Planar carbon electrodes for real-time quantification of hydrogen sulfide release from cells.

A planar electrode system was developed to permit the real-time, selective detection of hydrogen sulfide (H2S) from stimulated cells. Planar carbon electrodes were produced via stencil printing carbon ink through a laser cut vinyl mask. Electrodes were preconditioned using a constant potential amperometry methodology to prevent sensor drift resulting from elemental sulfur adsorption. Modification with a bilaminar coating (electropolymerized ortho-phenylenediamine and a fluorinated xerogel) facilitated high selectivity to H2S. To demonstrate the biological application of this planar sensor system, H2S released from 17ß-estradiol-stimulated human umbilical vein endothelial cells (HUVECs) was quantified in situ in real-time. Stimulated HUVECs released sustained H2S levels for hours before returning to baseline. Cellular viability assays demonstrated negligible cell cytotoxicity at the electrochemical potentials required for analysis.

Endophyte-Promoted Phosphorus Solubilization in Populus.

Phosphorus is one of the essential nutrients for plant growth, but it may be relatively unavailable to plants because of its chemistry. In soil, the majority of phosphorus is present in the form of a phosphate, usually as metal complexes making it bound to minerals or organic matter. Therefore, inorganic phosphate solubilization is an important process of plant growth promotion by plant associated bacteria and fungi. Non-nodulating plant species have been shown to thrive in low-nutrient environments, in some instances by relying on plant associated microorganisms called endophytes. These microorganisms live within the plant and help supply nutrients for the plant. Despite their potential enormous environmental importance, there are a limited number of studies looking at the direct molecular impact of phosphate solubilizing endophytic bacteria on the host plant. In this work, we studied the impact of two endophyte strains of wild poplar (Populus trichocarpa) that solubilize phosphate. Using a combination of x-ray imaging, spectroscopy methods, and proteomics, we report direct evidence of endophyte-promoted phosphorus uptake in poplar. We found that the solubilized phosphate may react and become insoluble once inside plant tissue, suggesting that endophytes may aid in the re-release of phosphate. Using synchrotron x-ray fluorescence spectromicroscopy, we visualized the nutrient phosphorus inside poplar roots inoculated by the selected endophytes and found the phosphorus in both forms of organic and inorganic phosphates inside the root. Tomography-based root imaging revealed a markedly different root biomass and root architecture for poplar samples inoculated with the phosphate solubilizing bacteria strains. Proteomics characterization on poplar roots coupled with protein network analysis revealed novel proteins and metabolic pathways with possible involvement in endophyte enriched phosphorus uptake. These findings suggest an important role of endophytes for phosphorus acquisition and provide a deeper understanding of the critical symbiotic associations between poplar and the endophytic bacteria.

Mode of nitric oxide delivery affects antibacterial action.

Nitric oxide (NO) is a broad-spectrum antibacterial agent, making it an attractive alternative to traditional antibiotics for treating infections. To date, a direct comparison of the antibacterial activity of gaseous NO (gNO) versus water-soluble NO-releasing biopolymers has not been reported. In this study, the bactericidal action of NO-releasing chitosan oligosaccharides was compared to gNO treatment against cystic fibrosis-relevant Gram-positive and Gram-negative bacteria. A NO exposure chamber was constructed to enable the dosing of bacteria with gNO at concentrations up to 800 ppm under both aerobic and anaerobic conditions. Bacteria viability, solution properties (i.e., pH, NO concentration), and toxicity to mammalian cells were monitored to ensure a thorough understanding of bactericidal action and reproducibility for each delivery method. The NO-releasing chitosan oligosaccharides required significantly lower NO doses relative to gNO therapy to elicit antibacterial action against Pseudomonas aeruginosa and Staphylococcus aureus under both aerobic and anaerobic conditions. Reduced NO doses required for bacteria eradication using water-soluble NO-releasing chitosan were attributed to the release of NO in solution, removing the need to transfer from gas to liquid phase and the associated long diffusion distances of gNO treatment.

Nitric oxide diffusion through cystic fibrosis-relevant media and lung tissue.

A simplified diffusion cell methodology was employed to measure the diffusion coefficient of nitric oxide (NO) through phosphate buffered saline (PBS) and artificial sputum medium (ASM)-an in vitro analog for airway mucus. Diffusion through the proteinaceous ASM yielded a significantly lower diffusion coefficient compared to PBS, which is attributed to both the physical obstruction by the mucin mesh and reactive nature of NO radicals towards the biological compounds in ASM. To further confirm that ASM was restricting NO from diffusing freely, a macromolecular propylamine-modified cyclodextrin donor (CD-PA) was employed to release the NO more slowly. The NO diffusion characteristics in ASM via the NO donor were also slower relative to PBS. As NO is likely to interact with lung cells after passing through the mucus barrier, the diffusion of both NO and the CD-PA macromolecular NO donor through differentiated lung tissue was investigated with and without an ASM layer. Comparison of NO diffusion through the three diffusion barriers indicated that the lung tissue significantly impeded NO penetration over the course of the experiment compared to PBS and ASM. In fact, the diffusion of CD-PA through the lung tissue was hindered until after the release of its NO payload, potentially due to the increased net charge of the NO donor structure. Of importance, the viability of the tissue was not influenced by the NO-releasing CD-PA at bactericidal concentrations.

A direct and selective electrochemical hydrogen sulfide sensor.

Continuous, in situ detection of hydrogen sulfide (H2S) in biological milieu is made possible with electrochemical methods, but direct amperometry is constrained by the generation of elemental sulfur as an oxidative byproduct. Deposition of a sulfur layer passivates the working electrode, reducing sensitivity and causing performance variability. Herein, we report on the use of a surface preconditioning procedure to deposit elemental sulfur on a glassy carbon electrode prior to measurement and evaluate performance with common analytical metrics. The lack of traditional anti-poisoning techniques (e.g. redox mediators, cleaning pulses) also allowed for facile surface modification with electropolymerized films. For the first time, a series of electropolymerized films were characterized for their H2S permselective behavior against common biological interferents. Highly selective, film-modified electrodes were then evaluated for their anti-biofouling ability in simulated wound fluid. The final optimized electrode was capable of measuring H2S with a low detection limit (i.e., <100 nM) and ∼80% of its initial sensitivity in proteinaceous media.

Direct Electrochemical Sensing of Hydrogen Sulfide without Sulfur Poisoning.

An electrochemical method capable of direct, real-time detection of hydrogen sulfide was developed using triple pulse amperometry (TPA) to mitigate sulfur poisoning and its related passivation of the working electrode surface. Through repeated cycles of discrete potential pulses, the electrooxidation of surface-adsorbed elemental sulfur to water-soluble sulfate ions was exploited to regenerate the glassy carbon electrode surface and maintain consistent sensor performance. Amperometric measurements and X-ray photoelectron spectroscopy surface analysis demonstrated that the TPA sensors provided enhanced analytical performance via decreased sulfur accumulation relative to low-potential (≤+0.7 V) constant potential amperometry. Sensors operated under optimized TPA parameters retained high sensitivity (57.4 ± 13.0 nA/µM), a wide linear dynamic range (150 nM-15 µM), fast response times (<10 s), and a submicromolar detection limit (<100 nM) upon consecutive calibration cycles. The sensitivity and response time achieved were comparable to or better than current electrochemical sensors. Moreover, the simplicity of the method eliminates the need for external redox mediators or semipermeable membranes.

Structure-function relationships affecting the sensing mechanism of monolayer-protected cluster doped xerogel amperometric glucose biosensors.

A systematic study of the structure-function relationships critical to understanding the sensing mechanism of 1st generation amperometric glucose biosensors with an embedded nanoparticle (NP) network is presented. Xerogel-based films featuring embedded glucose oxidase enzyme and doped with alkanethiolate-protected gold NPs, known as monolayer protected clusters (MPCs), exhibit significantly enhanced performance compared to analogous systems without NPs including higher sensitivity, faster response time, and extended linear/dynamic ranges. The proposed mechanism involves diffusion of the glucose to glucose oxidase within the xerogel, enzymatic reaction production of H2O2 with subsequent diffusion to the embedded network of MPCs where it is oxidized, an event immediately reported via fast electron transfer (ET) through the MPC system to the working electrode. Various aspects of the film construct and strategy are systematically probed using amperometry, voltammetry, and solid-state electronic conductivity measurements, including the effects of MPC peripheral chain length, MPC functionalization via place-exchange reaction, MPC core size, and the MPC density or concentration within the xerogel composite films. The collective results of these experiments support the proposed mechanism and identify interparticle spacing and the electronic communication through the MPC network is the most significant factor in the sensing scheme with the diffusional aspects of the mechanism that may be affected by film/MPC hydrophobicity and functionality (i.e., glucose and H2O2 diffusion) shown to be less substantial contributors to the overall enhanced performance. Understanding the structure-function relationships of effective sensing schemes allows for the employment of the strategy for future biosensor design toward clinically relevant targets.

Functional layer-by-layer design of xerogel-based first-generation amperometric glucose biosensors.

Xerogel-based first-generation amperometric glucose biosensors, constructed through specific layer-by-layer assembly of films featuring glucose oxidase doped xerogel, a diffusion-limiting xerogel layer, and capped with both electropolymerized polyphenol and blended polyurethane semipermeable membranes, are presented. The specific combination of xerogels formed from specific silane precursors, including propyl-trimethoxysilane, isobutyl-trimethoxysilane, octyl-trimethoxysilane, and hydroxymethyl-triethoxysilane, exhibit impressive dynamic and linear ranges of detection (e.g., ≥24-28 mM glucose) and low response times, as well as significant discrimination against common interferent species such as acetaminophen, ascorbic acid, sodium nitrite, oxalic acid, and uric acid as determined by selectivity coefficients. Additionally, systematic electrochemical and contact angle studies of different xerogel silane precursors, varying in structure, chain length, and/or functional group, reveal that sensor performance is more dependent on the tunable porosity/permeability of the layered interfaces rather than the hydrophobic character or functional groups within the films. While the sensing performance largely exceeds that of existing electrochemical glucose sensing schemes in the literature, the presented layered approach establishes the specific functionality of each layer working in concert with each other and suggests that the strategy may be readily adaptable to other clinically relevant targets and is amenable to miniaturization for eventual in situ or in vivo sensing.

Monolayer-protected nanoparticle doped xerogels as functional components of amperometric glucose biosensors.

First-generation amperometric glucose biosensors incorporating alkanethiolate-protected gold nanoparticles, monolayer protected clusters (MPCs), within a xerogel matrix are investigated as model systems for nanomaterial-assisted electrochemical sensing strategies. The xerogel biosensors are comprised of platinum electrodes modified with composite films of (3-mercaptopropyl)trimethoxy silane xerogel embedded with glucose oxidase enzyme, doped with Au225(C6)75 MPCs, and coated with an outer polyurethane layer. Electrochemistry and scanning/transmission electron microscopy, including cross-sectional TEM, show sensor construction, humidity effects on xerogel structure, and successful incorporation of MPCs. Analytical performance of the biosensor scheme with and without MPC doping of the xerogel is determined from direct glucose injection during amperometry. MPC-doped xerogels yield significant enhancement of several sensor attributes compared to analogous films without nanoparticles: doubling of the linear range, sensitivity enhancement by an order of magnitude, and 4-fold faster response times accompany long-term stability and resistance to common interfering agents that are competitive with current glucose biosensing literature. Ligand chain length and the MPC/silane ratio studies suggest the MPC-induced enhancements are critically related to structure-function relationships, particularly those affecting interparticle electronic communication where the MPC network behaves as a three-dimensional extension of the working electrode into the xerogel film, reducing the system's dependence on diffusion and maximizing efficiency of the sensing mechanism. The integration of MPCs as a functional component of amperometric biosensor schemes has implications for future development of biosensors targeting clinically relevant species.