肌酸酐 (Creatinine) 為診斷人體腎功能重要的因子，也是肌肉中肌酸代謝的產物，所以人體肌酸酐的含量為相當重要的一個指標，本實驗企圖利用4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) 進行鹵素取代反應生成4-(2-hydroxyethylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-1)， 再和ethyloyl chloride反應合成4-(2-acryloyloxyethylamino)-7-nitro-2,1,3-benzoxadiazole
(NBD-2)，將合成的螢光單體和中間產物利用FT-IR、1H NMR和13C NMR進行定性之鑑定，確定合成的各階段產物和預期之結構相同。另外合成的螢光單體亦進行螢光穩定性的探討，並且量測不同濃度的螢光單體之螢光強度，得知低濃度時，螢光強度會隨濃度之增加而上升；在高濃度時，螢光強度則因濃度上昇而下降，同時，此螢光單體在不同溶劑下之三維螢光光譜圖亦一倂討論。
將此螢光單體、肌酸酐與功能性單體甲基丙烯酸 (methacrylic acid)一起混合，在交聯劑乙二醇二甲基丙烯酸酯 (ethylene glycol dimethacrylate) 作用下，進行高分子聚合反應，以製備肌酸酐螢光模版高分子，並且利用高壓液相層析儀檢測模版高分子粉末之吸附，在單成份溶液中，模版高分子吸附肌酸酐的量為1.497 ± 0.088 mg creatinine/g MIP，非模版高分子吸附肌酸酐的量為0.652 ± 0.057 mg creatinine/g NIP，吸附肌酸的量0.361 ± 0.091 mg creatine/g MIP，模印因子為2.313 ± 0.242，吸附肌酸的量為0.397 ± 0.086 mg creatine/g NIP，選擇率為4.357 ± 0.918；在肌酸和肌酸酐雙成份溶液吸附中，模版高分子吸附肌酸酐的量為1.094 ± 0.052 mg creatinine/g MIP，吸附肌酸的量為0.222 ± 0.083 mg creatine/g MIP，模印因子2.513 ± 0.265，非模版高分子吸附肌酸酐的量為0.439 ± 0.043 mg creatinine/g NIP，吸附肌酸的量為0.217 ± 0.045 mg creatine/g NIP，選擇率為5.561 ± 1.836，顯示在雙成份溶液中進行吸附，模版高分子之選擇率和模印因子均較佳。
對此模版高分子研磨顆粒進行沉降實驗，並對沉降數據進行模擬。以螢光檢測模版高分子粉末之吸附，模版高分子吸附肌酸酐之螢光變化量為3.32% ± 0.70%，吸附肌酸後之螢光變化量為0.85% ± 0.27%，非模版高分子吸附肌酸肝之螢光變化量為0.92% ± 0.25%，則模印因子為3.91 ± 0.51，選擇率為3.60 ± 0.28，比較液相層析與螢光之檢測結果，顯示以螢光方式進行檢測之模印因子較高。
以不同條件進行模版高分子膜之製備，在熱聚合條件下，交聯比1：6 (功能性單體 (MAA) 對交聯劑)進行吸附，並以螢光檢測，得知模印因子為2.27 ± 0.44，選擇率為1.98 ± 0.21。在交聯比1：5條件下，模印因子則為2.63 ± 0.45，選擇率為2.48 ± 0.39。若在光聚合條件下，以交聯比1：6製備所得之模版螢光材料進行吸附，以螢光進行檢測，得知模印因子為2.45 ± 0.49，選擇率為1.77 ± 0.25，在交聯比1：5條件下之模印因子則為2.88 ± 0.64，選擇率為2.85 ± 0.64，顯示交聯比1：5的情況下，模印因子和選擇率都比交聯比1：6者佳。
綜合前述，已知以螢光單體進行聚合製備之模版材料，可有效地對肌酸酐進行較具專一性之吸附，在模印與選擇效應均顯著滿足的條件下，後續以螢光式進行檢測肌酸酐之可行性亦已確立。對於肌酸酐螢光檢測晶片之建立則具重大的突破性發展。

4-Chloro-7-nitro-2,1,3-benzoxadiazole (1, NBD-Cl) was used as the precursor for the synthesis of a fluorescent monomer, 4-(2-acryloyloxyethylamino)-7-nitro- 2,1,3-benzoxadiazole (3, NBD-2). Compound (1) was first reacted with ethanolamine for the irreversible addition of nucleophile to form 4-(2-hydroxyethylamino)-7-nitro- 2,1,3-benzoxadiazole (2, NBD-1). Then, compound (2) was further reacted with acryloyl chloride reversibly for the formation of the target monomer, which is the fluorescent monomer (3). To identify the chemical structure as well as the purity of compound (3), TLC, HPLC, FTIR, and NMR of 1H and 13C were used to analyze the spectrum of this key compound and the intermediate from different aspects. Compound (3) was thus accomplished and confirmed and was further used for the fabrication of the creatinine imprinted fluorescent polymer membrane as well as monolith. The fluorescent effect of the fluorescent monomer as prepared was comparably superior to the others. The fluorescent stability of this monomer was also investigated. How the solvents and concentration of the fluorescent monomer influence the emission intensity as well as shift of the peak wavelength were also discussed. The sedimentation behavior of the imprinted fluorescent particles in solvent can be well fitted by a response model derived from a second-order time system.
The fluorescent compound (3) was mixed with an initiator, 2,2-azobisisobutyronitrile (AIBN), another functional monomer, methacrylic acid (MAA), and the crosslinker, ethyl glycol dimethylacrylate (EGDMA) to form the pre-polymerization solution. The solution was then applied onto the surface of gold. Both heated polymerization and UV-irradiated polymerization were taken place to prepare the polymeric film imprinted with creatinine, respectively. After the extraction of creatinine templates by the porogen solvent, the imprinted fluorescent polymer film was prepared. Among all the polymerization conditions, the molar ratio of the functional monomer to the crosslinker affected the specific binding performance of the imprinted polymer matrix. It was discovered that the molar ratio of 1:5 (functional monomer: crosslinker) showed better results than the others. Consequently, 1:6 of this respective molar ratio was determined according to the comparison made from the experimental results. For the results from the fabrication of the imprinted polymer film, the imprinting effect and the specific binding capacity toward creatinine were both investigated. Thus, an imprinting factor of 2.63  0.45 and 2.88  0.64 could be achieved from the approach via heated polymerization and UV-polymerization, respectively. Additionally, a selectivity of 2.48  0.39 and 2.85  0.64 for creatinine against creatine could be obtained from both polymerizations. Therefore, both heated polymerization and UV-irradiated polymerization did show almost similar performance from the analytical results of creatinine.
Both the imprinting effect and the selectivity from the screened imprinted fluorescent polymer particles were also confirmed by comparing the reading from the absorbance and the fluorescence. After the uptake of creatinine by the imprinted polymer particles, the binding capacities both detected from an HPLC and a fluorescence spectrophotometer were compared. The results of imprinting factor and selectivity both appeared that to carry out the specific detection of the creatinine from the combination of the imprinted materials and the fluorescent approach could achieve better results. In fact, to perform the analysis of the creatinine via the fluorescent approach is much more efficient in the detection time as well. In addition, while the creatinine/ creatine mixture was uptaken by the imprinted polymer particles, the results appeared that the imprinting effect of the polymer toward creatinine template was enhanced due to the presence of creatine.
Concluded from the above results, the imprinted fluorescent polymer materials could specifically rebind creatinine molecules after the effective formation of the recognition cavity sites with respect to creatinine molecules. The feasibility to detect creatinine via the fluorescent approach was also verified. It is further beneficial to establish the fluorescent chip for the specific and efficient detection of creatinine.

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