Abstract

This study presents an electrochemical DNA biosensor designed for the direct, label-free, and sensitive detection of human immunodeficiency virus type 1 (HIV-1) DNA. The biosensor utilized pyrrolidinyl peptide nucleic acid (acpcPNA) probes immobilized on chitosan/glutaraldehyde-modified screen-printed carbon electrodes to facilitate the duplex invasion process with the target DNA. In the presence of the target DNA backbone, the formed duplex invasion triggered the electrostatic accumulation of a cationic hexaammineruthenium(III) redox indicator, resulting in an increased current response monitored by square wave voltammetry. The biosensor detected synthetic HIV-1 DNA within a linear range of 5–75 nM (limit of detection (LOD): 1.34 nM; limit of quantification (LOQ): 4.44 nM) and clinical HIV-1 cDNA within 10³–10⁵ copies/mL (LOD: 2.25 × 10² copies/mL; LOQ: 7.50 × 10² copies/mL), with correlation coefficients of 0.9988 and 0.9905, respectively. Importantly, the proposed platform eliminated the need for thermal denaturation and amplification while demonstrating strong selectivity against mismatched and non-target sequences, as confirmed by RT-PCR. In addition, this flexible platform enabled on-site analysis by integrating successfully with Bluetooth potentiostats and near-field communication reader chips. Given its advantages, this biosensor could serve as a promising prototype and a valuable tool for resource-limited settings requiring the detection of clinically relevant nucleic acid markers.