分子模版高分子技術 (Molecularly imprinted polymers，MIP)，乃是先將目標分子或稱模版分子 (template)，以共價鍵或非共價鍵作用力與功能性單體 (functional monomer) 形成穩定的複合物，再加入交聯劑 (cross-linker) 與單體起聚合作用，並將單體穩固的固定住，而與單體結合之模版分子也隨之定位，隨後利用適當的溶劑萃洗出模版分子，因而產生與目標分子大小相同、官能基互補之辨識位置，這也造就了分子模版高分子之特異性吸附能力。

　　膽紅素 (Bilirubin IXa) 為本實驗之模版分子，為膽汁的主要成份之一，亦存在血液中。由於膽汁是由肝細胞所生成，經由輸送、分泌膽汁的膽道系統所分泌。因此，膽紅素對於肝功能之鑑定具指標性意義。本研究以甲基丙烯酸 (methacrylic acid，MAA)、二甲基丙烯酸乙二醇酯 (ethylene glycol dimethacrylate，EGDMA) 分別作為功能性單體及交聯劑，聚合而得膽紅素模版高分子，其模版高分子顆粒之模印因子 (imprinting factor) 為 10.18 ± 4.63。

　　本研究以光接枝聚合方式，將模版高分子固定於氧化鋁金電極之表面，並以硫醇提高金電極與高分子薄膜間之作用力。以 SEM 對於模版高分子薄膜之性質進行觀察，所製備薄膜之厚度約 140 nm。膽紅素以電化學感測的方式進行，其靈敏度為 0.42 mA/(mg/dl)。並以膽紅素模版高分子吸附其結構相似物膽綠素，以決定模版高分子之選擇性。結果確認了此高分子材料之模印效果，亦確認了在相似物共存下對膽紅素之辨識吸附能力。同時亦確認了以此電極進行電化學式感測膽紅素之可行性。

　　Bilirubin was used as a template. Molecularly imprinted polymer (MIP) technology and electrochemical analyzer were to be utilized, to form a sensor with high selectivity between bilirubin and its structural isomers biliverdin. The sensor’s substrate was alumina plate. After clean procedures, gold was sputtered on the plate (gold electrode). Subsequently, thiol was used to bind with the Au electrode and the molecularly imprinted polymer thin film. A solution consisted of template, porogen, initiator, monomer and cross-linker were dropped on the Au electrode, later. Surface polymerization was applied, and a polymer film could be obtained, it’s film thickness was approximately 140 nm. With complementary functional group and molecular size, the MIP film had specific imprinted sites. To find the correlations between the concentration of bilirubin solution and response current. A electrochemical analysis system was used to observe the electrochemical reaction occurred in the reaction cell. The response current of bilirubin solution could be calibrate, relied on MIP electrode. Its sensitivity was 0.42 mA/(mg/dl). A linear correlation existed between the concentrations of bilirubin and response current, from 0 mg/dl to 5 mg/dl. The imprinting factor of MIP particle was 10.18 ± 4.63. Thus, both of the sensitivity and the selectivity were demonstrated. The reproducibility of MIP electrode was perceived, by several MIP electrodes which were prepared by the same protocol. Bilirubin in the real sample (bile) could also be find by the MIP particles. In conclusion, combined the MIP film electrode and electrochemical sensing system to detect concentration of bilirubin was successful.

[1] L.C. Clark, Jnr. Ann. NY Acad. Sci., 102, 29-45, 1962
[2] K. Hosoya, Y. shirasu, K. Kimata and N. Tanaka, Molecularly imprinted chiral stationary phase prepared with racemic template, Analytical Chemistry, 70, 943-945, 1998
[3] L. Schweit, L. I. Andersson, Molecular imprint-based stationary phases for capillary electrochromatography, Journal of Chromatography A, 817, 5-13, 1998
[4] D. Kriz, C. B. Kriz, L. I. Andersson and K. Mosbach, Thin-layer chromatography based on the molecular imprinting technique, Analytical Chemistry, 66, 2636-2639, 1994
[5] A. Zander, P. Findlay, T. Renner and B. Sellergren, Analysis of nicotine and its oxidation products in nicotine chewing gum by a molecularly imprinted solid-phase extraction, Analytical Chemistry, 70, 3304-3314, 1998
[6] S. Kroger, A.P.F. Turner, K. Mosbach, K. Haupt , Anal. Chem., 71, 1999
[7] A. Pizzariello, M. Stre'ansky, S. Stred'anska, S. Miertus, A solid binding matrix/molecularly imprinted polymer-based sensor system for the determination of clenbuterol in bovine liver using differential-pulse voltammetry, Sens. Actuators B, 76, 286-294, 2001
[8] D. Kriz, Mosbach K, Competitive amperometric morphine sensor based on an agarose immobilised molecularly imprinted polymer, Anal. Chim. Acta, 300, 71-75, 1995
[9] N. Kirsh, J.P. Hart, D.J. Bird, R.W. Luxton, D.V. McCalley, Towards the development of molecularly imprinted polymer based screen-printed sensors for metabolites of PAHs, Analyst, 126, 1936-1941, 2001
[10] T. Yamazaki, Z. Meng, K. Mosbach, K. Sode, Electrochemistry, 69, 969, 2001
[11] T. Panasyuk-Delaney, V.M. Mirsky, M. Ulbritch, O.S. Wolfbeis, Impedometric herbicide chemosensors based on molecularly imprinted polymers, Anal. Chim. Acta, 435, 157-162, 2001
[12] S.A. Piletsky, E.V. Piletskaya, A.V. Elgersma, K. Yano, I. Karube, Y.P. Parhometz, A.V. El'skaya, Atrazine sensing by molecularly imprinted membranes, Biosens. Bioelectron., 10, 959-964, 1995
[13] E. Hedborg, F. Winquist, I. Lundstrom, L.I. Andersson, K. Mosbach, Sens. Actuators A, 37-38, 796, 1993
[14] S.A. Piletsky, E.V. Piletskaya, T.L. Panasyuk, A.V. El'skaya, R. Levi, I. Karube, G. Wulff, Imprinted Membranes for Sensor Technology: Opposite Behavior of Covalently and Noncovalently Imprinted Membranes, Macromolecules, 31, 2137 - 2140, 1998
[15] Y. Yoshimi, R. Ohdaira, C. Iiyama, K. Sakai, “Gate effect” of thin layer of molecularly-imprinted poly(methacrylic acid-co-ethyleneglycol dimethacrylate), Sens. Actuators B, 73, 49-53, 2001
[16] M.C. Blanco-López, M.J. Lobo-Castañón, A.J. Miranda-Ordieres, P. Tuñón-Blanco, Voltammetric sensor for vanillylmandelic acid based on molecularly imprinted polymer-modified electrodes, Biosens. Bioelectron., 18, 353 - 362, 2003
[17] Z. Cheng, E. Wang, X. Yang, Capacitive detection of glucose using molecularly imprinted polymers, Biosens. Bioelectron., 16, 179-185, 2001
[18] T. Panayuk, V.M. Mirsky, S.A. Piletsky, O.S. Wolfbeis, Electropolymerized Molecularly Imprinted Polymers as Receptor Layers in Capacitive Chemical Sensors, Anal. Chem., 71, 4609 - 4613, 1999
[19] T. Panasyuk, V. Campo Dall'Orto, G. Marrazza, A. El'skaya, S. Piletsky, I. Rezzano, M. Mascini, Anal. Lett., 31, 1809, 1998
[20] R.S. Hutchins, L.G. Bachas, Nitrate-Selective Electrode Developed by Electrochemically Mediated Imprinting/Doping of Polypyrrole, Anal. Chem., 67, 1654 - 1660, 1995
[21] L.D. Spurlock, A. Jaramillo, A. Praserthdam, J. Lewis, A. Brajter-Toth, Selectivity and sensitivity of ultrathin purine-templated overoxidized polypyrrole film electrodes, Anal. Chim. Acta, 336, 37-46, 1996
[22] A. Jenkins, R. Yin, J.L. Jensen, Molecularly imprinted self-assembled films with specificity to cholesterol, Sens. Actuators B, 60, 216-220, 1999
[23] V. Mirsky, T. Hirsch, S.A. Piletsky, O.S. Wolfbeis, Angew. Chem. Int. Ed., 38, 1108, 1999
[24] M. Lahav, E. Katz, I. Willner, Photochemical Imprint of Molecular Recognition Sites in Two-Dimensional Monolayers Assembled on Au Electrodes: Effects of the Monolayer Structures on the Binding Affinities and Association Kinetics to the Imprinted Interfaces, Langmuir, 17, 7387-7395, 2001
[25] S. Gutiérrez-Fernández, M.J. Lobo-Castañón, A.J. Miranda-Ordieres, P. Tuñón-Blanco, G.A. Carriedo, F.J. Garca-Alonso, J.I. Fidalgo, Molecularly Imprinted Polyphosphazene Films as Recognition Element in a Voltammetric Rifamycin SV Sensor, Electroanalysis, 13, 1399-1404, 2001
[26] R. Makote, M. Collinson, Template Recognition in Inorganic-Organic Hybrid Films Prepared by the Sol-Gel Process, Chem. Mater., 10, 2440-2445, 1998
[27] M. Zayats, M. Lahav, A.B. Kharitonov, I. Willner, Imprinting of specific molecular recognition sites in inorganic and organic thin layer membranes associated with ion-sensitive field-effect transistors, Tetrahedron, 58, 815-824, 2002
[28] J. D Wilson, E. Braunwald, K. J. Isselbacher, R. G. Petersdorf, J. B. Martin, A. S Fauci, R. K. Root, Harrison’s Principles of Internal Medicine, McGRAW-HILL, Inc., New York, 264-266, 1991
[29] A. K. Tipton, D. A. Lightner, A. F. Mcdonagh, Synthesis and metabolism of the first thia-bilirubin, Journal of Organic Chemistry, 66, 1832-1838, 2001
[30] T. Dorner, B. Knipp, D. A. Lightner, Heteronuclear noe analysis of bilirubin solution conformation and intramolecular hydrogen bonding, Tetrahedron, 53, 2697-2716, 1997
[31] E. Sykes, E. Epstein, Laboratory measurement of bilirubin, Clinics in Perinatology, 17, 397-415
[32] K. L. J. Vink, W. Schuurman, R. V. Gansewinket, Direct spectrophotometry of bilirubin in serum of the newborn, with use of caffeine reagent, Clinical Chemistry, 34, 67-70, 1988
[33] B. T. Dumas, B. Perry, B. Jendrazejczak, L. Davis, Measurement of direct bilirubin by use of bilirubin oxidase, Clinical Chemistry, 38, 1349-1353, 1987
[34] A. Kosaka, C. Yamamoto, Y. Morishita, K. Nakane, Enzymatic determination of bilirubin fractions in serum, Clinical Biochemistry, 20, 451-458, 1987
[35] C. C. Kuenzle, M. Sommerhalder, J. R. Ruttner, Separation and quantitative estimation of four bilirubin fractions from serum and of three bilirubin fractions from bile, The Journal of Laboratory and Clinical Medicine, 67, 282-293, 1966
[36] J.J. Lauff, M. E. Kasper, Separation of bilirubin species in serum and bile by high performance reversed-phase liquid chromatography, Journal of Chromatography, 226, 391-637, 1982
[37] S. Blix, Studies on thr fibrinolytic system in the englobulin fraction of human plasma, The Scandinavian Journal of Clinical and Laboratory Investingation, 13, 1-80, 1961
[38] B. T. Doumas, B. W. Perry, E. A. Sasse, J. V. Straumfjord, Standardization in bilirubin assays: evaluation of selected methods and stability of bilirubin solutions, Clinical Chemistry, 19, 948-993, 1997
[39] 林碩彥, 電流式醋酸薄膜感測器, 國立成功大學化工所, 2003
[40] Mei-Jywan Syu, Jing-Hong Deng, You-Ming Nian, An-Hua Wu, Binding specificity of a-bilirubin imprinted poly(methacrylic acid-co-ethylene glycol dimethylacrylate) toward a-bilirubin, Biomaterials, 26(22), 4684-4692, 2005
[41] Mei-Jywan Syu, Jing-Hong Deng, and You-Ming Nian, Towards bilirubin imprinted poly(methacrylic acid-co-ethylene glycol dimethylacrylate) for the specific binding of a-bilirubin, Analytica Chimica Acta, 504(1), 167-177, 2004