Fluorescence Resonance Energy Transfer-Based Biosensor with Femtomolar Sensitivity for Culture-Independent Detection...

Bioaerosols containing pathogenic microorganisms contribute to the transmission of infectious diseases and pose public health risks. Traditional culture-based detection methods require long incubation times (typically 24-48 h) and often underestimate the microbial concentrations. In this study, we present a femtomolar-level sensitivity biosensor leveraging fluorescence resonance energy transfer (FRET) for detecting adenosine triphosphate (ATP) derived from airborne bacteria. This biosensor utilizes a fluorophore-labeled cDNA (DNA) strand, an unlabeled ATP-specific aptamer, and graphene oxide (GO) that preferentially adsorbs single-stranded DNA over double-stranded DNA. An indirect hybridization approach was employed to avoid the structural interference often observed with directly labeled aptamers, thus improving hybridization efficiency and fluorescence contrast. Under optimized conditions, the biosensor achieved a regression-based analytical limit of detection (LOD) of 0.52 fM ATP while experimentally enabling the detection of airborne bacteria down to 2.4 colony-forming units (CFU) per 1 mL. Importantly, the analytical assay time per measurement was approximately 15 min, which is markedly shorter than that of conventional culture-based methods. The effectiveness and sensitivity of the biosensor were validated through indoor field tests, demonstrating the ability to detect airborne bacteria at concentrations below 10² CFU per 1 m³ of air. This culture-independent method provides a rapid and practical approach for airborne bacterial monitoring and is directly applicable to indoor air quality evaluation and infectious disease management.