Nano differential scanning fluorimetry in drug discovery: advances, applications, and limitations

INTRODUCTION: Nano differential scanning fluorimetry monitors changes in the intrinsic tryptophan and tyrosine fluorescence of a target protein during thermal unfolding, providing a label-free approach to assessing protein stability and ligand engagement in early-stage drug discovery. AREAS COVERED: The authors examine the technical differences between nano differential scanning fluorimetry and dye-based differential scanning fluorimetry, including sample demand, throughput, buffer and solvent compatibility, and optical artifacts. In addition, the authors present applications in fragment screening, hit validation, and the characterization of oligomeric, allosteric, membrane, proximity-induced degradation modalities and RNA-binding targets. Finally, the limitations arising from reporter distribution, noncooperative unfolding, aggregation, and complex matrices are highlighted. This review is based on literature searches using PubMed and Google Scholar to identify articles published between 2001 and 2026. EXPERT OPINION: NanoDSF is most valuable as a context-dependent primary or orthogonal method rather than a universal high-throughput replacement. Its future impact will depend on automation, analysis of raw fluorescence and scattering, validated reconstituted systems, and integration with quantitative binding, structural, and functional assays. Expansion to cell-like matrices will require target-specific deconvolution.