Gerold A. Willing

Conductivity of ionic liquid-derived polymers with internal gold nanoparticle conduits

The transport properties of self-supporting Au nanoparticle–ionic liquid-derived polymer composites were characterized. Topographic AFM images confirm the perforated lamellar composite architecture determined by small-angle X-ray scattering (SAXS) and further show that the in situ synthesized Au nanoparticles are localized within the hydrophilic (water) domains of the structure. At low Au nanoparticle content, the images reveal incomplete packing of spherical particles (i.e., voids) within these columns. The confinement and organization of the Au nanoparticles within the hydrophilic columns give rise to a large manifold of optical resonances in the near-IR region. The bulk composite conductivity, Rb, was determined by ac electrochemical impedance spectroscopy (EIS) for samples prepared with increasing Au3+ content over a frequency range of 10 Hz to 1 MHz. A 100-fold increase was observed in the bulk conductivity at room temperature for composites prepared with the highest amount of Au3+ (1.58 ± 0.065 µmol) versus the no Au composite, with the former reaching a value of 1.3 × 10−4 S cm−1 at 25 °C. The temperature dependence of the conductivity recorded over this range was well-modeled by the Arrhenius equation. EIS studies on samples containing the highest Au nanoparticle content over a broader range of frequencies (2 × 10−2 Hz to 5 × 105 Hz) identified a low frequency component ascribed to electronic conduction. Electronic conduction due to aggregated Au nanoparticles was further confirmed by dc conductivity measurements. This work identifies a nanostructured composite that exhibits both ionic transport through the polymeric ionic liquid and electronic conduction from the organized encapsulated columns of Au nanoparticles.

Microscopic Studies of Various Sizes of Gold Nanoparticles and Their Cellular Localizations

Gold nanoparticles (GNPs) are widely used in biological and clinical applications due to their favorable chemical and optical properties. GNPs can be used for drug delivery to targeted cells. In addition, GNPs serve as ideal probes for biological and cell imaging applications. Recent studies indicate that the size diversity of GNPs plays an important role in targeting cellular components for biomedical applications. In this study, we conducted a series of studies using different sizes of gold nanoparticles, including 3, 10, 25, and 50 nm, to determine the effect of size variations on their intracellular localizations. Our cytotoxicity studies of GNPs into the HEp-2 cells using MTT assay indicated that 3 nm GNPs possess the highest toxicity. We exposed HEp-2 cells with various sizes of gold nanoparticles for different time intervals (1, 2, 4, 12, and 24 h) followed by imaging using scanning electron microscope (SEM) and atomic force microscope (AFM). Our SEM and AFM results showed that, after 1 hr incubation, 3 and 10 nm gold nanoparticles entered the nucleus, whereas 25 and 50 nm particles accumulated around the nucleus. As the time of exposure increased, GNPs entered the cells and accumulated in the cytosol and nucleus based solely on their sizes.

Scaled-up separation of cellobiohydrolase1 from a cellulase mixture by ion-exchange chromatography

Enzymatic hydrolysis of cellulose often involves cellulases produced by Trichoderma reesei, of which cellobiohydrolase1 (CBH1) is the most abundant (about 60% of total cellulases) and plays an important role in the hydrolysis of crystalline cellulose. A method for separating sufficient quantities from the bulk cellulase cocktail is highly desirable for many studies, such as those that aim to characterize binding and hydrolysis kinetics of CBH1. In this work, CBH1 was separated from other Spezyme CP cellulases by ion‐exchange chromatography using an efficient modification of a smaller scale process. The ion‐exchange column was connected to a vacuum manifold system to provide a steady flow through parallel columns and thus achieve scale‐up for enzyme separation. With five 5‐mL columns running in parallel, about 55 mg of CBH1 was separated from 145 mg of Spezyme CP in a single separation. Step elution was used to replace the continuous gradient used at smaller scale. The purified CBH1 was collected in the fraction eluted with a buffer containing 0.33 M salt and showed comparable purity and activity as the enzyme purified by a fast protein liquid chromatography system. The stability of separated CBH1 was studied for up to 2 days and good thermal stability was observed. Separated CBH1 also showed both high adsorption to bacterial microcrystalline cellulose with ∼4 μmol/g maximum adsorption and a Ka of 5.55 ± 2.34 μM−1, and good hydrolytic activity based on atomic force microscopy observations that show a reduction in fiber height.

An Effective Zeta Potential Fitting Model for Sphere-Plate Interaction Force in Nanoparticle Suspensions

The silica sphere-plate interaction forces in zirconia nanoparticle suspensions have been successfully measured to explain how negligibly charged silica microspheres can be stabilized through the addition of highly charged zirconia nanoparticles. However, the influence of nanoparticle volume fraction on the stabilization as well as how various forces (the attractive van der Waals force, repulsive electrostatic force, and depletion force) contribute to the total interaction force still remains unclear. Therefore, an effective zeta potential fitting model is developed to explain the experimental interaction force curves based on a variable effective Debye length and a measured effective zeta potential using a continuum assumption.

Adhesion and Mechanical Properties of RSV Infected Human Epithelial Cells

Respiratory syncytial virus is the leading cause of lower respiratory tract infection in infants and currently lacks an effective vaccine or treatment beyond symptom relief. The atomic force microscope is particularly well suited for imaging biological samples including cells, DNA, viruses and proteins. Analysis of fixed HEP-2 cells with the AFM after infection and incubation with RSV for periods of 0.5–24 h reveals several physical changes within the cells that may lead to a better understanding of the viral effects on living cells. Analysis of force–distance curves reveals changes in the mechanical properties of the cells throughout the infection period as well as changes in the surface chemistry, specifically in the presence of hydrophobic domains, of the cell membrane as shown by variability in the measured adhesion force. Elastic moduli of the fixed cells did not show a statistically significant trend, but hydrophobic molecular expression on the cellular surface decreased with increasing infection period. This change in the chemical nature of the cell membrane might be explained by the rapid removal, repair and replacement of the lipid bilayer occurring as a result of the exocytosis of RSV virions.

Atomic force microscopic investigation of respiratory syncytial virus infection in HEp-2 cells.

Respiratory syncytial virus (RSV) primarily causes bronchiolitis and pneumonia in infants. In spite of intense research, no safe and effective vaccine has been developed yet. For understanding its pathogenesis and development of anti‐RSV drugs/therapeutics, it is indispensable to study the RSV–host interaction. Although, there are limited studies using electron microscopy to elucidate the infection process of RSV, to our knowledge, no study has reported the morphological impact of RSV infection using atomic force microscopy. We report the cytoplasmic and nuclear changes in human epidermoid cell line type 2 using atomic force microscopy. Human epidermoid cell line type 2 cells, grown on cover slips, were infected with RSV and fixed after various time periods, processed and observed for morphological changes using atomic force microscopy. RSV infected cells showed loss of membrane integrity, with degeneration in the cellular content and cytoskeleton. Nuclear membrane was disintegrated and nuclear volume was decreased. The chromatin of the RSV infected cells was condensed, progressing towards degeneration via pyknosis and apoptosis. Membrane protrusions of ∼150–200 nm diameter were observed on RSV infected cells after 6 h, suggestive of prospective RSV budding sites. To our knowledge, this is the first study of RSV infection process using atomic force microscopy. Such morphological studies could help explore viral infection process aiding the development of anti‐RSV therapies.

Direct calibration of colloidal probe cantilevers via Derjaguin, Landau, Verwey, and Overbeek surface forces in electrolyte solution

The development of colloidal probe microscopy has made it possible to directly measure the interaction forces between two different surfaces in solution. Cantilever calibration is presently a subject of intense experimental and theoretical interest due to the need for accurate force measurement. We developed a novel and direct calibration method for colloidal probe cantilevers to which a silica microsphere has been previously attached based on fitting experimental force curves for the interaction between the silica sphere and a silica flat in dilute KBr solutions to the theoretical Derjaguin, Landau, Verwey, and Overbeek force curves using the measured zeta potential of the silica surfaces.