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Fluorescent biosensing method used for detecting low molecular ligand target protein and based on combination of inhibition of click chemistry reaction

A technology of click chemical reaction and small molecule ligand, which is applied in the field of fluorescent biosensing, and achieves wide application prospects, fast and easy operation, and strong specificity

Inactive Publication Date: 2015-07-22
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But at the same time, these new methods often rely on the terminal protection mechanism involving nucleic acid tool enzymes, and the activity of tool enzymes is easily affected or contaminated by acidity, ion concentration and other molecules in complex samples such as serum, which will seriously hinder the corresponding Practical application of the method to complex samples

Method used

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  • Fluorescent biosensing method used for detecting low molecular ligand target protein and based on combination of inhibition of click chemistry reaction
  • Fluorescent biosensing method used for detecting low molecular ligand target protein and based on combination of inhibition of click chemistry reaction
  • Fluorescent biosensing method used for detecting low molecular ligand target protein and based on combination of inhibition of click chemistry reaction

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment one: The fluorescence characterization of the click chemical reaction, specifically involves the click chemical reaction and the establishment of the "copper nanoparticle-graphene" fluorescence quenching nanoprobe system, the steps are as follows:

[0028]Add 18 μL of 10 μM P1 strand and 18 μL of 10 μM P2 strand to 81 μL of MOPS buffer, mix well and react at room temperature for 2.5 hours to make the P1 and P2 strands complementary hybridize to form a double strand. Subsequently, 90 μL of MOPS buffer, 18 μL of 0 or 10 μM P3 chain, and 5 μL of 100 mM ascorbic acid solution were added to the above solution, and after shaking well, 230 μL of MOPS buffer containing 200 μM copper sulfate was added to the reaction at room temperature. 30 minutes to perform click chemistry and synthesize copper nanoparticles. Finally, 40 μL of 200 μg / mL graphene oxide was added to the above solution, and after the fluorescence was quenched at room temperature for 0 or 40 minutes, ...

Embodiment 2

[0035] Embodiment two: Quantitative detection of folate receptors. Folate receptor is a cell surface receptor that binds and transcribes folate molecules (folate) overexpressed in a variety of malignant tumor cells, and its detection is of great significance for the treatment and diagnosis of related cancers. Therefore, the present invention takes the quantitative detection of folate receptor as an example to investigate the feasibility of the established fluorescent biosensing method. It specifically involves the specific recognition between the folic acid molecule modified at the end of the oligonucleotide DNA chain and the folate receptor, the binding inhibition of the click chemical reaction, and the analysis and detection based on the "copper nanoparticle-graphene" fluorescence quenching nanoprobe system, The steps are as follows:

[0036] Take 18 μL of 10 μM P1 chain and 18 μL of 10 μM P2 chain and mix with 81 μL of MOPS buffer. After reacting at room temperature for...

Embodiment 3

[0042] Embodiment three: Detection of folate receptors in complex samples

[0043] In order to study the detection effect of the fluorescent biosensing method established in the present invention in complex samples, we prepared serum samples by dissolving different concentrations of folic acid receptors in human serum (the concentration of folic acid receptors added was 0.8 nM, 3.2 nM and 6.4nM), and perform fluorescence detection according to the steps in Example 2. As shown in Table 1, the recovery rate of folate receptor detection in serum samples was 99.4-106.2%, indicating that this method can meet the needs of complex sample analysis.

[0044] Table 1 The recovery rate of detection of folate receptor in serum samples

[0045]

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Abstract

The invention discloses a fluorescent biosensing method used for detecting low molecular ligand target protein and based on combination of inhibition of a click chemistry reaction. The method comprises steps as follows: (1), specific molecular recognition of oligonucleotide DNA (deoxyribonucleic acid) chain end modified low molecular ligands and target protein of the low molecular ligands; (2), combination of inhibition of the click chemistry reaction; (3), fluorescent quantitation detection based on a fluorescence quenching nanoprobe system comprising copper nanoparticles and graphene oxide. The method has the advantages of high sensitivity, high specificity, simplicity and convenience in operation and good universality, thereby having great potential application value in fields of clinical diagnosis, drug research and development and the like.

Description

technical field [0001] The invention relates to a fluorescent biosensing method for detecting a small molecule ligand target protein, in particular to a fluorescent biosensing method for detecting a small molecule ligand target protein based on binding inhibition click chemical reaction. Background of the invention [0002] As the basic components of living organisms and important executors of cell functions, proteins are involved in almost all physiological and pathological processes, and these processes often involve the interaction between proteins and small molecule ligands. Therefore, the research on protein-small molecule ligand interaction, especially the analysis and detection of small molecule ligand target protein based on this is of great significance. It not only helps to reveal the complex cellular pathway mechanisms involved in proteins and small molecule ligands, but also provides necessary data information support for clinical diagnosis, new drug development...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 李根喜曹亚陈伟伟韩鹏
Owner SHANGHAI UNIV
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