Detection method of trace amount of target based on monomolecular fluorescent sensing

A detection method and fluorescence sensing technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as affecting repeatability and reliability, reducing solution fluorescence background, reducing sensitivity and specificity, etc. Highly selective and sensitive, improved sensitivity and specificity, easy-to-prepare effects

Active Publication Date: 2018-03-30
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 adsorp

Method used

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  • Detection method of trace amount of target based on monomolecular fluorescent sensing
  • Detection method of trace amount of target based on monomolecular fluorescent sensing
  • Detection method of trace amount of target based on monomolecular fluorescent sensing

Examples

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Embodiment 1

[0052] Example 1: Detection of Acetamiprid and PCB-77 by Single Molecule Fluorescence Sensing

[0053] (1) Surface modification of microscope slides

[0054] Disposable slides were sonicated in 1 M KOH solution for 20 min and rinsed with water. Boil in 14% hydrogen peroxide / ammonia for more than 20 minutes, rinse with water, then sonicate in methanol for 15 minutes and rinse with acetone. Put a certain amount of deionized water at the bottom of the empty pipette tip box to maintain a humid environment. Place the above blow-dried slides on the rack of the pipette tip box.

[0055] A 0.1 M sodium bicarbonate solution was prepared as PEG buffer. Prepare a solution containing 250mg / ml mPEG and 25mg / ml biotin-PEG with this buffer, immediately drop 120μL on each slide, and cover it with another slide without air bubbles. Reacted for 3 hours at room temperature, rinsed with water and blown dry with nitrogen, with the modified surface facing up on the rack of the pipette tip box. ...

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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-...

Claims

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Application Information

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IPC IPC(8): C12Q1/6834
CPCC12Q1/6834C12Q2525/205C12Q2563/107
Inventor 翁瑞苏昕邱静喻长远钱永忠楼圣婷
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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