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Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe

A nucleic acid aptamer, a technology in a sample, used in biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve problems such as difficulty in returning to the initial state, fragile tertiary conformation, and low stability and repeated use times.

Active Publication Date: 2014-12-03
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that the stable and reusable times are very limited, usually between 5-15 times
The reason is that the tertiary conformation of DNA is very fragile, and it is difficult to return to the initial state under various elution conditions.
Such a low number of stable repeated uses also greatly limits the development and scope of application of solid-phase DNA technology.

Method used

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  • Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe
  • Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe
  • Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the preparation of kit

[0053] 1. Preparation of optical fiber surface modified with desthiobiotin

[0054] See flow chart figure 2 .

[0055] 1. Take the optical fiber and make its surface hydroxylated.

[0056] Specific methods: (1) Immerse the optical fiber (570 / 600 / 900, purchased from Nanjing Chunhui Technology Industrial Co., Ltd.) in piraha solution (98% concentrated H 2 SO 4 :H 2 o 2 =3:1, volume ratio) and reacted at 120°C for 1 h; (2) After step (1), take out the optical fiber, wash it with ultrapure water until the pH value is neutral, dry it with nitrogen at room temperature, and then place it in Store in a vacuum oven at 70°C for 1 hour.

[0057] 2. After completing step 1, take the optical fiber and modify its surface with APTS.

[0058] Specific methods: (1) After completing step 1, take the optical fiber and place it in the APTS solution for 1 hour of reaction; (2) After completing step (1), take out the optical fiber, rinse it wit...

Embodiment 2

[0087] Embodiment 2, detection ochratoxin A

[0088] Using DMF as solvent, prepare 1 mg / mL stock solution of ochratoxin A.

[0089] 1. Take the functional magnetic bead suspension (containing about 20 mg magnetic beads) of Example 1, wash it twice with PBS buffer containing 2 mg / mL BSA, and discard the supernatant.

[0090] 2. After completing step 1, take the magnetic beads, add ochratoxin A stock solution, and add PBS buffer solution containing 2 mg / mL BSA to make the total volume of the reaction system 350 μL, and mix for 15 minutes. The ochratoxin A stock solution is set with different addition amounts, so that the concentration of ochratoxin A at the initial moment of the reaction system is 6.4nM, 38.2nM, 95.4nM, 158.7nM, 222.6nM, 445.3nM, 635.0nM, 1269.6nM , 3174.9nM or 3710.0nM, each concentration set three replicates.

[0091] 3. After completing step 2, perform magnetic separation and collect the supernatant (solution to be tested).

[0092] 4. The operation proced...

Embodiment 3

[0095] Embodiment 3, the number of reusable times of the optical fiber prepared in step 1 of embodiment 1

[0096] Using DMF as solvent, prepare 1 mg / mL stock solution of ochratoxin A.

[0097] 1. Take the functional magnetic bead suspension (containing about 20 mg magnetic beads) of Example 1, wash it twice with PBS buffer containing 2 mg / mL BSA, and discard the supernatant.

[0098] 2. After completing step 1, take the magnetic beads, add ochratoxin A stock solution, and add PBS buffer solution containing 2 mg / mL BSA, so that the total volume of the reaction system is 350 μL (the initial moment of the reaction system is ochratoxin A The concentration is 3μM), mixed for 15min.

[0099] 3. After completing step 2, perform magnetic separation and collect the supernatant (solution to be tested).

[0100] 4. The operating procedure of the all-fiber evanescent wave biosensor: turn on the laser, and the excitation wavelength is 650nm; wash the optical fiber prepared in step 1 of ...

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Abstract

The invention discloses a method for detecting a target substance in a to-be-detected sample based on fluorescent sensing analysis of an aptamer probe. The method comprises the following steps: (1) jointly incubating functional magnetic beads and the to-be-detected sample together, wherein the functional magnetic beads are obtained by hybridizing magnetic beads (a) of which the surfaces are modified with specifically recognized target substance and a probe (b) linked with streptavidin and a marker; (2), after the step (1) is completed, carrying out magnetic separation and collecting a supernatant; and (3) under the excitation effect of an excitation wavelength corresponding to the marker, detecting the supernatant obtained in the step (2) by virtue of a solid-phase chip of which the surface is modified with a dethiobiotin or a biotin and determining whether the target substance is contained in the to-be-detected sample according to the values of signals. The method disclosed by the invention is low in cost and simple and rapid and has the advantages of wide aptamer target molecules, good specificity and high sensitivity and the method can be stably used over 300 times.

Description

technical field [0001] The invention relates to a method for detecting target substances in samples to be tested based on nucleic acid aptamer probe fluorescence sensing analysis. Background technique [0002] Sensing analysis methods using nucleic acid aptamers as biorecognition materials have developed rapidly in the past two decades. Aptamers are RNA or single-stranded DNA fragments that specifically recognize target contaminants. As early as the 1990s, Nature and Science journals reported nucleic acid aptamer materials and their screening techniques for the first time almost simultaneously. After more than 20 years of development, the reported nucleic acid aptamer materials have been able to detect hundreds of substances in the environment. [0003] Sensing analysis methods using nucleic acid aptamers as biological recognition elements include two methods, homogeneous and heterogeneous (or heterogeneous) aptamer sensing and analysis. At present, the homogeneous nuclei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6837C12Q2525/205C12Q2565/501C12Q2563/131
Inventor 周小红向宇王若瑜施汉昌
Owner TSINGHUA UNIV
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