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.

Pt-Decorated MWCNTs-Ionic Liquid Composite-Based Hydrogen Peroxide Sensor To Study Microbial Metabolism Using Scanning Electrochemical Microscopy.

Hydrogen peroxide (H2O2) is a highly relevant metabolite in many biological processes, including the oral microbiome. To study this metabolite, we developed a 25 µm diameter, highly sensitive, nonenzymatic H2O2 sensor with a detection limit of 250 nM and a broad linear range of 250 nM to 7 mM. The sensor used the synergistic activity of the catalytically active Pt nanoparticles on a high surface area multiwalled carbon nanotube and conducting ionic liquid matrix to achieve high sensitivity (2.4 ± 0.24 mA cm-2 mM-1) for H2O2 oxidation. The unique composite allowed us to miniaturize the sensor and couple it with a Pt electrode (25 µm diameter each) for use as a dual scanning electrochemical microscopy probe. We could detect 65 ± 10 µM H2O2 produced by Streptococcus gordonii (Sg) in a simulated biofilm at 50 µm above its surface in the presence of 1 mM glucose and artificial saliva solution (pH 7.2 at 37 °C). Because of its high stability and low detection limit, the sensor showed a promising chemical image of H2O2 produced by Sg biofilms. We were also able to detect 30 µM H2O2 at 50 µm above the biofilm in the presence of the H2O2-decomposing salivary lactoperoxidase and thiocyanate, which would not otherwise be possible using an existing H2O2 assay. Thus, this sensor can potentially find applications in the study of other important biological processes in a complex matrix where circumstances demand a low detection limit in a compact space.

Carbon-Based Solid-State Calcium Ion-Selective Microelectrode and Scanning Electrochemical Microscopy: A Quantitative Study of pH-Dependent Release of Calcium Ions from Bioactive Glass.

Solid-state ion-selective electrodes are used as scanning electrochemical microscope (SECM) probes because of their inherent fast response time and ease of miniaturization. In this study, we report the development of a solid-state, low-poly(vinyl chloride), carbon-based calcium ion-selective microelectrode (Ca(2+)-ISME), 25 µm in diameter, capable of performing an amperometric approach curve and serving as a potentiometric sensor. The Ca(2+)-ISME has a broad linear response range of 5 µM to 200 mM with a near Nernstian slope of 28 mV/log[a(Ca(2+))]. The calculated detection limit for Ca(2+)-ISME is 1 µM. The selectivity coefficients of this Ca(2+)-ISME are log K(Ca(2+),A) = -5.88, -5.54, and -6.31 for Mg(2+), Na(+), and K(+), respectively. We used this new type of Ca(2+)-ISME as an SECM probe to quantitatively map the chemical microenvironment produced by a model substrate, bioactive glass (BAG). In acidic conditions (pH 4.5), BAG was found to increase the calcium ion concentration from 0.7 mM ([Ca(2+)] in artificial saliva) to 1.4 mM at 20 µm above the surface. In addition, a solid-state dual SECM pH probe was used to correlate the release of calcium ions with the change in local pH. Three-dimensional pH and calcium ion distribution mapping were also obtained by using these solid-state probes. The quantitative mapping of pH and Ca(2+) above the BAG elucidates the effectiveness of BAG in neutralizing and releasing calcium ions in acidic conditions.

Single-Cell Migration as Studied by Scanning Electrochemical Microscopy.

Scanning electrochemical microscopy (SECM) was used to study the migration of single live head and neck cancer cells (SCC25). The newly developed graphite paste ultramicroelectrode (UME) showed significantly less fouling in comparison to a 10 µm Pt-UME and thus could be used to monitor and track the migration pattern of a single cell. We also used SECM probe scan curves to measure the morphology (height and diameter) of a single live cancer cell during cellular migration and determined these dimensions to be 11 ± 4 µm and 40 ± 10 µm, respectively. The migration study revealed that cells within the same cell line had a heterogeneous migration pattern (migration and stationary) with an estimated migration speed of 8 ± 3 µm/h. However, serum-starved synchronized cells of the same line were found to have a non-heterogeneous cellular migration pattern with a speed of 9 ± 3 µm/h. Thus, this non-invasive SECM-based technique could potentially be expanded to other cell lines to study cellular biomechanics for improved understanding of the structure-function relationship at the level of a single cell.

Electrocatalyst on insulating support?: Hollow silica spheres loaded with Pt nanoparticles for methanol oxidation.

Electrocatalytic oxidation of methanol on silica hollow spheres, loaded with platinum nanoparticles (Pt-SiO2-HS), is reported. The functionalized hollow silica spheres were prepared by the surfactant (lauryl ester of tyrosine) template-assisted synthesis. These spheres were loaded with platinum nanoparticles by γ-radiolysis. Energy-dispersive X-ray analysis (EDAX) and X-ray photoelectron spectroscopy (XPS) analyses confirmed presence of Si and Pt in the composite. High-resolution transmission electron microscopy showed the formation of uniformly deposited Pt nanoparticles over the hollow spheres with a predominant Pt(111) lattice plane on the surface. In spite of the poor conducting nature of the silica support, the oxidation potential and current density per unit mass for methanol oxidation were noted to be ca. 0.72 V vs NHE and 270 mA mg(-1), respectively, which are among the best values reported in its class. The composite did not show any sign of a degradation even after repeated use. In fact, the anodic current was found to increase under constant polarization, which is attributed to a facile reaction between adsorbed CO with a surface hydroxyl group present on the silica support. These results are in favor of Pt-SiO2-HS as a promising electrocatalyst material in the direct methanol fuel cell (DMFC) applications.