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Reagent box for HRP activity determination and H2O2 concentration detection and application thereof

A kit and reagent technology, applied in the field of biochemical detection, to achieve the effects of high sensitivity, cheap and easy-to-obtain raw materials, and low cost

Active Publication Date: 2017-09-22
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] As mentioned above, suitable concentrations of surfactants and special ionic liquid reverse micellar systems can both improve the catalytic efficiency of enzymes. However, there are few reports on the use of the two compound solutions to improve the efficiency of enzyme-catalyzed reactions.

Method used

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  • Reagent box for HRP activity determination and H2O2 concentration detection and application thereof
  • Reagent box for HRP activity determination and H2O2 concentration detection and application thereof
  • Reagent box for HRP activity determination and H2O2 concentration detection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Enzyme activity assay method:

[0063] (1) Dissolving the substrate: Dissolve 0.0127g of B in 0.5mL of F to make a 0.1mol / L TMB solution.

[0064] (2) Preparation of enzyme solution: the selected samples can come from natural peroxidase of animal and plant tissue cells and commercially purified peroxidase. If the detection object is animal and plant tissue cells, it is necessary to add F to grind it, centrifuge, and separate the supernatant to obtain a crude enzyme extract; if the detection object is commercial peroxidase, it is necessary to use F to dissolve the solid enzyme protein Dissolve to obtain the enzyme solution to be tested. Store the prepared enzyme solution to be tested in a refrigerator at 4°C, and preheat it in a water bath at 25°C for 15 minutes before measurement.

[0065] (3) Activity measurement: Add 32 μL TMB (0.1mol / L) and 1 μL C (8.8mol / L) to A in turn to obtain 4 mL of mixed reaction solution; finally add a small amount of enzyme solution to be ...

Embodiment 2

[0074] To determine the activity of an unknown HRP sample:

[0075] In this embodiment, horseradish peroxidase produced by a reagent company is selected as the detection object. Dissolve it with F, and configure it as a test solution with a concentration of 0.1g / L. The volume of enzyme solution added for activity measurement was 2 μL. With the operation of Example 1, three groups of ΔA were measured 652nm , substituted into the formula (1) to calculate the enzyme activity, and the results are shown in Table 1.

[0076] Table 1 Enzyme Activity Determination Results

[0077]

Embodiment 3

[0079] h 2 o 2 concentration determination method

[0080] Determination of H 2 o 2 The standard curve of : use A to prepare C into a set of different concentrations of H 2 o 2 Standard sample, concentration range: 1 μmol / L-100 μmol / L; TMB solution is prepared as described in Example 1. Add 32 μL TMB (0.1mol / L) and 2 μL D to 4 mL standard sample in turn, shake and mix well, measure the absorbance of the product at 652 nm after 5 min, measure 3 times in parallel for each group, and take the average value. Do absorbance (A)-H 2 o 2 Concentration (C H2O2 / mmol L -1 ) standard curve, such as figure 1 As shown, the obtained standard curve equation is:

[0081]

[0082] Due to the high concentration of H 2 o 2 It has an inhibitory effect on HRP activity, and the measured object is a trace concentration of H 2 o 2 The sample can come from the metabolic process of glucose and cholesterol in biological tissue cells, or it can be diluted H 2 o 2 sample.

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Abstract

The invention relates to a biochemical detection reagent box. The reagent box comprises a reagent A, a reagent B and a reagent C, wherein the reagent A is a SDS / [C2mim][BF4] compound reaction solution, the reagent B is color rendering substrate 3,3'5,5'-tetramethyl benzidine (TMB) solid powder, and the reagent C is a H2O2 solution. According to needs, the reagent box further comprises a reagent D, a reagent E and a reagent F, wherein the reagent D is HRP enzyme liquid, the reagent E is a buffer solution PBS, and the reagent F is a substrate solvent dimethyl sulfoxide (DMSO-d6, 99.9%). According to the reagent box, on the basis of the principle that the SDS / [C2mim][BF4] compound reaction solution can significantly improve the color rendering efficiency of HRP-H2O2-TMB reaction, and the synergism effect is adopted to achieve HRP enzyme activity determination and H2O2 concentration detection. The reagent box has the advantages of being quick, sensitive and stable in color rendering, reagent organisms for detection are non-toxic, the operation is quick and convenient, the cost is low, and the reagent box has commercial potential for large-scale HRP activity determination and H2O2 concentration detection.

Description

technical field [0001] The invention relates to a biochemical detection method, in particular to a method for measuring HRP activity and HRP activity. 2 o 2 Concentration testing kits. Background technique [0002] Horseradish peroxidase (HRP) is one of the earliest commercialized and most widely used enzyme preparations. It is not only closely related to the metabolic process of life, but also has a wide range of applications in the fields of clinical medicine, organic synthesis, biosensors, wastewater treatment, environmental chemistry and food industry. Since HRP can catalyze H 2 o 2 Oxidation of a variety of aromatic substrates to produce color reactions has become a routine detection method for biochemical experiments and disease diagnosis. This method is simple to operate, visible to the naked eye, low in cost, and less polluting. Moreover, in recent years, there have been continuous researches to refresh its detection limit, which has greatly improved the sensiti...

Claims

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Application Information

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IPC IPC(8): C12Q1/28
CPCC12Q1/28C12Q2326/12
Inventor 尚亚卓李萌黄想容帅洁李芯蕊吴婧刘洪来
Owner EAST CHINA UNIV OF SCI & TECH
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