NextGen-O2k Technical developments

NextGen-O2k

K-Regio MitoFit

O2k-Innovation

Houska-Award 2012

K-Regio MitoCom

Bioblast 2012

NextGen-O2k Technical developments

The revolutionary all-in-one instrument to conquer mitochondrial disease.


WP1: Technical developments

Abstract

In this workpackage the Q- and PB-Modules are developed for the NextGen-O2k.

• Development of the Q-Module and PB-Module and integration in the NextGen-O2k
• Testing of the NextGen-O2k in collaboration with the Early testers
• Testing and development of the software for the NextGen-O2k Modules
• Integration of the new protocols in the DatLab software

Achievements

Project Year 1: In the first year of the NextGen-O2k project, the new Q- and PB-Modules supported by the new architecture of the upgraded DatLab software have been developed and integrated. Additionally, we have successfully partnered with Key Opinion Leaders to begin testing the Q-Module prototypes. Working closely with our subcontractors WGT and H-Tech, key components such as cyclic voltammetry have been integrated into the system.

Progress and next steps

2020-09-30: Tests on the PB-Sensors and PB-Module after upgrade of the firmware give positive results.

2020-09-11: Upgrade of the PB-Module firmware so the light intensity is corrected in real-time by the photodiode of the PB-Sensor tip

2020-07-16: Test of the new PB-Sensors give positive results.

2020-07-06: Delivery of the new PB-Sensor by WGT. Start of testing them at Oroboros.

2020-05-25: Project partner WGT begins designing a new type of sensor tip that protects the LEDs and helps with the maintenance of the PB-Sensors

2020-05-20: First tests of the PB-Module in the Q3-003 and Q3-004

2020-05-04: Third (Q3-003) and fourth (Q3-004) NextGen-O2k with the PB-Module integrated delivered for in-house testing

2020-04-27: First tests of the PB-Module in the Q3-002

2020-04-02: Start of the Quality Control tests in the Q3-002

2020-03-11: First tests of the PB-Module run in the Q3-001

2020-03-05: Second prototype (Q3-002) with integrated PB-Module received for in-house testing

2020-02-11: First prototype of NextGen-O2k (Q3-001) with integrated PB-Module received for in-house testing

2020-02-14: Dispatch of 2 prototypes for testing at Marten Szibor’s lab in Jena, DE and Carlo Viscomi’s Lab in Padua, IT

2020-02-11: Second set of feedback from the Key Opinion Leaders on the NextGen-O2k with the Q-Module received

2020-01-10: First feedback from the Key Opinion Leaders on the NextGen-O2k with the Q-Module received

2019-12-18: Final type of LEDs and wavelengths to be integrated in the PB-Module are confirmed

2019-11-31: New software for the quality control by cyclic voltammetry (Q-Module) sent to KOLs

2019-11-06: Dispatch of 1 prototype for testing at Anthony Molina’s Lab in San Diego, US

2019-10-29: Dispatch of 1 prototype for testing at Anthony Moore’s Lab in Sussex, UK

2019-10-16: Dispatch of 1 prototype for testing at Christos Chinopoulos’ Lab in Budapest, HU

2019-10-22: DatLab7.4 released

2019-10: Calibration of the light intensity provided by the PB-Sensors prototypes.

2019-09: 5 prototypes of the Q-Module ready for testing at our Key Opinion Leader’s labs

2019-08-07: Setting up of our lab for algae culturing activities started

2019-06-13: Start of testing the PB-Sensors prototypes at Oroboros

2019-06: Delivery of the first PB-Sensors prototypes by WGT to test at Oroboros

2019-06: Integration of cyclic voltammetry as a quality control step into the Q-Module

2019-06: Final version of the specific Q-Stopper (with integrated electronics) confirmed

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  Some of the Key Opinion Leader’s at the IOC141

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  Shaker (laboratory) for algae

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  Setting up our lab for algae culturing activities

MitoPedia: NextGen-O2k

» NextGen-O2k«

Term	Abbreviation	Description
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	Hydrogen peroxide, H2O2 or dihydrogen dioxide, is one of several reactive oxygen intermediates generally referred to as 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+ / F mtMP 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
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.
PB-Module	PB-Module	The PB-Module, developed both for conducting measurements of PhotoBiology, including photosynthesis, is an integral part of the NextGen-O2k and consists of the PB-Sensor, integrated electronic components in the O2k, and the DatLab software.
PB-Sensor		The PB-Sensor has been designed as a part of the PB-Module for providing with an external source of light, in the range of the Blue or Red wavelengths, allowing to carry out experiments for evaluating the production/consumption of O2 in the presence of light. The PB-Sensor consists on one LED and one photodiode mounted on the sensor tip behind a plastic cover. The LED provides with blue or red light to the sample and its intensity can be regulated from 0 to 5000 µEinsteins*m-2*s-1. The photodiode allows to measure in real-time the light intensity provided by the LED, so it can be adjusted continuously to the desired value, and also helps to compensate for the temperature rise due to the operation of the LED.
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.

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