title: Immobilization-free programmable hairpin probe for ultrasensitive electronic monitoring of nucleic acid based on a biphasic reaction mode.
abstract: This work designs a novel programmable hairpin probe (PHP) for the immobilization-free electrochemical detection of nucleic acid by coupling polymerase/nicking-induced isothermal signal amplification strategy with a biphasic reaction mode for the first time. The designed PHP (including a target-recognition region, a template sequence for enzymatic reaction and an inactivated anti-streptavidin aptamer) could program multiple isothermal reactions in the solution phase accompanying in situ amplified detectable signal at the electrode surface by the labeled ferrocene tag on the PHP. Upon addition of target analyte into the detection solution, target DNA initially hybridized with the recognition region on the PHP. Replication-induced strand-displacement generated an activated anti-streptavidin aptamer with the assistance of polymerase. Then, the polymerase/nicking enzymes could cleave and polymerize repeatedly the replication product, thus resulting in the formation of numerous template-complementary DNA initiator strands. The released initiator strands could retrigger the programmable hairpin probe to produce a large number of activated anti-streptavidin aptamers, which could be captured by the immobilized streptavidin on the electrode, thus activating the electrical contact between the labeled ferrocene and the electrode. Going after the aptamers, the carried ferrocene could produce the in situ amplified electronic signal within the applied potentials. Under optimal conditions, the electrochemical signal increased with the increasing target DNA concentration in the dynamic range from 5 fM to 10 pM with a detection limit (LOD) of 2.56 fM at the 3sblank criterion. Importantly, the methodology with high specificity was also validated and evaluated by assaying 6 target DNA-spiked human serum and calf thymus DNA samples, and the recoveries were 95-110%. Further work for the programmable hairpin probe could be even utilized in a real world sample to detect miRNA-21 at femtomol level.
authors.affiliation: Institute of Nanomedicine and Nanobiosensing, MOE Key Laboratory of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University , Fuzhou 350108, P.R. China.
authors.forename: Junyang
authors.initials: J
authors.lastname: Zhuang
authors.forename: Dianping
authors.initials: D
authors.lastname: Tang
authors.forename: Wenqiang
authors.initials: W
authors.lastname: Lai
authors.forename: Guonan
authors.initials: G
authors.lastname: Chen
authors.forename: Huanghao
authors.initials: H
authors.lastname: Yang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000032:analysis / Q000097:blood*; D015342:DNA Probes / Q000737:chemistry; D055664:Electrochemical Techniques / Q000379:methods*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D015342:DNA Probes; D004247:DNA; C427203:calf thymus DNA