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Preparation method and application of hybridization indicator 5,7-binitro-2-sulfo-acridone

A technology of acridone and dinitro, which is applied in the field of preparation of hybridization indicator 5,7-dinitro-2-sulfo-acridone, which can solve the problems of low sensitivity, high price and lack of economy , to achieve the effect of strong selectivity, high specificity detection and high electrochemical activity

Inactive Publication Date: 2015-01-28
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these sensing strategies can achieve sensitive and specific detection of DNA, there are still the following defects: 1) Complex and time-consuming, lack of economy
2) The existence of traditional indicators is expensive, and it will interact with both single and double-stranded DNA, and the specificity of hybridization detection is not high
3) Poor stability
4) Certain functional group labeling methods have large background signals and low sensitivity
5) False positives and false negatives are still inevitable
These electrical biosensing strategies still face great difficulties in replacing traditional clinical techniques in actual sample detection.

Method used

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  • Preparation method and application of hybridization indicator 5,7-binitro-2-sulfo-acridone
  • Preparation method and application of hybridization indicator 5,7-binitro-2-sulfo-acridone
  • Preparation method and application of hybridization indicator 5,7-binitro-2-sulfo-acridone

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Experimental program
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Embodiment 1

[0032] The characterization data of DSA are as follows:

[0033] Elemental analysis of DSA: C, 42.83 %; H, 1.99 %; N, 11.52 %. (Calculated value: C, 42.75 %; H, 1.93 %; N,11.50 %); Infrared analysis of DSA IR (KBr)ν: 3410(υNH), 1592(υC-C), 1642(υC =C), 1389 (υC-NO2), 1190 (υSO2OH).

[0034] Mass spectrometry of DSA FAB-MS: m / z 366 ([M+1]+).

[0035] DSA hydrogen spectrum analysis 1H NMR (CDCl3, δ): 9.04 (s, ArH), 8.76 (s, ArH), 8.13 (s, ArH), 7.85 (d, ArH), 6.87 (d, ArH), 4.21 ( s, NH).

Embodiment 2

[0037] The electrochemical sensor of the present invention comprises a glassy carbon electrode GCE, the surface is coated with a sensitive film, ExoIII enzyme, and auxiliary sequences AP1 and AP2. The sensitive membrane is composed of polylysine PLLy and immobilized hairpin probe gene P (5'-NH2-AAA AAT TTA TTT GAT AGG CGA ACT ATT TGT TTT TAA TAT CAA ATA ATG GTT-3'), which will immobilize the probe The glassy carbon electrode of DNA is immersed in PBS (phosphate pH 7.0) buffer solution containing a certain concentration of complementary DNA (T1) for hybridization reaction. Put the hybridized electrode in 10 U ExoIII enzyme buffer, and eliminate it by shearing at 37 °C, because ExoIII can gradually degrade the probe gene P from the blunt end of the 3′ end until the part that hybridizes with T1 to form a double strand is completely degraded , leaving some genes not hybridized with T1 remaining on the surface of the electrode. At the same time, T1 is released intact and can conti...

Embodiment 3

[0047] The above electrochemical sensor is used to detect c-erbB-2 related genes. The specific detection method is as follows: the glassy carbon electrode immobilized with the hairpin probe DNA is immersed in a solution containing a certain concentration of complementary DNA for hybridization reaction. The hybridized electrode was placed in 10 U of exonuclease III buffer, and cut and eliminated at 37 °C, the probe P transformed from a hairpin structure to a flexible short linear structure. Auxiliary probes AP1 and AP2 solutions were added dropwise on the electrode surface, and DNA long-distance self-assembly was carried out at room temperature. The long-distance self-assembled electrode was placed in a pH 7.0 PBS buffer solution containing 5,7-dinitro-2-sulfo-acridone to allow it to be embedded in the double helix structure of DNA. After the above-mentioned electrodes are washed and dried, they can be placed in PB buffer solution and detected by square wave voltammetry.

[00...

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Abstract

The invention provides a preparation method and application of a hybridization indicator 5,7-binitro-2-sulfo-acridone and provides a preparation method of an electrochemical biosensor based on a triple signal amplification technology of film modified electrode, exonuclease III auxiliary target sequence circulation and DNA long-range self-assembly by using the 5,7-binitro-2-sulfo-acridone as an indicator. The 5,7-binitro-2-sulfo-acridone is used for detecting extra-high sensitivity and high specificity of a gene relative to a target sequence c-erbB-2 relative to lung cancer. As the linear range of the electrochemical biosensor is 2 aM-50 fM, detection limit reaches 0.5 am, and sequences which are completely complementary and mismatched to each other can be better recognized, so that ultralow content of target sequence in an actual lung cancer serum sample can be clinically detected.

Description

technical field [0001] The invention relates to a preparation method and application of a hybridization indicator 5,7-dinitro-2-sulfo-acridone, belonging to the field of organic synthesis. Background technique [0002] Lung cancer is one of the major malignant tumors that threaten human life in the world. In recent years, its morbidity and mortality have increased year by year, ranking first among malignant tumors. Among them, non-small cell lung cancer (NSCLC) accounts for all lung cancers. 80% of. At present, research on the treatment of lung cancer has made great progress, but the overall 5-year survival rate of lung cancer is still only 15%. Even for patients with stage IA NSCL, the 5-year survival rate after surgery is only 80%. Therefore, early and rapid diagnosis of lung cancer is very important for the selection of treatment options and prognosis evaluation. [0003] In recent years, with the development of technologies such as biochemistry, molecular biology and i...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N27/327G01N27/26
CPCG01N27/26G01N27/327G01N33/6812
Inventor 陈敬华李春艳赵燕苹刘智晶吴冬枝陈梅章溪刘映昕
Owner FUJIAN MEDICAL UNIV
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