Sensitive electrochemical biosensor for Ustilaginoidea Virens short-stranded DNA segment via higher-ordered G-quadruplex multimerization induced by LAMP/H+-responsive i-motif folding

Abstract

By using variable loop-mediated isothermal amplification byproduct H+ ions (vLAMP/H+) as signal transducer, exploring the electrochemical assay of short-stranded DNA segment related to U. virens (uDNA, 21-nt) is intriguing. Herein, we report the first design of a sensitive electrochemical biosensor for uDNA as targeting input promoter of vLAMP, for which a discernible template hairpin and a precursor hairpin are well-designed. The specific recognition and complementary hybridization among them form a double-dumbbell DNA structure tethering two stem-loop motifs that is served as template precursor to operate vLAMP for DNA+1 polymerization via nucleotide incorporation, generating vLAMP/H+ to modulate pH-responsive i-motif folding of a C-rich strand. A ferrocene-tagged G-rich strand is dehybridized and incubated in modified electrode surface including an amino-labeled G-rich strand that adopts compact G-quadruplex (G4) conformation. This G4, as an initiator, guides the ‘head-to-tail’ π-π stacking of electroactive tags for developing higher-ordered wire-like G4 multimers (G4W), which are stably held together via interquadruplex multimerization. No involving additional nucleic acid amplifiers, numerous electroactive mediators are repetitively cascaded, thereby significantly amplifying the current readout signal and achieving sensitive electrochemical detection of U. virens. Benefited from simple and rapid vLAMP/H+-G4W route, our assay strategy would provide a new paradigm to create applicable electrochemical biosensors for diverse short-stranded DNA biomarkers by altering LAMP operation modes readily.