Detection Of Ochratoxin A By Aptamer Sensor

Ochratoxin A(OTA)is a mycotoxin that is highly toxic and widely present in food(such as alcohol,coffee,cereals,etc.)and feed,posing a greater threat to human food safety.Therefore,its low cost,high sensitivity,simple and fast detection is very necessary.Compared with traditional detection methods(instrumental analysis,immunoassay),in recent years,aptamers biosensors have become a research hot spot because aptamers have the advantages of chemical synthesis and good stability.In this paper,two aptamer sensor methods are constructed based on fluorescence and chemiluminescence for OTA detection,and a nucleic acid aptamer optimization method is developed.This thesis includes three parts:The first part:Based on guanine bases that can quench fluorescein(FAM)through photo-induced electron transfer,we designed a single fluorescence(FAM)labeled oligonucleoside sersor where guanine base as a quencher.In the sensor,the reaction of fluorescently labeled oligonucleotide probe A,OTA and nucleic acid aptamer sequence B is a competitive reaction.Under optimized experimental conditions,the measured OTA concentration is between 0-8 nM,the linear equation is F=6.55 × 103+1.14 × 102COTA(nM),R2=0.9996,and the detection limit(LOD)obtained is 2.0 nM.This method is simple and has good universality.Based on the principle of the sensor in the first part,the second part,we construct a simple and fast method for optimizing the sequence of aptamers.Aptamers are OTA biorecognition elements.Reasonably optimizing the structure of is of great significance for future research.Based on the work in the previous section,a set of methods for evaluating the affinity of new nucleic acid aptamers,So as to optimize the original nucleic acid aptamer.Finally,it was determined that AP-OTA31 with 5 bases removed at the 3 'end was the core sequence,and its structure was predicted to have three pairs of complementary bases at the ends and blunt ends.This structure is suitable for constructing new biosensors to detect OTA.This method is easy to operate,high in sensitivity,short in time,and universal.It can be applied to the optimization of other aptamer sequences.The third part,we construct a method for detecting OTA based on the intramolecular splitting G-quadruplex DNAzyme chemiluminescence method based on the screened OTA aptamers with blunt ends.Inserting the aptamer with even ends into the middle of the DNAzyme makes the tightness of the nucleic acid aptamer recognize the target molecule an important factor affecting the formation of DNAzyme.In this paper,7 single-stranded oligonucleotide sequences were designed based on the types of bases at both ends of the G-quartet,the position of adenine at the end of the G-quadruplex,and the number of adenine,and finally we proved that the DNAzyme activity is higher when the G-rich sequence split by mode 4-8,and when there are two A bases at the 5 'end.The addition of formamide further increased the sensitivity and lowered the detection limit,which reached 0.13 nM.This design provides a high-efficiency scheme for the split G-quadruplex DNAzyme as the detection response unit using the blunt-end aptamer as the recognition unit.