Preparation method and application of electrochemical sensor based on tyrosinase nanocapsule

A tyrosinase, nanocapsule technology, applied in the field of biosensors

Active Publication Date: 2021-06-08
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, no tyrosinase nanocapsules have been used to prepare electrochemical sensors a

Method used

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  • Preparation method and application of electrochemical sensor based on tyrosinase nanocapsule
  • Preparation method and application of electrochemical sensor based on tyrosinase nanocapsule
  • Preparation method and application of electrochemical sensor based on tyrosinase nanocapsule

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Preparation and Characterization of Tyrosinase Nanocapsules

[0053] Firstly, the alkenyl group is connected to the surface of the enzyme molecule by adsorption, covalent crosslinking, etc. to make it a polymerizable unimolecular enzyme form, and then a thin layer of polymer is formed on the enzyme surface by in situ free radical polymerization. Shell, that is, the formation of enzyme single-molecule nanocapsules.

[0054] (a) Surface modification of tyrosinase

[0055] Dissolve tyrosinase (Tyr) in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (50 mM, pH 7.0) at a concentration of 5 mg / mL; dissolve N-acryloyloxysuccinimide (NAS) In dimethyl sulfoxide (DMSO), the concentration was 10 mg / mL.

[0056] Take 20μL 10mg / mL NAS and slowly add to 200μL 5mg mL -1 The tyrosinase solution was reacted at 4 °C for 2 h. The reacted solution was transferred to a dialysis tube with a molecular weight cut-off of 12kDa, and dialyzed in disodium hydrogen phosphate-sodi...

Embodiment 2

[0064] Activity comparison of tyrosinase and surface-modified tyrosinase

[0065] Fabrication of electrochemical enzyme sensors

[0066] (1) Polish the surface of the glassy carbon electrode with aluminum oxide powder with a particle size of 1 μm, 0.3 μm, and 0.05 μm, and then repeatedly ultrasonically clean it in absolute ethanol and deionized water, and then clean the electrode surface with high-purity nitrogen gas. Blow dry and set aside. Put in 1mmol L -1 The cyclic voltammetry curve was scanned between -0.1 and +0.6V in the potassium ferricyanide / potassium ferrocyanide solution (molar ratio 1:1). The redox peak potential difference of the curve is less than 70mV, indicating that the redox reaction on the surface of the glassy carbon electrode is a completely reversible reaction, and the electrode polishing is in good condition, and the next experiment can be carried out.

[0067] (2) The biocompatible film-forming agent chitosan (its film-forming ability and adhesion i...

Embodiment 3

[0074] Thermostability of Tyrosinase Nanocapsules

[0075] In the tyrosinase nanocapsule solution that obtains in embodiment 1, add film-forming agent chitosan, fully mix, the composition of final dispersion liquid is that the concentration of tyrosinase nanocapsule is 2.5mg mL-1 ;Chitosan concentration is 1.5mg mL -1 , 5 μL of the final dispersion was dropped onto the surface of the glassy carbon electrode, and left to dry slowly at room temperature to obtain a homogeneous tyrosinase nanocapsule-chitosan electrode (nTyr-Chi / GCE).

[0076] Prepare Tyr-Chi and nTyr-Chi electrodes with free tyrosinase and tyrosinase nanocapsules. At room temperature, add 4 μL of 1 mM bisphenol A standard solution continuously (every 50 s) to the Tyr-Chi or nTyr electrode. In the detection solution of the three-electrode system composed of -Chi electrodes in 8mL phosphate buffer solution, the constant potential scanning was carried out under the condition of the working potential of -0.04V, and t...

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Abstract

The invention provides a preparation method and application of an electrochemical sensor based on a tyrosinase nanocapsule. The preparation method comprises the following steps: firstly, performing surface modification of tyrosinase: adding an alkenyl-containing group, and immobilizing alkenyl-containing small molecules on the surface of enzyme molecules in modes of adsorption, covalent crosslinking and the like to make the enzyme molecules become polymerizable enzyme single molecules; then carrying out in-situ free radical polymerization: adding a stabilizer, a monomer, a cross-linking agent and an initiator into a surface-modified tyrosinase solution, and carrying out in-situ free radical polymerization to enable the polymer to form a thin-layer network polymer on the surface of an enzyme molecule so as to form an enzyme single-molecule nanocapsule; freeze-drying to obtain the tyrosinase nanocapsule; and finally, preparing the electrochemical sensor based on the tyrosinase nanocapsule. The sensor is high in sensitivity, low in detection limit, high in selectivity, high in stability, reliable, portable, low in cost and simple in preparation method.

Description

technical field [0001] The invention belongs to the field of biosensors, in particular to a preparation method and application of an electrochemical sensor based on tyrosinase nanocapsules. Background technique [0002] Bisphenol A, as an environmental estrogen, can simulate natural hormones in the body, bind to hormone receptors, affect the amount of hormones in the body, and interfere with the endocrine, immune and reproductive systems. It is mainly used to synthesize polycarbonate, epoxy resin and other plastic products, and is an important monomer for synthetic plastics. It is also used in the production of some plasticizers, flame retardants and other fine chemical products. In the manufacturing process of plastic products, the addition of bisphenol A can make it colorless, transparent, durable, lightweight and outstanding impact resistance. As the inner coating of metal containers, it can effectively prevent acidic vegetables and fruits from corroding metal containers...

Claims

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Application Information

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IPC IPC(8): G01N27/327
CPCG01N27/308G01N27/327
Inventor 吴令霞卢宪波陈吉平
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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