Nanoconfinement and Crowding Enhanced Single-Molecule Detection of Small Molecules with Nanopipettes.

Glass nanopipettes have gained widespread use as a versatile single-entity detector in chemical and biological sensing, analysis, and imaging. Its advantages include low cost, easy accessibility, simplicity of use, and high versatility. However, conventional nanopipettes based on the volume exclusion mechanism have limitations in detecting small biomolecules due to their small volume and high mobility in aqueous solution. To overcome this challenge, we have employed a novel approach by capitalizing on the strong nanoconfinement effect of nanopipettes. This is achieved by utilizing both the hard confinement provided by the long taper nanopipette tip at the cis side and the soft confinement offered by the hydrogel at the trans side. Through this approach, we have effectively slowed down the exit motion of small molecules, allowing us to enrich and jam them at the nanopipette tip. Consequently, we have achieved high throughput detection of small biomolecules with sizes as small as 1 nm, including nucleoside triphosphates, short peptides, and small proteins with excellent signal-to-noise ratios. Furthermore, molecular complex formation through specific intermolecular interactions, such as hydrogen bonding between closely spaced nucleotides in the jam-packed nanopipette tip, has been detected based on the unique ionic current changes.