Quantitative Tracking of Apopotic Caspase‐3 In Vivo for Early Evaluation of Radiation Therapy Efficacy

Currently, disease‐associated markers are only qualitatively imaged in‐vivo by conventional probes in an activated on/off mode. However, accurate quantification of markers (especially protease’s dynamic concentration) in real‐time in‐vivo remains a formidable challenge. Herein, we developed a protease activated ratiometric fluorescent (FL) probe that emits both reference and responsive signals at 1550 nm, enabling the accurate quantification of disease‐associated protease in‐vivo after administration into living organisms. This probe was fabricated by conjugating FL molecules with Ln3+‐doped down‐conversion nanoparticles throughout a substrate peptide of caspase‐3. Caspase‐3 induces a response signal while ensuring a consistent reference signal, resulting in a ratiometric signal with a linear correlation with the caspase‐3 concentration and a limit of detection of 9.96 U/mL. An early efficacy assessment system established accordingly can predict the cancer therapeutic effect within 12 h after treatment, which would otherwise require several days or weeks to become evident, thus guiding the optimization of the therapeutic regimen. Thus, the ratiometric NIR‐II FL probe holds the potential for advancing from qualitative imaging to quantitative detection by enabling the in‐situ quantification of disease markers.