NextGen-O2k Technical developments

2022

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.

Project Year 2: During the second year of the project the testing of the PB-Module by algae KOLs began, while testing of the Q-Module continued. Feedback prompted a change in the PB-Sensor that will allow a wider spectrum of research questions to be addressed. Features of the DatLab 8 software were implemented and tested, and SUIT protocols were integrated.

Progress and next steps

See » Track record

Some of the Key Opinion Leader’s at the IOC141 Sept. 2019
Shaker (laboratory) for algae
Setting up our lab for algae culturing activities

MitoPedia: NextGen-O2k

» NextGen-O2k«

Term	Abbreviation	Description
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.
Light-enhanced dark respiration	LEDR	Light-enhanced dark respiration LEDR is a sharp (negative) maximum of dark respiration in plants in response to illumination, measured immediately after switching off the light. LEDR is supported by respiratory substrates produced during photosynthesis and closely reflects light-enhanced photorespiration (Xue et al 1996). Based on this assumption, the total photosynthetic oxygen flux TP is calculated as the sum of the measured net photosynthetic oxygen flux NP plus the absolute value of LEDR.
Mitochondrial membrane potential	mtMP, ΔΨ_p⁺, Δ_elF_ep⁺ [V]	The mitochondrial membrane potential difference, mtMP or ΔΨ_p⁺ = Δ_elF_ep⁺, is the electric part of the protonmotive force, Δp = Δ_mF_eH⁺. Δ_elF_ep⁺ = Δ_mF_eH⁺ - Δ_dF_eH⁺ ΔΨ_p⁺ = Δp - Δµ_H+·(z_H⁺·F)^-1 ΔΨ_p⁺ is the potential difference across the mitochondrial inner membrane (mtIM), expressed in the electric unit of volt [V]. Electric force of the mitochondrial membrane potential is the electric energy change per ‘motive’ charge or per charge moved across the transmembrane potential difference, with the number of ‘motive’ charges expressed in the unit coulomb [C].
NADH-Module		The NADH-Module, is a component of the NextGen-O2k for simultaneous measurement of oxygen consumption and NAD(P)H autofluorescence. NAD(P)H autofluorescence is used to evaluate the redox state of the NAD(P)H-pool. The NADH-Module incorporates an UV light and NADH-Sensors which include a photodiode and specific filters.
NADH-Sensor		The NADH-Sensor has been developed as a part of the NADH-Module for simultaneous monitoring of oxygen consumption and NADH redox state. The NADH-Sensor is composed of a photodiode and equipped with three supergel R370 Italian blue filters (Rosco, US).
NextGen-O2k
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 J_max and coupling state.
PB Light Source		The PhotoBiology Light Source (PBLS) has been designed as a part of the PB-Module to provide with an external source of light. This enables experiments for evaluating the production of O₂ in the presence of light. The PBLS consists of one LED and one photodiode mounted on the PBLS head protected by a PMMA plastic cover. Three pairs of PBLS (white, blue, and red) are provided with the PB-Module. The light intensity can be regulated from 0 to 2750 µmol·s^-1·m^-2 (red PBLS), from 0 to 3000 µmol·s^-1·m^-2 (blue PBLS), from 0 to 3500 µmol·s^-1·m^-2 (white PBLS). An integrated photodiode provides real-time measurement of the light intensity allowing for continuous adjustment to the desired value.
PB-Module		The PB-Module has been developed for conducting measurements of PhotoBiology, including photosynthesis. It consists of the PB Light Source and electronic components which are an integral part of the NextGen-O2k. Measurements are recorded and evaluated with the DatLab 8 software.
PhotoBiology	PB	PhotoBiology is the science of the effect of light on biological processes. This includes photosynthesis, photochemistry, photophysics, photomorphogenesis, vision, bioluminescence, circadian rhythms and photodynamic therapy. Phototoxicity results from non-ionizing radiation (i.e. ultraviolet, visible and infrared radiation). Non-ionizing 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 interact with molecules, the molecules can absorb the photon energy and become excited, reacting with surrounding molecules and stimulating "photochemical" and "photophysical" changes. Respiration may be affected by light during photosynthesis or in dark respiration, with the transient response of light-enhanced dark respiration.
Photosynthesis	PS	Photosynthesis is the process that converts light energy into chemical energy which is subsequently transformed to the physiological energy demand. Photosynthesis has a light-dependent and light-independent (dark) phase. In plants, algae, and cynobacteria, light energy is absorbed during the light phase by the pigment chlorophyll and used to split water and generate adenosine triphosphate (ATP) and reducing power - nicotinamide adenine dinucleotide phosphate (NADPH), with the net production of O₂ as a waste product. During the dark phase ATP and NADPH are used to synthesize carbohydrates from CO₂ through the metabolic pathway called Calvin-Benson cycle. Oxygenic photosynthesis is responsible for producing and maintaining the oxygen concentration of the Earth’s atmosphere. In bacteria such as cyanobacteria, photosynthesis involves the plasma membrane and the cytoplasm. In eukaryotic cells (plants and algae), photosynthesis takes place in the chloroplasts.
Q-Module	Q-Module	The Q-Module, developed for measuring the Q redox state and cyclic voltammetry, is supported by 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).