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G-quadruplet DNAzyme signal amplification strategy-based progesterone detection method for aptamer sensors

An aptamer sensor and signal amplification technology, used in instruments, measuring devices, scientific instruments, etc., can solve complex and expensive scientific instruments and professional operators, cannot achieve high-sensitivity detection of low-concentration targets, and is not easy to miniaturize and other problems, to achieve the effect of improving selectivity and stability, high chemical stability, and avoiding poor stability

Active Publication Date: 2019-07-09
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] (1) The methods used to detect progesterone, such as HPLC, LC / MS, enzyme-linked immunosorbent assay and radioimmunoassay, these methods are costly, time-consuming, require complex and expensive scientific instruments and professional operators, and do not easy to miniaturize
[0008] (2) At present, there are few studies on aptamer sensing strategies for progesterone detection, and the detection of progesterone in biological matrices is challenging because its concentration is within 1 ng mL -1 (1ng·mL -1 =0.325nM), or even lower
However, most aptasensors output signals according to a 1:1 signal:target ratio, which has low sensitivity and narrow detection range, and cannot achieve high-sensitivity detection of low-concentration targets.

Method used

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  • G-quadruplet DNAzyme signal amplification strategy-based progesterone detection method for aptamer sensors

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Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1: the PBS damping fluid of different pH is to the impact of detection progesterone

[0049] (1) Aptamer, cDNA, H1 and H2 were centrifuged at 10,000 rpm and 4°C for 3 minutes before opening the caps for the first time; they were dissolved in secondary water to make 100 μmol / L mother solutions, and then used respectively with pH 7.4 and 0.05mol / L Tris-HCl buffer (containing 1mmol / L MgCl 2 , 1mmol / L CaCl 2 , 5mmol / L KCl, 0.14mol / L NaCl) to a specific concentration and stored at 4°C for later use;

[0050] (2) Grinding and cleaning of glassy carbon electrodes: use Al with different particle sizes of 1 μm, 0.3 μm, and 0.05 μm in sequence 2 o 3 Polish the glassy carbon electrode with polishing powder, grind 50 times clockwise and 50 times counterclockwise, ultrasonically clean with acetone, absolute ethanol, and secondary water for 1 minute, and dry naturally for use;

[0051] (3) Electropolymerization of gold: immerse the glassy carbon electrode obtained in ...

Embodiment 2

[0056] Embodiment 2: the impact of different reaction times on the detection of progesterone

[0057] (1) Aptamer, cDNA, H1 and H2 were centrifuged at 10,000 rpm and 4°C for 3 minutes before opening the caps for the first time; they were dissolved in secondary water to make 100 μmol / L mother solutions, and then used respectively with pH 7.4 and 0.05mol / L Tris-HCl buffer (containing 1mmol / L MgCl 2 , 1mmol / L CaCl 2 , 5mmol / L KCl, 0.14mol / L NaCl) to a specific concentration and stored at 4°C for later use;

[0058](2) Grinding and cleaning of glassy carbon electrodes: use Al with different particle sizes of 1 μm, 0.3 μm, and 0.05 μm in sequence 2 o 3 Polish the glassy carbon electrode with polishing powder, grind 50 times clockwise and 50 times counterclockwise, ultrasonically clean with acetone, absolute ethanol, and secondary water for 1 minute, and dry naturally for use;

[0059] (3) Electropolymerization of gold: immerse the glassy carbon electrode obtained in step (2) i...

Embodiment 3

[0064] Embodiment 3: detect progesterone

[0065] Such as figure 1 As shown, the construction of the aptamer sensor based on the G-quadruplex DNAzyme signal amplification strategy provided by the embodiment of the present invention and its application in the detection of progesterone specifically include the following steps:

[0066] S101: aptamer, cDNA, H1, and H2 were centrifuged at 10,000 rpm at 4°C for 3 minutes before opening their caps for the first time; they were dissolved in secondary water to make a 100 μmol / L mother solution, and then used with pH 7.4 and 0.05mol / L respectively. L of Tris-HCl buffer (containing 1mmol / L MgCl 2 , 1mmol / L CaCl 2 , 5mmol / L KCl, 0.14mol / L NaCl) to a specific concentration and stored at 4°C for later use;

[0067] S102: Grinding and cleaning of glassy carbon electrodes: use Al with different particle sizes of 1 μm, 0.3 μm, and 0.05 μm in sequence 2 o 3 Polish the glassy carbon electrode with polishing powder, grind 50 times clockwis...

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Abstract

The invention discloses a G-quadruplet DNAzyme signal amplification strategy-based progesterone detection method for aptamer sensors. Compared with the traditional progesterone detection method, the method provided by the invention is capable of avoiding the establishment of an analysis method for precious instruments; and a nucleic acid aptamer is used as a recognition element, so that the selectivity and stability of target detection are improved. A signal amplification strategy and an electric analysis method are combined to construct an electrochemical sensing new method and new technologytaking progesterone as a detection object, so that the analysis sensitivity is improved. The progesterone detection method provided by the invention is more convenient, more sensitive and higher in selectivity and cost benefit, and has important practical significance and development prospect in the analysis of trace small biological molecules.

Description

technical field [0001] The invention relates to the construction of an aptamer sensor based on a G-quadruplex DNAzyme signal amplification strategy and its application in progesterone detection, belonging to the technical field of aptamer sensing. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] Progesterone is a typical endocrine disrupting substance, and its content in human or animal body is above or below a certain threshold, which will have adverse effects on animals and humans. In addition, when humans or animals ingest high doses of progesterone, usually very little is absorbed by the body, and the rest is discharged as waste into sewage and may have an impact on the environment. Therefore, it is crucial to monitor the concentration of progesterone in environmental or clinical samples within an appropriate range. At present, the methods for detecting progesterone are mainly HPLC, LC / MS, enzyme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/48
CPCG01N27/3277G01N27/3278G01N27/48
Inventor 刘冰倩石维平蔡杰杨娅妮
Owner GUIZHOU UNIV
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