肌酸酐 (Creatinine) 是為肌肉中肌酸代謝的產物，為診斷腎功能的重要因子，所以體液中如血液(血清)或尿液肌酸酐的濃度是相當重要生理指標。
本研究乃是在製備特定針對肌酸酐具模印記憶的模版高分子材料，同時，使此材料具螢光可調節性，即可依肌酸酐被吸附量與螢光強度變化比例的關係進行對肌酸酐分子之較具專一性之螢光式檢測。研究是由合成螢光功能性單體為主軸開始進行。以4-bromo-1,8-naphthalic anhydride (4-BNA) 為起始反應物，進行合成反應 : 首先先以乙烯苯胺基取代4-BNA中的酸酐合成4-bromo-N-vinylbenzylnaphthalimide (4-BVNI)，使之具可聚合之官能基；爾後再以甲基胺取代溴合成4-methylamino-N-vinylbenzylnaphthalimide (4-MAVNI)，合成最終產物具螢光性之單體，並對此螢光單體進行螢光性質之分析。繼之，合成專屬於肌酸酐的螢光模版高分子並以材料對肌酸酐進行吸附探討。模版材料之合成乃是以肌酸酐為模版分子，加入功能性單體 methylacrylic acid (MAA)、螢光單體4-methylamino-N-vinylbenzylnaphthalimide (4-MAVNI)及高比例的交聯劑ethylene glycol dimethacrylate (EGDMA) 進行聚合。此模版材料 (MIP) 對肌酸酐的平均吸附量為8.49 ± 0.64 mg/g MIP；而對應之非模印材料 (NIP) 則為2.04 ± 0.83 mg/g NIP，因此可得模印因子imprinted factor為4.16 ± 0.48，這是以 HPLC 進行肌酸酐成分分析所得的結果。而在螢光檢測部分，螢光模版高分子之螢光變化量為19.96 ± 1.13 %，螢光非模版高分子之螢光變化量為5.85 ± 1.17 %，其imprinting factor為3.41 ± 0.61，顯示此製備之螢光模版高分子可以螢光方式檢測未知樣本或檢體中肌酸酐濃度之可行性。
在製備過程中，4-MAVNI, MAA, EGDMA 之較佳莫耳比是首要的操作條件，而以模印效果做為比較之指標；此外在螢光模版高分子選擇性吸附感測中，皆顯示出有良好選擇率，在肌酸酐/N-羥基丁二醯亞氨雙成份水溶液中之選擇率為3.04 ± 0.21；而在螢光模版高分子之干擾性吸附測試，在肌酸酐濃度10 mg/dl，其螢光強度變化量為7.16 ± 0.56 %，平均吸附量為4.40 ± 0.66 mg/g MIP，皆呈現良好之辨識效果。
以製備之螢光模版高分子膜進行肌酸酐之吸附感測，以2 mg/dl肌酸酐測得之螢光強度變化是3.25 ± 0.07 %，模印因子則是1.37 ± 0.21，更進一步確証MIP之特異性吸附性質，而致造成不同於NIP之螢光變化。
綜合前述，以螢光單體進行模版高分子材料之製備以確認可行，且在對肌酸酐吸附效應上，無論專一性、干擾性、選擇性和靈敏性皆有良好之效果，顯示以螢光模版高分子進行肌酸酐螢光式感測之可行性已確立。

Creatinine, the metabolic product of creatine in muscle, is an important factor in diagnosis of renal function, so creatinine concentration in serum or urine is an important clinical health marker.
This study is in the manufacture of molecular imprinted polymer (MIP) that provides specific cavity of creatinine and fluorescent property specific target of creatinine by tuning the ratio between creatinine binding capacity and fluorescent intensity change. The study started from aiming fluorescent monomer as the main target. The fluorescent monomer was synthesized from the reaction of 4-bromo-1,8-naphthalic anhydride with 4-vinylaniline to form 4-bromo-N-vinylbenzylnaphthalimide, and further react with methylamine to form 4-methylamino-N-vinylbenzylnaphthalimide. When the fluorescent monomer was successfully synthesized, 4-methylamino-N-vinylbenzylnaphthalimide, EGDMA crosslinker, and AIBN initiator were then mixed together in the presence of creatinine template. The specific recognition cavity for creatinine will exist after the removal of creatinine by proper solvent from the prepared polymer matrix. The binding capacities of the MIP and the non-imprinted ones are 8.49 ± 0.64 and 2.04 ± 0.83 mg/g, respectively, and that ratio is 4.16 ± 0.48 which is defined as the imprinting factor. On the other hand, the ratios of corresponding fluorescence change are 19.96 ± 1.13 % and 5.85 ± 1.17 % respectively, thus the imprinted factor calculated from fluorescent detection is 3.41 ± 0.61. Both kinds of measurements confirmed that the thus-prepared MIP is practicable toward detecting the creatinine concentration of unknown samples or samples of serum or urine by fluorescent method. .
In the manufacture process, the composition of the solution for polymerization was studied. The moles of 4-MAVNI, MAA, EGDMA were considered as the vital role affecting the imprinting effect; in addition, the imprinting effect as well as selectivity from the creatinine containing mixture was also evaluated. The selectivity of MIP toward creatinine/N-hydroxysuccinimide mixture is 3.04 ± 0.21. Creatinine was also spiked into fetal bovine serum (FBS) for the investigation of MIP toward creatinine binding. The average binding capacity of MIP is 4.40 ± 0.66 mg/g MIP, and fluorescent change ratio is 7.16 ± 0.56 %. The effect of MIP toward creatinine recognition is confirmed to be fine.
The imprinting effect of the prepared fluorescent MIP films was studied by the fluorescent detection. The fluorescent change ratio from the creatinine uptake by the MIP films in 2 mg/dL creatinine solution was 3.25 ± 0.07 %, and that of the imprinting factor was 1.37 ± 0.21. The specific adsorption property of MIP was further confirmed and proofed to be right that causes the fluorescent change to be different from the results of NIP.
Combining the results above, it was confirmed that MIP made by adding fluorescent monomer in the manufacture process is feasible. The effect of creatinine adsorption by MIP is good no matter the reading from specific recognizable binding, selectivity test, sensitivity test, or the adsorption in the mixing solutions (serum). That confirms the practicable of using fluorescent MIP toward creatinine detection by fluorescent method.

1. George Vlatakis, Lars I. Andersson, Ralf Muller, KlausMosbach, Drug assay using antibody mimics made by molecular imprinting, Nature 361, 645 - 647 , 18 February 1993.
2. Hongyuan Yan, Kyung Ho Row, Characteristic and Synthetic Approach of Molecularly Imprinted Polymer, Int. J. Mol. Sci. 2006, 7, 155-178.
3. Giinter Wulff, Molecular Imprinting in Cross-Linked Materials with the Aid of Molecular Templates-A Way towards Artificial Antibodies, angew. Chem. Int. Ed. Engel. 34, 1812-1832, 1995.
4. Wulff, G.; Sharhan, A.; Zabrocki, K., Enzyme analogue built polymers and their use for the resolution of racements. Tetrahedron Lett.1973, 14, 4329-4332.
5. R. Cody Stringer, Shubhra Gangopadhyay, Sheila A. Grant, Comparison of molecular imprinted particles prepared using precipitation polymerization in water and chloroform for fluorescent detection of nitroaromatics, Analytica Chimica Acta, 2011.
6. Guoqing Pan, Yue Ma, Ying Zhang, Xianzhi Guo, Chenxi Li, Huiqi Zhang, Controlled synthesis of water-compatible molecularly imprinted polymer microspheres with ultrathin hydrophilic polymer shells via surface-initiated reversible addition-fragmentation chain transfer polymerization, Soft Matter, 2011, 7, 8428.
7. Shenguang Ge, Juanjuan Lu, Lei Ge, Mei Yan, Jinghua Yu, Development of a novel deltamethrin sensor based on molecularly imprinted silica nanospheres embedded CdTe quantum dots, Spectrochimica Acta Part A 79 , 2011 ,1704– 1709.
8. Yiqun Yang, Chenglin Yi, Jing Luo, Ren Liu, Junkang Liu, Jinqiang Jiang, Xiaoya Liu, Glucose sensors based on electrodeposition of molecularly imprinted polymeric micelles: A novel strategy for MIP sensors, Biosensors and Bioelectronics 26 , 2011, 2607–2612.
9. Alfonso Fernández-González , Rosana Badía-Laíño, Marta Elena Díaz-García, Improving the synthesis of a molecularly imprinted sol-gel for serine using a Plackett-Burman design, Microchim Acta , 2011, 172:351–356.
10. Paolo Lucci, Delphine Derrien, Florent Alix, Celine Perollier, Sami Bayoudh, Molecularly imprinted polymer solid-phase extraction for detection of zearalenone in cereal sample extracts, Analytica Chimica Acta 672 , 2010, 15–19.
11. Baojiao Gao, Yanbin Li, Zhenguo Zhang, Preparation and recognition performance of creatinine-imprinted material prepared with novel surface-imprinting technique, Journal of Chromatography B, 878, 2010, 2077–2086.
12. Xiaogang Hu,Jialiang Pan, Yuling Hu,Gongke Li, Preparation of Molecularly Imprinted Polymer Coatings with the Multiple Bulk Copolymerization Method for Solid-Phase Microextraction, Journal ofAppliedPolymer Science,Vol. 120, 1266–1277 , 2011.
13. Wulff, G.; Vesper, R.; Grobe, E.; Sarhan, A. Enzyme-analogue built polymers. 4. on the synthesis of polymers containing chiral cavities and their use for the resolution of racemates. Makromol. Chem. 1977, 178, 2799-2816.
14. Saman Azodi-Deilami, Majid Abdouss, Mehran Javanbakht, The Syntheses and Characterization of Molecularly Imprinted Polymers for the Controlled Release of Bromhexine, Appl Biochem Biotechnol , 2011, 164:133–147.
15. Joris P. Schillemans1, Cornelus F. van Nostrum1, Molecularly imprinted polymer particles: synthetic receptors for future medicine, Nanomedicine, 2006, 1(4), 437–447.
16. Jun Haginaka, Monodispersed, molecularly imprinted polymers as affinity-based chromatography media, Journal of Chromatography B, 866 , 2008, 3–13.
17. Yan Lu, Chang-Ling Yan, Shu-Yan Gao, Preparation and recognition of surface molecularly imprinted core–shell microbeads for protein in aqueous solutions., Applied Surface Science, 2009, 255, 6061–6066.
18. Ryh-Yaw Hsieh, Hsuan-Ang Tsai, Mei-Jywan Syu, Designing a molecularly imprinted polymer as an artificial receptor for the specific recognition of creatinine in serums, Biomaterials 27 , 2006, 2083–2089.
19. Toshifumi Takeuchi,Takashi Mukawa,Jun Matsui, Miho Higashi, Ken D. Shimizu, Molecularly Imprinted Polymers with Metalloporphyrin-Based Molecular Recognition Sites Coassembled with Methacrylic Acid, Anal. Chem. 2001, 73, 3869-3874.
20. Xianwen Kan, Qun Zhao, Zhong Zhang, Zhilin Wang , Jun-Jie Zhu, Molecularly imprinted polymers microsphere prepared by precipitation polymerization for hydroquinone recognition, Talanta 75 , 2008, 22–26.
21. Gianluca Ciardelli, Cristiana Borrelli, Davide Silvestri, Caterina Cristallini , Niccoletta Barbani , Paolo Giusti, Supported imprinted nanospheres for the selective recognition of cholesterol, Biosensors and Bioelectronics 21,2006, 2329–2338.
22. Olivier Y. F. Henry , David C. Cullen, Sergey A. Piletsky, Optical interrogation of molecularly imprinted polymers and development of MIP sensors: a review, Anal Bioanal Chem (2005) 382: 947–956.
23. Daniel J. Duffy, Kanad Das, Shaw L. Hsu, Jacques Penelle, Vincent M. Rotello, and Howard D. Stidham, Binding Efficiency and Transport Properties of Molecularly Imprinted Polymer Thin Films, J. Am. Chem. Soc., 2002, 124 (28), 8290-8296.
24. Michael Jakusch, M. Janotta, Boris Mizaikoff, Klaus Mosbach, Karsten Haupt, Molecularly Imprinted Polymers and Infrared Evanescent Wave Spectroscopy. A Chemical Sensors Approach, Anal. Chem., 1999, 71 (20), 4786-4791.
25. Sing Muk Ng & R. Narayanaswamy, Fluorescence sensor using a molecularly imprinted polymer as a recognition receptor for the detection of aluminium ions in aqueous media, Anal Bioanal Chem , 2006, 386:1235–1244.
26. Petra Turkewitsch,Barbara Wandelt, Graham D. Darling, William S. Powell, Fluorescent Functional Recognition Sites through Molecular Imprinting. A Polymer-Based Fluorescent Chemosensor for Aqueous cAMP, Anal. Chem. 1998, 70, 2025-2030.
27. T. Hien Nguyen, Richard J. Ansell, Fluorescent imprinted polymer sensors for chiral amines, Org. Biomol. Chem., 2009, 7, 1211–1220.
28. Cheuk-Fai Chow, Michael H.W. Lam, Mitch K.P. Leung, Fluorescent sensing of homocysteine by molecular imprinting, Analytica Chimica Acta 466, 2002, 17–30.
29. Mei-Hwa Lee, Yun-Chao Chen, Min-Hsien Ho, Hung-Yin Lin, Optical recognition of salivary proteins by use of molecularly imprinted poly(ethylene-co-vinyl alcohol)/quantum dot composite nanoparticles, Anal Bioanal Chem, 2010, 397:1457–1466.
30. 馬濤, 石英晶體微天平的原理及應用, 2008.
31. Hui Peng, Chengdu Liang, Anhong Zhou, Youyu Zhang, Qingji Xie, Shouzhuo Yao, Development of a new atropine sulfate bulk acoustic wave sensor based on a molecularly imprinted electrosynthesized copolymer of aniline with o-phenylenediamine, Analytica Chimica Acta 423 , 2000, 221–228.
32. Naoto Tsuru , Masashi Kikuchi, Haruma Kawaguchi, Seimei Shiratori, A quartz crystal microbalance sensor coated with MIP for‘‘Bisphenol A’’ and its properties, Thin Solid Films 499 , 2006, 380 – 385.
33. M. C. Blanco-Lo′ pez, M. J. Lobo-Castan˜ o′ n, A. J. Miranda-Ordieres, P. Tun˜ o′ n-Blanco, Electrochemical sensors based on molecularly imprinted polymers, Trends in Analytical Chemistry, 2004, 23, 36–48.
34. Dario Kriz, Klaus Mosbach, Competitive amperometric morphine sensor based on an agarose immobilised molecularly imprinted polymer, Analytica Chimica Acta 300 , 1995, 71-75.
35. T.A. Sergeyeva, S.A. Piletsky, A.A. Brovko, E.A. Slinchenko, L.M. Sergeeva, A.V. El'skaya, Selective recognition of atrazine by molecularly imprinted polymer membranes. Development of conductometric sensor for herbicides detection, Analytica Chimica Acta 392 , 1999, 105-111.
36. Michal Lahav, Eugenii Katz, Itamar 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 2001, 17, 7387-7395.
37. Sheshadri Narayanan, Harold D. Appleton, Creatinine: A Review, Clinical Chemistry, 1980, 26, 1119-1126.
38. Burtis, C. A., and Ashwood, E. R., Tietz testbook of clinical chemistry, Saunders, Philadelphia.
39. Kaplan, A., Jack, R., Opheim, K. E., Toivola, B., and Lyon, A. W., Clinical Chemistry-Interpretation and Techniques 4th, 1995, Williams & Wilkins
40. Spierto, F. W., Hannon, W. H., Gunter, E. W., and Smith, S. J., Stability of urine creatinine, Clinica Chimica Acta, 1997, 264, 227-232.
41. Bazari H. Approach to the patient with renal disease. In: Goldman L, Ausiello D, eds. Cecil Medicine. 23rd ed. Philadelphia, Pa: Saunders Elsevier, 2007, chap 115.
42. Dhana Lakshmi, Bhim Bali Prasad , Piyush Sindhu Sharma, Creatinine sensor based on a molecularly imprinted polymer-modified hanging mercury drop electrode, Talanta 70 , 2006, 272–280.
43. J.-C. Chen, A.S. Kumar, H.-H. Chung, S.-H. Chien , M.-C. Kuo , J.-M. Zen, An enzymeless electrochemical sensor for the selective determination of creatinine in human urine, Sensors and Actuators B 115 , 2006, 473–480.
44. Smith-Palmer T., Separation methods applicable to urinary creatine and creatinine, Journal of Chromatography B–Analytical Technologies in the Biomedical and Life Sciences, 2002, 781, 93-106.
45. A. Ramanavicius, Amperometric biosensor for the determination of creatine., Analytical and Bioanalytical Chemistry, 2007, 387, 1899-1906.
46. Jason A. Berberich ,1, Andy Chan , Mark Boden, Alan J. Russell, A stable three-enzyme creatinine biosensor. 3. Immobilization of creatinine amidohydrolase, Acta Biomaterialia 1 , 2005, 193–199.
47. Skoog.Holler.Nieman., Principle of instrumental analysis, fifth edition, 355-376.
48. 林敬二, 林宗義., 儀器分析(上), 第四版, 174-193