BPS2026 – Probing biopolymers’ conformational space using pipette-elastomer interfacial nanopores

Secretomics, a proteomics-based approach to identify and quantify the proteins secreted by a cell; while theoretically prodigious, currently lacks a gold standard sensing and analysis pathway for time-resolved localized detection of single-cell macromolecule (proteins, RNA etc.) secretions given the low sample size and complex parameter space (both in unique sample quantity and chemistries). The interfacial nanopore is a nascent but proven technique ideally suited to localized detection and fingerprinting of secretions; with dynamic pore sizing, clog-free stability, positionable with nanometer precision and functionalisable, interfacial nanopores provide in-situ aperture size control through the constriction of a solid-state micropore upon contact and depression with an elastic surface. Nanopore AFMs have already been used to study fibronectin secretion, and later an AFM-based interfacial nanopore was used to conduct localized recordings next to single neurons. Here, we present a novel pipette-elastomer interfacial nanopore (PEIN), whereby a nanopore is created at the interface of an elastomer (polydimethylsiloxane, PDMS) and a rigid micropipette (glass). Dynamically selecting and modifying aperture size with high resolution allows for size-specific sensing and probing of biopolymers’ conformational space. Indeed, in a solution containing single- and double-stranded DNA, we could reduce pore size such that only the single-stranded DNA could translocate, suggesting unparalleled specificity. PEINs can also induce conformational changes in biopolymers: we show DNA translocations in its bulk random-walk state, folded states and linearized state through aperture size modification. In addition to the dynamic benefits, PEINs have proven to increase biopolymer dwell times by orders of magnitude over glass nanopipettes and achieve reproducible nanopores of less than 2 nm, bringing solid-state nanopores a step closer to polymer sequencing. We aim to push PEINs toward molecular-level characterization and ultimately protein structure fingerprinting, as well as aiding our understanding of nanoscale biophysics.