A method for detecting trace target substances based on single-molecule fluorescence sensing

Abstract

The invention discloses a detection method of a trace amount of a target based on monomolecular fluorescent sensing. The detection method comprises the following steps of: (1) preparing a standard solution containing a target object; (2) fixing an auxiliary probe on a slide, adding an aptamer, and hybridizing the auxiliary probe with the aptamer; (3) adding the standard solution to the slide, andbinding the target object with the aptamer to separate the target object from the auxiliary probe; (4) adding a signal probe to the slide, and carrying out transient hybridization on the signal probeand the released auxiliary probe; (5) placing the slide on an object stage of a total internal reflection fluorescence microscope for detection, recording single-molecule fluorescence points with thenumber of times of change of the fluorescence state being more than 15 times, taking the single-molecule fluorescence points as an ordinate and taking the concentration of the target object in the standard solution as an abscissa to make a standard curve; and (6) repeating the steps (3) to (5) for a sample to be detected, recording single-molecule fluorescence points with the number of times of change of the fluorescence state being more than 15 times, and obtaining the concentration of the target object in the sample to be detected according to the standard curve.

Description

technical field [0001] The invention relates to a method for detecting a trace target, in particular to a method for detecting a trace target based on single-molecule fluorescence sensing, and belongs to the field of detection of a trace target. Background technique [0002] Trace analysis refers to analytical methods for very low concentrations of target substances. Pharmaceutical analysis, disease diagnosis, environmental analysis, food safety analysis, and public security fields all have extensive trace analysis requirements. For example, in the early stages of some diseases, some biomarkers are produced in the blood, and the concentrations of these markers are usually very low. [0003] Trace analysis is an important field of contemporary analytical chemistry, and there are more than one hundred techniques that can be used for trace analysis. For the trace detection of small molecular compounds and metal ions, the more traditional analytical methods are chromatography-...

AI Technical Summary

Problems solved by technology

In the excitation mode of total internal reflection, due to the existence of the evanescent field, only the fluorescent molecules within 100nm close to the interface are excited, thus forming a monolayer, which greatly reduces the fluorescence background of the solution, but the non-specific adsorption of fluorescent agents on the interface often gives Single-molecule detection results in a strong fluorescent background

However, subtracting the background through simple control experiments will not only reduce the sensitivity and specificity, but also affect the repeatability and reliability.

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