Biomarker and Bioorthogonality Dual‐Locked Chemiluminescent Probes for Cancer Urine Test

Abstract

ABSTRACT Bioorthogonality‐activatable optical imaging activates caged or quenched probes via bioorthogonal reactions for disease diagnosis. Although chemiluminescence enables low‐background detection, it has rarely been applied to bioorthogonality‐activatable imaging. Here, we synthesize chemiluminescent tetrazine probes (CLTZs) by coupling a tetrazine acceptor to a phenoxy‐1,2‐dioxetane donor through π‐conjugated vinylene linkers, enabling biomarker and bioorthogonality dual‐locked activation for cancer urine testing. Unlike conventional chemiluminophores, tetrazine in CLTZs mediates an internal‐conversion‐to‐a‐dark‐state quenching mechanism, suppressing chemiluminescence even after phenolate formation. Strong chemiluminescence is restored only when the phenolate form undergoes bioorthogonal reaction with bicyclononyne (BCN), generating pyridazine‐containing donor–π–acceptor chemiluminophores. This unique dark state enables development of an H 2 O 2 and bioorthogonality dual‐locked probe ( H BCP), emitting only when both H 2 O 2 and BCN are present. By engineering BCN into a cancer‐activatable bioorthogonal urinary reporter ( C BUR) that releases renal‐clearable BCN in tumors, a chemiluminescent bioorthogonal pair ( H BCP & C BUR) is created. This pair achieves in vivo tumor imaging with a signal‐to‐background ratio of 438 and detects lung metastasis in early urinary assays with up to 23.9‐fold signal enhancement over healthy mice. This work provides a molecular design for bioorthogonality‐activatable chemiluminescence toward noninvasive, sensitive disease diagnosis.