高效液相色谱-串联质谱法检测副溶血弧菌生物被膜形成过程中的N-酰基高丝氨酸内酯信号分子

摘要

N-acyl-homoserine lactones (AHLs)are typical quorum sensing signal molecules and regulate the expression of many physiological characteristic in V.parahaemolyticus.At present,there is a lack of accurate detection methods for AHLs in biofilms.Based on the existing conditions of HPLC-MS/MS,detection methods for AHLs could be optimized during the biofilm formation of V.parahaemolyticus.It was identified that absolute methanol:0.1％ ammonium acetate water as the mobile phase separation could help to obtain better results.If the collision energy was 10～ 15 eV,a higher proportion of characteristic fragment ions(m/z) 102.1 and 74.1 could be formed for each signal molecule.Good linear relationship was obtained in the wide range of 0～20 μg/L(R2 ＞0.995).This method could help to detect three different signal molecules,including C4-HSL,3-oxo-C6-HSL and 3-oxo-C14-HSL,which could be formed in the formation process of biofilms of V.parahaemolyticus.Among these signal molecules,the amount of C4-HSL was higher than others(＞91.67％).In the early stage of biofilm formation(0.5～3 d),the concentration of AHLs increased slowly and was positively relate to the biofilm formation.In the discursive stage(4～5 d),the concentration of signa molecules increased rapidly.Furthermore,a negative correlation could be found significouldtly between the concentration of signal molecules and biofilm formation.The optimization of this method could help to effectively detect the AHLs of biofilm and provided support for further study of regulative relations between biofilm and the quorumsensing as well.%N-酰基高丝氨酸内酯(AHLs)是副溶血弧菌的群体感应系统的信号分子,参与微生物的毒力调控,目前缺乏准确检测生物被膜中的信号分子的方法.在现有信号分子HPLC-MS/MS检测条件基础上,针对副溶血弧菌生物被膜生成过程中信号分子定性定量检测方法进行优化.结果显示:无水甲醇∶0.1％乙酸铵水为流动相分离效果较优;当选择碰撞能量为10～15 eV时,各信号分子均可形成较高比例的特征碎片离子(m/z)102.1和74.1,并在0～20 μg/L范围内均有良好的线性关系(R2＞0.995),采用该法能够检测副溶血性弧菌生物被膜形成过程中产生的C4-HSL、3-oxo-C6-HSL和3-oxo-C14-HSL三种信号分子,其中C4-HSL的量占绝对优势(＞91.67％).在生物被膜形成的前期(0.5～3d),信号分子的浓度缓慢增长,与生物被膜的生成量成正相关;当被膜进入消散期时(4～5 d),信号分子的浓度快速增长,与被膜生成量呈显著负相关.高效液相色谱-串联质谱法的优化有助于更加有效地检出生物被膜中的信号分子,为更好地认识生物被膜与群体感应信号分子间的调控关系提供支撑.