Multiple signaling probe-based ultrasensitive electrochemical DNA sensor integrated with NFC-enabled smartphone to diagnose leptospirosis

Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic disease. Due to the non-specific symptoms and low concentrations of leptospiral DNA in the blood of patients during the acute phase of leptospirosis, the development of effective biosensors for its detection remains a challenge. Here we introduced a multiple signaling probe-based electrochemical DNA sensor (E-DNA) that integrates near-field communication (NFC) with portable smartphones, enabling ultrasensitive and on-site detection of leptospiral DNA in clinical samples. Unlike conventional sandwich-hybridization assays which employ a single signaling probe, our E-DNA sensor used multiple signaling probes labeled with gold nanoparticles (AuNPs-DNA) to amplify the electrochemical signal in the presence of trace DNA targets. Hybridization between the first capture probe, coated on a magnetic bead, and the DNA target was pre-concentrated in a reaction tube. Subsequent conjugation with AuNPs-DNA significantly enhanced the gold current response. Both the increasing number of the AuNPs-DNAs and the capture probe binding sequences improved the signal by approximately 2.6-fold. This proposed sensor architecture achieved a sensitivity of 43 copies/mL, as a limit of detection (LOD), with a broad linearity ranging from 102 to 106 copies/ mL. Its specificity in clinical samples was verified by real-time polymerase chain reaction (real-time PCR). Integration of the NFC with a smartphone renders this E-DNA sensor portable for on-site diagnosis of leptospirosis. Our work provides practical guidelines for designing sensitive E-DNA sensors, especially when DNA targets are present at low concentrations.