A Chemical Timer Strategy Programs the Onset of Peroxidase Chemiluminescence

Peroxidase chemiluminescence (CL), particularly horseradish peroxidase (HRP)-mediated CL, is widely used in biochemical analysis and immunoassays. However, the onset of CL is typically instantaneous and difficult to control, imposing strict timing requirements for measurements and compromising reproducibility and imaging quality. Herein, we report a general chemical timer strategy that programs CL onset using catechol-type phenolics as signal delayers. Protocatechuic aldehyde (PCAL) and protocatechuic acid (PCA) enable concentration-dependent delays from seconds to minutes while largely preserving emission intensity and extending signal duration. The strategy is compatible with multiple luminol derivatives, a non-luminol acridan substrate, and other peroxidases. Applying the chemical timer, peroxidase-mediated CL encoding and dynamic patterning are demonstrated, and a class of time-resolved enhanced chemiluminescence (ECL) reagents is developed to provide defined imaging windows for immunoblots, enabling kinetic suppression of background and noise. Notably, this strategy is compatible with commercial ECL kits via simple additive introduction, providing a practical route to achieve time-resolved CL readout in routine bioassays.