The general theme of this dissertation is the application of novel carbon based micro sensors with the scanning electrochemical microscopy (SECM). SECM allows positioning of the probe without touching the substrate while mapping the chemical parameters in 3D space above the substrate. The main focus of the dissertation is the development of ion selective microelectrodes (ISME) to be used with SECM. Such ion selective microelectrodes are very challenging to manufacture, considering the high amount of PVC present in the ion selective membranes. Miniaturizing the membranes will only increase the resistance and make the voltage measurement across such membranes difficult. This problem was overcome by replacing a large amount of the PVC present in the membrane with the carbon content. The increased carbon content decreased the resistance of the membrane resulting in a fast response time of 0.2 s. Such a fast response is required for SECM where the tip is moving at a particular speed while measuring the analyte of interest. Finally the carbon paste based ISMEs were used as amperometric (current vs time) sensors to perform approach curve with the SECM to fix the probe-substrate distance. The carbon paste based ISMEs could be used with SECM to detect biologically important molecules like Ca2+ and for pH measurement locally. Both Ca2+-ISME and pH-ISME were used to study the local chemical effects caused by a novel dental material called as BAG. The Ca2+-ISME was also used for studying the calcification process occurring at the surface of the Sporosarcina pasteurii biofilm.
Sporosarcina pasteurii is known to produce calcite or biocement in the presence of urea and Ca2+. The Ca2+-ISME was used along with a SECM, to monitor a real-time, bacteria-mediated urea hydrolysis process. A fast depletion (10 min) of Ca2+ was observed from 85 mM to 10 mM the presence of a high urea (10 g/L) brine solution at 23°C. The Ca2+ concentration profiles were extended up to 600 μm from the biofilm surface, whereas the bulk chemical composition of the brine solution remained constant over the entire 4 h of SECM experiments. In addition, we observed a change in biofilm surface morphology and an increase in overall biofilm height of 50 µm after 4 h of precipitation. Electron microscopy confirmed the changes in surface morphology and formation of CaCO3 crystals. Development of the Ca2+ profile took 10 minutes, whereas that of the pH profile took 2 minutes. This finding indicates that the initial urea hydrolysis process is fast and limited by urease or number of bacteria, whereas later CaCO3 formation and growth of crystals is a slow chemical process. The ultramicrosensors and approaches employed here are capable of accurately characterizing bioremediation on temporal and spatial scales pertinent to the microbial communities and the processes they mediate.
The Ca2+-ISME was also used as an SECM probe to quantitatively map the chemical microenvironment produced by, bioactive glass (BAG) which is aimed at remineralizing the teeth by releasing Ca2+ ions. In acidic conditions (pH 4.5), BAG was found to increase the calcium ion concentration from 0.7 mM ([Ca2+] in artificial saliva) to 1.4 mM at 20 µm above the surface. In addition, a solid-state dual-tip 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 response time of 0.2 s was achieved by carbon paste based ISME. This allowed the ISME to perform real time measurement of the analytes, when the probe moves at a particular speed. The quantitative mapping of pH and Ca2+ above the BAG elucidates the effectiveness of BAG in neutralizing and releasing calcium ions in acidic conditions.
The measurements made on pure BAG were important to determine the chemical effectiveness of this smart dental filling material. But the pure BAG is fragile and cannot be used by itself as a dental filling material. The BAG particles need to be incorporated into the conventional resin based dental composites to get both mechanical strength and chemical microenvironment similar to pure BAG. The chemical microenvironment surrounding dental composites plays a crucial role in controlling bacteria grown on these specialized surfaces. To optimize the BAG particle distribution in the dental composites, SECM and solid-state H+ and Ca2+ ion-selective microelectrodes were used. The local Ca2+ concentration released by different composites ranged from 10-224 µM for a BAG particle size of <5 µm to 150 µm respectively in the presence of artificial saliva at pH 4.5. The local pH was constant above the composites in the same saliva solution. The released amount of Ca2+ was determined to be maximal for particles of less than 30 µm and a volume fraction of BAG to resin of 0.32. This optimized BAG-resin composite also showed significant inhibition of biofilm growth (24 µm ± 5 µm) in comparison to resin-only composites (53 µm ± 6 µm) after Streptococcus mutans bacteria were grown for 3 days in basal medium mucin solution. Biofilm morphology and its subsequent volume, as determined by the SECM imaging technique, was (0.59 ± 0.38) x 107 µm3 for BAG-resin composites and (1.29 ± 0.53) x 107 µm3 for resin-only composites. This study laid the foundation for a new analytical technique for designing dental composites that is based on the chemical microenvironment created by biomaterials to which bacteria have been exposed.
Overall, the newly developed carbon paste based Ion-Selective Microelectrodes (ISMEs) have very fast response time and an amperometric function which allowed to perform approach curves to fix the probe distance very close to the substrates. Both Ca2+- ISME and pH-ISME were used to characterize not only the pure BAG but also the dental composites incorporated with BAG particles. The Ca2+-ISME was also used to monitor a biological process with Sporosarcina pasteurii biofilm. The process of designing the Ca2+- ISME and pH-ISMEs was also used to make Na+ and K+ ISMEs to prove that the technique can be extended towards other analytes of interest and applied to study biomaterials and biological systems of relevance.
In another study, cancer cell migration at a single cell level was studied by SECM with live head and neck cancer cells (SCC25). The newly developed graphite paste ultra-microelectrode (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. I 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.
Ummadi, Jyothir Ganeshwar Reddy, 2018, “Development of Carbon-based Ion Selective Microsensors as Scanning Electrochemical Microscopy (SECM) Probes”, Doctor of Philosophy, Oregon State University, Corvallis.