Applications of scanning electrochemical microscopy in biological systems

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Koley, Dipankar

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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.




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