A Non-Enzyme and Non-Label Sensitive Fluorescent Aptasensor Based on Simulation-Assisted and Target-Triggered Hairpin Probe Self-Assembly for Ochratoxin a Detection

Abstract

The monitoring and control of mycotoxins has caused widespread concern due to their adverse effects on human health. In this research, a simple, sensitive and non-label fluorescent aptasensor has been reported for mycotoxin ochratoxin A (OTA) detection based on high selectivity of aptamers and amplification of non-enzyme hybridization chain reaction (HCR). After the introduction of OTA, the aptamer portion of hairpin probe H1 will combine with OTA to form OTA-aptamer complexes. Subsequently, the remainder of the opened H1 will act as an initiator for the HCR between the two hairpin probes, causing H1 and H2 to be sequentially opened and assembled into continuous DNA duplexes embedded with numerous G-quadruplexes, leading to a significant enhancement in fluorescence signal after binding with N-methyl-mesoporphyrin IX (NMM). The proposed sensing strategy can detect OTA with concentration as low as 4.9 pM. Besides, satisfactory results have also been obtained in the tests of actual samples. More importantly, the thermodynamic properties of nucleic acid chains in the monitoring platform were analyzed and the reaction processes and conditions were simulated before carrying out biological experiments, which theoretically proved the feasibility and simplified subsequent experimental operations. Therefore, the proposed method possess a certain application value in terms of monitoring mycotoxins in food samples and improving the quality control of food security.

Mycotoxins as Emerging Contaminants. Introduction to the Special Issue “Rapid Detection of Mycotoxin Contamination”

“…In addition to immunoanalytical (antibody-based) setups, the development of a label-free aptamer-based fluorescent sensor is reported by Qian et al [ 18 ] for the detection of OTA. The aptasensor utilizing a nucleotide recombination hybridization chain reaction amplification element allows high selectivity for OTA with a LOD of 2.0 pg/mL (4.9 pM).…”

Recent Progress and Development of G-Quadruplex-Based Luminescent Assays for Ochratoxin A Detection

“…In a recent study, an OTA detection method based on the amplification of non-enzyme hybridization chain reaction was described (Qian et al, 2020 ). In the presence of OTA, the fluorescent signals are significantly enhanced due to the interaction of G4 with N-methylmesoporphyrin IX.…”

Fluorescent Aptasensors: Design Strategies and Applications in Analyzing Chemical Contamination of Food

“…Fluorescent aptasensors based on DNA amplification techniques, such as nuclease-aided recycling amplification, , HCR, and RCA were also applied for the sensitive detection of OTA. For example, in a structure-switching aptamer approach, RNase H was applied to cleave the RNA in cDNA/RNA hybrid and release the cDNA, leading to cDNA recycling signal amplification .…”

“…In structure-switching aptasensors that utilized the adsorption affinity of dye-labeled ssDNA with GO, cDNA hybridized with a specifically designed hairpin DNA and Exo III was applied to digest dsDNA for cDNA release to generate a cyclic hybridization–hydrolysis process for signal amplification . Qian et al reported a simple, sensitive, and label-free fluorescent aptasensor for OTA detection through NMM binding with G4 structures generated in HCR products . The study combined computer simulation and biological experiments.…”