Ligand-Involved Chemiluminescence Using Gold Nanoclusters: Mechanistic Elucidation and Its Application for Ultrasensitive Trypsin Biosensing

Traditional molecular chemiluminescence (CL) often suffers from low photon yields because each event typically produces limited emission. Self-amplification using nanomaterial probes with high-density ligands can increase photon output per luminophore; however, widely used CdTe nanocrystals and perovskites remain constrained by poor stability and biocompatibility. Herein, we report a protein-engineered bovine serum albumin-stabilized gold nanocluster (BSA-Au NCs) CL probe that delivers high-intensity emission while addressing these limitations. The CL intensity depends on BSA concentration and the triggering oxidant, and structural analyses indicate that a more loosely folded secondary structure favors stronger CL. Notably, NaClO oxidizes the BSA ligand shell and provides sufficient energy to excite Au NCs, producing an ∼800-fold higher CL signal than H2O2 at the same concentration. The red-shifted CL spectrum relative to photoluminescence, together with X-ray photoelectron spectroscopy, supports a ligand-involved emission mechanism, which is further validated by BSA-specific recognition experiments. Leveraging ligand-shell disruption, we construct a signal-off CL assay for ultrasensitive trypsin detection with a linear range of 0.01–500 μg/mL and a detection limit of 3.3 ng/mL (S/N = 3). This work provides a robust trypsin sensing platform and mechanistic insight into ligand-involved CL in BSA-Au NCs, enabling the rational design of functional Au nanocluster-based luminescent materials.