Characterization of Hepatitis B Virus Capsids by Resistive-Pulse Sensing

Abstract

We report characterization of Hepatitis B virus (HBV) capsids by resistive-pulse sensing through single track-etched conical nanopores formed in poly(ethylene terephthalate) membranes. The pores were ~40 nm in diameter at the tip, and the pore surface was covalently modified with triethylene glycol to reduce surface charge density, minimize adsorption of the virus capsids, and suppress electroosmotic flow in the pore. The HBV capsids were assembled in vitro from Cp149, the assembly domain of HBV capsid protein. Assembled T=3 (90 Cp149 dimer) and T=4 (120 dimer) capsids are 31 and 36 nm in diameter, respectively, and were easily discriminated by monitoring the change in current as capsids passed through an electrically biased pore. The ratio of the number of T=3 to T=4 capsids transiting a pore did not reflect actual concentrations, but favored transport of smaller T=3 capsids. These results combined with longer transit times for the T=4 capsids indicated that the capsids must overcome an entropic barrier to enter a pore.Nanopores and nanochannels exhibit unique transport properties1 and have a number of potential applications.2 Of particular interest is developing label-free, nondestructive techniques for rapid sensing, characterization, and sorting of particles with nanometer dimensions. The resistive-pulse technique3 measures changes in ion current resulting from transit of particles through an electrically biased nanopore filled with electrolyte. As sensing elements, protein pores,4 e.g., α-hemolysin, exhibit highly reproducible pore composition and dimensions, but lack robustness when suspended in lipid bilayers. Alternatively, microand nanofabrication techniques are used to fabricate solid-state and synthetic nanopores5 , 6 with a wide range of well-defined geometries and dimensions. Forming these pores parallel or perpendicular to the substrate surface permits straightforward integration with other device features. Solid-state and synthetic nanopores exhibit ion depletion/concentration,7 -9 ion permittivity,10 enhanced channel conductance,11 ion current rectification,12 , 13 and pressure-induced salt flux rectification.14 , 15 The ability to control pore dimensions over a range of length scales permits analysis of a variety of samples, including DNA,16 -18 proteins,19 viruses,20 immune complexes,21 nanoparticles,22 and small molecules,23 and similarly designed pores may be used to sequence DNA.24 In some cases, the molecule of interest, e.g., DNA, must overcome an entropic barrier to enter nanoscale slits25 and pores. 26 Related to this work is the characterization of viruses with track-etched pores20 and immune complexes with femtosecond laser-machined pores.21 In both examples, the studied protein complexes are ~100-150 nm in diameter. The reassembly process is inherently of interest, and this system offers a unique opportunity to characterize capsid transport, capsid properties, and nanopore properties. The T=3 and T=4 capsids are similar in diameter, 31 and 36 nm, respectively, and have identic...

Pores with Longitudinal Irregularities Distinguish Objects by Shape

“…3 The resistive-pulse technique has been widely applied to size single molecules, viruses, particles and cells. [1][2][3][4][5][6][7][8][9][10][11][12] A single object passing through a pore causes a transient change of the system resistance, which is detected as a transient change of the transmembrane current, called a resistive-pulse. 6,7 The pulse amplitude is a measure of the object size while the pulse duration can be correlated with its surface charge.…”

Stiff filamentous virus translocations through solid-state nanopores

“…The possibility of applying nanopores to the analysis of nucleic acids, in particular DNA sequencing, has generated interest 3 , and motivated fundamental studies of the physics of nanopore translocations [4][5][6][7][8][9][10][11][12][13][14][15][16][17] . The uses of solid-state nanopores have recently expanded to include detecting single proteins 18 , mapping structural features along RecA-bound DNA-protein complexes 19 and detecting spherical and icosahedral virus strains [20][21][22] . Such advances underline the importance of expanding our understanding of nanopore translocations beyond the case of DNA.…”

A novel SU-8 nanofluidic chip fabrication technique based on traditional UV photolithography

“…Nowadays nanofluidic has become a fundamental and critical technique to study nano-scale fluidic, molecular and ion properties due to the special phenomena only occur in nanochannels (Sparreboom et al 2009;Freedman et al 2013). In recent 5 years, a substantial amount of research has been reported such as protein analysis (Chun et al 2013;Sang et al 2013), DNA stretching (van Kan et al 2012;Pedersen et al 2013), virus characterization (Zhou et al 2011) and ion separation (Tsukahara 2010;Gillespie and Pennathur 2013) based on nanofluidic chips.…”