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

Mengyao Qian,Luhui Wang,Yafei Dong,Yue Wang,Wenxiao Hu

doi:10.3390/toxins12060376

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.

Highlights

Ochratoxin A (OTA), another major contaminating mycotoxins in various food commodities such as grains, vegetables, nuts, spices, wine and animal feed [1,2], has attracted worldwide attention after aflatoxin [3]
It is essential to carry out computer simulations of the secondary structure is critical to the function of the nucleic acid strands, we introduced an algorithm method proposed in order to economize time and cost of subsequent operations
In secondary structure is critical to the function of the nucleic acid strands, we introduced an algorithm to general, the combination of thermodynamic models and dynamic programming algorithms can predict and analyze the thermodynamic properties of the strands designed in this paper

Summary

Introduction

Ochratoxin A (OTA), another major contaminating mycotoxins in various food commodities such as grains, vegetables, nuts, spices, wine and animal feed [1,2], has attracted worldwide attention after aflatoxin [3]. The mycotoxin has highly kidney and liver toxicity, teratogenicity, carcinogenicity, mutagenicity and immunosuppressive effects on animals and humans [4,5,6]. Many international agencies have specified the highest levels of OTA in different foodstuffs. Toxins 2020, 12, 376 in wine and grape-based beverages, the European Commission has set the maximum levels at 2 μg/kg. Cereal-derived products and dried vine fruit, the levels were set at 5 μg/kg,.

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