Affordable infectious pathogen detection using a dual-mode biosensor integrating exonuclease III-assisted target recycling amplification with high-throughput 96-well microplate format

The ongoing challenge of infectious pathogens highlights the need for accurate and accessible methods to discern their genetic signatures, especially in resource-limited settings. In response to this crucial requirement, we introduce an affordable large-scale screening platform for infectious pathogen detection, using Hepatitis B virus (HBV) as a fundamental model. This proposed biosensor integrates an exonuclease III-assisted target recycling amplification strategy within a high-throughput 96-well microplate format. The HBV DNA target binds to a capture probe DNA and exonuclease III digests the probe to release the target. This mechanism enables the target to engage in binding cycles with new probes, each digested in turn, increasing detection sensitivity for even small quantities of HBV DNA. The implemented approach incorporates a biotin-streptavidin interaction allowing the undigested capture probe DNA to bind to a 5′-biotin-modified detection probe for effective HBV DNA quantification. This interaction generates a signal that, following the enzyme-substrate reaction, can be detected on-site using a smartphone, offering either optical or electrochemical readouts. The developed biosensor was capable of detecting HBV DNA with a detection limit of 5.62 fM and provided a considerable linear range covering concentrations from 100 fM to 100 nM. The determination of HBV DNA quantities in spiked human serum was achieved with a recovery of 90.0 % – 107.4 % as well. The results suggest that the developed dual-mode biosensor offers an adaptable and cost-effective approach for detecting infectious diseases, with promising applications in medical diagnostics and environmental monitoring to support public health efforts.