| Ochratoxin A(OTA)is a class of secondary toxic metabolites produced by certain fungi of the genus Penicillin and Aspergillus under suitable conditions,especially in food and feed pollution is the most serious.A large number of studies have shown that OTA has strong nephrotoxicity,liver toxicity,immunotoxicity,and carcinogenicity,posing a huge threat to human and animal health.At present,although general detection methods can also perform quantitative detection of OTA,these methods have many problems,such as long detection time,complicated sample preparation,high cost,and the need for professional personnel to operate,which cannot meet the requirements of daily rapid detection.Therefore,it is necessary to establish an OTA detection method with simple operation,low cost,short time-consuming and high sensitivity,which is of great significance for protecting human health and food safety.Since nucleic acid aptamers have the advantages of high affinity,wide application range,simple screening,reversible reusability,and non-immunity,a series of quantitative detection methods have been developed using aptamers as identification elements.Based on this,this project uses OTA as the detection target,and uses the specific recognition,high affinity and conformational transformation of the aptamer to construct DNA aptamer chemiluminescence and electrochemical biosensors,which realizes the rapid detection of OTA in food.Quantitative testing.The main research contents and results of this topic are as follows:1.Research on the detection of OTA based on triple-helix DNA aptamer chemiluminescence method.In this study,a triple-helix DNA aptamer probe(TAP)was introduced.The structure of TAP includes an aptamer loop,two stem DNA fragments,and a signal probe that forms a triple-helix structure.When OTA does not exist,TAP always maintains a stable rigid structure.When OTA is present in the system,the aptamer sequence specifically binds to the OTA,causing the conformation of TAP to change,thereby releasing the signal probe.Subsequently,with the participation of heme and K+,the signal probe self-assembled to form a G-tetramer,which can catalyze the chemiluminescence(CL)of the luminol-H2O2 system.Under optimal conditions,the signal maintains a linear relationship with the target concentration in the range of 0.1-2.0 ng mL-1,and the limit of detection(LOD)is as low as 0.07 ng mL-1.This newly designed chemiluminescence biosensor has the advantages of high sensitivity,high specificity,low detection limit and no need to modify aptamers,and can quantitatively detect trace amounts of OTA.2.Research on electrochemical detection of OTA based on DNA aptamer and RecJf enzyme-assisted signal amplification.First,the aptamer DNA hybridizes with its complementary DNA(cDNA)to form double-stranded DNA(dsDNA),the 3’end of the DNA(cDNA)used as the probe is labeled with a methylene blue(MB)signal molecule.When OTA is present in the system,the aptamer is more inclined to combine with OTA to form an aptamer-OTA complex with a G-quadruplex conformation,rather than an aptamer-cDNA double-strand,resulting in the probe DNA from dsDNA Separated out.Then,RecJf exonuclease is added to destroy the aptamer-OTA complex and release OTA,The released OTA will continue to bind with the aptamer in dsDNA for a new round,thereby realizing the recycling of the target substance.At the same time,the cDNA modified with MB is digested into single nucleotides by RecJf exonuclease,and MB is released as a signal molecule.Due to the recognition of β-cyclodextrin and host and guest,MB can diffuse and be enriched on the electrode surface.The concentration of OTA was quantitatively detected by the measurement of DPV signal.Under the best experimental conditions,the signal and target concentration showed a linear logarithmic relationship in the range of 10 pg mL-1-10.0 ng mL-1,and the LOD was 3 pg mL-1.This new electrochemical biosensor has simple operation,low cost,good linear range and sensitivity,and can complete the quantitative detection of OTA. |
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