Method for determination of acid phosphatase with carbon quantum dots as photo active mimetic enzyme

A technology of acid phosphatase and assay method, which is applied in the field of photoactive nanomaterial imitating enzyme and its application in the detection of acid phosphatase activity, and can solve problems such as application limitations

Inactive Publication Date: 2018-06-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of the peroxide mimetic enzymes based on nanomaterials need the help of a large amount of destructi

Method used

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  • Method for determination of acid phosphatase with carbon quantum dots as photo active mimetic enzyme
  • Method for determination of acid phosphatase with carbon quantum dots as photo active mimetic enzyme
  • Method for determination of acid phosphatase with carbon quantum dots as photo active mimetic enzyme

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Embodiment 1

[0016] a. In a 100mL beaker, add 25mL 2.0mol / L sulfuric acid, 5mL 2.0mol / L nitric acid and 0.1g ascorbic acid to form a homogeneous solution under constant stirring; take 20mL of the mixture and add it to the reaction kettle. Heated for 3 hours, cooled to room temperature naturally, slowly added 80mL of deionized water, and then adjusted the pH of the solution to neutral with 1.0mol / L NaOH solution; the obtained solution was dialyzed for 3 days with a dialysis bag to obtain water-soluble carbon quantum point;

[0017] b. Determination of acid phosphatase activity: Mix 10 μL of acid phosphatase of different concentrations with 10 μL of 1.0 mmol / L sodium pyrophosphate and 30 μL of acetate buffer solution with pH=5.0, and incubate in a 96-well plate with shaking at 40°C for 2 hours; Then add 30μL LCDs, 20μL 5mmol / L 3,3',5,5'-tetramethylbenzidine, use 0.2mol / L acetic acid buffer solution with pH=3.0 to dilute the reaction solution to 200μL; mix well and place under visible light ...

Embodiment 2

[0019] a. In a 100mL beaker, add a mixed solution of 12.5mL 2.0mol / L sulfuric acid, 12.5mL 2.0mol / L nitric acid and 0.2g trisodium citrate to form a solution under constant stirring; take 20mL of the mixture and add it to the reaction In the kettle, heat and react at 80°C for 5 hours, after naturally cooling to room temperature, slowly add 80mL of deionized water, and then use 1.0mol / L NaOH solution to adjust the pH of the solution to neutral; the obtained solution is dialyzed for 3 days with a dialysis bag , to obtain water-soluble carbon quantum dots;

[0020] a. Determination of acid phosphatase activity: Mix 10 μL of acid phosphatase of different concentrations with 10 μL of 1.0 mmol / L o-phosphate-L-tyrosine and 30 μL of acetate buffer solution with pH=5.0, and put them in a 96-well plate Incubate with shaking at 40°C for 2 hours; then add 30 μL of CDs, 20 μL of 5 mmol / L 3,3',5,5'-tetramethylbenzidine, and dilute the reaction solution to 200 μL with 0.2 mol / L acetic acid b...

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Abstract

A method for determination of acid phosphatase with carbon quantum dots as a photo active mimetic enzyme is disclosed. A carbon-quantum-dot-based photo active mimetic enzyme is provided, and the activity of the mimetic enzyme is found to be inhibited / recovered in the presence of Ce<3 + >/ PO4<3->, so that a new method for determination of the acid phosphatase (ACP) is established. A simple hydrothermal method is used to synthesize the carbon quantum dots, and the carbon quantum dots show high mimetic enzyme activity under visible light (lambda >= 400nm), and can catalyze chromogenic reactionof 3, 3 ', 5, 5'-tetramethyl benzidine. The catalytic activity is weakened by aggregation of the carbon quantum dots due to complex reaction of the Ce <3+> with carboxyl groups on the surface of theCDs (carbon quantum dots); in the presence of the PO4<3-> which is a hydrolysate of the ACP, a precipitate is formed by the Ce <3+>, so that the mimetic enzyme inhibitory effect on the CDs is eliminated, and the enzyme like catalytic activity of the system is restored. The detection limit of the activity of the ACP detected by the method can reach 0.02U / L, and the method has the advantages of convenient operation, high sensitivity and good selectivity and the like.

Description

Technical field: [0001] The invention relates to the field of nano-biological analysis and detection, in particular to carbon quantum dots as novel photoactive nanomaterials to simulate enzymes and their application in the detection of acid phosphatase activity. Background technique [0002] Natural enzymes have significant advantages such as good substrate specificity and high catalytic activity, but their extraction costs are high, their variability is inactivated, and they are expensive, which greatly limit their applications. Therefore, in recent years, the development of nanomaterial-mimicking enzymes and their application in biosensing have attracted more and more attention. Compared with natural enzymes, mimetic enzymes based on nanostructures have many advantages: low cost, high catalytic activity, and tolerance to harsher environments, which indicates that nanomaterials have good prospects for sensing applications as mimetic enzymes [Gao L Z. ; Zhuang J.; Nie L.; Z...

Claims

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Application Information

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IPC IPC(8): C12Q1/42
CPCC12Q1/42
Inventor 王光丽李苹董玉明曹根霞
Owner JIANGNAN UNIV
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