A method for yeast-assisted synthesis of porous carbon / vanadium sulfide nanozymes and its application in serum creatinine detection

Abstract

The present invention discloses a method for yeast-assisted synthesis of porous carbon / vanadium sulfide nanozyme and its application in serum creatinine detection, belonging to the technical field of serum creatinine detection. The method comprises the following steps: (1) mixing yeast, vanadium chloride and ethanol, stirring until the liquid is completely volatilized after ultrasonic dispersion, and obtaining a first solid A; (2) mixing and grinding the first solid substance A, potassium carbonate and potassium chloride to obtain a second solid B; (3) placing the second solid substance B and thioacetamide in the grooves at the left and right ends of the graphite sample stage of the Joule heat device, respectively, and performing a rapid Joule heat shock to obtain a third solid C; (4) washing and drying the third solid C to obtain the porous carbon / vanadium sulfide nanozyme. Based on the nanozyme prepared by the present invention, a colorimetric sensing method is constructed for creatinine detection, which has an extremely low detection limit, with LOD as low as 0.07µM / mL, reflecting high specificity and sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of serum creatinine detection, and in particular to a method for yeast-assisted synthesis of porous carbon / vanadium sulfide nanozyme and application thereof in serum creatinine detection. Background Art

[0002] Biomarkers are biochemical indicators used to objectively measure or evaluate physiological, pathological or therapeutic processes. By detecting biomarkers, we can understand the patient's disease progression. In recent years, the detection of specific biomarkers has become an important part of disease identification, early diagnosis, prevention and treatment. Creatinine (CR) is a muscle metabolite, mainly produced by creatine phosphate metabolism, transported to the kidneys through the blood and excreted through glomerular filtration. Therefore, the creatinine level in the blood can reflect the filtration function of the kidneys and is an important basis for doctors to judge kidney health. When the blood creatinine level exceeds the normal range, it may indicate a decrease in glomerular filtration rate and kidney damage. In addition, the trend of blood creatinine changes can also reflect the progression of the disease, and monitoring its level helps to assess the severity of the disease and the effectiveness of treatment.

[0003] Clinically, serum creatinine is mainly detected by enzymatic method and picric acid method. The picric acid method is based on the reaction of creatinine with picric acid under alkaline conditions to generate an orange-red complex, and the creatinine concentration is determined by colorimetry. However, this method is easily interfered by a variety of substances and has poor specificity. It is not suitable for jaundice, hemolysis and other conditions. In contrast, the enzymatic method has the advantages of small sample requirements, fast speed and high specificity. Creatine is generated by creatinine under the catalysis of creatinase, followed by a series of reactions to generate urea and other products, and finally the creatinine content is determined by colorimetry. However, natural enzymes are difficult to prepare and purify, costly, sensitive to the environment, and easy to inactivate. Nanozymes are a class of nanomaterials with similar enzyme catalytic properties. Compared with natural enzymes, nanozymes are low in cost, high in stability and more durable, but the activity of artificially synthesized nanozymes currently used for creatinine detection is relatively low, which limits its application. Therefore, it is of great clinical significance to develop an economical and effective method to synthesize highly active nanozymes to replace natural enzymes for blood creatinine detection.

[0004] Transition metal sulfides have been widely reported to have enzyme-mimicking activity due to their weaker MS bonds, which make enzyme-catalyzed reactions more kinetically favorable. They have also been successfully used in colorimetric platforms for detecting H2O2, ascorbic acid, and other small molecules. The catalytic activity of nanozymes can be optimized by introducing surface defects, which enhance the activity by affecting the electronic and geometric properties of the catalytic active sites. However, the metal sulfide nanozymes prepared by traditional methods have the problem of complex defect introduction processes and a limited variety of defects. Therefore, it is particularly important to develop a simple and effective method to introduce abundant defects into nanozymes to significantly enhance their catalytic activity. Summary of the invention

[0005] The purpose of the present invention is to provide a method for yeast-assisted synthesis of porous carbon / vanadium sulfide nanozymes and its application in serum creatinine detection, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a method for yeast-assisted synthesis of porous carbon / vanadium sulfide nanozymes, comprising the following steps:

[0008] (1) mixing yeast, vanadium chloride and ethanol, and stirring them after ultrasonic dispersion until the liquid is completely volatilized to obtain a first solid material A;

[0009] (2) mixing and grinding the first solid material A, potassium carbonate and potassium chloride to obtain a second solid material B;

[0010] (3) placing the second solid substance B and thioacetamide in the grooves at the left and right ends of the graphite sample stage of the Joule heat device, respectively, and subjecting them to rapid Joule heat shock to obtain a third solid substance C;

[0011] (4) The third solid object C is washed and dried to obtain the porous carbon / vanadium sulfide nanozyme.

[0012] The second technical solution of the present invention is the porous carbon / vanadium sulfide nanozyme prepared by the method.

[0013] The third technical solution of the present invention is the application of the porous carbon / vanadium sulfide nanozyme in creatinine detection.

[0014] A fourth technical solution of the present invention is a method for quantitatively detecting creatinine for purposes other than disease diagnosis or treatment, which utilizes the porous carbon / vanadium sulfide nanozyme to quantitatively detect creatinine in a sample to be tested.

[0015] A fifth technical solution of the present invention is the use of the porous carbon / vanadium sulfide nanozyme in the preparation of products for creatinine detection.

[0016] Based on the above technical solution, the present invention has the following technical effects:

[0017] 1. The present invention adopts a yeast-assisted high-temperature solid-phase vulcanization reaction, in which the synergistic etching of potassium carbonate and potassium chloride promotes the doping and modification of porous carbon generated by yeast pyrolysis by S elements to form a porous carbon material rich in defects. At the same time, these defect-rich porous carbons provide a large number of growth sites for the growth of vanadium sulfide crystals, inhibiting the agglomeration of crystals, and part of the vanadium ions are reduced to vanadium carbide by porous carbon, thereby forming vanadium defects in vanadium sulfide crystals. In addition, the P element in the yeast partially replaces the S element in the vanadium sulfide crystal at high temperature, forming S ion defects. These defects can optimize the geometric and electronic structure of the nanozyme and enhance the intrinsic activity of the enzyme catalytic active site.

[0018] 2. Porous carbon as a carrier helps to disperse the active substance nanozyme, thereby exposing more enzyme catalytic active sites. At the same time, porous carbon can enrich reactants, provide a reactant-enriched microenvironment for the active sites, and promote the adsorption of reactants at the active sites.

[0019] 3. Based on the nanozyme prepared by the present invention, a colorimetric sensing method was constructed for creatinine detection. The method has an extremely low detection limit, with LOD as low as 0.07µM / mL, which reflects high specificity and sensitivity.

[0020] 4. The preparation method of the nanozyme of the present invention is simple, and it also has high stability and low cost, which can overcome the shortcomings of natural enzymes that are easily inactivated and expensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only 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.

[0022] Figure 1 2 is the scanning electron microscope image of Example 1 and the corresponding element distribution map.

[0023] Figure 2 This is the nitrogen adsorption-desorption isotherm curve of Example 1.

[0024] Figure 3 This is the Raman spectrum of Example 1.

[0025] Figure 4 The electron paramagnetic resonance spectra of Example 1, Comparative Example 1 and Comparative Example 3 are shown.

[0026] Figure 5 This is the X-ray photoelectron spectroscopy (XPS) graph of Example 1.

[0027] Figure 6The figure is a comparison chart of the peroxidase-like activity of the samples of Example 1 and Comparative Examples 1-3.

[0028] Figure 7 The UV absorption spectra of the CR / TMB / H2O2 / PC / V3S4-1 nanozyme system with different creatinine (CR) concentrations.

[0029] Figure 8 This is the relationship between UV absorption value and creatinine concentration. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0036] The embodiment of the present invention provides a method for yeast-assisted synthesis of porous carbon / vanadium sulfide nanozymes, comprising the following steps:

[0037] (1) mixing yeast, vanadium chloride and ethanol, and stirring them after ultrasonic dispersion until the liquid is completely volatilized to obtain a first solid material A;

[0038] (2) mixing and grinding the first solid material A, potassium carbonate and potassium chloride to obtain a second solid material B;

[0039] (3) placing the second solid substance B and thioacetamide in the grooves at the left and right ends of the graphite sample stage of the Joule heat device, respectively, and subjecting them to rapid Joule heat shock to obtain a third solid substance C;

[0040] (4) The third solid object C is washed and dried to obtain the porous carbon / vanadium sulfide nanozyme.

[0041] In some specific embodiments, the mass volume ratio of the yeast, vanadium chloride and ethanol is 1 mg: (0.05-0.2) mg: (0.5-10) mL.

[0042] In some specific embodiments, the mass ratio of the first solid substance A, potassium carbonate and potassium chloride is 1 mg: (1-5) mg: (1-10) mg.

[0043] In some specific embodiments, the mass ratio of the second solid substance B to thioacetamide is 1:(1-10);

[0044] The conditions of the rapid Joule thermal shock are: in a nitrogen atmosphere, the temperature is 600-1600° C., the Joule thermal shock heating rate is 1500-2500° C. / s, and the number of Joule thermal shocks is 1-6 times.

[0045] The embodiments of the present invention also provide a porous carbon / vanadium sulfide nanozyme prepared by the method.

[0046] The embodiments of the present invention also provide the application of the porous carbon / vanadium sulfide nanozyme in creatinine detection.

[0047] The embodiment of the present invention also provides a method for quantitatively detecting creatinine for non-disease diagnosis or treatment purposes, using the porous carbon / vanadium sulfide nanozyme to quantitatively detect creatinine in a sample to be tested.

[0048] In some specific embodiments, the steps include:

[0049] Mix the buffer with creatinase, creatininase, sarcosine oxidase and the sample to be tested and incubate;

[0050] After the incubation, NaAc-HAc buffer, porous carbon / vanadium sulfide nanozyme suspension, and 3,3',5,5'-tetramethylbenzidine were added for a second incubation;

[0051] The creatinine in the solution was quantitatively detected by absorbance.

[0052] In some specific embodiments, the buffer is 0.015 M PBS, pH 7.5; the concentration of creatinase is 0.1 mg / mL; the concentration of creatininease is 0.2 mg / mL; the concentration of sarcosine oxidase is 0.2 mg / mL;

[0053] The volume ratio of the buffer, creatinase, creatininase, sarcosine oxidase and the sample to be tested is 8:1:1:1:2;

[0054] The incubation condition is 37°C for 30 min;

[0055] The concentration of the NaAc-HAc buffer is 0.2 M, pH 4.5; the concentration of the porous carbon / vanadium sulfide nanozyme suspension is 2 mg / mL; the concentration of the 3,3',5,5'-tetramethylbenzidine is 1 mM; the volume ratio of the NaAc-HAc buffer, the porous carbon / vanadium sulfide nanozyme suspension, the 3,3',5,5'-tetramethylbenzidine and the sample to be tested is 285:1:1:2;

[0056] The secondary incubation condition is incubation at room temperature for 20 min;

[0057] The absorbance is the absorbance measured by a UV-visible spectrophotometer at 652 nm.

[0058] The embodiments of the present invention also provide the use of the porous carbon / vanadium sulfide nanozyme in preparing a product for creatinine detection.

[0059] Example 1

[0060] Step S1, adding 25 mg of yeast, 2.5 mg of vanadium chloride and 30 mL of ethanol into a beaker, and stirring until the liquid is completely volatilized after ultrasonic dispersion to obtain a first solid A.

[0061] Step S2: Add 20 mg of the first solid material A, 30 mg of potassium carbonate, and 30 mg of potassium chloride into a mortar and grind for ten minutes to obtain a second solid material B.

[0062] Step S3, placing 20 mg of the second solid B and 100 mg of thioacetamide in the grooves at the left and right ends of the graphite sample stage of the Joule heat device, respectively, and performing a rapid Joule heat shock in a nitrogen atmosphere, the rapid Joule heat shock temperature is 900°C, the Joule heat shock heating rate is 2000°C / s, and the number of Joule heat shocks is 3 times. Obtain a third solid C. In the present invention, the Joule heat shock is heating from room temperature to the target temperature, and then naturally cooling to room temperature, which is represented as one time.

[0063] Step S4: The third solid C was washed three times by centrifugation with deionized water and dried to obtain a porous carbon / vanadium sulfide nanozyme with abundant defects, named PC / V3S4-1.

[0064] Step S5, prepare PC / V3S4-1 nanozyme suspension (2 mg / mL), TMB (1 mM), and H2O2 (10 mM) solutions with pure water, then mix 10 μL of porous carbon / vanadium sulfide nanozyme, 10 μL of H2O2 and 10 μL of TMB in 2970 μL of sodium acetate-acetic acid (NaAc-Hac, pH=4.5, 0.2 M) buffer solution, incubate at room temperature for 20 min, and measure the absorbance of the reaction solution at 652 nm using a UV-visible spectrophotometer to evaluate the activity of the porous carbon / vanadium sulfide nanozyme.

[0065] Step S6: 80 μL PBS (0.015 M, pH 7.5) was mixed with 10 μL creatinase (0.1 mg / mL), 10 μL creatinine enzyme (0.2 mg / mL), 10 μL sarcosine oxidase (0.2 mg / mL), and 20 μL creatinine (0-550 μM), and incubated at 37°C for 30 min. Then, 2850 μL NaAc-HAc buffer (0.2 M, pH 4.5), 10 μL PC / V3S4-1 nanozyme suspension (2 mg / mL), and 10 μL TMB (1 mM) were added to the above solution and incubated at room temperature for 20 min. Finally, the absorbance of the reaction solution was measured at 652 nm using a UV-visible spectrophotometer to quantitatively detect the creatinine in the solution.

[0066] Example 2

[0067] The only difference from Example 1 is that in step S1, 25 mg of yeast is replaced by 30 mg of yeast, and the rest of the method is the same as Example 1, which is denoted as PC / V3S4-2.

[0068] Example 3

[0069] The only difference from Example 1 is that in step S2, 30 mg potassium carbonate and 30 mg potassium chloride are replaced by 20 mg potassium carbonate and 40 mg potassium chloride respectively, and the rest of the method is the same as that of Example 1, which is recorded as PC / V3S4-3.

[0070] Example 4

[0071] The only difference from Example 1 is that in step S2, 30 mg potassium carbonate and 30 mg potassium chloride are replaced by 40 mg potassium carbonate and 40 mg potassium chloride respectively, and the rest of the method is the same as that of Example 1, which is recorded as PC / V3S4-4.

[0072] Example 5

[0073] The only difference from Example 1 is that in step S3, 100 mg of thioacetamide is replaced by 30 mg of thioacetamide, and the rest of the method is the same as that of Example 1, which is denoted as PC / V3S4-5.

[0074] Example 6

[0075] The only difference from Example 1 is that in step S3, 100 mg of thioacetamide is replaced by 150 mg of thioacetamide, and the rest of the method is the same as that of Example 1, which is denoted as PC / V3S4-6.

[0076] Comparative Example 1

[0077] The only difference from Example 1 is that in step S1, 25 mg of yeast is replaced by 5 mg of porous carbon, and the rest of the method is the same as that of Example 1, which is denoted as PC / V3S4-D1.

[0078] Comparative Example 2

[0079] The only difference from Example 1 is that potassium carbonate and potassium chloride are not used in step S2, and the rest of the method is the same as Example 1, which is denoted as PC / V3S4-D2.

[0080] Comparative Example 3

[0081] The difference from Example 1 is that in step S3, the Joule heat heating method is replaced by tube furnace heating. The specific steps are as follows: 20 mg of the second solid substance B is placed in the center of the temperature zone of the high-temperature tube furnace, and 100 mg of thioacetamide is placed upstream of the gas. In a nitrogen atmosphere, the heating rate is 2°C / min, heated to 800°C, and kept warm for 4 hours. The rest of the method is the same as in Example 1, recorded as PC / V3S4-D3.

[0082] Experimental Results

[0083] Figure 1The scanning electron micrograph and element distribution diagram of Example 1 are shown. The results show that the PC / V3S4-1 prepared in Example 1 mainly presents a rough and porous spherical structure with a size of about 5 μm. The C, S, V and P elements are evenly distributed in PC / V3S4-1, indicating that the vanadium sulfide active substance is evenly dispersed on the yeast-derived porous carbon matrix, and the phosphorus element is evenly doped inside the vanadium sulfide crystal. This dispersion characteristic is conducive to exposing more active sites, and the uniform phosphorus doping improves the intrinsic nanozyme activity of the active substance, thereby improving the creatinine detection performance.

[0084] To further analyze the pore structure of PC / V3S4-1, N2 adsorption-desorption tests were carried out. Figure 2 The characteristic hysteresis loop is shown, indicating that the material forms a mesoporous structure after high temperature treatment. The Barrett-Joyner-Halenda (BJH) analysis results in the illustration show a wide pore size distribution, further proving that the material is mainly a mesoporous structure. The rich pore structure helps to disperse the active substances and expose more enzyme active sites. At the same time, porous carbon can enrich the reactants and provide a good reaction environment for the active sites, thereby improving the activity of the nanozyme.

[0085] Figure 3 Table 1 shows the Raman spectra of Example 1 and the comparative example. D / I G The ratio is the peak area ratio of the D band (disordered carbon structure, about 1353 cm⁻¹) to the G band (ordered sp² hybridized carbon, about 1587 cm⁻¹). D / I G The ratios are as follows: Example 1 (1.68) > Comparative Example 3 (1.25) > Comparative Example 2 (1.15) > Comparative Example 1 (1.08). This indicates that Example 1 has more carbon defects, mainly due to the corrosion of potassium carbonate and potassium chloride during pyrolysis and the rapid heating and cooling effect of Joule heat treatment. The formed carbon defects enhance the adsorption of reactants, provide an enriched microenvironment for active sites, and improve the sensitivity of creatinine detection.

[0086] Table 1 Raman test results

[0087] name Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 <![CDATA[I D / I G ]]> 1.68 1.08 1.15 1.25

[0088] I D / I G : The peak intensity ratio of the D band of disordered carbon structure and the G band of ordered sp2 hybridized carbon in the Raman spectrum.

[0089] Figure 4The electron paramagnetic resonance (EPR) spectra of Example 1 and Comparative Examples 1 and 3 are shown. A strong symmetrical EPR signal appears at g = 1.961, and the order of intensity is: Example 1 > Comparative Example 3 > Comparative Example 1. This signal originates from vanadium (V) vacancy defects, indicating that in the yeast-assisted rapid high-temperature solid-phase sulfurization reaction, part of the vanadium ions are in situ reduced to vanadium carbide by porous carbon to form V defects.

[0090] Figure 5 The XPS spectrum of Example 1 shows that there are abundant sulfur defects in the PC / V3S4-1 nanozyme. Figure 1 Elemental analysis shows that sulfur defects are caused by partial substitution of sulfur by phosphorus. Figure 4 The nanozyme prepared in Example 1 is rich in S anion defects and V cation defects, which optimize the electronic and geometric structures of the active sites, reduce the adsorption, activation and conversion energy barriers of the reactants, and thus significantly improve the catalytic activity.

[0091] Figure 6 The peroxidase-like activity of Example 1 and Comparative Examples 1-3 samples was compared. The absorbance data measured by ultraviolet spectrophotometer showed that using TMB as a chromogenic substrate, in the presence of H2O2 and defect-rich porous carbon / vanadium sulfide nanozymes, the nanozymes catalyzed the decomposition of H2O2 to generate hydroxyl radicals (‧OH) and superoxide radicals (‧O2⁻), and then oxidized TMB to generate ox-TMB, causing the solution color to change from colorless to blue, with an absorption peak at 652 nm. The color depth is proportional to the enzyme activity, and the material in Example 1 exhibits the highest nanozyme activity.

[0092] Creatinine can be oxidized in the body in sequence by three natural enzymes: creatinase, creatininase, and sarcosine oxidase, ultimately generating H2O2. Subsequently, H2O2 generates a variety of free radicals under the catalytic action of defect-rich porous carbon / vanadium sulfide nanozymes. Colorless TMB is converted into blue ox-TMB under the oxidation of free radicals. Obviously, as the concentration of creatinine in the system increases, the absorbance of ox-TMB at 652nm also increases. Therefore, the absorbance can directly reflect the peroxidase-like catalytic activity of defect-rich porous carbon / vanadium sulfide nanozyme materials, as well as the concentration of creatinine in the solution. Figure 7 The UV absorption spectra of the CR / TMB / H2O2 / PC / V3S4-1 system under different creatinine (CR) concentrations are shown. The results show that as the creatinine concentration increases from 0.1 to 550µM / mL, the absorbance value of the TMB solution increases steadily. Figure 7 The absorbance data was converted to Figure 8The relationship between UV absorption and creatinine concentration. In the range of 0.1-100µM / mL, the UV absorption intensity and creatinine concentration show a good linear relationship (see Figure 8 Illustration), the linear equation is: y=0.00682x+0.0719 (R 2 =0.997). Therefore, the detection limit (LOD) of the defect-rich porous carbon / vanadium sulfide nanozyme material is as low as 0.07 µM / mL, indicating that the colorimetric sensing method based on this material has excellent detection sensitivity for creatinine.

[0093] The rapid Joule thermal shock technology in the embodiment of the present invention comes from a Joule thermal rapid heating device, which is composed of an electrode, a 316L vacuum chamber, a gas path device, a vacuum pump, a graphite sample stage, a temperature measurement module, and a data collection system; the output voltage is 0-40V, the output current is 0-375A, the current ramp time is 1000ms, the model is JH3.2, and the power supply is single-phase 220V / 40A.

[0094] In summary, the present invention provides a method for yeast-assisted synthesis of porous carbon / vanadium sulfide nanozymes and its application in serum creatinine detection. The metal sulfide nanozymes prepared by traditional methods have the problems of complex defect introduction process and insufficient defect types. The yeast-assisted Joule heat calcination method proposed in the present invention can form a porous carbon template in situ, inhibit the agglomeration of vanadium sulfide crystals, and is conducive to the exposure of nanozyme catalytic sites. At the same time, the P source and C source provided by the yeast induce the vanadium sulfide crystals to produce a large number of S defects and vanadium defects, thereby improving the intrinsic activity of the nanozyme catalytic site. The Joule heat drives the doping of heteroatom S in the porous carbon, forming abundant C defects, which prompts the formation of a reactant-enriched microenvironment near the nanozyme. When this porous carbon / vanadium sulfide with rich defects is used as a nanozyme for blood creatinine detection, creatinine is oxidized by three natural enzymes (creatinase, creatininase and sarcosine oxidase) to generate H2O2 in sequence, and H2O2 is then catalyzed by the nanozyme, and the colorless 3,3',5,5'-tetramethylbenzidine (TMB) indicator is oxidized to blue oxidized TMB (ox-TMB). Due to the synergistic effect of enzymes and nanozymes, the detection limit (LOD) of this colorimetric sensor is as low as 0.07µM / mL, which is of great significance for the trace determination of creatinine in actual samples.

[0095] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for yeast-assisted synthesis of porous carbon / vanadium sulfide nanozymes, characterized in that: The following steps are involved: (1) mixing yeast, vanadium chloride and ethanol, and stirring them after ultrasonic dispersion until the liquid is completely volatilized to obtain a first solid material A; (2) mixing and grinding the first solid material A, potassium carbonate and potassium chloride to obtain a second solid material B; (3) placing the second solid substance B and thioacetamide in the grooves at the left and right ends of the graphite sample stage of the Joule heat device, respectively, and subjecting them to rapid Joule heat shock to obtain a third solid substance C; (4) The third solid object C is washed and dried to obtain the porous carbon / vanadium sulfide nanozyme.

2. The method according to claim 1, characterized in that The mass volume ratio of the yeast, vanadium chloride and ethanol is 1 mg: (0.05-0.2) mg: (0.5-10) mL.

3. The method according to claim 1, characterized in that The mass ratio of the first solid substance A, potassium carbonate and potassium chloride is 1 mg: (1-5) mg: (1-10) mg.

4. The method according to claim 1, characterized in that The mass ratio of the second solid substance B to thioacetamide is 1:(1-10); The conditions of the rapid Joule thermal shock are: in a nitrogen atmosphere, the temperature is 600-1600° C., the Joule thermal shock heating rate is 1500-2500° C. / s, and the number of Joule thermal shocks is 1-6 times.

5. Application of the porous carbon / vanadium sulfide nanozyme prepared by the method according to any one of claims 1 to 4 in creatinine detection.

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