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

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 pseudocritical 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.

Single Particle Observation of SV40 VP1 Polyanion-Induced Assembly Shows That Substrate Size and Structure Modulate Capsid Geometry.

Simian virus 40 capsid protein (VP1) is a unique system for studying substrate-dependent assembly of a nanoparticle. Here, we investigate a simplest case of this system where 12 VP1 pentamers and a single polyanion, e.g., RNA, form a T = 1 particle. To test the roles of polyanion substrate length and structure during assembly, we characterized the assembly products with size exclusion chromatography, transmission electron microscopy, and single-particle resistive-pulse sensing. We found that 500 and 600 nt RNAs had the optimal length and structure for assembly of uniform T = 1 particles. Longer 800 nt RNA, shorter 300 nt RNA, and a linear 600 unit poly(styrene sulfonate) (PSS) polyelectrolyte produced heterogeneous populations of products. This result was surprising as the 600mer PSS and 500-600 nt RNA have similar mass and charge. Like ssRNA, PSS also has a short 4 nm persistence length, but unlike RNA, PSS lacks a compact tertiary structure. These data indicate that even for flexible substrates, shape as well as size affect assembly and are consistent with the hypothesis that work, derived from protein-protein and protein-substrate interactions, is used to compact the substrate.

AC Electroosmotic Pumping in Nanofluidic Funnels.

We report efficient pumping of fluids through nanofluidic funnels when a symmetric AC waveform is applied. The asymmetric geometry of the nanofluidic funnel induces not only ion current rectification but also electroosmotic flow rectification. In the base-to-tip direction, the funnel exhibits a lower ion conductance and a higher electroosmotic flow velocity, whereas, in the tip-to-base direction, the funnel has a higher ion conductance and a lower electroosmotic flow velocity. Consequently, symmetric AC waveforms easily pump fluid through the nanofunnels over a range of frequencies, e.g., 5 Hz to 5 kHz. In our experiments, the nanofunnels were milled into glass substrates with a focused ion beam (FIB) instrument, and the funnel design had a constant 5° taper with aspect ratios (funnel tip width to funnel depth) of 0.1 to 1.0. We tracked ion current rectification by current-voltage (I-V) response and electroosmotic flow rectification by transport of a zwitterionic fluorescent probe. Rectification of ion current and electroosmotic flow increased with increasing electric field applied to the nanofunnel. Our results support three-dimensional simulations of ion transport and electroosmotic transport through nanofunnels, which suggest the asymmetric electroosmotic transport stems from an induced pressure at the junction of the nanochannel and nanofunnel tip.

Conductivity-based detection techniques in nanofluidic devices.

This review covers conductivity detection in fabricated nanochannels and nanopores. Improvements in nanoscale sensing are a direct result of advances in fabrication techniques, which produce devices with channels and pores with reproducible dimensions and in a variety of materials. Analytes of interest are detected by measuring changes in conductance as the analyte accumulates in the channel or passes transiently through the pore. These detection methods take advantage of phenomena enhanced at the nanoscale, such as ion current rectification, surface conductance, and dimensions comparable to the analytes of interest. The end result is the development of sensing technologies for a broad range of analytes, e.g., ions, small molecules, proteins, nucleic acids, and particles.

Single-particle electrophoresis in nanochannels.

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) cm(2) V(-1) s(-1)) in the nanopores and in the nanochannel with the larger cross-sectional area.

The abilities of selenium dioxide and selenite ion to coordinate DNA-bound metal ions and decrease oxidative DNA damage.

Several transition metals react with H2O2 and produce reactive oxygen species (ROS) responsible for oxidative damage linked to many diseases and disorders, and species that form coordination complexes with these metal ions show promise as antioxidants. The present study demonstrates that metal-mediated radical and non-radical oxidative DNA damage decreases when selenium dioxide (SeO2) and sodium selenite (Na2SeO3) are present. Radical-induced damage is associated with production of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), which arises from ROS generated at or near the guanine base, and the selenium compounds reduce Fe(II)-, Cr(III)- and Cu(II)-mediated radical damage to differing degrees based on the identity of the metal ion and the order in which the metals, selenium compounds and DNA are combined. Radical damage arising from Fe(II) and Cr(III) decreases substantially when they are pre-incubated with the selenium compounds prior to adding DNA. Non-radical damage is associated with oxidation of the adenine base in the presence of high H2O2 concentrations through an ionic mechanism, and this type of damage also decreases significantly when the selenium compounds are allowed to interact with the metal ions before adding DNA. Fluorescence studies using dihydrodichlorofluorescein diacetate (DCF-DA) to probe ROS formation indicate that the majority of the SeO2- and SeO3(2-)-metal systems in combination with H2O2 (no DNA present) produce ROS to the same degree as the metal/H2O2 systems in the absence of the selenium compounds, suggesting that selenium-metal complexes react with H2O2 in a sacrificial manner that protects DNA from oxidative damage.