Recent advancements in parallel and tandem reaction-based fluorogenic probes for tracing enzymatic activities

Abstract

Optical imaging is a non-invasive and indispensable technique for tracing enzymatic activities at a molecular level. However, unintended activation or off-target accumulation of imaging probes often leads to false-positive signals. Moreover, the use of multiple fluorophores to detect distinct biomarkers is limited by variability in cellular uptake and subcellular localization within complex biological environments. To address these deficiencies, multi-locked fluorogenic probes have been developed to target multiple biomarkers simultaneously. These probes typically employ parallel or tandem-locked configuration, wherein enzyme-specific substrates quench the fluorescence of reporter motifs until activation occurs. Recent advancements in multi-locked probes have revealed many opportunities for advancing non-invasive diagnostic chemical tools. This review systematically compiles recent progress in the development of enzyme-responsive dually locked probes for bio-imaging applications. It provides a comprehensive analysis of their underlying mechanisms and design strategies. The review concludes with a summary of current achievements and future perspectives in this rapidly evolving field.