DNA‐Barcoded Plasmonic Nanostructures for Activity‐Based Protease Sensing

• Published in: Angewandte Chemie Vol. 136; no. 2
• Main Authors: Pandit, Subrata, Duchow, Mark, Chao, Wilson, Capasso, Anna, Samanta, Devleena
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley Subscription Services, Inc 08-01-2024
• Subjects: Activity-Based Sensing; Apoptosis; Biological properties; Biological samples; Biosensors; Caspase-3; Cathepsin B; Chemistry; Colorectal carcinoma; CRISPR; Deoxyribonucleic acid; Detection limits; DNA; DNA probes; Gold; Multiplexing; Nanoparticles; Nanostructure; Peptide; Peptides; Plasmonic Nanostructure; Plasmonics; Probes; Protease; Proteinase; Room temperature; Severe acute respiratory syndrome coronavirus 2; Substrates
• ISSN: 0044-8249; 1521-3757
• DOI: 10.1002/ange.202310964

We report the development of a new class of protease activity sensors called DNA‐barcoded plasmonic nanostructures. These probes are comprised of gold nanoparticles functionalized with peptide‐DNA conjugates (GPDs), where the peptide is a substrate of the protease of interest. The DNA acts as a barcode identifying the peptide and facilitates signal amplification. Protease‐mediated peptide cleavage frees the DNA from the nanoparticle surface, which is subsequently measured via a CRISPR/Cas12a‐based assay as a proxy for protease activity. As proof‐of‐concept, we show activity‐based, multiplexed detection of the SARS‐CoV‐2‐associated protease, 3CL, and the apoptosis marker, caspase 3, with high sensitivity and selectivity. GPDs yield >25‐fold turn‐on signals, 100‐fold improved response compared to commercial probes, and detection limits as low as 58 pM at room temperature. Moreover, nanomolar concentrations of proteases can be detected visually by leveraging the aggregation‐dependent color change of the gold nanoparticles. We showcase the clinical potential of GPDs by detecting a colorectal cancer‐associated protease, cathepsin B, in three different patient‐derived cell lines. Taken together, GPDs detect physiologically relevant concentrations of active proteases in challenging biological samples, require minimal sample processing, and offer unmatched multiplexing capabilities (mediated by DNA), making them powerful chemical tools for biosensing and disease diagnostics.
We report a new class of activity‐based protease sensors, termed DNA‐barcoded plasmonic nanostructures. These probes afford over 100‐fold increase in sensitivity compared to conventional fluorophore‐quencher‐based probes, allow for multiplexed detection of disease‐relevant proteases, and enable the monitoring of active proteases in cancer cells obtained from colorectal cancer patients.