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Selective fluorescence labeling and sensitive determination of Staphylococcus aureus by microchip electrophoresis with a multiple-concentration approach

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Chinese Chemical Letters 2018年第9期29卷 1383-1386页

作者： Yan Zhang Yi Zhang Yating Zhang Luqi Zhu Pingang He Qingjiang Wang School of Chemistry and Molecular Engineering East China Normal University

The detection of Staphylococcus aureus （S. aureus） is very important as it is responsible for bacterial infectious diseases and food poisoning. In this paper we explored the application of fluorescently labelled vancomycin to specifically bind and detect S. aureus. In view of the specificity of vancomycin towards bacterial cell surfaces, we utilized Cy5 to label vancomycin （CyS-Van） for the identification of S. aureus. Our experiments were designed to examine in greater detail the specificity of the reaction between CyS- Van and S. aureus. Detection parameters such as the derivatization conditions, concentrations of buffer, pH value, response performance of CyS-Van to bacterial surface, injection time and reversed-polarity time have been investigated and optimized. To develop a simple and quick assay for the detection of S. aureus at low concentrations, we propose to use the Cy5-Van for labeling the 5. aureus coupled with an on-line multiple-concentration in microcbip electrophoresis. Under the optimized conditions, the detection orS. aureus was achieved within 150 s with limit of detection （S/N = 3） of 981 CFU/mL, and 350- fold enhancement was obtained for S. aureus as compared to using the no concentration step. It is self- evident that this approach has great potential in the future for the analysis of S. aureus.

关键词： Bacteria Vancomycin microchip electrophoresis Multiple-concentration

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Rapid detection of high-risk HPV16 and HPV18 based on microchip electrophoresis

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Journal of Pharmaceutical Analysis 2020年第4期10卷 329-333页

作者： Zhaoxuan Fan Xiao Feng Weifei Zhang Xueji Zhang Jin-Ming Lin Research Center for Bioengineering and Sensing Technology School of Chemistry and Biological EngineeringUniversity of Science and Technology BeijingBeijing100083PR China Department of Chemistry Beijing Key Laboratory of Microanalytical Methods and InstrumentationMOE Key Laboratory of Bioorganic Phosphorus Chemistry&Chemical BiologyTsinghua UniversityBeijing100084PR China State Key Laboratory of Chemical Resource Engineering Beijing University of Chemical TechnologyBeijing100029PR China School of Biomedical Engineering Shenzhen UniversityShenzhenGuangdong 518060PR China

Researches on detection of human papillomavirus(HPV)high-risk samples were carried out by polymerase chain reaction(PCR)coupled with microchip electrophoresis(MCE).Herein,we introduced a simple,rapid,automated method for detecting high-risk samples HPV16 and *** this research,general primers were initially selected to obtain sufficient detectable yield by PCR to verify feasibility of MCM method for HPV detection,then type-specific primers were further used to evaluate the specificity of MCE *** results indicated MCE method was capable of specifically detecting high-risk HPV16 and HPV18,and also enabled simultaneous detection of multiplex *** MCE method described here has been successfully applied to HPV detection and displayed excellent reliability demonstrating by sequencing *** inherent capability of MCE facilitated HPV detection conducted in a small chip with automated,high throughput,massive parallelized *** envision that MCE method will definitely pave a way for clinical diagnosis,and even on-site screening of cervical cancer.

关键词： Human papillomavirus DNA analysis Polymerase chain reaction microchip electrophoresis Detection

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Rapid Determination of Free Amino Acids in Milk by microchip electrophoresis Coupled with Laser-induced Fluorescence Detection

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Chinese Journal of Chemistry 2012年第10期30卷 2517-2521页

作者：吴敏成双李刚汪志芳王清江何品刚方禹之 Department of Chemistry East China Normal University Shanghai 200062 China

A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis （MCE） coupled with laser-induced fluorescence （LIF） detection was developed. Seven kinds of standard amino ac- ids were derivated with sulfoindocyanine succinimidyl ester （Cy5） and then perfectly measured by MCE-LIF within 150 s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions： 100 mmolo L^-1 sodium borate solution （pH 10.0） as running buffer solution, 0.8 kV as injection voltage, 2.2 kV as separation voltage etc. The linear range of the detection of amino acids was from 0.01 μmol·L^-1 to 1.0 μmol·L^-1 and the detection limit was as low as about 1.0 μmol·L^-1. This MCE-LIF method was applied to the measurements of free amino acids in actual milk samples and satisfactory experimental results were achieved.

关键词： free amino acids microchip electrophoresis laser-induced fluorescence detection milk

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A Simple Evaluation System for Microbial Property in Soil and Manure

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Advances in Microbiology 2016年第2期6卷 88-97页

作者： Naoto Horinishi Kunimasa Matsumoto Katsuji Watanabe Department of Life Environment and Materials Science Graduate School of Engineering Fukuoka Institute of Technology Fukuoka Japan

Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and the enormous population of soil microorganisms [1], the other was an existence of numerically dominant unculturable microorganisms which comprise 99% of soil habitat [2]. We evaluated whether our newly developed method, by which taxonomies and their number of each bacterial groups were estimated, could be used as evaluation method of microbial properties of soils and manures. In the forest soil, β-Proteobacteria, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacteria (3.64 × 106 MPN g-1 dry soil), followed by γ-Proteobacteria (1.32 × 106 MPN), δ-Proteobacteria (0.006 × 106 MPN), and the other gram negative bacteria (0.006 × 106 MPN). In the commercial manure, Actinobacteria, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 106 MPN), followed by α-Proteobacteria (26.0 × 106 MPN), β-Proteobacteria (17.1 × 106 MPN), δ-Proteobacteria (11.2 × 106 MPN), the other Firmicutes (1.71 × 106 MPN), γ-Proteobacteria (0.5 × 106 MPN), and the other gram negative bacteria (0.05 × 106 MPN). In the upland field, the other Firmicutes, which included Paenibacillus sp., was numerically dominant bacteria (4.41 × 106 MPN), followed by Actinobacteria (2.14 × 106 MPN), Bacillus sp. (2.14 × 106 MPN), and γ-Proteobacteria (0.35 × 106 MPN). Although the precision of the affiliations became lower because of higher diversity of samples and the number of some Antinobacteria and Firmicutes might be underestimated by the used PCR condition, the method was

关键词： Evaluation System Microbial Property Soil and Manure Multiple Enzyme Restriction Fragment Length Polymorphism Analysis The Most Probable Number Method microchip electrophoresis

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Identification and Enumeration Method of Both Eukaryotic and Prokaryotic Microorganisms in Food Sample

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Food and Nutrition Sciences 2016年第5期7卷 345-354页

作者： Katsuji Watanabe Naoto Horinishi Kunimasa Matsumoto Yuji Sogabe Department of Life Environment and Materials Science Graduate School of Engineering Fukuoka Institute of Technology Fukuoka Japan Global Application Development Center Shimadzu Co. Kyoto Japan

The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g-1, and Penicillium spp., was enumerated to be 1.5 × 106 MPN g-1. In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g-1. In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g-1) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g-1), and the other Firmicutes (3.7 × 106 MPN g-1). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g-1), Burkhokderia sp. (1.5 × 106 MPN g-1), and the other α,β,γ-proteobacteria (0.12 × 106 MPN g-1). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.

关键词： Eukaryotic Microorganisms Prokaryotic Microorganisms Multiple Enzyme Restriction Fragment Length Polymorphism Analysis the Most Probable Number Method microchip electrophoresis

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人膀胱癌脱落细胞捕获及其基因检测分析

人膀胱癌脱落细胞捕获及其基因检测分析

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作者：邓勇北京化工大学

学位级别：硕士

膀胱癌是人泌尿系统中一种常见的癌症,非侵入性早期检测膀胱癌在临床诊断上具有重要的意义,并且得到了广泛的研究。本文通过研究尿液中脱落细胞捕获和研究肿瘤相关基因检测两方面内容,提高了细胞的捕获效率,增强了检测的灵敏度,希望应... 详细信息

膀胱癌是人泌尿系统中一种常见的癌症,非侵入性早期检测膀胱癌在临床诊断上具有重要的意义,并且得到了广泛的研究。本文通过研究尿液中脱落细胞捕获和研究肿瘤相关基因检测两方面内容,提高了细胞的捕获效率,增强了检测的灵敏度,希望应用于膀胱癌的早期临床诊断或者预后中。研究的主要内容和结论如下：第一章综述了体液中脱落肿瘤细胞的分离捕获方法,详细阐述了膀胱癌早期检测的方法及最新进展。在此基础上提出了论文的研究思路和方案。第二章膀胱癌发生发展过程中肿瘤细胞会脱落到尿液中,为非侵入性检测膀胱癌提供了可能。本文通过采用聚碳酸酯微孔膜和镍微孔筛快速高效的捕获尿液中的膀胱癌细胞,并且利用二氯甲烷溶膜的特性,简单快捷的回收捕获在聚碳酸酯微孔滤膜上的细胞。微孔过滤法捕获细胞效率>85%,分离富集时间<20min,明显优于临床上传统的离心分离富集方法。本实验较为系统的考察了滤膜类型、固定剂以及流速对肿瘤细胞捕获效率的影响,该方法速度快,成本低,并且环境友好,为今后应用于临床诊断研究提供了可能性及理论依据。第三章体细胞突变和甲基化在肿瘤发生过程中起着重要作用。本文设计了通过基因分析非侵入性检测膀胱癌的标志物,该方法包括体细胞突

关键词： bladder cancer cell isolation microfiltration somaticmutation DNA methylation microchip electrophoresis

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