Analysis of intact bacteria, bacterial DNA and mutagenic alkaloids by capillary electrophoresis

ANALYSIS OF INTACT BACTERIA, BACTERIAL DNA AND MUTAGENIC ALKALOIDS BY CAPILLARY ELECTROPHORESIS YU LIJUN (M. Sc., Xiamen University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements Foremost, I would like to extend my sincere thanks to my supervisor, Professor Sam Fong Yau Li for his invaluable guidance and encouragement throughout this study. Under his guidance, not only did I gain precious research experience, but also the attitude to be a researcher. I am grateful to my colleagues, Dr. Qin Weidong, Dr. Feng Huatao, Dr. Yuan Lingling, Xu Yan, Law Waisiang, Lau Hiufung, Tay Teng Teng Elaine, Jiang Zhangjian and Junie Tok who gave me their hands during my candidature. Without their favor, my research could not go ahead successfully. I am also thankful to the staff in the department of chemistry, in particular Mrs Lim Francis and Ms Tang Chui Ngoh for their kind help. I thank the National University of Singapore for providing me the financial support to carry out the research. Lastly, I would like to appreciate my family for the love, support and encouragement. I Table of Contents Acknowledgements I Table of Contents II Summary VII Chapter Introduction 1.1 Electrophoresis 1.2 History of capillary electrophoresis 1.3 Basic principles of capillary electrophoresis 1.4 Different modes of capillary electrophoresis 19 1.5 Instrumentation for capillary electrophoresis 24 1.6 Scope of research 32 Part I 35 Analysis of intact bacteria and bacterial DNA Chapter Electrophoretic behavior analysis of intact bacteria by capillary electrophoresis 35 2.1 Introduction 35 2.2 Experimental section 44 2.2.1 Chemicals and materials 44 II 2.2.2 Apparatus 45 2.2.3 Capillary electrophoresis 46 2.2.4 Bacterial sample preparation 47 2.2.5 Fluorescence labeling with SYTO 13 dye 48 2.3 Results and discussion 50 2.3.1 Basic theory 50 2.3.2 Electrophoretic behavior study of bacteria by CE methods 51 2.3.2.1 Effect of bacterial sample pretreatment 51 2.3.2.2 Effect of ionic strength on electrophoretic mobilities of bacteria 54 2.3.2.3 Separation of bacteria with UV and fluorescence detection 57 2.3.3 Determination of pathogenic bacteria (Edwardsiella tarda) in fish species by capillary electrophoresis with blue LED induced fluorescence 61 2.3.3.1 Effect of pH on EOF and migration behavior of bacteria 61 2.3.3.2 Quantitative analysis 65 2.3.3.3 Fish fluids analysis after direct injection of bacteria 66 69 2.4 Conclusions Chapter Analysis of DNA by capillary electrophoresis with laser induced fluorescence detection 70 3.1 Introduction 70 3.2 Experimental section 74 III 3.2.1 Equipment 74 3.2.2 Chemicals 74 3.2.3 Capillary coating 75 3.2.4 Viscosity measurement 76 3.2.5 CE performance 76 3.2.6 PCR products from bacteria EHEC gene 77 3.3 Results and discussion 77 3.3.1 Mechanism of DNA movement in entangled polymer solutions 77 3.3.2 Effect of YO-PRO-1 dye on resolution of DNA 80 3.3.3 PVP as a sieving matrix 83 3.3.4 Comparison of PVP and other polymers 85 3.3.5 Effect of PVP polymer concentration 89 3.3.6 Optimization of MES/TRIS/PVP system 91 3.3.7 Fitting of DNA separation models 96 3.3.8 Analysis of PCR products 97 3.4 Conclusions 100 Part II Analysis of mutagenic pyrrolizidine alkaloids 101 Chapter Analysis of mutagenic pyrrolizidine alkaloids in traditional Chinese by capillary electrophoresis 101 IV 4.1 Introduction 101 4.2 Experimental section 109 4.2.1 Materials 109 4.2.2 Instrumentation and methods 110 4.2.2.1 HPLC conditions 110 4.2.2.2 CE conditions 111 4.2.3 Standard sample and run buffer preparation 111 4.2.4 Extraction of pyrrolizidine alkaloids in plant 112 4.3 Results and discussion 112 4.3.1 Analysis of pyrrolizidine alkaloids by micellar electrokinetic chromatography 112 4.3.1.1 Comparison of different methods for the separation of four pyrrolizidine alkaloids (PAs) 112 4.3.1.2 Effect of borate buffer concentration on separation of PAs 117 4.3.1.3 Effect of SDS concentration on separation of PAs 118 4.3.1.4 Effect of methanol concentration 119 4.3.1.5 Linearity, reproducibility and limit of detection 122 4.3.1.6 Application 124 4.3.2 Analysis of pyrrolizidine alkaloids by dynamic pH junction-sweeping 4.3.2.1 Dynamic pH junction-sweeping on-line preconcentration strategy V 126 126 4.3.2.2 Comparison of different on-line preconcentration strategies 128 4.3.2.3 Effect of sample matrix type and conductivity on dynamic pH junction-sweeping performance 132 4.3.2.4 Effect of pH of sample matrix on pH junction-sweeping performance 135 4.3.2.5 Effect of sample plug length on performance of pH junction-sweeping 137 4.3.2.6 Linearity, reproducibility and limit of detection 142 4.3.2.7 Real sample analysis 144 4.4 Conclusions 146 Chapter Conclusions and outlook 147 References 150 List of publications 165 VI Summary Capillary electrophoresis (CE) has become popular due to its high efficiency, high selectivity, high throughput screening ability, and simplicity in nature and operation. In this thesis, efforts were dedicated to the development of various CE methods as well as their applications in the analysis of biological and biomedical samples. Analysis of intact bacteria based on diluted polymer addition into run buffer by CE were performed and demonstrated to be feasible. Besides, the methods were successfully applied to bacterial pathogen determination using fish fluid as matrix. Being an alternative to slab gel electrophoresis, a CE method with laser induced fluorescence detection using poly(vinylpyrrolidone) as a sieving matrix was developed for the separation of DNA and mutated genes from bacteria. In addition, a micellar electrokinetic chromatography and an on-line preconcentration dynamic pH junction-sweeping method were developed for the analysis of mutagenic pyrrolizidine alkaloids in traditional Chinese medicine. Keywords: Intact bacteria, DNA, alkaloid, capillary electrophoresis VII Chapter Introduction Chapter Introduction 1.1 Electrophoresis Electrophoresis is a separation technique based on the different mobilities of charged molecules in a conductive medium (usually aqueous solution) under an applied voltage. It was first introduced by Tiselius in the 1930s as a separation technique and later on he was awarded the Nobel Prize for his pioneering work [1]. Since then, historical advances of electrophoresis in paper, cellulose and gel electrophoresis have been made. Electrophoresis was performed on a support medium (i.e. semisolid slab-gel) or in nongel support medium (i.e. paper and cellulose acetate). The support medium provides physical support and mechanical stability for the fluidic buffer system. In some modes of electrophoresis, the gel participates in the mechanism of separation by serving as a molecular sieve. Despite impressive success of such kinds of electrophoresis, they have reached their limits with regard to analysis speed, separation efficiency and resolution etc. [2, 3]. Capillary electrophoresis (CE) has emerged as an alternative form of electrophoresis, where the capillary wall provides the mechanical stability for the carrier electrolyte and it represents a merging of technologies derived from traditional electrophoresis and high performance liquid chromatography (HPLC). The arrival of CE solved many experimental problems of gels and microchromatographic separations and it has made great advances in the past few decades. Its distinctive feature over other forms of Chapter Introduction electrophoresis is that smaller capillaries which have large area-to-volume ratio are used. Due to highly efficient Joule heat dissipation of such kind of smaller capillary, high voltages of up to 30 kV can be used in electrophoresis. Compared with normal separation techniques such as gas chromatography (GC), high performance liquid chromatography (HPLC), μ-LC, or slab gel electrophoresis (SGE), CE has several advantages, such as shorter analysis time, higher separation efficiency and smaller sample consumed as well as higher throughput ability. Table 1-1 Comparison of Slab-Gel, μ-LC, HPLC and CE [4] Slab-Gel μ-LC HPLC Speed Slow Moderate Moderate Instrumentation cost Low High Moderate Sensitivity CLOD Poor Poor Excellent MLOD Poor Good Poor Efficiency Moderate Moderate Moderate Automation Little Yes Yes Precision Poor Good Excellent Quantitation Difficult Easy Easy Selectivity Moderate Moderate Moderate Methods development Slow Moderate Moderate Reagent consumption Low Low High Preparative mode Good Fair Excellent Ruggedness Good Good Excellent Separations DNA Excellent Fair Fair Proteins Excellent Good Good Small molecules poor Excellent Excellent CLOD: Concentration limit of detection MLOD: Mass limit of detection CE Fast Moderate Poor Excellent High Yes Good Easy High Rapid Minimal Poor Good Excellent Excellent Excellent Table 1-1 provides a comparison of SGE, μ-LC, HPLC and CE. Although CE shows merits compared to conventional HPLC, there are two disadvantages of CE. 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Lijun Yu, Lingling Yuan, Huatao Feng, Sam Fong Yau Li, “Determination of the bacterial pathogen Edwardsiella tarda in fish species by capillary electrophoresis with blue light-emitting diode-induced fluorescence”, Electrophoresis 25 (18-19): 3139-3144, 2005. 2. Lijun Yu, Yan Xu, Huatao Feng, Sam Fong Yau Li, “Separation and determination of pyrrolizidine alkaloids in traditional Chinese herbal medicine by micelle electrokinetic chromatography with organic modifier”, Electrophoresis 26 (17): 3397-3404, 2005. 3. Lijun Yu, Sam Fong Yau Li, “Dynamic pH junction-sweeping capillary electrophoresis for on-line preconcentration of toxic pyrrolizidine alkaloids in chinese herbal medicine”, Electrophoresis 26 (22):4360-4367, 2005. 4. Lijun Yu, Weidong Qin and Sam Fong Yau Li, “Ionic liquids as additives for separation of benzoic acid and chlorophenoxy acid herbicides by capillary electrophoresis”, Analytica Chimica Acta 547:165-171, 2005. 5. Lijun Yu, Sam Fong Yau Li, “Electrophoretic Behavior Study of Bacteria of Pseudomonas aeruginosa, Edwardsiella tarda and Enteropathogenic escherichia coli by Capillary Electrophoresis with UV and Fluorescence Detection”, Chromatographia 62(708):401-407, 2005. 165 [...]... the basis of mobility differences as small as 0.05% Due to the existence of EOF, the simultaneous separation of cations, neutral analytes and anions is possible EOF can be used to not only adjust analysis time and separation efficiency, but also serve as an electrokinetic pump to move solutions in electrophoretic techniques Figure 1-3 Flow profiles of EOF and laminar flow 1.3.3 Measurement of EOF Routine... or electroosmotic velocity at the center of the capillary is greater than the velocity near the walls of the capillary The temperature differential of the buffer between the middle and the wall of the capillary can be estimated from ΔT = 0.24 Wr 2 4K (1-14) Where W is power, r is capillary radius, and K is thermal conductivity of the buffer, capillary wall and polyimide cladding It is clear that the... the use of higher applied voltage [6] In 1981, Jorgenson and Lukacs [7-9] employed 75 μm i.d glass capillaries and excellent separations with symmetrical peaks and efficiencies in excess of 400, 000 theoretical plates per meter were observed Clearly their advances have promised the start of the era of CE In the 1980s, rapid growth of CE took place Adaptation of capillary gel electrophoresis [10] and isoelectric... the viscosity of the buffer, and ζ is the zeta potential of the liquid-solid interface A further key feature of EOF is that it has flat flow profile, which is shown in Figure 1-3, alongside the parabolic flow profile generated by an external pump, as used for HPLC EOF has a flat profile because its driving force (i.e., charge on the capillary wall) is uniformly distributed along the capillary, which... 1.4.3 Capillary gel electrophoresis (CGE) CGE can be also considered as CZE which is performed in electrophoretic media of gels giving a size sieving effect [67] CGE is well known for its extremely high efficiency and is a powerful analytical technique for the separation of double-stranded DNA, single-stranded DNA and polymerase chain reaction products Initially, CGE was carried out by converting gel electrophoresis. .. inside of the capillary can be estimated by dH IV = dT LA (1-12) Where L is capillary length and A is the cross-sectional area Rearranging this equation using I=V/R, where the resistance R=L/kA and k is the conductivity, dH kV 2 = dT L2 (1-13) The amount of heat that must be removed is proportional to the conductivity of the buffer, as well as the square of the field strength In CE, the center of the capillary. .. Introduction high-throughput DNA separation In this case, the ease of automation, precision and ruggedness of CE have superceded the slab-gel 1.2 History of capillary electrophoresis From a historical perspective, Tiselius was the first researcher who made great contributions to the development of electrophoresis in both theoretical and experimental aspects [1] He demonstrated that electrophoresis could be... to the square of the capillary radius Hence, the use of narrow capillaries facilitates high resolution On the other hand, the use of dilute buffers permits the use of wider bore capillaries, but loading capacity of the separation is reduced 1.4 Different modes of CE CE comprises a family of related techniques with different mechanisms of separation Table 1-2 Usually, only modifications of the background... Basically, CE modes include capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEF) and capillary isotachophoresis (CITP) In the following section, CZE, CGE and MEKC modes were simply described since they were employed for the analysis of different biomedical samples... protein and DNA passage of larger molecules Micellar electrokinetic chromatography (MEKC) Separation mechanism is based on the differential partition Neutral and charged of the solutes between the hydrophobic interior of a molecules charged micelle and the aqueous phase Capillary electrochromatography (CEC) Capillary is packed with a stationary phase that can be Neutral and charged capable of retaining . ANALYSIS OF INTACT BACTERIA, BACTERIAL DNA AND MUTAGENIC ALKALOIDS BY CAPILLARY ELECTROPHORESIS YU LIJUN (M. Sc., Xiamen. principles of capillary electrophoresis 6 1.4 Different modes of capillary electrophoresis 19 1.5 Instrumentation for capillary electrophoresis 24 1.6 Scope of research 32 Part I Analysis of intact. 3.3.7 Fitting of DNA separation models 96 3.3.8 Analysis of PCR products 97 3.4 Conclusions 100 Part II Analysis of mutagenic pyrrolizidine alkaloids 101 Chapter 4 Analysis of mutagenic