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Visual rapid detection method of biological enzymes, proteins and their inhibitors based on nano-mimetic enzymes

A technology for the detection of biological enzymes and biological enzymes, applied in the field of biological analysis, can solve the problems of time-consuming, complicated operation, and poor sensitivity, and achieve the effects of simple operation, good selectivity, and high sensitivity

Active Publication Date: 2019-08-06
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Purpose of the invention: The present invention aims at the complex operation, time-consuming and poor sensitivity of some current biological enzyme / protein activity detection methods, and develops new principles to realize the detection of related biological enzymes by using some nanomaterials that can simulate natural enzyme activity. / Rapid, highly selective, highly sensitive detection of protein activity

Method used

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  • Visual rapid detection method of biological enzymes, proteins and their inhibitors based on nano-mimetic enzymes
  • Visual rapid detection method of biological enzymes, proteins and their inhibitors based on nano-mimetic enzymes
  • Visual rapid detection method of biological enzymes, proteins and their inhibitors based on nano-mimetic enzymes

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Experimental program
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Effect test

Embodiment 1

[0034] 1) Preparation of nano-mimetic enzyme nanoceria:

[0035] 2.52g Ce(NO 3 ) 3 Dissolve in 100mL of 1:1 mixed solution of water and ethylene glycol, and heat and stir to 60°C. Then, 16 mL of fresh ammonia water was quickly added under stirring conditions, and stirring was continued at 60° C. for 3 hours. The product was centrifuged and successively washed with water and centrifuged 3 times to obtain the crude product of nanoceria. Disperse the nanoceria crude product in 100mL of sodium citrate solution with a concentration of 15mg / mL, and ultrasonicate for 30 minutes. After the nanoceria and sodium citrate have fully reacted, add 100mL of ethanol, stir and centrifuge, and the obtained product is sequentially washed with 1:1 The mixture of water and ethanol was washed and centrifuged three times, and dispersed in pure water. After calculation and measurement, the nanoceria concentration in the dispersion is 14 mg / mL, and the obtained nanoceria dispersion with a concentr...

Embodiment 2

[0039] Example 2 Application of Nano-mimetic Enzyme Technology in the Detection of Acetylcholinesterase Inhibitors Methylphosphine and Tacrine

[0040] First, 1.5U acetylcholinesterase was mixed with different concentrations of methyl-paraphosphine (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 μM) or tacrine (0, 0.5, 5, 50 μM) were mixed with phosphate buffer (pH 7.0) with a concentration of 5 mM to make up to 50 μL, and reacted at room temperature for 30 minutes. After the enzyme activity is fully inhibited, dilute the mixture to 180 μL with 5 mM phosphate buffer (pH 7.0), then add 4 μL TMB solution (25 mM), 2 μL ATP solution (100 mM), 10 μL acetylcholine solution (1.0 M ) and 1.5 μL nanoceria stock solution (14 mg / mL). After mixing evenly, the reagent was placed in a water bath at 37° C. for reaction, and the change of the absorbance value at 652 nm was recorded in real time by an ultraviolet-visible spectrophotometer.

[0041] from figure 2 In A, we can see that with...

Embodiment 3

[0043] Application of embodiment 3 nano-mimetic enzyme technology in urease detection

[0044] Add 4 μL of TMB solution (25 mM), 2 μL of ATP solution (100 mM), 10 μL of urea (1.0M) into 180 μL of the prepared phosphate buffer solution with a concentration of 5 mM and a pH of 4.5, mix well and then add different amounts of urease (0, 7.5, 15, 37, 5, 75, 187.5, 375, 750, 1500mU) and 3μL nanoceria stock solution (2.5mg / mL), after mixing evenly, place the reagent in a water bath at 37°C for reaction, and use UV-Vis The spectrophotometer records the change of the absorbance value at 652nm in real time.

[0045] from Figure 4 It can be seen that in the presence of urease, the absorbance value at 652nm of the detection solution decreases continuously. This is because urease hydrolyzes urea to produce ammonia and consumes H + , the pH of the solution system increased gradually, the catalytic oxidation ability of the nanomimetic enzyme nanoceria was gradually inhibited, and the cat...

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Abstract

The invention discloses a detection reagent containing nano mimic enzyme. The invention further discloses a visual detection method of bio-enzyme or protein and its inhibitor on the basis of nano mimic enzyme technology. The method shows good pH dependence for the oxidization reaction of many developing agents on the basis of some nano materials capable of simulating natural oxidase, and many oxidase or proteins can release / consume H+ characteristics in the biological catalysis process, and uses the in-site change of pH of reaction system to influence the catalytic oxidization ability of the nano material, thus the detection reagent has color change at different levels, and the visual detection of bio-enzyme or protein activity is realized, and further the visual detection of corresponding inhibitor is realized. The physical signal output by the detection method can show a very good linear relationship for the bio-enzyme or protein activity within a certain concentration scale, and thereby realizing the high-sensitivity detection of a target to be tested.

Description

technical field [0001] The invention belongs to the field of biological analysis, in particular to a method for visually and rapidly detecting biological enzymes, proteins and inhibitors thereof based on nanometer simulated enzymes. Background technique [0002] A series of biochemical reactions in organisms all have the participation of corresponding biological enzymes. It is the regulation of these biological enzymes with high selectivity and high catalytic activity that organisms can complete complex advanced life activities. For example, acetylcholinesterase (AChE) is a very important biological enzyme in the central nervous system, which can catalyze the hydrolysis of acetylcholine in the brain to generate acetate and choline. Studies have shown that low activity of acetylcholinesterase in the central nervous system can lead to a large accumulation of acetylcholine, which can cause organ damage and even death. And if the activity of acetylcholinesterase is too high, i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/46C12Q1/34
CPCC12Q1/34C12Q1/46
Inventor 魏辉程翰俊
Owner NANJING UNIV
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