The Study On The Detection Of Patulin Based On Aptamer Recognition

Patulin is a mycotoxin produced by Penicillium claviforme, Aspergillus clavatus, Byssochlamys fulva, which reported to have acute toxicity and chronic toxicity to human body. Recently, reported detection methods for patulin such as HPLC, ELISA may display some limits in the aspects on detection limis, sensitivity, cost, ease of operation, on-site test, etc. Aptamers are single-stranded oligonucleotides with high affinity and high specifity binding to the target molecules. They can be obtained by systematic evolution of ligands by exponential enrichment(SELEX) in vitro screening. In this work, aptamers for patulin with high affinity and high specifity have been obtained by GO(graphene oxide)-SELEX without immobilization, then applied to visual detections for patulin.Firstly, graphene oxide was used for the separation for bound and unbound single-stranded oligonucleotides(ss DNA) because of its strong adsorption to ss DNA, which skiped of target molecules activating and connecting to the solid material. There were 15 rounds of selections for the small molecule toxin patulin, and finally aptamer PAT-11 with high affinity and high specifity was obtained after the cloning, sequencing and analysis of the primary and secondary structrures of all obtained ligonucleotide sequences. The dissociation constant of PAT-11 was 21.83±5.022 nmol/L.Secondly, a visial detection method was developed based on the aptamer and the color changing effect on the gold nanoparticles, which can be protected by aptamers in the high salt solution. Under the optimal conditions, the correlation between concentration of target and A650/A521 was observed to be linear within the range of 0.1 ng/m L to 10 ng/m L. The linear regression equation was y=0.22026x+0.13648(R2=0.9909), and the detection limit of the proposed method was 0.086 ng/m L. The specificity experiment and detection of the apple juice samples showed that this developed method could be used for detection of the real sample, recovery of standard was 95.00~106.20%.Lastly, a visial detection method was developed based on the aptamer and color changing effect of squaric acid and IronⅢ, using aptamer-conjugated magnetic nanoparticles for seperation and aptamer-conjugated gold nanoparticles for recognition. Under the optimal conditions, the correlation between concentration of target and A469 UV-visible absorption was observed to be linear within the range of 0.05 ng/m L to 25 ng/m L. The linear regression equation was y = 0.15606 x + 0.05182(R2= 0.9905),and the detection limit of the proposed method was 0.048 ng/m L. The specificity experiment and detection of the apple juice samples showed that this developed method could be used for detection of the real sample, recovery of standard was 91.20~108.88%.