Proteins that are nearly identical can still signal the difference between a healthy cell and a cancerous one, but existing tests cannot reliably tell them apart. This project builds a sensor that reads the electrical signature of individual protein molecules as they pass through a tiny pore, distinguishing between closely related variants—called isoforms—that current antibody-based methods miss. The researchers will engineer the pore’s entrance loops, mutating them to create a library of binders, then use cell sorting and parallel electrical screening to identify which pores recognise specific isoforms of medically relevant proteins such as interleukin-8 and KRAS4A. If successful, the method would allow clinicians to measure multiple protein isoforms simultaneously from a single sample, improving early disease diagnosis and treatment monitoring. This is fundamental biosensor engineering: it does not yet deliver a diagnostic device, but it establishes a platform that could be adapted to any protein target. Past work on single-molecule sensing has already transformed DNA sequencing; this project aims to do the same for proteins.
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Protein isoforms share substantial similarity in their sequences, structures, and functions, which play vital roles in numerous life activities. Their dysregulation may lead to disease development and progression. Therefore, the ability to discriminate and quantify protein isoforms as biomarkers in a high-throughput and multiplexed manner is highly desirable for disease diagnosis and prognostic analysis, therapeutic development, and the fundamental understanding of biology. Conventional methods for detecting protein isoforms face challenges in dynamic range and the availability of reliable reagents (e.g., antibodies) and equipment. In addition, these ensemble measurements do not reveal the intrinsic heterogeneity within a protein population. To this end, we envision a technological upgrade in isoform-specific detection achieved by developing a single-molecule technique that simultaneously targets a set of protein isoforms through binding to their common epitopes and discriminates between these isoforms with high structural sensitivity by an alternative means. Nanopore technology emerges as a versatile single-molecule parallel sensing platform, which holds the potential in isoform-specific sensing based on their electrical fingerprints. Inspired by affirmers, a non-antibody binding scaffold which recognizes specific proteins through two variable loops connecting four ß strands, we aim to engineer analyte-specific nanopore binders by mutating the extracellular loops of monomeric porins. We will streamline the construction and high-throughput screening of these nanopore libraries to develop bespoke single-molecule nanopore biosensors for isoform-specific detection and quantification. In particular, our strategy is to leverage both affinity-based cell sorting and parallel electrical recording-based screening to identify suitable nanopore biosensors for model proteins of biological relevance (e.g., interleukin-8 and KRAS4A). Ultimately, multiplexed and ratiometric analysis of different protein targets and isoforms in a mixed sample will be demonstrated. Upon successful establishment of this method, we will be able to readily develop single-molecule nanopore biosensors for any targets of interest, which promises to advance fundamental biomedical research and clinical diagnosis.
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