title: Exonuclease-Catalyzed Target Recycling Amplification and Immobilization-free Electrochemical Aptasensor.
abstract: A simple, sensitive, and selective immobilization-free electrochemical aptasensor had been developed which combines the advantages of the discrimination of the aggregation of long and short DNA on a negatively charged indium tin oxide (ITO) electrode, high selectivity of the aptamer, and high efficiency of exonuclease-catalyzed target recycling amplification. Ochratoxin A (OTA), a type of mycotoxin, has been chosen as the model target. Methylene blue (MB) labeled probe DNA had been hybridized with the OTA aptamer first, which cannot diffuse freely to the negative charged ITO electrode surface due to the repulsion of the negative charges, since the hybridized DNA contains large negative charges. In the presence of target (OTA), the aptamer prefers to form an OTA-aptamer complex in lieu of an aptamer-DNA duplex, which results in the dissociation of probe DNA from the probe DNA-aptamer complex. The released probe DNA could be digested into mononucleotides, including a MB-labeled electroactive mononucleotide (eT), due to the employment of the RecJf exonuclease, a single-stranded DNA specific exonuclease. Since the eT contains little negative charge, it can diffuse easily to the negative charged ITO electrode surface, which results in the enhanced electrochemical response detected. At the same time, the aptamer in the OTA-aptamer complex can be digested by RecJf exonuclease also to liberate the target, which can participate in the next reaction cycling and realize the electrochemical signal amplification. Based on this strategy, an ultrasensitive homogeneous immobilization-free electrochemical aptasensor for OTA can be developed with a low detection limit (LOD) of 0.004 ng mL(-1) (S/N = 3). The proposed biosensor combines the advantages of the simplicity of immobilization-free homogeneous ITO based electrochemical determination, high efficiency of exonuclease-catalyzed target recycling, and high selectivity of the aptamer. The fabricated biosensor has been applied to detect OTA in real samples with satisfactory results. The same strategy can be applied to develop biosensors for diverse targets.
authors.affiliation: Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Fuzhou University , Fuzhou, Fujian 350116, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Tan
authors.affiliation: Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Fuzhou University , Fuzhou, Fujian 350116, China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Wei
authors.affiliation: Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Fuzhou University , Fuzhou, Fujian 350116, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Agrifood Safety and Quality, Ministry of Agriculture, Institute of Quality Standards & Testing Technology for Agriculture Products, China Agricultural Academy of Science , Beijing 100081, P.R. China.
authors.forename: Peilong
authors.initials: P
authors.lastname: Wang
authors.affiliation: Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Fuzhou University , Fuzhou, Fujian 350116, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Qiu
authors.affiliation: Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Fuzhou University , Fuzhou, Fujian 350116, China.
authors.forename: Longhua
authors.initials: L
authors.lastname: Guo
authors.affiliation: Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Fuzhou University , Fuzhou, Fujian 350116, China.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Fuzhou University , Fuzhou, Fujian 350116, China.
authors.forename: Huang-Hao
authors.initials: HH
authors.lastname: Yang
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D055162:Biocatalysis; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry; D055664:Electrochemical Techniques / Q000295:instrumentation; D004566:Electrodes; D005092:Exonucleases / Q000378:metabolism*; D008751:Methylene Blue / Q000737:chemistry; D009793:Ochratoxins / Q000096:biosynthesis* / Q000737:chemistry; D017971:Tin Compounds / Q000737:chemistry
chemical_list: D052157:Aptamers, Nucleotide; D009793:Ochratoxins; D017971:Tin Compounds; C025589:ochratoxin A; C109984:indium tin oxide; D004247:DNA; D005092:Exonucleases; D008751:Methylene Blue