Broadband Dielectric Spectroscopy as a Potential Label-Free Method to Rapidly Verify Ultraviolet-C Radiation Disinfection.

Microwave (MW) sensing offers noninvasive, real-time detection of the electromagnetic properties of biological materials via the highly concentrated electromagnetic fields, for which advantages include wide bandwidth, small size, and cost-effective fabrication. In this paper, we present the application of MW broadband dielectric spectroscopy (BDS) coupled to a fabricated biological thin film for evaluating ultraviolet-C (UV-C) exposure effects. The BDS thin film technique could be deployed as a biological indicator for assessing whole-room UV-C surface disinfection. The disinfection process is monitored by BDS as changes in the electrical properties of surface-confined biological thin films photodegraded with UV-C radiation. Fetal bovine serum (FBS, a surrogate for protein) and bacteriophage lambda double-stranded deoxyribonucleic acid (dsDNA) were continuously monitored with BDS during UV-C radiation exposure. The electrical resistance of FBS films yielded promising yet imprecise readings, whereas the resistance of dsDNA films discernibly decreased with UV-C exposure. The observations are consistent with the expected photo-oxidation and photodecomposition of protein and DNA. While further research is needed to characterize these measurements, this study presents the first application of BDS to evaluate the electrical properties of solid-state biological thin films. This technique shows promise toward the development of a test method and a standard biological test to determine the efficacy of UV-C disinfection. Such a test with biological indicators could easily be applied to hospital rooms between patient occupancy for a multipoint evaluation to determine if a room meets a disinfection threshold set for new patients.

Lens-Free Imaging as a Sensor for Dynamic Cell Viability Detection Using the Neutral Red Uptake Assay.

Neutral red is a low-cost supravital stain for determining cell viability. The standard protocol relies on a destructive extraction process to release the accumulated dye for endpoint spectrophotometric quantification. We report a non-destructive, live-cell quantification of neutral red uptake using a compact lens-free system. Two light sources indentify the cell perimeter and quantify neutral red uptake. The quantification occurs during staining, thus eliminating the destructive extraction process and reducing assay time. Our system enables live quantification for continuous high-throughput screening of cell viability within confined spaces such as incubators.

Uncovering the Contribution of Microchannel Deformation to Impedance-Based Flow Rate Measurements.

Changes in electrical impedance have previously been used to measure fluid flow rate in microfluidic channels. Ionic redistribution within the electrical double layer by fluid flow has been considered to be the primary mechanism underlying such impedance based microflow sensors. Here we describe a previously unappreciated contribution of microchannel deformation to such measurements. We found that flow-induced microchannel deformation contributes significantly to the change in electrical impedance of solutions, in particular to those solutions producing an electrical double layer in the order of a few tens of nanometers (i.e., containing relatively high ionic strength). Since the flow velocity at the measurement surface is near zero, due to the laminar nature of the flow, the contribution of the double layer under the conditions mentioned above should be negligible. In contrast, an increase in the fluid flow rate results in an increase in the microchannel cross-sectional area (because of higher local pressure), therefore, producing a decrease in solution resistance between the two electrodes. Our results suggest that microflow sensors based on the concept of elastic deformation could be designed for in situ monitoring and fine control of fluid flow in flexible microfluidics. Finally, we show that purposefully engineering a larger deformability of the microchannel, by changing the geometry and the Young's modulus of the microchannel, enhances the sensitivity of this flow rate measurement.

Dimensional reduction of duplex DNA under confinement to nanofluidic slits.

There has been much interest in the dimensional properties of double-stranded DNA (dsDNA) confined to nanoscale environments as a problem of fundamental importance in both biological and technological fields. This has led to a series of measurements by fluorescence microscopy of single dsDNA molecules under confinement to nanofluidic slits. Despite the efforts expended on such experiments and the corresponding theory and simulations of confined polymers, a consistent description of changes of the radius of gyration of dsDNA under strong confinement has not yet emerged. Here, we perform molecular dynamics (MD) simulations to identify relevant factors that might account for this inconsistency. Our simulations indicate a significant amplification of excluded volume interactions under confinement at the nanoscale due to the reduction of the effective dimensionality of the system. Thus, any factor influencing the excluded volume interaction of dsDNA, such as ionic strength, solution chemistry, and even fluorescent labels, can greatly influence the dsDNA size under strong confinement. These factors, which are normally less important in bulk solutions of dsDNA at moderate ionic strengths because of the relative weakness of the excluded volume interaction, must therefore be under tight control to achieve reproducible measurements of dsDNA under conditions of dimensional reduction. By simulating semi-flexible polymers over a range of parameter values relevant to the experimental systems and exploiting past theoretical treatments of the dimensional variation of swelling exponents and prefactors, we have developed a novel predictive relationship for the in-plane radius of gyration of long semi-flexible polymers under slit-like confinement. Importantly, these analytic expressions allow us to estimate the properties of dsDNA for the experimentally and biologically relevant range of contour lengths that is not currently accessible by state-of-the-art MD simulations.

Anthrax toxin-induced rupture of artificial lipid bilayer membranes.

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

Theory for polymer analysis using nanopore-based single-molecule mass spectrometry.

Nanometer-scale pores have demonstrated potential for the electrical detection, quantification, and characterization of molecules for biomedical applications and the chemical analysis of polymers. Despite extensive research in the nanopore sensing field, there is a paucity of theoretical models that incorporate the interactions between chemicals (i.e., solute, solvent, analyte, and nanopore). Here, we develop a model that simultaneously describes both the current blockade depth and residence times caused by individual poly(ethylene glycol) (PEG) molecules in a single alpha-hemolysin ion channel. Modeling polymer-cation binding leads to a description of two significant effects: a reduction in the mobile cation concentration inside the pore and an increase in the affinity between the polymer and the pore. The model was used to estimate the free energy of formation for K(+)-PEG inside the nanopore (approximately -49.7 meV) and the free energy of PEG partitioning into the nanopore ( approximately 0.76 meV per ethylene glycol monomer). The results suggest that rational, physical models for the analysis of analyte-nanopore interactions will develop the full potential of nanopore-based sensing for chemical and biological applications.

Sizing the Bacillus anthracis PA63 channel with nonelectrolyte poly(ethylene glycols).

Nonelectrolyte polymers of poly(ethylene glycol) (PEG) were used to estimate the diameter of the ion channel formed by the Bacillus anthracis protective antigen 63 (PA(63)). Based on the ability of different molecular weight PEGs to partition into the pore and reduce channel conductance, the pore appears to be narrower than the one formed by Staphylococcus aureus alpha-hemolysin. Numerical integration of the PEG sample mass spectra and the channel conductance data were used to refine the estimate of the pore's PEG molecular mass cutoff (approximately 1400 g/mol). The results suggest that the limiting diameter of the PA(63) pore is <2 nm, which is consistent with an all-atom model of the PA(63) channel and previous experiments using large ions.

Single molecule measurements within individual membrane-bound ion channels using a polymer-based bilayer lipid membrane chip.

The measurement of single poly(ethylene glycol) (PEG) molecules interacting with individual bilayer lipid membrane-bound ion channels is presented. Measurements were performed within a polymer microfluidic system including an open-well bilayer lipid membrane formation site, integrated Ag/AgCl reference electrodes for on-chip electrical measurements, and multiple microchannels for independent ion channel and analyte delivery. Details of chip fabrication, bilayer membrane formation, and alpha-hemolysin ion channel incorporation are discussed, and measurements of interactions between the membrane-bound ion channels and single PEG molecules are presented.

Miniaturized glucose biosensor modified with a nitric oxide-releasing xerogel microarray.

An enzyme-based glucose biosensor modified to release nitric oxide (NO) via a xerogel microarray is reported. The biosensor design is as follows: (1) glucose oxidase (GOx) is immobilized in a methyltrimethoxysilane (MTMOS) xerogel layer; (2) a blended polyurethane/hydrophilic polyurethane coating prevents enzyme leaching and imparts selectivity for glucose; and (3) micropatterned xerogel lines (5 microm wide) separated by distances of 5 or 20 microm provide NO-release capability. This configuration allows for increased glucose sensitivity relative to sensors modified with NO-releasing xerogel films since significant portions of the sensor surface remain unmodified. Glucose diffusion to the GOx layer is thus less inhibited. The micropatterned NO-releasing biosensors generate sufficient NO levels to reduce both Pseudomonas aeruginosa and platelet adhesion without significantly compromising the enzymatic activity of GOx. The glucose response, linearity and stability of the NO-releasing micropatterned sensors are reported.

Inhibition of implant-associated infections via nitric oxide release.

The in vivo antibacterial activity of nitric oxide (NO)-releasing xerogel coatings was evaluated against an aggressive subcutaneous Staphylococcus aureus infection in a rat model. The NO-releasing implants were created by coating a medical-grade silicone elastomer with a sol-gel-derived (xerogel) film capable of storing NO. Four of the bare or xerogel-coated silicone materials were subcutaneously implanted into male rats. Ten rats were administered 10 microl of a 10(8) cfuml(-1)S. aureus colony directly into the subcutaneous pocket with the implant prior to wound closure. Infection was quantitatively and qualitatively evaluated after 8d of implantation with microbiological and histological methods, respectively. A 82% reduction in the number of infected implants was achieved with the NO-releasing coating. Histology revealed that the capsule formation around infected bare silicone rubber controls was immunoactive and that a biofilm may have formed. Capsule formation in response to NO-releasing implants had greater vascularity in comparison with uninoculated or untreated controls. These results suggest that NO-releasing coatings may dramatically reduce the incidence of biomaterial-associated infection.

In vitro cytotoxicity of nitric oxide-releasing sol-gel derived materials.

The cytotoxicity of bare and PU-coated nitric oxide (NO)-releasing sol-gel derived materials (sol-gels) was investigated using L929 mouse fibroblasts in both direct and indirect contact models to differentiate between the biological impact of the sol-gel matrix and NO release. The flux of NO was varied up to 150 pmol cm(-2) s(-1) using N-(6-aminohexyl)-aminopropyltrimethoxysilane (balance iso-butyltrimethoxysilane) diazeniumdiolate (NO donor)-modified sol-gels. The addition of a polyurethane (PU) outer membrane greatly improved the stability of the sol-gel matrix without significantly suppressing the NO flux. Direct contact studies demonstrated a cytotoxic effect that was dependent on the aminosilane content of the sol-gel. The use of the thin PU overcoat eliminated this effect. A direct cytotoxicity dependence of NO release for L929 fibroblasts was discovered from indirect contact studies, where 24 h exposure to NO fluxes in excess of 50 pmol cm(-2) s(-1) was cytotoxic.

Nitric oxide-releasing sol-gels as antibacterial coatings for orthopedic implants.

To assess the benefits of nitric oxide (NO)-releasing sol-gels as potential antibacterial coatings for orthopedic devices, medical-grade stainless steel is coated with a sol-gel film of 40% N-aminohexyl-N-aminopropyltrimethoxysilane and 60% isobutyltrimethoxysilane. Upon converting the diamine groups in these films to diazeniumdiolate NO donors, the NO release from the sol-gel-coated stainless steel is evaluated at both ambient and physiological temperature. Sol-gel films incubated at 25 degrees C have a lower NO flux over the first 24 h compared to those at 37 degrees C, but release more than five times longer. The bacterial adhesion resistance of NO-releasing coatings is evaluated in vitro by exposing bare steel, sol-gel, and NO-releasing sol-gel-coated steel to cell suspensions of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis at 25 degrees C and 37 degrees C. Cell adhesion to bare and sol-gel-coated steel is similar, while NO-releasing surfaces have significantly less bacterial adhesion for all species and temperatures investigated.

Poly(vinyl chloride)-coated sol-gels for studying the effects of nitric oxide release on bacterial adhesion.

Nitric oxide (NO) releasing sol-gel materials coated with poly(vinyl chloride) (PVC) films exhibit increased stability at ambient and physiological temperatures. The polymer overcoat, however, reduces the NO fluxes by 5-35% over the initial week of release. The variation in NO fluxes between unmodified and PVC-coated sol-gels is negligible after 7 days. The PVC polymeric layer provides controlled surface chemistry for systematic studies of the effects of NO release on bacterial adhesion. As an example, the adhesion of Pseudomonas aeruginosa and Proteus mirabilis at PVC-coated NO-releasing sol-gels is investigated. A direct NO dependence on the reduction of P. aeruginosa adhesion is observed for NO fluxes up to 20 pmol cm(-2) s(-1). Although decreased by 50% in the presence of NO release, P. mirabilis adhesion does not appear to correlate to the flux of NO release. PVC-coated NO-releasing sol-gels may prove useful for studying the effects of localized NO release on other biological and chemical systems.

Antibacterial properties of nitric oxide-releasing sol-gels.

The antibacterial characteristics of nitric oxide (NO)-releasing sol-gel coatings are described. The NO release from these surfaces is steady over short periods (approximately 1 h) and measurable over several days. The ability of NO to prevent bacterial adhesion is evaluated by exposing controls and NO-releasing sol-gels to approximately 10(8) colony-forming units (cfu)/mL saline suspensions of Pseudomonas aeruginosa. Pseudomonas aeruginosa adhesion to sol-gel controls varies depending on the sol-gel formulation. Sol-gel surfaces capable of NO release decrease bacterial adhesion by 30% to 95% relative to controls. The contact angle measurements of control and NO-releasing surfaces are similar, supporting NO's action as an antibacterial agent against bacterial adhesion.