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Monitoring of Cellular Dynamics with Focus on Electrochemical Detection Techniques

Heiskanen, Arto LU (2009)
Abstract
This thesis presents work in two main areas of focus, the development of electroanalytical techniques to monitor the dynamics of cellular processes as well as design and fabrication of microelectrode chips for electrochemical detection as well as a prototype microfluidic system to be used for cell analysis and culturing. The electrochemically monitored cellular parameters were the cellular redox environment (CRE), cell adhesion and exocytosis.

CRE is a combination of the redox states of individual cellular redox couples. A mediated amperometric method based on the function of the double-mediator system, ferricyanide-menadione, was developed for probing intracellular menadione reducing enzymes (MREs) in Saccharomyces cerevisiae.... (More)
This thesis presents work in two main areas of focus, the development of electroanalytical techniques to monitor the dynamics of cellular processes as well as design and fabrication of microelectrode chips for electrochemical detection as well as a prototype microfluidic system to be used for cell analysis and culturing. The electrochemically monitored cellular parameters were the cellular redox environment (CRE), cell adhesion and exocytosis.

CRE is a combination of the redox states of individual cellular redox couples. A mediated amperometric method based on the function of the double-mediator system, ferricyanide-menadione, was developed for probing intracellular menadione reducing enzymes (MREs) in Saccharomyces cerevisiae. These enzymes utilize the reduced cofactors, NADH and NADPH; changes in the obtained current responses collectively reflect the dynamics in the level of these cofactors. The utilization of fermentative cells of a phosphoglucose isomerase deletion mutant strain of S. cerevisiae, EBY44, facilitated selective probing of the catabolic cytosolic pathways, the pentose phosphate pathway (PPP) and glycolytic pathway (GP), predominantly producing NADPH and NADH, respectively. Together with metabolic flux analysis, menadione was found to increase the cytosolic NADPH production through the action of the PPP and the enzyme aldehyde dehydrogenase that concomitantly produces acetate. The results indicated that NADPH was the preferred cofactor for cytosolic menadione reduction. The method was also applied to probing the effect of dicoumarol, a cytosolic and mitochondrial inhibitor and uncoupler, on fermentative and respiratory cells of EBY44 and its parental strain ENY.WA. In the study, cells with different phenotype and genotype were immobilized on fabricated microelectrode arrays to demonstrate the possibility of simultaneous monitoring, using array technology and a multi-channel potentiostat. In an additional study, the effect of dicoumarol on the cytosolic and mitochondrial functions in EBY44 was compared to results obtained in crude cell extracts. The obtained results indicated that the IC50 in crude cell extracts could be up to 50 times higher than the corresponding value in live cells. The effect of 5-hydroxymethyl furfural (HMF) on S. cerevisiae was studied using cells overexpressing the gene encoding for alcohol dehydrogenase6 (ADH6 strain) and the corresponding control strain. The results showed that the ADH6 strain had less NADPH available as a consequence of the increased NADPH-dependent HMF reduction. The enzyme kinetic parameters, KM,apparent and VMAX of ADH6 were determined as a demonstration of the capabilities of mediated amperometry.

The adherence of S. cerevisiae cells on thiol-modified microelectrodes was characterized using electrochemical impedance spectroscopy (EIS), which was then applied for monitoring S. cerevisiae growth on microelectrodes. PC12 cell growth was monitored using impedance measurements for 144 hours on microelectrodes modified with physisorbed polyethyleneimine (PEI), which was found to be a highly suitable adhesion factor to be used when culturing PC12 cells on electrochemical transducers.

K+ evoked dopamine exocytosis from both single PC12 cells and populations of cultured cells was monitored using amperometric detection. The results showed that planar gold microelectrodes combined with amperometry at 10-kHz sampling rate were able to fully reveal the temporal resolution of single-vesicle exocytotic events. Exocytosis from cell populations showed proportionality to the size of the population. Exocytosis from a population of neuronal growth factor-differentaited PC12 cells was also monitored and shown to significantly differ from that of a population of non-differentiated cells. The rising time and the descending portion of the current-time trace for differentiated cells was steeper. This was attributed to closer proximity of the differentiated cells to the electrode surface and that the exocytotically active zones are not distributed thoughout the cell body as is the case with non-differentiated cells. (Less)
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author
supervisor
opponent
  • Professor Verpoorte, Sabeth, Department of Pharmacy, University of Groningen, The Netherlands
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Microfluidic system, Microchip, PC12, Saccharomyces cerevisiae, Electrochemical impedance spectroscopy, Cell adhesion, Amperometry, Exocytosis, Redox environment, Cell-based assay
pages
300 pages
publisher
Analytical Chemistry, Lund University
defense location
Lecture Hall B, Center for Chemistry and Chemical Engineering, Getingevägen 60, Lund
defense date
2009-04-03 10:30
ISBN
978-91-7422-220-3
language
English
LU publication?
yes
id
1ff53308-8f67-412e-ab5b-75a177f9e241 (old id 1359482)
date added to LUP
2009-03-13 15:30:12
date last changed
2016-09-19 08:45:03
@misc{1ff53308-8f67-412e-ab5b-75a177f9e241,
  abstract     = {This thesis presents work in two main areas of focus, the development of electroanalytical techniques to monitor the dynamics of cellular processes as well as design and fabrication of microelectrode chips for electrochemical detection as well as a prototype microfluidic system to be used for cell analysis and culturing. The electrochemically monitored cellular parameters were the cellular redox environment (CRE), cell adhesion and exocytosis.<br/><br>
CRE is a combination of the redox states of individual cellular redox couples. A mediated amperometric method based on the function of the double-mediator system, ferricyanide-menadione, was developed for probing intracellular menadione reducing enzymes (MREs) in Saccharomyces cerevisiae. These enzymes utilize the reduced cofactors, NADH and NADPH; changes in the obtained current responses collectively reflect the dynamics in the level of these cofactors. The utilization of fermentative cells of a phosphoglucose isomerase deletion mutant strain of S. cerevisiae, EBY44, facilitated selective probing of the catabolic cytosolic pathways, the pentose phosphate pathway (PPP) and glycolytic pathway (GP), predominantly producing NADPH and NADH, respectively. Together with metabolic flux analysis, menadione was found to increase the cytosolic NADPH production through the action of the PPP and the enzyme aldehyde dehydrogenase that concomitantly produces acetate. The results indicated that NADPH was the preferred cofactor for cytosolic menadione reduction. The method was also applied to probing the effect of dicoumarol, a cytosolic and mitochondrial inhibitor and uncoupler, on fermentative and respiratory cells of EBY44 and its parental strain ENY.WA. In the study, cells with different phenotype and genotype were immobilized on fabricated microelectrode arrays to demonstrate the possibility of simultaneous monitoring, using array technology and a multi-channel potentiostat. In an additional study, the effect of dicoumarol on the cytosolic and mitochondrial functions in EBY44 was compared to results obtained in crude cell extracts. The obtained results indicated that the IC50 in crude cell extracts could be up to 50 times higher than the corresponding value in live cells. The effect of 5-hydroxymethyl furfural (HMF) on S. cerevisiae was studied using cells overexpressing the gene encoding for alcohol dehydrogenase6 (ADH6 strain) and the corresponding control strain. The results showed that the ADH6 strain had less NADPH available as a consequence of the increased NADPH-dependent HMF reduction. The enzyme kinetic parameters, KM,apparent and VMAX of ADH6 were determined as a demonstration of the capabilities of mediated amperometry.<br/><br>
The adherence of S. cerevisiae cells on thiol-modified microelectrodes was characterized using electrochemical impedance spectroscopy (EIS), which was then applied for monitoring S. cerevisiae growth on microelectrodes. PC12 cell growth was monitored using impedance measurements for 144 hours on microelectrodes modified with physisorbed polyethyleneimine (PEI), which was found to be a highly suitable adhesion factor to be used when culturing PC12 cells on electrochemical transducers.<br/><br>
K+ evoked dopamine exocytosis from both single PC12 cells and populations of cultured cells was monitored using amperometric detection. The results showed that planar gold microelectrodes combined with amperometry at 10-kHz sampling rate were able to fully reveal the temporal resolution of single-vesicle exocytotic events. Exocytosis from cell populations showed proportionality to the size of the population. Exocytosis from a population of neuronal growth factor-differentaited PC12 cells was also monitored and shown to significantly differ from that of a population of non-differentiated cells. The rising time and the descending portion of the current-time trace for differentiated cells was steeper. This was attributed to closer proximity of the differentiated cells to the electrode surface and that the exocytotically active zones are not distributed thoughout the cell body as is the case with non-differentiated cells.},
  author       = {Heiskanen, Arto},
  isbn         = {978-91-7422-220-3},
  keyword      = {Microfluidic system,Microchip,PC12,Saccharomyces cerevisiae,Electrochemical impedance spectroscopy,Cell adhesion,Amperometry,Exocytosis,Redox environment,Cell-based assay},
  language     = {eng},
  pages        = {300},
  publisher    = {ARRAY(0x8ce9b80)},
  title        = {Monitoring of Cellular Dynamics with Focus on Electrochemical Detection Techniques},
  year         = {2009},
}