Hejna Monika, Kapuścińska Dominika, Aksmann Anna
Department of Biotechnology and Nutrigenomics, Institute of Genetics and Animal Biotechnology of the Polish Academy of Sciences, Magdalenka, Poland.
Department of Plant Physiology and Biotechnology, Faculty of Biology, University of Gdańsk, Gdańsk, Poland.
J Phycol. 2024 Dec;60(6):1356-1370. doi: 10.1111/jpy.13524. Epub 2024 Nov 25.
One of the reactive forms of oxygen is hydrogen peroxide (HO), which has been investigated as a key component of growth processes and stress responses. Different methods for the determination of HO production by animal and bacterial cells exist; however, its detection in algal cell cultures is more complicated due to the presence of photosynthetic pigments in the cells and the complex structure of cell walls. Considering these issues, a reliable, quick, and simple method for HO detection is needed in phycological research. The aim of this methodological study was to optimize an Amplex UltraRed method for the fluorometric detection of HO produced by microalgae cells, using a wild-type strain of Chlamydomonas reinhardtii as a model. The results showed that (i) potassium phosphate is the most suitable reaction buffer for this method, (ii) a 560 nm wavelength variant is the most appropriate as the excitation wavelength for fluorescence spectra measurement, (iii) a 50:50 ratio for the reaction mixture to sample was the most suitable, (iv) the fluorescence signal was significantly influenced by the density of the microalgae biomass, and (v) sample fortification with HO allowed for an increase of the method's reliability and repeatability. The proposed protocol of the Amplex UltraRed method for the fluorometric detection of HO produced by microalgae cells can yield a sensitive and accurate determination of the content of the test compound, minimizing measurement errors, eliminating chlorophyll autofluorescence problem, and compensating for the matrix effect. This method can be applied to the study of other microalgae species.
活性氧的一种形式是过氧化氢（HO），它已被作为生长过程和应激反应的关键成分进行研究。存在多种用于测定动物和细菌细胞中HO产生量的方法；然而，由于藻类细胞中存在光合色素以及细胞壁结构复杂，在藻类细胞培养物中检测HO更为复杂。考虑到这些问题，藻类学研究需要一种可靠、快速且简单的HO检测方法。本方法学研究的目的是优化一种Amplex UltraRed方法，以荧光法检测微藻细胞产生的HO，使用莱茵衣藻野生型菌株作为模型。结果表明：（i）磷酸钾是该方法最合适的反应缓冲液；（ii）560 nm波长变体是荧光光谱测量最合适的激发波长；（iii）反应混合物与样品的比例为50:50最合适；（iv）荧光信号受微藻生物量密度的显著影响；（v）用HO强化样品可提高该方法的可靠性和可重复性。所提出的用于荧光检测微藻细胞产生的HO的Amplex UltraRed方法方案能够灵敏且准确地测定被测化合物的含量，将测量误差降至最低，消除叶绿素自发荧光问题，并补偿基质效应。该方法可应用于其他微藻物种的研究。
