A highly sensitive electrochemical biosensor via short-stranded DNA activating LAMP(H+) to regulate i-motif folds for signal transduction

Exploring loop-mediated isothermal amplification (LAMP)-based electrochemical assay for short-stranded DNA biomarkers is intriguing. Herein, a HIV-specific DNA segment (tDNA) was introduced to implement a variant LAMP reaction, where the generated by-products H+, vLAMP(H+), acted as signal transducers for developing a sensitive electrochemical biosensor. As proof-of-concept, the tDNA-recognizable complement was programmed in a template hairpin (TH) that was ended with a 3′-terminus phosphate group to avoid non-specific elongation. Upon TH simultaneously hybridizing with tDNA and a precursor hairpin (PH), the PH underwent a loop-mediated extension with Bst polymerase and dNTPs, forming a new stem-loop hairpin in another terminus. Then the elongated product containing a double stem-loop dumbbell-like DNA structure (dDDS) was displaced and released. Using dDDS as an initiator, the vLAMP reaction occurred via consecutively cycled strand displacement for self-primed DNA incorporation. The resulting tDNA-dependent vLAMP(H+) was introduced in the modified electrode surface to guide the pH-sensitive i-motif (iM) folding. Thus, a signaling hairpin labeled with electroactive ferrocene (Fc) was dehybridized and closed, thereby bringing Fc close to the electrode interface for increased current signal readout that was highly sensitive to tDNA down to 0.034 fM. Our electrochemical biosensor would be applicable for detecting various short-stranded DNA or miRNA.