A method for detecting cocaine based on polycytosine-based nucleic acid aptamers

A nucleic acid aptamer, polycytosine technology, applied in the field of biotechnology detection, can solve the problems of strong background signal, low detection limit, complicated operation, etc., and achieves the effects of simple method, reduced synthesis cost, and improved detection sensitivity

Active Publication Date: 2022-03-22
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These sensors also have some problems, such as strong background signal, low detection limit, complicated operation and high cost

Method used

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  • A method for detecting cocaine based on polycytosine-based nucleic acid aptamers
  • A method for detecting cocaine based on polycytosine-based nucleic acid aptamers
  • A method for detecting cocaine based on polycytosine-based nucleic acid aptamers

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Experimental program
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Effect test

Embodiment 1

[0025] Poly-C modification is carried out on the cocaine nucleic acid aptamer fragment, and the cocaine nucleic acid aptamer fragment sequence after Poly-C modification is shown in SEQ ID NO.1, which is: 5'-CCCCCCCCCCCCCCCCATAGGGAGACAAGGATAAATCCTTCAATGAAGTGGGTCTCCC-3, the sequence is composed of Shanghai Sangon synthesized and modified FAM fluorescence (FAM-modified DNA has a characteristic absorption peak at 520nm), and then added phosphate-buffered saline (PBS) to configure the PBS buffer of Poly-C cocaine nucleic acid aptamer with a final concentration of 10 μM , stored at -20°C;

[0026] Add 5 µg / ml GO to 600 µL of PBS buffer containing 20 nM Poly-C cocaine aptamer, and react at room temperature for half an hour to obtain the Poly-C-GO cocaine aptamer;

[0027] Add Tween 20 with a concentration of 0.05% to the Poly-C-GO cocaine nucleic acid aptamer solution containing 6 µg / mLGO and 20nMPoly-C modification, and measure its fluorescence intensity after reacting at room tempe...

Embodiment 2

[0030] A method for detecting cocaine based on a nucleic acid aptamer of polycytosine, comprising the following steps:

[0031] (1) Poly-C modification was performed on the cocaine nucleic acid aptamer fragment. The sequence of the poly-C modified cocaine nucleic acid aptamer fragment is shown in SEQ ID NO.1, which was synthesized by Shanghai Sangong and FAMed Fluorescent modification (FAM-modified DNA has a characteristic absorption peak at 520nm), then add phosphate buffered saline (PBS) to configure the PBS buffer solution of Poly-C cocaine nucleic acid aptamer with a final concentration of 10 μM, and store at -20°C;

[0032] (2) Add 5 µg / ml GO to 600 µL of PBS buffer containing 20 nM Poly-C cocaine aptamer, and react at room temperature for half an hour to obtain Poly-C-GO cocaine aptamer;

[0033] (3) Add Tween 20 at a concentration of 0.01% to the Poly-C-GO cocaine nucleic acid aptamer for modification, and perform fluorescence detection after 30 minutes of reaction at r...

Embodiment 3

[0037] Poly-C modification was performed on the cocaine nucleic acid aptamer fragment, and the nucleotide sequence of the cocaine nucleic acid aptamer fragment modified by Poly-C is shown in SEQ ID NO.1, and the sequence was synthesized by Shanghai Sangong and FAM Fluorescent modification (FAM-modified DNA has a characteristic absorption peak at 520nm), then add phosphate buffered saline (PBS) to configure the PBS buffer solution of Poly-C cocaine nucleic acid aptamer with a final concentration of 10 μM, and store at -20°C;

[0038] Add GO at concentrations of 0, 2, 4, 5, 6, and 7 μg / mL to 600 µL of PBS buffer containing 20 nM Poly-C cocaine nucleic acid aptamer, react at room temperature for half an hour, and measure the fluorescence intensity (a.u.) . figure 2 It is the comparison graph of fluorescence intensity after quenching of Poly-C cocaine nucleic acid aptamer by different concentrations of GO; figure 2 It can be seen that as the concentration of GO increases, the f...

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Abstract

The invention belongs to the field of biotechnology detection, in particular to a polycytosine-based nucleic acid aptamer detection method for cocaine. The present invention uses polycytosine to modify the cocaine nucleic acid aptamer and fluorescently labels it, graphene oxide physically adsorbs the modified cocaine nucleic acid aptamer, and Tween 20 removes the adsorption of cocaine on the GO surface to detect cocaine; The recovery of fluorescence after adding Tween 20 is significantly better than that without adding Tween 20. Using GO to adsorb cocaine aptamers with Poly-C does not require additional chemical modification of DNA, and can effectively immobilize DNA for quenching. Poly-C modified The cocaine aptamer can effectively reduce the cost of synthesis, and the method is simple, fast, accurate and highly sensitive.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, in particular to a polycytosine-based nucleic acid aptamer detection method for cocaine. Background technique [0002] Cocaine is a stimulant that can seriously endanger human physical and mental health, causing a series of mental illnesses and even death. Finding highly sensitive detection methods is of great significance for treatment and prognosis. At present, traditional cocaine detection mainly uses chromatography, spectroscopy, and electrochemical techniques. However, these methods have disadvantages such as complex procedures, long time consumption, and high cost. With the development of science and technology, DNA-functionalized nanomaterials have attracted extensive research interest. The detection of cocaine in the field of sensors includes electrochemical sensors, enzyme-linked immunosorbent sensors, piezoelectric sensors, field-effect transistor sensors, etc. These sensors als...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/94G01N33/58C12N15/10C12N15/115
Inventor 高力相文文时海霞刘城
Owner JIANGSU UNIV
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