Characterization of Flagellar Filaments and Flagellin through Optical Microscopy and Label-Free Nanopore Responsiveness

Abstract

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 ∼65 °C (most commonly reported temperature), ∼70 °C, ∼75 °C, and ∼80 °C to investigate the effect of temperature (T depol) on depolymerization. The change in conductance (ΔG) profiles corresponding to T depol ∼65 °C and ∼70 °C were bracketed within the flagellin monomer profile whereas those of ∼75 °C and ∼80 °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 T depol of ∼65 °C, ∼70 °C, ∼75 °C, and ∼80 °C yielded ∼4.2 ± 0.2 nm, ∼4.3 ± 0.3 nm, ∼4.1 ± 0.2 nm, and ∼4.7 ± 0.5 nm, respectively. This, along with ΔG (plateaued values) and translocation time profiles, points to the possibility of flagellin misfolding at ∼80 °C.

Modulation of electrophoresis, electroosmosis and diffusion for electrical transport of proteins through a solid-state nanopore

“…When an appropriate voltage bias is applied across the membrane, a molecule translocates from one chamber (cis) to the other (trans), perturbing the open pore current and generating resistive pulses that are characteristic of the molecule under analysis. The applications of nanopores span a wide range of elds such as genomics, 1,2 proteomics, [3][4][5][6] glycomics, 7,8 virology, [9][10][11] and lipid nanoparticles. [12][13][14] Analyte transport generally occurs through electrophoresis, electroosmosis, dielectrophoresis or diffusion mechanisms.…”

Capture of rod-like molecules by a nanopore: Defining an “orientational capture radius”

“…Previous studies of the translocation of charged rodlike objects mainly focused on how rods enter a nanopore and then translocate 17,19,24,25 . In this short paper, we examine the capture of a short rod-like object by the field gradient extending outside a nanopore, and in particular the impact of rod orientation on the capture process.…”

Assessment of 1/f noise associated with nanopores fabricated through chemically tuned controlled dielectric breakdown

“…4A and 4B)—the two extreme concentrations used in this study. Although it is possible to go to lower concentrations, nanopore experiments are generally done in this concentration range [48,49]. The with applied voltage was then fitted using Eq.…”

“…In contrast, the operational range of CDB is mostly limited to <40 nm, while laser‐assisted CDB could reach diameters around ∼50 nm [50]. Studies with larger diameter pores (in the ∼40 nm range) are seldom reported [49,51] and are mostly done in the <30 nm diameter range [3,52]. To produce larger diameter pores through CDB, a higher voltage must be applied.…”