Detection Of Ochratoxin A By A Guanine Base Fluorescence Quenching Nucleic Acid Aptamer Sensor

Ochratoxin A(OTA),ubiquitous member of mycotoxins,is produced by various species of Aspergillus and Penicillium.Potentially,it can contaminate cereals(wheat,corn)and wines.The studies have introduced OTA as nephrotoxicity,hepatotoxicity and DNA damage and inhibition of RNA and protein synthesis.Instrumental analysis represented by highPerformance liquid chromatography(HPLC)and immunoassays represented by antibodies are traditional methods for detecting OTA.However,they are usually time-consuming,labor-intensive and expensive.Therefore,these parameters limited their usage.So,there is an urgent need for a simple,rapid,highly sensitive,and highly selective detection method for determining OTA.The emerging method of detection,aptasensor,has attracted more attention for OTA detection,due to distinctive advantages including high affinity,selectivity and chemical stabilization.Meanwhile,guanine has a certain quenching ability for fluorescein(FAM).The use of guanine as the quenching group can effectively reduce the difficulty of design and reduce the detection cost.This paper is based on the specific recognition ability of OTA aptamer,and has established a new method to detect OTA by using guanine quenching FAM fluorescence.According to the experimental data and the literature analysis,we forecast that the OTA with the aptamer formed the G-quadruplexes structure.Removing the 3 'end 5 bases of the aptamer sequence as the core structure we design two new single OTA aptamer sequences OTXAsF1 and OTXAsF2,tagging on the 3' end with FAM and three prominent G bases at the 5'end as quenching group.OTXAsF2 added a pair of complementary bases to increase recognition structure stability,which is the difference of two sequences.When the target molecule OTA is present,the aptamer forms a recognition structure and the terminal bases complement each other.Then,FAM is close to the 5'-end guanine,and OTA is quantitatively detected based on the relationship between the fluorescence fluorescence value and the OTA concentration.The detection limit of OTXAsF1 biosensor is 0.2 nM,the linear range is 0.2-2 nM.The OTXAsF2 sequence has one more pair of complementary bases than OTXAsF1,which makes the structure more stable and the quenching efficiency is higher.Adding complementary chains,the detection signal can be amplified without reducing the affinity of the OTXAsF2 aptamer.As the number of complementary bases increases,the EC50 value of the working curve also increases.To balance the relationship between the response value and the EC50 value,the complementary strand A11 is finally selected for detection.The linear range is 0.5-7.8 nM and the detection limit is 0.48 nM.This method is rapid,with low detection limit,high sensitivity,and good specificity.The entire detection process requires only 30 minutes.During the experiment,it was found that the increased 3 G bases at the terminus may affect the formation of antiparallel G-quadruplexes.Therefore,we deleted the prominent G bases,thereby constructing two novel single-labeled aptamers OTXAsF10 and OTXAs F20.Because there is no steric hindrance caused by the tail chain,the FAM is closer to the plane formed by the G-quadruplexes when the aptamer recognizes OTA.So that the EC50 value of the working curve is smaller,the detection limit is lower,and the quenching efficiency is better.So the EC50 values of OTXAsF10 and OTXAsF20 sequences are 15.04±1.9 nM and 9.47±2.6 nM,respectively,and the OTXAsF20 sequence has a 20-fold higher affinity than the 1.12.2 sequence.