In situ fluorogenesis enabled by bioorthogonal cycloadditions with dye-free reagents

The integration of bioorthogonal chemistry with fluorescence imaging has enabled bioorthogonal fluorescent probes with high spatiotemporal resolution for real-time biological applications. More recently, dye-free bioorthogonal fluorescent probes have emerged as an attractive strategy, in which two essentially non-emissive reagents react in situ to generate a fluorescent product, offering favorable biocompatibility and potentially high fluorescence turn-on ratios. A major challenge in developing such probes lies in deconstructing high-performance fluorophore scaffolds into suitable bioorthogonal reaction partners and efficiently reassembling them into emissive products, which requires sophisticated molecular design and synthetic expertise. We envision that theoretical calculations, together with artificial intelligence, will further accelerate the discovery and optimization of dye-free probes. This review focuses on the mechanisms of representative fluorogenic bioorthogonal reactions, strategies for tuning their photophysical properties, and the development of functional dye-free probes, with the aim of providing design insights and perspectives for future advances in this emerging field.