Oxygen Consumption Rate Measurements in Closed Microrespirators

Applied to seeds, as shown as example in this application note, oxygen dynamics reveal the metabolic switch that marks the transition from quiescence to active growth. This format captures the seeds’ imbibition lag, the onset of respiratory activation, and sustained O₂ consumption during germination—metrics that quantify vigor, detect early damage, and evaluate priming or stress treatments in a scalable, reproducible way via assessing the respective Oxygen Consumption Rate (OCR).

Individual Broccoli and White Cabbage seeds act as sample in this respiration study. 100 µL of a 80 °C 2% (w/v) Phytagel solution was added to each well of a 96 well PhoxyPlate 96 O. Phytagel formed a stable gel layer upon cooling to room temperature. After cooling of the gel one individual seed was placed onto the Phytagel in each well. The plate was vertically split in half portions for the two seed types. One row acted as blank control (Fig. 2). The hydrogel serves several purposes in this study: it provides a defined water reservoir that is needed to activate the seeds, keeps the humidity in the chambers constant, and prevents direct contact of the seed to the sensor material on the bottom of the well hampering potential strong local on-sensor variations that would generate a measurement bias. After pacing the seeds into the wells, the plate was sealed with aluminum foil sealing film for well plates to prevent ambient air ingress. The samples were placed in a temperature-controlled cabinet at 18 °C at 1 atm air pressure and monitored every 5 minutes for a period of ~ 6 days in total.

Seeds undergo an initial imbibition phase (day 0 - 2) where they soak up some water and get activated. Seeds that start their germination process (day 2 - 3), thus activate their metabolism which results in stronger O₂ consumption due to respiration. O₂ levels in the closed respiration chambers drop over time. 5 out of 42 investigated White Cabbage seeds (in wells A5, E4, E5, F3, F5) reported no oxygen drop after their imbibition phase, which is a clear sign of a failure to germinate (see Fig. 3). The non-germinating rate of the investigated White Cabbage seeds was found to be 88 % compared to a 100 % germination rate with the Broccoli seed samples investigated.

This study focuses on the main respiration effect (OCR) during the germination phase showing a higher metabolic activity compared to the imbibition phase.  From each parallel respiration measurement, the steepest O₂ change in % a.s. per hour can be determined. Therefore, the maximum OCR per well is calculated (Fig. 4) from the oxygen decline between two individual measurement points in 4 hours’ time interval. The chosen time interval can be adapted for samples showing higher or lower respiration activity. For relatively slow small-seed respiration a 4 hour interval is a good basis.

The pO₂ in % a.s. can be transferred to amounts of O₂ in moles or nmoles which is an amount of substance based unit by a conversion factor. For a one phase system that is straightforward using either ideal gas law (gaseous system) or Henry’s law (aqueous phase system). In the shown example, each well contained 100µl of Phytagel Hydrogel and 260µl of air in the headspace and therefore is a two phase system. Assuming an equilibrium between two phases the total amount of oxygen at 18°C and 1atm pressure can be calculated from the ideal gas law for the air phase (1 % a.s. pO₂ = 22.3nmol) + the dissolved oxygen in 100µl aqueous phase (1 % a.s. pO₂ = 0,254 nmol) from Henry’s law. The total conversion factor for the two-phase system was calculated to be 1 % a.s. pO₂ = 22.6 nmol O₂. The average OCR for the two investigated sample types are 49,8 ± 15 nmol/hour per White Cabbage seed (excluding the 5 non-germinating samples) and 69,6 ± 20 nmol/hour per Broccoli seed.

The 96‑well, closed‑chamber format demonstrated here with PhoxyCube along with PhoxyPlate 96 O sensor equipped well plates enables true parallel, time‑resolved respiration measurements—each well functioning as an independent respirometer—while preventing ambient gas exchange and cross‑contamination. This configuration yields scalable, quantitative metrics (e.g., maximum O₂ decline, converted to nmol (O₂) per h) and supports adaptable sampling intervals to match different respiration rates. Applied beyond seeds, the approach can be used with diverse small biological samples or treatments for high‑throughput phenotyping, dose–response testing, and temporal kinetic studies.