Virus assembly is a coordinated process in which typically hundreds of subunits react to form complex, symmetric particles. We use resistive-pulse sensing to characterize the assembly of Hepatitis B Virus core protein dimers into T = 3 and T = 4 icosahedral capsids. This technique counts and sizes intermediates and capsids in real time, with single particle sensitivity, and at biologically relevant concentrations. Other methods are not able to produce comparable real-time, single-particle observations of assembly reactions below, near, and above the pseudocritical dimer concentration, at which the dimer and capsid concentrations are approximately equal. Assembly reactions across a range of dimer concentrations reveal three distinct patterns. At dimer concentrations as low as 50 nM, well below the pseudo-critical dimer concentration of 0.5 µM, we observe a switch in the ratio of T = 3 to T = 4 capsids, which increases with decreasing dimer concentration. Far above the pseudo-critical dimer concentration, kinetically trapped, incomplete T = 4 particles assemble rapidly, then slowly anneal into T = 4 capsids. At all dimer concentrations tested, T = 3 capsids form more rapidly than T = 4 capsids, suggesting distinct pathways for the two forms.

We report fabrication of nanofluidic channels with asymmetric features (e.g., funnels) that were cast in high modulus poly(dimethylsiloxane) and had well-defined geometries and dimensions. Masters used to cast the funnels were written in the negative tone resist SU-8 by electron beam lithography. Replicated funnels had taper angles of 5, 10, and 20 degrees and were 80 nm wide at the tip, 1 microm wide at the base, and 120 nm deep. The planar format permitted easy coupling of the funnels to microfluidic channels and simultaneous electrical and optical characterization of ion transport. All three designs rectified ion current, and the 5 degrees funnel exhibited the highest rectification ratio. Fluorescence measurements at the funnel base showed that an anionic probe was enriched and depleted in the high and low conductance states, respectively.

We report fabrication and characterization of nanochannel devices with two nanopores in series for resistive-pulse sensing of hepatitis B virus (HBV) capsids. The nanochannel and two pores are patterned by electron beam lithography between two microchannels and etched by reactive ion etching. The two nanopores are 50-nm wide, 50-nm deep, and 40-nm long and are spaced 2.0-μm apart. The nanochannel that brackets the two pores is 20x wider (1 μm) to reduce the electrical resistance adjacent to the two pores and to ensure the current returns to its baseline value between resistive-pulse events. Average pulse amplitudes differ by <2% between the two pores and demonstrate the fabrication technique is able to produce pores with nearly identical geometries. Because the two nanopores in series sense single particles at two discrete locations, particle properties, e.g., electrophoretic mobility, are determined from the pore-to-pore transit time.

Electrophoretic mobilities and particle sizes of individual Hepatitis B Virus (HBV) capsids were measured in nanofluidic channels with two nanopores in series. The channels and pores had three-dimensional topography and were milled directly in glass substrates with a focused ion beam instrument assisted by an electron flood gun. The nanochannel between the two pores was 300 nm wide, 100 nm deep, and 2.5 μm long, and the nanopores at each end had dimensions 45 nm wide, 45 nm deep, and 400 nm long. With resistive-pulse sensing, the nanopores fully resolved pulse amplitude distributions of T = 3 HBV capsids (32 nm outer diameter) and T = 4 HBV capsids (35 nm outer diameter) and had sufficient peak capacity to discriminate intermediate species from the T = 3 and T = 4 capsid distributions in an assembly reaction. Because the T = 3 and T = 4 capsids have a wiffle-ball geometry with a hollow core, the observed change in current due to the capsid transiting the nanopore is proportional to the volume of electrolyte displaced by the volume of capsid protein, not the volume of the entire capsid. Both the signal-to-noise ratio of the pulse amplitude and resolution between the T = 3 and T = 4 distributions of the pulse amplitudes increase as the electric field strength is increased. At low field strengths, transport of the larger T = 4 capsid through the nanopores is hindered relative to the smaller T = 3 capsid due to interaction with the pores, but at sufficiently high field strengths, the T = 3 and T = 4 capsids had the same electrophoretic mobilities (7.4 × 10–5 cm2 V–1 s–1) in the nanopores and in the nanochannel with the larger cross-sectional area.

We report the measurement of electroosmotic mobilities in nanofluidic channels with rectangular cross sections and compare our results with theory. Nanofluidic channels were milled directly into borosilicate glass between two closely spaced microchannels with a focused ion beam instrument, and the nanochannels had half-depths (h) of 27, 54, and 108 nm and the same half-width of 265 nm. We measured electroosmotic mobilities in NaCl solutions from 0.1 to 500 mM that have Debye lengths (κ–1) from 30 to 0.4 nm, respectively. The experimental electroosmotic mobilities compare quantitatively to mobilities calculated from a nonlinear solution of the Poisson–Boltzmann equation for channels with a parallel-plate geometry. For the calculations, ζ-potentials measured in a microchannel with a half-depth of 2.5 μm are used and range from −6 to −73 mV for 500 to 0.1 mM NaCl, respectively. For κh > 50, the Smoluchowski equation accurately predicts electroosmotic mobilities in the nanochannels. However, for κh < 10, the electrical double layer extends into the nanochannels, and due to confinement within the channels, the average electroosmotic mobilities decrease. At κh ≈ 4, the electroosmotic mobilities in the 27, 54, and 108 nm channels exhibit maxima, and at 0.1 mM NaCl, the electroosmotic mobility in the 27 nm channel (κh = 1) is 5-fold lower than the electroosmotic mobility in the 2.5 μm channel (κh = 100).

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.

Virus self-assembly is a critical step in the virus lifecycle. Understanding how viruses assemble and disassemble provides needed insight into developing antiviral pharmaceuticals. Few tools offer sufficient resolution to study assembly intermediates that differ in size by a few dimers. Our goal is to improve resistive-pulse sensing on nanofluidic devices to offer better particle-size and temporal resolution to study intermediates and capsids generated along the assembly pathway. To increase the particle-size resolution of the resistive-pulse technique, we measured the same, single virus particles up to a thousand times, cycling them back and forth across a series of nanopores by switching the polarity of the applied potential, i.e., virus ping-pong. Multiple pores in series provide a unique multipulse signature during each cycle that improves particle tracking and, therefore, identification of a single particle and reduces the number of cycles needed to make the requisite number of measurements. With T = 3 and T = 4 hepatitis B virus (HBV) capsids, we showed the standard deviation of the particle-size distribution decreased with the square root of the number of measurements and approached discriminating particles differing in size by single dimers. We then studied in vitro assembly of HBV capsids and observed that the ensemble of intermediates shift to larger sizes over 2 days of annealing. On the contrary, assembly reactions diluted to lower dimer concentrations an hour after initiation had fewer intermediates that persisted after the 2 day incubation and had a higher ratio of T = 4 to T = 3 capsids. These reactions indicate that labile T = 4 intermediates are formed rapidly, and dependent on conditions, intermediates may be trapped as metastable species or progress to yield complete capsids.

The large-scale manufacture of complex synthetic peptides is challenging due to many factors such as manufacturing risk (including failed product specifications) as well as processes that are often low in both yield and overall purity. To overcome these liabilities, a hybrid solid-phase peptide synthesis/liquid-phase peptide synthesis (SPPS/LPPS) approach was developed for the synthesis of tirzepatide. Continuous manufacturing and real-time analytical monitoring ensured the production of high-quality material, while nanofiltration provided intermediate purification without difficult precipitations. Implementation of the strategy worked very well, resulting in a robust process with high yields and purity.