Browsing by Subject "Electroanalytical techniques"
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Item Applications of scanning electrochemical microscopy in biological systems(2011-08) Koley, Dipankar; Bard, Allen J.; Crooks, Richard M.; Stevenson, Keith J.; Mullins, Charles B.; Humphrey, Simon M.The main theme in this dissertation is to develop Scanning Electrochemical Microscopy (SECM) based electroanalytical techniques to study quantitative biology in real time. The multidrug resistance (MDR) phenomenon in live cancer cells was studied using mimic drug molecules such as menadione with the aid of SECM. Real time quantitative detection of thiodione (menadione-conjugate) pumped out of the cells was determined to be 140 μM due to exposure of 500 μM menadione to the cells. Selective blocking of these MDR pumps in live intact cells was also achieved by small molecules such as MK571 as well as by the MDR specific antibody. An approximately 50% drop in thiodione flux was observed in both cases of MDR pumps inhibition. This SECM technique was also extended to measure the permeability of a highly charged hydrophilic molecule passing through the membrane of a single living cell. The permeability was measured to be 6.5 ± 2.0 × 10-6 m/s. Real time monitoring of morphological changes in a live HeLa cell due to addition of varying concentration of surfactant such as Triton X-100 was also demonstrated by SECM. This electroanalytical technique was also expanded to study quantitative microbiology. Real time quantitative detection of pyocyanin produced by Pseudomonas aeruginosa (PA14 strain) biofilm locally was determined to be 2.5 μM after 6 h. Pyocyanin (PYO) was also observed to be reduced by PA14 biofilm, thus maintaining a reduced atmosphere above the biofilm even in presence of oxygen. Spatial mapping of this reduced PYO showed that this reduced zone was only formed up to 500 μm above the biofilm. The cells are also able to modulate the height of the reduced PYO zone in accordance to the availability of Fe(III/II) in the solution to scavenge iron from the surrounding environment. Real time spatial mapping hydrogen peroxide across polymicrobial biofilm (Sg and Aa) was also achieved with the aid of SECM. The local peroxide concentration produced by Sg was measured to be 1 mM, which is significantly higher than the bulk peroxide concentration. This study also showed that the local concentration across the microbial film is more important than the bulk concentration since bacteria communicate locally in real world.Item The use of scanning electrochemical microscopy for the detection and quantification of adsorbed intermediates at electrodes(2010-08) Rodriguez Lopez, Joaquin, 1983-; Bard, Allen J.; Crooks, Richard M.; Stevenson, Keith J.; Mullins, C. B.; Henkelman, GraemeScanning electrochemical microscopy (SECM) was used for the study and characterization of catalytic and electrocatalytic processes occurring at electrodes. The Surface Interrogation mode (SI-SECM) was introduced for the detection and quantification of adsorbed intermediates and products of catalyzed chemical and electrochemical reactions at noble metals (Pt, Au). In SI-SECM two micro electrodes (i.e. an SECM tip and a substrate of the desired material) are aligned concentrically at a micrometric distance where SECM feedback effects operate. A contrast mechanism based on feedback effects allows for the detection of reactive adsorbed intermediates at the substrate: the SECM tip generates a reactive homogeneous species that “micro-titrates” the substrate adsorbates to yield an electrochemical signal that contains information about the amount of intermediate and about its kinetics of reaction with the redox mediator. The technique was used for the study of the reactivity of three model small adsorbates: 1) the reactivity of adsorbed oxygen on Au and Pt with a reducing mediator was explored and suggested the detection of “incipient oxides” at these surfaces; kinetic parameters of the reactivity of Pt oxides with mediators were obtained, fit to theory and used to explain observations about the electrocatalytic behavior of Pt under anodizing conditions; 2) the reactivity of oxidizing mediators with adsorbed hydrogen on Pt was studied and showed the cation of N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) to be a successful interrogation agent, the detection of hydrogen generated by the decomposition of formic acid on Pt at open circuit was investigated; 3) electrogenerated bromine was used to catalytically interrogate carbon monoxide at Pt, this reaction was previously unreported. The mentioned applications of SECM were validated through the use of digital simulations of diffusion in the complex SECM geometry through flexible commercial finite element method software.