Rapid Detection For Ochratoxin A Based On Functional Nucleic Acids Of DNA Aptamer And G-quadruplex

Ochratoxin A（Ochratoxin A,OTA）is a mycotoxin produced by different strains of Aspergillus and Penicillium.Because of its strong teratogenicity,neurotoxicity,immunotoxicity and carcinogenic effects,it seriously endangers public health and food safety.Therefore,the rapid and accurate detection and research of OTA has received great attention.Commonly used OTA detection methods are mass spectrometry,high performance chromatography,and antibody-based immunoassay techniques.These methods have good stability,accuracy and reliability for OTA measurement,but they are time-consuming,complicated,and require a large number of precision equipment and highly skilled professional operators.For this reason,it is essential to develop a fast,sensitive and easy-to-operate OTA detection method.In recent years,the biosensor technology of nucleic acid aptamers has received extensive attention in the field of edible agricultural products safety testing due to its high efficiency,flexibility,and ease of operation.With the rapid development of analytical methods and nanotechnology,research teams at home and abroad have reported a large number of aptamer-based sensing and rapid detection methods for the analysis of a series of targets such as metal ions,biomolecules,viruses,and cells.Based on this,this study uses OTA as the detection object,transforms nucleic acid aptamer and assembles triple-helix DNA molecular switches to construct a label-free colorimetric biosensor.Furthermore,we use aptamers and OTA to identify the changes in conformation before and after the combination.Thioflavin T（Th T）constructs a label-free fluorescent biosensor,and finally realizes fast and sensitive detection of OTA in colorimetric and fluorescent dual modes.The main content and related results of this research are as follows:（1）According to the base pairing principles of Watson-Crick and Hoogsteen,a triple helix molecular switch（THMS）colorimetric biosensing platform was constructed to quickly detect OTA.THMS is assembled from an appropriately modified aptamer and an oligonucleotide sequence rich in guanine.In the presence of OTA,the aptamer sequence recognizes and binds to the target,so that THMS undergoes structural transformation and releases guanine-rich oligonucleotide sequences.This sequence forms a G-quadruplex in the presence of K⁺,and combines with hemin to form a DNAzyme with horseradish peroxidase activity,which catalyzes the oxidation of ABTS^2-with hydrogen peroxide,produces a color change,and outputs a detection signal.On the contrary,in the absence of OTA,the triple helix structure is stable and there is only low background signal.Under the optimal reaction conditions,the detection signal maintains a linear relationship with the concentration of the target OTA,ranging from 10μg·kg^-1to 1 500μg·kg^-1,and the detection limit is as low as 4 ppb.This design utilizes the appropriately modified aptamer and G-rich sequence,which has the characteristics of high efficiency,specificity and speed,and provides a new idea for the visual and rapid detection of OTA.（2）Further,we use the aptamer and OTA to recognize the conformational changes before and after,combined with Thioflavin T（Th T）to construct a label-free fluorescent biosensor,which can detect OTA quickly and sensitively.In the presence of the fluorescent small molecule Th T,the OTA aptamer turns from antiparallel G-quadruplex to parallel G-quadruplex and produces strong fluorescence.As the concentration of OTA increases gradually,it transforms into a parallel G-quadruplex.The body is gradually induced into an anti-parallel structure,and the fluorescence gradually decreases.This method is simple,rapid,and highly sensitive to OTA.Under the optimal reaction conditions,the linear range of OTA concentration is 0.05μg·kg^-1～600μg·kg^-1,and the detection limit is 0.01μg·kg^-1.It has good specificity for OTA and is used in food safety.The field has good application prospects.