| 1. |
- Heiskanen, Arto, et al.
(författare)
-
Bioelectrochemical probing of intracellular redox processes in living yeast cells-application of redox polymer wiring in a microfluidic environment
- 2013
-
Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 405:11, s. 3847-3858
-
Tidskriftsartikel (refereegranskat)abstract
- Conventionally, microbial bioelectrochemical assays have been conducted using immobilized cells on an electrode that is placed in an electrochemical batch cell. In this paper, we describe a developed microfluidic platform with integrated microelectrode arrays for automated bioelectrochemical assays utilizing a new double mediator system to map redox metabolism and screen for genetic modifications in Saccharomyces cerevisiae cells. The function of this new double mediator system based on menadione and osmium redox polymer (PVI-Os) is demonstrated. "Wiring" of S. cerevisiae cells using PVI-Os shows a significant improvement of bioelectrochemical monitoring in a microfluidic environment and functions as an effective immobilization matrix for cells that are not strongly adherent. The function of the developed microfluidic platform is demonstrated using two strains of S. cerevisiae, ENY. WA and its deletion mutant EBY44, which lacks the enzyme phosphoglucose isomerase. The cellular responses to introduced glucose and fructose were recorded for the two S. cerevisiae strains, and the obtained results are compared with previously published work when using an electrochemical batch cell, indicating that microfluidic bioelectrochemical assays employing the menadione-PVI-Os double mediator system provides an effective means to conduct automated microbial assays.
|
|
| 2. |
- Heiskanen, Arto, et al.
(författare)
-
Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells.
- 2009
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 384:1, s. 11-19
-
Tidskriftsartikel (refereegranskat)abstract
- This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pentose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric in vitro assays show no difference between the cofactors. Respiratory competent cells show cytosolic inhibition only when NADPH is produced, whereas production of NADH reveals uncoupling at low dicoumarol concentrations and inhibition of complexes III and IV at higher concentrations. Spectrophotometric assays only indicate the presence of cytosolic inhibition regardless of the reduced cofactor used. This article shows the applicability of the amperometric method and emphasizes the significance of determining biological effects of chemicals in living cells.
|
|
| 3. |
- Heiskanen, Arto, et al.
(författare)
-
Monitoring of Saccharomyces cerevisiae Cell Proliferation on Thiol-Modified Planar Gold Microelectrodes Using Impedance Spectroscopy.
- 2008
-
Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 24:16, s. 9066-9073
-
Tidskriftsartikel (refereegranskat)abstract
- An impedance spectroscopic study of the interaction between thiol-modified Au electrodes and Saccharomyces cerevisiae of strain EBY44 revealed that the cells formed an integral part of the interface, modulating the capacitive properties until a complete monolayer was obtained, whereas the charge transfer resistance ( R ct) to the redox process of [Fe(CN)6] (3-/4-) showed a linear relationship to the number of cells even beyond the monolayer coverage. R ct showed strong pH dependence upon increasing the pH of the utilized buffer to 7.2. Upon addition of S. cerevisiae cells at pH 7.2, the obtained value of R ct showed over 560% increase with respect to the value obtained on the same thiol-modified electrode without cells. It was demonstrated that real-time monitoring of S. cerevisiae proliferation, with frequency-normalized imaginary admittance (real capacitance) as the indicator, was possible using a miniaturized culture system, ECIS Cultureware, with integrated planar cysteamine-modified Au microelectrodes. A monolayer coverage was reached after 20-28 h of cultivation, observed as an approximately 15% decrease in the real capacitance of the system.
|
|
| 4. |
|
|
| 5. |
- Kostesha, Natalie
(författare)
-
PROBING NAD(P)H AVAILABILITY IN LIVING CELLS USING MINIATURIZED ELECTROCHEMICAL SYSTEMS
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in this thesis demonstrates the application of electrochemical methods for probing the dynamics of cellular metabolism in prokaryotic and eukaryotic cells. The main part of the work has been done using genetically modified strains of yeast. Employing baker’s yeast, S. cerevisiae, as a model organism, it was demonstrated how an amperometric method can be utilized for real-time in vivo screening of intracellular NAD(P)H availability in relation to redox enzyme activities. NAD(P)H availability was evaluated using the amperometric method based on a menadione-ferricyanide double-mediator system. Mediated amperometry is based on the ability of menadione, a lipophilic quinone, to freely diffuse through the plasma membrane and accept electrons from certain intracellular NAD(P)H-dependent enzymes. The reduced form of menadione is extracellularly oxidized by ferricyanide. The ferrocyanide formed is in turn oxidized at the gold microband electrode, giving rise to a bioelectrocatalytic current proportional to the activity of the menadione reducing enzyme. The bioelectrocatalytic current was measured by immobilizing living S. cerevisiae cells on gold microband electrode chips (Si/SiO2 substrate) in Ca-alginate gel. The microelectrodes (width/length 25/1000 µm) were fabricated using micromachining technology for real-time amperometric probing of NAD(P)H-dependent redoxprocesses and pathways in living cells. The dependence of menadione-reducing enzyme activity on the main metabolic pathways was investigated by introducing different carbon sources (glucose and fructose). It was found that the amplitude of the yeast-catalysed amperometric signal was significantly higher when carbon sources were administered, primarily resulting in the formation of NAD(P)H through the pentose phosphate pathway and glycolytic pathway. The crucial effect of inhibitors on menadione-reducing activity was surveyed using the method. The results obtained during this work demonstrate the significance of enzyme activity determination in living cells as the source of information on cellular responses, reflecting different metabolic pathways and regulatory mechanisms. This gives further support to the potential of mediated amperometry as a tool in metabolic profiling.
|
|
| 6. |
- Kostesha, Natalie, et al.
(författare)
-
Probing the redox metabolism in the strictly anaerobic, extremely thermophilic, hydrogen-producing Caldicellulosiruptor saccharolyticus using amperometry
- 2011
-
Ingår i: Extremophiles. - : Springer Science and Business Media LLC. - 1433-4909 .- 1431-0651. ; 15:1, s. 77-87
-
Tidskriftsartikel (refereegranskat)abstract
- Changes in the redox metabolism in the anaerobic, extremely thermophilic, hydrogen-forming bacterium Caldicellulosiruptor saccharolyticus were probed for the first time in vivo using mediated amperometry with ferricyanide as a thermotolerant external mediator. Clear differences in the intracellular electron flow were observed when cells were supplied with different carbon sources. A higher electrochemical response was detected when cells were supplied with xylose than with sucrose or glucose. Moreover, using the mediated electrochemical method, it was possible to detect differences in the electron flow between cells harvested in the exponential and stationary growth phases. The electron flow of C. saccharolyticus was dependent on the NADH- and reduced ferredoxin generation flux and the competitive behavior of cytosolic and membrane-associated oxidoreductases. Sodium oxamate was used to inhibit the NADH-dependent lactate dehydrogenase, upon which more NADH was directed to membrane-associated enzymes for ferricyanide reduction, leading to a higher electrochemical signal. The method is noninvasive and the results presented here demonstrate that this method can be used to accurately detect changes in the intracellular electron flow and to probe redox enzyme properties of a strictly anaerobic thermophile in vivo.
|
|
| 7. |
- Kostesha, Natalie, et al.
(författare)
-
Real-time detection of cofactor availability in genetically modified living Saccharomyces cerevisiae cells - Simultaneous probing of different geno- and phenotypes.
- 2009
-
Ingår i: Bioelectrochemistry. - : Elsevier BV. - 1878-562X .- 1567-5394. ; 76, s. 180-188
-
Tidskriftsartikel (refereegranskat)abstract
- This work describes a mediated amperometric method for simultaneous real-time probing of the NAD(P)H availability in two different phenotypes, fermentative and respiratory, of the phosphoglucose isomerase deletion mutant strain of S. cerevisiae, EBY44 [ENY.WA-1A pgi1-1D::URA3], and its parental strain, ENY.WA-1A. The developed method is based on multichannel detection using microelectrode arrays. Its versatility was demonstrated by using four microelectrode arrays for simultaneously monitoring the NAD(P)H availability of both geno- and phenotypes under the influence of two different carbon sources, glucose and fructose, as well as the cytosolic and mitochondrial inhibitor and uncoupler, dicoumarol. The obtained results indicate that the method is capable of accurately and reproducibly (overall relative standard error of mean 3.2%) mapping the real-time responses of the cells with different genotype-phenotype combinations. The ENY.WA cells showed the same response to glucose and fructose when dicoumarol was used; fermentative cells indicated the presence of cytosolic inhibition and respiratory cells a net effect of mitochondrial uncoupling. EBY44 cells showed cytosolic inhibition with the exception of respiratory cells when fructose was used as carbon source.
|
|
| 8. |
|
|
