LAMP-H-responsive electrochemical ratiometric biosensor with minimized background signal for highly sensitive assay of specific short-stranded DNA.

Herein, the sequence-specific short-stranded biomarker DNA (hDNA, 21-nt) is acted as targeting out-primer to implement the loop-mediated isothermal amplification for releasing hydrogen ions (LAMP-H). Using LAMP-H as signaling transducer, we report a highly sensitive electrochemical ratiometric biosensor for hDNA with minimized background signal, which is achieved via magnetic separation using AuNPs-modified FeO (Au@FeO) as micro-reactor. In Au@FeO, a double-stranded complex of a pH-responsible strand (I*) and a substrate strand (S*) is bound via Au-N bonds, where the treatment with LAMP-H leads to I* folding into i-motif conformation and S* dehybridization. The S* further hybridizes a catalytic strand (C*) to assemble Mg-DNAzymes that are cleaved by Mg, releasing C* for repeated formation and robust nicking of Mg-DNAzymes. The resultant output fuel strands (F*) are introduced in a modified electrode to drive the strand displacement of two hairpins individually labeled with two electron mediators. Through F*-mediated recycled amplification, the ratio of their electrochemical currents changed in opposite is highly sensitive to the varied hDNA down to 2.1 fM. By integrating LAMP-H-stimulated i-motif switching with Mg-DNAzyme cleavage, this logic transduction of LAMP-H(i-motif/Mg-DNAzyme)F* efficiently minimizes the inherent background of traditional LAMP-based assays. Resultantly, our electrochemical ratiometric strategy would be applicable to diverse short-stranded DNAs or even RNAs as targeting primers of LAMP.