Method for high-sensitivity detection of ofloxacin based on nucleic acid aptamer sensor

Abstract

The invention discloses a method for high-sensitivity detection of ofloxacin based on a nucleic acid aptamer sensor, and belongs to the field of detection of fluoroquinolone antibiotics by biosensors. The method comprises the following steps of: (1) combining a Cy3-aptamer with BHQ2-cDNA, and quenching fluorescence of Cy3 by BHQ2; (2) adding a to-be-detected sample, making the to-be-detected sample stand still for reaction, when a target object OFL exists, carrying out affinity competition to form a nucleic acid aptamer / OFL mixture, releasing and recovering fluorescence through a Cy3 fluorophore, and at the same time, enhancing the fluorescence of Cy3 by OFL; and (3) determining the concentration of OFL in the to-be-detected sample according to the recovery and enhancement degree of the fluorescence. The OFL has an enhancement effect on the fluorescence intensity of the fluorescent dye Cy3, so that the sensitivity of the method can be remarkably enhanced, and the detection limit is as low as 0.2 nM; and the detection is rapid and convenient, the dependence on large equipment instruments is not needed, and the requirements on the detection environment and time are not strict.

Description

technical field [0001] The invention belongs to the technical fields of biochemistry, molecular biology and analytical chemistry, and relates to a biosensor for detecting fluoroquinolone antibiotics, in particular to a highly sensitive method for detecting ofloxacin based on a nucleic acid aptamer sensor. Background technique [0002] Quinolones constitute a large class of synthetic antibiotic drugs that have remarkable effects in the treatment of various diseases, especially their fluorinated derivatives, called fluoroquinolones (FQs). FQs are potentially highly polluting substances that not only pollute the aquatic environment but also cause toxic effects on surface water at low concentrations. Ofloxacin (OFL), as a typical and widely used fluoroquinolone antibiotic, has a wide range of medicinal activities against most Gram-negative bacteria and many Gram-positive bacteria. Extensive use of ofloxacin in the body leads to incomplete metabolism in the body, and it has anti...

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Problems solved by technology

[0004] Although Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay [J]. Antibiotics, 2020, 9(12): 860. discloses a method for detecting ofloxacin, its 100 μM OFL can only The fluorescence of the modified fluorescent group FAM in the nucleic acid aptamer is recovered by about 40%, and the sensitivity is not high enough; moreover, in the prior art, OFL cannot enhance the fluorescence of the FAM labeled on the aptamer, and trace OFL in the sample limited detection

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