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OTA (Ochratoxin A) chemiluminescence detection method based on label-free aptasensor

A chemiluminescence detection, aptamer sensor technology, applied in the field of analytical chemistry, can solve the problems of cumbersome detection steps, poor sensitivity, unstable results, etc.

Active Publication Date: 2017-01-04
绍兴叁讯科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides an OTA chemiluminescence detection method based on an unlabeled aptamer sensor to solve the technical defects of poor sensitivity and unstable results of the OTA detection method in the prior art
[0006] Another technical problem to be solved by the present invention is that the OTA chemiluminescence detection method based on the aptamer technology in the prior art generally requires additional markers to improve the luminescence sensitivity, resulting in a technology with cumbersome detection steps

Method used

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  • OTA (Ochratoxin A) chemiluminescence detection method based on label-free aptasensor
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  • OTA (Ochratoxin A) chemiluminescence detection method based on label-free aptasensor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1 Experimental principle

[0037] In this experiment, carboxyl magnetic microspheres were used as separation carriers to construct OTA aptamer sensors on the surface of magnetic microspheres. The negative correlation between the immobilized amount of OTA aptamer on the surface of magnetic microspheres and the concentration of OTA and the G base in the OTA aptamer sequence were used. The transient derivatization reaction of chemiluminescence reagent PG realizes the label-free chemiluminescence detection of OTA. Firstly, amino capture probes were immobilized on the surface of carboxyl magnetic microspheres by aminocarboxyl binding reaction; then different concentrations of OTA were taken to specifically bind to equal and excess OTA aptamers, and then the excess free OTA aptamers after the reaction were passed through Complementary hybridization with capture probes to immobilize the surface of magnetic microspheres; finally, the magnetic microspheres were separated by a ma...

Embodiment 2

[0063] A kind of OTA chemiluminescence detection method based on label-free aptamer sensor, comprises the following steps:

[0064] 1) immobilizing the amino-modified capture probe on the surface of the carboxyl magnetic microsphere through an aminocarboxyl reaction to obtain a magnetic microsphere-capture probe complex;

[0065] 2) Mix the sample to be tested with excess OTA aptamer and react fully;

[0066] 3) adding excess magnetic microsphere-capture probe complex to the product obtained in step 2) to capture free OTA aptamers, and taking the solid phase after the reaction to obtain the OTA aptamer sensor;

[0067] 4) Mix the OTA aptasensor described in step 3) with the PG solution, and then detect the CL value of the luminescence signal.

[0068] On the basis of the above technical solutions, the following conditions are met:

[0069] Step 1) includes the following operations: take the magnetic microspheres, wash them with 0.1M imidazole buffer solution, then take the s...

Embodiment 3

[0077] A kind of OTA chemiluminescence detection method based on label-free aptamer sensor, comprises the following steps:

[0078] 1) immobilizing the amino-modified capture probe on the surface of the carboxyl magnetic microsphere through an aminocarboxyl reaction to obtain a magnetic microsphere-capture probe complex;

[0079] 2) Mix the sample to be tested with excess OTA aptamer and react fully;

[0080] 3) adding excess magnetic microsphere-capture probe complex to the product obtained in step 2) to capture free OTA aptamers, and taking the solid phase after the reaction to obtain the OTA aptamer sensor;

[0081] 4) Mix the OTA aptasensor described in step 3) with the PG solution, and then detect the CL value of the luminescent signal.

[0082] On the basis of the above technical solutions, the following conditions are met:

[0083] Step 1) includes the following operations: take the magnetic microspheres, wash them with 0.05M imidazole buffer solution, then take the s...

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Abstract

The invention provides an OTA (Ochratoxin A) chemiluminescence detection method based on a label-free aptasensor. According to the technical scheme, the OTA chemiluminescence detection method comprises: fixing an aptamer capturing probe on the surface of a magnetic microsphere; combining the capturing probe and OTA with a competition relationship of an OTA aptamer respectively; obtaining a quantitative relationship between the quantity of magnetic microsphere surface detection probes and the concentration of OTA to be detected based on the fact that the specific combining capability of the aptamer is stronger than the binding force of complementary strands; finally, carrying out chemiluminescence detection on the aptasensor. A chemiluminescence detection mechanism of the OTA chemiluminescence detection method is realized based on a principle that a guanine base (G) on an OTA aptamer strand and a chemiluminescence reagent phenylglyoxal (PG) are subjected to an instantaneous derivative reaction to generate chemiluminescence, so that a labeling link of other chemiluminescence markers is omitted; operation steps are simplified on the basis of guanrateeing the sensitivity and the detection time is shortened; a chemiluminescence anslysis method has the advantages of wide linear range, simple equipment and easiness of operation, so that full-automatic detection is hopeful to realize.

Description

technical field [0001] The invention relates to the technical field of analytical chemistry, and further relates to nucleic acid aptamer technology, in particular to an OTA chemiluminescent detection method based on an unlabeled aptamer sensor. Background technique [0002] Ochratoxin A (ochratoxin A, OTA) is a highly toxic mycotoxin, and its toxicity ranks second in the mycotoxin family, second only to aflatoxin. OTA is widely distributed in nature, which not only pollutes grain food, but also seriously pollutes animal food, posing a great threat to human beings. In view of the fact that food safety is related to the safety of the whole society and is a major concern of the country and the people, the establishment of OTA's highly sensitive, simple and rapid analysis method has attracted more and more attention from researchers. [0003] At present, many grass-roots food testing departments mostly use thin-layer chromatography for OTA quantitative detection. This is also a...

Claims

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Application Information

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IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 严喜鸾刘杰肖义陂
Owner 绍兴叁讯科技股份有限公司
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