FeCuZn nano-alloy, colorimetric biosensor and application

Abstract

The invention provides a FeCuZn nano-alloy, a colorimetric biosensor and application, and relates to the field of biosensors. The FeCuZn nano-alloy is prepared through a solvothermal method, three metal salts are used as precursors, citric acid is used as a reducing agent, Mo (CO) 6 is used as an auxiliary reducing agent, polyvinylpyrrolidone is used as a stabilizer, and N, N-dimethylformamide is used as a solvent; the metal salt is composed of iron salt, copper salt and zinc salt. The prepared FeCuZn nano-alloy belongs to a novel multi-metal nano-enzyme, consumption of precious metal is avoided, and meanwhile, the FeCuZn nano-alloy has the catalytic characteristic similar to that of natural catalase; the constructed sensor has excellent detection performance on glucose and glutathione, has the advantages of low cost, simplicity and convenience in operation and rapidness in detection, and has a wide application prospect in the actual production fields of medicine, biology and the like.

Description

Technical Field

[0001] The present invention relates to the field of biosensors, and in particular to a FeCuZn nano alloy, a colorimetric biosensor and applications. Background Art

[0002] Compared to natural enzymes, nanozymes combine the unique properties of nanomaterials with the high catalytic performance of enzymes. In addition, the stable physical / chemical properties and low cost of nanozymes make them an attractive enzyme mimic. Because the metal active center can mimic the process of electron redox catalysis by natural enzymes, most existing nanozymes are metals and metal oxides. Among them, multi-metal nanozymes exhibit excellent enzymatic activity due to their rich active sites. The superior chemical stability and electrochemical activity of multi-metal nanozymes are attributed to the efficient charge transfer ability and synergistic effect between different elements. Through the synergistic interaction of various elements, synergistic effects between different sites can be achieved and enzyme activity can be optimized. In addition, the catalytic efficiency and activity of multi-metal nanozymes can be tuned by changing the composition and structure, which is closely related to the synthesis conditions, including the oxidation state of the metal center, the reducing agent, and the temperature. Currently, nanozymes based on precious metals (including platinum (Pt), palladium (Pd), and gold (Au)) are widely used in various fields due to their inherent properties. However, the high price of precious metals has limited their practical application to a certain extent. Therefore, the development of non-precious metal-based nanozymes with high catalytic activity is necessary.

[0003] Glucose (GLU) is an indispensable component of body fluids, and its concentration and structural changes are crucial for various physiological activities. Too low a concentration may lead to stroke or other vascular diseases. High concentrations of GLU may lead to obesity, diabetes, kidney disease, heart disease, and nerve damage. In order to monitor metabolism in the body and assess the course of disease, it is crucial to accurately measure the glucose concentration in body fluids. Currently, the main methods for detecting glucose include glucokinase assay, photochemical method, and electrochemical method. As the main method for glucose quantification, glucokinase assay is performed by detecting enzymatic byproducts produced by glucose oxidation. Due to its simple operation, rapidity, and low cost, the colorimetric glucokinase assay is the most widely used method.

[0004] Glutathione (GSH) is an important intracellular substance that promotes the metabolism of substances in the human body. Glutathione is also an essential antioxidant with the ability to inhibit free radicals and plays a vital role in human physiological activities. However, the disruption of GSH homeostasis may also lead to a series of diseases, including cardiovascular disease, cancer, aging, cystic fibrosis and excessive oxidative stress. Therefore, accurate and sensitive detection of GSH plays an important role in biomedical research, clinical monitoring and diagnosis. Currently, methods used for quantitative analysis of GSH include mass spectrometry, high performance liquid chromatography, electrochemical methods and molecular spectroscopy. Among them, colorimetry is a sensitive, simple and rapid method for detecting GSH. Summary of the Invention

[0005] In view of this, the present invention proposes a FeCuZn nanoalloy, a colorimetric biosensor and applications.

[0006] The technical solution of the present invention is achieved as follows:

[0007] A FeCuZn nanoalloy is prepared by a solvothermal method, using three metal salts as precursors, citric acid as a reducing agent, Mo(CO)6 as an auxiliary reducing agent, polyvinyl pyrrolidone as a stabilizer, and N,N-dimethylformamide as a solvent; the metal salts are composed of iron salt, copper salt, and zinc salt.

[0008] The molar ratio of the iron salt, the copper salt and the zinc salt is 9-11:9-11:9-11; the molar ratio of the iron salt, the copper salt and the zinc salt is 9-11:9-11:9-11.

[0009] Furthermore, the iron salt is FeSO4·7H2O, the copper salt is CuCl2·2H2O, and the zinc salt is Zn(CH3COO)2·2H2O.

[0010] Furthermore, the preparation method of the FeCuZn nanoalloy includes the following steps: mixing iron salt, copper salt and zinc salt; adding Mo(CO)6, citric acid, polyvinyl pyrrolidone and N,N-dimethylformamide, and ultrasonically treating; after dissolving, transferring it to a polytetrafluoroethylene reactor for reaction; after natural cooling, centrifuging, washing with anhydrous ethanol, and drying to obtain the alloy.

[0011] Furthermore, the mass ratio of the iron salt to Mo(CO)6, citric acid and polyvinyl pyrrolidone is 46:25-35:500-700:1500-2500.

[0012] Furthermore, the ultrasonic time is 20-40 minutes.

[0013] Furthermore, the reaction temperature is 180-220° C., and the reaction time is 6-10 h.

[0014] Furthermore, the centrifugal speed is 10000-15000 r / min, and the centrifugal time is 10-20 minutes.

[0015] A colorimetric biosensor is prepared using the FeCuZn nanoalloy described in any one of the embodiments of the present invention.

[0016] The FeCuZn nano alloy described in any one of the items of the present invention is used in detecting glucose and glutathione.

[0017] Furthermore, the glucose detection method includes the following steps: incubating glucose solutions of different concentrations with glucose oxidase; then adding FeCuZn nanoalloy solution, TMB solution and acetate buffer to obtain a mixed solution, placing it in a constant temperature shaker for reaction, and detecting the absorbance after the reaction.

[0018] Furthermore, the glutathione detection method includes the following steps: adding HAc-NaAc buffer solution, FeCuZn nanoalloy solution, TMB solution, H2O2 solution and GSH solutions of different concentrations to obtain a mixed solution, placing it in a constant temperature shaker for reaction, and detecting the absorbance after the reaction.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] A novel non-precious metal FeCuZn nanoalloy was successfully prepared, and a highly sensitive and selective colorimetric sensor for the detection of glucose (GLU) and glutathione (GSH) was constructed. Experimental results showed that the FeCuZn nanoalloy exhibited superior peroxidase-like activity compared to the FeCuCe nanoalloy, oxidizing 3,3,5,5-tetramethylbenzidine (TMB) to blue oxTMB in the presence of H2O2, resulting in a change in the absorbance of the system at 652 nm. Glucose can produce H2O2 under the catalysis of glucose oxidase (GOD), while the reducing activity of glutathione can eliminate oxTMB oxidized by hydroxyl radicals. Therefore, the FeCuZn nanoalloy-based colorimetric sensor can be used to detect GLU and GSH. The sensor had detection limits of 3.10 μmol / L for GLU and 0.212 μmol / L for GSH, with linear ranges of 10-800 μmol / L and 0.7-50 μmol / L, respectively. The feasibility of the sensor was verified by qualitative detection in human serum and saliva samples.

[0021] The FeCuZn nanoalloy prepared by the present invention is a new type of multi-metal nanozyme that avoids the consumption of precious metals and has catalytic properties similar to natural catalase. The constructed sensor has excellent detection performance for GLU and GSH, and has the advantages of low cost, simple operation and rapid detection, indicating that it has broad application prospects in actual production fields such as medicine and biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Schematic diagram of the detection mechanism of H2O2, GLU and GSH.

[0023] Figure 2 : (a) Transmission electron microscopy (TEM) image of FeCuZn nanoalloy, (bg) energy dispersive spectroscopy (EDS) elemental distribution map, (h) X-ray photoelectron spectroscopy (XPS) spectrum of FeCuZn nanoalloy.

[0024] Figure 3 : (a) Transmission electron microscopy (TEM) image of FeCuCe nanoalloy, (bf) energy dispersive spectroscopy (EDS) element distribution maps.

[0025] Figure 4 : (a) X-ray photoelectron spectroscopy (XPS) spectrum of FeCuCe nanoalloy, high-resolution (b) Fe 2p and (c) Cu 2p XPS spectra.

[0026] Figure 5 : High-resolution (a) Fe 2p, (b) Cu 2p, and (c) Zn 2p XPS spectra of FeCuZn nanoalloy.

[0027] Figure 6 : UV-visible absorption spectra of FeCuZn nanoalloy under different conditions.

[0028] Figure 7 :(a) UV-visible absorption spectrum with changes in H2O2 concentration, (b) linear relationship between absorbance value at 652nm and H2O2 concentration (in the range of 0.1-50μmol / L).

[0029] Figure 8 : (a) Standard curve of absorbance at 652 nm versus GLU concentration (in the range of 10-800 μmol / L), (b) shows the absorption response of the proposed probe to various biomolecules and ions.

[0030] Figure 9:(a) UV-visible absorption spectrum as a function of GSH concentration, (b) standard curve of absorbance at 652 nm and GSH concentration, (c) absorption response of the constructed probe to various biomolecules and ions, (d) standard curve of absorbance at 652 nm and GSH concentration in human saliva. DETAILED DESCRIPTION

[0031] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0032] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0033] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0034] Table 1 Chinese meanings of English or abbreviations

[0035] English or abbreviation Chinese name PVP Polyvinylpyrrolidone DMF N,N-Dimethylformamide TMB 3,3',5,5'-Tetramethylbenzidine GSH Glutathione GLU glucose GOD Glucose oxidase Nanoalloy Nano alloys Intensity strength Binding Energy Binding energy Wavelength wavelength Starch starch Maltose maltose Levulose fructose Sucrose sucrose Glycine Glycine L-arginine L-Arginine

[0036] Example

[0037] In this invention, non-precious metal-based multi-metal nanozymes, FeCuZn and FeCuCe nanoalloys, were successfully prepared for the first time. These multi-metal nanozymes exhibited excellent peroxidase-like activity and extremely high affinity for H2O2, and then oxidized TMB to produce ox-TMB, which increased the absorbance at 652nm. Based on the excellent activity of FeCuZn nanoalloys and the change in colorimetric signal, H2O2 was used as an intermediate to construct a biosensor with high selectivity and sensitivity for GLU and GSH ( Figure 1 ).

[0038] Experimental part

[0039] 1.1 Reagents and Instruments

[0040] CuCl2·2H2O, FeSO4·7H2O, Zn(CH3COO)2·2H2O, NaCH3COO, Mo(CO)6, CH3CH2OH, CH3COOH, H2O2 (30% by mass), citric acid, polyvinylpyrrolidone (PVP), N,N-dimethylformamide (DMF), 3,3',5,5'-tetramethylbenzidine (TMB), glucose (GLU), glutathione (GSH), and N-ethylmaleimide were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). All the above reagents were of analytical grade and used without further purification. Double-distilled water was used in all experiments.

[0041] UV-visible absorption spectra were measured using a Hitachi U-2600 spectrophotometer. The morphology of FeCuZn and FeCuCe nanoalloys was characterized by a JEOL Jem-2100F field emission electron microscope. X-ray photoelectron spectroscopy (XPS) characterization of FeCuZn and FeCuCe nanoalloys was performed at Thermo Scientific. TM K-Alpha TM+ On the spectrometer.

[0042] 1.2 Preparation of FeCuZn and FeCuCe nanoalloys

[0043] First, 4.6 mg of FeSO₄·7H₂O, 2.8 mg of CuCl₂·2H₂O, and 3.6 mg of Zn(CH₃COO)₂·2H₂O were mixed in a glass beaker. Then, 3.0 mg of Mo(CO)₆, 60 mg of citric acid, 200 mg of PVP, and 10 mL of DMF were added sequentially, and the mixture was sonicated for 30 minutes. After dissolution, the material was transferred to a polytetrafluoroethylene reactor and reacted at 200°C for 8 hours. After cooling naturally, the mixture was centrifuged at 12,000 rpm for 15 minutes and washed three times with anhydrous ethanol. The centrifuged product was dried in an oven at 60°C and stored for subsequent use.

[0044] The synthesis method of FeCuCe nanoalloy was the same except that 3.6 mg Zn(CH3COO)2·2H2O was replaced by 7.2 mg Ce(NO3)3.

[0045] 1.3 Steady-state kinetic analysis of FeCuZn and FeCuCe nanoalloys

[0046] 1.3.1 TMB as a substrate

[0047] To a HAc-NaAc buffer solution (pH 4.5, 0.1 mol / L), 250 μL of the nanoalloy solution (0.2 g / L), various H₂O₂ solutions (10, 15, 20, 25, and 30 mmol / L), and 75 μL of TMB solution (24 mmol / L) were added to create a 2.7 mL solution. The absorbance at 652 nm was measured every 20 seconds, and data were collected and analyzed over a five-minute period. Three replicates were performed for each group.

[0048] 1.3.2 Using H2O2 as substrate

[0049] In HAc-NaAc buffer solution (pH = 4.5, 0.1 mol / L), 250 μL of nanoalloy solution (0.2 g / L), 50 mL of H2O2 (50 mmol / L) and different concentrations of TMB (0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.6, 0.8, 1 mmol / L) were added to form a 2.7 mL system, and the absorbance of each group was measured three times within five minutes.

[0050] 1.4 Colorimetric detection of H2O2

[0051] A HAc-NaAc buffer solution (pH 4.5, 0.1 mol / L), 250 mL of the nanoalloy solution (0.2 g / L), various H2O2 concentrations, and 75 μL of TMB solution (24 mmol / L) were sequentially added to the test tube. The H2O2 concentrations in the mixture were 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.5, 5.0, 10, 20, 30, 40, and 50 μmol / L, respectively. After reacting at 35°C for 5 minutes, the absorbance at 652 nm was measured, and the absorption spectrum from 300 to 700 nm was measured at 2 nm intervals.

[0052] 1.5 Colorimetric detection of GLU and GSH

[0053] 100 μL of glucose solutions of varying concentrations and 100 μL of glucose oxidase (1 mg / mL) were incubated at 37°C for 30 minutes. Then, 100 μL of FeCuZn nanoalloy solution (0.25 g / L), 50 μL of 24 mM TMB solution, and acetate buffer (pH 3.5) were added. The solutions were mixed and placed in a thermostatic shaker set at 35°C for 5 minutes. The absorbance was then measured, with each group repeated three times.

[0054] A solution system was formed by adding HAc-NaAc buffer solution (pH = 4.5, 0.1 mol / L), 250 mL of FeCuZn nanoalloy solution (0.2 g / L), 75 μL of TMB solution (24 mmol / L), 50 mL of H2O2 solution (50 mmol / L), and different concentrations of GSH solution (1, 10, 20, 30, 40, 50, 60, 70, and 80 μmol / L). After reacting in a 35°C constant temperature shaker for 5 minutes, the absorbance at 652 nm was measured. Three parallel experiments were performed for each group.

[0055] 1.6 Detection of GLU and GSH in actual samples

[0056] Human blood samples were obtained from the First Affiliated Hospital of Hainan Medical University. After centrifugation and filtration, the samples were diluted 100-fold with distilled water and spiked with various concentrations of GLU. The prepared samples were then tested according to the above procedures.

[0057] Saliva samples were collected as follows: volunteers rinsed their mouths with drinking water, minimizing swallowing. Saliva was collected in centrifuge tubes, filtered through a 0.22 μm syringe filter, and stored at -20°C for later use. To each 100 μL saliva sample, 1 μL of a GSH standard solution at 25, 50, 75, 100, 150, or 200 μmol / L was added. The prepared samples were then tested according to the above steps.

[0058] 2 Results and Discussion

[0059] 2.1 Characterization of FeCuZn and FeCuCe nanoalloys

[0060] FeCuZn and FeCuCe nanoalloys were prepared by a solvothermal method using equimolar amounts of various metal salts as precursors, citric acid as a reducing agent, Mo(CO)6 as an auxiliary reducing agent, PVP as a stabilizer, and DMF as a solvent. Figure 2 As shown in a, a single FeCuZn nanoalloy is composed of several nanosheets. The nanoalloy is irregular in shape and has a lateral size of about 170±3nm. In contrast, the FeCuCe nanoalloy is a nearly circular nanosheet with a diameter of about 200nm. Many nanosheets are clustered together ( Figure 3 a). Energy dispersive elemental distribution spectroscopy (EDS) images of FeCuZn nanoalloys show that Fe and Cu are evenly distributed throughout the nanomaterial, while the Zn content is relatively low. C and O still exist in the nanomaterial ( Figure 2 bg). Fe, Cu and Ce are evenly distributed in the FeCuCe nanoalloy, indicating that the multi-metal nanozyme was successfully prepared ( Figure 3 bf).

[0061] Figure 2 The XPS results of h show that the atomic ratios of Fe, Cu, Zn, C and O in the FeCuZn nanoalloy are 9:2:1:34:54. The peaks at 711.0 and 724.7 eV correspond to Fe 2+ 2p 3 / 2 and 2p 1 / 2 ( Figure 5 a). Cu 2p 3 / 2 , the peaks at ≈932.2 eV and 933.9 eV are attributed to Cu + and Cu 2+ The shock peaks at 940.6, 943.7 and 962.6 eV further indicate the presence of divalent copper ( Figure 5 b). The peaks at ≈1021.5 and 1044.6 eV correspond to Zn 2+ 2p 3 / 2 and 2p 1 / 2 ( Figure 5 c). Figure 4 a shows that the atomic ratios of Fe, Cu, Ce, C, and O in the FeCuCe nanoalloy are 2.5%, 0.5%, 5%, 51%, and 41%, respectively. The peaks at 709.2 and 710.4 eV are attributed to Fe 2+ 2p 3 / 2 and Fe 3+ 2p 3 / 2 Multiple shock peaks further indicate the presence of trivalent iron ( Figure 4 b) Cu XPS curve fitting proves that Cu + and Cu 2+ The existence of Figure 4 c).

[0062] 2.2 Analysis of peroxidase-like activity of FeCuZn and FeCuCe nanoalloys

[0063] When TMB is used as a substrate, nanozymes can catalyze the reduction of H2O2 to ·OH, further oxidizing colorless TMB to blue ox-TMB. Figure 6 ) showed that in the mixture of FeCuZn nanoalloy and TMB, the mixture of FeCuZn nanoalloy and H2O2, and the system containing only H2O2 and TMB, no characteristic absorption peak at 652nm appeared, and the solution did not appear blue. However, in the case of the simultaneous presence of FeCuZn nanoalloy, H2O2 and TMB, the absorbance at 652nm was significantly higher than that of the other three groups, and the solution was dark blue, indicating that a large amount of ox-TMB was produced in the system. FeCuCe nanoalloy showed a similar phenomenon. In summary, FeCuZn and FeCuCe nanoalloys have peroxidase-like activity, which can catalyze H2O2 to produce ·OH and further oxidize TMB to ox-TMB.

[0064] In order to further explore the peroxidase-like activity of FeCuZn and FeCuCe nanoalloys, the absorbance at 652 nm was detected by changing the concentrations of H2O2 and TMB, and the kinetic parameters were calculated by the Michaelis-Menten equation and the Linewever-Burk equation. m The smaller the value, the greater the affinity of the enzyme for the substrate, while the larger the ν m The value indicates that the higher the initial reaction rate of the enzyme, the stronger the catalytic activity. As shown in Table 2, the Km The value is close to horseradish peroxidase (HRP), while the ν m The value is higher than that of HRP, indicating that the catalytic activity of FeCuZn nanoalloy is close to that of natural enzyme. Compared with FeCuZn nanoalloy, the catalytic activity of FeCuCe nanoalloy is weaker, which may be affected by the lower Fe(Ⅱ) content.

[0065] Table 2 Steady-state kinetic parameters of FeCuZn and FeCuCe nanoalloys

[0066]

[0067] 2.3 Determination of H2O2

[0068] Based on the peroxidase-like activity of FeCuZn and FeCuCe nanoalloys, a simple, low-cost and sensitive colorimetric biosensor was developed for the determination of H2O2. After optimizing the detection conditions (including the concentration of FeCuZn nanoalloy and TMB, pH value, incubation temperature and time), Figure 7 The absorption spectra of systems containing different concentrations of H2O2 are shown. The experimental results show that with the addition of H2O2, the color of the solution gradually deepens and the absorbance value increases. Figure 7 As shown in Figure b, the absorbance at 652 nm is proportional to the H2O2 concentration. In the range of 0.1-50 μmol / L, there is a good linear relationship between the H2O2 concentration and the absorbance, and the linear equation is A=0.0124C H2O2 +0.169 (R=0.999). The detection limit of H2O2 was calculated as 0.0283 μmol / L by the ratio of three times the standard deviation to the slope of the calibration curve (3σ / s, n=11).

[0069] Using the same experimental steps, the detection performance of a biosensor based on FeCuCe nanoalloy for H2O2 was also demonstrated. The detection limit for H2O2 using the FeCuCe nanoalloy biosensor was 0.320 μmol / L, more than 10 times higher than that of the FeCuZn nanoalloy-based biosensor. This result was attributed to its weaker catalytic performance compared to the FeCuZn nanoalloy.

[0070] 2.4 Colorimetric detection of GLU and GSH

[0071] FeCuZn nanoalloys catalyze the conversion of H₂O₂ to ·OH. Under the catalysis of glucose oxidase, GLU produces H₂O₂, which in turn generates more ox-TMB. Due to the sulfhydryl groups in GSH, the ox-TMB generated by the ·OH reaction is reduced, rendering the system colorless. Therefore, FeCuZn nanoalloy-based biosensors can be used for the indirect determination of GLU and GSH.

[0072] like Figure 8 As shown in a, as the concentration of GLU in the standard solution increases, the absorbance at 652nm increases. In the range of 10-800μmol / L, a good linear relationship was obtained between absorbance and GLU concentration, and the standard curve was A=0.00554C GLU +0.0312 (R = 0.991), with a detection limit of 3.10 μmol / L. The low absorbance values ​​of starch, maltose, sucrose, potassium chloride, sodium chloride, calcium chloride, glycine, and L-arginine at 652 nm indicate that the biosensor based on FeCuZn nanoalloy has good selectivity and anti-interference ability, and can meet the high selectivity requirements of glucose colorimetry and actual sample detection ( Figure 8 b).

[0073] In the standard solution, as the concentration of GSH increases, the characteristic absorption peak at 652 nm gradually decreases ( Figure 9 a). In the ranges of 0.7-15 μmol / L and 15-50 μmol / L, the absorbance value was linearly related to the GSH concentration, and the linear equations were A=-0.0417C GSH +1.138 (R=0.989) and A=-0.0079C GSH +0.722(R=0.992)( Figure 9 b), with a detection limit of 0.212 μmol / L. In order to study the effect of possible interfering substances on GSH determination, different concentrations of amino acids (L-histidine, D-histidine, L-arginine, D-arginine, glycine) and common ions (Na + , K + , Ca 2+ ). Figure 9 c verified that there was no significant change in the absorbance in the presence of interfering substances, indicating that the FeCuZn nanoalloy-based biosensor has excellent anti-interference ability for GSH.

[0074] Table 3 shows the performance comparison of the FeCuZn nanoalloy-based biosensor and other colorimetric sensors in terms of detection limit. As can be seen from the table, the FeCuZn nanoalloy-based biosensor developed in the present invention exhibits the highest sensitivity to GLU and GSH.

[0075] Table 3 Comparison of analytical performance of the biosensor constructed by the present invention and the determination method reported in the literature

[0076]

[0077] 2.5 Detection of GLU and GSH in actual samples

[0078] A colorimetric method for the detection of glucose in serum was established using a FeCuZn nanoalloy-based biosensor. As shown in Table 4, the glucose concentrations in the diluted serum samples were 26.36 and 25.21 μmol / L (relative standard deviations (RSDs) of 2.1% and 2.6%, respectively). Spiked recoveries for human serum samples ranged from 95.9% to 106.6%, demonstrating the potential of this biosensor for analyzing complex samples.

[0079] Different concentrations of GSH standard solution were added to the pretreated saliva samples. The relationship between the absorbance value at 652nm and the concentration of GSH mixed solution is as follows: Figure 9 d. The higher the GSH concentration in saliva, the lower the absorbance value. There is a good linear relationship between the absorbance value and the concentration value, and the linear equation is A = -6.300C GSH +1.1804 (R=0.999), indicating that the biosensor can also be used to determine the GSH content in complex matrices.

[0080] Table 4 Determination of GLU content in human serum samples (n=3, at 95% confidence level)

[0081]

[0082] in conclusion

[0083] The present invention establishes a colorimetric biosensor for GLU and GSH analysis, using FeCuZn as a highly active nanozyme, TMB as a color developer, and H2O2 as an intermediate. The peroxidase-like activity of the FeCuZn nanoalloy is similar to that of HRP. Under the influence of H2O2, TMB can be oxidized by the FeCuZn nanoalloy to blue ox-TMB, increasing the absorbance at 652nm. Because the nanoalloy does not use precious metals, the sensor is not only simple to operate but also has significant cost advantages. The colorimetric biosensor has been successfully applied to the determination of GLU and GSH in human serum and saliva, demonstrating high sensitivity. This method opens up new avenues for the design and application of probes based on FeCuZn nanoalloys. In addition, the sensor developed by the present invention can also be used to detect other analytes related to H2O2.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the scope of the present invention should be included in the scope of protection of the present invention.

Claims

1. A FeCuZn nanoalloy, characterized in that: The FeCuCe nanoalloy is prepared by a solvothermal method, using three metal salts as precursors, citric acid as a reducing agent, Mo(CO)6 as an auxiliary reducing agent, polyvinyl pyrrolidone as a stabilizer, and N,N-dimethylformamide as a solvent; the metal salts are composed of iron salts, copper salts, and zinc salts.

2. The FeCuZn nanoalloy according to claim 1, characterized in that The molar ratio of the iron salt, copper salt and zinc salt is 9-11:9-11:9-11; the iron salt is FeSO4·7H2O, the copper salt is CuCl2·2H2O, and the zinc salt is Zn(CH3COO)2·2H2O.

3. The FeCuZn nanoalloy according to claim 1 or 2, characterized in that The preparation method of the FeCuZn nano alloy comprises the following steps: mixing iron salt, copper salt and zinc salt; adding Mo(CO)6, citric acid, polyvinyl pyrrolidone and N,N-dimethylformamide, and ultrasonically treating; after dissolving, transferring the mixture into a polytetrafluoroethylene reactor for reaction; naturally cooling, centrifuging, washing with anhydrous ethanol, and drying to obtain the alloy.

4. The FeCuZn nanoalloy according to claim 3, characterized in that The mass ratio of the iron salt to Mo(CO)6, citric acid and polyvinyl pyrrolidone is 46:25-35:500-700:1500-2500.

5. The FeCuZn nanoalloy according to claim 3, characterized in that The ultrasonic time is 20-40 minutes.

6. The FeCuZn nanoalloy according to claim 3, characterized in that The reaction temperature is 180-220° C., and the reaction time is 6-10 h.

7. The FeCuZn nanoalloy according to claim 3, characterized in that The centrifugal speed is 10000-15000 r / min, and the centrifugal time is 10-20 minutes.

8. A colorimetric biosensor, characterized in that It is prepared using the FeCuZn nano alloy described in any one of claims 1 to 7.

9. Use of the FeCuZn nanoalloy according to any one of claims 1 to 7 in detecting glucose and glutathione.

10. The use according to claim 9, characterized in that The glucose detection method comprises the following steps: incubating a glucose solution with glucose oxidase; then adding a FeCuZn nano alloy solution, a 3,3',5,5'-tetramethylbenzidine solution and an acetate buffer to obtain a mixed solution, placing the solution in a constant temperature shaker for reaction, and detecting the absorbance after the reaction; The glutathione detection method comprises the following steps: adding HAc-NaAc buffer solution, FeCuZn nano alloy solution, 3,3',5,5'-tetramethylbenzidine solution, H2O2 solution and glutathione solution to obtain a mixed solution, placing the solution in a constant temperature shaker for reaction, and detecting the absorbance after the reaction.

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