Rapid and Sensitive Quantification of Bacterial Viability Using Ratiometric Fluorescence Sensing

化学 荧光 色谱法 光学 物理
作者
Shengbin He,Yajing Chen,Jingtong Wang,Jian Sun,Xinyi Zhang,Quanzhi Chen
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:96 (27): 11018-11025 被引量:7
标识
DOI:10.1021/acs.analchem.4c01737
摘要

Bacterial viability assessment plays an important role in food-borne pathogen detection and antimicrobial drug development. Here, we first used GelRed as a DNA-binding stain for a bacterial viability assessment. It was found that live bacteria were able to exclude GelRed, which however could easily penetrate dead ones and be absorbed nonspecifically on the bacterial periplasm. Cations were used to reduce the nonspecific adsorption and greatly increase the red fluorescence ratio of dead to live bacteria. Combined with SYTO 9 (a membrane-permeable dye) for double-staining, a ratiometric fluorescent method was established. Using Escherichia coli O157:H7 as a bacteria model, the ratiometric fluorescent method can probe dead bacteria as low as 0.1%. A linear correlation between the ratiometric fluorescence and the theoretical ratio of dead bacteria was acquired, with a correlation coefficient R2 of 0.97. Advantages in sensitivity, accuracy, and safety of the GelRed/SYTO9-based ratiometric fluorescent method against traditional methods were demonstrated. The established method was successfully applied to the assessment of germicidal efficacy of different heat treatments. It was found that even 50 °C treatment could lead to the death of minor bacteria. The as-developed method has many potential applications in microbial researches, and we believe it could be expanded to the viability assessment of mammalian cells.
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