EAGER Proposal: Crowding-assisted biomolecular sensing with natural nanopores

University Of Massachusetts Amherst, Amherst MA

Investigators

Abstract

Parsegian 1249199 Concerns over health and safety motivate the development of detectors to sense and to identify toxic substances at concentrations of one part in a billion. For reasons of robustness, reliable construction, abundance, accessibility, and atomic characterization, natural membrane channels will be used for the sensor of the intended detector. Already designed by evolution to withstand the strong electric fields that drive materials through pores, natural channels offer a practical alternative to artificial pores that can wear down under the stress of electric fields applied to them. Intellectual Merit: Using simple polymers and salts as "molecular crowding" agents to drive suspect solutes into detector channels, the objective is to identify these substances by the changes they make in electrical current so as to create sensitive single-molecule-based detectors and to develop a strategy for detecting and characterizing biologically relevant water-soluble molecules: sugars, peptides, proteins, and other low-molecular and polymeric materials at sub-nanomolar concentrations. Early results reveal a striking and unexpected increase in rates of interaction between membrane-reconstituted ion channels and various types of biologically relevant proteins and synthetic polymers as various solution ?crowders,? including simple salts, are added. By monitoring the small-ion current passing through these pores, it will be possible to detect these interactions as individual, reproducible events of current interruption, with characteristic times specific for each kind of material. The proposed crowding method enhances the sensitivity of detection enough to allow identification of species at sub-nanomolar concentrations. Such ?crowding-driven? nanopore detection is expected to lead to construction of practical versatile devices to identify minute amounts of water-soluble agents, including harmful molecules, pathological agents, immune markers etc., in mixed samples that reflect real situations. Broader Impact: This project will explore how different classes of biologically relevant materials, from simple polymers to complex protein assemblies, interact with protein-based nanopores reconstituted into membranes to realize two large goals: (i) Creation of a new class of single-molecule detectors by using solution conditions to control sensitivity. These nanodevices will be applicable to a broad range of bio- and nano-particle sensing and are expected to allow exploration of mixed systems of polymers and salts or of polymers of different types and sizes. (ii) Transport of large solutes driven from concentrated solutions reveals unexpected properties of polymer and molecular packing in the confined volumes typical of biological cells. In particular, it will become possible to discriminate among mixtures of compounds under crowded conditions so as to guide design for molecular separation and reaction at sub-nanomolar concentrations.