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Preparation method of triple artificial mimic enzyme as well as product and application of triple artificial mimic enzyme

A technology of artificial simulation and seeds, applied in the field of preparation of triple artificial simulated enzymes, can solve the problems of complicated operation, long detection time and low sensitivity, etc.

Pending Publication Date: 2022-07-29
SHANDONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these traditional detection methods generally have problems such as long detection time, complicated operation, and low sensitivity.

Method used

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  • Preparation method of triple artificial mimic enzyme as well as product and application of triple artificial mimic enzyme
  • Preparation method of triple artificial mimic enzyme as well as product and application of triple artificial mimic enzyme
  • Preparation method of triple artificial mimic enzyme as well as product and application of triple artificial mimic enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0039] Example 1 Preparation of triple artificial simulated enzymes

[0040] The graphene-gold composite (GO-Au) was first synthesized.

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

[0042] 1mL of 0.12mM HAuCL 4 Mix with 1mL of 0.048MCTAB solution, add 0.12mL of freshly prepared 2.4mM NaBH with constant stirring 4 , the color of the solution changed from yellow to brown, and the seed solution of gold nanorods was obtained. The seed solution was placed at 27°C for 2h before use.

[0043] (2) Preparation of growth solution

[0044] 50mL of 0.2mM HAuCL 4 Mix with 50mL of 0.048M cetyltrimethylammonium bromide (CTAB) solution, then add 2.5mL of 0.96mM AgNO at room temperature 3 solution and 3.4 mg of H-RGO. After that, 0.7 mL of 0.01 M ascorbic acid was added, at which point the color of the solution changed from dark yellow to colorless. Then, 0.12 mL of the seed solution prepared in step (1) was added, and the reaction was carried out at 27° C. for 20 min. Th...

Embodiment 2

[0049] Example 2 Detection of homocysteine ​​in plasma

[0050] (1) Draw a standard curve

[0051] Homocysteine ​​was added to the plasma with known homocysteine ​​concentration, and the concentrations were respectively 10 μmol / L, 20 μmol / L, 50 μmol / L, 70 μmol / L, 150 μmol / L, 200 μmol / L homocysteine Mixed solutions of acids, numbered a-g in sequence. Add the same volume of hydrogen peroxide to the mixed solutions of a-g respectively, react for 1 hour to inactivate other enzymes in the plasma, and then add the same volume of the above-mentioned mixed solution of homocysteine ​​to a concentration of 0.002g / L The dithiothreitol solution was reacted at 4°C for 12 hours, and finally the same volume of triple artificial simulated enzyme (GO-Au-MP-11) synthesized in Example 1 was added to react for 6 hours to prepare a detection solution. Take 1 mL of a mixed solution of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide, add 200 μL of the detection solution to react for 1 h...

Embodiment 3

[0055] Using two natural peroxidase (horseradish peroxidase HRP and microperoxidase MP-11) as a comparative experiment, it was found that the catalytic activity of HRP was insensitive to the concentration of homocysteine, and it was impossible to achieve homocysteine detection of amino acids. The sensitivity of pure MP-11 for the detection of homocysteine ​​is also low, and it can only detect homocysteine ​​at a minimum of 80 μmol / L.

[0056] In summary, the present invention prepares a triple artificial simulated enzyme, and the synergistic enhancement between graphene, gold nanoflowers and MP-11 in the material greatly increases the catalytic performance, thereby improving the detection sensitivity. When the synthetic material was combined with sulfhydryl-containing compounds such as homocysteine, the sulfhydryl group specifically bound to the iron center in MP-11, which played a key role, resulting in greatly reduced catalytic performance. When thiol-bound and unbound thio...

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Abstract

The invention discloses a preparation method of a triple artificial mimic enzyme as well as a product and application of the triple artificial mimic enzyme, and relates 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 the gold nanorod; (2) mixing a HAuCL4 solution and a hexadecyl trimethyl ammonium bromide solution, adding an AgNO3 solution and H-RGO, then adding a reducing agent, finally adding the seed solution of the gold nanorod, and reacting to obtain GO-Au; and (3) connecting MP-11 to the GO-Au, so as to obtain the triple artificial mimic enzyme. The invention designs and synthesizes a triple artificial mimic enzyme based on graphene, MP-11 and gold nanoflowers, and the artificial mimic enzyme can replace natural peroxidase to detect the concentration of homocysteine in human plasma with high sensitivity.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a preparation method of a triple artificial simulated enzyme and its product and application. Background technique [0002] Homocysteine, also known as homocysteine, is an amino acid containing a sulfhydryl group and an important intermediate product of human metabolism. Under normal circumstances, homocysteine ​​is catabolized by the body and the concentration stays at a low level. However, due to some primary and secondary reasons, it will accumulate in the body, resulting in a substantial increase in the incidence of cardiovascular and cerebrovascular diseases. Therefore, homocysteine ​​is an important indicator of human health. At present, the detection of homocysteine ​​is mainly by fluorescence polarization or immunoassay, and isotope and chromatography are also used. However, these current traditional detection methods generally have problems such as long d...

Claims

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Application Information

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IPC IPC(8): C12N9/08G01N33/68G01N21/33
CPCC12N9/0065G01N33/6815G01N21/33
Inventor 刘静张童王茹可王博
Owner SHANDONG UNIV OF SCI & TECH
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