Mechanistic analysis of the photolytic decomposition of solid-state S-nitroso-N-acetylpenicillamine.

S-Nitroso-N-acetylpenicillamine (SNAP) is among the most common nitric oxide (NO)-donor molecules and its solid-state photolytic decomposition has potential for inhaled nitric oxide (iNO) therapy. The photochemical NO release kinetics and mechanism were investigated by exposing solid-state SNAP to a narrow-band LED as a function of nominal wavelength and intensity of incident light. The photolytic efficiency, decomposition products, and the photolytic pathways of the SNAP were examined. The maximum light penetration depth through the solid layer of SNAP was determined by an optical microscope and found to be within 100-200 µm, depending on the wavelength of light. The photolysis of solid-state SNAP to generate NO along with the stable thiyl (RS·) radical was confirmed using Electron Spin Resonance (ESR) spectroscopy. The fate of the RS· radical in the solid phase was studied both in the presence and absence of O2 using NMR, IR, ESR, and UPLC-MS. The changes in the morphology of SNAP due to its photolysis were examined using PXRD and SEM. The stable thiyl radical formed from the photolysis of solid SNAP was found to be reactive with another adjacent thiyl radical to form a disulfide (RSSR) or with oxygen to form various sulfonyl and sulfonyl peroxyl radicals {RS(O)xO·, x = 0 to 7}. However, the thiyl radical did not recombine with NO to reform the SNAP. From the PXRD data, it was found that the SNAP loses its crystallinity by generating the NO after photolysis. The initial release of NO during photolysis was increased with increased intensity of light, whereas the maximum light penetration depth was unaffected by light intensity. The knowledge gained about the photochemical reactions of SNAP may provide important insight in designing portable photoinduced NO-releasing devices for iNO therapy.

Real-Time Metabolic Interactions between Two Bacterial Species Using a Carbon-Based pH Microsensor as a Scanning Electrochemical Microscopy Probe.

We have developed a carbon-based, fast-response potentiometric pH microsensor for use as a scanning electrochemical microscopy (SECM) chemical probe to quantitatively map the microbial metabolic exchange between two bacterial species, commensal Streptococcus gordonii and pathogenic Streptococcus mutans. The 25 µm diameter H+ ion-selective microelectrode or pH microprobe showed a Nernstian slope of 59 mV/pH and high selectivity against major ions such Na+, K+, Ca2+, and Mg2+. In addition, the unique conductive membrane composition aided us in performing an amperometric approach curve to position the probe and obtain a high-resolution pH map of the microenvironment produced by the lactate-producing S. mutans biofilm. The x-directional pH scan over S. mutans also showed the influence of the pH profile on the metabolic activity of another species, H2O2-producing S. gordonii. When these bacterial species were placed in close spatial proximity, we observed an initial increase in the local H2O2 concentration of approximately 12 ± 5 µM above S. gordonii, followed by a gradual decrease in H2O2 concentration (>30 min) to almost zero as lactate was produced, and a subsequent decrease in pH with a more pronounced metabolic output of S. mutans. These results were supported by gene expression and confocal fluorescence microscopic studies. Our findings illustrate that H2O2-producing S. gordonii is dominant while the buffering capacity of saliva is valid (∼pH 6.0) but is gradually taken over by S. mutans as the latter species slowly starts decreasing the local pH to 5.0 or less by producing lactic acid. Our observations demonstrate the unique capability of our SECM chemical probes for studying real-time metabolic interactions between two bacterial species, which would not otherwise be achievable in traditional assays.

Dendritic Hydrogel Bioink for 3D Printing of Bacterial Microhabitat.

A glucose-modified dendritic hydrogel is used as a bioink for bacterial encapsulation. This biocompatible hydrogel is a potentially suitable alternative to conventional alginate hydrogel for bacterial encapsulation, as it readily forms gel in the presence of Na+ or K+ ions without any additional stimuli such as pH, temperature, sonication, or the presence of divalent metal ions. We created a bacterial microhabitat by adding the gelator to phosphate-buffered saline containing live bacteria at physiological pH and using an additive three-dimensional (3D) printing technique. The bacteria remained viable and metabolically active within the 3D printed bacterial microhabitat, as shown with confocal laser scanning microscopy (CLSM) and scanning electrochemical microscopy (SECM).