An Engineered E. Coli ‐Based Living Material Platform for Protease Biosensing

ABSTRACT Accurate quantification of protease activity is crucial for clinical diagnostics and fundamental biological research. Here, we present a programmable living material engineered from an optimized Escherichia coli surface‐display system. As a dynamic and self‐adapting biological material, the engineered cells assemble a responsive sensing interface that integrates metabolically balanced promoters, efficient membrane‐anchoring scaffolds, protease‐specific cleavable peptide substrates (PCPs), and high‐brightness fluorescent reporters. This modular architecture enables the material to autonomously process extracellular cues, maintain stable functionality, and achieve high‐throughput and ultrasensitive single‐cell analysis of diverse proteases. By plug‐and‐play substitution of PCPs, the system quantitatively detects matrix metalloproteinase 2, thrombin, and prostate‐specific antigen with detection limits of 26.4, 2.16, and 3.8 fg/mL, respectively, exceeding the sensitivity of commercial assays by more than three orders of magnitude. Beyond its exceptional sensitivity, the living material exhibits high specificity, low production cost, scalable manufacturing, and excellent storage stability. Successfully applied to two normal and five cancer‐derived cell lines, the platform accurately detected protease activity, confirming its outstanding environmental adaptability. As a universal and expandable active‐material framework, this platform provides a powerful tool for protease biomarker profiling and demonstrates the broad potential of engineered living materials in next‐generation diagnostic and biosensing technologies.