Method for detecting cocaine based on dimercapto aptamer

A technology of bis-mercapto nucleic acid and nucleic acid aptamer, which is applied in biological tests, measuring devices, material inspection products, etc., can solve the problems of cumbersome operation process, weak system stability, poor portability, etc., and achieves simple operation process and system stability. , easy to carry effect

Active Publication Date: 2019-01-01
JIANGSU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the defects of high cost, poor portability, weak system stability, low sensitivity, cumbersome operation process and the like in the prior art. The present invention provides a method for detecting cocaine based on a dithiol nucleic acid aptamer

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  • Method for detecting cocaine based on dimercapto aptamer
  • Method for detecting cocaine based on dimercapto aptamer
  • Method for detecting cocaine based on dimercapto aptamer

Examples

Experimental program
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Embodiment 1

[0045] (1) Synthetic specific DNA sequence:

[0046] DNA 1, DNA 2, and nucleic acid aptamer 2 were obtained using an exponential enrichment screening system and prepared by Shanghai Bioengineering Co., Ltd. The nucleotide sequences of DNA1, DNA2, and nucleic acid aptamer 2 are as follows:

[0047] The nucleotide sequence (SEQ.ID.NO.1) of DNA1 is: 5'-SH-CCATAGGGAGACAAGGATAAATCCTTCAATGAAGTGGGTCTCCC-3';

[0048] The nucleotide sequence (SEQ.ID.NO.2) of DNA 2 is: 3'-SH-GGTATCCCT-5';

[0049] The nucleotide sequence (SEQ.ID.NO.5) of the nucleic acid aptamer 2 is: 5'-CCATAGGGAGACAAGATAAATCCTTCAATGAAGTGGGTCTCCC-FAM-3';

[0050] Among them, DNA1 has no fluorescence, and a sulfhydryl group is modified at the 5' end; DNA 2 has no fluorescence, and a sulfhydryl group is modified at the 3' end, which is partially complementary to DNA 1; FAM in nucleic acid aptamer 2 is carboxyfluorescein, which is a fluorescent modification group. The fluorescent group is modified at the 3' end without ...

Embodiment 2

[0076] In this example, the concentration of cocaine and its analogs adenosine, uridine, and uric acid in the specificity verification experiment of step (7) in Example 1 was changed to 200nM, and the concentration of cocaine in human serum was added to step (8). Change to 0.05nM, 0.5nM, 50nM; other steps are the same as in Example 1.

[0077] The experimental result of this embodiment is:

[0078] (1) Established fluorescence growth rate F / F 0 The linear regression equation between -1 and cocaine concentration x is still y=12.28497x+0.58782 with embodiment 1, R 2 =0.96752, where y represents the fluorescence growth rate F / F 0 -1. According to the 3S / N principle, the calculated detection limit of cocaine is about 0.54pM.

[0079] (2) The fluorescence signal of cocaine is the highest in the specificity verification experiment, which shows that only cocaine can cause the fluorescence of the detection system to be significantly enhanced, and also shows that the detection meth...

Embodiment 3

[0083] In this example, the water bath condition in step (2) of Example 1 was changed to 4°C for half an hour, and other methods and steps were the same as in Example 1.

[0084] Experimental results:

[0085] (1) Established fluorescence growth rate F / F 0 The linear regression equation between -1 and cocaine concentration x is y=11.78529x+0.89573, R 2 =0.97846, where y represents the fluorescence growth rate F / F 0 -1, the calculated detection limit for cocaine is about 0.54pM.

[0086] (2) The fluorescence signal of cocaine is the highest in the specificity verification experiment, which shows that only cocaine can cause the fluorescence of the detection system to be significantly enhanced, and also shows that the detection method designed in the present invention has obvious selectivity.

[0087] (3) The cocaine concentration actually detected in human serum is close to the concentration of 0.01nM, 0.1nM, and 10nM added before the detection. For reliable data), the recov...

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Abstract

The invention belongs to the field of biomolecular detection methods, and particularly relates to a method for detecting cocaine based on a dimercapto aptamer. The method for detecting the cocaine, which is provided by the invention, particularly comprises the following steps of mixing a DNA (Deoxyribonucleic Acid) sequence 1 solution with a DNA sequence 2 solution, and culturing in a water bath,so as to obtain a dimercapto aptamer 1 solution; fixing a dimercapto aptamer 1 on the surface of MoS2@AuNPs; adding a cocaine solution, so as to obtain a cocaine solution 1; adding an aptamer 2, so asto obtain a cocaine solution 2; determining the fluorescence intensity of the cocaine solution 2 in the former step, calculating the fluorescence growth rate of the cocaine solution 2, and establishing a linear regression equation between the fluorescence growth rate and the concentration of the cocaine; and determining the concentration of a to-be-detected cocaine solution. According to the method, the MoS2@AuNPs-dimercapto aptamer 1, the aptamer 2 and the cocaine are adopted to form a sandwich-like structure; a fluorescence signal can be amplified, is convenient to capture and detect by a detection system; and the effect of quickly, accurately and specifically detecting the to-be-detected substance cocaine is achieved.

Description

technical field [0001] The invention belongs to the field of biomolecular detection methods, and in particular relates to a method for detecting cocaine based on a bisthiol nucleic acid aptamer. Background technique [0002] So far, laboratory analysis of cocaine mainly uses techniques such as chromatography, spectroscopy, and mass spectrometry, such as liquid chromatography-dual mass spectrometry (LC / MS / MS). Traditional drug analysis methods also include electrochemical methods, surface plasmon resonance methods, quartz microbalance methods, nanopore methods, and enzyme-linked immunoassays. However, these methods still have some disadvantages, such as poor selectivity of electrochemical methods, inaccurate analysis of the composition and content of the substance to be measured, and easy polarization, which makes the electrode potential value and equilibrium when current flows through the electrode The electrode potential deviates, resulting in a large error in the detectio...

Claims

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Application Information

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IPC IPC(8): G01N33/94G01N21/64
CPCG01N21/64G01N21/6428G01N33/946G01N2021/6417
Inventor 高力夏妮邓泽斌时海霞
Owner JIANGSU UNIV
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