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柱前衍生-固相萃取-高效液相色谱荧光检测法测定生物脱氮菌群中痕量信号分子AI-2
黄晓遇, 谭炳琰, 李淳峰, 陈雨佳, 韦童, 荣宏伟, 储昭瑞
0
(广州大学土木工程学院)
摘要:
为揭示污水生物脱氮工艺中污泥菌群间的群体感应作用,建立了柱前衍生-固相萃取-高效液相色谱荧光检测法定量检测介导细菌种间群体感应信号分子AI-2的方法。取反应器的泥水混合液,经0.45 μm滤膜过滤后,用氨基磺酸掩蔽NO2-干扰,并与2,3-二氨基萘(DAN)发生衍生化反应,衍生化产物用C18固相萃取柱进行固相萃取,经氮吹浓缩后上机分析。采用C18色谱柱(4.6 mm×250 mm,5 μm)进行分离,乙腈与水(含0.1%甲酸)作为流动相进行梯度洗脱,使用荧光检测器(激发和发射波长分别为271 nm和503 nm)进行检测。本方法在1~200 ng/ml范围内呈现良好的线性关系,检出限为1ng/ml,回收率为55.08%~59.25%,相对标准误差为2.98%~10.41%。本方法适用于杂质干扰多的痕量信号分子AI-2 定量分析,可为生物脱氮工艺中信号分子AI-2介导群体感应研究提供有效分析方法。
关键词:  生物脱氮  群体感应  信号分子AI-2  高效液相色谱荧光检测法
DOI:
投稿时间:2018-08-07修订日期:2018-12-24
基金项目:广州大学大学生创新训练项目(201711078014);广东省教育厅青年创新人才项目(2016KQNCX121)
Detection of AI-2 Signal Molecules of Quorum Sensing in Biological Nitrogen Removal Processes by Pre-column Derivatization-Solid Phase Extraction-High Performance Liquid Chromatography with Fluorescence Detector
Huang Xiaoyu, Tan Bingyan, Li Chunfeng, Chen Yujia, Wei Tong, Rong Hongwei, Chu Zhaorui
(Department of Civil Engineering,Guangzhou University)
Abstract:
To reveal the mechanisms of quorum sensing in biological nitrogen removal processes, a method using HPLC-FLD with pre-column derivatization and solid-phase extraction for the quantitative determination of AI-2 signal molecules was established. Water-sludge mixtures sampled from the reactors which filtered with 0.45 μm filter membrane reacted with Amino-sulfonic acid to mask nitrite and derived with 2,3-diaminonaphthalene(DAN). Then, the derived products were extracted and concentrated by solid phase extraction (SPE). Thesamples were chromatographed by HPLC (C18 column, 4.6 mm×250 mm, 5 μm) with fluorescence detector (excitation wavelength and emission wavelength were 271 nm and 503 nm , respectively). Gradient elution was carried by acetonitrile and water (containing 0.1% formic acid) as mobile phases. The results presented an excellent linearity with the concentration ranging from 1 to 200 ng/ml while the detection limits were 1ng/ml. The recovery and relative standard deviation ranged from 55.08% to 59.25% and 2.98% to 10.41%, respectively. This method is used for the quantitative determination of trace signal molecule AI-2 with many
Key words:  Biological nitrogen removal  Quorum sensing  Autoinducer-2  HPLC-FLD