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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. / text

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Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Allen J. Bard. Vita. Includes bibliographical references.

This thesis focuses on in-situ studies at the solid-liquid interface by combining scanning electrochemical microscopy (SECM) with gold and platinum mercury amalgam microelectrodes. It is shown that stripping voltammetry experiments at imaging amalgam microelectrodes provide laterally resolved insight on the electrochemistry of biogeochemically relevant processes. SECM provides information on electroactive surface processes with high spatial resolution, and offers the opportunity to study heterogeneous electron-transfer reactions. Thereby, chemical species of interest, such as metal ions, can be electrochemically detected at mercury amalgam electrodes. Platinum and gold mercury amalgam microelectrodes were developed for the detection of biogeochemically relevant analytes such as manganese and iron during SECM imaging experiments at the mineral/water interface establishing the fundamental basis of SECM imaging with amalgam microelectrodes. SECM experiments were performed for the quantitative determination of Mn2+ during the dissolution of microstructured manganese carbonate (rhodochrosite) precipitates at mildly acidic conditions. SECM images along with spatially resolved quantitative data on the Mn2+ concentration were obtained. This measurement concept was then extended to the investigation of the corrosion behavior of diamond-like carbon (DLC) protected zinc selenide (ZnSe) waveguides applied in mid-infrared attenuated total reflectance spectroscopy at strongly oxidizing conditions. The corrosion behavior of DLC coated and uncoated ZnSe crystals was studied obtaining laterally resolved information on the oxidative degradation of ZnSe at defects of the DLC layer utilizing SECM in combination with square wave anodic stripping voltammetry (SWASV) at gold amalgam microelectrodes. Thereby, insight on the corrosion behavior of ZnSe and concentration profiles of Zn2+ at oxidizing conditions was obtained. These results corroborate the utility of SECM imaging with amalgam microelectrodes for addressing relevant analytical questions. Finally, the developed amalgam microelectrodes were applied for SECM studies of iron-reducing proteins separated from Shewanella microbes in native polyacrylamide gels. After calibration of Pt/Hg microelectrodes in bulk solution for the targeted analytes (iron and sulfur species), SECM approach curves recorded above the native gel enabled positioning of the amalgam electrode in close proximity above protein bands with suspected iron-reducing activity. This technique enabled the (semi)quantitative determination of the anaerobic respiratory activity associated with microbial proteins/protein complexes responsible for the reductive dissolution of manganese and iron oxides above microbial protein bands separated in a native gel matrix.

Unstable Sn(III) intermediates generated in the Sn(IV)/Sn(II) redox reaction in 2 M HBr + 4 M NaBr media were detected by scanning electrochemical microscopy (SECM) and cyclic voltammetry (CV). In CV, the underpotential deposition of Sn(0) and its stripping peaks severely perturbed the analysis of diffusional reactions. In SECM, however, the detection of diffusional Sn(III) bromide species was clearly observed due to the absence of the perturbation from the surface reactions. The ECEC-DISP mechanism in both the reduction and oxidation reactions was proposed via Sn(III) bromide intermediates. CVs at different concentrations of Sn(IV) and at various scan rates were fit by numerical simulations based on the proposed mechanism with good agreement. Enhanced electrochemical reversibility in the Sn(IV)/Sn(II) redox reaction was observed at the elevated temperature of 80 °C. We attributed such observation to changes in the rate of bromide loss from Sn(IV)Br₆²⁻ to Sn(IV)Br₅⁻ based on the CV simulation. In a similar approach, a short-lived intermediate, presumably bromine anion radical Br₂⁻·, was detected in the Br⁻ /Br₃⁻ electro-oxidation reaction in nitrobenzene solution by SECM and CV. The reaction mechanism was proposed based on a detected Br₂⁻· intermediate as follows: (1) the one electron transfer of Br⁻ to Br·, (2) the dimerization of 2Br· to Br₂, (3) the bromide addition reaction of Br₂ to Br₃⁻ , (4) the bromide addition reaction of Br· to Br₂⁻·, and (5) the Br· addition reaction of Br₂⁻· to Br₃⁻. The simulation based on the proposed mechanism fitted well with the experimental SECM and CV results. At last, the applicability of the Sn/Br system as electrolyte for electrochemical energy storage was tested. A redox flow battery was constructed, where the Sn(IV)/Sn(II) reduction was carried out on the negative electrode, while the Br· /Br₂ oxidation was carried out on the positive electrode during charging. Cyclability was tested up to 35 charge/discharge cycles, and 100 % coulombic efficiency was observed in all cycles. However, only 40 % of voltage efficiency was obtained, mainly due to the large irreversibility of the Sn(IV)/Sn(II) redox reaction in the bromide media.

Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2006. / Levent Degertekin, Committee Member ; William P. King, Committee Member ; Boris Mizaikoff, Committee Member ; Mark G. Allen, Committee Member ; Peter J. Hesketh, Committee Chair.

A presente tese versa sobre resultados obtidos na aplicação da microscopia eletroquímica de varredura no estudo de sistemas micelares e no estudo de bicamadas lipídicas. Os estudos envolvendo sistemas micelares foram realizados utilizando a SECM no modo substrato-gerador/microeletrodo-coletor. Neste modo de operação, um microeletrodo de platina foi posicionado próximo a um substrato de platina e utilizado para monitorar espécies eletrogeradas nesse substrato. Conhecendo o tempo necessário para a espécie eletrogerada difundir do substrato até o microeletrodo, foi possível aplicar a equação de Einstein-Smoluchowski para determinar o coeficiente de difusão da espécie eletroativa e de micelas de surfactantes. Como as micelas não são eletroativas, o ferroceno eletrogerado no substrato e incorporado nas micelas foi utilizado como sonda para a estimativa do tempo de difusão. Os resultados obtidos para o surfactante brometo de tetradecil trimetil amônio (C14TABr) corroboram dados reportados na literatura, demonstrando a utilidade da metodologia proposta no estudo de sistema micelares. Também foram realizados experimentos envolvendo micelas do surfactante cloreto de 1-alquil-3-metilimidazólio, CxMelmCl (x = 10, 12, 14, 16) e com os resultados obtidos foi possível evidenciar o efeito da cadeia carbônica no coeficiente de difusão das espécies. Os experimentos envolvendo a permeação de substâncias através de bicamadas lipídicas foram realizados em duas etapas. Os primeiros ensaios foram realizados utilizando modelo de membrana semipermeável (papel celofane) com o intuito de verificar a aplicabilidade da SECM no monitoramento de espécies eletroativas que permeiam através da membrana. Na segunda etapa, apresentou-se metodologia para a obtenção de microfuros em folhas de poliestireno utilizados para a formação das bicamadas lipídicas, assim como detalhes sobre a construção da célula de medidas utilizadas nos experimentos de permeação. Foram realizados experimentos envolvendo o uso de bicamadas lipídicas planas obtidas pelo método de Miller preparadas com lecitina de soja. Esses experimentos foram realizados com o intuito de avaliar a estabilidade e para verificar a permeabilidade de algumas substâncias nas bicamadas formadas. Os experimentos de permeação foram realizados posicionando um microeletrodo próximo à membrana, com posterior detecção amperométrica da espécie eletroativa que atravessa a membrana. / This thesis shows results on the use of scanning electrochemical microscopy in the study of micellar systems and lipid bilayers. Studies involving micellar systems were performed by using SECM in the substrate-generator/tip-collector mode. In this operation mode a platinum microelectrode was positioned close to a platinum substrate and used to monitor electrogenerated species on this surface. Taking into account the time for the electrogenerated species to diffuse from the substrate to the microelectrode, the diffusion coefficient of the electroactive species and of the micelles can be calculated by applying the Einstein-Smoluchowski equation. As micelles are not electroactive, ferrocene electrogenerated on the substrate and incorporated into the micelles was used as a probe to estimate the diffusion time. The results obtained for tetradecyl trimethyl ammonium bromide (C14TABr) corroborate those reported in the literature, demonstrating the applicability of the proposed methodology in the study of micellar systems. Experiments with micelles obtained from 1-alkyl-3-methylimidazolium, CxMelmCl (x = 10, 12, 14, 16) chloride surfactants were also performed and results showed the effect of the carbon chain in the diffusion coefficient. Experiments involving the permeation of chemical species through lipid bilayers were carried out in two steps. A membrane model (cellophane) was preliminary used in order to investigate the possibility of using SECM as a tool for monitoring the permeation of electroactive species through the membrane. Then, a methodology for obtaining microholes in polystyrene sheets used to form lipid bilayers was presented, as well as details about the design of an electrochemical cell used in the permeation experiments. Experiments involving the use of planar lipid bilayers obtained by the method of Miller prepared using soybean lecithin were performed. These experiments were carried out in order to evaluate the stability and to check the permeation of some substances through the prepared bilayers. Permeation experiments were performed by placing the microelectrode close to the membrane with subsequent amperometric detection of any electroactive species that cross the membrane

This research is dedicated to develop novel batch fabrication procedures for two distinct AFM (Atomic Force Microscope) probes integrated with electrodes enabling electrical sample characterization and electrochemical sample surface profiling respectively. These AFM probes allow for highly accurate control of the probe positioning, low contact force and sample surface imaging with high lateral resolution. As an electrical characterization tool, a nickel micro four-point probe integrated with solid nickel tips was developed. Low electrical resistance of the probe and contact resistance were achieved due to the solid nickel cantilever and tips. Low aspect ratio solid metal tips reduced contact resistance resulting in stable electrical measurement. Conductivity loss easily experienced while using metal coated AFM cantilevers was overcome by solid nickel tip integration to the electrically conductive AFM cantilevers. The fabrication method introduces selective conical nickel tip etching in silicon dioxide etching chambers. A novel batch fabrication method for SECM-AFM (Scanning Electrochemical Microscope-Atomic Force Microscope) tip integrated with a ring electrode was developed as a tool for electrochemical imaging as well as topological imaging. The electroactive area at an exactly defined distance above the apex of the AFM tip is fabricated using an inverse silicon mold technique. The electrode at a deliberately chosen distance from the end of a scanning probe tip allowing electrochemical sample imaging separated from sample topology imaging. The ring electrode coated with polymer entrapping enzymes enabled the probe to detect ATP from living epithelial cells.

The goal of this thesis was the advanced design, fabrication, and application of combined atomic force microscopy - scanning electrochemical microscopy (AFMSECM) probes for high-resolution topographical and electrochemical imaging. The first part of the thesis describes innovative approaches for the optimization of AFM-SECM probe fabrication with recessed frame electrodes. For this purpose, commercial silicon nitride AFM cantilevers were modified using optimized critical fabrication processes including improved metallization for the deposition of the electrode layer, and novel insulation strategies for ensuring localized electrochemical signals. As a novel approach for the insulation of AFM-SECM probes, sandwiched layers of PECVD SixNy and SiO2, and plasma-deposited PFE films were applied and tested. Using sandwiched PECVD SixNy and SiO2 layers, AFM-SECM probes providing straight (unbent) cantilevers along with excellent insulation characteristics facilitating the functionality of the integrated electrode were reproducibly obtained. Alternatively, PFE thin films were tested according to their utility for serving as a mechanically flexible insulating layer for AFM-SECM probes. The electrochemical characterization of PFEinsulated AFM-SECM probes revealed excellent insulating properties at an insulation thickness of only approx. 400 nm. Finally, AFM-SECM cantilevers prepared via both insulation strategies were successfully tested during AFM-SECM imaging experiments. In the second part of this thesis, disk-shaped nanoelectrodes were for the first time integrated into AFM probes for enabling high-resolution AFM-SECM measurements. Disk electrodes with an electrode radius < 100 nm were realized, which provides a significantly improved lateral resolution for SECM experiments performed in synchronicity with AFM imaging. Furthermore, the developed fabrication scheme enables producing AFM-SECM probes with integrated disk nanoelectrodes at significantly reduced time and cost based on a highly reproducible semi-batch fabrication process providing bifunctional probes at a wafer scale. The development of a detailed processing strategy was accompanied by extensive simulation results for developing a fundamental understanding on the electrochemical properties of AFM-SECM probes with nanoscale electrodes, and for optimizing the associated processing parameters. Thus fabricated probes were electrochemically characterized, and their performance was demonstrated via bifunctional imaging at model samples. The third part of this thesis describes the development and characterization of the first AFM tip-integrated potentiometric sensors based on solid-state electrodes with submicrometer dimensions enabling laterally resolved pH imaging. Antimony and iridium oxides were applied as the pH sensitive electrode material, and have been integrated into the AFM probes via conventional microfabrication strategies. The pH response of such AFM tip-integrated integrated pH microsensors was tested for both material systems, and first studies were performed demonstrating localized pH measurements at a model system.