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Fluorescence detection signal amplification system

A signal amplification and fluorescence detection technology, applied in the field of fluorescence detection signal method system, can solve problems such as false positives, sequence specificity is not absolute, and samples are easily contaminated.

Pending Publication Date: 2020-06-16
HUBEI UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, nucleic acid amplification techniques such as PCR also have various problems. The sequence specificity of nucleic acid amplification techniques is not absolute, samples are easily contaminated, different nucleic acid sequences have amplification differences, and there are also non-specific amplification and off-target effects. resulting in false positive results

Method used

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  • Fluorescence detection signal amplification system
  • Fluorescence detection signal amplification system
  • Fluorescence detection signal amplification system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0143] Embodiment 3, olefinic fluorescent monomers are applied to the detection of thrombin.

[0144] Configure 25mM HEPES buffer, pH=8, containing: KCl, 1mM; heme, concentration 0.001mM; acetylacetone, 0.001mM; hydrogen peroxide, 0.001mM; DNA of G quadruplex sequence, sequence: 5'- GGTTGGTGTGGTTGG-3'(SEQ ID NO.2), concentration 0.0005mM, the sequence is also a thrombin aptamer sequence; fluorescent monomer, 0.1mM, the fluorescent monomer is:

[0145]

[0146] The above solution can be used for the detection of thrombin. Add 0.0005mM thrombin and mix well. After 20 minutes, the fluorescence is enhanced ten times.

[0147] The detection principle is attached figure 2Shown: In the absence of thrombin 103, DNA sequence 101 is difficult to form a stable G quadruplex, so it cannot bind heme. When there is thrombin, DNA sequence 101 combines with thrombin 103 to form a stable G quadruplex The body structure, combined with heme 102, can catalyze hydrogen peroxide to generate...

Embodiment 4

[0160] Implementation example 4, mercaptoenyne click reaction applied to DNA detection.

[0161] The mercaptoenyne click reaction is slightly different from the olefin polymerization reaction. The mercapto group acts as a free radical transfer during the reaction, so a very small amount of free radicals can trigger the reaction of a large number of functional groups until the mercapto group is consumed.

[0162] Configure 25mM HEPES buffer, pH=7, containing: 100mM KCl; complexes, concentration 0.001mM; mercapto compound monomers, 0.05mM; olefin fluorescent monomers, 0.05mM; DNA probe sequence: 5'-GGGTAGGGCGGGTTGGGAGTTAGCACCCAACCC- 3' (SEQ ID NO. 3), concentration 0.0005 mM.

[0163] Among them, the complex is manganese phthalocyanine, the structure is

[0164] The monomer structure of mercapto compound is:

[0165] Fluorescent monomers are:

[0166] The above solution can be used for target DNA detection. The target sequence: 5'-TGGGTGCTAACT-3' (SEQ ID NO.4), is com...

Embodiment 5

[0183] Example 5, mercaptoenyne click reaction applied to DNA detection.

[0184] Configure 25mM HEPES buffer solution, pH=7, containing: 100mM KCl; heme, concentration 0.001m; hydrogen peroxide, concentration 0.0005mM; fluorescent monomer, 0.05mM;

[0185] DNA probe sequence 1: 5'-ATGACTATCTTTAAT GGGTAGGG-3' (SEQ ID NO.6), concentration 0.001 mM; DNA probe sequence 2: 5'-GGGTTGGG CGTATGGAAAATGAG-3' (SEQ ID NO.7), concentration 0.001 mM.

[0186] Wherein the fluorescent monomer molecule is:

[0187] The above solution can be used for target DNA detection, the detectable target sequence is: 5'-CTCATTTTCCATACATTAAAGATAGTCAT-3' (SEQ ID NO.8), its 5' terminal sequence CTCATTTTCCATACA can be formed with the 3' terminal sequence CGTATGGAAAATGAG of probe sequence 2 Complementary pairing; its 3' terminal sequence TTAAAGATAGTCAT can form a complementary pairing with the 5' terminal sequence ATGACTATCTTTAAT of probe sequence 1.

[0188] Add 0.001 mM target sequence to the solutio...

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Abstract

The invention provides a novel signal amplification system and a method thereof, which can be used for chemical and biomedical detection. According to the invention, peroxidase and G-quadruplex peroxidase are utilized to construct a free radical initiation system for initiating free radical polymerization, and aggregation-induced emission is utilized to realize fluorescence signal amplification. The system can realize detection of nucleic acid and detection of an aptamer target. The method can be used on a fluorescent quantitative PCR instrument, and the concentration of a detected object is obtained through a fluorescence signal. Compared with nucleic acid amplification methods such as PCR, rolling circle amplification and the like, the method does not involve a nucleic acid amplificationprocess, so that expensive reagents such as DNA polymerase and the like are not needed, and the problem of non-specific amplification of nucleic acid does not exist. The method can also be combined with an enzyme-linked immunosorbent assay, ELISA, nucleic acid amplification and other methods to realize multiple rounds of signal amplification, and can be used for antibody antigen detection.

Description

[0001] The present invention is a divisional application of the invention patent with the application number CN201610122276.6, the invention title: Enzyme-induced free radical polymerization and detection application; the application date is March 3, 2016. technical field [0002] The invention belongs to the technical fields of biological monitoring and fluorescence detection, and in particular relates to a fluorescence detection signal method system. Background technique [0003] Fluorescence sensing is widely used in chemical, biological detection and clinical medical diagnosis because of its high sensitivity and low sample consumption. Fluorescent sensing is inseparable from the design and synthesis of fluorescent probes. Fluorescent probes can combine with target molecules in the sample or undergo chemical reactions, thereby changing the fluorescence of the probe molecules, and then the samples can be judged by instrument or naked eye observation. content of the target ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/28C12Q1/6804C12Q1/682G01N21/64
CPCC12Q1/6804C12Q1/682C12Q1/28G01N21/6428G01N2021/6432C12Q2525/205C12Q2563/107
Inventor 徐黎朱泽策
Owner HUBEI UNIV OF CHINESE MEDICINE
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