Planar electrochromotography and electromembrane protein separation using nanoporous alumina materials

PLANAR ELECTROCHROMOTOGRAPHY AND ELECTROMEMBRANE PROTEIN SEPARATION USING NANOPOROUS ALUMINA MATERIALS HE LIN A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERISTY OF SINGAPORE 2008 -1- ACKNOWLEDGEMENT Firstly, I would like to extend my deepest gratitude to my supervisor, Asst Prof Toh Chee Seng, for his endless support, encouragement, and stimulating discussions During my work I enjoyed the untiring discussion with my supervisor Many helpful advices and the moral support from Dr Toh lead to the success of my dissertation work His passion and preciseness in academic work inspirit me in my work and I believe that the invaluable knowledge, the serious attitude for research and the way to solve scientific problem I learned from him would benefit me immensely in my future work And I thank to my vice supervisor Asso Prof Chin Wee Shong for her continuous encouragement and great help I would like to thank Prof Sam Li Fong Yau for providing microchip and helpful suggestion My thanks also go to Mrs Tay Teng Teng Elaine, Mr Law Wai Siang for the valuable advices rendered Thank Asso Prof Li Tian Hu for his great help and helpful discussion I would like to thank the ladies and gentlemen in NUSNNI and IMRE for instrument utilization I have benefited immensely from the great help and support of colleagues: Dr Shuchi Agarwal, Ms Liu LingYan, Ms Cheow Pui Sze, Ms Ridha Wivanius, Ms Chen LiYan, Ms Kok Guiwan, Mr Eugene Ting In addition, thanks to all lab technologists of Department of Chemistry, NUS The financial support of this work is provided by the National University of Singapore in the form of a research scholarship, which is gratefully acknowledged here.I am also grateful to all my friends in the Department of Chemistry, National University of Singapore for their companionships and support throughout my graduate study In the end, I would like to thank my dearest families for their continuous love, understanding and encouragement during my entire graduate studies in Singapore -2- TABLE OF CONTENTS TITLE PAGE ACKNOWLEDGEMENT TABLE OF CONTENTS SUMMARY LIST OF TABLES LIST OF FIGURES CHAPTER Introduction 11 1.1 Electrophoresis 12 1.1.1 Principle of electrophoresis 13 1.1.2 Gel electrophoresis 14 1.1.3 SDS-PAGE 15 1.1.4 Microchip 17 1.1.5 Capillary electrophoresis 17 1.2 Chromatography 19 1.2.1 Principle of chromatography 19 1.2.2 Three kinds of Chromatography 20 a) Ion-exchange Chromatography 21 b) Gel-filtration Chromatography 22 c) Affinity Chromatography 22 1.2.3 High Performance Liquid Chromatography 1.3 Membrane separation 23 25 1.3.1 Mechanisms for membrane separation 25 1.3.2 Transport phenomena of membrane chromatography 26 1.3.3 Three types of membrane absorbers 27 1.4 Scope of research 28 CHAPTER Fabrication and modification of alumina surface 30 2.1 Introduction 31 2.2 Materials 32 -3- 2.3 Preparation of nanoporous alumina films 2.3.1 Deposition of Al films 32 32 a) Sputtering of Al films 32 b) Evaporation of Al films 33 2.3.2 Electrochemical anodization and surface etching of 34 glass-supported alumina 2.4 Characterization of glass-supported anodized alumina 35 2.5 Mechanism of forming of anodized alumina 38 2.6 Chemically grafted nanoporous alumina surface 39 2.6.1 Preparation of chemically grafted etched glass- 39 supported alumina films 2.6.2 Contact angle analysis of chemically treated alumina films 40 2.6.3 XPS study of chemical treated glass-supported alumina film 42 CHAPTER Planar electrochromatography using self-organized 47 nanoporous Alumina material 3.1 Mechanism for planar electrochromotography protein separation 48 3.2 Off-line detection of protein separation on nanoporous 49 alumina surface 3.2.1 Layout of experiment set-up 50 3.2.2 Correlation of apparent protein concentration 50 3.2.3 Off-line protein separation under different conditions 51 a) Variation of surface and pH 51 b) Variation of pore size and pore depth 56 3.3 On-line detection of electrochromatography protein separation 3.3.1 Fluorescence dye labeling of proteins 58 58 a) Labeling method of Lysozyme with Atto 425 NHS ester 59 b) Labeling Protocol of BSA with Alexa Fluor 430 59 c) Separate the conjugate from unreacted labeling reagent 59 d) Storage of protein conjugates 59 3.3.2 Fluorescence spectra of labeled protein 60 -4- 3.3.3 Planer electrochromatography protein separation 3.3.3.1 Planer electrochromatography protein separation 61 61 by broad channel a) Variation of electronic field strength 61 b) Variation of separation channel surfaces 65 3.3.3.2 Planer electrochromatography protein separation 66 by thin channel CHAPTER Multi nano-channel electromembrane protein separation 70 4.1 Introduction 71 4.2 Experimental 72 4.2.1 Reagents and materials 72 4.2.2 Membrane Coating 72 4.2.3 Membrane holder modification 75 4.3 Protein Transport 75 4.3.1 Multi Nano-channel Electromembrane Separation 75 (MNES) set-up 4.3.2 Detection method of MNES separation 4.4 Results and discussion 77 77 4.4.1 Single Protein Transport 77 4.4.2 Bovine Serum Albumin transport 79 a) Protein concentration & injection amount 79 b) Separation potential polarity & magnitude 82 4.4.3 Lysozyme transport 85 a) Protein concentration 85 b) Separation potential polarity & magnitude 86 4.4.4 Separation of protein mixture CHAPTER CONCLUSION AND PROSPECTIVE WORK 88 95 5.1 Conclusion 96 5.2 Prospective work 97 REFERENCES 99 -5- SUMMARY The fabrication and application of anodized alumina material has attracted much attention because of its technological importance in nanoscience and nanoengineering By electronic anodization, the pore size and pore intensity of nanoporous alumina was precisely adjusted Surface treatment with different organic acids was carried out on home-made glass supported alumina surface X-ray Photoelectron Spectroscopy AXIS Instrument was used to characterize the surface modification Successful grafting on etched glass-supported alumina surface was achieved The variation of surface property after chemical treatment was studied by contact angle measurement Application of nano-porous alumina has been explored for planar electrochromatography protein separation Off-line and on-line detection of protein separation on nanoporous alumina surface was designed and protein separation efficiency was compared by varying alumina pore size, pore depth, surface properties as well as the pH value of buffer solution Proteins separation condition was optimized The mechanism of planar electrochromatography protein separation was studied Multi Nano-channel Electromembrane Separation (MNES) technique with an on-site horizontal set-up was also demonstrated With this method, the possibility of having differential control of transportation and separation of charged proteins were established Different factors affecting separation efficiency were studied and MNES protein separation mechanism was proposed and studied Separation of BSA and LYS across polarized alumina membrane has been achieved The focus of this work is to create and control the nanostructure and properties of anodized alumina and to utilize this structure to investigate effect of nanostructure on protein electrophoresis separation process and to investigate the separation mechanism and at last to develop novel devices/techniques which will allow effective and low cost protein separation -6- LIST OF TABLES Table 2.1 Types of acid used for modification Table 2.2 Contact angle measurement on glass-supported alumina films with different pores sizes Table 2.3 Contact angle measurement of glass-supported alumina films modified by different organic acids Table 4.1 Protein Properties Table 4.2 Onset time and retention time under different separation potentials -7- LIST OF FIGURES Figure 1.1 Ion-exchange Chromatography Figure 1.2 Gel-filtration Chromatography Figure1.3 Affinity Chromatography Figure1.4 Solute transport in packed bed chromatography and membrane chromatography Figure 1.5 Flow in three types of membrane adsorbers Figure 2.1 SEM micrograph of the aluminum by (a) sputtering, (b) evaporation Figure 2.2 SEM pictures of glass-supported alumina film with different pore sizes Figure 2.3 SEM pictures of anodized alumina (a) with vacuum oven treatment (etching time 30mins, 50nm) (b) without vacuum oven treatment (etching time 30mins, 50nm) Figure 2.4 Mechanism of electrochemical anodization and etching process Figure 2.5 Chemical modification of alumina surface Figure 2.6 XPS spectra of the N (1s) region of 6-aminohexanoic acid modified alumina film Figure 2.7 Survey scans for unmodified, 6-aminohexanoic acid and heptanediolic acid modified alumina film Figure 2.8 XPS spectra of C (1s) region of bare alumina surface, pimelic-acid-modified alumina and 6-aminohexanoic-acid-modified surface Figure 3.1 Schematic of separation mechanism Figure 3.2 Layout of electrophoresis experiment set-up Figure 3.3 Protein concentrations against time (bare alumina surface) Figure 3.4 Selectivity ratios at pH 8.3 Figure 3.5 Selectivity ratios at pH 5.5 Figure 3.6 Selectivity ratios obtained with different sputtering time and etching time -8- Figure 3.7 Fluorescence emission spectra of labeled BSA, lysozyme and the mixture Figure 3.8 Electropherogram under voltage of 400V Figure 3.9 Fluorescent spectrums under 400V Figure 3.10 Electropherogram under voltage of 200V Figure 3.11 Fluorescent spectrum under 200V Figure 3.12 Electropherogram under voltage of 100V Figure 3.13 Fluorescent spectrum at elution peaks under 100V Figure 3.14 Electropherogram on 300µm depth channel at 200V Figure 3.15 Fluorescent spectrum at elution peak by 300µm depth channel at 200V Figure 3.16 Electropherogram under voltage of 200V by 50µm gap channel Figure 3.17 Fluorescent spectrum at elution peaks under 200V by 50µm depth channel Figure 4.1 Unsputtered membrane (left) and sputtered one (right) Figure 4.2 SEM of membrane sputtered for different duration a) unsputtered, b) 10min, c) 20 Figure 4.3 Modified membrane holder Figure 4.4 Schematic of horizontal MNES set-up Figure 4.5 BSA elute current under different HPLC pump flow rate Figure 4.6 Current of elute with different BSA concentration Figure 4.7 Current of elute with different BSA injection amount Figure 4.8 Current of elute with different separation potentials Figure 4.9 Current of elute with different LYS concentration Figure 4.10 Current of LYS elute under different potential polarity Figure 4.11 Current of LYS elute under different separation potentials Figure 4.12 a) Electronic field direction and the electronic force direction b) Solution flow direction and electronic force direction c) Net protein migration velocity Figure 4.13 Current of LYS and mixture elute with different BSA concentration -9- Figure 4.14 Current of mixture elute under different separation potential Figure 4.15 Current of mixture elute under -3.0v and +3.0v - 10 - the second protein BSA Current/A E-6 46 LYS 40ppm Rt=39.5sec 46 46 LYS 20ppm LYS 20ppm BSA 400ppm BSA160ppm Rt=39sec;61sec 44 Rt=39.3sec;61.5sec 44 44 42 42 42 40 40 40 38 38 38 36 36 36 34 34 34 32 60 80 100 32 120 14060 80 100 32 120 14060 80 100 120 140 Time/sec Figure 4.13 Current of LYS and mixture elute with different BSA concentration Current curves of protein mixture at E=+1.15v and E=-1.15v were compared while keeping all other conditions the same The mixture contained 20ppm LYS and 300ppm BSA As shown in Fig 4.14, solid dot curve showed the elute current at E=+1.15v and the one with hollow dot gave that at E=-1.15v It is obvious that under E=-1.15v the retention time difference for the two proteins was bigger than that under positive potential The reason was elaborated by Fig 4.12 above As shown above, separation of the protein mixture was partly achieved when a negative potential on receiver side of the membrane was applied Positive LYS would be transported towards the receiver side solution faster than BSA due to electrostatic attraction towards the receiver solution However, if we reverse the polarity of the applied electric field, separation was not as obvious as that under negative potential - 92 - 45 44 43 Positive LYS elutes faster with positive potential +1.15v -1.15v Negative BSA elutes slower with negative potential Current/A E-6 42 41 40 39 38 37 36 35 34 90 100 110 120 130 140 150 160 170 180 Time/sec Figure 4.14 Current of mixture elute under different separation potential In order to further improve the separation efficiency, a higher potential of -3.0v was applied This is because enhanced potential on the membrane would further enlarge the retention time difference of two proteins As shown in Fig 4.15, the protein mixture was separated under potential of -3.0v with all other operation conditions the same Two well separated peaks and second retention time difference of BSA and LYS was detected Separation was not observed with positive potential +3.0v This again indicated that the most effective protein mixture separation was achieved only when the feed side of the membrane was negatively charged and at the same time higher potential would benefit the separation of the two proteins because of the increased electric field and electrostatic force on charged protein molecules - 93 - 34.0 -3.0v 33.5 LYS BSA 34.0 -3.0v LYS BSA 33.5 33.0 +3.0v 32.5 33.0 33.0 32.5 32.5 32.0 32.0 31.5 31.5 31.0 31.0 30.5 30.5 30.5 30.0 30.0 30.0 29.5 Current/A E-6 32.0 29.5 100 29.5 200 31.5 31.0 100 29.0 200 100 200 Time/sec Figure 4.15 Current of mixture elute under -3.0v and +3.0v In summary, with all the other conditions the same and under separation potential of -3.0v, individual protein retention time was determined in order to identify the two separated peaks obtained in the elute mixture LYS eluted significantly faster (with retention time of 51 second) than that of the former (with retention time of 59 second), which was exactly the same as that of the two peaks respectively When E = +3.0V, the electrostatic interaction between the protein and the membrane wall was balanced by the higher diffusion flux of the smaller LYS protein Hence, separation could not be achieved It was clear that the transport of both BSA and LYS was markedly influenced by the polarity of the potential applied on the alumina membrane Electrochemically controlled transport of proteins using platinum coated alumina membrane under different experimental conditions has been demonstrated with this novel multi nano-channel electromembrane technique The transport of proteins across the membrane can be electrochemically manipulated by applying different magnitude and polarity of constant potential The greatest BSA and LYS separation was obtained under E = -3.0V - 94 - Chapter Conclusion and prospective work - 95 - 5.1 Conclusion With the well defined pores sizes, nano-porous alumina manufactured by electrochemical method has its potential applications in a variety of fields such as template for fabricating other nano-porous structures; dielectrics in capacitors; mechatronic system and so on In this work, electrochemically anodized nano-porous alumina surfaces under different condition as well as chemically grafted nano-porous alumina with different surface properties have been produced and utilized in protein separation Nano-porous alumina surfaces on glass substrate with different pore sizes and depth were manufactured using electrochemical anodization method based on sputtered and evaporated aluminum by different wet etching period and sputtering or evaporation time Three kinds of carboxylic acids, 6-aminohexanoic acid, heptanediolic acid, and hexanoic acid were grafted on home-made glass-supported alumina surface Surface modification effect and hydrophobicity properties of alumina surface were studied by X-ray Photoelectron Spectroscopy AXIS Instrument as well as contact angle measurements Application of nano-porous alumina has been explored for planar electrochromatography protein separation Off-line and on-line detection systems of protein separation on nanoporous alumina surface were designed and protein separation efficiency was compared by varying alumina pore size, pore depth, surface properties as well as the pH value of buffer solution With the use of anionic surfactant sodium dodecyl sulfate (SDS), proteins were denatured and the separation condition was optimized The mechanism of Size-Exclusion Electrochromatography (SEEKC) separation was proposed and studied Multi Nano-channel Electromembrane Separation (MNES) technique with an on-site horizontal set-up was demonstrated Continuous flow operation was employed to - 96 - detect the sample over time thus decreasing the detection limit With this method, the possibility of having differential control of transportation and separation of charged proteins were established Different factors affecting separation efficiency were studied and MNES protein separation mechanism was proposed and studied Separation of BSA and LYS across polarized alumina membrane has been achieved 5.2 Prospective work The hydrophilic alumina surface can be modified to exhibit properties Other functional groups such as carboxylic group, amino group and so on can be grafted on both etched glass supported alumina and commercial alumina membrane surface with freely controlled nanopores These diverse functional groups can change alumina surface property to allow it to react with other molecules, for instance, bio-molecules, thus producing covalent binding on nanoporous alumin surface with broad promising applications such as biosensors Protein separation by nanoporous alumina film on microchip could be applied to increase the separation efficiency of planar electrochromatography protein separation technique Future work would seek to apply the principles of SEEKC onto a μ-total analysis system On the micro channel, pre-coated aluminum film will be covered on the glass bottom and further anodizad into nanoporous alumina film By using this alumina covered microchip channel, protein separation can be carried out and higher separation efficiency could be expected Further studies on Multi Nano-channel Electromembrane Separation (MNES) technique to have additional control in the transportation and separation could be done through salt elution, isocratic elution and increasing the thickness of the membrane Because the absorption of protein on alumina pores may result in low separation efficiency in the former study as well as membrane fouling, modification of alumina membrane by using organic carboxylic group and polyethylene glycol could be - 97 - carried out to further reduce protein absorption on the alumina surface to enhance the protein separation efficiency - 98 - REFERENCES A Manz, N.P., D Iossifidis, Bioanalytical Chemistry 2004, London: Imperial College Press Blanco, D., et al., Capillary zone electrophoretic separation of proteins using coated capillaries Journal of Liquid Chromatography & Related Technologies, 2002 25(8): p 1171-1185 Ho, A.K., et al., Measurement and theoretical modeling of protein mobility through membranes Aiche Journal, 1999 45(7): p 1434-1450 Radovich, J.M., N.S Mason, and R.E Sparks, Coupling Electrophoresis with Ultrafiltration for Improved Processing of Plasma-Proteins Separation Science and Technology, 1980 15(8): p 1491-1498 Tiselius, A., Electrophoresis of serum globin Biochemical Journal, 1937 31: p 313-17 Andreas Manz ; Nicole Pamme ; Dimitri Iossifidis, Bioanalytical Chemistry 2004, London: Imperial College Press Freeman, S.E., L.L Larcom, and B.D Thompson, Electrophoretic Separation of Nucleic-Acids - Evaluation by Video and Photographic Densitometry Electrophoresis, 1990 11(5): p 425-431 Merrick, B.A., et al., Separation and Sequencing of Familiar and Novel Murine Proteins Using Preparative 2-Dimensional Gel-Electrophoresis Electrophoresis, 1994 15(5): p 735-745 Vandergeer, P and T Hunter, Phosphopeptide Mapping and Phosphoamino Acid Analysis by Electrophoresis and Chromatography on Thin-Layer Cellulose Plates Electrophoresis, 1994 15(3-4): p 544-554 10 Larmann, J.P., et al., 2-Dimensional Separations of Peptides and Proteins by Comprehensive Liquid-Chromatography Capillary-Electrophoresis Electrophoresis, 1993 14(5-6): p 439-447 11 Tsugita, A., et al., Separation and Characterization of Rice Proteins Electrophoresis, 1994 15(5): p 708-720 12 Novakova, Z., et al., Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis Electrophoresis, 2006 27(7): p 1277-1287 13 Manabe, T., et al., Correlation of plasma protein separation patterns obtained by two-dimensional polyacrylamide gel electrophoresis and by capillary electrophoresis Electrophoresis, 1998 19(8-9): p 1319-1324 14 Pan, S.Q., X.S Ye, and J Kuc, A Technique for Detection of Chitinase, Beta-1,3-Glucanase, and Protein-Patterns after a Single Separation Using Polyacrylamide-Gel Electrophoresis or Isoelectrofocusing Phytopathology, 1991 81(9): p 970-974 15 Kunz, C and B Lonnerdal, Human-Milk Proteins - Separation of Whey Proteins and Their Analysis by Polyacrylamide-Gel Electrophoresis, Fast Protein Liquid-Chromatography (Fplc) Gel-Filtration, and Anion-Exchange Chromatography American Journal of Clinical Nutrition, 1989 49(3): p 464-470 16 Siegmann, M and G Thomas, Separation of Multiple Phosphorylated Forms of 40 S-Ribosomal Protein-S6 by Two-Dimensional Polyacrylamide-Gel Electrophoresis Methods - 99 - in Enzymology, 1987 146: p 362-369 17 Faye, L., A New Enzymatic Staining Method for the Detection of Radish Beta-Fructosidase in Gel-Electrophoresis Analytical Biochemistry, 1981 112(1): p 90-95 18 Rivoal, J., et al., A method for activity staining after native polyacrylamide gel electrophoresis using a coupled enzyme assay and fluorescence detection: Application to the analysis of several glycolytic enzymes Analytical Biochemistry, 2002 300(1): p 94-99 19 Luo, S.Q., J.M Feng, and H.M Pang, High-throughput protein analysis by multiplexed sodium dodecyl sulfate capillary gel electrophoresis with UV absorption detection Journal of Chromatography A, 2004 1051(1-2): p 131-134 20 Garcia-Canas, V., R Gonzalez, and A Cifuentes, Detection of genetically modified maize by the polymerase chain reaction and capillary gel electrophoresis with UV detection and laser-induced fluorescence Journal of Agricultural and Food Chemistry, 2002 50(5): p 1016-1021 21 Westbrook, J.A., et al., A combined radiolabelling and silver staining technique for improved visualisation, localisation, and identification of proteins separated by two-dimensional gel electrophoresis Proteomics, 2001 1(3): p 370-376 22 Lemkin, P., et al., Software Aids for the Analysis of 2d Gel-Electrophoresis Images Computers and Biomedical Research, 1979 12(6): p 517-544 23 Giddings, J.C., Two-Dimensional Separations - Concept and Promise Analytical Chemistry, 1984 56(12): p 1258-& 24 Liu, Y.M and J.V Sweedler, Two-dimensional separations: Capillary electrophoresis coupled to channel gel electrophoresis Analytical Chemistry, 1996 68(22): p 3928-3933 25 Opiteck, G.J., J.W Jorgenson, and R.J Anderegg, Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides Analytical Chemistry, 1997 69(13): p 2283-2291 26 Krause, I., U Muller, and H.D Belitz, Characterization of Glutenins in Wheat-Varieties by Sds-Polyacrylamide Gel-Electrophoresis (Sds-Page) and Two-Dimensional Electrophoresis (2d-Page) Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung, 1988 186(5): p 398-406 27 Krylov, S.N and N.J Dovichi, Capillary electrophoresis for the analysis of biopolymers Analytical Chemistry, 2000 72(12): p 111R-128R 28 Heller, C., Capillary Electrophoresis of Proteins and Nucleic-Acids in Gels and Entangled Polymer-Solutions Journal of Chromatography A, 1995 698(1-2): p 19-31 29 Li, Y., et al., Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfludic network Analytical Chemistry, 2004 76(3): p 742-748 30 Manz, A., et al., Planar Chips Technology for Miniaturization of Separation Systems - a Developing Perspective in Chemical Monitoring Advances in Chromatography, 1993 33: p 1-66 31 Dolnik, V., S.R Liu, and S Jovanovich, Capillary electrophoresis on microchip Electrophoresis, 2000 21(1): p 41-54 32 Soper, S.A., et al., Polymeric microelectromechanical systems Analytical Chemistry, 2000 72(19): p 642A-651A 33 Nagata, H., et al., High-speed separation of proteins by microchip electrophoresis using a - 100 - polyethylene g glycol-coated plastic chip with a sodium dodecyl sulfate-linear polyacrylamide solution Electrophoresis, 2005 26(14): p 2687-2691 34 Cohen, A.S and B.L Karger, High-Performance Sodium Dodecyl-Sulfate Polyacrylamide-Gel Capillary Electrophoresis of Peptides and Proteins Journal of Chromatography, 1987 397: p 409-417 35 Tsuji, K., High-Performance Capillary Electrophoresis of Proteins - Sodium Dodecyl-Sulfate Polyacrylamide Gel-Filled Capillary Column for the Determination of Recombinant Biotechnology-Derived Proteins Journal of Chromatography, 1991 550(1-2): p 823-830 36 Karger, B.L., F Foret, and J Berka, Capillary electrophoresis with polymer matrices: DNA and protein separation and analysis, in High Resolution Separation and Analysis of Biological Macromolecules, Pt B 1996 p 293-319 37 Scholz, E., et al., Separation of the unique proteins of wheat protein fractions by capillary electrophoresis Chromatographia, 2000 51: p S130-S134 38 Manabe, T., H Oota, and J Mukai, Size separation of sodium dodecyl sulfate complexes of human plasma proteins by capillary electrophoresis employing linear polyacrylamide as a sieving polymer Electrophoresis, 1998 19(13): p 2308-2316 39 Chang, W.W.P., et al., Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings Electrophoresis, 2005 26(11): p 2179-2186 40 Gamble, T.N., C Ramachandran, and K.P Bateman, Phosphopeptide isomer separation using capillary zone electrophoresis for the study of protein kinases and phosphatases Analytical Chemistry, 1999 71(16): p 3469-3476 41 Shieh, P., et al., Fast Protein Separation by Capillary Sds Gel-Electrophoresis Using a Uv Transparent Polymer Network Abstracts of Papers of the American Chemical Society, 1994 207: p 91-ANYL 42 Guo, X., et al., UV embossed polymeric chip for protein separation and identification based on capillary isoelectric focusing and MALDI-TOF-MS Analytical Chemistry, 2006 78(10): p 3249-3256 43 Mao, Y and X.M Zhang, Comprehensive two-dimensional separation system by coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection Electrophoresis, 2003 24(18): p 3289-3295 44 Martinovic, S., et al., Separation and detection of intact noncovalent protein complexes from mixtures by on-line capillary isoelectric focusing-mass spectrometry Analytical Chemistry, 2000 72(21): p 5356-5360 45 Ofverstedt, L.G., et al., Protein-Concentration and Recovery from Gel Slabs by Displacement Electrophoresis (Isotachophoresis) and the Effects of Elctroosmosis and Counter Flow Electrophoresis, 1981 2(3): p 168-173 46 Beattie, J.H and M.P Richards, Analysis of Metallothionein Isoforms by Capillary Electrophoresis - Optimization of Protein Separation Conditions Using Micellar Electrokinetic Capillary Chromatography Journal of Chromatography A, 1995 700(1-2): p 95-103 47 Zhang, X., et al., Separation of proteins by microemulsion electrokinetic chromatography Chemical Journal of Chinese Universities-Chinese, 2000 21(8): p 1183-1186 48 Ishihama, Y., Y Oda, and N Asakawa, Hydrophobicity of cationic solutes measured by - 101 - electrokinetic chromatography with cationic microemulsions Analytical Chemistry, 1996 68(23): p 4281-4284 49 Packer, N.H., et al., A general approach to desalting oligosaccharides released from glycoproteins Glycoconjugate Journal, 1998 15(8): p 737-747 50 Tivesten, A., A Lundqvist, and S Folestad, Selective chiral determination of aspartic and glutamic acid in biological samples by capillary electrophoresis Chromatographia, 1997 44(11-12): p 623-633 51 Bandilla, D and C.D Skinner, Protein separation by monolithic capillary electrochromatography Journal of Chromatography A, 2003 1004(1-2): p 167-179 52 Yang, W.C., E.S Yeung, and M.J Schmerr, Detection of prion protein using a capillary electrophoresis-based competitive immunoassay with laser-induced fluorescence detection and cyclodextrin-aided separation Electrophoresis, 2005 26(9): p 1751-1759 53 Nguyen, A.T and L Yengoyan, New approach in surface-modified capillaries: Characterization and protein separation by capillary electrophoresis Abstracts of Papers of the American Chemical Society, 2000 219: p U365-U365 54 Iki, N and E.S Yeung, Non-bonded poly(ethylene oxide) polymer-coated column for protein separation by capillary electrophoresis Journal of Chromatography A, 1996 731(1-2): p 273-282 55 Kato, M., et al., Integration of on-line protein digestion, peptide separation, and protein identification using pepsin-coated photopolymerized sol-gel columns and capillary electrophoresis/mass spectrometry Analytical Chemistry, 2004 76(7): p 1896-1902 56 Albarghouthi, M.N., T.M Stein, and A.E Barron, Poly-N-hydroxyethylacrylamide as a novel, adsorbed coating for protein separation by capillary electrophoresis Electrophoresis, 2003 24(7-8): p 1166-1175 57 Jiang, T.F., et al., Dynamically coating the capillary with cationic polymer for basic protein separation by capillary electrophoresis Chinese Journal of Analytical Chemistry, 2002 30(2): p 144-147 58 Dawson, J.B., et al., Separation of Protein-Bound Copper and Zinc in Human-Plasma by Means of Gel-Filtration Ion-Exchange Chromatography Analyst, 1981 106(1259): p 153-159 59 Matsukura, H., et al., Automated Separation and Measurement of Urinary Isoenzymes and Protein by Ion-Exchange Liquid-Chromatography Journal of Chromatography-Biomedical Applications, 1987 414(1): p 47-54 60 Kang, K and B.J McCoy, Protein Separation by Ion-Exchange Chromatography - a Model for Gradient Elution Biotechnology and Bioengineering, 1989 33(6): p 786-790 61 Wu, Z.P., B.J Xu, and Z.Y Chen, Separation and purification of Bacillus thuringiensis(Bt) toxic protein CrylAb from transgenic rice by ion-exchange chromatography Chromatographia, 2001 53(9-10): p 571-573 62 Zhang, Y., et al., Separation and purification of annexin B1, a novel Ca2+-dependent phospholipid binding protein, expressed in Escherichia coli by ion-exchange chromatography Chromatographia, 2003 58(11-12): p 713-716 63 Chakravarthy, M., et al., Protein structure and chromatographic behavior - The separation and characterization of four proteins using gel filtration and ion-exchange chromatography and gel electrophoresis Journal of Chemical Education, 1996 73(3): p 268-272 - 102 - 64 Dalton, J.C., et al., Separation of recombinant human protein C from transgenic animal milk using immobilized metal affinity chromatography, in Oxygen Transport to Tissue Xviii 1997 p 419-428 65 Tadepalli, S.S., et al., Separation of protein C from fraction IV of the Cohn process using immobilized metal affinity chromatography, in Oxygen Transport to Tissue Xix 1997 p 639-644 66 Wu, H.P., D.F Bruley, and K.A Kang, Protein C separation from human blood plasma Cohn fraction IV-1 using immobilized metal affinity chromatography, in Oxygen Transport to Tissue Xx 1998 p 697-704 67 Dave, A.S and D.F Bruley, Separation of Human Protein C from components of transgenic milk using immobilized metal affinity chromatography, in Oxygen Transport to Tissue Xxi 1999 p 639-647 68 Wu, H.P and D.F Bruley, Homologous human blood protein separation using immobilized metal affinity chromatography: Protein C separation from prothrombin with application to the separation of factor IX and prothrombin Biotechnology Progress, 1999 15(5): p 928-931 69 Qi, Y., Z Yan, and J.X Huang, Chromatography on DEAE ion-exchange and Protein G affinity columns in tandem for the separation and purification of proteins Journal of Biochemical and Biophysical Methods, 2001 49(1-3): p 263-273 70 Xu, F., et al., A novel hierarchical nanozeolite composite as sorbent for protein separation in immobilized metal-ion affinity chromatography Advanced Materials, 2003 15(20): p 1751-+ 71 Kerlavage, A.R., et al., Reversed-Phase High-Performance Liquid-Chromatography of Escherichia-Coli Ribosomal-Proteins - Characteristics of the Separation of a Complex Protein Mixture Journal of Chromatography, 1983 266(AUG): p 225-237 72 Asakawa, N., et al., High-Performance Liquid-Chromatography of Protein Separation of Proteins by Reversed Phase High-Performance Liquid-Chromatography Yakugaku Zasshi-Journal of the Pharmaceutical Society of Japan, 1981 101(8): p 708-712 73 Asakawa, N., et al., High-Performance Liquid-Chromatography of Protein Separation of Insulin from Different Species (Equine, Porcine, Bovine and Ovine) by Reversed Phase High-Performance Liquid-Chromatography Yakugaku Zasshi-Journal of the Pharmaceutical Society of Japan, 1982 102(1): p 43-48 74 Iwakiri, S., et al., High-Performance Liquid-Chromatography of Protein Separation of Myoglobin from Different Species (Sperm Whale, Horse) by Reversed Phase High-Performance Liquid-Chromatography Yakugaku Zasshi-Journal of the Pharmaceutical Society of Japan, 1986 106(3): p 265-267 75 Itoh, H., et al., Fast Protein Separation by Reversed-Phase High-Performance Liquid-Chromatography on Octadecylsilyl-Bonded Nonporous Silica-Gel Improvement in Recovery of Hydrophobic Proteins Analytical Biochemistry, 1991 199(1): p 7-10 76 Chong, B.E., et al., Rapid screening of protein profiles of human breast cancer cell lines using non-porous reversed-phase high performance liquid chromatography separation with matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis Rapid Communications in Mass Spectrometry, 1999 13(18): p 1808-1812 77 Wall, D.B., et al., Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human - 103 - erythroleukemia cell-line Rapid Communications in Mass Spectrometry, 2001 15(18): p 1649-1661 78 Iberer, G., et al., Improved performance of protein separation by continuous annular chromatography in the size-exclusion mode Journal of Chromatography A, 2001 921(1): p 15-24 79 Chiantore, O and M Guaita, High-Performance Size Exclusion Chromatography with Microparticulate Porous Silica Spheres - Influence of Flow-Rate, Solute Mass-Transfer and Polydispersity of Samples Journal of Liquid Chromatography, 1982 5(4): p 643-667 80 Nice, E.C., C.J Lloyd, and A.W Burgess, The Role of Short Microbore High-Performance Liquid-Chromatography Columns for Protein Separation and Trace Enrichment Journal of Chromatography, 1984 296(JUL): p 153-170 81 Hwang, S.a.K.K., Membranes in Separation 1975: John Wiley and Sons 99-123 82 Ghosh, R., Protein separation using membrane chromatography: opportunities and challenges Journal of Chromatography A, 2002 952(1-2): p 13-27 83 Zhou, D., et al., Protein transport and separation using polypyrrole coated, platinised polyvinylidene fluoride membranes Reactive & Functional Polymers, 2000 45(3): p 217-226 84 Devereux, N., M Hoare, and P Dunnill, Membrane Separation of Protein Precipitates Unstirred Batch Studies Biotechnology and Bioengineering, 1986 28(1): p 88-96 85 Sannier, F., et al., Stability of a Mineral Membrane Ultrafiltration Reactor for Peptic Hydrolysis of Hemoglobin Journal of Chemical Technology and Biotechnology, 1994 61(1): p 43-47 86 R.W Baker, E.L.C., W Eykamp, W.J Koros, R.L Riley, H Strathmann, Membrane Separation Systems: Recent Developments and Further Directions 1991: N.J: Noyes Data Corporation 87 Nakao, S., et al., Separation of Proteins by Charged Ultrafiltration Membranes Desalination, 1988 70(1-3): p 191-205 88 Hwang, T.S and J.H Lee, Synthesis of sulfonated hollow PP-g-styrene fibrous ion-exchange membrane and separation of BSA protein Polymer-Korea, 2002 26(4): p 415-421 89 Dallbauman, L and C.F Ivory, Protein Separation Via Affinity-Mediated Membrane-Transport Acs Symposium Series, 1990 419: p 188-211 90 Zeng, X.F and E Ruckenstein, Membrane chromatography: Preparation and applications to protein separation Biotechnology Progress, 1999 15(6): p 1003-1019 91 Ghosh, R., Fractionation of human plasma proteins by hydrophobic interaction membrane chromatography Journal of Membrane Science, 2005 260(1-2): p 112-118 92 Ghosh, R and L Wang, Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography Journal of Chromatography A, 2006 1107(1-2): p 104-109 93 Li, Y., J.W Cooper, and C.S Lee, Miniaturized membrane-based reversed-phase chromatography and enzyme reactor for protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry Journal of Chromatography A, 2002 979(1-2): p 241-247 94 Zhang, J.X., et al., Polarization properties of ordered copper nanowire microarrays embedded in anodic alumina membrane Chemical Physics Letters, 2004 400(1-3): p 158-162 - 104 - 95 Martin, C.R., Nanomaterials - a Membrane-Based Synthetic Approach Science, 1994 266(5193): p 1961-1966 96 Zong, R.L., et al., Linear and nonlinear optical properties of Ag nanorods/AAM composite films Chemical Physics Letters, 2004 398(1-3): p 224-227 97 Moueddeb, H., et al., Membrane bioreactor for lactic acid production Journal of Membrane Science, 1996 114(1): p 59-71 98 Popat, K.C., et al., Surface modification of nanoporous alumina surfaces with poly(ethylene glycol) Langmuir, 2004 20(19): p 8035-8041 99 Belyavskii, S.G., P.G Mingalev, and G.V Lisichkin, Chemical modification of gamma-Al2O3 surface with aryl silanes Colloid Journal, 2004 66(2): p 128-136 100 Karaman, M.E., D.A Antelmi, and R.M Pashley, The production of stable hydrophobic surfaces by the adsorption of hydrocarbon and fluorocarbon carboxylic acids onto alumina substrates Colloids and Surfaces a-Physicochemical and Engineering Aspects, 2001 182(1-3): p 285-298 101 Toh, C.S., et al., Fabrication of free-standing nanoscale alumina membranes with controllable pore aspect ratios Nano Letters, 2004 4(5): p 767-770 102 Koh, G., et al., Characterization of the barrier layer of nanoporous alumina films prepared using two different contact configurations Electrochimica Acta, 2007 52(8): p 2815-2821 103 Chu, P.K., et al., Plasma-surface modification of biomaterials Materials Science & Engineering R-Reports, 2002 36(5-6): p 143-206 104 Liu LY, T.C., Chemical modification of nanoporous alumina membranes 2006 105 Cheow, P.S., L.Y Liu, and C.S Toh, Grafting of nanoporous alumina membranes and films with organic acids Surface and Interface Analysis, 2007 39(7): p 601-610 106 L.Y., L., Chemically Grafted Alumina Surface And Applications 2006 107 Mahapatro, A., et al., Surface modification of functional self-assembled monolayers on 316L stainless steel via lipase catalysis Langmuir, 2006 22(3): p 901-905 108 Coman, V and S Kreibik, Planar dielectrochromatography - A perspective technique Jpc-Journal of Planar Chromatography-Modern Tlc, 2003 16(5): p 338-346 109 Berezkin, V.G., et al., Use of low volatility mobile phases in electroosmotic thin-layer chromatography Journal of Chromatography A, 2005 1084(1-2): p 13-17 110 A, F.J.R., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience 2003, Goergetown, TX 111 Yu, S.F., et al., Size-based protein separations in poly(ethylene glycol)-derivatized gold nanotubule membranes Nano Letters, 2001 1(9): p 495-498 112 Lee, S.W., et al., Transport and functional behaviour of poly(ethylene glycol)-modified nanoporous alumina membranes Nanotechnology, 2005 16(8): p 1335-1340 113 Chu, S.Z., et al., Fabrication of TiO2-Ru-(O-2)/Al2O3 composite nanostructures on glass by Al anodization and electrodeposition Journal of the Electrochemical Society, 2004 151(1): p C38-C44 114 Yamauchi, T., E Kokufuta, and Y Osada, Electrically Controlled Protein Permeation through a Polyelectrolyte Gel Membrane Abstracts of Papers of the American Chemical Society, 1993 205: p 105-COLL 115 Zhao, H., W.E Price, and G.G Wallace, Electrochemically Controlled Transport of Potassium-Chloride across a Conducting Electroactive Polymer Membrane Journal of - 105 - Electroanalytical Chemistry, 1992 334(1-2): p 111-120 116 Guizard, C., et al., Electronically Conductive Mineral Membranes Designed for Electro-Ultrafiltration Journal of Membrane Science, 1989 41: p 127-142 117 Wagener, K.B., Electrically Conductive Membranes Based on Polyacetylene Chemistry Acs Symposium Series, 1985 269: p 429-445 118 Wagener, K.B., Electrically conductive porous synthetic polymeric compositions, method for making them, and their use an electrodialysis process 1987, U.S 19 119 Lloyd, D.R., Materials Science of Synthetic Membranes - Preface Acs Symposium Series, 1985 269: p R9-R9 - 106 - [...]... mechanism investigation and research focusing on the areas of:  Off-line and on-line detection of planar electrochromatography protein separation using self-organized nanoporous alumina material  On-site detection of multi nano-channel alumina electromembrane protein separation - 29 - Chapter 2 Fabrication and modification of alumina surface - 30 - 2.1 Introduction Nanoporous alumina membranes are... By electronic anodization and surface treatment with different organic acids, successful grafting on both etched glass-supported alumina surface and alumina membrane was achieved Our research program is an effort to pursue novel and efficient protein separation devices/techniques while developing optimized condition to minimize protein separation time and improve protein separation efficiency This... research activity is the understanding of the physicochemical - 28 - principles that govern pore size, pore intensity and pore properties of nanoporous alumina in the fabrication and chemical treatment process This aim is realized by characterization of the nanoporous alumina through experiments and theoretical studies of principles and kinetics in nanoporous alumina anodization and chemical treatment systems... production Proteins are very diverse They differ by size, shape, charge, hydrophobicity, and their affinity for other molecules Currently, there are many methods in the field of protein separation and extraction The mechanism of protein separation can be based on size, shape, charge, hydrophobicity, and affinity for other molecules Basic principles and recent development of the protein separation techniques... maintaining high separation efficiency and resolution Microchip separation systems have been used successfully for the separation of DNA, proteins, and small molecule species Sano et al have miniaturized the SDS-PAGE system on a plastic chip coated with polyethylene glycol.[33] Separation of trypsin inhibitor, BSA and β-galactosidase (21.5, 66.5 and 116.0kDa respectively) was achieved within 8 seconds using field... was utilized in order to separate proteins by capillary electrophoresis.[53, 54] Integration of on-line protein digestion, peptide separation, and protein identification was achieved by using pepsin-coated photopolymerized sol-gel columns and capillary electrophoresis/mass spectrometry.[55] Poly-N-hydroxyethylacrylamide as adsorbed coating was applied on CE for protein separation [56] Cationic polymer... protein and the matrix depends on the pH and ionic strength of the solution passing down the column, which can be varied in a controlled way to achieve an effective separation Figure 1.1 Ion-exchange Chromatography Protein- bound copper and zinc was separated in human-plasma by means of Gel-Filtration Ion-Exchange Chromatography in1981.[58] Separation and measurement of urinary isoenzymes and protein. .. separate proteins according to their size The matrix consists of tiny porous beads Protein molecules that are small enough to enter the holes in the beads are delayed and travel more slowly through the column Proteins that cannot enter the beads are washed out of the column first Such columns also allow an estimate of protein size Four proteins were separated and the protein structure was characterized using. .. biotechnology have seen protein research attracting great interest as it plays a fundamental role in future development of drugs Hence, there is an increasing need for the effective separation of proteins and peptide drugs from biological broths and blood Chemical separations constitute some of the most important stages in most chemical, pharmaceutical and petrochemical processes and it contributes towards... acids, proteins, oligonucleotides) with charge/mass ratios differing by ... electrochromatography protein separation Off-line and on-line detection of protein separation on nanoporous alumina surface was designed and protein separation efficiency was compared by varying alumina pore... electrochromatography protein separation using self-organized nanoporous alumina material  On-site detection of multi nano-channel alumina electromembrane protein separation - 29 - Chapter Fabrication and modification... transportation and separation of charged proteins were established Different factors affecting separation efficiency were studied and MNES protein separation mechanism was proposed and studied Separation