Serum glucose detection method based on MOFs nano-enzyme

Abstract

The invention discloses a serum glucose detection method based on MOFs nano-enzyme, and belongs to the technical field of serum glucose detection.The method comprises the following steps that GOD and human serum are mixed and react at 37 DEG C for 10 minutes, and GOD catalyzes glucose in the serum to react to generate H2O2; then, a mixed solution of MIL-53Fe and TMB is added into the system, and the reaction continues to be carried out for 10 minutes at the temperature of 37 DEG C; in the process, MIL-53Fe plays a catalytic role, H2O2 is promoted to be subjected to a reduction reaction to generate. OH, meanwhile, TMB is oxidized into oxTMB, the solution is turned into blue from colorless, and a remarkable absorption peak is formed at the wavelength of 652 nm. The concentration of glucose in serum is in positive correlation with the amount of generated. OH, so that under the catalytic action of MIL-53Fe, the amount of generated oxTMB is increased along with the increase of the concentration of glucose, and the blue color of the solution is deepened; the serum glucose detection method based on the MOFs nano-enzyme can be used for detection in a neutral solution, and has excellent accuracy, precision, sensitivity and reproducibility.

Description

Technical Field

[0001] The present invention specifically relates to a serum glucose detection method based on MOFs nanozymes, and belongs to the technical field of serum glucose detection. Background Art

[0002] Glucose is the main energy source and key metabolic intermediate for most organisms. Its abnormal levels in the body have been proven to be closely related to the occurrence and development of diseases such as diabetes, obesity, and cancer. Therefore, accurate monitoring of glucose levels in the human body plays a vital role in the diagnosis and treatment of diseases such as diabetes. Such monitoring processes often require the participation of natural enzymes. However, although natural enzymes (such as POD) have high catalytic activity, their protein chemical nature leads to high production costs and they are easily inactivated in extreme environments (such as high temperature, high humidity, and extreme pH values), which leads to many limitations in production, transportation, storage, and use. In view of the above challenges, it is particularly important to explore alternatives with POD activity for the determination of serum glucose. This alternative is designed to circumvent the limitations of the use of natural enzymes and improve the convenience, rapidity, high sensitivity, and high selectivity of the detection process. Therefore, the development of a serum glucose detection method based on nanozymes has extremely high practical value and scientific significance.

[0003] At present, a variety of low-cost, high-stability nanomaterials have been widely studied and reported as potential POD mimetics, including precious metals, metal oxides, metal sulfides, carbon-based materials (such as graphene oxide and graphene dots), metal organic frameworks (MOFs) and their composite derivatives, etc. However, it is worth noting that most nanozymes exhibit excellent catalytic activity in neutral acidic environments (pH 3-4), but their catalytic activity is significantly reduced under neutral conditions close to the normal pH value of biological systems (usually pH 5-7.4), which greatly restricts their application potential in biological systems.

[0004] Based on the excellent catalytic activity of MIL-53Fe under neutral pH conditions, the present invention proposes a new method for serum glucose detection. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting serum glucose based on MOFs nanozymes to address the deficiencies of the prior art. The method detects serum glucose based on MOFs nanozymes. The nanozymes have a large specific surface area (367.99 m 2 / g) and pore volume (0.24cm 3 / g), these characteristics greatly promote the exposure of active sites, thereby accelerating the reaction process and effectively increasing the contact area with the substrate. Its kinetic parameters have reached a level similar to that of natural horseradish peroxidase, which can not only significantly improve the stability of the detection reagent and extend its storage life, but also greatly improve the accuracy of the detection.

[0006] In addition, the nanozyme has excellent catalytic ability, and the serum glucose detection method of the present invention does not require additional adjustment of the pH value. It has the significant advantages of simple operation, rapid response, high sensitivity and strong selectivity, which makes it show broad application prospects in practical applications, especially in serum glucose determination.

[0007] A method for detecting serum glucose based on MOFs nanozymes. The implementation route of this method is described in detail as follows: First, glucose oxidase (GOD) is mixed with serum, and hydrogen peroxide (H2O2) is generated through the catalytic effect of GOD on glucose. Subsequently, a composite solution of MIL-53Fe and 3,3',5,5'-tetramethylbenzidine (TMB) is added to the mixture. In this step, MIL-53Fe acts as a catalyst to promote the reduction reaction of H2O2, thereby generating hydroxyl radicals (·OH). At the same time, the generated ·OH oxidizes TMB and converts it into oxidized TMB (oxTMB), causing the solution color to change from colorless to blue and present an ultraviolet absorption peak at 652nm. As the glucose concentration in serum increases, the amount of H2O2 produced by GOD catalyzing glucose also increases accordingly. Under the catalytic effect of H2O2, the amount of oxTMB generated also increases, thereby deepening the blue color of the solution and increasing the absorbance value. By comparing the content of standard glucose in serum, we established a serum glucose detection method based on the peroxidase-like catalytic properties of MIL-53Fe at neutral pH. In addition, thanks to the high selectivity of GOD for glucose catalysis and the excellent catalytic activity of MIL-53Fe for H2O2, this method can achieve sensitive and selective detection of serum glucose, providing strong technical support for clinical diagnosis and medical research.

[0008] The present invention adopts the following technical solutions:

[0009] (1) Preparation of MIL-53Fe: MIL-53Fe was prepared by solvothermal method. The specific preparation process is as follows: Weigh 0.81g of ferric chloride FeCl3·6H2O and 0.49g of terephthalic acid HOOCC6H4COOH (H2BDC) and dissolve them in 61mL N,N-dimethylformamide DMF, ultrasonicate for 30min and then stir magnetically for 1h until completely dissolved. The mixed solution was placed in a 100mL stainless steel reactor with a polytetrafluoroethylene inner layer, sealed and reacted at 150°C for 15h, and naturally cooled to room temperature. The precipitate was washed by centrifugation with DMF and anhydrous ethanol for multiple times and then dried in a 60°C forced air drying oven for 6h, which is the nanozyme used in the method of the present invention.

[0010] Serum glucose detection: GOD and human serum were mixed and reacted at 37°C for 10 minutes. GOD catalyzed the glucose reaction in the serum to produce H2O2. Subsequently, a mixed solution of MIL-53Fe and TMB was added to the system and the reaction continued at 37°C for 10 minutes. In this process, MIL-53Fe played a catalytic role, prompting H2O2 to undergo a reduction reaction to generate ·OH, while oxidizing TMB to oxTMB. The solution changed from colorless to blue, and a significant absorption peak was formed at a wavelength of 652nm. The concentration of glucose in serum is positively correlated with the amount of ·OH produced. Therefore, under the catalytic action of MIL-53Fe, the amount of oxTMB generated increases with the increase in glucose concentration, and the blue color of the solution also deepens. This color change is directly related to the increase in absorbance, and in the range of glucose concentration of 1-100μM, the increase in absorbance is linearly related to the glucose concentration. The detection limit and quantification limit of the detection method are 0.45 μM and 1.35 μM respectively, the spike recovery rate is between 97.1% and 103.5%, and the relative standard deviation (RSD) is between 1.30 and 3.45%.

[0011] Beneficial effects:

[0012] (1) The preparation process of the MIL-53Fe nanozyme of the present invention is simple, the yield is abundant, and the cost-effectiveness is significant, providing an economical and efficient solution for practical applications;

[0013] (2) The MIL-53Fe nanozyme prepared by the present invention exhibits excellent stability. Its easy preparation and storage characteristics enable it to be stored for several years at room temperature and in the dark. This characteristic ensures that the stability of the reagent and the activity of the enzyme can be effectively maintained even under harsh transportation and storage conditions, thereby significantly extending the storage time of the detection reagent and improving the accuracy of the detection;

[0014] (3) The MOFs material of the present invention has a unique porous structure, which helps to promote the improvement of reaction rate, thereby significantly saving detection time and improving detection efficiency;

[0015] (4) The method of the present invention can be carried out under neutral pH conditions without the need for additional adjustment of the pH value during the reaction, making the entire reaction process faster, simpler and easier to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a basic flow chart of the present invention;

[0017] Figure 2 This is a scanning electron microscope (SEM) image of the MIL-53Fe nanozyme prepared by the present invention;

[0018] Figure 3 This is the X-ray diffraction pattern XRD pattern of the MIL-53Fe nanozyme prepared by the present invention;

[0019] Figure 4 This is an X-ray electron spectrum XPS diagram of the MIL-53Fe nanozyme prepared by the present invention;

[0020] Figure 5 This is a Fourier transform infrared spectrum FT-IR diagram of the MIL-53Fe nanozyme prepared by the present invention;

[0021] Figure 6 The specific surface area and pore size distribution diagram of the MIL-53Fe nanozyme prepared by the present invention;

[0022] Figure 7 Four different reaction systems of the MIL-53Fe nanozyme prepared by the present invention are used to study the peroxidase-like activity of the MIL-53Fe nanozyme, including (a) MIL-53Fe+H2O2+TMB; (b) H2O2+TMB; (c) MIL-53Fe+TMB and (d) TMB;

[0023] FIG8 is a steady-state kinetic study of the MIL-53Fe nanozyme prepared by the present invention, FIG8(a) maintaining the TMB concentration at 0.1 mM and changing the H2O2 concentration, FIG8(b) maintaining the H2O2 concentration at 0.8 mM and changing the TMB concentration; FIG8(c) is a Michaelis-Menten diagram under changes in H2O2 concentration; FIG8(d) is a Michaelis-Menten diagram under changes in TMB concentration;

[0024] Fig. 9 The selectivity analysis diagram of the detection method of the present invention for glucose detection (under neutral pH conditions). DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0026] See also Figure 1-Figure 9 As shown, a method for detecting serum glucose based on MOFs nanozymes comprises the following steps:

[0027] Preparation of MIL-53Fe nanozyme: MIL-53Fe was prepared by solvothermal method. The specific preparation process is as follows: Weigh 0.81g of ferric chloride FeCl3·6H2O and 0.49g of terephthalic acid HOOCC6H4COOH (H2BDC) and dissolve them in 61mL N,N-dimethylformamide DMF, ultrasonicate for 30min and then stir magnetically for 1h until completely dissolved. The mixed solution was placed in a 100mL stainless steel reactor with a polytetrafluoroethylene inner layer, sealed and reacted at 150°C for 15h, and naturally cooled to room temperature. The precipitate was washed by centrifugation with DMF and anhydrous ethanol for multiple times and then dried in a 60°C forced drying oven for 6h, which was the MIL-53Fe nanozyme used in the method of the present invention.

[0028] Characterization of MIL-53Fe nanozyme: The prepared MIL-53Fe nanozyme was characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray electron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR), and N2 adsorption-desorption.

[0029] Figure 2 This is the SEM image of MIL-53Fe nanozyme. Figure 2 It can be seen that MIL-53(Fe) has a regular octahedral structure.

[0030] Figure 3 This is the XRD diffraction pattern of MIL-53Fe nanozyme. It can be seen from the figure that the characteristic peaks of the sample are consistent with those reported in the literature, indicating that MIL-53Fe nanozyme was successfully prepared by the solvothermal method.

[0031] Figure 4 This is the XPS spectrum of MIL-53Fe nanozyme. It can be seen from the figure that MIL-53Fe nanozyme is composed of Fe, C and O elements. Figure 4 b is the C1s high-resolution XPS spectrum, in which two peaks appear at 284.6 and 288.6 eV, which are attributed to the benzene ring and carboxylic acid group in the organic ligand, respectively. Figure 4 c is the high-resolution XPS spectrum of O1s. Two signal peaks can be observed from the figure, located at 531.0 and 531.9 eV, which are the Fe-O group in MIL-53Fe nanozyme and the carboxylic acid group in the organic ligand, respectively. The high-resolution XPS spectrum of Fe2p is shown in Figure 4 d, the signal peaks at 712.6 and 726.3 eV correspond to Fe 2p 3 / 2 and Fe 2p 1 / 2 , the splitting energy is equal to 13.7 eV, indicating that the Fe element contained in the sample is Fe(Ⅲ).

[0032] Figure 5 This is the FI-IR spectrum of MIL-53Fe nanozyme. It can be seen from the figure that MIL-53Fe has a high concentration of 500-1700 cm -1 There are six typical absorption peaks in the wavenumber range, at 3435cm -1 The absorption peak is mainly caused by the OH vibration generated by the adsorption of moisture in the air; at 1657cm -1 The characteristic absorption peaks of C=O correspond to the stretching vibration of C=O; at 1539 and 1390 cm -1 The characteristic absorption peaks at 750cm- -1 The characteristic absorption peak at 547 cm corresponds to the CH vibration of the benzene ring in H2BDC; -1 The characteristic absorption peak at 1657cm -1 The characteristic absorption peaks of C=O correspond to the stretching vibration of C=O; at 1539 and 1390 cm -1 The characteristic absorption peaks at 750cm- -1 The characteristic absorption peak at 547 cm corresponds to the CH vibration of the benzene ring in H2BDC; -1 The characteristic absorption peak at corresponds to the Fe-O stretching vibration, proving that Fe-O in the material exists between carboxylic acid and trivalent Fe. The above characterization shows that the nanozyme prepared by the present invention is an Fe-based MOF, MIL-53Fe.

[0033] Figure 6 The specific surface area and pore size distribution of the MIL-53Fe nanozyme prepared by the present invention show that the nanozyme prepared by the present invention has a large specific surface area (367.99 m 2 / g) and pore volume (0.24cm 3 / g), which is beneficial to the exposure of active sites, making the reaction faster, increasing the contact area with the substrate, and shortening the time required for the detection method.

[0034] Nanozyme activity analysis: In order to study the catalytic activity of the sample peroxidase, MIL-53Fe nanozyme was used to catalyze the oxidation of TMB by H2O2. First, the MIL-53Fe nanozyme was added to deionized water and ultrasonically treated for 15 minutes to make it evenly dispersed. Then, 10μL of MIL-53Fe nanozyme (1.0mg / mL) was added to a mixed solution of H2O2 (10μL, 10mM) and TMB (100μL, 10mM). Subsequently, the system of the mixed solution was adjusted to 3mL and reacted at 37°C for 3 minutes, and then quickly used for UV-visible absorption experiments. The experimental results are shown in Figure 7 .

[0035] When only TMB and H2O2 are present in the solution, the solution has almost no color change, and no absorption peak is observed at 652nm. When only TMB and MIL-53Fe nanozyme are present in the solution, due to the lack of the co-oxidant H2O2, MIL-53Fe nanozyme has a slight catalytic oxidation effect on TMB, the color of the solution changes from colorless to light blue, and the absorption peak of the solution at 652nm is very weak. When H2O2 and MIL-53Fe nanozyme are present in the solution, after TMB is oxidized, the color of the solution changes to a darker blue-green, and the absorption at 652nm increases significantly. This experimental phenomenon further proves that MIL-53Fe nanozyme has peroxidase (POD)-like activity under neutral conditions.

[0036] Steady-state kinetic analysis: The steady-state kinetics of the enzymatic reaction of MIL-53Fe nanozyme as a POD mimetic under neutral conditions was analyzed by changing the concentrations of TMB and H2O2. The concentration of TMB was fixed at 0.1mM, and the concentration of H2O2 varied in the range of 0.05mM to 0.9mM. The H2O2 concentration was fixed at 0.8mM, the TMB concentration was 0.1 to 1.0mM, and the other conditions were the optimal catalytic conditions. Within a certain concentration range, the enzyme catalytic reaction usually follows the typical Michaelis-Menten kinetic model.

[0037]

[0038] Where, V represents the initial rate of the reaction; [S] is the substrate concentration (TMB or H2O2), V max is the maximum reaction rate, K m Corresponding to Michaelis constant. Enzyme steady-state kinetic parameter V max and K m It can be calculated based on the Line-Weaver-Burk line graph (double reciprocal plotting method) of the above model. As shown in Figure 8, it is calculated that the Km of TMB is 0.1139 mM, and V max =3.2×10-8Ms-1 ; K of H2O2 m =0.0426mM, V max =1.5×10 -8 Ms -1 .

[0039] Selective analysis of glucose detection: MIL-53(Fe) was dispersed in water to obtain a 1.0 mg / mL dispersion. 10 μL of MIL-53(Fe) suspension, 100 μL of 10 mM TMB solution, 10 μL of 1.0 mg / mL GOD solution and 100 μL of glucose solutions of different concentrations were placed in a 3 mL centrifuge tube, and pure water was added to make the reaction system 3 mL of solution. Mix well, incubate at 37°C for 10 min, and measure the UV absorption peak intensity at 652 nm. + , K + , Ca 2+ , Zn 2+ , NO3 - The same experiment was carried out using aqueous solutions of interfering substances such as vitamin C, glutathione and dopamine instead of glucose solution to verify the selectivity of the detection method of the present invention.

[0040] Detection of glucose in spiked serum samples: Take 10 μL of serum samples, 9 portions, 3 portions in a group, and add different concentrations of glucose standard solutions to each group. Then add 10 μL MIL-53 (Fe) suspension, 100 μL 10mM TMB solution and 10 μL 1mg / mL GOD solution in a 3mL centrifuge tube, add pure water to make the reaction system 3mL solution, mix well, incubate at 37 ° C for 10min, and measure the ultraviolet absorption peak intensity at 652nm. At the same time, compare the blank tube and the standard tube (glucose standard solution), calculate the concentration of glucose in serum, the concentration of glucose after spike recovery, and obtain the spike recovery rate and RSD value of the detection method of the present invention. The results are shown in Table 1. As shown in Table 1, the spike recovery rate of the method of the present invention is 101.8%, and the RSD value is 1.5%, indicating that the accuracy and precision of the method are good.

[0041] Table 1 Detection of glucose in spiked serum samples

[0042]

[0043]

[0044] Detection of human serum glucose: This method was used to detect the glucose concentration in actual human serum samples (5 samples) and compared with the test results of a commercial blood glucose meter. The results are shown in Table 2. It can be seen from Table 2 that compared with the commercial blood glucose meter, the results measured by this method are more accurate.

[0045] Table 2 Detection of human serum glucose

[0046]

[0047] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0048] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for detecting serum glucose based on MOFs nanozymes, characterized in that: The following steps are involved: S1. Mixing glucose oxidase (GOD) and serum, and using the catalytic reaction of glucose oxidase (GOD) on glucose to produce hydrogen peroxide (H2O2); S2, adding a mixed solution of MOFs nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB), wherein the MOFs nanozyme is specifically MIL-53Fe nanozyme, and the MIL-53Fe nanozyme catalyzes the reduction of hydrogen peroxide (H2O2) to produce hydroxyl radicals (··OH), and at the same time, the hydroxyl radicals (··OH) oxidize TMB to oxidized TMB (oxTMB), and the solution changes from colorless to blue, and this solution produces an ultraviolet absorption peak at 652nm; S3. Add pure water, mix well, incubate for 10 minutes, measure the ultraviolet absorption peak intensity at 652nm, compare the blank tube and the standard tube, determine the absorbance value, and calculate the concentration of glucose in the serum.

2. The method for detecting serum glucose based on MOFs nanozymes according to claim 1, characterized in that: The MIL-53Fe nanozyme was prepared by solvothermal method, and the specific preparation process is as follows: (1) Weigh 0.81 g of ferric chloride (FeCl3·6H2O) and 0.49 g of terephthalic acid (HOOCC6H4COOH (H2BDC)) and dissolve them in 61 mL of N,N-dimethylformamide (DMF). Ultrasonicate for 30 min and then stir magnetically for 1 h until they are completely dissolved. (2) The mixed solution was placed in a 100 mL stainless steel reactor with a polytetrafluoroethylene inner layer, sealed, reacted at 150° C. for 15 h, naturally cooled to room temperature, and the precipitate was washed several times by centrifugation with DMF and anhydrous ethanol, and then dried in a 60° C. forced air drying oven for 6 h to obtain MIL-53Fe nanozyme; (3) MIL-53Fe nanozyme was dispersed in water and ultrasonicated for 15 min to obtain a 1.0 mg / mL dispersed suspension.

3. The method for detecting serum glucose based on MOFs nanozymes according to claim 1, characterized in that: The specific dosage of each substance is: 10 μL of serum sample, 10 μL of MIL-53 (Fe) suspension, 100 μL of TMB solution, 10 μL of 1 mg / mL GOD solution, the total amount of solution is controlled at 3 ml, and pure water is used for the final addition to make the reaction system 3 mL of solution.

4. The method for detecting serum glucose based on MOFs nanozymes according to claim 1, characterized in that: In step S3, the incubation temperature is 37°C.

5. The method for detecting serum glucose based on MOFs nanozymes according to claim 1, characterized in that: In step S3, the blank tube is filled with distilled water, and the standard tube is filled with glucose standard solution.

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