Benjamin J. Privett

Antibacterial Fluorinated Silica Colloid Superhydrophobic Surfaces

A superhydrophobic xerogel coating synthesized from a mixture of nanostructured fluorinated silica colloids, fluoroalkoxysilane, and a backbone silane is reported. The resulting fluorinated surface was characterized using contact angle goniometry, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Quantitative bacterial adhesion studies performed using a parallel plate flow cell demonstrated that the adhesion of Staphylococcus aureus and Pseudomonas aeruginosa was reduced by 2.08 ± 0.25 and 1.76 ± 0.12 log over controls, respectively. This simple superhydrophobic coating synthesis may be applied to any surface, regardless of geometry, and does not require harsh synthesis or processing conditions, making it an ideal candidate as a biopassivation strategy.

Electrochemical nitric oxide sensors for physiological measurements

The important biological roles of nitric oxide (NO) have prompted the development of analytical techniques capable of sensitive and selective detection of NO. Electrochemical sensing, more than any other NO detection method, embodies the parameters necessary for quantifying NO in challenging physiological environments such as blood and the brain. In this tutorial review, we provide a broad overview of the field of electrochemical NO sensors, including design, fabrication, and analytical performance characteristics. Both electrochemical sensors and biological applications are detailed.

Nitric oxide-releasing S-nitrosothiol-modified xerogels

The synthesis, material characterization, and in vitro biocompatibility of S-nitrosothiol (RSNO)-modified xerogels are described. Thiol-functionalized xerogel films were formed by hydrolysis and co-condensation of 3-mercaptopropyltrimethoxysilane (MPTMS) and methyltrimethoxysilane (MTMOS) sol-gel precursors at varying concentrations. Subsequent thiol nitrosation via acidified nitrite produced RSNO-modified xerogels capable of generating nitric oxide (NO) for up to 2 weeks under physiological conditions. Xerogels also exhibited NO generation upon irradiation with broad-spectrum light or exposure to copper, with NO fluxes proportional to wattage and concentration, respectively. Xerogels were capable of storing up to approximately 1.31 micromol NO mg(-1), and displayed negligible fragmentation over a 2-week period. Platelet and bacterial adhesion to nitrosated films was reduced compared to non-nitrosated controls, confirming the antithrombotic and antibacterial properties of the NO-releasing materials. Fibroblast cell viability was maintained on the xerogel surfaces illustrating the promise of RSNO-modified xerogels as biomedical device coatings.

Examination of Bacterial Resistance to Exogenous Nitric Oxide

While much research has been directed to harnessing the antimicrobial properties of exogenous NO, the possibility of bacteria developing resistance to such therapy has not been thoroughly studied. Herein, we evaluate potential NO resistance using spontaneous and serial passage mutagenesis assays. Specifically, Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa were systematically exposed to NO-releasing 75mol% MPTMS-TEOS nitrosothiol particles at or below minimum inhibitory concentration (MIC) levels. In the spontaneous mutagenesis assay, bacteria that survived exposure to lethal concentrations of NO showed no increase in MIC. Similarly, no increase in MIC was observed in the serial passage mutagenesis assay after exposure of these species to sub-inhibitory concentrations of NO through 20 d.

Synergy of nitric oxide and silver sulfadiazine against gram-negative, gram-positive, and antibiotic-resistant pathogens.

The synergistic activity between nitric oxide (NO) released from diazeniumdiolate-modified proline (PROLI/NO) and silver(I) sulfadiazine (AgSD) was evaluated against Escherichia coli, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis using a modified broth microdilution technique and a checkerboard-type assay. The combination of NO and AgSD was defined as synergistic when the fractional bactericidal concentration (FBC) was calculated to be <0.5. Gram-negative species were generally more susceptible to the individual antimicrobial agents than the Gram-positive bacteria, while Gram-positive bacteria were more susceptible to combination therapy. The in vitro synergistic activity of AgSD and NO observed against a range of pathogens strongly supports future investigation of this therapeutic combination, particularly for its potential use in the treatment of burns and chronic wounds.

Efficacy of surface-generated nitric oxide against Candida albicans adhesion and biofilm formation

This report details the efficacy of nitric oxide (NO)-releasing xerogel surfaces composed of N-(6-aminohexyl)aminopropyl trimethoxysilane (AHAP3) and isobutyltrimethoxysilane (BTMOS) against Candida albicans adhesion, viability, and biofilm formation. A parallel plate flow cell assay was used to examine the effect of NO on planktonic fungal cells. Nitric oxide fluxes as low as 14 pmol cm−2 s−1 were sufficient to reduce fungal adhesion by ∼49% over the controls after 90 min. By utilizing a fluorescence live/dead assay and replicate plating, NO flux was determined to reduce fungal viability in a dose-dependent manner. The formation of C. albicans biofilms on NO-releasing xerogel-coated silicon rubber (SiR) coupons was impeded when compared to control (non-NO-releasing) and bare SiR surfaces. The synergistic efficacy of NO and silver sulfadiazine against adhered fungal cells and biofilms is reported with increased killing and biofilm inhibition over NO alone.

Microfluidic Amperometric Sensor for Analysis of Nitric Oxide in Whole Blood

Standard photolithographic techniques and a nitric oxide (NO) selective xerogel polymer were utilized to fabricate an amperometric NO microfluidic sensor with low background noise and the ability to analyze NO levels in small sample volumes (~250 μL). The sensor exhibited excellent analytical performance in phosphate buffered saline, including a NO sensitivity of 1.4 pA nM(-1), a limit of detection (LOD) of 840 pM, and selectivity over nitrite, ascorbic acid, acetaminophen, uric acid, hydrogen sulfide, ammonium, ammonia, and both protonated and deprotonated peroxynitrite (selectivity coefficients of -5.3, -4.2, -4.0, -5.0, -6.0, -5.8, -3.8, -1.5, and -4.0, respectively). To demonstrate the utility of the microfluidic NO sensor for biomedical analysis, the device was used to monitor changes in blood NO levels during the onset of sepsis in a murine pneumonia model.

In Vivo Analytical Performance of Nitric Oxide-Releasing Glucose Biosensors

The in vivo analytical performance of percutaneously implanted nitric oxide (NO)-releasing amperometric glucose biosensors was evaluated in swine for 10 d. Needle-type glucose biosensors were functionalized with NO-releasing polyurethane coatings designed to release similar total amounts of NO (3.1 μmol cm–2) for rapid (16.0 ± 4.4 h) or slower (>74.6 ± 16.6 h) durations and remain functional as outer glucose sensor membranes. Relative to controls, NO-releasing sensors were characterized with improved numerical accuracy on days 1 and 3. Furthermore, the clinical accuracy and sensitivity of rapid NO-releasing sensors were superior to control and slower NO-releasing sensors at both 1 and 3 d implantation. In contrast, the slower, extended, NO-releasing sensors were characterized by shorter sensor lag times (<4.2 min) in response to intravenous glucose tolerance tests versus burst NO-releasing and control sensors (>5.8 min) at 3, 7, and 10 d. Collectively, these results highlight the potential for NO release to enhance the analytical utility of in vivo glucose biosensors. Initial results also suggest that this analytical performance benefit is dependent on the NO-release duration.