Target-regulated proximity hybridization with three-way DNA junction for in situ enhanced electronic detection of marine biotoxin based on isothermal cycling signal amplification strategy.
A new signal amplification strategy based on target-regulated DNA proximity hybridization (TRPH) reaction accompanying formation of three-way DNA junction was designed for electronic detection of Microcystin-LR (MC-LR used in this case), coupling with junction-induced isothermal cycling signal amplification. Initially, a sandwiched-type immunoreaction was carried out in a low-cost PCR tube between anti-MC-LR mAb1 antibody-labeled DNA1 (mAb1-DNA1) and anti-MC-LR mAb2-labeled DNA2 (mAb2-DNA2) in the presence of target to form a three-way DNA junction. Then, the junction could undergo an unbiased strand displacement reaction on an h-like DNA nanostructure-modified electrode (labeled with methylene blue redox tag on the short DNA strand), thereby resulting in the dissociation of methylene blue-labeled signal DNA from the electrode. The newly formed double-stranded DNA could be cleaved again by exonuclease III, and the released three-way DNA junction retriggered the strand-displacement reaction with h-like DNA nanostructures for junction recycling. During the strand-displacement reaction, numerous methylene blue-labeled DNA strands were far away from the electrode, thus decreasing the detectable electrochemical signal within the applied potentials. Under optimal conditions, the TRPH-based immunosensing system exhibited good electrochemical responses for detecting target MC-LR at a concentration as low as 1.0ngkg(-1) (1.0ppt). Additionally, the precision, reproducibility, specificity and method accuracy were also investigated with acceptable results.
State Key Laboratory of Photocatalysis on Energy and Environment, Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China.
Bingqian
B
Liu
State Key Laboratory of Photocatalysis on Energy and Environment, Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China.
Jinfeng
J
Chen
State Key Laboratory of Photocatalysis on Energy and Environment, Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China.
Qiaohua
Q
Wei
State Key Laboratory of Photocatalysis on Energy and Environment, Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China.
Bing
B
Zhang
State Key Laboratory of Photocatalysis on Energy and Environment, Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China.
Lan
L
Zhang
State Key Laboratory of Photocatalysis on Energy and Environment, Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China. Electronic address: dianping.tang@fzu.edu.cn.
Dianping
D
Tang
D003217:Conductometry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007118:Immunoassay / Q000295:instrumentation*; D017403:In Situ Hybridization / Q000295:instrumentation*; D008387:Marine Toxins / Q000032:analysis*; D052998:Microcystins / Q000032:analysis*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*
D008387:Marine Toxins; D052998:Microcystins; C057862:cyanoginosin LR
DNA proximity hybridization; Electrochemical immunoassay; Isothermal cycling signal amplification; Microcystin-LR; Three-way DNA junction