臺灣博碩士論文加值系統

本研究製備磁性分子印模聚合物(MMIP)吸附床，應用其高度選擇性及隨著磁場方便收集的特性，用於分散式微固相萃取(d-μSPE)食物樣品中的黃麴毒素(aflatoxins)，做為高效液相層析-紫外光偵測器(HPLC-UV)分析檢測之前處理。
研究先在超純水中加入三氯化鐵和二氯亞鐵，在3 %氨水的催化下進行反應，合成磁性粒子。反應結束後以超純水洗滌至中性，即完成磁性粒子的製備。再於磁性粒子表面利用Tetraethylorthosilicate (TEOS)及3-(trimethoxysilyl) propyl mthacrylate (MPS)來修飾，使其具有與可分子印模聚合物反應連接之官能基。 MMIP的製備，係利用乙醇水溶液為溶劑及製孔劑，以5,7-dimethoxycoumarin (DMC)為模板分子，苯乙烯(styrene)及甲基丙烯酸(methacrylic acid, MAA)為功能性單體，加入修飾的磁性粒子，以超音波震盪使均勻分散，通入氮氣去除氧氣後，再加入交聯劑(EGDMA)與起始劑(AIBN)進行聚合反應。反應結束後利用磁鐵收集MMIP，即完成MMIP的製備。以乙氰清洗MMIP，去除模板分子後即可使用。
在以MMIP萃取黃麴毒素前，對於固態樣品須先以甲醇水溶液將黃麴毒素預萃取出為樣品溶液。為達到最佳萃取效果，對影響萃取效率的參數，如MIP合成條件、樣品溶液pH值、鹽析作用影響、及脫附溶劑選擇等進行詳細的測試。試驗結果顯示，將10毫升pH 7的樣品溶液，以MMIP材料在800 rpm轉速磁石攪拌下進行微固相萃取10分鐘後，以磁鐵吸附MMIP粒子及去除基質溶液，以1毫升超純水潤洗後，再以1毫升的乙氰脫附黃麴毒素，可達最佳萃取效果。萃取液先以氮氣吹乾，再以100μL乙氰回溶，注入HPLC-UV分析。偵測訊號對濃度的線性範圍為0.2 ng/mL-25 ng/mL，線性相關係數達0.9986以上，偵測極限(S/N = 3)為0.04 ng/mL，定量極限(S/N = 10) 為0.1 ng/mL。以此方法分析市售牛奶樣品，經添加不同濃度的黃麴毒素標準溶液進行偵測，黃麴毒素B1與B2回收率可達89.5 %至102.9 %，RSD低於7.0 %。所製備的MMIP，材料性質穩定(可連續使用50次以上)，及具有良好的再現性(RSD小於3.8%)。研究結果顯示，利用所製備的MMIP以d-μSPE方式萃取樣品中的黃麴毒素是一個簡單、快速、高選擇性及環境友善的樣品前處理方法。

In this study, a magnetic molecularly imprinted polymer (MMIP) was prepared as the sorbent to achieve dispersive micro-solid phase extraction (d-μ-SPE) of aflatoxins B1 and B2 in aqueous food samples for high performance liquid chromatography-ultraviolet detection (HPLC-UV).
The magnetic particles were prepared by adding ferric chloride and ferrous chloride in 3 % NH4OH under stirring for 1 hour, and then washing by pure water. The surface of magnetic particles were then modified with tetraethyl orthosilicate (TEOS) and 3-(trimethoxysilyl) propyl methacrylate (MPS) to build-up functional groups for bonding with the MIPs which prepared by using 5,7-dimethoxycoumarin (DMC) as the template molecule, styrene and methacrylic acid (MAA) as the functional monomers, ethylene glycol dimethacrylate (EGDMA) as the cross linker, azobisisobutyronitrile (AIBN) as the initiator, and ethanol-water(4:1, v/v) as the porogen and solvent,
Parameters influenced the extraction efficiencies of aflatoxins B such as synthesis of MMIP, sample pH, salting out effect, and solution for eluting were investigated thoroughly and optimized. After a series of tests, for a 10 mL aqueous sample solution at pH 7.0 the best extraction efficiency was obtained by using MMIP-d-μ-SPE to collect aflatoxins B for 10 min under 800 rpm stirring, and then using a magnet to separate the MMIPs particles from matrix solution. After rinsing with water, aflatoxins B were desorbed with acetonitrile for HPLC-UV analysis. Under the optimal condition, the detections were linear in 0.2 - 25 ng/mL with correlation coefficients above 0.9986 for both aflatoxin B1 and B2. The detection limits (based on S/N = 3) were 0.04 ng/mL and quantitative limits (based on S/N = 10) were 0.1 ng/mL for both aflatoxin B1 and B2. The aflatoxin B1 and B2 in milk and spiked samples were analyzed with the present method and characterized by mass spectrometry. Recoveries were ranged 89.5 – 102.9 % with relative standard deviations of less than 7.0 %. The prepared MMIP is stable and can be used for over 50 times. These results have proven the proposed MMIPs-d-μ-SPE method is a simple, rapid, selective, and eco-friendly sample pretreatment method to extraction aflatoxins in aqueous samples for HPLC-UV analysis.

摘要 i
Abstract ii
目錄 iii
圖目錄 vi
表目錄 viii
第一章 緒論 1
1.1 前言 1
1.2黃麴毒素簡介 1
1.3黃麴毒素之相關分析技術回顧 2
1.4 分子印模聚合物之概述 7
1.4.1 分子印模聚合物之簡述 7
1.4.2 分子印模聚合物原理 8
1.4.3 分子印模聚合物之鍵結作用力 11
1.4.4 分子印模聚合物之製備 16
1.4.5 分子印模聚合物之合成 23
1.5 磁性分子聚合物之概述 26
1.5.1磁性材料之概述 26
1.5.2磁性分子印模聚合物 26
1.6 研究目的 27
第二章 研究方法 29
2.1 藥品、實驗器材與儀器設備 29
2.1.1藥品 29
2.1.2器材 30
2.2 藥品配置 31
2.2.1緩衝溶液之配置 31
2.2.2黃麴毒素標準儲備溶液之配置 32
2.2.4牛奶樣品前處理 33
2.2.5添加黃麴毒素於牛奶樣品 33
2.2.6麥片樣品前處理 34
2.2.7添加黃麴毒素於麥片樣品 34
2.2.8花生粉樣品前處理 34
2.2.9添加黃麴毒素於花生粉樣品 34
2.3玻璃裝置矽烷化 35
2.4 儀器操作之參數 35
2.4.1高效能液相層析儀之參數 35
2.4.2磁性分子印模具合物固相萃取法之最佳化參數 35
2.5實驗方法及流程 36
2.5.1磁性分子印模聚合物合成與條件 36
2.5.2磁性分子印模聚合物特性測試 37
2.5.3磁性分子印模聚合物固相萃取條件 38
2.5.4固相萃取之條件探討步驟 39
2.6方法可行性評估 40
2.6.1儀器檢量線製作 40
2.6.2方法檢量線製作 41
2.6.3磁性分子印模聚合物之重複使用性測試 41
2.6.4磁性分子印模聚合物製備批次間之比較 41
2.7真實樣品之分析及回收率 41
2.7.1牛奶樣品分析 41
2.7.2牛奶樣品回收率測定 41
2.7.3麥片樣品分析 42
2.7.4麥片樣品回收率測定 42
2.7.5花生粉樣品分析 42
2.7.6花生粉樣品回收率測定 42
第三章 結果與討論 43
3.1 磁性分子印模聚合物之特性測試 44
3.1.1磁性分子印模聚合物之SEM圖 44
3.1.2磁性分子印模聚合物之IR光譜 44
3.1.3磁性分子印模聚合物之吸附量 46
3.2固相萃取之最佳化 49
3.2.1功能性單體比例之探討 49
3.2.2樣品萃取時間之探討 49
3.2.3樣品pH值之探討 51
3.2.4無機鹽類添加對萃取效率之探討 54
3.2.5磁石攪拌子轉速對萃取效率之探討 56
3.2.6脫附溶劑之探討 56
3.2.7脫附溶劑體積之探討 58
3.3方法可行性評估與真實樣品之測定 62
3.3.1直接注入之校正曲線及偵測極限 62
3.3.2經方法之校正曲線及偵測極限 62
3.3.3真實樣品之測定 63
3.3.4磁性分子印模聚合物之穩定性及再現性 74
3.3.5磁性分子印模聚合物固相萃取與其他方法比較 77
結論 79
參考文獻 80

1.Wogan, G.N., Chemical nature and biological effects of the aflatoxins. Bacteriological reviews 1966, 30(2), 460.
2.Mateles, R. I.; J. C. Adye., Production of aflatoxins in submerged culture." Applied microbiology 1965, 13(2), 208-211.
3. Davis, N. D.; Diener, U. L.; Eldridge D. W., Production of aflatoxins B1 and G1 by Aspergillus flavus in a semisynthetic medium. Applied microbiology 1966, 14(3), 378-380.
4.Gourama, H.; Lloyd, B. B., Aspergillus flavus and Aspergillus parasiticus: aflatoxigenic fungi of concern in foods and feeds†: a review. Journal of Food protection 1995, 58(12), 1395-1404.
5.Pitt, J. I.; Ailsa D. H.; Diane R. G., An improved medium for the detection of Aspergillus flavus and A. parasiticus. Journal of Applied Bacteriology 1983, 54(1), 109-114.
6.Diener, U.L., Epidemiology of aflatoxin formation by Aspergillus flavus. Annual review of phytopathology 1987, 25(1), 249-270.
7.Butler, W. H.; Greenblatt M.; Lijinsky, W., Carcinogenesis in rats by aflatoxins B1, G1, and B2." Cancer research 1969, 29(12), 2206-2211.
8.Trail, F.; Nibedita M.; John L., Molecular biology of aflatoxin biosynthesis. Microbiology 1995, 141(4), 755-765.
9.E U Commission, Commission Regulation (EC) No. 466/2001 of 8 March 2001 setting maximum levels for certain contaminants in foodstuffs, Official Journal European Communities L77 2001.
10.Stubblefield, R. D.; ODETTE L. S., Reverse phase analytical and preparative high pressure liquid chromatography of aflatoxins. Journal of the Association of Official Analytical Chemists 1977, 60(4), 784-790.
11.Manabe; Masaru; Tetsuhisa G.T.; Shinji M., High-performance liquid chromatography of aflatoxins with fluorescence detection. Agricultural and Biological Chemistry 1978, 42(11), 2003-2007.
12.Moss; Elizabeth J., Some mass-spectral and nmr analytical studies of a glutathione conjugate of aflatoxin B1. Biochemical Journal 1983, 210(1), 227-233.
13.Robertson; James A.; Walter A. P.; Goldblatt. L. A., Preparation of aflatoxins ad determination of their ultraviolet and fluorescent characteristics. Journal of Agricultural and Food Chemistry 1967, 15(5), 798-801.
14.Sobolev; Victor S.; Joe W. D., Cleanup procedure for determination of aflatoxins in major agricultural commodities by liquid chromatography. Journal of AOAC International 2002, 85(3), 642-645.
15.Fu, Zhaohui; Xuexiang H.; Shungeng M., Rapid determination of aflatoxins in corn and peanuts. Journal of Chromatography A 2008, 1209(1) 271-274.
16.Thurman; Earl M.; Margaret S. M., Solid-phase extraction: principles and practice. New York: Wiley 1998; Vol. 16.
17.Camel, V., Solid phase extraction of trace elements. Spectrochimica Acta Part B: Atomic Spectroscopy 2003, 58(7), 1177-1233.
18.Blesa, J., Determination of aflatoxins in peanuts by matrix solid-phase dispersion and liquid chromatography. Journal of Chromatography A 2003, 1011(1), 49-54.
19.Tozzi, C., A combinatorial approach to obtain affinity media with binding properties towards the aflatoxins. Analytical and bioanalytical chemistry 2003, 375(8), 994-999.
20.Lattanzio; Veronica M.T., Improved method for the simultaneous determination of aflatoxins, ochratoxin A and Fusarium toxins in cereals and derived products by liquid chromatography–tandem mass spectrometry after multi-toxin immunoaffinity clean up. Journal of Chromatography A 2014, 1354, 139-143.
21.Baggiani; Claudio; Laura A.; Cristina G., Molecular imprinted polymers as synthetic receptors for the analysis of myco-and phyco-toxins. Analyst 2008, 133(6) 719-730.
22.Fischer, E., Einfluss der Configuration auf die Wirkung der Enzyme. European Journal of Inorganic Chemistry 1894, 27(3), 2985-2993.
23.Wulff, G., Molecular imprinting in cross‐linked materials with the aid of molecular templates—a way towards artificial antibodies. Angewandte Chemie International Edition in English 1995, 34(17), 1812-1832.
24.Sellergren, B., Molecularly imprinted polymers: man-made mimics of antibodies and their application in analytical chemistry. Elsevier:2000; Vol. 23.
25.Andersson, L. I., Molecular imprinting for drug bioanalysis: a review on the application of imprinted polymers to solid-phase extraction and binding assay." Journal of Chromatography B: Biomedical Sciences and Applications 2000, 739(1), 163-173.
26.Mosbach, Klaus. "Molecular imprinting." Trends in biochemical sciences 1994, 19(1), 9-14.
27.Yan, M., Molecularly imprinted materials: science and technology. CRC press: 2004.
28.Arvand, M.; Masoumeh H., Synthesis by precipitation polymerization of a molecularly imprinted polymer membrane for the potentiometric determination of sertraline in tablets and biological fluids. Journal of the Brazilian Chemical Society 2012, 23(3), 392-402.
29.Whitcombe, M. J., A new method for the introduction of recognition site functionality into polymers prepared by molecular imprinting: synthesis and characterization of polymeric receptors for cholesterol. Journal of the American Chemical Society 1995, 117(27), 7105-7111.
30.da Silva, M. S., Supercritical fluid technology as a new strategy for the development of semi-covalent molecularly imprinted materials. Rsc Advances 2012, 2(12), 5075-5079.
31.Kim, H.; David A. S., New insight into modeling non-covalently imprinted polymers."Journal of the American Chemical Society 2003, 125(37), 11269-11275.
32.Yan, H,; Kyung H. R., Characteristic and synthetic approach of molecularly imprinted polymer. International journal of molecular Sciences 2006, 7(5), 155-178.
33.Sellergren, B., Direct drug determination by selective sample enrichment on an imprinted polymer. Analytical Chemistry 1994, 66(9), 1578-1582.
34.Tamayo, F. G.; Turiel E.; Martín-Esteban A., Molecularly imprinted polymers for solid-phase extraction and solid-phase microextraction: recent developments and future trends. Journal of Chromatography A 2007, 1152(1), 32-40.
35.Sajonz, P., Study of the thermodynamics and mass transfer kinetics of two enantiomers on a polymeric imprinted stationary phase. Journal of Chromatography A 1998, 810(1), 1-17.
36.Haginaka, J., Molecularly imprinted polymers for solid-phase extraction. Analytical and bioanalytical chemistry 2004, 379(3), 332-334.
37.Mayes, A. G.; and Klaus M., Molecularly imprinted polymer beads: suspension polymerization using a liquid perfluorocarbon as the dispersing phase. Analytical Chemistry 1996, 68(21), 3769-3774.
38.Ye, L.; Peter A. C.; Klaus M., Molecularly imprinted monodisperse microspheres for competitive radioassay. Analytical Communications 1999, 36(2) 35-38.
39.Wang, J., Monodisperse, molecularly imprinted polymer microspheres prepared by precipitation polymerization for affinity separation applications. Angewandte Chemie International Edition 2003, 42(43), 5336-5338.
40.Turiel, E.; Martin-Esteban. A., Molecularly imprinted polymers: towards highly selective stationary phases in liquid chromatography and capillary electrophoresis. Analytical and bioanalytical chemistry 2004, 378(8), 1876-1886.
41.Sergeyeva; Tatiana A., Molecularly imprinted polymer membranes for substance-selective solid-phase extraction from water by surface photo-grafting polymerization. Journal of Chromatography A 2001, 907(1), 89-99.
42.Matsui, J., Molecular recognition in continuous polymer rods prepared by a molecular imprinting technique. Analytical Chemistry 1993, 65(17), 2223-2224.
43.Li, H., Separation and purification of chlorogenic acid by molecularly imprinted polymer monolithic stationary phase. Journal of Chromatography A 2005, 1098(1), 66-74.
44.Šafařı́ková, M.; Šafařı́k, I., Magnetic solid-phase extraction." Journal of Magnetism and Magnetic Materials 1999, 194(1), 108-112.
45.Li, X. S., Synthesis and applications of functionalized magnetic materials in sample preparation. TrAC Trends in Analytical Chemistry 2013, 45, 233-247.
46.Stark, D. D., Superparamagnetic iron oxide: clinical application as a contrast agent for MR imaging of the liver. Radiology 1988, 168(2), 297-301.
47.Gupta; Ajay K.; Mona G., Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 2005, 26(18), 3995-4021.
48.Abu, R.; Raed, Metal supported on dendronized magnetic nanoparticles: highly selective hydroformylation catalysts. Journal of the American Chemical Society 2006, 128(15), 5279-5282.
49.Jung, J. H.; Ji H. L.; Seiji S., Functionalized magnetic nanoparticles as chemosensors and adsorbents for toxic metal ions in environmental and biological fields. Chemical Society Reviews 2011, 40(9), 4464-4474.
50.Chen, L.; Ting W.; Jia T., Application of derivatized magnetic materials to the separation and the preconcentration of pollutants in water samples. TrAC Trends in Analytical Chemistry 2011, 30(7), 1095-1108.
51.Deng, Y.; Superparamagnetic high-magnetization microspheres with an Fe3O4@ SiO2 core and perpendicularly aligned mesoporous SiO2 shell for removal of microcystins. Journal of the American Chemical Society 2008, 130(1), 28-29.
52.Villanueva, A., The influence of surface functionalization on the enhanced internalization of magnetic nanoparticles in cancer cells. Nanotechnology 2009, 20(11), 115103.
53.Shu, Z.; Shulin W., Synthesis and characterization of magnetic nanosized Fe 3 O 4/MnO 2 composite particles. Journal of Nanomaterials 2009, 2009, 2.
54.Qi, Y., Preparation of Magnetic Molecularly Imprinted Polymer for Melamine and its application in milk sample analysis by HPLC. Journal of Biomedical Sciences 2016.
55.Szumski, M., Monolithic molecularly imprinted polymeric capillary columns for isolation of aflatoxins. Journal of Chromatography A 2014, 1364, 163-170.
56.Blesa, J., Determination of aflatoxins in peanuts by matrix solid-phase dispersion and liquid chromatography. Journal of Chromatography A 2003, 1011(1), 49-54.
57.Yu, L., Graphene oxide: an adsorbent for the extraction and quantification of aflatoxins in peanuts by high-performance liquid chromatography. Journal of Chromatography A 2013, 1318, 27-34.
58.Scaglioni, P. T., Aflatoxin B 1 and M 1 in milk. Analytica chimica acta 2014 829, 68-74.