Preparation method of novel colorimetric biosensor and method for detecting hydrogen peroxide by using novel colorimetric biosensor

Abstract

The invention discloses a preparation method of a novel colorimetric biosensor and a method for detecting hydrogen peroxide by using the colorimetric biosensor. The colorimetric biosensor comprises a Gly (Fe) 2 modified metal organic framework prepared from ZrCl4, 5, 10, 15, 0-tetraferriporphyrin and Gly (Fe) 2, and a colorimetric probe; the metal organic framework is composed of ZrCl4 and 5, 10, 15, 0-tetraferriporphyrin, and is named as PCN222 (Fe); the colorimetric probe is TMB (Tetramethylbenzidine); the Gly (Fe) 2 modified metal organic framework is composed of Gly (Fe) 2 and PCN222 (Fe). According to the method, H2O2 in biological cells can be visually and quantitatively detected in real time / on site, the sensitivity is high, false influence on experimental results is avoided, the colorimetric result is more stable, and the detection time is shorter. In an on-site detection environment, a H2O2 content detection result can be obtained within 100 seconds and is not influenced by interfering substances.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, in particular to a method for preparing a novel colorimetric biosensor and detecting hydrogen peroxide. Background Art

[0002] Hydrogen peroxide (H2O2) is an important type of reactive oxygen molecule. Normal levels of H2O2 in the human body will not cause damage to the body. However, the continuous overproduction of H2O2 in cells may cause oxidative damage to cells and biomolecules, trigger multiple functional disorders in organisms, and lead to the occurrence of multiple diseases. Therefore, visualizing, dynamically, and real-time targeting and accurately quantifying H2O2 in cells is of great significance for understanding its role in cell physiology and then for pathological diagnosis. However, H2O2 is constantly produced, transformed, and consumed in living organisms, which makes it extremely difficult to quantify and distinguish it. At present, the analysis methods of H2O2 include electrochemical methods, high-performance liquid chromatography-mass spectrometry, and mass spectrometry imaging technology. Although these methods have a wide detection range and a low detection limit, these methods require the destruction of cells or tissues and are not suitable for real-time and dynamic cell detection. Therefore, developing visualizing, dynamically, and real-time targeting and accurate quantification of H2O2 in cells is crucial to understanding its role in cell physiology.

[0003] In recent years, H2O2 colorimetric analysis has attracted much attention due to its advantages such as high selectivity, rapid response, and no need for complex sample pretreatment before detection. Although different colorimetric probes can detect H2O2 in cells, they are not widely used. This is mainly because in complex physiological environments, probes are easily interfered with, producing false positive or false negative signals, and the detection limit is also high; secondly, visualization of cell structure requires the probe to be biocompatible, and most of the current probes for monitoring H2O2 have high cytotoxicity, and there are few strategies for efficiently targeting important organelles, which greatly limits the study of the physiological effects of H2O2 at the cellular level; finally, most H2O2 probes have a long signal output reaction time, which is not conducive to rapid quantitative analysis of biological samples, as well as rapid, highly sensitive, and highly selective real-time monitoring of the cell environment.

[0004] At present, many iron ion probes are often designed for living cell detection because of their good cell membrane permeability and binding ability. As a green and cheap biocatalyst, chelated iron compounds have high catalytic efficiency and high sensitivity. Their internal circulation catalytic mechanism can well meet the detection needs under complex physiological environments. However, under conditions such as excessive acidity, excessive alkali, and high temperature, the stability and sensitivity are poor, which limits practical applications. Compared with traditional supported catalysts, iron-containing metal organic frameworks (MOFs) catalysts are efficient and stable, with well-dispersed active sites and stable structures. The porous structure and strong adsorption allow the reactants to contact the active sites more fully, avoiding aggregation and blockage, and improving the utilization and stability of the catalyst. Therefore, the combination of chelated iron compounds and MOFs whose ligands are chelated iron porphyrins is a new probe method worthy of exploration, aiming to have efficient catalytic ability under extreme conditions, significantly expand the colorimetric sensitivity, and shorten the detection time. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a novel colorimetric biosensor for detecting biological cells and a method for detecting H2O2. The colorimetric biosensor is non-cytotoxic and has good biocompatibility. It can detect H2O2 in real time / on-site visual quantitative manner with high sensitivity. It produces a Fenton cycle inside the compound structure and will not produce a false effect on the experimental results. The reaction rate and detection effect are improved by synergistic effects, and the colorimetric results are more stable and the detection time is shorter. The linear range of H2O2 is 1~100 μM, and the equation is ΔA=0.0102 [H2O2](μM)+0.0887. R2=0.9955, n =11). The detection limit (LOD, 3σ) for H2O2 is 0.26 nM. In the field detection environment, the H2O2 content detection result can be obtained within 100 s and is not affected by interfering substances. The detection limit of the colorimetric method is 0.26 μM, and the detection process is convenient, fast and low-cost.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a colorimetric biosensor, which includes a metal organic framework modified with ferrous glycine prepared from ZrCl4, 5,10,15,0-tetrakis(4-aminophenyl)porphyrin and ferrous glycine Gly(Fe)2 and a colorimetric probe; the metal organic framework is composed of ZrCl4 and 5,10,15,0-tetrakis(4-aminophenyl)porphyrin, which is named PCN222(Fe) and can be used as a modified carrier of Gly(Fe)2; the colorimetric probe is 3,3',5,5'-tetramethylbenzidine (TMB); the metal organic framework modified with Gly(Fe)2 is composed of Gly(Fe)2 and PCN222(Fe), which catalyzes the hydroxyl free radical •OH produced by H2O2, and can oxidize the colorless TMB into a blue product oxTMB and produce an ultraviolet absorption peak at 652 nm.

[0007] As a preferred embodiment, Gly(Fe)2 and PCN222(Fe) are mixed and stirred, and the peroxidase-like enzyme PCN222(Fe)-Gly(Fe)2 is obtained under the action of the cross-linking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and then the PCN222(Fe)-Gly(Fe)2 solution and the colorimetric probe TMB solution are physically mixed to obtain a colorimetric biosensor. PCN222(Fe)-Gly(Fe)2 can quickly catalyze H2O2 to produce •OH, and convert the colorless TMB into the blue oxide oxTMB. However, oxTMB will be affected by the concentration of the analyte, changing the ultraviolet absorption peak (colorimetric signal intensity), resulting in a good linear relationship between the colorimetric signal of the sensor at 652 nm and the H2O2 concentration, thereby realizing rapid quantitative detection of the H2O2 concentration in the analyte. The colorimetric biosensor of the present invention has a detection limit of 0.26 μM for H2O2, can accurately detect H2O2 in a concentration range of 1 to 100 μM, and can visually and quantitatively detect H2O2 in real time / on-site. The colorimetric biosensor constructed based on PCN222(Fe)-Gly(Fe)2 has good detection and selectivity for H2O2, and the detection process is portable, fast and low-cost, which meets the detection requirements of food safety and can be used for actual environment detection.

[0008] In a second aspect, the present invention also provides a method for preparing the above-mentioned colorimetric fluorescence dual-mode sensor, comprising the following steps: Step 1: Mix a DMF solution of ZrCl4 and a DMF solution of 5,10,15,0-tetrakis(4-aminophenyl)iron porphyrin, and perform a thermal reaction to obtain a solution A; Step 2: The solution A is repeatedly washed and dried to obtain a metal organic framework (PCN222(Fe)), 50 mg is taken, and phosphate buffer is added to obtain a solution B; Step 3: Add EDC and NHS to solution B and stir for 2 h to obtain solution C; Step 4: Weigh 150 mg of Gly(Fe)2 and add it to phosphate buffer to obtain solution D; Step 5: Physically mix solution C and solution D, stir and react for 8 h to obtain solution E; Step 6: The solution E is repeatedly washed and dried to obtain a metal organic framework loaded with Gly(Fe)2 (PCN222(Fe)-Gly(Fe)2), which is added to a phosphate buffer to obtain a solution F, which is stored at 4°C; Step 7: Weigh 20 mg of TMB and add anhydrous ethanol to obtain solution G; Step 8: Physically mix solutions F and G, and adjust the volume with buffer to obtain the colorimetric biosensor, which is stored at 4°C.

[0009] As a preferred embodiment, in the step 1, the molar mass ratio of ZrCl4 and 5,10,15,0-tetrakis(4-aminophenyl)iron porphyrin in solution A is 6:1, the total volume of DMF is 8 mL, and the solution mixing method is to slowly drop the DMF solution of ZrCl4 into the DMF solution of 5,10,15,0-tetrakis(4-aminophenyl)iron porphyrin, the heating temperature is 120°C, the concentration of the DMF solution of ZrCl4 is 0.6 mol / L, and the concentration of the DMF solution of 5,10,15,0-tetrakis(4-aminophenyl)iron porphyrin is 0.1 mol / L; the detergent used in the washing in the step 2 is anhydrous ethanol, the drying method adopts oven drying, the temperature range is 60-80°C, and the volume of solution B is 20 mL; the mass ratio of PCN222(Fe), EDC and NHS in the step 3 is 10:1:1; the volume of the phosphate buffer in the step 4 is 20 mL; the stirring behaviors in steps 3 and 5 were all carried out at room temperature at a speed of 150 rpm.

[0010] As a preferred embodiment, the detergent used for washing in step 6 is phosphate buffer, the drying method adopts oven drying, and the volume of solution F is 20 mL; the concentration of solution G in step 7 is 1 mM; the buffer in step 8 is acetic acid-sodium acetate buffer solution; the volume ratio of solution F and G is 2:1, the pH of the acetic acid-sodium acetate buffer solution is 4, and the system is fixed to 1.8 mL with acetic acid-sodium acetate buffer solution, that is, the colorimetric biosensor system in step 8 is composed of F, G and acetic acid-sodium acetate buffer solution, and their volumes are 0.2 mL, 0.1 mL and 1.5 mL, respectively.

[0011] In a third aspect, the present invention also provides the use of the above colorimetric biosensor or the colorimetric biosensor obtained by the above colorimetric biosensor preparation method in H2O2 detection.

[0012] In a fourth aspect, the present invention also provides a method for detecting H2O2 using the above colorimetric biosensor, comprising: Step S1: the colorimetric biosensor is mixed with a series of H2O2 of different concentrations in proportion to prepare a reaction system, reacted at 25-40°C for 1-3 min, and then spectral detection is performed using a UV-visible spectrophotometer. Based on the change in UV absorption of the colorimetric biosensor at 652 nm, a linear regression equation is plotted with the H2O2 concentration as the abscissa and the UV absorption peak of the colorimetric biosensor at 652 nm as the ordinate; Step S2: The colorimetric biosensor and the H2O2 solution to be tested are mixed in proportion to prepare a reaction system. After reacting at 25-40°C for 1-3 min, a UV-visible spectrophotometer is used for spectral detection. The H2O2 concentration in the sample is calculated based on the linear regression equation obtained in step 1 through the change in the UV absorption peak of the colorimetric biosensor at 652 nm, thereby realizing the detection of H2O2.

[0013] As a preferred embodiment, the reaction system preparation method includes: adding 0.2 mL of H2O2 solution of different concentrations to the colorimetric biosensor system in step 8 for reaction. The actual specific optimization operation is that after adding 0.2 mL of PCN222(Fe)-Gly(Fe)2 solution, 0.1 mL of TMB solution and 0.2 mL of H2O2 solution of different concentrations in sequence, the volume is fixed to 2 mL using NaAc-HAc buffer with a pH value of 4.0 to prepare a 2 mL reaction system; then incubate at 35°C for 1.5 min, and record the absorbance of the solution at 652 nm. The concentration of the PCN222(Fe)-Gly(Fe)2 solution is 0.5 mg / mL, and the solvent is phosphate buffer solution; the concentration of the TMB solution is 1 mM, and the solvent is anhydrous ethanol; the H2O2 solutions of different concentrations are obtained by diluting a 10 mM H2O2 solution with ultrapure water.

[0014] As a preferred embodiment, a UV-visible spectrophotometer is used for spectral detection, and the UV absorption peak at 652 nm is recorded. The linear regression equation is obtained by using the functional relationship between the UV absorption peak and the H2O2 concentration, and the concentration of H2O2 to be measured can be obtained by the linear regression equation. The colorimetric biosensor has a light yellow or colorless color when there is no H2O2. This is the color of the colorimetric biosensor itself. However, as the H2O2 concentration increases, the UV absorption intensity at 652 nm increases, and the color of the reaction system gradually changes from light yellow or colorless to blue. After the color no longer changes, the UV absorption value at 652 nm can be detected, and then the concentration of H2O2 can be obtained by using the linear regression equation.

[0015] As a preferred embodiment, under acidic conditions, PCN222(Fe)-Gly(Fe)2 can catalyze H2O2 to produce •OH, oxidize colorless TMB into blue oxTMB, and then utilize the linear relationship between the ultraviolet absorption peak at 652nm of the colorimetric biosensor and the concentration of H2O2 to be detected, thereby realizing the detection of the concentration of H2O2 in the substance to be tested; the conditions of the ultraviolet-visible spectrum include: the observation range of the ultraviolet-visible spectrum is 500-750 nm.

[0016] The technical principle of the present invention is that PCN222(Fe)-Gly(Fe)2 can quickly catalyze H2O2 to produce •OH, and convert the colorless TMB into blue oxide oxTMB. oxTMB can be used as a colorimetric detection signal to construct a colorimetric medium of a colorimetric biosensor based on PCN222(Fe)-Gly(Fe)2. oxTMB is affected by the concentration of H2O2, the substance to be tested, and the ultraviolet absorption peak (colorimetric signal intensity) changes, resulting in a good linear relationship between the colorimetric signal of the sensor at 652 nm and the H2O2 concentration, thereby realizing rapid quantitative detection of the H2O2 concentration in the substance to be tested.

[0017] Advantages of the present invention: 1. The present invention uses PCN222(Fe), a peroxidase-like zirconium-based metal-organic framework with a porous structure, as a carrier and modifies glycine ferrous iron (Gly(Fe)2) on its surface, thereby solving the problem of insufficient stability and sensitivity of the colorimetric probe.

[0018] 2. The colorimetric method of the present invention is non-cytotoxic, has good biocompatibility, can be used for living cell detection, and has good cell membrane permeability and binding ability.

[0019] 3. The colorimetric biosensor constructed based on PCN222(Fe)-Gly(Fe)2 of the present invention integrates the excellent synergistic catalytic ability of chelated iron and the adsorption catalytic properties of PCN222(Fe) into one system, which has simple design, convenient operation, stable detection, and greatly reduces the detection time and cost.

[0020] 4. The present invention utilizes the porous structure and efficient adsorption function of PCN222 (Fe) to further accelerate the speed at which H2O2 enters the catalytic center, improve the catalytic efficiency, and truly realize "double catalytic activity amplification", and the internal Fenton effect will not have a false impact on the experimental results.

[0021] 5. The H2O2 detection method established in the present invention has high detection sensitivity, a colorimetric detection limit of 0.26 μM, and good anti-interference and selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 These are the SEM and TEM images of PCN222(Fe) in Example 1 and PCN222(Fe)-Gly(Fe)2 in Example 2 of the present invention.

[0024] Figure 2 It is the Fourier transform infrared spectrum and X-ray diffraction pattern of PCN222(Fe)-Gly(Fe)2 and its synthetic raw materials in Example 2 of the present invention.

[0025] Figure 3 This is a cytotoxicity graph of PCN222(Fe)-Gly(Fe)2 in Verification Example 1 of the present invention.

[0026] Figure 4 This is a verification diagram of the catalytic ability of PCN222(Fe)-Gly(Fe)2 in Verification Example 2 of the present invention.

[0027] Figure 5 This is a stability test chart of PCN222(Fe) and PCN222(Fe)-Gly(Fe)2 in Verification Example 3 of the present invention.

[0028] Figure 6 These are images of the colorimetric sensor solution after adding H2O2 at different concentrations in Example 4 of the present invention.

[0029] Figure 7It is the UV-visible spectrum of the colorimetric sensor solution after adding different concentrations of H2O2 in Example 4 of the present invention.

[0030] Figure 8 It is a linear graph of the ultraviolet absorption peak value and the H2O2 concentration value of the colorimetric sensor solution after adding different concentrations of H2O2 in Example 4 of the present invention.

[0031] Fig. 9 This is an analysis diagram of the anti-interference performance of the colorimetric biosensor in Verification Example 4 of the present invention.

[0032] Fig.10 This is a selectivity analysis diagram of the colorimetric biosensor in Verification Example 4 of the present invention.

[0033] Fig.11 This is a graph showing the ability of PCN222(Fe)-Gly(Fe)2 to catalyze H2O2 to produce •OH in Verification Example 5 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, all raw materials used in the examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art. Example 1

[0036] This embodiment first provides a zirconium-based metal organic framework material, and its synthesis method comprises the following steps: 47 mg ZrCl4 and 28 mg 5,10,15,0-tetrakis(4-aminophenyl)iron porphyrin were dissolved in 4 mL DMF and sonicated until completely dissolved.

[0037] The 5,10,15,0-tetrakis(4-aminophenyl)iron porphyrin solution was poured into a 50 mL eggplant bottle, placed in silicone oil at 120° C., and stirred at 200 rpm with a magnetic stirrer.

[0038] 1.803 g of benzoic acid was added and after it was completely dissolved, the DMF solution of ZrCl4 was slowly added dropwise into the flask.

[0039] After the addition was completed, the mixture was stirred for 30 min, the magnetic stirrer was turned off, and heating was maintained at 120 °C for 48 h.

[0040] After heating, extract the eggplant bottle from the silicone oil bath. After the solution cools to room temperature, transfer the solution equally into two 50 mL centrifuge tubes, 4 mL of solution in each tube, and then add 16 mL of anhydrous ethanol to each tube, and centrifuge at 12000 rpm at room temperature for 20 min.

[0041] Remove the supernatant, add 10 mL of anhydrous ethanol, centrifuge, and repeat this step 3 times.

[0042] The final precipitate was placed in an oven at 70°C until completely dried to obtain PCN222 (Fe) material.

[0043] Characterization of PCN222(Fe) Figure 1 a and Figure 1 b in the figure are SEM and TEM images of PCN222(Fe), respectively. It can be seen from the figure that the synthesized PCN222(Fe) has a stick structure with uniform size of about 1-2 μm. Example 2

[0044] This embodiment further provides a method for synthesizing PCN222(Fe)-Gly(Fe)2, which specifically comprises the following steps: Take 50 mg PCN222(Fe) in a 50 mL brown glass sample bottle, add 20 mL phosphate buffer solution, and ultrasonicate for 1 h to completely disperse it. Add 5 mg of crosslinking agent EDC and NHS to the above solution, stir at 150 rpm for 2 h, and obtain solution C.

[0045] Take 150 mg of Gly(Fe)2 in a 50 mL centrifuge tube and add 20 mL of phosphate buffer to obtain solution D. The above solution D was added into solution C at one time, and the mixture was stirred at 150 rpm for 1.5 h to obtain solution E.

[0046] After stirring, transfer solution E equally into four 50 mL centrifuge tubes, 10 mL of solution in each tube, and then add 10 mL of phosphate buffer solution to each tube, and centrifuge at 12000 rpm at room temperature for 20 min.

[0047] The supernatant was removed, and 10 mL of phosphate buffer solution was added, followed by centrifugation. This step was repeated three times.

[0048] The final precipitate was placed in an oven at 70°C until completely dried to obtain PCN222(Fe)-Gly(Fe)2 material.

[0049] Characterize PCN222(Fe)-Gly(Fe)2. Figure 1c and Figure 1 The d in the figure are the SEM and TEM images of PCN222(Fe)-Gly(Fe)2, respectively. Figure 1 c and Figure 1 As can be seen from image d, there is a large amount of loading on the surface of the prepared PCN222(Fe)-Gly(Fe)2, confirming that Gly(Fe)2 is fixed on PCN222(Fe).

[0050] Figure 2 a in the figure is the Fourier transform infrared spectra of PCN222(Fe)-Gly(Fe)2, PCN222(Fe) and Gly(Fe)2. Figure 2 b in the figure is the X-ray diffraction pattern of PCN222(Fe)-Gly(Fe)2 and PCN222(Fe). Figure 2 a and Figure 2 In b, the characteristic peak of Gly(Fe)2 appears on PCN222(Fe)-Gly(Fe)2, and the crystal of PCN222(Fe)-Gly(Fe)2 has changed, all of which reflect the successful preparation of PCN222(Fe)-Gly(Fe)2. Verification Example 1

[0051] In order to verify the biocompatibility of PCN222(Fe)-Gly(Fe)2. HUVECs cells in the logarithmic growth phase with good growth status were selected, and cell suspensions were prepared and counted. 100 μL of cell suspension was added to each well of a 96-well culture plate, which was divided into a blank group with no cells and only culture medium; a control group with complete culture medium containing serum but without PCN222(Fe)-Gly(Fe)2 in Example 2; and an experimental group with cells and different concentrations of PCN222(Fe)-Gly(Fe)2. Three parallel wells were set in each group, and then fresh culture medium containing different concentrations of PCN222(Fe)-Gly(Fe)2 (200, 100, 50, 25, 10, 5 and 0 μg / mL) was added to each well of the experimental group. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for 24 h. After the incubation, 10 μL of CCK-8 solution was added to each well. Continue to incubate in the incubator for 1-4 h until the solution turns orange-yellow. Use a microplate reader to detect the absorbance (OD value) of each well at a wavelength of 450 nm. Finally, the toxicity of PCN222(Fe)-Gly(Fe)2 to HUVECs cells was evaluated by calculating the survival rate of the treatment groups with different concentrations. If the survival rate is >80%, it means that the cytotoxicity is low at this concentration. If the survival rate is >90%, it means that the concentration is non-toxic or slightly toxic to the cells.

[0052] like Figure 3The figure shows the survival rate of HUVECs after incubation with different concentrations of PCN222(Fe)-Gly(Fe)2 for 24 h. It can be seen from the figure that the effect of PCN222(Fe)-Gly(Fe)2 on HUVECs is negligible, and even at a high concentration of 200 μg / mL, the cell survival rate exceeds 90%, indicating that PCN222(Fe)-Gly(Fe)2 has good biocompatibility. Verification Example 2

[0053] In order to verify the catalytic activity of PCN222(Fe)-Gly(Fe)2, 5 mg of PCN222(Fe)-Gly(Fe)2 obtained in Example 2 was dissolved in 45 mL of phosphate buffer to a concentration of 0.5 mg / mL.

[0054] Then, 0.2 mL PCN222(Fe)-Gly(Fe)2 solution, 0.1 mL TMB solution and 0.2 mL 0.4 mM H2O2 solution were added in sequence, and the volume was adjusted to 2 mL using NaAc-HAc buffer with a pH value of 4.0 to prepare a 2 mL reaction system; after incubation for 1.5 min, the UV absorption peak at 652 nm was recorded using a UV-visible spectrophotometer. At the same time, the absorbance values ​​of TMB, TMB and H2O2 mixed solution, PCN222(Fe)-Gly(Fe)2 and H2O2 mixed solution, and PCN222(Fe)-Gly(Fe)2 and TMB mixed solution at 652 nm were measured, and the reaction systems measured above were also 2 mL.

[0055] Then, Gly(Fe)2 and PCN222(Fe) were used to replace PCN222(Fe)-Gly(Fe)2 respectively, and other conditions were the same. The reaction system was configured and the absorbance at 652 nm was recorded. The absorbance of PCN222(Fe)-Gly(Fe)2 was used as a control to compare the activity of Gly(Fe)2, PCN222(Fe) and the blank group without material.

[0056] like Figure 4 The a in the figure shows that PCN222(Fe)-Gly(Fe)2 has obvious ultraviolet absorption at 652 nm, indicating that it has colorimetric ability. Figure 4 Figure b shows that PCN222(Fe)-Gly(Fe)2 has a higher catalytic activity, and its catalytic activity exceeds that of PCN222(Fe) and Gly(Fe)2. Its ability comes from its synergistic ability with Gly(Fe)2. Verification Example 3

[0057] In order to verify the stability of PCN222(Fe)-Gly(Fe)2, 5 mg of PCN222(Fe) and PCN222(Fe)-Gly(Fe)2 obtained in Examples 1 and 2 were dissolved in 45 mL of phosphate buffer to a concentration of 0.5 mg / mL.

[0058] First, PCN222(Fe) and PCN222(Fe)-Gly(Fe)2 solutions were placed in a temperature range of 0℃ to 100℃ (0, 25, 50, 75 and 100℃) and a pH range of 2.0 to 9.0 (2.0, 3.0, 5.0, 7.0 and 9.0) for 12 h, and then 0.2 mL PCN222(Fe)-Gly(Fe)2 solution, 0.1 mL TMB solution and 0.2 mL 0.4 mM H2O2 solution were added in sequence. The volume was adjusted to 2 mL using NaAc-HAc buffer with a pH value of 4.0 to prepare a 2 mL reaction system. After incubation for 1.5 min, the UV absorption peak at 652 nm was recorded using a UV-visible spectrophotometer. PCN222(Fe) and PCN222(Fe)-Gly(Fe)2 solutions were then placed at 35℃ for 90 d. A 2 mL reaction system was prepared using the same method as above. After incubation for 1.5 min, the UV absorption peak at 652 nm was recorded using a UV-visible spectrophotometer.

[0059] like Figure 5 The results showed that PCN222(Fe)-Gly(Fe)2 was resistant to temperature, acid and alkali, and could be stored for a long time without affecting its skeleton stability. The enhanced storage stability was attributed to the presence of carboxylic acid and amino groups in the modified Gly(Fe)2. Even after 90 days of storage, PCN222(Fe)-Gly(Fe)2 did not undergo significant changes. Example 3

[0060] This embodiment further provides a method for preparing a colorimetric biosensor, which specifically comprises the following steps: 5 mg of PCN222(Fe)-Gly(Fe)2 obtained in Example 2 was dissolved in 45 mL of phosphate buffer to a concentration of 0.5 mg / mL.

[0061] Subsequently, 0.2 mL of PCN222(Fe)-Gly(Fe)2 solution, 0.1 mL of TMB solution and 1.5 mL of NaAc-HAc buffer with a pH value of 4.0 were added in sequence to prepare a 1.8 mL colorimetric biosensor system; that is, the colorimetric biosensor system consists of PCN222(Fe)-Gly(Fe)2, TMB and acetic acid-sodium acetate buffer solution, and their volumes are 0.2 mL, 0.1 mL and 1.5 mL, respectively.

[0062] The concentration of TMB solution is 1 mM, and the solvent is anhydrous ethanol. The concentration of acetic acid-sodium acetate buffer solution can be 0.2 M or other concentrations, depending on the actual choice of other researchers. Example 4

[0063] This embodiment provides a colorimetric detection method for detecting H2O2, which specifically includes the following steps: 0.2 mL of H2O2 solution of different concentrations to be tested was added to the colorimetric sensor prepared in Example 3 to prepare a 2 mL reaction system. After 1.5 min, the color change of the solution was observed under sunlight. Then, a UV-visible absorption spectrum was measured using a UV-visible spectrophotometer. The observation range of the UV-visible spectrum was 500-750 nm. There was a functional relationship between the UV absorption peak at 652 nm and the H2O2 concentration, that is, the UV absorption peak in the reaction system corresponded to the H2O2 concentration value.

[0064] The functional relationship between the UV-visible absorption peak value and the H2O2 concentration is: y=0.0102x+0.0887(R 2 =0.99), where y represents the value of the UV absorption peak at 652 nm and x represents the H2O2 concentration.

[0065] Figure 6 The following are images of the colorimetric sensor solution after adding different concentrations of H2O2. It can be seen from the figure that the blue color becomes more and more obvious as the concentration of H2O2 increases. Figure 5 is Figure 4 UV-visible spectra of the colorimetric sensor solution after adding different concentrations of H2O2; the concentrations of the different concentrations of H2O2 in the reaction system are 0 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 150 μM and 200 μM, respectively.

[0066] based on Figure 7 UV-visible spectra of the colorimetric sensor solution after adding different concentrations of H2O2. Through the linear regression equation, we can get Figure 6, that is, the linear graph of the ultraviolet absorption peak value of the colorimetric sensor solution and the H2O2 concentration value after adding different concentrations of H2O2.

[0067] Figure 8 It shows that as the H2O2 concentration increases, the ultraviolet absorption peak of the colorimetric sensor solution at 652 nm becomes larger. Figure 6 The regression coefficient is 0.99, which indicates that the functional relationship between the ultraviolet absorption peak at 652 nm and the H2O2 concentration is good, and it is feasible and accurate to detect the H2O2 concentration by the colorimetric sensor of the present invention. Example 5

[0068] This embodiment further provides a method for verifying the ability of PCN222(Fe)-Gly(Fe)2 to catalyze H2O2 to produce •OH, which specifically includes the following steps: 5 mg of PCN222(Fe)-Gly(Fe)2 obtained in Example 2 was dissolved in 45 mL of phosphate buffer to a concentration of 0.5 mg / mL.

[0069] Subsequently, 0.2 mL PCN222(Fe)-Gly(Fe)2 solution, 0.2 mL H2O2 solution, and 0.1 mL phosphate buffer solution were added in sequence, and then the volume was adjusted to 2 mL with NaAc-HAc buffer with a pH value of 4.0 to prepare 2 mL of H2O2 catalytic system; after 1.5 min of reaction, the •OH intensity was measured using an EPR instrument.

[0070] The concentration of H2O2 solution is 0.4 mM, and the diluent is ultrapure water. The concentration of acetic acid-sodium acetate buffer solution can be 0.2 M or other concentrations, depending on the actual choice of other researchers. Verification Example 4

[0071] The colorimetric biosensor was prepared according to Example 3, and K 2+ 、Zn 3+ , Mn 2+ , Ca 2+ 、Na 2+ As interfering ions, HCO3 - , Cl - 、CH3COO - Acidic ions such as thr, lys, his, gly, ala, fru, suc and other amino acid interfering substances were used to explore the anti-interference and selectivity of the colorimetric biosensor for detecting H2O2. The concentration of the interfering ions was 10 times that of the H2O2 solution. After reacting at 25-40 ° C for 1-3 min, the spectral detection was carried out by UV-visible spectrophotometer and compared with the UV absorption change of the colorimetric biosensor at 652 nm. Fig. 9 and Fig.10 They are the anti-interference analysis and selectivity analysis of the colorimetric sensor. It can be seen from the figure that the ultraviolet absorption value of the colorimetric biosensor changes significantly only in the presence of H2O2, and the interfering ions will not affect the result change, indicating that the sensor has good anti-interference and selectivity for H2O2 detection in vivo. Verification Example 5

[0072] The H2O2 catalytic system was prepared according to Example 5. Fig.11 This is the ability diagram of PCN222(Fe)-Gly(Fe)2 to catalyze the production of •OH from H2O2 measured by EPR instrument. It can be seen from the figure that the peak signal with an intensity of 1:2:2:1 is attributed to the formation of DMPO / •OH adduct, which shows that PCN222(Fe)-Gly(Fe)2 can indeed catalyze the production of •OH from H2O2.

[0073] It is worth noting that the •OH signal intensity catalyzed by PCN222(Fe)-Gly(Fe)2 is much higher than that of PCN222(Fe), which indicates that PCN222(Fe)-Gly(Fe)2 formed by modifying PCN222(Fe) with Gly(Fe)2 can solve the problem of insufficient stability and sensitivity of chelated iron Gly(Fe)2. Therefore, the colorimetric biosensor constructed based on PCN222(Fe)-Gly(Fe)2 integrates the excellent synergistic catalytic ability of chelated iron Gly(Fe)2 and the adsorption catalytic properties of PCN222(Fe) into one system, greatly improving the catalytic ability of PCN222(Fe)-Gly(Fe)2.

[0074] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A colorimetric biosensor, characterized in that: The colorimetric biosensor includes a metal organic framework modified with ferrous glycine prepared from ZrCl4, 5,10,15,0-tetrakis(4-aminophenyl)porphyrin and ferrous glycine Gly(Fe)2 and a colorimetric probe; the metal organic framework is composed of ZrCl4 and 5,10,15,0-tetrakis(4-aminophenyl)porphyrin and is named PCN222(Fe), which serves as a modified carrier of Gly(Fe)2; the colorimetric probe is 3,3',5,5'-tetramethylbenzidine (TMB); the metal organic framework modified with Gly(Fe)2 is composed of Gly(Fe)2 and PCN222(Fe), which catalyzes the hydroxyl radical •OH produced by H2O2, oxidizes the colorless TMB into a blue product oxTMB and produces an ultraviolet absorption peak at 652 nm.

2. A colorimetric biosensor according to claim 1, characterized in that: Gly(Fe)2 and PCN222(Fe) were mixed and stirred, and under the action of cross-linking agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), a peroxidase-like enzyme PCN222(Fe)-Gly(Fe)2 was obtained. Subsequently, the PCN222(Fe)-Gly(Fe)2 solution and the colorimetric probe TMB solution were physically mixed to obtain a colorimetric biosensor.

3. The method for preparing a colorimetric biosensor according to claim 1, characterized in that: The steps include: Step 1: Mix a DMF solution of ZrCl4 and a DMF solution of 5,10,15,0-tetrakis(4-aminophenyl)iron porphyrin, and perform a thermal reaction to obtain a solution A; Step 2: The solution A is repeatedly washed and dried to obtain a metal organic framework (PCN222(Fe)), 50 mg is taken, and phosphate buffer is added to obtain a solution B; Step 3: Add EDC and NHS to solution B and stir for 2 h to obtain solution C; Step 4: Weigh 150 mg of Gly(Fe)2 and add it to phosphate buffer to obtain solution D; Step 5: Physically mix solution C and solution D, stir and react for 8 h to obtain solution E; Step 6: The solution E is repeatedly washed and dried to obtain a metal organic framework loaded with Gly(Fe)2 (PCN222(Fe)-Gly(Fe)2), which is added to a phosphate buffer to obtain a solution F, which is stored at 4°C; Step 7: Weigh 20 mg of TMB and add anhydrous ethanol to obtain solution G; Step 8: Physically mix solutions F and G, and adjust the volume with buffer to obtain the colorimetric biosensor, which is stored at 4°C.

4. The method for preparing a colorimetric biosensor according to claim 3, characterized in that: In the step 1, the molar mass ratio of ZrCl4 and 5,10,15,0-tetrakis(4-aminophenyl)iron porphyrin in solution A is 6:1, the total volume of DMF is 8 mL, and the solution mixing method is to slowly drop the DMF solution of ZrCl4 into the DMF solution of 5,10,15,0-tetrakis(4-aminophenyl)iron porphyrin, the heating temperature is 120°C, the concentration of the DMF solution of ZrCl4 is 0.6 mol / L, and the concentration of the DMF solution of 5,10,15,0-tetrakis(4-aminophenyl)iron porphyrin is 0.1 mol / L; the detergent used in the washing in the step 2 is anhydrous ethanol, the drying method is oven drying, the temperature range is 60-80°C, and the volume of solution B is 20 mL; in the step 3, the mass ratio of PCN222(Fe), EDC and NHS is 10:1:1; the volume of the phosphate buffer in the step 4 is 20 mL; the stirring behaviors in steps 3 and 5 were all carried out at room temperature at a speed of 150 rpm.

5. The method for preparing a colorimetric biosensor according to claim 3, characterized in that: The detergent used for washing in step 6 is phosphate buffer, the drying method adopts oven drying, and the volume of solution F is 20 mL; the concentration of solution G in step 7 is 1 mM; the buffer in step 8 is acetic acid-sodium acetate buffer solution; the volume ratio of solution F and G is 2:1, the pH of acetic acid-sodium acetate buffer solution is 4, and the system is fixed to 1.8 mL with acetic acid-sodium acetate buffer solution, that is, the colorimetric biosensor system in step 8 is composed of F, G and acetic acid-sodium acetate buffer solution, and their volumes are 0.2 mL, 0.1 mL and 1.5 mL respectively.

6. Use of the colorimetric biosensor according to claim 1 or 2 or the colorimetric biosensor obtained by the preparation method of the colorimetric biosensor according to claim 3 in the detection of H2O2 in a living body.

7. The method for detecting H2O2 by a colorimetric biosensor according to claim 1, characterized in that: The steps include: Step S1: the colorimetric biosensor is mixed with a series of H2O2 of different concentrations in proportion to prepare a reaction system, reacted at 25-40°C for 1-3 min, and then spectral detection is performed using a UV-visible spectrophotometer. Based on the change in UV absorption of the colorimetric biosensor at 652 nm, a linear regression equation is plotted with the H2O2 concentration as the abscissa and the UV absorption peak of the colorimetric biosensor at 652 nm as the ordinate; Step S2: The colorimetric biosensor and the H2O2 solution to be tested are mixed in proportion to prepare a reaction system. After reacting at 25-40°C for 1-3 min, a UV-visible spectrophotometer is used for spectral detection. The H2O2 concentration in the sample is calculated based on the linear regression equation obtained in step 1 through the change in the UV absorption peak of the colorimetric biosensor at 652 nm, thereby realizing the detection of H2O2.

8. The method for detecting H2O2 by a colorimetric biosensor according to claim 7, characterized in that: The preparation method of the reaction system of step S1 and step S2 comprises: 0.2 mL of H2O2 solution of different concentrations was added to the colorimetric biosensor system in step 8 for reaction. The actual specific optimization operation was as follows: after adding 0.2 mL of PCN222(Fe)-Gly(Fe)2 solution, 0.1 mL of TMB solution and 0.2 mL of H2O2 solution of different concentrations in sequence, the volume was fixed to 2 mL using NaAc-HAc buffer with a pH value of 4.0 to prepare a 2 mL reaction system; then, after incubating at 35°C for 1.5 min, the absorbance of the solution at 652 nm was recorded. The concentration of the PCN222(Fe)-Gly(Fe)2 solution was 0.5 mg / mL, and the solvent was phosphate buffer solution; the concentration of the TMB solution was 1 mM, and the solvent was anhydrous ethanol; and the H2O2 solutions of different concentrations were obtained by diluting a 10 mM H2O2 solution with ultrapure water.

9. The method for detecting H2O2 by a colorimetric biosensor according to claim 7, characterized in that: The spectrum was detected by UV-visible spectrophotometer, and the UV absorption peak at 652 nm was recorded. The linear regression equation was obtained using the functional relationship between the UV absorption peak and the H2O2 concentration.

10. The method for detecting H2O2 by a colorimetric biosensor according to claim 7, characterized in that: Under acidic conditions, PCN222(Fe)-Gly(Fe)2 can catalyze H2O2 to produce •OH, oxidizing the colorless TMB to blue oxTMB. Then, the linear relationship between the ultraviolet absorption peak at 652nm of the colorimetric biosensor and the concentration of H2O2 to be detected is used to detect the concentration of H2O2 in the substance to be tested. The conditions of the UV-visible spectrum include: the observation range of the UV-visible spectrum is 500-750 nm.

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