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Peroxide mimic enzyme and preparation and applications thereof

A technology of peroxides and simulating enzymes, applied in the fields of biochemical equipment and methods, physical/chemical process catalysts, organic compounds/hydrides/coordination complex catalysts, etc. problems such as mass production and application, to achieve the effect of low manufacturing cost, good biocompatibility, and high labeling efficiency

Inactive Publication Date: 2014-09-03
NANJING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] (1) Nucleic acid is expensive, its structure is not stable enough, and its storage conditions are harsh. It is not suitable for large-scale industrial synthesis, and its catalytic activity is still difficult to match that of proteases. For example, document 1
[0005] (2) The cost of graphene is expensive, the difficulty of biological functionalization and the easy pollution caused by graphene production restrict its mass production and application, as document 2
[0007] The above defects have made it difficult to obtain peroxide mimetic enzymes with stable properties, low prices, structures close to natural enzymes, green environmental protection, and comparable catalytic activity to natural enzymes by using existing processes, which limits its further application in the field of biological analysis.

Method used

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  • Peroxide mimic enzyme and preparation and applications thereof
  • Peroxide mimic enzyme and preparation and applications thereof
  • Peroxide mimic enzyme and preparation and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The peroxide mimetic enzyme structure is as figure 1 The carbon nitride nanosheet is used as the carrier, and the peroxide mimetic enzyme is prepared through the axial coordination bond between the abundant pyridinic nitrogen atom in its skeleton and the cobalt ion in the cobalt porphyrin center. The specific preparation process is as follows:

[0055] (1) Use cheap industrial raw material melamine as the reactant, program the temperature in the muffle furnace to raise the temperature by 3-5 °C per minute to 500-600 °C, and react for 4 hours to produce yellow granular b-C 3 N 4 . Then g-C was prepared by a simple thermal oxidation-liquid phase exfoliation tandem scheme 3 N 4 , 500~600℃, thermal oxidation time is 2~4h, ultrasonic time is 30-60min; finally disperse and dilute with secondary water to a mass concentration of 1.0mg·mL -1 of aqueous solution.

[0056] (2) Take 1.0mg·mL -1 g-C 3 N 4 aqueous solution and 1mMCo II Mix and stir the methanol solution of -...

Embodiment 2

[0059] VerificationCo II -hemin with g-C 3 N 4 Through the binding mode and verify the catalytic properties and structural features:

[0060] Firstly, the electrochemical properties of peroxidase are verified, and the detection steps are as follows:

[0061] (1) Use 0.3 and 0.05 μm γ-Al for GCE respectively 2 o 3 Polished, ultrasonically cleaned in absolute ethanol and secondary water, and finally dried with high-purity nitrogen, stored at room temperature for later use.

[0062] (2) Take the equivalent amount of Co respectively II -hemin solution and peroxide mimetic enzyme solution were dropped on the surface of two bare electrodes and allowed to dry naturally. The catalytic current of the above-mentioned modified electrode in the nitrogen-saturated detection solution is measured by electrochemical technology, and a certain amount of H is added to the nitrogen-saturated detection solution. 2 o 2 catalytic current.

[0063] (3) A standard three-electrode configuratio...

Embodiment 3

[0066] Select respectively the peroxide mimetic enzyme prepared by the present invention (Co II -Heming-C 3 N 4 ) and several other peroxide mimetic enzymes are used as catalysts to catalyze the oxidation reaction of pyrogallic acid, and carry out kinetic determination. The determination method is as follows:

[0067]

[0068] After the prepared peroxide mimetic enzyme was dispersed in PBS, the kinetic parameters of the enzymatic reaction as a catalyst were measured based on the pyrogallol oxidation reaction. The specific measurement method is as follows: Figure 8 Add standard aqueous solutions of different concentrations of pyrogallic acid in batches as shown, 2.0, 1.0, 0.5, 0.25, 0.125mM, according to the classic Lineweaver-Burk equation:

[0069] 1 / υ=(K M / V max )·1 / [S]+1 / V max

[0070] Where υ is the instantaneous reaction rate catalyzed by the enzyme, K M is the Michaelis-Menten constant, [S] is the enzyme catalytic substrate concentration, V max is the maxim...

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Abstract

The invention discloses a peroxide mimic enzyme and preparation and applications thereof, and relates to a horse radish peroxidase bio-mimetic technology, namely a technology of orderly fixing natural enzyme prothetic groups that are porphyrin molecules on the surface of a mass-produced carbon nitride nanosheet in an axial coordination manner so as to produce a mimic enzyme with a structure approaching a natural enzyme protein structure. The various catalytic kinetics indexes of the mimic enzyme are equivalent with that of a natural enzyme. On this basis, a general peroxide mimic enzyme CoII-Hemin@g-C3N4 nanometer biological probe is designed, and a high-sensitivity high-specificity biosensor used for detection of the H7N9 avian influenza virus is constructed, wherein the biosensor has characteristics of stable electrogenerated chemiluminescence output signals and a low detection lower limit. The mimic enzyme can be used in the aspects of biomedicine, clinical diagnosis, environment monitoring, judicial expertise, and the like.

Description

technical field [0001] The invention relates to the field of bioelectric analysis chemistry, in particular to a peroxide mimetic enzyme, its preparation and its application. Background technique [0002] Peroxidase is a kind of redox enzyme produced by microorganisms or plants, which can catalyze many reactions. It is based on H 2 o 2 Enzymes that catalyze the oxidation of substrates for electron acceptors. It mainly exists in the peroxisomes of cells. With iron porphyrin as a prosthetic group, it can catalyze the reaction of oxidized phenolic and amine compounds with hydrogen peroxide, and has the effect of eliminating the toxicity of hydrogen peroxide, phenolic and amines. double effect. Peroxide mimetic enzymes simulate the structure, characteristics, principle of action of peroxidase and the chemical reaction process of enzymes in organisms through artificially synthesized organic / inorganic composite substances. The main purpose of studying simulated enzymes is to s...

Claims

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Application Information

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IPC IPC(8): B01J31/22C08G73/06C07D487/22C12Q1/68
Inventor 邓盛元袁培新季旭波单丹黄清怡
Owner NANJING UNIV OF SCI & TECH
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