pH敏感型荧光纳米胶束用于烟用纸张中大肠杆菌的快速检测
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摘要
为了建立烟用纸张中大肠杆菌的快速检测方法,使用pH敏感型荧光染料异硫氰酸荧光素对氨基修饰的磷脂聚乙二醇进行共价修饰制备纳米胶束单体,通过自组装在磷酸盐缓冲溶液中形成荧光纳米胶束,加入到热琼脂溶液中制备荧光传感凝胶,荧光传感凝胶和大肠杆菌液体培养基通过水相滤膜隔离构建大肠杆菌荧光传感器,大肠杆菌荧光传感器通过检测大肠杆菌生长过程中pH的变化实现大肠杆菌的快速检测。结果表明:(1)制备的纳米胶束的粒径为100～500 nm,激发和发射波长分别在480和520 nm;传感凝胶在pH 4.5～9.0之间随pH的升高,荧光信号快速增加。(2)构建的大肠杆菌荧光传感器基于传统培养理论可以实现烟用纸张中大肠杆菌的快速检测,检测时间相比传统平板计数法大幅缩短,可以用来评估烟用纸张中微生物的数量。
In order to develop a quick method for detecting the Escherichia coli(E. coli) in cigarette paper,pH-sensitive fluorescent dye fluorescein isothiocyanate(FITC) was coupled with amino modified DSPE-PEG2000 to form fluorescent self-assembled nano-micelles in phosphate buffer saline(PBS)and used to prepare pH-sensitive fluorescent sensory gel. The fluorescent sensors were thus developed by separating fluorescent sensory gel from liquid culture medium for E. coli using aqueous phase filter membrane, and the quick detection of E. coli was realized via monitoring the change of physiological pH values during the growing procedure of E. coli. The results showed that: 1) The sizes of nano-micelles ranged from 100 to 500 nm,excitation and emission wavelengths were 480 and 520 nm respectively. The fluorescent intensity of the sensory gel heightened when pH values raised from 4.5 to 9.0. 2)On the basis of traditional culture theory, fluorescent sensors could be used for the quick detection of E. coli in cigarette paper within much shorter screening time compared with traditional plate counting method. This method is suitable for assessing the amount of E. coli in cigarette paper.
In order to develop a quick method for detecting the Escherichia coli(E. coli) in cigarette paper,pH-sensitive fluorescent dye fluorescein isothiocyanate(FITC) was coupled with amino modified DSPE-PEG2000 to form fluorescent self-assembled nano-micelles in phosphate buffer saline(PBS)and used to prepare pH-sensitive fluorescent sensory gel. The fluorescent sensors were thus developed by separating fluorescent sensory gel from liquid culture medium for E. coli using aqueous phase filter membrane, and the quick detection of E. coli was realized via monitoring the change of physiological pH values during the growing procedure of E. coli. The results showed that: 1) The sizes of nano-micelles ranged from 100 to 500 nm,excitation and emission wavelengths were 480 and 520 nm respectively. The fluorescent intensity of the sensory gel heightened when pH values raised from 4.5 to 9.0. 2)On the basis of traditional culture theory, fluorescent sensors could be used for the quick detection of E. coli in cigarette paper within much shorter screening time compared with traditional plate counting method. This method is suitable for assessing the amount of E. coli in cigarette paper.
引文
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[3]陈樑.水环境中大肠杆菌PCR快速检测体系的研究[J].环境科技,2010,23(2):52-54,58.CHEN Liang.Research on PCR-based rapid detection system of Escherichia coli in water environment[J].Environmental Science and Technology,2010,23(2):52-54,58.
[4]Puligundla P,Jung J,Ko S.Carbon dioxide sensors for intelligent food packaging applications[J].Food Control,2012,25(1):328-333.
[5]张峻峰,刘道亮,赵占民,等.流式细胞术在微生物快速检测领域的研究进展[J].食品工程,2010(3):19-22.ZHANG Junfeng,LIU Daoliang,ZHAO Zhanmin,et al.Research development on flow cytometry in the rapid detection of microorganisms[J].Food Engineering,2010(3):19-22.
[6]Shin J,Braun P V,Lee W.Fast response photonic crystal pH sensor based on templated photopolymerized hydrogel inverse opal[J].Sensors and Actuators B:Chemical,2010,150(1):183-190.
[7]Nopwinyuwong A,Trevanich S,Suppakul P.Development of a novel colorimetric indicator label for monitoring freshness of intermediate-moisture dessert spoilage[J].Talanta,2010,81(3):1126-1132.
[8]Wang X D,Meier R J,Wolfbeis O S.Fluorescent pH-sensitive nanoparticles in an agarose matrix for imaging of bacterial growth and metabolism[J].Angewandte Chemie International Edition,2013,52(1):406-409.
[9]Pfannebecker J,Schiffer-Hetz C,Fr?hlich J,et al.Culture medium optimization for osmotolerant yeasts by use of a parallel fermenter system and rapid microbiological testing[J].Journal of Microbiological Methods,2016,130:14-22.
[10]Sch?ferling M.Nanoparticle-based luminescent probes for intracellular sensing and imaging of pH[J].Wiley Interdisciplinary Reviews:Nanomedicine and Nanobiotechnology,2016,8(3):378-413.
[11]Schreml S,Meier R J,Wolfbeis O S,et al.2Dluminescence imaging of pH in vivo[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(6):2432-2437.
[12]Schulz A,Wotschadlo J,Heinze T,et al.Fluorescent nanoparticles for ratiometric pH-monitoring in the neutral range[J].Journal of Materials Chemistry,2010,20(8):1475-1482.
[13]Wu D Q,Lu B,Chang C,et al.Galactosylated fluorescent labeled micelles as a liver targeting drug carrier[J].Biomaterials,2009,30(7):1363-1371.
[14]Rein J,Zimmermann B,Hille C,et al.Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly[J].Journal of Experimental Biology,2006,209(9):1716-1724.
[15]陈瑞希,周宏福,刘秀丽,等.pH敏感的荧光探针及在活细胞中的应用[J].激光生物学报,2016,25(6):491-500.CHEN Ruixi,ZHOU Hongfu,LIU Xiuli,et al.pH-sensitive fluorescence probes and their applications in living cells[J].Acta Laser Biology Sinica,2016,25(6):491-500.