RESUMO
Solid-state nanopores (SSNs) are single-molecule resolution sensors with a growing footprint in real-time bio-polymer profiling-most prominently, but far from exclusively, DNA sequencing. SSNs accessibility has increased with the advent of controlled dielectric breakdown (CDB), but severe fundamental challenges remain: drifts in open-pore current and (irreversible) analyte sticking. These behaviors impede basic research and device development for commercial applications and can be dramatically exacerbated by the chemical complexity and physical property diversity of different analytes. We demonstrate a SSN fabrication approach attentive to nanopore surface chemistry during pore formation, and thus create nanopores in silicon nitride (SiNx) capable of sensing a wide analyte scope-nucleic acid (double-stranded DNA), protein (holo-human serum transferrin) and glycan (maltodextrin). In contrast to SiNx pores fabricated without this comprehensive approach, the pores are Ohmic in electrolyte, have extremely stable open-pore current during analyte translocation (>1 h) over a broad range of pore diameters ([Formula: see text]3- â¼30 nm) with spontaneous current correction (if current deviation occurs), and higher responsiveness (i.e. inter-event frequency) to negatively charged analytes (â¼6.5 × in case of DNA). These pores were fabricated by modifying CDB with a chemical additive-sodium hypochlorite-that resulted in dramatically different nanopore surface chemistry including â¼3 orders of magnitude weaker Ka (acid dissociation constant of the surface chargeable head-groups) compared to CDB pores which is inextricably linked with significant improvements in nanopore performance with respect to CDB pores.
RESUMO
In this study, we investigated the translocation characteristics of flagellar filaments (Salmonella typhimurium) and flagellin subunits through silicon nitride nanopores in tandem with optical microscopy analysis. Even though untagged flagella are dark to the optical method, the label-free nature of the nanopore sensor allows it to characterize both tagged (Cy3) and pristine forms of flagella (including real-time developments). Flagella were depolymerized to flagellin subunits at â¼65 °C (most commonly reported temperature), â¼70 °C, â¼75 °C, and â¼80 °C to investigate the effect of temperature (Tdepol) on depolymerization. The change in conductance (ΔG) profiles corresponding to Tdepol â¼65 °C and â¼70 °C were bracketed within the flagellin monomer profile whereas those of â¼75 °C and â¼80 °C extended beyond this profile, suggesting a change to the native protein state. The molecular radius calculated from the excluded electrolyte volume of flagellin through nanopore-based ΔG characteristics for each Tdepol of â¼65 °C, â¼70 °C, â¼75 °C, and â¼80 °C yielded â¼4.2 ± 0.2 nm, â¼4.3 ± 0.3 nm, â¼4.1 ± 0.2 nm, and â¼4.7 ± 0.5 nm, respectively. This, along with ΔG (plateaued values) and translocation time profiles, points to the possibility of flagellin misfolding at â¼80 °C.