Immobilization-Free Programmable Hairpin Probe for
Ultrasensitive Electronic Monitoring of Nucleic Acid Based on a Biphasic
Reaction Mode
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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 <i>anti</i>-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 <i>anti</i>-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 <i>anti</i>-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 3<i>s</i><sub>blank</sub> 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