NextGen-O2k Technical developments

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NextGen-O2k Technical developments

NextGen-O2k

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

2020-10-15: Dispatch of 2 prototypes with integrated PB-Modules for testing at Tomas Morosinotto’s lab in Padua, IT and Yagut Allahverdiyeva-Rinne’s Lab in Turku, FI
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


Links and references

MitoPedia: NextGen-O2k

» NextGen-O2k«
TermAbbreviationDescription
Hydrogen peroxideH2O2
Hydrogen peroxide
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 potentialmtMP, 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 InstrumentNextGen-O2k Instrument
NextGen-O2k Technical developments
Oxygen kineticsOxygen 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-ModulePB-ModuleThe PB-Module has been developed for conducting measurements of PhotoBiology, including photosynthesis. It consists of the PB-Sensor and electronic components which are an integral part of the NextGen-O2k. Measurements are recorded and evaluated with the DatLab 8 software.
PB-SensorThe PB-Sensor has been designed as a part of the PB-Module to provide with an external source of light, in the range of the blue (451 nm) or red (634 nm) wavelengths. This enables 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 emits with blue or red light on the sample and its intensity can be regulated from 0 to 5000 µEinsteins*m-2*s-1. The photodiode provides real-time measurement of the light intensity allowing for continuous adjustment to the desired value, which can also help compensate for the temperature rise due to the operation of the LED.
Q-ModuleQ-ModuleThe 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-SensorThe 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).
Template NextGen-O2k.jpg


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Bioblast links: NADH - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Mitochondrial NADH and respiration

» Nicotinamide adenine dinucleotide
» NADH electron transfer-pathway state
» N-junction
» Complex I
» Rotenone
» Amytal
» Piericidin

Mitochondrial NADH production

» Tricarboxylic acid cycle
» Fatty acid oxidation

NextGen-O2k

» NextGen-O2k




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Bioblast links: Hydrogen peroxide - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>



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Bioblast links: ATP production - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Phosphorylation pathway

» Phosphorylation pathway
» ATP synthase
» Adenine nucleotide translocase
» Phosphate carrier

Phosphorylation pathway substrates

» ADP
» ATP
» Inorganic phosphate

Phosphorylation pathway inhibitors

» Oligomycin
» Carboxyatractyloside
» Atractyloside
» Bongkrekik acid

Coupling control

» Oxidative phosphorylation
» OXPHOS capacity
» P-L control efficiency
» ROUTINE respiration
» R-L control efficiency

Respiratory complexes and coupling

» Complex I
» Complex III
» Complex IV
» Proton pump
» Electron-transfer-pathway state

ATP production measurement

» Magnesium Green



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Bioblast links: Calcium - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>



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Bioblast links: PhotoBiology and plant physiology - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

PhotoBiology: photosynthesis

» PhotoBiology
» Photosynthesis

Plant physiology: respiration

» Photorespiration
» Chlororespiration

NextGen-O2k and PB-Module

» NextGen-O2k
» PB-Module
» PB-Sensor



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MitoPedia:NextGen-O2k