Understanding the mechanism of direct electrochemistry of mitochondria-modified electrodes from yeast, potato and bovine sources at carbon paper electrodes

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One of the main issues of enzymatic biofuel cells is the need to develop stable bioelectrodes. However, there is no standard method to study bioelectrode stability. In this study, laccase and anthracene modified multiwall carbon nanotubes (anth-MWCNTs) were used in conjunction with different immobilization polymers in order to increase the stability of the biocathodes. A series of stability assays were used to understand the operational stability of the biocathode in a biofuel cell environment. The immobilization matrices used in this study were tetrabutyl ammonium bromide modified Nafion (TBAB modified Nafion), octyl modified linear polyethylenimine (C 8 -LPEI), and vapor deposited tetramethyl orthosilicate (TMOS) gels. The decrease in activity of the galvanostatic and potentiostatic measurements over a 24 hour period of the TBAB modified Nafion were 4.0 ± 0.6% and 4.1 ± 0.4%; C 8 -LPEI were 0.7 ± 0.1% and 9.6 ± 0.5%; and TMOS were 4.1 ± 0.1% and 10 ± 2.0%, respectively. This data show that potentiostatic measurements provide a harsher environment for the enzyme and result in lower stability, as well as showing that the TBAB modified Nafion offers a more stabilizing immobilization strategy compared to the C 8 -LPEI or TMOS matrices. The increasing interest in producing electrical energy from renewable resources has caused the field of biofuel cells to flourish. Enzymatic biofuel cell systems are capable of producing energy from renewable fuels. Basically, enzymatic biofuel cells (EBFC) generate electricity from the oxidation of the fuel at the anode and the reduction of the oxidant at the cathode using biological catalysts (enzymes) instead of traditional metal catalysts. More detailed descriptions of such biofuel cells can be found in reviews that discuss the fundamental concepts of this technology.1,2 Applications of EBFCs range from energy sources for small electronic devices, 3 self-powered sensors 4 and more recently as implantable power sources. [5][6][7] To improve this technology, much research has been focused on engineering and modifying the cathode half of these biofuel cells. [8][9][10][11][12][13][14][15][16][17][18] One such engineered bioelectrode system, previously developed by our group, employs anthracene modified multi-walled carbon nanotubes (anth-MWCNTs) for docking of the oxidoreductase enzyme laccase to the current collector. This biocathode uses anthracene covalently modified to the ends of the multi-walled carbon nanotubes (MWCNTs) in order to favorably orient the laccase enzyme to the ends of the carbon nanotubes, since laccase has a hydrophobic binding site for anthracene and the aromatic structure allows for improved conductivity between the active site and the carbon surface. The steric orientation provides increased catalytic reduction of oxygen to water and favorable distances such that the laccase performs direct electron transfer (DET). 8In biofuel cells, DET occurs through the enzyme's ability to oxidize or reduce a substrate while transferring the necessary electrons to or fro...

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confidence: 99%

Operational Stability Assays for Bioelectrodes for Biofuel Cells: Effect of Immobilization Matrix on Laccase Biocathode Stability

Shrier

Giroud

Rasmussen

2014

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“…15 Known Complex I inhibitors permethrin and rotenone [21][22][23] were tested on mitochondrial electrodes, while permethrin also interrupts mitochondrial sodium channels. None of those inhibitors are electrochemically active in the potential window of the experiment (Blue line, Figures 1A, 1B and 1C).…”

Section: Resultsmentioning

confidence: 99%

“…4 Minteer et al suggest that ubiquinone is the key species for mitochondrial electrochemistry where research was based on the individual depletion of ubiquinone and cytochrome c to determine which species contributed to the resulting electrochemical response. 15,16 All of these electrochemical measurements have been done on carbon electrode surfaces, * Electrochemical Society Student Member.* * Electrochemical Society Fellow. z E-mail: minteer@chem.utah.edu because there is good adhesion of the mitochondria to the carbon electrode surfaces compared to gold, ITO, etc.…”

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confidence: 99%

Effect of Riboflavin Metabolites on Mitochondrial Electrochemistry

Wang

2016

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Mitochondrial deficiency is the cause of many diseases and the determination of changes in metabolic rates usually requires lysing of the mitochondria and isolating individual mitochondrial proteins. Alternatively, mitochondria can be immobilized on electrode surfaces to utilize electroanalytical evaluation of metabolic rates of intact mitochondria. However, the redox mechanisms are still poorly understood. In this paper, the riboflavin cycle of mitochondria is studied electrochemically and its impact on mitochondrial voltammetry is discussed. The inhibition mechanism of mitochondria by three different inhibitors (rotenone, carboxin, and permethrin) is discussed and it is found that the inhibition behavior observed electrochemically is due to not only ubiquinone, which is the electrochemical communicating species of mitochondrial electrochemistry. It is also shown that riboflavin derivatives interact with ubiquinone leading to a change in the intensity of ubiquinone voltammetry peaks. This interaction is affected by altering the choice of solvent used during the electrode preparation process. Finally, it is concluded that the observed voltammetry of mitochondrial inhibition is due to a change in riboflavin metabolism within the intact mitochondria immobilized on carbon electrodes. Mitochondria are the primary source of respiration and ATP synthesis for all eukaryotic cells. The mitochondrial electron transport chain (ETC), which is responsible for mitochondrial respiration, contains four proteins (three for fungal mitochondria) and ATP synthase as well as multiple electrochemically active species such as ubiquinone and cytochrome c. Mitochondria contain the proteins of the Krebs cycle 1-3 that are capable of metabolizing sugar metabolites, fatty acids 4 and amino acids 5 and have therefore been considered as attractive catalysts for bioelectrocatalysis applications such as biofuel cells, due to their high volumetric catalytic activity. The metabolism integrity and complexity also allows mitochondria to be responsive to many different toxins, such as inhibitors (rotenone, carboxin, etc.), uncouplers (dinitrophenol, dicumarol) and other toxins (cyanide, azide, CO), with different electrochemical responses toward different toxins. 6,7 Since mitochondrial ETC deficiency has been recognized as the reason for many diseases and disorders, like early age hypertonia, Kearns-Sayre syndrome, Alzheimer's and Leber's hereditary optic neuropathy, 8 an electrochemical biosensor incorporating mitochondria could possibly be an alternative biosensor technology to the current mitochondria deficient study that is based on mouse models.The electrochemistry of mitochondria has been extensively studied. In terms of practical application, mitochondrial fuel cells with advantageous fuel adaptation, power output and complete biofuel oxidation as well as mitochondrial biosensors with universality have been reported. 6,7,[9][10][11][12][13][14] Mitochondria contain many different electrochemically active species, like flavin adenine dinucl...

“…The cut Toray carbon paper was placed in methanol in a sonic bath for 10 minutes. 11 The methanol was changed and bathed again and then for a third time. 11 The electrodes were rinsed with water multiple times and laid to dry, covered, in between clean weigh boats.…”

Section: Methodsmentioning

confidence: 99%

“…[1][2][3][4][5] The source of mitochondrial electrochemical response has also been debated. [6][7][8][9][10][11] We hypothesize this disagreement is due to variation in the mitochondrial isolation causing a different primary communicating redox species to be observed on the electrode. This hypothesis is supported by literature which describes major differences in mitochondrial electrochemical communication.…”

mentioning

confidence: 99%

Understanding the Role of Mitochondrial Health in the Mechanism of Mitochondrial Bioelectrocatalysis

Koepke

Watkins

2016

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Mitochondria are an alternative biocatalyst to microbes and enzymes in bioelectrochemical applications, including biosensors and biofuel cells. However, the bioelectrocatalytic mechanism is not well understood and there are contradictory reports in the literature. The electrochemical communicating species for mitochondria has previously been reported to be ubiquinone, cytochrome c, or cytochrome c oxidase. In this paper, it is determined that the primary electrochemical communicating species in mitochondria is ubiquinone by comparing yeast wild-type and mutants without cytochrome c and cytochrome c oxidase. The electrochemical results compared healthy and unhealthy yeast mitochondria, as established by a Seahorse oxygen consumption assay. Healthy mitochondrial electrochemical response increased by 20 ± 7% for reduction and 51 ± 3% for oxidation while unhealthy mitochondrial response increased 3 ± 50% for reduction and 39 ± 3% for oxidation when uncoupled with DNP. These responses provide a better understanding of mitochondrial systems in vitro and their relationship to in vivo systems as well as insights into the mechanism of electrochemical response.