磁性微粒在生物科技的領域及臨床醫學等分面有廣泛的用途，其商業價值與潛力逐漸受到重視。在各種生物技術的研發上，純化技術一直是不可或缺的重要關鍵，磁性高分子微粒的應用，除了可以作為選擇性的吸附劑，用來檢測與純化藥物外，應用在分離純化細胞、DNA、RNA及蛋白質的實例更是不勝枚舉。
本研究致力於開發磁性高分子顆粒，首先是表面有孔的苯乙烯與二乙烯苯的磁性共聚物，利用三步驟腫脹聚合方法，包埋磁性奈米顆粒。實驗結果發現經由三步驟合成法可以製備出平均粒徑3.2 μm的均一顆粒。此球形高分子顆粒為表面有孔，比表面積44.5 m2/g。但因表面有孔，會有使用時磁性奈米從球形流失的缺失，所以轉而著手於研究製備無孔且具有磁性的高分子顆粒。
在無孔的磁性高分子顆粒上，我們發展一套有別於以往製備磁性高分子顆粒的方法。首先是以分散聚合法製備微米大小的聚苯乙烯(PS)為粒種，後加入適當的溶劑浸泡使其腫脹，同時加入適量的磁性奈米粉體，使之擴散進入腫大的顆粒中，得到具有超順磁性之高分子微粒。所製成的磁性高分子顆粒，粒徑均一，平均大小約為1.1 μm，比表面積為4.8 m2/g。不管是孔隙特性或是飽和磁化量，都相當不錯，不輸目前廣泛使用的商品─Dynabeads顆粒，因此有很高的應用價值。
將單股的低聚核甘酸(oligonucleotide)固定在此磁性高分子微米顆粒，可作為固相基因重組、DNA雜合及固相PCR等用途。本研究提出一個在顆粒表面作基因重組的的方法，主要的目的是在解決傳統重組質體操作時多步驟純化的缺點，因往往造成所製備的質體或基因片段濃度過低，而使重組失敗。以固相重組的方式，通常只要經過簡單的磁鐵吸引的，就可以輕易的將產物給分離出來，重組效率因而提高。

Functionalized polymeric microparticles have been widely applied in biotechnological and clinical areas and gained great interest recently due to their great commercially potential. Superparamagnetic magnetic particles play an essential role in the bioseparation, since they can easily and gently be collected by using a magnetic field. Besides as bio-specific adsorbents and carriers for clinical assays, they are widely applied for the isolation and purification of cells, DNA, RNA and proteins.
In this study, methods for preparing magnetic polymer particles were developed. First, styrene-divinylbenzene copolymer particles entrapping magnetic materials were prepared by a three-step swelling and polymerization method. Results from the measurement of particle porosity by Micromeritics (ASAP2000) and SEM observation suggested that the obtained particles have a superficially porous structure and mono-dispersed size distribution. The averaged particle diameter and specific surface area of the particles were determined to be 3.2 μm and 44.5 m2/g, respectively. Since magnetic material might be lost from the porous PS-DVB particles during use, non-porous magnetic particles are thus more suitable for a long-time application.
In the present study, microsized polystyrene (PS) beads were prepared, swollen with a proper solvent, and then incubated with a solution of magnetic nanoparticles. Superparamagnetic magnetic particles with a higher magnetism were obtained after the magnetic nanoparticles were diffused into the PS beads. These magnetic particles have a mono-dispersed size distribution with an average diameter of 1.1 μm and a specific surface area of 4.8 m2/g. Also these magnetic PS beads were non-porous and very comparable with well-known Dynabeads. Their potential in biological applications is expected.
Immobilization of oligonucleotides on the magnetic PS beads could be applied for solid-phase gene cloning, solid-phase PCR and DNA hybridization. After coating with an oxirane-containing polymer, the prepared magnetic particles with aldehyde groups were used for DNA recombination and PCR, both in solid phase modes. When the magnetic beads were employed, the protocol of solid-phase PCR can be simplified by reducing the isolation steps. The PCR products on the magnetic particles are reusable since the collected is simple by using a magnetic field. A method for DNA recombination using macrosized magnetic particles as the carriers was proposed in the present work. This method can reduce the purification steps in the conventional DNA cloning method and improve the efficiency by preventing lose of plasmid and cloned DNA fragment, The successful recombinant DNA could be easily collected due to the magnetic property of the carrier.

摘要……………………………………………………………………....I
Abstract………………………………………………………………..III
目錄……………………………………………………………………..VI
圖目錄……………………..…………………………………………...XI
表目錄……………………………………………………………….XIII
第一章 緒論……………………………………………………………..1
1.1研究動機與目的….…………………………………………….1
1.2文獻回顧……….……………………………………………….4
1.2.1 聚苯乙烯顆粒的製備……………………………………..6
1.2.2苯乙烯與二乙烯苯共聚物顆粒的合成……………………7
1.2.3包埋磁性顆粒的高分子顆粒………………………………8
1.2.4固定DNA…………………………………………………...9
1.2.5親和性吸附固定DNA……………………………………10
1.2.6化學反應共價鍵結DNA………………………………….11
1.2.7固相基因重組……………………………………………..12
第二章 實驗藥品與設備………………………………………............14
2.1實驗藥品……………………………………………………….14
2.1.1高分子顆粒聚苯乙烯（PS）之製備………………………14
2.1.2高分子顆粒苯乙烯與二乙烯苯共聚物之製備………….14
2.1.3包埋磁性奈米顆粒的苯乙烯與二乙烯苯共聚物顆粒的製備…………………………………………………………..15
2.1.4包埋磁性奈米顆粒的聚苯乙烯顆粒的製備……….…….16
2.1.5 苯乙烯與二乙烯苯共聚物顆粒表面之醛基改質……....17
2.1.6 苯乙烯與二乙烯苯共聚物顆粒上醛基含量的測定..…..17
2.1.7 聚苯乙烯顆粒表面之醛基改質…………………………18
2.1.8 聚苯乙烯顆粒上醛基含量的測定………………...…….18
2.1.9固定Avidin………..............................................................18
2.1.10大腸桿菌K12染色體DNA的製備…………………….19
2.1.11 PCR合成DNA反應……………………………………..20
2.1.12 PCR純化………………………………………………...20
2.1.13瓊脂凝膠電泳分析………………………………………20
2.1.14固定在顆粒上的DNA用EcoRI digest………………….21
2.1.15質體pGEX-1λT之製備………………………………...21
2.1.16質體pGEX-1λT之純化………………………………...22
2.1.17將質體用EcoRI和BamHI digest………………………22
2.1.18固相重組質體……………………………………………23
2.1.19將重組質體與顆粒分離…………………………………23
2.2實驗設備……………………………………………………….24
第三章 實驗方法與步驟………………………………………………27
3.1高分子顆粒的製備………………………………………….....27
3.1.1 聚苯乙烯粒種的製備……………………………............27
3.1.2 Polymeric porogen顆粒的製備…………………………..27
3.1.3苯乙烯與二乙烯苯磁性共聚物顆粒的製備……………..28
3.1.4 磁性聚苯乙烯顆粒的製備………………………………29
3.1.5 磁性苯乙烯與二乙烯苯共聚物顆粒表面醛基化..……..29
3.1.6 磁性苯乙烯與二乙烯苯共聚物顆粒上醛基含量的測定30
3.1.7磁性聚苯乙烯顆粒表面醛基化…………..………………32
3.1.8 磁性聚苯乙烯顆粒上醛基含量的測定…………………32
3.2固相重組質體………………………………………………….35
3.2.1固定卵白素（avidin）…….……………………………...35
3.2.2大腸桿菌K12染色體DNA的製備………………………37
3.2.3 PCR放大反應…………………………………………….38
3.2.4純化PCR放大之DNA……………………………………39
3.2.5將PCR放大之片段固定在顆粒上……………………….40
3.2.6將固定在顆粒上之PCR放大之片段用EcoRⅠdigest…..40
3.2.7微量抽取質體DNA ( plasmid DNA mini preparation)…..41
3.2.8將環狀質體用EcoRI和BamHI enzyme digest成linear…42
3.2.9接合反應（Ligation）..……………………………………42
3.2.10將重組質體從顆粒上取出………………………………43
3.2.11將重組好的質體ligation成環狀……………………….43
3.2.12瓊脂凝膠膠電泳…………………………………………44
第四章 實驗結果與討論………………………………………………45
4.1 磁性苯乙烯與二乙烯苯共聚物顆粒之製備………………...45
4.1.1聚苯乙烯粒種的合成…………………………………….45
4.1.2包埋磁性奈米顆粒的苯乙烯與二乙烯苯共聚物顆粒….46
4.1.3 磁性苯乙烯與二乙烯苯共聚物顆粒表面醛基化………47
4.1.4 磁性苯乙烯與二乙烯苯共聚物顆粒表面醛基定量……47
4.1.5 磁性苯乙烯與二乙烯苯共聚物顆粒比表面積的量測....53
4.2 磁性聚苯乙烯顆粒製備之結果…….………...……………...55
4.2.1包埋磁性奈米顆粒的聚苯乙烯顆粒…………….………55
4.2.2 磁性聚苯乙烯顆粒比表面積的量測……………………55
4.2.3 磁性聚苯乙烯顆粒磁化量的量測………………………55
4.2.4 磁性聚苯乙烯顆粒表面醛基化…………………………59
4.3固相重組質體………………………………………………….59
4.3.1定量固定上顆粒之卵白素的量………...………………...64
4.3.2聚合酶連鎖反應(PCR)…………………………………...65
4.3.3固定PCR產物DNA………………………………………68
4.3.4製備環狀質體……………………………………………..69
4.3.5 環狀質體用BamHI和EcoRI digest……………………..71
4.3.6 接合反應（ligation）............................................................71
4.3.7將重組好之pGEX-nan基因從顆粒中取出……………...74
第五章 結論與建議……………………………………………………77
參考文獻………………………………………………………………..80

林慧蓉, ”固定單股低聚核苷酸於無孔高分子微米顆粒及其在DNA雜合與基因重組上的應用” 國立中正大學化學工程研究所碩士論文(2002)
高震于, ”表面有孔之苯乙烯與二乙烯苯共聚物顆粒之製備與其在生化上之應用” 國立中正大學化學工程研究所碩士論文（2000）
陳志浩, ”無孔性甲基丙烯酸甲酯-甲基丙烯酸環氧丙酯共聚物之製備及其蛋白質層析之應用” 國立中正大學化學工程研究所碩士論文(1998)
廖雅玲, ”磁性奈米微粒與DNA複合體之製備及應用研究” 國立中正大學化學工程研究所碩士論文 (2001)
Agatha, F., Sirdhar, R., and Sylvia D.,”Interaction of Immobilized Avidin with an Aequorin-Biotin Conjugate: An Aequorin-Linked Assay for Biotin.”, Analytical Biochemistry., 254, 62-68 (1997)
Asha, A. O.,Svend, E. R., Henrik, N. R., Soren, R. R., Barbara, M. S., and Johansson, A.,”Detection of Immobilized Amplicons by ELISA-like technique”, Clinical Chemistry, 42, 1547-1555 (1996)
Bauer, S., Ziv, E.,”Dense Growth of Aerobic Bacteria in a Bench-Scale Fermentor”, Biotechnology and Bioengineering, 18,81-94 (1976)
Bertrand, R. J., “Large-Scale Expression Measurement by Hybridization Methods : From High-Density Membranes to “DNA Chips”, The Biochemistry Journal, 124, 251-258 (1998)
Brett, A. S., and John, L. T., “Expression Microarray Hybridization Kinetics Depend on Length of the Immobilized DNA but Are Independent of Immobilization Substrate”, Analytic Biochemistry, 295, 149-157 (2001)
Cereghino, J. L.,and Cregg, J. M.,”Heterologous Protein Expression in the Methylotrophic Yeast Pichia pastoris”, Ferms Microbiology Reviews, 24, 45-66 (2000)
Cheng, C., Huang, Y. L., and Yang, S. T., ” A Novel Feeding Strategy for Enchanced Plasmid Stability and Protein Production in Recombinant Yeast Fedbatch Fermentation”, Biotechnology and Bioengineering, 56,23-31 (1997)
Chrisey, L. A., Lee, G. U., and O’Ferrall, C. E., “Covalent attachment of synthetic DNA to self-assembled monolayer films”, Nucleic Acids Research, 24, 3031-3039 (1996)
Christof, M. N., Larissa, B., Bulent, C., and Dietmar, B., “DNA-Directed Immobilization: Efficient, Reversible, and Site-Selective Surface Binding of Proteins by Means of Covalent DNA-Streptavidin Conjugates”, Analytical Biochemistry, 268, 54-63 (1999)
Chun, X. H. C., Pingang, H., and Yuzhi, F., “Electrochemical Detection of Sequence-Specific DNA Using a DNA Probes Labeled with Aminoferrocene and Chitosan Modified Electrode Immobilized with ssDNA”, The Royal Society of Chemistry, 126, 62-65 (2001)
Cynthia, J. B., Mark, S. S., David, A. H., Jay, W. G., Fred, J. B., and Darrell, P. C., “ Rotating Rod Renewabel Microcolumn for Automated, Solid-Phase DNA Hybridization Studies”, Analytic Chemistry, 71, 4135-4141 (2000)
Day, P. J. R., Flora, P. S., Fox, J. E., and Walker, M. R., “Immobilization of Polynucleotides on Magnetic Particals. Factors Influencing Hybridization Efficiency”, The Biochemistry Journal, 278,735-740 (1990)
Heinrich, F. A, Margaret, N. K., and Xiao, Q. G., “Multiplexed DNA Sequencing and Diagnostics by Hybridization with Enriched Stable Isotope Labels”, Analytic Chemistry, 1510-1517 (1997)
Fahy, E., Davis, G. R., DiMichele, L. J., and Ghosh, S. S.,”Design and Synthesis of Polyacrylamide-based oligonucleotide supports for use in nucleic acid diagnostics”, Nucleic Acids Research, 21, 1819-1826 (1993)
Fan, Z. H., Mangru, S., Granzow, R., Heaney, P., Ho, W., Dong, Q., and Kumar, R.,”Dynamic DNA Hybridization on a Chip Using Paramagnetic Beads”, Analytical Chemistry, 71, 4851-4859 (1999)
Gilham, P. T.,” The Synthesis of Celluloses Containing Covalently Bound Nucleotides, Polynucleotides, and Nucleic Acids”, Biochemistry, 7, 2809-2813 (1968)
Heinrich, F. A., Margaret, N. K., and Xiao, Q. G., “Multiplexed DNA Sequencing and Diagnostics by Hybridization with Enriched Stable Isotope Labels”, Analytical Chemistry, 69, 1510-1517 (1997)
Hitoshi, K. and Dennis, A. C., ”Solid-phase Polymerase Chain Reaction”, Journal of Clinical Laboratory Analysis, 8, 452-455 (1994)
Imai, T., Sumi, Y., Hatakeyama, M., Fujimoto, K., Kawaguchi, H., Hayashida, N., Shiozaki, K., Terada, K., Yajima, H., and Handa, H., “Selective Isolation of DNA or RNA Using Single-Stranded DNA Afinity Latex Particles”, Journal of Colloid and Interface Science, 177, 245-249 (1996)
Jane, G. M., Gregory, M. D., Sabrina, E. R., Linda, M. H., and Michael, L., “The Selective Isolation of Novel cDNA Encoded by the Regions Surrounding the Human Interleukin 4 and 5 Genes”, Nucleic Acids Research, 20, 5173-5179 (1992)
Joanne, D. A., and Linda, A. C., ”Use of immobilized PCR primers to generate covalently immobilized DNAs for in vitro transcription /translation reactions”, Nucleic Acids Research, 28, E5 (2000)
Joanthan, N. K., Joseph, L. W., Joseph, P. D., Rachel, E. M., Mary, C., and Eugene, L. B., “Immobilization of DNA via Oligonucleotides containing an Aldehyde or Carboxylic Acid Group at The 5’-Terminus”, Nucleic Acids Research, 7, 2891-2909 (1987)
Kalim, U. M., and Edwin, M. S.,”Determining the Influence of Structure on Hybridization using Oligonucleotide Arrays”, Nature Biotechnology, 17, 788-792 (1999)
Kazunobu, F., and Jonathan,S.,“Surprising Lability of Biotin-Streptavidin Bond During Transcription of Biotinylated DNA Bound to Paramagnetic Streptavidin Beads”, Biotechniques, 14, 608-617 (1993)
Khudyakov, Y. E., Gaur, L., Singh, J., Patel, P., and Fields, H. A.,” Primer Specific Solid-Phase Detection of PCR Products”, Nucleic Acids Research, 22, 1320-1321 (1994)
Linda, A. C., Gil, U. L., and Eilzabeth, C. E., “Covalent Attachment of Synthetic DNA to Self-Assembled Monolayer Films”, Nucleic Acids Research, 24, 3031-3039 (1996)
Lund, V., Schmid, R., Rickwood, D., and Hornes, E., “Assessment of Methods for Covalent Binding of Nucleic Acids to Magnetics Beads, DynabeadsTM, and Characteristics of the Bound Nucleic Acids in Hybridization Reaction”, Nucleic Acids Research, 16, 10861-10880 (1998)
Magnus, J., Simon, F., Anthony, J. B., and Ulf, L.,” Effect of Oligonucleotide Truncation on Single-Nucleotide Distinction by Solid-Phase Hybridization”, Analytical Chemistry, 74, 199-202 (2002)
Maria, S. T., Peter, T., Ray, H., Morris, C. P., and Angela, V. D., “Solid-Phase Amplification and Detection: A Single-Tube Diagnostic Assay for Infectious Agents”, Molecular Diagnosis, 6, 131-136 (2001)
Marie, K.W., Xinwen, W., Bart, and C. W., “Optimizing the immobilization of single-stranded DNA onto Glass Beads”, Journal of Biochemical and Biophysical Methods, 47, 221-231 (2001)
Mark, S. T., Sarah, P., Angela, S., Valentine, J. H.,Ray, H.,Morris, C.P., and Angela, V. D., “Solid-Phase Amplification for Detection of C282Y and H63D Hemochromatosis (HFE) Gene Mutations”, Clinical Chemistry, 47, 1384-1389 (2001)
Mikhail, A. L., and Andrei, D. M.,”Theoretical Analysis of the Kinetics of DNA Hybridization with Gel-Immobilized Oligonucleotides”, The Journal of Biophysical, 71, 2795-2801 (1996)
Minna, S., Jorma, I., and Timo, L., “Solid-Phase PCR with Hybridization and Time resolved Fluorometry for Detection of HLA-B27”, Clinical Chemistry, 47, 498-504 (2001)
Nathalie, Z., Christophe, G., Dominique, D., Jean, J. P., and Jose R.,”Amination of Polystyrene Microwells : Application to the Covalent Grafting of DNA Probes for Hybridization Assays”, Analytical Biochemistry , 236 , 85-94 (1996)
Nikiforov, T. T., Rendle, R. B., Kotewicz, M. L., and Rogers, Y. H., ” The Use of Phosphorothioate Primers and Exonuclease Hydrolysis for the Preparation of Single-stranded PCR Products and their Detection by Solid-phase Hybridization”, PCR Methods and Applications, 3, 285-291 (1994)
Parker, C., and DiBiasio, D., “Effect of Growth Rate and Expression Level on Plasmid Stability in Saccharomyces cerevisiae”, Biotechnology and Bioengineering, 29, 215-221 (1987)
Riccelli, P. V., Merante, F., Leung, K. T., Bortolin, S., Zastawny, R. L.,
Wilchek, M. and Bayer, E. A.,”Application of Avidin-Biotin Technology: Literature Survey”, Methods in Enzymology, 184, 14-45 (1990)
Rasmussen, S. R., Larsen, M. R., and Rasmussen, S. E., “Covalent Immobilization of DNA onto Polystyrene Microwells : The Molecules Are Only Bound at the 5’ End”, Analytical Biochemistry, 198, 138-142 (1991)
Stanley, F. W., Lora, H., Judith, F., Jonathan, N. K., Joseph, P. D., and Kenneth, J.,”Rapid Hybridization Kinetics of DNA Attached to Submicron Latex Particles”, Nucleic Acids Research, 15, 2911-2926 (1987)
Thomas, H., Staffan, B., Tomas, M., and Mathias, U.,”Bidirectional Solid-Phase Sequencing of In Vitro-Amplified Plasmid DNA”, Biotechniques, 10, 84-93 (1991)
Thomas, H., Stefan, S., Erok, H., and Mathias, U., “Direct Soild Phase Sequencing of Genomic and Plasmid DNA using Magnetic Beads as Solid Support”, Nucleic Acids Research, 17, 4937-4945 (1989)
Tomoko A., Tetsushi, Y., Masaru, I., Kyoko, S., Sadao, S., Mikio, N., Minoru, N., and Yoshizo, A., “Quantitation of Human Herpesvirus 6 DNA in Infant with Exanthem Subitum by Microplate PCR-Hybridization Assay”, Pediatrics International, 43, 372-378 (2001)
Troels, K., Nana, J., Jef, F., Ulrich, B., Mogens, F., and Mogens, H. J.,” Photochemical Immobilization of Anthraquinone Conjugated Oligonucleotides and PCR Amplicons on Solid Surfaces”, Bioconjugate Chemistry, 11, 474-483 (2001)
Uhlen, M., Hultman, T., Wahlberg, J., Lundeberg, J., Bergh, S., Pettersson, B., Holmberg, A., Stahl, S., Moks, T., “Semi-automated solid-phase DNA Sequencing”, Trends Biotechnol, 10,52-55 (1992)
Voshiaki, T. I., Mitsuru, I., and Hirano, K. I., “Investigation of the Hydrolysis of Single DNA Molecules Using Fluorescence Video Microscopy”, Analytical Chemistry, 72, 1649-1656 (2000)
Wolf, S. F., Haines, L., Fosh, J., Kremsky, J. N., Dougherty, J. P., and Jacobs, K., “Rapid Hybridization Kinetics of DNA Attached to Submicron Latex Particles”, Nucleic Acids Research, 15, 2911-2926 (1987)
Yee, L., and Blanch, H. W., ”Recombinant Trypsin Production in High Cell Density Fed-Batch Culture in Escherichia coli”, Biotechnology and Bioengineering , 41, 781-790 (1992)
Zammatteo, N., Girardeaux, C., Delforge, D., Pireaux, J. J., and Remacle, J., “Amination Polystyrene Microwells : Application to the Covalent Grafting of DNA Probes for Hybridization Assays”, Analytical Biochemistry, 236, 85-94(1996)