Short-stranded DNA segment regulates alterable LAMP(H+) to switch pH-responsive intramolecular hairpins for amplified electrochemical biosensing

It is intriguing to integrate alterable loop-mediated isothermal amplification byproduct H+ ions, aLAMP(H+), with hybridization chain reaction (HCR) for translating and amplifying electrochemical sensing to a specific short-stranded DNA segment (sDNA). The sDNA-recognizable hairpin template concurrently hybridized sDNA and a hairpin precursor to form a bi-hairpin DNA structure (bhDS) as a scaffolding initiator for operating aLAMP via nucleotide incorporation, on which the blocking terminated phosphate minimized nonspecific background leakage for generating sDNA-dependent aLAMP(H+). A pH-sensitive motif strand (pH-MS5) with five non-perfectly complementary adenine/cytosine mismatches adopted intramolecular hairpin conformation at slight acidic conditions, since adenine was protonated and then formed A+-C base pairs with cytosine. The pH-MS5 complemented a -NH2-labeled inhibiting strand (NH2-IS) to form a metastable dsDNA complex. Upon intruding by aLAMP(H+), the complex was dehybridized to release NH2-IS, pointing to its Au-N immobilization in electrode surface. The next incubation of ferrocene (Fc)-tagged signaling hairpin and helping hairpin activated HCR assembly, rationally cumulating electroactive Fc for amplifiable current readout. Without tedious design of long DNA templates and primers as a big challenge in conventional LAMP, our strategy was the first example to our best understanding by coupling pH-sensitive intramolecular hairpin folding with HCR for signal transduction and amplification, achieving highly sensitive assay of short DNA segment. This would be potential for more applicable LAMP-based bioanalysis and therapeutics.