Detection of Single Proteins in a Complex Biofluid using a Selective Nanopore: Finding the Needle in a Haystack

Abstract

In this work, we have used a highly selective engineered protein nanopore for recruiting targeted proteins from a complex biological fluid. Our sensing strategy circumvented traditional nanopore approaches of protein sensing that rely on analysis of current blockages. Instead, protein detection occurred outside the protein nanopore and involved a binding protein bait fused to a polypeptide adaptor that facilitated the real-time measurement of protein-protein interactions at single-molecule precision. We optimized these single-molecule electrical recording conditions for unambiguous separation of targeted protein detection events from those current blockages produced by the biofluid constituents. Finally, our approach can provide a direct and quantitative determination of a certain protein in heterogeneous solutions, and in a manner that fundamentally and conceptually distinguishes from those used by existing detectors. Our method will likely impact medical biotechnology by providing a new class of selective biosensors for protein identification and quantification in molecular biomedical diagnostics.