Bridging biological samples to functional nucleic acid biosensor applications: current enzymatic-based strategies for single-stranded DNA generation

The escalating threat of emerging diseases, often stemming from contaminants and lethal pathogens, has precipitated a heightened demand for sophisticated diagnostic tools. Within this landscape, the functional nucleic acid (FNA) biosensor, harnessing the power of single-stranded DNA (ssDNA), has emerged as a preeminent choice for target analyte detection. However, the dependence on ssDNA has raised difficulties in realizing it in biological samples. Therefore, the production of high-quality ssDNA from biological samples is critical. This review aims to discuss strategies for generating ssDNA from biological samples for integration into biosensors. Several innovative strategies for ssDNA generation have been deployed, encompassing techniques, such as asymmetric PCR, Exonuclease-PCR, isothermal amplification, biotin–streptavidin PCR, transcription-reverse transcription, ssDNA overhang generation, and urea denaturation PAGE. These approaches have been seamlessly integrated with biosensors for biological sample analysis, ushering in a new era of disease detection and monitoring. This amalgamation of ssDNA generation techniques with biosensing applications holds significant promise, not only in improving the speed and accuracy of diagnostic processes but also in fortifying the global response to deadly diseases, thereby underlining the pivotal role of cutting-edge biotechnology in public health and disease prevention.