Comparative study of nanopore phenylalanine clamp variants reveals unique peptide biosensing and classification properties

The rapid, label-free, and low-cost detection of peptides is critical for the development of next-generation diagnostics, drug discovery, and environmental monitoring. Nanopore-based biosensing offers a promising platform to address this need by leveraging single-molecule analysis. In this study, we utilize protein engineering to create a series of novel peptide biosensors from the anthrax toxin protective antigen (PA) nanopore by targeting its central phenylalanine clamp constriction (residue F427), a key site known to interact dynamically with translocating molecules. This series of engineered variants were evaluated for their performance in both unsupervised clustering and supervised classification of a diverse set of seven guest-host peptides. Intriguingly, we found that the engineered variants exhibited a broad range of unique biosensing and classification properties. There was a notable divergence between the ability of the variants to intrinsically separate peptides (unsupervised clustering) and their performance in supervised classification tasks. Notably, PA F427A nanopores showed enhanced specificity for small molecular weight peptides that were challenging for WT nanopores to classify, achieving exceptionally high performance (accuracy of 0.93). These findings challenge the assumption that a single unmodified biosensor is sufficient for complex discrimination. Instead, our results highlight the potential for a more robust approach: leveraging the unique, complementary strengths of multiple sensor variants in an ensemble or multiplexed array. Such a system can achieve high and balanced performance across diverse peptide classes, representing a significant step forward in the development of sophisticated nanopore biosensors.