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Bioelectrochemical sensor for detecting nuclear factor-kappab, its preparation method and application

A bioelectrochemical and sensor technology, applied in the field of new bioelectrochemical sensors, achieves the effects of broad application prospects, high sensitivity, rapid quantitative detection and convenience

Inactive Publication Date: 2011-11-30
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many articles on electrochemical biosensors that study the interaction between DNA-binding proteins and their target DNA, but DNA biosensors targeting NF-κB have not been reported

Method used

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  • Bioelectrochemical sensor for detecting nuclear factor-kappab, its preparation method and application
  • Bioelectrochemical sensor for detecting nuclear factor-kappab, its preparation method and application
  • Bioelectrochemical sensor for detecting nuclear factor-kappab, its preparation method and application

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Effect test

Embodiment 1

[0031] Example 1: Modification of gold electrodes

[0032] The gold electrode was first ground on 5000-mesh metallographic sandpaper, and then polished with a suspension of alumina powder with a particle size ranging from 1.0 μm, 0.3 μm, and 0.05 μm to make the surface of the gold electrode into a smooth mirror surface. Sonicate in water ethanol and ultrapure water for 5 min, then put the gold electrode in 0.5 M H 2 SO 4 Cyclic voltammetry scan (0 - 1.5 V voltage range) was carried out in , the scan rate was set at 100 mV / s, and the stability was reached in about 20 cycles. Then dry the electrode in nitrogen to obtain a bare gold electrode with a clean surface, which can be used for the next step of DNA modification. The gold electrode was immersed in a solution containing 2 μM Capture DNA (10 mM Tris-HCl, 1 mM EDTA, 0.1 M NaCl, and 10 mM TCEP, pH 7.4) for 16 hours, and then immersed in 1 mM sulfhydryl Hexanol solution for 2 hours, then rinse the electrode with a sufficient...

Embodiment 2

[0033] Example 2: Preparation of gold nanoparticles and modification of Report DNA

[0034] The glass instruments used in the preparation of gold nanoparticles were soaked in aqua regia (hydrochloric acid: nitric acid = 3:1 (v / v)) for 30 minutes, rinsed with a large amount of tap water, washed with double distilled water and dried. Gold nanoparticles with a diameter of 13 nm were prepared by the sodium citrate reduction method (References: Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995 , 67 , 735-743.). In this method, sodium citrate is used as a reducing agent and a stabilizing agent to reduce chloroauric acid to generate gold nanoparticles under heating and boiling conditions. The specific method is to install a condenser tube in a round bottom flask with a volume of 250 ml, and add 100 ml of 0.01% HAuCl 4 , be heated to boiling, add 3.5 ml concentration rapidly and be 1% sodium citrate when vigorously stirring solution, continue stirrin...

Embodiment 3

[0036] Embodiment three: the preparation of the sample to be tested

[0037] Mix the Target DNA (1 μL, 2 μM) containing the NF-κB binding site and the complementary Complementary DNA (1 μL, 0.2 μM) in an Eppendorf tube, and hybridize at 37 °C for 30 min. Then, different concentrations of NF-κB (1 μL) were added to the sample system and incubated at 37 °C for 30 min. Finally, NEase (1 μL, 2 U) was added, and the final volume of the reaction sample was 10 μL, and incubated at 58 °C for 30 min.

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Abstract

The invention relates to a novel bioelectrochemical sensor for detecting nuclear factor-kappaB (NF-κB), its preparation method and its application. The novel bioelectrochemical sensor is a three-electrode system sensor, which is characterized in that the counter electrode in the three electrodes is a platinum electrode, the reference electrode is a saturated calomel electrode, and the working electrode is a gold electrode, and the gold electrode is decorated with strand of DNA. The sensor can successfully identify the lowest concentration of NF-κB at 0.1nM, and the linear detection range is 0.1nM~10nM. The novel bioelectrochemical sensor of the present invention is based on the specific combination of NF-κB and target DNA, and comprehensively uses the dual signal amplification characteristics of Nicking Enzyme (NEase) and gold nanoparticles, as well as the high sensitivity of the electrochemical detection method, so that NF-κB The detection of κB is simple and feasible, and the same principle can also be extended to the detection of other biomolecules, with broad application prospects.

Description

technical field [0001] The invention relates to a novel bioelectrochemical sensor, its preparation method and application, especially a novel bioelectrochemical sensor for detecting nuclear factor-kappaB (NF-κB), its preparation method and its application. Background of the invention [0002] Nuclear factor-κB (Nuclear Factor-kappa B, NF-κB) is an important and conserved protein with multidirectional transcriptional regulation in higher animals. In 1986, Baltimore et al. first discovered that mouse B lymphocytes contain a nuclear protein that can specifically bind to the enhancer B site of the immunoglobulin κ light chain gene, so they named it nuclear factor-κB. NF-κB is widely distributed in higher animals, and participates in the expression regulation of various proteins such as cytokines, chemokines, adhesion molecules, growth factors, immune receptors, oxidative kinase-related enzymes, transcription factors, etc., and controls many Important physiological and patholog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/403G01N27/48C12N15/10C12Q1/68
Inventor 李根喜朱小立赵洪喜郭超陈阳阳吴瑶
Owner SHANGHAI UNIV
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