Bioorthogonal Molecular Turn-On Optical Imaging and Therapy

Bioorthogonal chemistry was introduced in the context of the molecular turn-on optical imaging and therapy over 20 years. A pivotal innovation in this field is the development of bioorthogonal turn-on optical probes (BioTOPs), which integrate bioorthogonal handles with imaging agents to leverage selective bioorthogonal reactions for activating optical signals, enabling high-contrast and real-time visualization of biomolecules in living systems. The precise spatiotemporal control over signal activation also enables minimal off-target therapy. This review summarizes recent advances in the design and application of BioTOPs. We first discuss the two-step pretargeted strategy of bioorthogonal turn-on optical imaging. Then, we categorize the bioorthogonal reactions that have been employed for signal activation, including Staudinger ligations, strain-promoted cycloadditions, metal-catalyzed reactions, ketone/aldehyde condensations, boron compound-enabled reactions, and thiol-selective bioconjugations, alongside the activation mechanisms, such as click-to-release, energy/electron dequenching, steric hindrance dequenching, bioorthogonal handle decaging, fluorogenic handle conjugation, and fluorogenic self-assembly, which collectively offer tunable kinetics and high signal turn-on folds. We conclude with applications in diagnostic and therapeutic contexts, from biomolecule profiling and mapping to cancer diagnosis and disease-specific interventions while offering a perspective on the future transformative potential of this technology for molecular diagnostics.