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A kind of bifunctional electroactive probe for recognizing cgg trinucleotide repeat sequence, preparation of probe and kit

A nucleic acid sequence, dual-function technology, applied in electrochemical variables of materials, biochemical equipment and methods, determination/inspection of microorganisms, etc., can solve the problems of complex operation, high redox rate, small volume of ferrocene, etc.

Inactive Publication Date: 2016-08-17
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, the electrochemical method requires labeled nucleic acid to analyze and detect nucleic acid. This method is costly and complicated to operate. Therefore, the label-free method has been highly praised by experts.
Electrochemical detection of label-free nucleic acids is usually based on probes that can interact with nucleic acids and have electrical activity at the same time. Metal complexes are generally used, such as ferrocene, cobalt complexes, ruthenium complexes, etc. Ferrocene is a commonly used electroactive labeling molecule, which has been widely used to label DNA. Due to the small size, high reversibility, high redox rate and high biological stability of ferrocene, the derivative of ferrocene Although these probes have good electrical activity, they have poor selectivity to nucleic acid sequences.

Method used

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  • A kind of bifunctional electroactive probe for recognizing cgg trinucleotide repeat sequence, preparation of probe and kit
  • A kind of bifunctional electroactive probe for recognizing cgg trinucleotide repeat sequence, preparation of probe and kit
  • A kind of bifunctional electroactive probe for recognizing cgg trinucleotide repeat sequence, preparation of probe and kit

Examples

Experimental program
Comparison scheme
Effect test

experiment example 2

[0065] Experimental Example 2: Electrochemical performance of a novel dual-functional electrochemical probe

[0066] Cyclic voltammetry curves of bare gold electrodes in 50 μM NCD and 50 μM FecNCD in pH 7.0 phosphate buffer solution respectively, with a scan speed of 0.1 Vs -1 , whose cyclic voltammetry is as figure 2 (A) shown. At -0.1V to 0.7V, compound NCD did not find any redox peaks, while the bifunctional electrochemical probe of the present invention showed clear oxidation peaks and reduction peaks at 0.464V and 0.409V (E 1 / 2 =0.4365V,△E p =0.055V). This result shows that the novel bifunctional electrochemical probe we designed and synthesized has good electrical activity. Further, we placed the CGG electrochemical sensor in phosphate buffer solution (pH7.0), 20μM NCD, 20μM ferrocene and 20μM FecNCD phosphate buffer solution (pH7.0), respectively, and obtained square wave voltammetry curves. Such as Figure 7 As shown, the CGG electrochemical sensor has obvious e...

Embodiment 3

[0068] Example 3: Preparation of CGG trinucleotide repeat modified electrode

[0069] Put the polished gold electrode at 0.5MH 2 SO 4 Electrochemical cleaning and activation were carried out, and then soaked in 2 μM SH-DNA (5'-HS-(CH 2 ) 6 -GGC CAC GAG TTG ACA-3') after 12 hours, take it out, wash, and then use 1mM 6-mercaptohexanol to seal the vacant electrochemical active sites on the surface of the gold electrode. The gold electrode was then immersed in 4 μM d(CGG) 10 (5'-CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG TGT CAA CTC GTG GCC-3') for 7 hours, so that d(CGG) 10 The 3' end of the electrode is hybridized with the SH-DNA base, and finally the electrode modified by the CGG trinucleotide repeat sequence is obtained. The modified electrode is characterized by electrochemical impedance, such as Figure 4 As shown in B, the electrochemical AC impedance gradually increases after DNA modification, indicating that the SH-DNA and CGG repeat sequences are successfully assemble...

Embodiment 4

[0071] Example 4: Selectivity of the novel dual-functional electrochemical probe FecNCD to the CGG triple repeat nucleotide sequence

[0072] Each prepared trinucleotide repeat sensor was placed in 20 μM novel bifunctional electrochemical probe phosphate buffer solution (pH7.0) of the present invention and incubated for 30 minutes, then washed with phosphate buffer solution to remove non-specificity Interacting probe molecules. The incubated sensor was characterized by square wave voltammetry (SWV) curves (SWV) in the phosphate buffer solution to characterize the selective recognition of the trinucleotide repeat sequence CGG by the novel bifunctional electrochemical probe of the present invention. Such as Figure 5 As shown in A, the CGG sensor exhibits obvious electrochemical signals at around 0.46V, while other trinucleotide repeat (TGG, CCG, GAA, CAG, CTG, ATT and CGG) sensors have no obvious electrochemical signals . These results indicate that the novel bifunctional el...

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Abstract

The invention relates to a difunctional electroactive probe FecNCD with electric signal transduction and nucleotide sequence recognition functions, a preparation method for the FecNCD and a kit used for detecting a CGG trinucleotide repetitive sequence and prepared from the FecNCD. The novel difunctional electroactive probe designed and synthesized in the invention integrates a recognition function and an electrochemical signal into one and enriches application of label-free nucleic acid electrochemical probes, a novel label-free electrochemical sensor used for detecting a neurological disease fragile X chromosome syndrome marker--the CGG repetitive sequence is constructed, and a satisfactory result is obtained.

Description

technical field [0001] The invention relates to the field of triple repeat nucleotide repeat sequence detection, in particular to a bifunctional electroactive probe for recognizing CGG trinucleotide repeat sequence, preparation of the probe and kit. Background technique [0002] Genetic diagnosis is a method of diagnosing diseases by directly detecting the presence or defect of genes, and the detection target is usually nucleic acid. In recent years, it has been found that neuropsychiatric diseases that are difficult to diagnose and treat are related to specific trinucleotide repeat sequences, such as fragile X syndrome and CGG repeat sequences, Huntington's disease (HD) and CAG, Friedreich co- Friedreich' is associated with GAA, and myotonic dystrophy (MD) is associated with CTG, so a lot of research has been done on the use of these genes to diagnose these neuropsychiatric diseases. In the methods of genetic diagnosis, there are mainly restriction endonuclease zymography ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C07D471/04C07F17/02G01N27/26
Inventor 何汉平夏静平常钢彭小倩孟哲张修华王升富
Owner HUBEI UNIV