Amplified and label-free electrochemical detection of a protease biomarker by integrating proteolysis-triggered transcription

Cell-free synthetic biology provides a promising strategy for developing high-performance biosensors by integrating with advanced testing technologies. However, the combination of synthetic biology with electrochemical testing techniques is still underdeveloped. Here, we proposed an electrochemical biosensor for the label-free and ultrasensitive detection of target protease biomarker by coupling a protease-responsive RNA polymerase (PR) for signal amplification. Taking tumor biomarker matrix metalloprotease-2 (MMP-2) as a model protease, we employed PR to transduce each proteolysis reaction mediated by MMP-2 into multiple programmable RNA outputs that can be captured by the DNA probes immobilized on a gold electrode. Moreover, the captured RNAs are designed to contain a guanine-rich sequence that can form G-quadruplex and bind to hemin in the presence of potassium ions. In this scenario, the activity of MMP-2 is converted and amplified into the electrochemical signals of hemin. Under the optimal conditions, this PR-based electrochemical biosensor enabled the sensitive detection of MMP-2 in a wide linear dynamic range from 10 fM to 1.0 nM, with a limit of detection of 7.1 fM. Moreover, the proposed biosensor was further applied in evaluating MMP-2 activities in different cell cultures and human tissue samples, demonstrating its potential in the analysis of protease biomarkers in complex clinical samples.