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基于异硫氰酸荧光素标记溶菌酶和荷正电纳米金构建荧光共振能量转移平台检测广谱细菌
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作者单位
王玲,张亚青,陶晓奇,宋尔群 1. 西南大学 食品科学学院重庆 4007152. 西南大学 药学院重庆 400715 
基金项目:The National Natural Science Foundation of China(22174116,21974110);Chongqing Science Funds for Distinguished Young Scientists(cstc2021jcyj-jqx0024);The Innovation Research Group at Higher Education Institutions in Chongqing,Chongqing Education Committee(CXQT21006)
中文摘要:通过异硫氰酸荧光素标记的溶菌酶(FITC-Lys)和聚乙烯亚胺修饰的荷正电纳米金粒子(PEI-AuNPs)与细菌结合,构建了一个基于荧光共振能量转移(FRET)的平台用于广谱细菌检测。待检样本中存在细菌时,荷正电的FITC-Lys(能量供体)通过与细胞壁肽聚糖的识别作用和静电作用与细菌结合,荷正电的PEI-AuNPs(能量受体)也通过静电作用与细菌结合,使能量供体和受体之间的距离缩短,荧光猝灭,显示出低荧光强度的FRET“开”的信号读出模式。采用水热法合成了PEI-AuNPs,通过紫外光谱、透射电镜、激光粒度仪等对PEI-AuNPs进行了表征。结果表明,PEI-AuNPs在526.5 nm处有最大紫外吸收,直径约为9.6 nm,荷正电。该方法可对10种细菌(如金黄色葡萄球菌、肺炎链球菌、粪肠球菌、表皮葡萄球菌、李斯特菌、大肠杆菌、鼠伤寒沙门氏菌、甲型副伤寒沙门氏菌、副溶血弧菌和铜绿假单胞菌)进行检测。考察了实际样本(以牛奶和果汁为例)中可能的干扰组分对检测的干扰情况。结果表明,将样本稀释10倍后,样本中蛋白、离子等不会对分析结果产生干扰。该研究构建的广谱细菌检测方法,对于致病菌的防控具有积极意义。
中文关键词:细菌  ?溶菌酶  ?纳米金颗粒  ?荧光共振能量转移
 
Detection of Broad Spectrum Bacteria Using a FITC-Lysozyme and Positively Charged AuNPs Constructed FRET Platform
Abstract:This manuscript demonstrated a universal platform for bacteria assay by using a fluorescein isothiocyanate-labeled lysozyme(FITC-Lys) and polyethylenimine-modified positively charged gold nanoparticles(PEI-AuNPs) constructed fluorescence resonance energy transfer(FRET) sensor based on its electrostatic binding with bacteria. In the presence of the bacteria,the energy donor of positively charged FITC-Lys bond with the bacteria through both the recognition to peptidoglycan of cell wall and electrostatic interaction while the energy acceptor of positively charged PEI-AuNPs bond with the bacteria based on the electrostatic interaction,which decreases the distance between energy donor and acceptor and then causes fluorescence quenching,showing FRET“on”signal readout mode with the low fluorescence intensity. PEI-AuNPs were synthesized by hydrothermal method and characterized by ultraviolet spectrum,transmission electron microscope,and laser particle size analyzer. The results shown that PEI-AuNPs with diameter about 9.6 nm had maximum ultraviolet absorption at 526.5 nm,and they were positively charged. The method developed in this study could detect 10 different kinds of bacteria such as?Staphylococcus aureus,Streptococcus pneumoniae,Enterococcus faecalis,Staphylococcus epidermidis,Listeria monocytogenes,Escherichia coli,Salmonella typhimurium,Salmonella paratyphoid A,Vibrio parahaemolyticus,and?Pseudomonas aeruginosa. Then,the interference of possible interfering components in actual samples(taking milk and juice as examples) on the detection was further discussed. The results showed that under our experimental conditions,the interfering components such as proteins,ions,and others in the samples would not interfere with the analysis results after ten times dilution. This strategy showed feasibility as a universal assay platform for broad spectrum bacteria,which is interesting for the control of pathogenic bacteria.
Key Words:bacteria  ?lysozyme  ?gold nanoparticle  ?FRET
引用本文:王玲,张亚青,陶晓奇,宋尔群.基于异硫氰酸荧光素标记溶菌酶和荷正电纳米金构建荧光共振能量转移平台检测广谱细菌[J].分析测试学报,2022,41(4):639-645.
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