Hewitt, E. | bioRxiv Science

bioRxiv2024

Single molecule delivery into living cells

Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform which uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform the nanopipette is used as both a scanning ion conductance microscope (SICM) scanning probe and as an injection probe. The SICM is used to position the nanopipette above the cell surface, before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modelling, we also show that macromolecular crowding in the cell increases the signal to noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.

biophysics↗

bioRxiv2022

Asymmetric Ion Mobility and Interface Displacement Drive the Signal Enhancement in a Polymer-electrolyte Nanopore

Solid-state nanopores have been widely employed in the detection of biomolecules, but low signal-to-noise ratios still represent a major obstacle to enable the discrimination of short nucleic acid and protein sequences. The addition of 50% polyethylene glycol (PEG) to the bath solution was recently demonstrated as a simple way to enhance the detection of such biomolecules translocating through a model solid-state nanopore. Here, we provide a comprehensive description of the physics describing a nanopore measurement carried out in 50% PEG that is supported by finite-element modelling and experiments. We demonstrate that the addition of PEG to the external solution introduces a strong imbalance in the transport properties of cations and anions, drastically affecting the characteristic current response of the nanopore. We further show that the strong asymmetric current response is due to a polarity-dependent ion distribution and transport at the nanopipette tip region, leading to either ion depletion or enrichment for few tens of nanometers across the aperture. Under negative potential, when double-stranded DNA molecules translocate, the depleted region (sensing region) significantly improves the sensitivity compared to systems without PEG. We then introduce a displacement of the interface between pore and external solution to simulate the mechanical interactions between analyte and PEG molecules. We found that this displacement affects the ion distribution in the sensing region, enhancing the detection current during the translocation of biomolecules.

biophysics↗