Preparation method, product and application of a triple artificial mimic enzyme

Abstract

The present invention discloses a preparation method, product and application of a triple artificial mimic enzyme, relating to the technical field of biological detection. The preparation method comprises the following steps: (1) Mixing a HAuCL4 solution and a CTAB solution, and then adding a NaBH4 solution to obtain a seed solution of gold nanorods; (2) After mixing a HAuCL4 solution and a cetyltrimethylammonium bromide solution, adding an AgNO3 solution and H-RGO, then adding a reducing agent, and finally adding the seed solution of the gold nanorods, and reacting to obtain GO-Au; (3) Connecting MP-11 to the GO-Au to obtain the triple artificial mimic enzyme. The present invention designs and synthesizes a triple artificial mimic enzyme based on graphene, MP-11 and gold nanoflowers. This artificial mimic enzyme can replace natural peroxidase to highly sensitively detect the concentration of homocysteine in human plasma.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly relates to a preparation method, product and application of a triple artificial mimic enzyme. Background Art

[0002] Homocysteine, also known as homocysteine, is an amino acid containing a mercapto group and is an important intermediate product of human metabolism. Under normal circumstances, homocysteine will be decomposed and metabolized by the human body, and its concentration stays at a relatively low level. However, due to some primary and secondary reasons, it will accumulate in the body, leading to a significant increase in the incidence of cardiovascular and cerebrovascular diseases. Therefore, homocysteine is an important human health indicator. At present, for the detection of homocysteine, fluorescence polarization method or immunoassay is mainly used, and isotope method and chromatography are also used. However, these current traditional detection methods generally have problems such as long detection time, complex operation, and low sensitivity.

[0003] In recent years, it has been found that some nanomaterials have catalytic activities similar to natural enzymes, which have attracted great interest as artificial mimic enzymes. For example, the initially discovered iron oxide nanoparticles have catalytic activities similar to horseradish peroxidase (HRP), and can catalyze enzyme substrates such as 3,3’,5,5’-tetramethylbenzidine (TMB) and hydrogen peroxide to undergo redox reactions, generating blue oxidation products. Subsequently, it has been successively found that many nanomaterials have mimic enzyme activities, including transition metal nanomaterials, noble metal nanomaterials, carbon nanomaterials, etc. Compared with natural enzymes, nanomaterials have the advantages of high stability, high catalytic activity and being cheap and easy to obtain. In particular, the unstable and variable characteristics of natural enzymes are avoided, increasing their application prospects in the fields of process catalysis and enzyme kinetics. Therefore, these nanomaterials with mimic enzyme activities have important significance in analytical chemistry. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method, product and application of a triple artificial mimic enzyme to solve the problems existing in the above-mentioned prior art. The triple artificial mimic enzyme prepared by the present invention can achieve highly sensitive detection of homocysteine.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a preparation method of a triple artificial mimic enzyme, including the following steps:

[0007] (1) Mix an HAuCL4 solution and a CTAB solution, and then add a NaBH4 solution to obtain a seed solution of gold nanorods;

[0008] (2) After mixing the HAuCL4 solution and the cetyltrimethylammonium bromide solution, add the AgNO3 solution and H-RGO, then add the reducing agent, and finally add the seed solution of the gold nanorods, and react to obtain GO-Au;

[0009] (3) Connect MP-11 to the GO-Au to obtain the triple artificial mimic enzyme.

[0010] Furthermore, the reducing agent is ascorbic acid.

[0011] The present invention also provides a triple artificial mimic enzyme prepared by the above preparation method.

[0012] The present invention also provides the application of the above triple artificial mimic enzyme in the detection of homocysteine.

[0013] The present invention also provides a method for detecting homocysteine according to the above triple artificial mimic enzyme, comprising the following steps:

[0014] (1) Draw a standard curve

[0015] Add homocysteine to plasma with a known homocysteine concentration, respectively prepare mixed solutions of homocysteine with different gradient concentrations, and then after detection operations, use a UV spectrophotometer to detect the UV absorption signal at 620 nm, and draw a standard curve;

[0016] The detection operation includes:

[0017] a Add hydrogen peroxide to the mixed solution of homocysteine, after reaction, add dithiothreitol solution, after reaction, add the above triple artificial mimic enzyme, and make a detection solution after reaction;

[0018] b Mix 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide, add the detection solution, and after reaction, it can be detected by a UV spectrophotometer;

[0019] (2) Sample detection

[0020] After the sample to be tested is subjected to the detection operation described in step (1), use a UV spectrophotometer to detect the UV absorption signal at 620 nm, and obtain the homocysteine concentration of the sample to be tested according to the standard curve.

[0021] Furthermore, in step a, the reaction time after adding the hydrogen peroxide is 1 h.

[0022] Furthermore, in step a, the reaction time after adding the dithiothreitol solution is 12 h.

[0023] Further, in step a, the reaction time after adding the triple artificial mimic enzyme is 6 h.

[0024] Further, in step b, the reaction time is 1 h.

[0025] The present invention discloses the following technical effects:

[0026] The present invention designed and synthesized a triple artificial mimic enzyme based on graphene, MP-11 and gold nanoflowers. This artificial mimic enzyme has excellent peroxidase-like catalytic properties and can efficiently catalyze the reaction of 3,3',5,5'-tetramethylbenzidine (TMB) with hydrogen peroxide to generate a blue oxide. When combined with homocysteine, the catalytic activity of this artificial mimic enzyme will be significantly reduced, and the reduction amplitude is linearly related to homocysteine. Therefore, by measuring the ultraviolet absorbance of the blue oxide, the concentration of homocysteine in plasma can be evaluated.

[0027] Based on the catalytic effect of this triple artificial mimic enzyme on the substrate 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide and its binding effect with homocysteine, the present invention constructs a method for highly sensitive detection of the concentration of homocysteine in human plasma by replacing natural peroxidase with artificial mimic enzyme. The present invention utilizes the excellent catalytic performance of the triple artificial mimic enzyme to efficiently catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine by hydrogen peroxide to produce a blue oxidation product, which has an obvious absorption peak at 620 nm under the detection of an ultraviolet spectrophotometer. The catalytic iron center of MP-11 can specifically bind to the sulfhydryl group of homocysteine. When the triple artificial mimic enzyme binds to homocysteine in human plasma, the catalytic activity of MP-11 will be sharply reduced, and the concentration of the blue oxidation product will decrease, and the absorption peak at 620 nm will be significantly reduced under the detection of an ultraviolet spectrophotometer. By this method, highly sensitive detection of homocysteine can be achieved, which has the advantages of short detection time, simple operation and high sensitivity compared with traditional detection methods. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 It is a transmission electron microscope characterization diagram of graphene and gold nanoflowers, where (a) is graphene and (b) is a graphene-gold nanoflower composite;

[0030] Figure 2 It is a schematic diagram of the process of MP-11 binding GO-Au;

[0031] Figure 3 For the comparison of the catalytic signals before and after the reaction of GO-Au-MP-11 with homocysteine, where Curve 1 is before the reaction and Curve 2 is after the reaction; the two solution bottles in the upper right corner are the physical solutions corresponding to the reaction before and after the curve, 1 is blue, and 2 is colorless and transparent;

[0032] Figure 4 For the comparison of the oxidation of TMB by H2O2 catalyzed after the binding of homocysteine with different concentrations to GO-Au-MP-11, from a-g, the concentration of homocysteine increases continuously, and a linear equation can be obtained. Among them, (a) is the signal curve at different homocysteine concentrations, and (b) is the linear equation. Detailed implementation manners

[0033] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0036] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0037] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0038] In the following examples, the instrument devices used included: FEI Talos F200S transmission electron microscope (Thermo Scientific); UV-vis spectrophotometer (Agilent Technologies Co., Ltd., USA); SZ-93 automatic double pure water distiller (Shanghai Yarong Biochemical Instrument Factory); KQ-50B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); the reagents used included: carboxylated graphene dispersion (GO, Nanjing Xianfeng Nano Material Technology Co., Ltd.); NaBH4 (Shanghai Chemical Reagent Co., Ltd.); TMB mixture (Biosharp Biotechnology Co., Ltd.). Chloroauric acid, microperoxidase-11 (MP-11), horseradish peroxidase (HRP) and dithiothreitol were all purchased from Sigma. All reagents used in the experiments were of analytical grade, the water was all secondary distilled water, and the PBS buffer solution (pH 7.4) was prepared with disodium hydrogen phosphate, sodium dihydrogen phosphate and secondary distilled water.

[0039] Example 1 Preparation of triple artificial mimetic enzyme

[0040] First, synthesize graphene-gold composite (GO-Au).

[0041] (1) Preparation of seed solution of gold nanorods

[0042] Mix 1 mL of 0.12 mM HAuCl4 and 1 mL of 0.048 M CTAB solution, and add 0.12 mL of freshly prepared 2.4 mM NaBH4 while stirring continuously. At this time, the color of the solution changes from yellow to brownish-yellow, obtaining the seed solution of gold nanorods. The seed solution was placed in an environment of 27 °C for 2 h before use.

[0043] (2) Preparation of growth solution

[0044] Mix 50 mL of 0.2 mM HAuCl4 and 50 mL of 0.048 M cetyltrimethylammonium bromide (CTAB) solution, then add 2.5 mL of 0.96 mM AgNO3 solution and 3.4 mg of H-RGO at room temperature. Then, add 0.7 mL of 0.01 M ascorbic acid. At this time, the color of the solution changes from dark yellow to colorless. Then add 0.12 mL of the seed solution prepared in step (1), and react at 27 °C for 20 min. The above solution was transferred to a condition of 30 °C and reacted for another 12 h. After the reaction product was centrifuged at 8500 rpm for 25 min, the supernatant was removed, and the precipitate was redispersed in 10 mL of ultrapure water to obtain the GO-Au solution. The electron micrograph of GO-Au is shown in Figure 1 .

[0045] (3) Obtain triple artificial mimetic enzyme (GO-Au-MP-11) by connecting MP-11 on the basis of GO-Au

[0046] First, prepare a 0.186 mg / mL MP-11 buffer solution with PBS buffer solution. Take 1 mL of the MP-11 buffer solution and mix it with 1 mL of the synthesized GO-Au solution, then dilute it to 10 mL with PBS buffer solution and stir for 12 hours at 4 °C. Wash away the excess MP-11 and redissolve it in 10 mL of PBS buffer solution to obtain the triple artificial mimic enzyme (GO-Au-MP-11). The schematic diagram of the process of MP-11 binding to GO-Au is shown in Figure 2 .

[0047] Effect verification

[0048] Homocysteine is an amino acid containing a sulfhydryl group, which can specifically bind to the catalytic center iron atom of MP-11, resulting in a decrease or disappearance of the catalytic activity of MP-11. Prepare a 0.0011 g / mL homocysteine solution with PBS buffer solution. Take the same volume of the homocysteine solution and mix it with the triple artificial mimic enzyme (GO-Au-MP-11), and stir for 3 hours at 4 °C. As Figure 3 shown, when homocysteine is added, the catalytic activity of the mimic enzyme decreases significantly and the solution color turns colorless, indicating that GO-Au-MP-11 has a very good effect on detecting homocysteine.

[0049] Example 2 Detection of homocysteine in plasma

[0050] (1) Plot the standard curve

[0051] Add homocysteine to plasma with known homocysteine concentration to prepare mixed solutions of 10 μmol / L, 20 μmol / L, 50 μmol / L, 70 μmol / L, 150 μmol / L, and 200 μmol / L homocysteine, numbered a - g in sequence. Add the same volume of hydrogen peroxide to the mixed solutions of a - g and react for 1 hour to inactivate other enzymes in the plasma. Then add the same volume of a 0.002 g / L dithiothreitol solution as that of the above homocysteine mixed solution and react for 12 hours at 4 °C. Finally, add the same volume of the triple artificial mimic enzyme (GO-Au-MP-11) synthesized in Example 1 and react for 6 hours to prepare the detection solution. Take 1 mL of the mixed solution of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide, add 200 μL of the detection solution and react for 1 hour, then detect it at 620 nm using an ultraviolet spectrophotometer and plot the standard curve (see Figure 4 ).

[0052] (2) Detection of actual samples

[0053] Take out the test samples 1 and 2, and measure the ultraviolet absorption signals as 0.75 and 0.415 respectively by the method of step (1). According to the standard curve and linear relationship, the concentrations of the analytes are measured as 7.83 μmoL / L and 12.01 μmoL / L respectively, which are consistent with the chemiluminescence results (7.8 μmoL / L and 12.9 μmoL / L respectively).

[0054] Example 3

[0055] Taking two natural peroxidases (horseradish peroxidase HRP and microperoxidase MP-11) as comparative experiments, it is found that the catalytic activity of HRP is not sensitive to the concentration of homocysteine, and the detection of homocysteine cannot be achieved. The sensitivity of pure MP-11 for the detection of homocysteine is also relatively low, and the lowest concentration of homocysteine that can be detected is only 80 μmol / L.

[0056] In summary, the present invention prepares a triple artificial mimic enzyme. The synergistic enhancement effect among graphene, gold nanoflowers and MP-11 in the material greatly increases the catalytic performance, thereby improving the detection sensitivity. When the synthetic material binds to a thiol-containing compound such as homocysteine, the thiol will specifically bind to the iron center that plays a key role in MP-11, resulting in a great reduction in catalytic performance. When the materials with bound and unbound thiols meet TMB and H2O2, there will be an obvious difference in catalytic activity, and the concentration of homocysteine is reflected by the depth of the blue color of the solution. The concentration of homocysteine can be detected by ultraviolet spectrophotometer detection and calculation.

[0057] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting homocysteine according to a triple artificial mimic enzyme, characterized in that, It includes the following steps: (1) Plot a standard curve Homocysteine is added to plasma with a known homocysteine concentration to prepare mixed solutions with different gradient concentrations of homocysteine respectively. After performing detection operations respectively, the ultraviolet absorption signal is detected at 620 nm using an ultraviolet spectrophotometer, and a standard curve is plotted; The detection operation includes: a. Hydrogen peroxide is added to the mixed solution of homocysteine. After reaction, dithiothreitol solution is added. After reaction, triple artificial mimic enzyme is added, and a detection solution is prepared after reaction; b. After 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide are mixed, the detection solution is added, and after reaction, it can be detected using an ultraviolet spectrophotometer; In step a, the concentration of the dithiothreitol solution is 0.002 g / L; the volume ratio of the mixed solution of homocysteine, the hydrogen peroxide and the dithiothreitol solution is 1:1:1; In step b, the volume ratio of the mixed solution of 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide to the detection solution is 1:0.2; (2) Sample detection After the sample to be tested undergoes the detection operation described in step (1), the ultraviolet absorption signal is detected at 620 nm using an ultraviolet spectrophotometer, and the homocysteine concentration of the sample to be tested is obtained according to the standard curve; The preparation method of the triple artificial mimic enzyme includes the following steps: (A) HAuCl4 solution and CTAB solution are mixed, and then NaBH4 solution is added to obtain a seed solution of gold nanorods; the seed solution of gold nanorods is placed in an environment of 27 °C for 2 h before use; (B) HAuCl4 solution and cetyltrimethylammonium bromide solution are mixed, then AgNO3 solution and H-RGO are added, then a reducing agent solution is added, and finally the seed solution of gold nanorods is added, and a GO-Au solution is obtained after reaction; (C) MP-11 is connected to the GO-Au to obtain the triple artificial mimic enzyme; In step (A), the concentration of the HAuCl4 solution is 0.12 mM; the concentration of the CTAB solution is 0.048 M; the concentration of the NaBH4 solution is 2.4 mM; the volume ratio of the HAuCl4 solution, the CTAB solution and the NaBH4 solution is 1:1:0.12; In step (B), the concentration of the HAuCl4 solution is 0.2 mM; the concentration of the cetyltrimethylammonium bromide solution is 0.048 M; the concentration of the AgNO3 solution is 0.96 mM; the reducing agent solution is ascorbic acid solution, and the concentration of the ascorbic acid solution is 0.01 M; the addition ratio of the HAuCl4 solution, the cetyltrimethylammonium bromide solution, the AgNO3 solution, the reducing agent solution, the H-RGO and the seed solution of gold nanorods is 50 mL:50 mL:2.5 mL:0.7 mL:3.4 mg:0.12 mL; In step (C), the step of connecting MP-11 to the GO-Au includes: Prepare an MP-11 buffer solution with a concentration of 0.186 mg / mL using PBS buffer solution. Mix the MP-11 buffer solution with the GO-Au solution to obtain a mixture, and dilute it with PBS buffer solution to 5 times the volume of the mixture submitted. Stir for 12 hours under the condition of 4 °C.

2. The method according to claim 1, characterized in that, In step a, the reaction time after adding the hydrogen peroxide is 1 h.

3. The method according to claim 1, characterized in that, In step a, the reaction time after adding the dithiothreitol solution is 12 h.

4. The method according to claim 1, characterized in that, In step a, the reaction time after adding the triple artificial mimic enzyme is 6 h.

5. The method according to claim 1, characterized in that, In step b, the reaction time is 1 h.

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