A sensitive and reliable method for the quantitative determination of hydrogen peroxide produced by microalgae cells

Area of Science:

• Phycology
• Biochemistry
• Cellular Biology

Background:

• Hydrogen peroxide (H2O2) is a key reactive oxygen species involved in cellular processes.
• Detecting H2O2 in microalgae is challenging due to pigments and cell walls.
• Existing methods are insufficient for accurate H2O2 determination in algal cultures.

Purpose of the Study:

• To optimize the Amplex UltraRed method for H2O2 detection in microalgae.
• To develop a reliable, quick, and simple H2O2 detection protocol for phycological research.
• To use Chlamydomonas reinhardtii as a model organism for method optimization.

Main Methods:

• Fluorometric detection using the Amplex UltraRed assay.
• Optimization of reaction buffer (potassium phosphate), excitation wavelength (560 nm), and sample-to-reagent ratio (50:50).
• Assessment of microalgae biomass density and sample fortification effects on fluorescence signal.

Main Results:

• Potassium phosphate buffer and a 560 nm excitation wavelength were optimal.
• A 50:50 reaction mixture to sample ratio yielded the best results.
• Microalgae biomass density significantly influenced the fluorescence signal.
• Sample fortification improved method reliability and repeatability.

Conclusions:

• The optimized Amplex UltraRed method provides sensitive and accurate H2O2 detection in microalgae.
• This protocol effectively minimizes errors, eliminates chlorophyll autofluorescence, and compensates for matrix effects.
• The method is applicable to various microalgae species for H2O2 studies.