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Real-time in-situ quantitative analysis method for hydrogen peroxide based on bipolar nano-electrode array

A nano-electrode array, hydrogen peroxide technology, applied in the field of biosensing, can solve the problems of insufficient sensitivity, low sensitivity, expensive instruments, etc., and achieve the effects of avoiding mutual interference, improving sensitivity, and easy operation

Pending Publication Date: 2022-05-13
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The chemiluminescent method is based on using hydrogen peroxide as a co-reactant and luminol as a luminous body to detect hydrogen peroxide, but because the method hydrogen peroxide and luminous body are mixed together, it is easy to cause interference, and luminol and hydrogen peroxide The sensitivity of the system is relatively low, and some biological enzymes, enzyme-like catalysts or metal nanoparticles are needed to catalyze the reaction for signal amplification, and then used for analysis
However, the fluorescent reagents required by the fluorescence method have insufficient sensitivity due to the existence of autofluorescence and photobleaching of the fluorescence.
Chemical methods usually require micro-nano electrodes to be inserted into cells, causing certain damage to cells, and the preparation process of micro-nano electrodes is relatively cumbersome and the equipment required is relatively expensive

Method used

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  • Real-time in-situ quantitative analysis method for hydrogen peroxide based on bipolar nano-electrode array
  • Real-time in-situ quantitative analysis method for hydrogen peroxide based on bipolar nano-electrode array
  • Real-time in-situ quantitative analysis method for hydrogen peroxide based on bipolar nano-electrode array

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: the preparation of gold nanoelectrode array

[0031] Using the template-assisted electrodeposition method, gold nanoelectrode arrays were obtained. Briefly, anodized aluminum oxide (AAO) was used as a template, which was mounted on a specific holder as a working electrode. Before electrodeposition, a thin silver film with a thickness of ~1 μm was evaporated on one side of the AAO film by electron beam vapor deposition (Kurt J. Lesker). In containing 25g / L chloroauric acid solution, 80g / L triammonium citrate, 150g / L anhydrous sodium sulfite (Na 2 SO 3 ) and 60g / L ethylenediaminetetraacetic acid (EDTA) self-made electrolyte, using repeated chronopotentiometry at 8.512mA / cm 2 Gold nanoelectrode arrays were obtained at a constant current density. Electrodeposition experiments were carried out in a conventional three-electrode system, Hg / Hg 2 SO4 electrode and platinum electrode were used as reference electrodes, and Reference600 constant current potentios...

Embodiment 2

[0032] Example 2: Cell Culture

[0033] HeLa cells (human cervical cancer cells) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% penicillin, 1% streptomycin and 10% fetal bovine serum at 37°C, containing 5% CO 2 . LO2 cells (normal hepatocytes) were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin.

[0034] First, pre-polish, clean and sterilize the nano-electrode array, then add 20 microliters of polylysine solution (PLL, 1 mg / mL) to one side of the gold nano-electrode array to promote cell adhesion and growth, and incubate at 37 ° C for 2 h Finally, digest the cells from the culture dish with trypsin, collect by centrifugation, and take a certain amount of suspension in fresh medium. A portion of the cell suspension was then added to one end of the gold nanoelectrode array. Finally, they were incubated in an incubator for 8 h for subsequent experiments.

Embodiment 3

[0035] Example 3: Principle Verification

[0036] 1) First, verify that the bipolar electrode system constructed by the gold nanoelectrode array is used to realize the photoelectric conversion function. Add (a) 5mM potassium ferricyanide, (b) 5μM hydrogen peroxide, and (c) PBS (0.01M, pH 7.0) solution to the cathode end of the bipolar electrode, and add a luminescent system (ruthenium bipyridine, 1mM Ru(bpy) 3 2+ ) and co-reactant (dibutylaminoethanol, 20mM DBAE), potassium ferricyanide is obtained by cyclic voltammetry, and the reduction potentials of hydrogen peroxide and water are respectively 1.0V, 1.8V and 2.2V, and at this potential, A stronger electrochemiluminescent signal was obtained. The results show that the photoelectric conversion can be effectively realized by using the gold nanoelectrode array. See image 3 a.

[0037] 2) Secondly, it is verified that the gold nanoelectrode array can be further used in the imaging of experimental electrochemiluminescence....

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Abstract

The invention discloses a real-time in-situ quantitative analysis method for hydrogen peroxide based on a bipolar nano-electrode array. The method comprises the following steps: preparing a gold nano-electrode array by using a template-assisted electrochemical deposition method; preparing a driving electrode Ag / AgCl; constructing a closed bipolar electrode by using the nano electrode array and the driving electrode; adding polylysine to the cathode end of the closed bipolar electrode, adding the cell suspension for in-situ incubation, and adding a stimulant to induce release of hydrogen peroxide in cells; an ECL luminescent system is added to the anode end of the closed bipolar electrode, and the content of hydrogen peroxide released by living cells at the cathode end is quantified through the ECL intensity of the anode under the external potential. Release of hydrogen peroxide in cells is induced by a label-free method, sensitive electrochemical signals are converted into visual optical signals in combination with a bipolar electrode, and imaging analysis of endogenous hydrogen peroxide in cells is achieved.

Description

technical field [0001] The invention belongs to the technical field of biosensing, and relates to an electrochemiluminescence method used for imaging hydrogen peroxide, in particular to a method for real-time in-situ quantitative analysis of hydrogen peroxide based on a bipolar nanometer electrode array. Background technique [0002] At present, there are mainly chemiluminescence, fluorescence and chemical methods for the analysis of intracellular hydrogen peroxide. The chemiluminescent method is based on using hydrogen peroxide as a co-reactant and luminol as a luminous body to detect hydrogen peroxide, but because the method hydrogen peroxide and luminous body are mixed together, it is easy to cause interference, and luminol and hydrogen peroxide The sensitivity of the system is relatively low, and some biological enzymes, enzyme-like catalysts or metal nanoparticles are needed to catalyze the reaction for signal amplification, and then used for analysis. However, the flu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N27/30
CPCG01N21/76G01N27/30
Inventor 刘松琴李秀秀
Owner SOUTHEAST UNIV
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