes of reaction. The RGB analysis of the hydrogel after color development was performed using a portable colorimeter. The color change value (ΔC) was calculated using the Euclidean distance equation.

[0078]

[0079] ΔR, ΔG, and ΔB are the changes in R, G, and B relative to the blank hydrogel, respectively. Fig.13 , indicating that the DAAO / HRP@MAF-7 hydrogel sensor has almost no fluorescence response to 14 common L-AAs. Only D-AAs can be oxidized by DAAO to produce H2O2, and the subsequent H2O2 can quickly oxidize the substrate TMB to blue-green oxTMB under the catalysis of HRP, proving that the sensor is reliable and convenient for chiral recognition of various D-AAs.

[0080] (1) Drawing of the standard curve for D-alanine (D-Ala) detection

[0081] In the experiment, D-Ala was used as the target molecule. 1 mL of D-Ala solution with different concentrations was added to a centrifuge tube, and the cap of the centrifuge tube with the hydrogel fixed was fastened. The centrifuge tube was inverted and turned over after 30 minutes of reaction. The hydrogel was analyzed by RGB using a portable colorimeter, and the color change value (ΔC) was calculated using the Euclidean distance equation.

[0082]

[0083] ΔR, ΔG, and ΔB are the changes in R, G, and B relative to the blank hydrogel. Before quantitative detection, the pH of the reaction solution was optimized. Fig.14 It can be seen that the optimal pH value is 7.0. Then the detection time was optimized. Fig.15 It can be seen that the signal intensity of the reaction system increases with the increase of reaction time and tends to be stable after 30 minutes, indicating that the detection of D-Ala can be basically completed within 30 minutes.

[0084] The quantitative function of DAAO / HRP@MAF-7 hydrogel sensor for D-AAs was further studied. Finally, the standard curve for detecting the target was obtained. Fig.16 The signal intensity increases with the increase of D-Ala concentration. In the range of 0.5μM-300μM (ΔC=1.11X+1.95, R 2 =0.998) had a good linear relationship and the limit of detection (LOD) was 0.35 μM.

[0085] (2) Stability test

[0086] 1 mL of 0.3 mM D-Ala solution was added to a centrifuge tube, and the tube cap was closed. The tube was inverted and reacted for 30 minutes before flipping over. The hydrogel was analyzed by RGB using a portable colorimeter. The color change value (ΔC) was calculated using the Euclidean distance equation. The stability of the test results was investigated. The devices were divided into two groups, one stored at room temperature and the other placed in a refrigerator at 4°C. Fig.17 It can be seen that the detection results of the device under different storage conditions within one week are almost the same. The detection signal decreases after 15 days of storage at room temperature, while it can be stored longer at 4°C, indicating that the device can be stored in a refrigerator to extend the use time of the sensor.

[0087] (3) Actual sample testing - determination of the D-Ala content in saliva samples

[0088] After the volunteers agreed to collect the saliva samples, they were asked not to smoke, drink, or chew gum within 30 minutes before collecting the saliva samples. The saliva samples were obtained by gargling with warm water. For the saliva samples, a 20% saliva sample was first prepared by adding 1mL of saliva sample to 5mL PBS 20mM pH=7. Then, 1mL of the prepared 20% saliva sample was used for D-Ala detection. The standard addition method was used to add D-Ala solutions with concentrations of 10μM, 30μM, and 50μM in the actual samples, and the recovery rate was 95-98.2%. The results obtained are shown in Table 1, which show that the constructed hydrogel sensor has good practicality in saliva sample applications.

[0089] Table 1 Analysis of D-Ala content in human saliva and determination of spike recovery

[0090]

[0091] (4) Actual sample testing - determination of the D-Ala content in serum samples

[0092] Serum samples were collected after the volunteers agreed. For serum samples, 20% serum samples were first prepared by adding 1 mL of serum sample to 5 mL of PBS 20 mM pH = 7. Then, 1 mL of the prepared 20% serum sample was used for D-Ala detection. D-Ala solutions with concentrations of 10 μM, 30 μM, and 50 μM were added to the actual samples in sequence using the standard addition method and analyzed, and the recovery rate was 99.4-103%. The results obtained are shown in Table 2, which show that the hydrogel sensor can realize the detection of D-Ala in serum samples.

[0093] Table 2 Analysis of D-Ala content in human serum and determination of spike recovery

[0094]

[0095] The present invention immobilizes DAAO and HRP in a hydrophilic metal organic framework MAF-7, and then the prepared DAAO / HRP@MAF-7 biocomposite is co-loaded with the color developer TMB in a PEG-DA hydrogel. The functionalized hydrogel is then fixed on the lid of a test tube, which can be conveniently operated in a test tube and reliably quantified by a handheld colorimeter. The nanopore size of the PEG-DA hydrogel (polyethylene glycol diacrylate) can be adjusted by changing the molecular weight of the hydrogel precursor, which makes the PEG-DA hydrogel promising to become the next generation of "molecular sieves" that can prevent macromolecules or cells in complex components from entering the hydrogel matrix while not affecting the diffusion of small molecules. More importantly, the enzyme fixation ability of the PEG-DA hydrogel and the water-rich three-dimensional mesh matrix make it an excellent carrier for enzymes and reagents in biological detection systems. In addition, the good adhesion of the hydrogel enables it to be integrated with other devices.

Claims

1. A method for preparing DAAO / HRP@MAF-7 hydrogel, characterized in that: The hydrogel is formed by immobilizing horseradish peroxidase HRP and D-amino acid oxidase DAAO in a hydrophilic metal organic framework MAF-7, and then co-loading the prepared DAAO / HRP@MAF-7 biocomposite with a color developer TMB in a PEG-DA hydrogel; The preparation method of the DAAO / HRP@MAF-7 hydrogel comprises the following steps: (1) Synthesis of DAAO / HRP@MAF-7 biocomposites HRP and DAAO were added to the Zn(NO3)2 solution, mixed evenly, and then mixed with a 3-methyl-1,2,4-triazole Hmtz aqueous solution, stirred at room temperature for reaction, and after the reaction was completed, the precipitate was collected by centrifugation, washed with deionized water for multiple times, and freeze-dried to obtain the DAAO / HRP@MAF-7 biocomposite material; (2) Preparation of functionalized hydrogel PEG-DA, synthesized DAAO / HRP@MAF-7 biocomposite and TMB ethanol solution were dissolved in pH=7, 20mM phosphate buffer PBS to obtain a precursor solution, and a photoinitiator 651 with a mass concentration of 10% w / w and a solvent of 1-vinyl-2-pyrrolidone were added, and the DAAO / HRP@MAF-7 hydrogel was obtained by polymerization under ultraviolet light. The DAAO / HRP@MAF-7 hydrogel is used as a biosensor in detecting D-amino acids. The detection method is as follows: the prepared DAAO / HRP@MAF-7 hydrogel is fixed on the cap of a centrifuge tube, the sample solution to be tested is added to the centrifuge tube, and the cap of the centrifuge tube with the hydrogel fixed is buckled. After reacting for 30 minutes, the centrifuge tube is turned over, and the RGB analysis of the hydrogel after color development is performed using a portable colorimeter. The color change value (ΔC) is calculated using the Euclidean distance equation. ΔR, ΔG, and ΔB are the changes of R, G, and B relative to the blank hydrogel, respectively, to achieve quantitative detection of D-amino acids.

2. The method for preparing the DAAO / HRP@MAF-7 hydrogel according to claim 1, characterized in that: In step (1), the amount of Zn(NO3)2 and 3-methyl-1,2,4-triazole is 1:(4-10).

3. The method for preparing the DAAO / HRP@MAF-7 hydrogel according to claim 1, characterized in that: In step (1), the stirring reaction time is 4-24 hours.

4. The method for preparing the DAAO / HRP@MAF-7 hydrogel according to claim 1, characterized in that: The mass ratio of the horseradish peroxidase to the D-amino acid oxidase is 1:(4-0.25).

5. The method for preparing the DAAO / HRP@MAF-7 hydrogel according to claim 1, characterized in that: In step (2), the mass ratio of PEG-DA and DAAO / HRP@MAF-7 biocomposite was 8:

1.

6. The method for preparing the DAAO / HRP@MAF-7 hydrogel according to claim 1, characterized in that: The molecular weight of the PEG-DA is 400.

7. The method for preparing the DAAO / HRP@MAF-7 hydrogel according to claim 1, characterized in that: The photopolymerization conditions are: irradiation with 365nm ultraviolet light, irradiance of 20mW / cm², and polymerization for 5 minutes.

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