近年來，許多研究顯示出另外一種快速有效預測心血管疾病的生物指標 – C-反應蛋白(C- reactive protein,CRP)，以人體血液中CRP的濃度，作為臨床上心血管疾病評估的因子。一般臨床上的分析方式是採用免疫分析法來定量CRP的濃度，但該分析方法需使用價格昂貴之抗體且有耗時及由於多重操作步驟所引起的人為誤差等缺點。

　　分子模版具有與抗體類似的高選擇性、親和力及立體構形等之特異性，這些特性為其他物質所欠缺的；加上分子模版具有良好的物性和化性、優良的機械性質，亦能耐高溫、高壓、抗酸鹼、穩定、便宜，基於這些特點，可引入分子模版於一般免疫分析法中取代高價之天然抗體。
　　
　　本研究主要是以微接觸法並輔以高分子聚合反應的方式製備C-反應蛋白分子模版。微接觸法的優點在於不需要使用溶劑，同時可在進行聚合反應時於分子模板表面形成緻密且對目標物分子具辨識能力的活性孔洞。
　　
　　實驗設計是以CRP為目標物，O-4-nitrophenylphosphorylcholine (O-4NPPC)為辨識性單體分子，2,2’-dimethoxy-2-phenyl-acetophenone為起始劑。實驗初期先利用不同交聯劑種類對CRP吸附程度的影響，找到一組對目標物有最低吸附值的交聯劑。實驗所使用的單體有親油性的Pentaerythritol tetraacrylate (PATTA)，二乙烯基苯（divinylbenzene，DVB），TMPTMA 以及親水性的交聯劑如乙二醇二甲基丙烯酸酯(ethylene glycol dimethacrylate, EGDMA)、乙三醇二甲基丙烯酸酯（Triethylene glycol dimethacrylate，TEGDMA）、聚乙二醇二甲基丙烯酸酯(Poly(ethylene glycol)dimethacrylate)等。由實驗結果得知當使用PEG400DMA所製備之薄膜對CRP無明顯之吸附量，因而PEG400DMA為最適此系統之交聯劑。本實驗中是利用UV光來起始光起始劑以產生自由基進行聚合以合成目標物之模版，再用胰蛋白脢及鹼性清潔劑作為脫附劑進行目標物之脫附，即完成分子模版。完成分子模版製備後，以製成分子模版薄膜浸入含目標物C-反應蛋白標準液中，進行目標物選擇性吸附功能的研究。由酵素固定法（ELISA）進行目標物含量分析偵測，由冷光發光強度計算目標物被吸附的效率。此外，也用AFM及SEM得到表面型態之資訊。

　　在本研究中，有一系列分子模版與非分子模版被合成出用以尋找最小之分子模版訊號。由結果得知，在多種不同交聯劑的選擇中，發現當以PEG400DMA作為交聯劑時，所合成出的分子模版有最低的背景吸附值；並以O-4NPPC模擬自然界中辨識CRP的phosphorylcholine site (pc site)且在有鈣離子存在的條件下所合成出的模版對目標物CRP、干擾物如人類白蛋白（human Albumin）、G型球蛋白(IgG)進行選擇性的吸附測試，結果顯示出，在個別單一環境下，C-反應蛋白再吸附於CRP分子模版的能力約為白蛋白的2倍，而為G型球蛋白的5.3倍。在競爭性吸附的測試，結果顯示當CRP分子模版在與C-反應蛋白、白蛋白同時存在的環境下，C-反應蛋白的辨識度約為白蛋白的8.7倍，而C-反應蛋白、G型球蛋白同時存在的環境下，C-反應蛋白的辨識度約為G型球蛋白的1.6倍。
　　
　　同樣的方法亦可應用於他種目標物分子上，如人體白蛋白感測。
本研究利用微接觸分子模版技術不但成功地辨識多環性的蛋白質分子；也成功突破了過去科學家認為分子模版技術無法應用在大分子如蛋白質、DNA研究上的瓶頸。

　In recent years, a lot of research has demonstrated that C-reactive protein (CRP) in blood can act as a predictor of cardiovascular disease risk. Immunoassay analysis is the traditional analytical method for determining CRP levels in body fluids; its shortcomings are that it is time consuming and requires complicated operating procedures.

　Molecularly imprinted polymers (MIPs) have high selectivities and affinities and for these reasons are sometimes known as the artificial antibodies. Additionally, they possess both stable physical and chemical properties and are relatively cheap to prepare. For some analysis, we are able to use MIPs to replace natural antibodies.

　The micro-contact CRP imprinting technique has been employed and further developed in this study. The benefit of this method is that no porogen is used and the MIPs produced show an excellent recognition for the template.

　The purpose of this study was to synthesize a CRP -imprinted polymer. O-(-4-nitrophenylphosphoryl)choline (O-4NPPC), being a commercially available analogue of the natural ligand, was used as the function monomer, and 2,2’-dimethoxy- 2-phenylacetophenone as the initiator. Radical polymerization was induced by UV light to form the polymer containing the template. A series of non-imprinted polymers made without template, but with varying crosslinker, were made in order to produce a control polymer with minimal non-specific recognition. Trypsin and detergent (SDS) were used in base conditions to remove the template. The rebinding efficiency of CRP species on the CRP MIP was determined by the intensity of luminescence in an enzyme-linked immunosorbent assay (ELISA). Also, the surface morphology was examined by both atomic force (AFM) and scanning electron microcopy. (SEM)

　In this study, a series of non-imprinted polymers made without template, but with varying cross-linking agents were made in order to produce a control polymer with minimal non-specific recognition. The result indicated that affinity of the CRP imprinted polymers when incubated together with Ca2+ ions for CRP albumin (alb) and Immunoglobulin G (IgG) were examined and the CRP/albumin and CRP/IgG binding ratios were found to be 2 and 5.3; respectively.

　A competitive binding experiment showed the CRP imprinted material to retain good selectivity for its template when jointly incubated with albumin or IgG. The affinity ratios of the imprinted polymers for CRP when in competition with albumin or IgG, were examined and found to be 8.7 and 1.6 for CRP/albumin and CRP/IgG; respectively.

　Using the developed method for CRP imprinting, this technique can be applied to another protein and form a micro-contact imprint of human serum albumin which demonstrated relatively good recognition for its own template.

　This work, which has resulted in the first successful imprint of a multi-subunit protein, shows that the micro-contact MIP technique is able to be extended to templates, such as proteins and DNA, it’s a breakthrough of imprinting big or macromolecular bottleneck.

參考文獻
[1]A. Bossi, M. D'Acunto, P. Righettivol, "Controlled enzyme-immobilisation on capillaries for microreactors for peptide mapping", Anal Bio Chem, vol. 378, pp. 1722-1728, 2004.
[2]P. Tarkkinen, T. Lovgren," Ultrarapid, Ultrasensitive One-Step Kinetic Immunoassay for C-Reactive Protein (CRP) in Whole Blood Samples: Measurement of the Entire CRP Concentration Range with a Single Sample Dilution", Clin Chem, vol. 48, pp.269-277 2002.
[3]A. Douglas, J. Linderman, "Receptors model for binding, trafficking, and signaling". New York Oxford: Oxford university press, Chapter 1-2, 1993.
[4]趙世斌、周大中等人編譯, 生物化學: 藝軒圖書出版社,台灣, 2000.
[5]D. Baltimore, "The brain of a cell", Science, vol. 84, pp.324-328, 1984.
[6]http://juang.bst.ntu.edu.tw/BCbasics/Enzyme34.htm.
[7] C. Fillee, M. Mourad, JP. Squifflet, J. Malaise ," Evaluation of a new immuno-assay to measure sirolimus blood concentrations compared to a tandem mass-spectrometric chromatographic analysis" Theory Drug Moni, 27 (2): 258-258
[8]N. Holland, Y. Qiu, M. Ruegsegger, R. Marchant, "Biomimetic engineering of non-adhesive glycocalyx-like surfaces using oligosaccharide surfactant polymers", Nature, vol. 392, pp. 799-801, 1998.
[9]http://www.smi.tu-berlin.De/syory/MIT.htm, 2002.
[10]B. Sellergren, "Molecularly Imprinted Polymers , Man-made mimics of antibodies and their applications in analytical chemistry",Elservier. New York, Chapter1, 2001.
[11]H. Dickey, "The Preparation of Specific Adsorbents", proceed. of Nat. Acad of Sci, vol. 35, pp. 227-229, 1940.
[12]K. Mosbach, "Toward the next generation of molecular imprinting with emphasis on the formation, by direct molding, of compounds with biological activity (biomimetics)", Anal Chim Acta, vol. 435, pp. 3-8, 2001.
[13]奈米生醫醫學網http://nano.cyut.net.tw/4.htm#2.
[14]G. Wulff, A. Steinert, O. Holler ,"Modification of amylose and investigation of its inclusion behavior". Carbohyd Res,307 (1-2): 19-31
[15]C. Muehleman, M. Whiteside, Z. Zhong, J. Mollenhauer, M. Aurich, K. E. Kuettner, L. D. Chapman, "Diffraction enhanced imaging for articular cartilage", Biophy Journal, vol. 82, pp. 470a-476a, 2002.
[16]A. Bernard, B. Hens, E. Delamarche, "Microcontact Printing of Proteins", Adv Mater, vol. 12, pp. 1067-1074, 2000.
[17]L.Anderson, "Molecularly imprinted polymers",Elsevier, New York, chapter 14, 2001
[18]M. Maeda. R. Barsch, "Molecular and ionic Recognition with Imprinted polymers", American Chemical Society, Washington, DC.Chapter1-2, 1988.
[19]林玉娟、李昆峰, "分子模印技術之介紹與發展契機," 工業技術研究院. http://www.itri.org.tw/chi/services/ieknews/h-B01-500324
[20]D. Kriz, O. Ramstrom, K. Mosbach,"Molecular imprinting - New possibilities for sensor technology", Anal Chem, vol. 69, pp. A345-A349, 1997.
[21]S. Piletsky, "Atrazine sensing by molecularly imprinted membranes", Biosens Bioelectron, vol. 10, pp. 959-964, 1995.
[22]J. Matsui, Y. Miyoshi, T. Takeuchi, "A molecularly imprinted synthetic polymer receptor selective for atrazine", Anal Chem, vol. 67, pp. 4404-4408, 1995.
[23]P. Turkewitsch, B. Wandelt, G. Darling, S. Powell, "Fluorescent functional recognition sites through molecular imprinting. A polymer-based fluorescent chemosensor for aqueous cAMP", Anal Chem, vol. 70, pp. 2771-2771, 1998.
[24]G. Vlatakis, L. Andersson, R.Muller, K.Mosbach, "Drug assay using antibody mimics made by molecular imprinting", Nature, vol. 361, pp. 645-647, 1993.
[25]D. Kriz, L. Andersson, K. Mosbach, "A theory of the low-frequency dielectroic dispersion of colloidal particles on electrolyte solution1", Anal Chem, vol. 66, pp. 2636-2639, 1994.
[26]A. Mayes, K. Mosbach, "Sugar Binding Polymers Showing High Anomeric and Epimeric Discrimination Obtained by Noncovalent Molecular Imprinting", Anal Biochem, vol. 222, pp. 483, 1994.
[27]L.I. Andersson, G. Vlatakis, K. Mosbach, Proc. Natl. Acad. Sci, USA, vol. 92, pp. 4788, 1995.
[28]L. Andersson,"Application of molecular imprinting to the development of aqueous buffer and organic solvent based radioligand binding assays for (S)-propranolol", Anal Chem, vol. 68, pp. 111-117, 1996.
[29]D. Kriz, A. Svensson, K. Mosbach," A Biomimetic Sensor Based on a Molecularly Imprinted Polymer as a Recognition Element Combined with Fiber-Optic Detection", Anal Chem, vol. 67, pp. 2142-2144, 1995.
[30]S. Piletsky, E. Piletskaya, T. Panasyuk, A. El'skaya, R. Levi, I. Karube, G. Wulff," Imprinted membranes for sensor technology: Opposite behavior of covalently and noncovalently imprinted membranes", Macro, vol. 31, pp. 2137-2140, 1998.
[31]A. Peter," Molecular Imprinting : Recent Developments and The Road Ahead", React Func Polym, vol. 41, 1999.
[32]http://www.doh.gov.tw/statistic/data/死因摘要/93年/統計圖/2.主要死因死亡率圖.xls
[33]林世鐸、杜思德、謝芳傑、許上人、林國川、許惠恆,"高敏感度C-反應蛋白(hsCRP)在偵測及預防心血管疾病方面的臨床應用", 內科學誌, vol. 15, pp. 4-8, 2004.
[34]薛樹清、呂旭峰、黃志堅、劉嘉又, "高敏感性C-反應蛋白(hs-CRP)試驗與冠心病之關係", 臨床醫學, vol. 53, pp. 422-429, 2004.
[35]J. Volanakis, "Human C-reactive protein: expression, structure, and function", Mol Immuno, vol. 38, pp. 189-197, 2001.
[36]S. Black, I. Kushner, and D. Samols, "C-reactive protein", J Bio Chem, vol. 279, pp. 48487-48490, 2004.
[37]K.Lagrand, A. Vieesr, J. Wolbink," C-reactive protein as a cardiovascular risk factor: more than an epiphenomenon?" Circul, vol. 100, pp. 96-102, 1999.
[38]R. Robert ,K. Wolfgang , "High-sensitivity C-reactive protein and cardiovascular risk in patients with coronary heart disease", Curr Opin Car, vol. 17, pp. 325-331, 2002.
[39]B. Christine, D.John, A.Lawrence, G. Janon," Loss of pentameric symmetry of C-repromotion of neutrophil-endothelial cell adhesion", J of Immun, vol. 167, pp. 5355-561, 2001.
[40]J.Alexanser, "The antimicrobial activity of C-reactive protein", Micro Infec, vol. 4, pp. 201-205, 2002.
[41]J. Michael, "A hypothesis resolving the apparently disparate activities of native and altered forms of human C-reactive protein", Immuno Res, vol. 12, pp. 37-47, 1993.
[42]T. Gary, "News focus: Does inflammation cut to the heart of the matter?" Science, vol. 296, pp. 242-245.2002
[43] N .Rifai, "C-reactive protein: a new and strong predictor of cardiovascular disease", Clin Lab news, vol. 27, pp. 12-14, 2001.
[44]K. Shigeru et al.," Immunosensors using a quartz crystal microbalance", Mat Sci Technol, vol. 14, pp. 1882-1887, 2003.
[45]詹子欣, "Rapid Identification of C-reactive Protein by Use of Fiber-optic Biosensor", 碩士論文，國立陽明醫學院，台北， 2003.
[46] 陳威志, “To Design the Creatinine Imprinted Polymers Based on the Information from Microcalorimeter”. 碩士論文，國立成功大學, 2004.
[47]M. Pepys and G. Hirschfield,”C-reactive protein: a critical update”, J of Clin Inves, vol. 111, pp. 1805-1812, 2003.
[48] D. Thompson, M. Pepys, B. Wood, “The physiological structure
of human C-reactive protein and its complex with phosphocholine”
,Structure Fold, vol.7 pp.169,1999
[49] J. Liberda, M.Ticha,”Immobilization of L-glyceryl phosphorylcholine: isolation of phosphorylcholine-binding proteins from seminal plasma”, J. of Chrom B, vol.770 pp 701-110, 2002
[50] 王春山，”polymers for microelectronics”國立成功大學化工所，台灣，ch 3,2004
[51] 喻凱，陳治清，”人體硬組織替代材料表面吸附白蛋白方法之研究”，生物醫學工程學雜誌，15（2），pp 155-157。
[52]http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/InfraRed/infrared.htm