Abstract
Fluorescence-based DNA quantification is essential for molecular diagnostics and environmental monitoring, yet conventional instruments remain inaccessible for field deployment due to high cost, operational complexity, and infrastructure requirements. This study presents a portable smartphone-based fluorescence detection system integrating light-emitting diode (LED) excitation, paper-based sample platforms, and smartphone imaging for rapid on-site DNA semi-quantification as a proof-of-concept demonstration. The system achieved a model-extrapolated estimated limit of detection (LOD) of approximately 7.63 × 10⁻⁴ ng/μL (below the NanoDrop reference instrument quantification limit of 2 ng/μL), for double-stranded DNA (dsDNA) using SYBR Green I, with the complete detection workflow accomplished within 30 minutes. Optimization of LED driving current identified 30 mA as the optimal condition, providing the best balance between detection sensitivity and dynamic range. The coefficient of variation (CV%) ranged from 1.02% to 4.44%, demonstrating good measurement reproducibility. Cross-platform evaluation demonstrated consistent performance across different smartphone models, supporting practical field deployment. The system functions as a semi-quantitative screening tool over an effective working range of 0.1-10 ng/μL and is not intended for absolute quantification. The system was validated through water quality testing using Escherichia coli DH5α as a model indicator organism, successfully differentiating sterile and bacteria-contaminated samples at concentrations of about 10⁴ to 10⁸ colony-forming units per milliliter (CFU/mL) in deionized (DI), bottled and tap water. Compared with existing portable DNA detection methods requiring nucleic acid amplification or enzymatic processing, the present system offers comparable sensitivity with significantly simplified operation, requiring only direct mixing of sample with fluorescent dye. The developed platform addresses the need for accessible fluorescence-based detection meeting the ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, Deliverable) for resource-limited settings. This system holds potential for preliminary screening in point-of-care diagnostics, environmental screening, and food safety applications.