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Fluorescent aptamer sensor based on hybridization chain reaction and ribozyme and application of fluorescent aptamer sensor

A hybrid chain reaction and aptamer sensor technology, applied in the field of chemical and biological sensing, can solve the problems of harsh experimental conditions, poor selectivity, and low sensitivity, so as to improve selectivity and sensitivity, realize quantitative detection, and shorten detection time. the effect of time

Pending Publication Date: 2021-02-02
SHANXI DATONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Prostate-specific antigen (PSA) is a broad-spectrum tumor marker, which has important clinical value for the differential diagnosis of malignant tumors, disease monitoring, and efficacy evaluation. However, in actual samples, the concentration of PSA is too low , it is difficult to achieve high sensitivity and high selectivity detection
[0003] At present, the commonly used PSA detection methods mainly include: electrochemical immunoassay, chemiluminescence immunoassay, colorimetric immunoassay, etc., but these methods have problems such as high cost, time-consuming, poor selectivity, low sensitivity, and harsh experimental conditions. , so that these methods are restricted in practical applications

Method used

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  • Fluorescent aptamer sensor based on hybridization chain reaction and ribozyme and application of fluorescent aptamer sensor
  • Fluorescent aptamer sensor based on hybridization chain reaction and ribozyme and application of fluorescent aptamer sensor
  • Fluorescent aptamer sensor based on hybridization chain reaction and ribozyme and application of fluorescent aptamer sensor

Examples

Experimental program
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Effect test

Embodiment 1

[0056] The nucleic acid aptamer probe, the first hairpin probe and the second hairpin probe were centrifuged at 10,000 rpm and 4°C for 30 seconds before opening the cover for the first time, and were dissolved in secondary water to make a concentration of 100 μM. Mother solution, then diluted with Tris-HCl buffer solution to form nucleic acid aptamer probe solution, first hairpin probe solution and second hairpin probe solution, stored at 4°C for later use; nucleic acid aptamer probe solution 5 μM 1. The first hairpin probe solution 5 μM and the second hairpin probe solution 5 μM were respectively heated to 95° C., reacted for 10 minutes, and cooled to room temperature for use.

[0057] Among them, in figure 2 In (a), add Tris-HCl buffer solution with a pH of 7.4 to the centrifuge tube, and react at 37°C for 6 hours; 2 HPO 4 -NaOH buffer, react at room temperature for 25 minutes.

[0058] exist figure 2 In (b), Tris-HCl buffer solution with a pH of 7.4 was added to the c...

Embodiment 2

[0064] The nucleic acid aptamer probe, the first hairpin probe and the second hairpin probe were centrifuged at 10,000 rpm and 4°C for 30 seconds before opening the cover for the first time, and then dissolved in secondary water to make a 100 μM mother solution, and then Dilute with Tris-HCl buffer and store at 4°C until use; heat the aptamer probe solution 5 μM, the first hairpin probe solution 5 μM and the second hairpin probe solution 5 μM to 95°C and react for 10 minutes , and cool to room temperature.

[0065] Among them, in image 3 In (a), add 50nM nucleic acid aptamer probe solution, 50nM first hairpin probe solution and 50nM second hairpin probe solution to the centrifuge tube in Tris-HCl buffer solution with pH 7.4, and react at 37°C 6 hours to obtain a mixed solution; then add 0.5 μM hemin, react at room temperature for 2 hours to obtain a reaction solution; then take out 100 μL of the reaction solution and add 5 mM thiamine solution and 25 mM hydrogen peroxide sol...

Embodiment 3

[0080] The nucleic acid aptamer probe, the first hairpin probe and the second hairpin probe were centrifuged at 10,000 rpm and 4°C for 30 seconds before opening the cover for the first time, and then dissolved in secondary water to make a 100 μM mother solution, and then Dilute with Tris-HCl buffer and store at 4°C until use; heat the aptamer probe solution 5 μM, the first hairpin probe solution 5 μM and the second hairpin probe solution 5 μM to 95°C and react for 10 minutes , and cool to room temperature.

[0081] exist Figure 5 In (a), add 50nM nucleic acid aptamer probe solution, 50nM first hairpin probe solution, 50nM second hairpin probe solution and 0.1nM immunoglobulin G (IgG) to the centrifuge tube at pH 7.4 In Tris-HCl buffer solution, react at 37°C for 6 hours to obtain a mixed solution; then add 0.5 μM hemin, react at room temperature for 2 hours to obtain a reaction solution; then take out 100 μL of the reaction solution and add 5 mM thiamine solution and 25 mM h...

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Abstract

The invention provides a fluorescent aptamer sensor based on a hybridization chain reaction and ribozyme and application of the fluorescent aptamer sensor. The fluorescent aptamer sensor comprises a nucleic acid aptamer probe and hairpin probes, wherein the nucleic acid aptamer probe comprises a nucleic acid aptamer sequence, an initiating chain sequence and a complementary sequence; the hairpin probes comprise a first hairpin probe and a second hairpin probe; and the first hairpin probe and the second hairpin probe comprise G-quadruplex ribozyme base sequences. The nucleic acid aptamer probespecifically recognizes a prostate specific antigen through the nucleic acid aptamer sequence, so that the nucleic acid aptamer probe is subjected to interconversion of conformations; and therefore, the first hairpin probe and the second hairpin probe form double-chain DNA. Under the condition of existence of hemin, the first hairpin probe and the second hairpin probe are self-assembled to form hemin / G-quadruplex ribozyme; the hemin / G-quadruplex ribozyme can catalyze and oxidize hydrogen peroxide mediated thiamine and give out fluorescence; the prostate specific antigen can be quantitatively detected; and the prostate specific antigen detection selectivity and sensitivity are improved.

Description

technical field [0001] The invention relates to the technical field of chemical and biological sensing, in particular to a fluorescent aptamer sensor based on hybridization chain reaction and ribozyme and its application. Background technique [0002] Prostate-specific antigen (PSA) is a broad-spectrum tumor marker, which has important clinical value for the differential diagnosis of malignant tumors, disease monitoring, and efficacy evaluation. However, in actual samples, the concentration of PSA is too low , it is difficult to achieve high sensitivity and high selectivity detection. [0003] At present, the commonly used PSA detection methods mainly include: electrochemical immunoassay, chemiluminescence immunoassay, colorimetric immunoassay, etc., but these methods have problems such as high cost, time-consuming, poor selectivity, low sensitivity, and harsh experimental conditions. , so that these methods are restricted in practical applications. Contents of the invent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825C12Q1/682G01N33/574
CPCC12Q1/6825C12Q1/682G01N33/57434G01N33/57484G01N2333/47C12Q2525/205C12Q2525/301C12Q2521/337C12Q2563/107
Inventor 白云峰赵璐赵瑞瑞冯海弟陈晓亮张慧琳冯锋
Owner SHANXI DATONG UNIV
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