Spatiotemporal Sequential Fluorescence and Electrochemistry Bimodal Quantitation of Enzyme Activity in a Living Cell

Accurate spatiotemporal single-cell quantification of intracellular enzyme activity is critical for understanding cellular heterogeneity and disease screening. However, current methods relying on single-signal modalities lack precision, and spatiotemporal multimodal strategies remain unexplored. Here, we develop a spatiotemporal sequential fluorescence-electrochemistry bimodal platform for both spatial mapping and precise quantitation of asparaginyl endopeptidase (AEP) activity in living single cells. A dual-aggregation-induced emission (AIE) probe, pyTPE-CBT, is delivered into single cells via a θ-nanopipette, yielding an 11.18-fold fluorescence enhancement to visualize AEP spatial distribution. Guided by fluorescence imaging, the nanopipette's detection channel is positioned at target subcellular regions. AEP-mediated cleavage of probe molecules alters charge density and effective channel size, modulating the ionic current for quantitative detection with a limit of detection (LOD) of 3.83 ng/mL. It overcomes the drawbacks of traditional single-mode methods and offers a powerful tool for studying enzyme heterogeneity, subcellular localization, and dynamic enzymatic activity in single cells.