臺灣博碩士論文加值系統

氨酸氨肽酶（LAP）是一種生物必需的蛋白酶，被認為有機會成為下一個肝臟惡性腫瘤的生物標誌物，其過度的表達造成致命生理和病理障礙有直接的關聯。因此我們設計了一種創新的電化學底物亮氨酸-氨基二茂鐵（Lap-FcP），能直接分析細胞中的LAP活性及選擇性。LAP迅速水解Lap-FcP的Leu殘基，通過設計潛伏式自我斷裂機制經由電子共振轉出氨基二茂鐵（AFc）。探針LAP-FcP對LAP具有高特異性並且沒有受其他電化學活性生物分子干擾。探針Lap-FcP賦予電化學傳感器強大的動態範圍和令人矚目的檢測極限2.14ng/ml。除此之外， Lap-FcP用於追踪活細胞（HepG2）表面及內部LAP活性，並透過抑制劑bestatin驗證確認。Lap-FcP電化學生物傳感器顯示出在監測HepG2細胞中的LAP活性表現出色可靠性。

Leucine aminopeptidase (LAP) is an essential proteolytic enzyme, and potential biomarker for liver malignancy; overexpression of LAP is directly linked with some fatal physiological, and pathological disorders. In this regard, we designed an innovative electrochemical substrate leucine-benzyl ferrocene carbamate (LAP-FcP) for direct activity based selective profiling of LAP activity in live cells. In practice, LAP instantaneously hydrolyze the Leu residue of the electrochemical substrate LAP-FC to eliminate the masked electrochemical reporter amino ferrocene (AF) via predefined self-immolative cascade. The probe LAP-FcP is very specific for LAP, and free of other electroactive biological interference. The probe LAP-FcP empowered electrochemical sensor displayed broad dynamic range and admirable detection limits2.14ng/ml. On top of this, the electrochemical substrate LAP-FcP was employed in real time tracking of endogenous LAP activity in live cells (Hep G2) on supplemented with LAP inhibitor bestatin. The Lap-FC substrate based electrochemical sensor showcased an excellent reliability towards monitoring cellular LAP activity in Hep G2

摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 vii
表目錄 ix
附圖目錄 x
第一章 前言 1
第二章文獻回顧 2
2.1即时檢测(Point-of-Care Testing) 2
2.2生物分子探針 3
2.2.1生物分子探針 3
2.2.2隱藏式自我斷裂 4
2.3亮氨酸氨肽酶(Leucine aminopeptidase) 5
2.4亮氨酸氨肽酶生理表現 7
2.4.1植物 7
2.4.2動物 7
2.4.2.1肝臟膽囊疾病 8
2.4.2.2懷孕疾病 8
2.4.2.3白內障疾病 9
2.5檢測平台 9
2.6電化學法 10
2.6.1電化學法原理及優勢 10
2.6.2循環伏安法 11
2.6.3微分脈衝伏安法 13
2.6.4 實驗裝置 14
第三章研究目的與策略 15
3.1實驗研究目的 15
3.2實驗研究策略 15
3.2.1探針設計概念 15
3.2.2電化學隱藏式探針LAPFcP 16
第四章 實驗方法與討論 17
4.1實驗藥品 17
4.1.1合成之藥品 17
4.1.2合成之溶劑 17
4.1.3緩衝溶液之藥品 18
4.1.4.干擾物之藥品 18
4.1.4真實樣品 19
4.2實驗儀器 19
4.2.1實驗使用之儀器 19
4.3探針合成設計 20
4.3.1 Compound(2)合成方法 20
4.3.2 Ferrocene Azide合成方法 21
4.3.3 LAP- Fc (3)合成方法 21
4.3.4 LAP- FcP(4)合成方法 22
4.4樣品配置 24
4.4.1緩衝溶液配置 24
4.4.2 LAP-FcP、Leucine Aminopeptidase儲備溶液 24
4.4.3電化學分析CV及DPV機制驗證 24
4.4.3.1電極清洗 24
4.4.4電化學分析線性檢量線 25
4.4.5電化學分析酵素動力學 25
4.4.6電化學分析干擾物 25
4.4.7電化學分析抑制劑Bestatin 25
4.4.8高效液相層析儀 26
4.4.9 真實樣品HepG2細胞 26
第五章 結果與討論 27
5.1 LAPFcP合成與設計機制 27
5.2 LAPFcP電化學探針機制驗證 28
5.2.1循環伏安法Cyclic voltammetry(CV)驗證 28
5.2.2微分脈衝伏安法Differential pulse voltammetry (DPV)驗證 29
5.2.3抑制劑Bestatin驗證 31
5.3檢測之最佳化參數 32
5.3.1最佳化參數-ph值 32
5.3.2檢測極限 33
5.3.3酵素動力學 34
5.3.4特異性測試 36
5.3.5真實樣品-細胞測試 38
第六章 結論 41
參考文獻 42
附錄 47

[1]C. P. Price, "Point of care testing," Bmj, vol. 322, pp. 1285-1288, 2001.
[2]V. Gubala, L. F. Harris, A. J. Ricco, M. X. Tan, and D. E. Williams, "Point of care diagnostics: status and future," Analytical chemistry, vol. 84, pp. 487-515, 2011.
[3]F. L. Kiechle, "The impact of continuous glucose monitoring on hospital point-of-care testing programs," Diabetes technology & therapeutics, vol. 3, pp. 647-650, 2001.
[4]C. Sawyers, "Targeted cancer therapy," Nature, vol. 432, p. 294, 2004.
[5]S. V. Frye, "The art of the chemical probe," Nature chemical biology, vol. 6, p. 159, 2010.
[6]Y. Meyer, J.-A. Richard, M. Massonneau, P.-Y. Renard, and A. Romieu, "Development of a new nonpeptidic self-immolative spacer. Application to the design of protease sensing fluorogenic probes," Organic letters, vol. 10, pp. 1517-1520, 2008.
[7]J.-A. Gu, V. Mani, and S.-T. Huang, "Design and synthesis of ultrasensitive off–on fluoride detecting fluorescence probe via autoinductive signal amplification," Analyst, vol. 140, pp. 346-352, 2015.
[8]M.-H. Wang, J.-A. Gu, V. Mani, Y.-C. Wu, Y.-J. Lin, Y.-M. Chia, et al., "A rapid fluorescence detecting platform: applicable to sense carnitine and chloramphenicol in food samples," RSC Advances, vol. 4, pp. 64112-64118, 2014.
[9]K. Manibalan, V. Mani, P.-C. Chang, C.-H. Huang, S.-T. Huang, K. Marchlewicz, et al., "Electrochemical latent redox ratiometric probes for real-time tracking and quantification of endogenous hydrogen sulfide production in living cells," Biosensors and Bioelectronics, vol. 96, pp. 233-238, 2017.
[10]R. J. Delange and E. L. Smith, "3 Leucine Aminopeptidase and Other N-Terminal Exopeptidases," in The enzymes. vol. 3, ed: Elsevier, 1971, pp. 81-118.
[11]S. R. Himmelhoch, "Leucine aminopeptidase: a zinc metalloenzyme," Archives of biochemistry and biophysics, vol. 134, pp. 597-602, 1969.
[12]M. Cappiello, V. Alterio, P. Amodeo, A. Del Corso, A. Scaloni, C. Pedone, et al., "Metal ion substitution in the catalytic site greatly affects the binding of sulfhydryl-containing compounds to leucyl aminopeptidase," Biochemistry, vol. 45, pp. 3226-3234, 2006.
[13]M. Matsui, J. H. Fowler, and L. L. Walling, "Leucine aminopeptidases: diversity in structure and function," Biological chemistry, vol. 387, pp. 1535-1544, 2006.
[14]D. BARTLING and E. W. WEILER, "Leucine aminopeptidase from Arabidopsis thaliana: molecular evidence for a phylogenetically conserved enzyme of protein turnover in higher plants," European journal of biochemistry, vol. 205, pp. 425-431, 1992.
[15]J. G. Scandalios, "Genetic control of multiple molecular forms of enzymes in plants: a review," Biochemical genetics, vol. 3, pp. 37-79, 1969.
[16]A. Mikkonen, "Purification and characterization of leucine aminopeptidase from kidney bean cotyledons," Physiologia Plantarum, vol. 84, pp. 393-398, 1992.
[17]C. Dammann, E. Rojo, and J. J. Sánchez‐Serrano, "Abscisic acid and jasmonic acid activate wound‐inducible genes in potato through separate, organ‐specific signal transduction pathways," The Plant Journal, vol. 11, pp. 773-782, 1997.
[18]W. S. Chao, Y.-Q. Gu, V. Pautot, E. A. Bray, and L. L. Walling, "Leucine aminopeptidase RNAs, proteins, and activities increase in response to water deficit, salinity, and the wound signals systemin, methyl jasmonate, and abscisic acid," Plant Physiology, vol. 120, pp. 979-992, 1999.
[19]W. S. Chao, V. Pautot, F. M. Holzer, and L. L. Walling, "Leucine aminopeptidases: the ubiquity of LAP-N and the specificity of LAP-A," Planta, vol. 210, pp. 563-573, 2000.
[20]Y. Q. Gu and L. L. Walling, "Identification of residues critical for activity of the wound‐induced leucine aminopeptidase (LAP‐A) of tomato," European journal of biochemistry, vol. 269, pp. 1630-1640, 2002.
[21]Y.-Q. Gu, W. S. Chao, and L. L. Walling, "Localization and post-translational processing of the wound-induced leucine aminopeptidase proteins of tomato," Journal of Biological Chemistry, vol. 271, pp. 25880-25887, 1996.
[22]S. KAWATA, T. IMAMURA, K. NINOMIYA, and S. MAKISUMI, "Purification and Characterization of an Aminopeptidase from Porcine Liver."
[23]N. Sträter, L. Sun, E. Kantrowitz, and W. N. Lipscomb, "A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase," Proceedings of the National Academy of Sciences, vol. 96, pp. 11151-11155, 1999.
[24]F. J. Behal, B. Asserson, F. Dawson, and J. Hardman, "A study of human tissue aminopeptidase components," Archives of biochemistry and biophysics, vol. 111, pp. 335-344, 1965.
[25]C. Jösch, L.-O. Klotz, and H. Sies, "Identification of cytosolic leucyl aminopeptidase (EC 3.4. 11.1) as the major cysteinylglycine-hydrolysing activity in rat liver," Biological chemistry, vol. 384, pp. 213-218, 2003.
[26]M. Cappiello, A. Lazzarotti, F. Buono, A. Scaloni, C. D'ambrosio, P. Amodeo, et al., "New role for leucyl aminopeptidase in glutathione turnover," Biochemical Journal, vol. 378, pp. 35-44, 2004.
[27]H. Kohno, S. Kanda, and T. Kanno, "Immunoaffinity purification and characterization of leucine aminopeptidase from human liver," Journal of Biological Chemistry, vol. 261, pp. 10744-10748, 1986.
[28]C. Bardawill and C. Chang, "Serum lactic dehydrogenase, leucine aminopeptidase and 5-nucleotidase activity: Observations in patients with carcinoma of the pancreas and hepatobiliary disease," Canadian Medical Association Journal, vol. 89, p. 755, 1963.
[29]A. M. Rutenburg, B. M. Banks, E. P. Pineda, and J. A. Goldbarg, "A comparison of serum aminopeptidase and alkaline phosphatase in the detection of hepatobiliary disease in anicteric patients," Annals of internal medicine, vol. 61, pp. 50-55, 1964.
[30]J. J. Reichling and M. M. Kaplan, "Clinical use of serum enzymes in liver disease," Digestive diseases and sciences, vol. 33, pp. 1601-1614, 1988.
[31]O. D. Kowlessar, L. J. Haeffner, and M. H. Sleisenger, "Localization of leucine aminopeptidase in serum and body fluids by starch gel electrophoresis," The Journal of clinical investigation, vol. 39, pp. 671-675, 1960.
[32]H. P. Oosterbaan and D. F. Swaab, "Amniotic oxytocin and vasopressin in relation to human fetal development and labour," Early human development, vol. 19, pp. 253-262, 1989.
[33]S. Mizutani, S. Sumi, K. Oka, R. Yamada, O. Kurauchi, H. Taira, et al., "In vitro degradation of oxytocin by pregnancy serum, placental subcellular fractions and purified placental aminopeptidases," Experimental and Clinical Endocrinology & Diabetes, vol. 86, pp. 310-316, 1985.
[34]T. Rogi, M. Tsujimoto, H. Nakazato, S. Mizutani, and Y. Tomoda, "Human Placental Leucine Aminopeptidase/Oxytocinase A NEW MEMBER OF TYPE II MEMBRANE-SPANNING ZINC METALLOPEPTIDASE FAMILY," Journal of Biological Chemistry, vol. 271, pp. 56-61, 1996.
[35]H. Kozaki, A. Itakura, M. Okamura, Y. Ohno, K. Wakai, and S. Mizutani, "Maternal serum placental leucine aminopeptidase (P‐LAP)/oxytocinase and preterm delivery," International Journal of Gynecology & Obstetrics, vol. 73, pp. 207-213, 2001.
[36]S. Mizutani, K. Shibata, F. Kikkawa, A. Hattori, M. Tsujimoto, M. Ishii, et al., "Essential role of placental leucine aminopeptidase in gynecologic malignancy," Expert opinion on therapeutic targets, vol. 11, pp. 453-461, 2007.
[37]L. Haakova, D. Cibula, K. Rezabek, M. Hill, M. Fanta, and J. Zivny, "Pregnancy outcome in women with PCOS and in controls matched by age and weight," Human Reproduction, vol. 18, pp. 1438-1441, 2003.
[38]K. Lamont, N. W. Scott, G. T. Jones, and S. Bhattacharya, "Risk of recurrent stillbirth: systematic review and meta-analysis," bmj, vol. 350, p. h3080, 2015.
[39]S. Goyal, J. Hardin, S. H. Uwaydat, A. A. Ellabban, D. B. Warner, and A. B. Sallam, "Review and update of cataract surgery in the diabetic eye," Expert Review of Ophthalmology, vol. 12, pp. 359-371, 2017.
[40]J. H. Kim, S. B. Han, S. J. Lee, and M. S. Kim, "Postoperative refractive errors after posterior capsulectomy during combined vitrectomy and cataract surgery," Journal of the Korean Ophthalmological Society, vol. 56, pp. 709-714, 2015.
[41]A. Taylor, M. Daims, J. Lee, and T. Surgenor, "Identification and quantification of leucine aminopeptidase in aged normal and cataractous human lenses and ability of bovine lens LAP to cleave bovine crystallins," Current eye research, vol. 2, pp. 47-56, 1982.
[42]W. Z. Zhang and R. C. Augusteyn, "Ageing of glutathione reductase in the lens," Experimental eye research, vol. 59, pp. 91-96, 1994.
[43]D. E. Bloom, D. Canning, and A. Lubet, "Global population aging: Facts, challenges, solutions & perspectives," Daedalus, vol. 144, pp. 80-92, 2015.
[44]K. T. Mills, Y. Xu, W. Zhang, J. D. Bundy, C.-S. Chen, T. N. Kelly, et al., "A systematic analysis of worldwide population-based data on the global burden of chronic kidney disease in 2010," Kidney international, vol. 88, pp. 950-957, 2015.
[45]E. Bakker, "Electrochemical Sensors," Anal. Chem., vol. 76, pp. 3285-3298, 2004.
[46]S. K. Burley, P. R. David, and W. N. Lipscomb, "Leucine aminopeptidase: bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis," Proceedings of the National Academy of Sciences, vol. 88, pp. 6916-6920, 1991.
[47]H. Suda, T. Aoyagi, T. Takeuchi, and H. Umezawa, "Inhibition of aminopeptidase B and leucine aminopeptidase by bestatin and its stereoisomer," Archives of biochemistry and biophysics, vol. 177, pp. 196-200, 1976.
[48]Q. Gong, W. Shi, L. Li, and H. Ma, "Leucine aminopeptidase may contribute to the intrinsic resistance of cancer cells toward cisplatin as revealed by an ultrasensitive fluorescent probe," Chemical science, vol. 7, pp. 788-792, 2016.
[49]Z. Zhou, F. Wang, G. Yang, C. Lu, J. Nie, Z. Chen, et al., "A ratiometric fluorescent probe for monitoring leucine aminopeptidase in living cells and zebrafish model," Analytical chemistry, vol. 89, pp. 11576-11582, 2017.
[50]W. Zhang, F. Liu, C. Zhang, J.-G. Luo, J. Luo, W. Yu, et al., "Near-infrared fluorescent probe with remarkable large Stokes shift and favorable water solubility for real-time tracking leucine aminopeptidase in living cells and in vivo," Analytical chemistry, vol. 89, pp. 12319-12326, 2017.
[51]S. Xu, H.-W. Liu, X.-X. Hu, S.-Y. Huan, J. Zhang, Y.-C. Liu, et al., "Visualization of endoplasmic reticulum aminopeptidase 1 under different redox conditions with a two-photon fluorescent probe," Analytical chemistry, vol. 89, pp. 7641-7648, 2017.
[52]T.-Y. Chen, V. Mani, S.-T. Huang, P.-C. Chang, C.-H. Huang, and N. M. Huang, "Synthesis of robust electrochemical substrate and fabrication of immobilization free biosensors for rapid sensing of salicylate and β-hydroxybutyrate in whole blood," Analytica chimica acta, vol. 990, pp. 78-83, 2017.
[53]M. Su, M. Wei, Z. Zhou, and S. Liu, "Application of capillary electrophoresis coupling with electrochemiluminescence detection to estimate activity of leucine aminopeptidas," Biomedical Chromatography, vol. 27, pp. 946-952, 2013.
[54]S. Huang, Y. Wu, F. Zeng, J. Chen, and S. Wu, "A turn-on fluorescence probe based on aggregation-induced emission for leucine aminopeptidase in living cells and tumor tissue," Analytica chimica acta, vol. 1031, pp. 169-177, 2018.
[55]T. Aoyagi, M. Nagai, M. Iwabuchi, W.-S. Liaw, T. Andoh, and H. Umezawa, "Aminopeptidase activities on the surface of mammalian cells and their alterations associated with transformation," Cancer research, vol. 38, pp. 3505-3508, 1978.
[56]G. Leyhausen, D. Schuster, P. Vaith, R. Zahn, H. Umezawa, D. Falke, et al., "Identification and properties of the cell membrane bound leucine aminopeptidase interacting with the potential immunostimulant and chemotherapeutic agent bestatin," Biochemical pharmacology, vol. 32, pp. 1051-1057, 1983.