CRISPR-enhanced biosensor for ultrasensitive and multiplexed enzyme activity sensing

Proteases play essential roles in numerous biological and pathological processes, including inflammation, cancer metastasis, neurodegeneration, and infectious disease. Specific protease activity serves as a valuable biomarker for disease progression, treatment monitoring, and drug development. However, existing detection methods are often limited by poor sensitivity, complex sample preparation, high instrumentation costs, and inability to monitor multiple proteases simultaneously. These challenges hinder early diagnosis and the adoption of protease assays in point-of-care and high-throughput applications.

This biosensing technology combines DNA-barcoded gold nanoparticles (AuNPs) with a CRISPR/Cas12a amplification system for ultrasensitive and multiplexed protease detection. Each AuNP is functionalized with a peptide-DNA conjugate, where the peptide is specific to a target protease and linked to a unique DNA barcode. Upon protease cleavage of the peptide, the DNA barcode is released and activates Cas12a to cleave fluorescently labeled reporter DNA, producing a quantifiable fluorescence signal. At higher protease concentrations, the cleavage also triggers nanoparticle aggregation, leading to a visible color change. This dual-mode readout—fluorescence and colorimetric—provides high sensitivity, broad dynamic range, and instrument-free visual detection. The modular DNA barcoding enables multiplexed analysis of several proteases within the same sample and can be easily adapted for new targets.

PCT/US2025/020426