Patrick A. Johnson

Polysaccharide-based polyelectrolyte complex nanoparticles from chitosan, heparin, and hyaluronan.

The formation of polyelectrolyte complex nanoparticles (PCN) was investigated at different charge mixing ratios for the chitosan-heparin (chi-hep) and chitosan-hyaluronan (chi-ha) polycation-polyanion pairs. The range of 0.08-19.2 for charge mixing ratio (n(+)/n(-)) was examined. The one-shot addition of polycation and polyanion solutions used for the formation of the PCN permitted formation of both cationic and anionic particles from both polysaccharide pairs. The influence of the charge mixing ratio on the size and zeta potential of the particles was investigated. The morphology and stability of the particles when adsorbed to surfaces was studied by scanning electron microscopy (SEM). For most conditions studied, colloidally stable, nonstoichiometric PCN were formed in solution. However, PCN formation was inhibited by flocculation at charge mixing ratios near 1. When adsorbed to surfaces and dried, some formulations resulted in discrete nanoparticles, while others partially or completely aggregated or coalesced, leading to different surface morphologies.

Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles

A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibody recognition using Raman reporter-coated Au nanoparticles (GNPs) and paramagnetic nanoparticles (PMPs) conjugated with polyclonal antibodies specific for each antigen target, followed by 785nm laser excitation of magnetically concentrated GNP/antigen/PMP complexes. The discrimination of WNV and RVFV antigen detection in mixed Raman spectra was achieved by SERS enhancement of Raman spectra specific for the Raman reporter dyes Infrared 792 (IR-792) and Nile Blue (NB), respectively. Assay reactions containing dilutions of both target antigens yielded a reduction in the intensification of IR-792 and NB signature spectrum peaks and provided a conservative limit of detection of ∼5fg/ml for assays conducted in phosphate buffered saline buffer (PBS) and ∼25pg/ml for assays containing PBS spiked with fetal bovine serum. Based on the inherent simplicity of the assay, magnetic capture-based SERS assays afford promise as a biosensor platform that provides high-level multiplex detection sensitivity and can be adapted for portable diagnostic applications in limited resource settings.

A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G

A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite green (MG)-conjugated protein A/G (MG-pA/G) as a bi-functional Raman tag/antibody binding reporter. Upon incubation of these reagents with serum collected from rabbits inoculated with E antigen, laser interrogation of the sandwiched immunocomplex revealed a SERS signaling response diagnostic for MG. The intensification of signature spectral peaks is shown to be proportionate to the concentration of added serum and the limit of antibody detection is 2 ng/ml of serum. To assess assay performance relative to more a traditional immunoassay, indirect enzyme-linked immunosorbent assays conducted using the same concentrations of reagents were found to be >400-fold less sensitive. Quartz crystal microbalance with dissipation (QCM-D) monitoring of immunocomplex film deposition on solid Au surfaces also confirmed the formation of antigen-antibody-protein A/G trilayers and provided quantitative measurements of film thickness which likely position MG within the sensing distance of laser-elicited, enhanced electromagnetic fields. The sensitivity and inherent versatility of the assay, which is provided by the binding of pA/G to a broad spectrum of immunoglobulins in different mammalian species, suggest that it could be developed as an alternative immunoassay format to the ELISA.

Synthesis and characterization of enzyme-magnetic nanoparticle complexes: effect of size on activity and recovery.

The influence of particle size on the activity and recycling capabilities of enzyme conjugated magnetic nanoparticles was studied. Co-precipitation and oxidation of Fe(OH)(2) methods were used to fabricate three different sizes of magnetic nanoparticles (5 nm, 26 nm and 51 nm). Glucose oxidase was covalently bound to the magnetic nanoparticles by modifying the surfaces with 3-(aminopropyl)triethoxysilane (APTES) and a common protein crosslinking agent, glutaraldehyde. Analysis by Transmission Electron Microscopy (TEM) showed that the morphology of the magnetic nanoparticles to be spherical and sizes agreed with results of the Brunauer, Emmett, and Teller (BET) method. Magnetic strength of the nanoparticles was analyzed by magnetometry and found to be 49 emu g(-1) (5 nm), 73 emu g(-1) (26 nm), and 85 emu g(-1) (51 nm). X-ray photoelectron spectroscopy (XPS) confirmed each step of the magnetic nanoparticle surface modification and successful glucose oxidase binding. The immobilized enzymes retained 15-23% of the native GOx activity. Recycling stability studies showed approximately 20% of activity loss for the large (51 nm) and medium (26 nm) size glucose oxidase-magnetic nanoparticle (GOx-MNP) bioconjugate and about 96% activity loss for the smallest GOx-MNP bioconjugate (5 nm) after ten cycles. The bioconjugates demonstrated equivalent total product conversions as a single reaction of an equivalent amount of the native enzyme after the 5th cycle for the 26 nm nanoparticles and the 7th cycle for the 51 nm nanoparticles.

Surface-enhanced Raman scattering detection of DNAs derived from virus genomes using Au-coated paramagnetic nanoparticles.

A magnetic capture-based, surface-enhanced Raman scattering (SERS) assay for DNA detection has been developed which utilizes Au-coated paramagnetic nanoparticles (Au@PMPs) as both a SERS substrate and effective bioseparation reagent for the selective removal of target DNAs from solution. Hybridization reactions contained two target DNAs, sequence complementary reporter probes conjugated with spectrally distinct Raman dyes distinct for each target, and Au@PMPs conjugated with sequence complementary capture probes. In this case, target DNAs were derived from the RNA genomes of the Rift Valley Fever virus (RVFV) or West Nile virus (WNV). The hybridization reactions were incubated for a short period and then concentrated within the focus beam of an interrogating laser by magnetic pull-down. The attendant SERS response of each individually captured DNA provided a limit of detection sensitivity in the range 20-100 nM. X-ray diffraction and UV-vis analysis validated both the desired surface plasmon resonance properties and bimetallic composition of synthesized Au@PMPs, and UV-vis spectroscopy confirmed conjugation of the Raman dye compounds malachite green (MG) and erythrosin B (EB) with the RVFV and WNV reporter probes, respectively. Finally, hybridization reactions assembled for multiplexed detection of both targets yielded mixed MG/EB spectra and clearly differentiated peaks which facilitate the quantitative detection of each DNA target. On the basis of the simple design of a single-particle DNA detection assay, the opportunity is provided to develop magnetic capture-based SERS assays that are easily assembled and adapted for high-level multiplex detection using low-cost Raman instrumentation.

Enzyme Nanoparticle Fabrication: Magnetic Nanoparticle Synthesis and Enzyme Immobilization

Immobilized enzymes are drawing significant attention for potential commercial applications as biocatalysts by reducing operational expenses and by increasing process utilization of the enzymes. Typically, immobilized enzymes have greater thermal and operational stability at various pH values, ionic strengths and are more resistant to denaturation that the soluble native form of the enzyme. Also, immobilized enzymes can be recycled by utilizing the physical or chemical properties of the supporting material. Magnetic nanoparticles provide advantages as the supporting material for immobilized enzymes over competing materials such as: higher surface area that allows for greater enzyme loading, lower mass transfer resistance, less fouling effect, and selective, nonchemical separation from the reaction mixture by an applied a magnetic field. Various surface modifications of magnetic nanoparticles, such as silanization, carbodiimide activation, and PEG or PVA spacing, aid in the binding of single or multienzyme systems to the particles, while cross-linking using glutaraldehyde can also stabilize the attached enzymes.

Layer-by-layer assembly of polysaccharide-based nanostructured surfaces containing polyelectrolyte complex nanoparticles

Nanoscale chemical and topographical features have been demonstrated to influence a variety of significant responses of mammalian cells to biomaterials surfaces. Thus, an important goal for biomaterials scientists is the ability to engineer the nanoscale surface features of biologically active materials. The goal of the current work is to demonstrate that polyelectrolyte complex nanoparticles (PCNs) in polyelectrolyte multilayers (PEMs) can be combined to create surfaces with controlled nanoscale surface topography and nanoscale presentation of surface chemistry. The polysaccharides used in this work are the biomedically relevant chitosan, heparin, and hyaluronan. Nanostructured surface coatings were characterized on both modified gold substrates and tissue-culture polystyrene surfaces. PCNs were adsorbed to oppositely charged PEMs, and were also embedded within PEMs. The construction of the surface coatings was characterized by quartz crystal microbalance with dissipation (QCM-D). The surface morphology was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The chemistry of the coatings was confirmed by both X-ray photoelectron spectroscopy (XPS) and polarization modulation infra-red reflection absorption spectroscopy (PM-IRRAS). Morphologically, we found that PCNs were colloidally stable and homogeneously distributed when adsorbed on or in the PEMs. Chemical analysis confirms that the PCNs adsorbed to PEMs significantly altered the surface chemistry, indicating significant surface coverage. Furthermore, the position of the PCNs normal to the surface can be adjusted by adding PEMs on top of adsorbed PCNs. Thus, PCNs can be used to introduce discrete nanoscale surface topographical features and varying surface chemistry into PEM surface coatings in a controlled way.

Interplay of anionic charge, poly(ethylene glycol), and iodinated tyrosine incorporation within tyrosine‐derived polycarbonates: Effects on vascular smooth muscle cell adhesion, proliferation, and motility

Regulation of smooth muscle cell adhesion, proliferation, and motility on biomaterials is critical to the performance of blood-contacting implants and vascular tissue engineering scaffolds. The goal of this study was to examine the underlying substrate-smooth muscle cell response relations, using a selection of polymers representative of an expansive library of multifunctional, tyrosine-derived polycarbonates. Three chemical components within the polymer structure were selectively varied through copolymerization: (1) the content of iodinated tyrosine to achieve X-ray visibility; (2) the content of poly(ethylene glycol) (PEG) to decrease protein adsorption and cell adhesivity; and (3) the content of desaminotyrosyl-tyrosine (DT), which regulates the rate of polymer degradation. Using quartz crystal microbalance with dissipation, we quantified differential serum protein adsorption behavior because of the chemical components DT, iodinated tyrosine, and PEG: increased PEG content within the polymer structure progressively decreased protein adsorption but the simultaneous presence of both DT and iodinated tyrosine reversed the effects of PEG. The complex interplay of these components was next tested on the adhesion, proliferation, and motility behavior cultured human aortic smooth muscle cells. The incorporation of PEG into the polymer reduced cell attachment, which was reversed in the presence of iodinated tyrosine. Further, we found that as little as 10% DT content was sufficient to negate the PEG effect in polymers containing iodinated tyrosine, whereas in non-iodinated polymers, the PEG effect on cell attachment was reversed. Cross-functional analysis of motility and proliferation revealed divergent substrate chemistry related cell response regimes. For instance, within the series of polymers containing both iodinated tyrosine and 10% of DT, increasing PEG levels lowered smooth muscle cell motility without a change in the rate of cell proliferation. In contrast, for non-iodinated tyrosine and 10% of DT, increasing PEG levels increased cell proliferation significantly while reducing cell motility. Clearly, the polycarbonate polymer library offers a sensitive platform to modulate cell adhesion, proliferation, and motility responses, which, in turn, may have implications for controlling vascular remodeling in vivo. Additionally, our data suggests unique biorelevant properties following the incorporation of iodinated subunits in a polymeric biomaterial as a potential platform for X-ray visible devices.

A major role for calcium-dependent potassium current in action potential repolarization in adrenal chromaffin cells

To determine the extent which Ca dependent K current (IKCa) contributes during an action potential (AP), bovine chromaffin cells were voltage-clamped using a pre-recorded AP as the command voltage waveform. Based on (1) differential sensitivity of IKCa and Ca-independent K current (IK) to tetraethylammonium; (2) measurements of AP currents under conditions where Ca activation of IKCa had been abolished; and (3) blockade by charybdotoxin, IKCa comprised 70-90% of the outward K current during AP repolarization. In addition, observations are made concerning the form of AP-evoked Ca current.

Stabilization of CO2 foam using by-product fly ash and recyclable iron oxide nanoparticles to improve carbon utilization in EOR processes

Fly ash nanoparticles (NPs), a by-product of coal combustion power plants, and recyclable iron oxide NPs are used to stabilize CO2 foam for use in enhanced oil recovery (EOR) processes as part of carbon capture, utilization and storage. The mean size of fly ash NPs is reduced to the nano-scale (40–80 nm) by a two stage grinding process. Iron oxide (IO) NPs are synthesized through Fe2+ and Fe3+ co-precipitation. The size and uniformity of the dispersion of NPs are characterized via dynamic light scattering and transmission electron microscopy. The stability and formability of NP-stabilized foam are investigated via a modified bulk foam test. The resulting improvement in oil recovery is investigated using flow experiments using a microfluidic device fabricated based on a two-dimensional representation of a Berea sandstone. The effects of the type of NP (fly ash and IO) on CO2 foam stability and the resulting incremental oil recovery are investigated and discussed. The results show that fly ash NPs with an Alpha-Olefin Sulfonate (AOS)–Lauramidopropyl betaine (LAPB) surfactant mixture provide excellent formability and stability (half-life 280 min) and resulted in over 90% recovery of the incremental oil after a waterflood in microfluidic experiments. Moreover, the material cost of fly ash NPs is significantly less than commonly used silica NPs. IO NP stabilized foam has an inferior foam stability and formability compared to fly ash stabilized foam; IO NPs can, however, be recycled using a magnetic field – the average recovery from the outflow of the microfluidic experiments was approximately 76%.