Simple-in-Design, Powerful-in-Amplification, and Direct-Signal-Output Sensing Strategy for Ultrasensitive Ota Analysis by Both Recognition-Activated Multiple Isothermal Cyclic Strand Displacement Amplification and Dnazyme-Mediated Cyclic Signal Reporting Mechanism

Abstract

We propose a recognition-driven ultrasensitive fluorescence sensing strategy for accurate analysis of ochratoxin A (OTA) by taking Mg2+-dependent DNAzyme based catalysis as the final signal output module. Recognition of target OTA destroys the simple double-strand recognition structure and contributes to the liberation of primer probe, which can trigger and activate the primer-mediated multiple isothermal cyclic strand displacement amplification (M-CSDA) process. The designed dual templates (T-P, T-A) can contribute to the generation of enormous nicked products (DNAzyme and primer), which will take effect in the triggering of M-CSDA. Meanwhile, one of the nicked products (DNAzyme) will also take part in the cyclically cleavage of designed hairpin probes (HP) with the presence of the cofactor Mg2+, inducing the recovery of fluorescent signal of quenched hairpin probes. Only the trace amount of target OTA could generate drastic fluorescent signal and ultrasensitive detection of OTA could be well achieved. Under optimized conditions, the detection limit of this method for OTA is 0.34 fg/mL. The characteristics of this method including the simplicity in dual signal amplification design, isothermal operation process and easy fluorescence measurement model exhibit the superior sensitivity and specificity for OTA detection. And this strategy can be further expanded for the design of ultrasensitive aptamer-sensing methods for series targets.

Keywords: OTA, Aptamer, Isothermal multiple cyclic amplification, DNAzyme, Fluorescence Sensing