Cyclic voltammetry (CV) is a type of electrochemical measurement which is applied in the Q-Module as a quality control step to determine the redox potential of Coenzyme Q in the specific experimental conditions used. In cyclic voltammetry, the Q-Sensor with the three-electrode system is used to obtain information about the analyte (CoQ) by measuring the current (I) as the electric potential (V) between two of the electrodes is varied. In CV the electric potential between the glassy carbon (GC) and the Ag/AgCl reference electrode changes linearly versus time in cyclical phases, while the current is detected between GC and platinum electrode (Pt). The detected current is plotted versus the applied voltage to obtain the typical cyclic voltammogram trace (Figure 1). The presence of substances that are oxidized/reduced will result in current between GC and Pt, which can be seen as characteristic peaks in the voltammogram at a defined potential. The oxidation or the reduction peak potential values are used to set the GC (integrated into the Q-Sensor) for a separate experiment to measure the Q redox state of a biological sample. The oxidation and reduction peak potentials can be influenced by 1) the respiration medium, 2) type of CoQ, 3) polarization window, 4) scan speed, 5) number of cycles, 6) concentration of the analyte (CoQ),and 7) initial polarization voltage. For further information, see: MiPNet24.12 NextGen-O2k: Q-Module.
Figure 1. Cyclic voltammogram of Coenzyme Q2 measured with the Q-Module. Measurements were carried out in non-stirred MiR05-Kit medium, at 37 °C using the NextGen-O2k. Initial potential: +30 mV, polarization window: between -500 mV and +500 mV, scanning speed: 100 mV/s, gain: 1; 30 µM Q2 was used for each test. The oxidation peak potential shows the maximum rate of quinol oxidation and the reduction peak potential is the point of maximum rate of quinone reduction.
Cyclic voltammetry is part of the Q-Module and the NextGen-O2k project
In the NextGen-O2k project we are working on developing the Q-Module for the new series of our O2k-FluoRespirometer, the NextGen-O2k. The Q-Module allows us to monitor the Q redox state of the Q-junction using the specific Q-Stoppers with the integrated three-electrode system and the modified electronics inside the O2k housing.
Communicated by Komlodi T, Cardoso LHD 2020-07-28
- » NextGen-O2k«
|Chlororespiration||Chlororespiration is the phenomenon by which oxygen is consumed by a putative respiratory electron transfer chain (ETC) within the thylakoid membrane of the chloroplasts and ATP is produced. It is a process that involves the interaction with the photosynthetic ETC in which the NAD(P)H dehydrogenase enzyme transfers electrons to oxygen molecules with the assistance of the photosynthetic Plastoquinone (PQ), which acts as a non-photochemical redox carrier. Initially described in the unicellular alga Chlamydomonas reindhartdii, chlororespiration was highly disputed for years until the discovery of a NAD(P)H-dehydrogenase (Ndh) complex (plastidic encoded) and plastid terminal oxidase (PTOX) (nuclear encoded) in higher-plant chloroplasts. The PTOX, which is homologous to the plant mitochondrial alternative oxidase, has the role of preventing the over-reduction of the PQ pool while the Ndh complexes provide a gateway for the electrons to form the ETC and consume oxygen. As a result of this process there is a cyclic electron flow around the Photosystem I (PSI) that has been reported to be activated under stress conditions acting as a photoprotection mechanism and could be involved in protecting against any other stress that implies the increase of ROS formation.|
|Cyclic voltammetry||CV||Cyclic voltammetry (CV) is a type of electrochemical measurement which is applied in the Q-Module as a quality control step to determine the redox potential of Coenzyme Q in the specific experimental conditions used. In cyclic voltammetry, the Q-Sensor with the three-electrode system is used to obtain information about the analyte (CoQ) by measuring the current (I) as the electric potential (V) between two of the electrodes is varied. In CV the electric potential between the glassy carbon (GC) and the Ag/AgCl reference electrode changes linearly versus time in cyclical phases, while the current is detected between GC and platinum electrode (Pt). The detected current is plotted versus the applied voltage to obtain the typical cyclic voltammogram trace (Figure 1). The presence of substances that are oxidized/reduced will result in current between GC and Pt, which can be seen as characteristic peaks in the voltammogram at a defined potential. The oxidation or the reduction peak potential values are used to set the GC (integrated into the Q-Sensor) for a separate experiment to measure the Q redox state of a biological sample. The oxidation and reduction peak potentials can be influenced by 1) the respiration medium, 2) type of CoQ, 3) polarization window, 4) scan speed, 5) number of cycles, 6) concentration of the analyte (CoQ),and 7) initial polarization voltage. For further information, see: MiPNet24.12 NextGen-O2k: Q-Module.|
|Hydrogen peroxide||H2O2||reactive oxygen species (ROS). It is formed in various enzyme-catalyzed reactions (e.g., superoxide dismutase) with the potential to damage cellular molecules and structures. H2O2 is dismutated by catalase to water and oxygen. H2O2 is produced as a signaling molecule in aerobic metabolism and passes membranes more easily compared to other ROS.|
|Mitochondrial membrane potential||mtMP, Δψ [V]||The mitochondrial membrane potential, mtMP, is the electric part of the protonmotive force, ΔpH+.
Δψ = ΔpH+ - ΔµH+ / FmtMP or Δψ is the potential difference across the inner mitochondrial (mt) membrane, expressed in the electric unit of volt [V]. Electric force of the mitochondrial membrane potential is the electric energy change per ‘motive’ electron or per electron moved across the transmembrane potential difference, with the number of ‘motive’ electrons expressed in the unit coulomb [C].
|NextGen-O2k Instrument||NextGen-O2k Instrument|
|NextGen-O2k Technical developments|
|Oxygen kinetics||Oxygen kinetics describes the dependence of respiration of isolated mitochondria or cells on oxygen partial pressure. Frequently, a strictly hyperbolic kinetics is observed, with two parameters, the oxygen pressure at half-maximum flux, p50, and maximum flux, Jmax. The p50 is in the range of 0.2 to 0.8 kPa for cytochrome c oxidase, isolated mitochondria and small cells, strongly dependent on Jmax and coupling state.|
|PhotoBiology||PB||PhotoBiology is the scientific study of the beneficial or harmful effects of light, understood as non-ionizing radiation (i.e. ultraviolet, visible and infrared radiation) on living organisms. It includes topics such as the study of photosynthesis, photochemistry, photophysics, photomorphogenesis, vision, bioluminescence, circadian rhythms and photodynamic therapy. Non-ionizing (or non-ionising) radiation is any type of electromagnetic radiation that does not carry enough energy per quantum (photon energy below 10 eV) to completely remove an electron from an atom or molecule. When photons contact molecules, the molecules can absorb the photon energy and become excited, reacting with surrounding molecules and stimulating "photochemical" and "photophysical" changes.|
|Photorespiration||Photorespiration is the process by which the enzyme RuBisCo oxygenates the Ribulose Biphosphate (RuBP) instead of carboxylating it as part of the Calvin-Benson cycle, thus wasting the energy produced by photosynthesis (in the form of a direct cost in ATP and NAD(P)H) and creating a product that cannot be used within this cycle, phosphoglycolate. It is estimated that approximately 25 % of RuBisCo reactions are photorespiration, meaning a potential 25 % reduction in photosynthetic output due to the carbon fixed by photorespiration being released as carbon dioxide and nitrogen as ammonia, while the other product, 3-phosphoglycerate (G3P), requires a higher metabolic cost. This process involves a complex network of enzyme and metabolite exchanges between the chloroplasts, peroxisomes and mitochondria. It is also known as the oxidative photosynthetic carbon cycle or C2 photosynthesis and abiotic conditions tend to affect it such as temperature and the atmospheric partial pressures of oxygen and carbon dioxide. Certain type of plants (C4 plants and CAM plants) and algae have biochemical and biophysical mechanisms to overcome the photosynthetic losses due to photorespiration making them more photosynthetically efficient than C3 plants. Recent plant biotechnology advances have been focused on increasing plant photosynthetic carbon fixation by reducing photorespiration loses.
|Photosynthesis||PS||Photosynthesis is the process used by plants and other organisms that converts light (mostly solar) energy into chemical energy which is subsequently released to fuel organisms' activities. It has two phases: the light-dependent phase and the light-independent (dark) phase. In plants, algae, and cynobacteria, light energy is absorbed during the light phase by the pigment called Chlorophyll and used to split water and generate short-term stores of chemical energy - adenosine triphosphate (ATP), and reducing power - nicotinamide adenine dinucleotide phosphate (NADPH), with the net production of O2 gas as a waste product. And during the dark phase this chemical energy and reducing power are used to synthesize organic matter from the atmospheric CO2 in the form of carbohydrates or sugars through the metabolic pathway called Calvin-Benson cycle. The whole process is what is called oxygenic photosynthesis and is responsible for producing and maintaining the oxygen concentration of the Earth’s atmosphere. In bacteria such as the cyanobacteria photosynthesis involves the plasma membrane and the cytoplasm, and in Eukaryotic cells (plants and algae) photosynthesis takes place inside organelles called chloroplasts.|
|Q redox state||Qr/Qt||The Q redox state reflects the redox status of the Q-junction in the mitochondrial or chloroplast electron transfer system (ETS). Ubiquinones, also known as coenzyme Q, and plastoquinones are essential mobile components of the mitochondria and chloroplasts that transfer electrons between the respiratory or photosynthetic complexes of the ETS. The Q redox state is dependent on the relative activities of the ETS enzymes that reduce and oxidize the quinones. Therefore, deficiencies in the mitochondrial ETS, originating from e.g. the malfunction of respiratory enzymes (complexes), can be detected by measuring the changes of the Q redox state with respect to respiratory activity.|
|Q-Module||Q-Module||The Q-Module, developed both for measuring the Q redox state and for cyclic voltammetry measurements, is an integral part of the NextGen-O2k and consists of the Q-Sensor, integrated electronic components in the O2k, and the DatLab software.|
|Q-Sensor||The Q-Sensor has been designed as a part of the Q-Module for measurements with cyclic voltammetry and voltammetry, allowing for analysis of the Q redox state. The Q-Stopper with the reference electrode is called Q-Sensor, which is plugged in the NextGen-O2k. A three-electrode system is used to detect the Q redox state. Two of the three electrodes (glassy carbon and platinum electrode) are built into the Q-Stopper, while the reference electrode is removable (Reference-Electrode\2.4 mm).|
|Three-electrode system||A three-electrode system is the setup used in the Q-Sensor, which is an integral part of the Q-Module. This system is used in voltammetry (including cyclic voltammetry) to study the current as a function of the applied potential using three different electrodes: 1) the working electrode 2) the reference electrode, and 3) the counter electrode. The working or detecting electrode is a glassy carbon (GC) electrode that is set to a given potential and makes contact with the analyte. The potential of the working electrode is controlled by the constant potential of the a silver/silver chloride (Ag/AgCl) reference electrode, which does not pass any current. The applied potential on the surface of the GC should be sufficient to either oxidize reduced analyte (in our case Coenzyme Q) or to reduce oxidized CoQ. Thus, the counter electrode is a platinum electrode (Pt) that passes a current to counter these redox events by completing the circuit that is rate-limited by electron transfer on the GC. To determine the Q redox ratio the GC electrode is set at the oxidation peak potential, which can be determined with cyclic voltammetry.|