Photo-PISA Driven <i>In Situ</i> Encapsulation of Nanocluster-Based Sensors within Stimuli-Responsive Polymersomes for AND Logic Gate Sensing

Colorimetric sensing is a widely utilized analytical technique due to its simplicity, accessibility, rapid response, and broad applicability in medical diagnostics. However, improving the sensitivity and specificity of these assays remains a critical challenge in complex disease states, especially when sensing endogenous enzymes as biomarkers. In this study, we have developed a hierarchical AND logic gate dual-sensing platform that integrates peptide-templated, catalytically active gold nanoclusters (AuNCs), acting as nanozymes tethered to a carrier protein (AuNC-protein complex nanosensor), and loads them within pH-responsive polymersomes synthesized via <i>in situ</i> photoinitiated polymerization-induced self-assembly (photo-PISA). Under physiological conditions, the AuNC-protein complex is stably encapsulated within the enzyme-impermeable polymersome but becomes released under acidic conditions. In the presence of a target enzyme, the AuNCs can then be cleaved from the supramolecular protein complex, separated, and quantified by a colorimetric readout, yielding a positive signal only when the sensor encounters both an acidic environment and the target enzyme. This AND logic gate design minimizes background signals and enhances specificity, making it particularly suitable for complex biological environments. We envision future use of this system for dual-responsive <i>in vivo</i> sensing of overexpressed enzymes in acidic tumor or inflammatory microenvironments, with a simple colorimetric urinary readout.