Triple-Response Peptide Probe for Simultaneous Monitoring the Activity of Three Proteases through a Single Nanopore

Accurate disease diagnosis requires analyzing multiple biomarkers in a single sample to improve diagnostic precision and guide treatment. However, the simultaneous detection of three or more proteases in a sample, especially with a single probe, remains a major challenge. Herein, we developed a triple-response peptide probe for monitoring three proteases via a single aerolysin nanopore. The T-shaped probe contains three independent arms each with a target-specific cleavage sequence. Upon protease recognition, short peptide fragments are released and generate characteristic current signatures during nanopore translocation. The enzymes themselves are sterically excluded, ensuring signals originate solely from cleavage products. Each peptide yields a distinct signature, enabling simultaneous, label- and amplification-free quantification with detection limits extending to the femtomolar range. Integrated with a machine learning classifier, the system achieves 97.7% accuracy in signal discrimination. This strategy enables accurate and multiplexed quantification of multiple enzymes in complex biological fluids, such as serum, which is important for disease diagnosis based on liquid biopsies.