A Novel Chemiluminescence Probe for Sensitive Detection of Fibroblast Activation Protein-Alpha In Vitro and in Living Systems

Fibroblast activation protein-alpha (FAPα) is a key modulator of the microenvironment in multiple pathologies and is becoming the next pan-cancer target for cancer diagnostics and therapeutics. Chemiluminescence (CL) luminophores are considered as one of the most sensitive families of probes for detection and imaging applications due to their high signal-to-noise ratio. Until now, however, no such effective CL probe was reported for FAPα detection. Herein, we developed a novel CL probe for the detection of endogenous FAPα activity by incorporating FAPα-specific dipeptide substrates (glycine-proline) to the improved Schaap’s adamantylidene-dioxetane. In this manner, we designed three CL probes (CFCL, BFCL, and QFCL) with the dipeptide substrate blocked by N-terminal benzyloxycarbonyl, N-tert-butoxycarbonyl or N-quinoline-4-carboxylic acid, respectively, which was used as the masking group to restrain the chemiexcitation energy. Probe CFCL exhibited the optimal specificity for the discrimination of FAPα from dipeptidase IV and prolyl oligopeptidase, which was elucidated by molecular docking simulation. Upon FAPα cleavage, CFCL was turned on for the highly selective and sensitive detection of FAPα with a limit of detection of 0.785 ng/mL. Furthermore, the ability of CFCL to image FAPα was effectively demonstrated in vitro, including various biological samples (plasma and tissue preparations), and in living systems (tumor cells and tumor-bearing mice). Furthermore, this newly established probe could be easily extended to evaluate FAPα inhibitors. Overall, we anticipate that probe CFCL will offer a facile and cost-effective alternative in the early detection of pathologies, individual tailoring of drug therapy, and drug screening.