Nanofluidic Devices with 8 Pores in Series for Real-Time, Resistive-Pulse Analysis of Hepatitis B Virus Capsid Assembly

Abstract

To improve the precision of resistive-pulse measurements, we have used a focused ion beam instrument to mill nanofluidic devices with 2, 4, and 8 pores in series and compared their performance. The in-plane design facilitates the fabrication of multiple pores in series, which, in turn, permits averaging of the series of pulses generated from each translocation event. The standard deviations (σ) of the pulse amplitude distributions decrease by 2.7-fold when the average amplitudes of eight pulses are compared to the amplitudes of single pulses. Similarly, standard deviations of the pore-to-pore time distributions decrease by 3.2-fold when the averages of the seven measurements from 8-pore devices are contrasted to single measurements from 2-pore devices. With signal averaging, the inherent uncertainty in the measurements decreases; consequently, the resolution (mean/σ) improves by a factor equal to the square root of the number of measurements. We took advantage of the improved size resolution of the 8-pore devices to analyze in real time the assembly of Hepatitis B Virus (HBV) capsids below the pseudo-critical concentration. We observe that abundances of assembly intermediates change over time. During the first hour of the reaction, the abundance of smaller intermediates decreased, whereas the abundance of larger intermediates with sizes closer to a T = 4 capsid remained constant.

A molecular breadboard: Removal and replacement of subunits in a hepatitis B virus capsid

“…The translocation of a particle through the pore results in a change in the recorded current (pulse). The amplitude of the current pulse is proportional to the volume of the electrolyte solution displaced from the pore by the translocating particle . In‐plane nanofluidic devices enable the fabrication of multiple pores connected in series, which increase the resolution of particle size measurements.…”

“…The amplitude of the current pulse is proportional to the volume of the electrolyte solution displaced from the pore by the translocating particle. [12][13][14][15][16] In-plane nanofluidic devices enable the fabrication of multiple pores connected in series, which increase the resolution of particle size measurements. CDMS is a gas phase technique, where individual ions oscillate in a trap and their mass to charge ratio (m/z) and charge (z) are determined from the frequency and amplitude of the measured signal, respectively.…”

Characterization of Virus Capsids and Their Assembly Intermediates by Multicycle Resistive-Pulse Sensing with Four Pores in Series

“…6 Other configurations with multiple pores in series include devices with two membrane-based nanopores stacked vertically, 36–37 up to 11 micrometer-scale pores in series for node-pore sensing, 38 and up to 8 nanopores in series for real-time analysis of virus assembly. 39…”

Analytical Techniques to Characterize the Structure, Properties, and Assembly of Virus Capsids

“…Two-dimensional, in-plane designs for nanopore fabrication offer unique advantages including straightforward coupling of nanofluidic components with microfluidic networks and fabrication of nanopores in-series or in-parallel. 80 Fabrication of multiple pores in series is advantageous, because individual particles can be measured multiple times (Figure 3a). For example, Figure 3b shows a nanofluidic device that consists of eight pores connected in series.…”

“…To further increase the measurement precision, e.g., resolution of the particle size distributions, devices with up to 8 pores in series were fabricated. 80 On these devices, assembly intermediates were characterized in real time (Figure 4b,c). During the first hour of the HBV assembly reaction, the relative abundance of smaller intermediates decreased, whereas the relative abundance of larger intermediates with sizes closer to that of the T = 4 HBV capsid remained constant.…”