FRET-based nanoprobes for nucleic acid sensing and disease diagnosis: materials, probe architectures, and analytical...

Förster resonance energy transfer (FRET) translates nanometre-scale changes in donor-acceptor separation into ratiometric fluorescence signals, enabling homogeneous and isothermal detection of nucleic acid biomarkers. In this review, we critically survey FRET-based nanoprobes for nucleic acid sensing and disease diagnosis, spanning organic dyes and cationic conjugated polymers to metallic nanoparticles, quantum dots, upconversion nanoparticles, carbon nanostructures, MXenes and polymeric nanoantennae. We organise probe designs by architectures, including sandwich hybridisation, disassembly hairpins, molecular beacons, dynamic junction sensors and enzyme-free DNA circuits, and analyse how these platforms achieve attomolar limits of detection, single-nucleotide discrimination and multiplexing in complex matrices. Representative case studies highlight applications in viral (e.g. SARS-CoV-2), bacterial and fungal pathogens, circulating cancer-related miRNAs and mRNAs, and neurodegenerative disease markers, as well as integration of FRET readouts with PCR and isothermal amplification workflows. We then compare FRET nanoprobes with established nucleic-acid assays in terms of analytical figures of merit, instrumentation, and compatibility with point-of-care (POC) formats. Remaining challenges, including photobleaching, spectral crowding, matrix autofluorescence, nanocarrier toxicity, and inter-laboratory reproducibility, are discussed in the context of emerging solutions such as near-infrared and lifetime-based FRET, CRISPR-coupled assays, DNA-origami nanoantennas, and microfluidic or smartphone-enabled devices. Finally, we outline key trends that could drive translation of FRET-based nucleic acid nanoprobes from proof-of-concept demonstrations to clinically actionable diagnostics.