Detection Of OTA In Rice By A Colorimetric Method Based On Nucleic Acid Amplification Combined With DNAzyme

As one of the most toxic mycotoxins,Ochratoxin A（OTA）has carcinogenicity,teratogenicity and nephrotoxicity,which is very harmful to human body.The OTA detection method based on colorimetric sensing technique has the advantages of visible to the naked eye and simple operation,but the disadvantage is that the sensitivity is not high.Therefore,it is crucial to construct a highly sensitive and visualized OTA detection method.This paper takes OTA as the detection target,based on the Apt recognizing ability and combined with nucleic acid isothermal amplification technology,constructs three different colorimetric sensing technique for the detection of OTA in food.The details are as follows:1.Based on the Mg²⁺-dependent DNAzyme（MNAzyme）cleavage strategy,a colorimetric sensing technique was constructed to improve the sensitivity of colorimetric assays and applied to detect OTA in rice.We designed a four-chain complex（Apt-DNA1-DNA2-MNAzyme）by the principle of complementary base pairing.In the presence of OTA,Apt combines with OTA,which destroy the structure of four-chain complex and releases MNAzyme chain.When the hairpin substrate（HP）is added,MNAzyme recognizes the ribonucleic acid（r A）site which modified on HP and cleaves HP,releasing a G-rich sequence which closed on HP.In the presence of K⁺,this sequence combines with hemin to form hemin/G-quadruplex DNAzyme,which catalyzes the oxidation of 2,2’-azino-bis-[3-ethylbenzothiazolin-6-sulfonic acid]-diammonium salt（ABTS）with hydrogen peroxide（H₂O₂）to produce a color change.In the range of 0.5 n M-25 n M,there is a good linear relationship between the concentration of OTA,and the detection limit is 0.41 n M.The recovery of OTA in rice is in the range of 93.6%-108.6%,indicating that the method can identify OTA in complex system.2.A single nucleic acid isothermal amplification technique has limited improvement in the sensitivity of colorimetric methods.Therefore,a colorimetric sensing technique based on cascaded signal amplification technique is constructed by using Apt specific combination with OTA to further improve the sensitivity of colorimetric methods,and the detection of OTA in rice is successfully realized.When OTA exists in the system,it combines with Apt,the released DNA1 combines with DNA2 in the solution to form MNAzyme,and catalyzes the cleavage of hairpin substrate（H1）to produce DNA3 and DNA4.And then,DNA3 opens the hairpin H2,releases a section of MNAzyme single chain which closed on H2,and continues the cyclic cracking of H1.The resulting DNA4 by cleavage further initiates the EDC cycle,releasing a large number of G-rich sequences to form hemin/G-quadruplex DNAzyme,and then catalyzes the color development of the chromogenic substrate ABTS mediated by H₂O₂.The method shows the characteristics of high sensitivity and strong selectivity.The detection limit is 48.97 f M and the linear range is 100 f M-10 n M.The method has been successfully applied to the determination of OTA in rice samples,and has great application value.3.Rapid visual detection is of great significance for the early monitoring of OTA,therefore,based on an enzyme-free and cascade amplification strategy,a colorimetric sensing technique was constructed for the rapid detection of OTA in rice.Among them,the cascade amplification process is composed of EDC,catalytic hairpin assembly（CHA）and MNAzyme.Through the specific binding of OTA and Apt,a starting chain is released to start the upstream reaction（EDC）,and then a new trigger unit is generated to promote the downstream reaction（CHA）to generate MNAzyme,further cleave the substrate chain（H4）,and induce the formation of hemin/G-quadruplex DNAzyme as a signal reading.The colorimetric sensing technology based on cascade amplification has the advantages of good selectivity and easy operation.The linear detection range of this method is 10 f M-100 p M,and the detection limit is as low as 8.7 f M.In addition,the solution had obvious color change when the reaction time was 20 min.