藉由新穎之表面電漿共振儀表面改質方式於人類血清白蛋白的偵測之研究

簡易檢索 / 詳目顯示

回結果列表

研究生：	林勃伸 Po-Shen Lin
論文名稱：	藉由新穎之表面電漿共振儀表面改質方式於人類血清白蛋白的偵測之研究 A novel method for detecting microalbuminuria by surface plasmon resonance
指導教授：	陳文逸 Wen-Yin Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程與材料工程學系 Department of Chemical & Materials Engineering
畢業學年度：	96
語文別：	中文
論文頁數：	150
中文關鍵詞：	人類血清白蛋白、染料分子、表面電漿共振儀
外文關鍵詞：	HSA, SPR, Cibacron Blue
相關次數：	點閱：6 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

人類血清白蛋白為人體血漿中含量最豐富的蛋白質。在健康的腎臟中，腎小球會將其過濾使其不會隨著尿酸跟廢棄物經由尿液排出；但是一個不健康的腎臟，因腎小球被破壞造成無法過濾的情形，白蛋白則會隨著尿液排出。依程度不同分成微蛋白尿、蛋白尿等，所以尿液中的白蛋白含量在臨床診斷則為一個重要的生理訊息。
本研究以仿生染料(Cibacron Blue F3GA)做為觸手，將其和具有抗非特異性吸附特性的PEG結合，以自我組裝技術固定在生物晶片上。結果發現，此種生物晶片在表面電漿共振儀之系統中，當檢測環境為緩衝溶液pH值4、外加鹽離子濃度為0.05M時對人類血清白蛋白具有高親和性以及高吸附量的特性及可重覆性，此外，對照於CD圖譜人類血清白蛋白峰值偏移的結果，亦發現pH值的改變會對人類血清白蛋白的結構產生變化，此顯示在同一緩衝溶液的鹽濃度環境下，pH值能影響人類血清白蛋白結構，而此結構改變可能也是造成親和性常數升高的重要原因之ㄧ。在酵素鏈結免疫分析確認了，CB對人類血清白蛋白之高特異性。定量方面，找出了適宜之檢測濃度範圍為0.01 mg/ml ~0.1mg/ml，可用來偵測人類血清白蛋白，增加了應用的價值。
最後，配合熱力學數據的探討，計算△H、△S、△Cp，說明CB與人類血清白蛋白之間的交互作用之機制，實驗證實人類血清白蛋白的結構會隨著溫度而改變，且與CB之吸附反應為自發性行為，為一種entropy driven與enthalpy cost的程序。ΔH之所以為正值，推測吸附反應可能包含了人類血清白蛋白的劇烈結構變化、疏水作用力、靜電作用力與氫鍵的破壞等。

Human serum albumin (HSA) is the most abundant protein in human blood plasma. It is produced in the liver. Albumin comprises about half of the blood serum protein. It is soluble and monomeric. In the healthy kidney, albumin''s size and negative electric charge exclude it from excretion in the glomerulus. But in some diseases including diabetic nephropathy, it can cross the glomerulus and stockpile in the bladder. Depending on the amount of albumin in the urine, a patient may have normal renal function, microalbuminuria, or albuminuria.So urine HSA is an important bio-marker to diagnose nephropathy. Cibacron Blue F3G-A is an anthraquinone dyes which has special affinity for HSA and it`s structure is similar to bilirubin. In this study, Cibacron Blue F3G-A was covalently attached onto PEG monolayer provided with low-fouling property which was established by self-assembled monolayer. The goals of this study used surface plasmon resonance (SPR) to detect HSA and to establish the optimum condition. The results exhibited the chip had high affinity, high capacity and could regeneration in the condition of pH 4. The results also show the pH value can change the HSA’s structure and the structure change is an important factor to raise the affinity constant. The same result also proved by ELISA. In addition, linear response appeared in concentration of HSA in the range of 0.01~0.1mg/ml. The detection limit was 4μg/ml . In other words, SPR biosensor is used as a sensitive technique for direction of diagnosis is powerful.

目錄
中文摘要..................................................I
Abstract................................................III
誌謝....................................................IV
目錄.....................................................V
圖目錄...................................................IX
表目錄..................................................XV
第一章	序論............................................1
第二章	文獻回顧........................................3
1 生物晶片之簡介.......................................3
2 對於人類血清白蛋白之研究與介紹.......................7
2.1 微量尿白蛋白定義及量測.............................7
2.2 人類血清白蛋白結構、特性之描述.....................9
2.3白蛋白與觸手(receptor)之關係.......................11
2.4現行使用之檢測系統.................................16
2.5 動機..............................................18
3 其他實驗室所發展之分離、檢測方式....................20
4 表面改質與分子固定化................................28
4.1 自我組裝單層膜....................................30
4.1.1 硫醇............................................32
4.1.2 矽烷或矽氧類....................................33
4.1.3 混合性自我組裝膜................................35
4.2 表面分子之固定化..................................39
4.2.1仿生染料之固定化.................................39
4.2.2去氧核醣核酸固定化...............................42
4.2.3蛋白質和抗體固定化...............................42
5 偵測生物分子間交互作用力之儀器......................44
5.1表面電漿共振儀.....................................44
5.2石英晶體微天平.....................................49
第三章 實驗藥品與儀器設備...............................51
1實驗藥品.............................................51
2儀器設備.............................................53
3實驗步驟.............................................54
3.1 衝溶液的配置......................................54
3.2生物晶片的改質.....................................54
3.3 表面電漿共振儀偵測實驗............................55
3.3.1 儀器設定及操作..................................55
3.3.2實驗設計.........................................56
3.3.2.1 最佳吸附條件的找尋............................56
3.3.2.2檢量線的製作...................................57
3.3.2.3 恆溫吸附曲線的製作............................57
3.4 圓二色光譜儀測量實驗..............................58
3.5 原子力顯微鏡測量實驗..............................58
3.6接觸角測量器.......................................59
3.7 BCA Protein Assay Kit.............................60
3.8 酵素連結免疫吸附分析(ELISA) ......................60
3.9 SPR數據之處理與軟體分析...........................61
第四章 實驗結果.........................................63
1 表面鑑定............................................63
2 檢測條件最佳化......................................72
3 特異性實驗之實驗結果................................77
3.1親和性測試.........................................77
3.2特異性比較實驗.....................................78
3.3競爭反應...........................................79
4酵素連結免疫吸附分析之實驗結果.......................85
5人類血清白蛋白之結構變化.............................89
6熱力學性質之演算.....................................93
7偵測人類血清白蛋白之檢量線的製備....................101
第五章 討論............................................107
1 緩衝溶液pH值的影響.................................107
2鹽離子濃度的影響....................................110
3 人類血清白蛋白與CB之間的交互作用過程...............112
第六章 結論............................................113
第七章 參考文獻........................................116
附件--投稿中之文章初稿.................................124

                                

[1] Rogers, K. R. (2006). "Recent advances in biosensor techniques for environmental monitoring." Analytica Chimica Acta 568(1-2): 222-231.
[2] Cooper, M. A. (2002). "Optical biosensors in drug discovery." Nature Reviews Drug Discovery 1(7): 515.
[3] Anthony P. F. T., U. Bilitewski (2000) ."Biosensors for Environmental Monitoring " Harwood Acad. Publ
[4] Zhang S., Sun Y. (2001). "Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents." Biotechnology and Bioengineering 75(6): 710-717
[5] Lonnie, J. L., N. C. Jennine, , J.Y. Yoon. (2007). "Lab-on-a-chip immunoassay for multiple antibodies using microsphere light scattering and quantum dot emission." Biosensors and Bioelectronics 23(5): 675-681.
[6] Boozer, C., J. Ladd, S. Chen, S. Jiang. (2006). "DNA-Directed Protein Immobilization for Simultaneous Detection of Multiple Analytes by Surface Plasmon Resonance Biosensor." Anal. Chem. 78(5): 1515-1519.
[7] Ladd, J., C. Boozer, Q. Yu, S. Chen, J. Homola, S. Jiang. (2004). "DNA-Directed Protein Immobilization on Mixed Self-Assembled Monolayers via a Streptavidin Bridge." Langmuir 20(19): 8090-8095.
[8] Boozer, C., J. Ladd, S. Chen, Q. Yu, J. Homola, S. Jiang (2004). "DNA Directed Protein Immobilization on Mixed ssDNA/Oligo(ethylene glycol) Self-Assembled Monolayers for Sensitive Biosensors." Anal. Chem. 76(23): 6967-6972.
[9] Huang, S.H., Y.C. Shih, C.Y. Wu, C.J. Yuan, Y.S. Yang, Y.K. Li , T.K. Wu (2004). "Detection of serum uric acid using the optical polymeric enzyme biochip system." Biosensors and Bioelectronics 19(12): 1627-1633.
[10] Poetz, O., J. M. Schwenk, S. Kramer, D. Stoll, M. F. Templin ,T. O. Joos (2005). "Protein microarrays: catching the proteome." Mechanisms of Ageing and Development 126(1): 161-170.
[11] Ozaki, H., A. Nishihira, M. Wakabayashi, M. Kuwahara, H. Sawai (2006). "Biomolecular sensor based on fluorescence-labeled aptamer." Bioorganic & Medicinal Chemistry Letters 16(16): 4381-4384.
[12] McCauley, T. G., N. Hamaguchi, M. Stanton (2003). "Aptamer-based biosensor arrays for detection and quantification of biological macromolecules." Analytical Biochemistry 319(2): 244-250.
[13] Balamurugan, S., A. Obubuafo, S. A. Soper, R. L. McCarley, D. A. Spivak (2006). "Designing Highly Specific Biosensing Surfaces Using Aptamer Monolayers on Gold." Langmuir 22(14): 6446-6453.
[14] Cho, E. J., J. R. Collett, A. E. Szafranska, A. D. Ellington (2006). "Optimization of aptamer microarray technology for multiple protein targets." Analytica Chimica Acta 564(1): 82-90.
[15] Campbell, C. T., G. Kim (2007). "SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics." Biomaterials 28(15): 2380-2392.
[16] Hawe, A., Frie, szlig ,Wolfgang (2007). "Formulation Development for Hydrophobic Therapeutic Proteins." Pharmaceutical Development and Technology 12(3): 223 - 237.
[17] Dockal, M., D. C. Carter, F. Ruker (1999). "The Three Recombinant Domains of Human Serum Albumin. STRUCTURAL CHARACTERIZATION AND LIGAND BINDING PROPERTIES." J. Biol. Chem. 274(41): 29303-29310.
[18] Dockal, M., D. C. Carter, F. Ruker (2000). "Conformational Transitions of the Three Recombinant Domains of Human Serum Albumin Depending on pH." J. Biol. Chem. 275(5): 3042-3050.
[19] He, X. M., D. C. Carter (1992). "Atomic structure and chemistry of human serum albumin." Nature 358(6383): 209-215.
[20] Chuang, V. T. G., M. Hijioka, M. Katsuki, K. Nishi, T. Hara, K.-i. Kaneko, M. Ueno, A. Kuniyasu, H. Nakayama, M. Otagiri (2005). "Characterization of benzodiazepine binding site on human [alpha]1-acid glycoprotein using flunitrazepam as a photolabeling agent." Biochimica et Biophysica Acta (BBA) - General Subjects 1725(3): 385-393.
[21] Adachi, K., H. Watarai (2006). "Binding Behavior of Subphthalocyanine-Tagged Testosterone with Human Serum Albumin at the n-Hexane/Water Interface." Anal. Chem. 78(19): 6840-6846.
[22] Gotti, R., C. Bertucci, V. Andrisano, R. Pomponio, V. Cavrini (2003). "Study of donepezil binding to serum albumin by capillary electrophoresis and circular dichroism." Analytical and Bioanalytical Chemistry 377(5): 875-879.
[23] Santra, M. K., A. Banerjee, O. Rahaman, D. Panda (2005). "Unfolding pathways of human serum albumin: Evidence for sequential unfolding and folding of its three domains." International Journal of Biological Macromolecules 37(4): 200-204.
[24] Barrett,D. A. , G.M. Power, M. A. Hussain, I. D. Pitfield, P. N. Shaw, M. C. Davies (2005). "Protein interactions with model chromatographic stationary phases constructed using self-assembled monolayers." Journal of Separation Science 28(5): 483-491.
[25] Zhang, S., Y. Sun (2004). "A Model for the Salt Effect on Adsorption Equilibrium of Basic Protein to Dye-Ligand Affinity Adsorbent." Biotechnol. Prog. 20(1): 207-214.
[26] Bird, J., N. Brough, S. Dixon, S. N. Batchelor (2006). "Understanding Adsorption Phenomena: Investigation of the Dye-Cellulose Interaction." J. Phys. Chem. B 110(39): 19557-19561.
[27] Qian, H. F., W. Huang (2006). "An azo dye molecule having a pyridine-2,6-dione backbone." Acta Crystallographica Section C 62(2): o62-o64.
[28] Yampolskaya, G., D. Platikanov (2006). "Proteins at fluid interfaces: Adsorption layers and thin liquid films." Advances in Colloid and Interface Science 128-130: 159-183.
[29] Douglas, E , R. C Atkins. "The detection and measurement of microalbuminria: a challenge for clinical chemistry." Medical Laboratory Observer; Feb 2005; 37,2; ProQuest Medical Library; U.S.A.
[30] Kumbhat, S., D. R. Shankaran, S. J. Kim, K. V. Gobi, V. Joshi, N. Miura (2007). "Surface plasmon resonance biosensor for dopamine using D3 dopamine receptor as a biorecognition molecule." Biosensors and Bioelectronics 23(3): 421-427.
[31] Yang, M., C. C. Lim, R. Liao, X. Zhang (2007). "A novel microfluidic impedance assay for monitoring endothelin-induced cardiomyocyte hypertrophy." Biosensors and Bioelectronics 22(8): 1688-1693.
[32] Kassab, A., H. Yavuz, M. Odabasi, A. Denizli (2000). "Human serum albumin chromatography by Cibacron Blue F3GA-derived microporous polyamide hollow-fiber affinity membranes." Journal of Chromatography B: Biomedical Sciences and Applications 746(2): 123-132.
[33] Cohn, E. J., E. Strong, W. L. Hughes, Jr., D. J. Mulford, N. Ashworthm, M. Melin, H.L. Taylor.(1946). "Preparation and Properties of Serum and Plasma Proteins. IV. A System for the Separation into Fractions of the Protein and Lipoprotein Components of Biological Tissues and Fluids1a,b,c,d." J. Am. Chem. Soc. 68(3): 459-475.
[34] Boyer, P., J. Hsu (1993). Protein purification by dye-ligand chromatography. Chromatography: 1-44.
[35] Handan Yavuz, A. D. (2004). "Dye Affinity Hollow Fibers for Albumin Purification." Macromolecular Bioscience 4(2): 84-91.
[36] Ma, Z., K. Masaya and S. Ramakrishna (2006). "Immobilization of Cibacron blue F3GA on electrospun polysulphone ultra-fine fiber surfaces towards developing an affinity membrane for albumin adsorption." Journal of Membrane Science 282(1-2): 237-244.
[37] Ma, Z.-Y., Y.-P. Guan, H.-Z. Liu (2006). " Affinity adsorption of albumin on Cibacron Blue F3GA-coupled non-porous micrometer-sized magnetic polymer microspheres " Reactive and Functional Polymers 66(6): 618-624.
[38] Bayramoglu, G., F. B. Senkal, G. Celik, M. Y. Arica (2007). "Preparation and characterization of sulfonyl-hydrazine attached poly(styrene-divinylbenzene) beads for separation of albumin." Colloids and Surfaces A: Physicochemical and Engineering Aspects 294(1-3): 56-63.
[39] AltIntas, E. B., A. Denizli (2006). "Efficient removal of albumin from human serum by monosize dye-affinity beads." Journal of Chromatography B 832(2): 216-223.
[40] Bonini, F., S. Piletsky, A. P. F. Turner, A. Speghini, A. Bossi (2007). "Surface imprinted beads for the recognition of human serum albumin." Biosensors and Bioelectronics 22(9-10): 2322-2328.
[41] Sun, W., K. Jiao (2002). "Linear sweep voltammetric determination of protein based on its interaction with Alizarin Red S." Talanta 56(6): 1073-1080.
[42] Wei, Q., D. Wu, Y. Li, B. Du, J. H. Wang (2006). "A Highly Sensitive and Rapid Assay for Protein Determination in Human Urine and Penicillin Using a Rayleigh Light-Scattering Technique with Benzeneazo-8-acetylamino-1-naphthol-3,6-disulfonic Acid Sodium Salt." Analytical Sciences 22(2): 275-279.
[43] Kanchana, W.i., T. Sakai, N. Teshima, S. Katoh, K. Grudpan (2007). "Successive determination of urinary protein and glucose using spectrophotometric sequential injection method." Analytica Chimica Acta 604(2): 139-146
[44] Jiang, C., L. Luo (2004). "Spectrofluorimetric determination of human serum albumin using a doxycycline-europium probe." Analytica Chimica Acta 506(2): 171-175.
[45] Zor, T., Z. Selinger (1996). "Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies." Analytical Biochemistry 236(2): 302-308.
[46] Bier, F. F., F. W. Scheller (1996). "Label-free observation of DNA-hybridisation and endonuclease activity on a wave guide surface using a grating coupler." Biosensors and Bioelectronics 11(6-7): 669-674.
[47] Senaratne, W., L. A, C. K. Ober (2005). "Self-Assembled Monolayers and Polymer Brushes in Biotechnology: Current Applications and Future Perspectives." Biomacromolecules 6(5): 2427-2448
[48] Gizeli E., C.R. Lowe, "Biomolecular Sensors" CRC Press ; 2002
[49] Ulman, A. (1996). "Formation and Structure of Self-Assembled Monolayers." Chem. Rev. 96(4): 1533-1554.
[50] Wang, R., S. L. Wunder (2001). "Thermal Stability of Octadecylsilane Monolayers on Silica: Curvature and Free Volume Effects." J. Phys. Chem. B 105(1): 173-181.
[51] Yu, J. j; Tan, Y. H.; Li, X.; Kuo, P. K.; Liu, G. y (2006). "A Nanoengineering Approach to Regulate the Lateral Heterogeneity of Self-Assembled Monolayers." J. Am. Chem. Soc. 128(35): 11574-11581.
[52] Vallant, T., H. Brunner, U. Mayer, H. Hoffmann, T. Leitner, R. Resch, G. Friedbacher (1998). "Formation of Self-Assembled Octadecylsiloxane Monolayers on Mica and Silicon Surfaces Studied by Atomic Force Microscopy and Infrared Spectroscopy." J. Phys. Chem. B 102(37): 7190-7197.
[53] Chen, S., L. Li, C. L. Boozer,S. Jiang (2000). "Controlled Chemical and Structural Properties of Mixed Self-Assembled Monolayers of Alkanethiols on Au(111)." Langmuir 16(24): 9287-9293.
[54] Li, L., S. Chen, S. Jiang (2003). "Nanoscale Frictional Properties of Mixed Alkanethiol Self-Assembled Monolayers on Au(111) by Scanning Force Microscopy: Humidity Effect." Langmuir 19(3): 666-671.
[55] Wang, H., S. Chen, L. Li , S. Jiang (2005). "Improved Method for the Preparation of Carboxylic Acid and Amine Terminated Self-Assembled Monolayers of Alkanethiolates." Langmuir 21(7): 2633-2636.
[56] Jin, G., L. Zhang, Q. Yao (2007). "Novel method for human serum albumin adsorption/separation from aqueous solutions and human plasma with Cibacron Blue F3GA-Zn(II) attached microporous affinity membranous capillaries." Journal of Membrane Science 287(2): 271-279.
[57] Nie, H.-L., L.-M. Zhu (2007). "Adsorption of papain with Cibacron Blue F3GA carrying chitosan-coated nylon affinity membranes." International Journal of Biological Macromolecules 40(3): 261-267.
[58] Chien, F.C., J.S. Liu, H.J. Su, L.A. Kao, C.F. Chiou, W.Y. Chen, S.-J. Chen (2004). "An investigation into the influence of secondary structures on DNA hybridization using surface plasmon resonance biosensing." Chemical Physics Letters 397(4-6): 429-434.
[59] Vakov, R., A. Gaudinov, H. S senbekov, P. Dobrev, M. Strnad, J. Hol, J. Lenfeld (1998). "Comparison of oriented and random antibody immobilization in immunoaffinity chromatography of cytokinins." Journal of Chromatography A 811(1-2): 77-84.
[60] KijBlinger, C., E. Uttenthaler, S. Drost, F. Aberl, H. Wolf, G. Brink, A. Stanglmaier, E. Sackmann (1995). "Comparison of the QCM and the SPR method for surface studies and immunological applications." Sensors and Actuators B: Chemical 24(1-3): 107-112.
[61] Boozer, C., Q. Yu, S. Chen, C.Y. Lee, J. Homola, S. S. Yee , S. Jiang (2003). "Surface functionalization for self-referencing surface plasmon resonance (SPR) biosensors by multi-step self-assembly." Sensors and Actuators B: Chemical 90(1-3): 22-30.
[62] Campbell, C. T., G. Kim (2007). "SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics." Biomaterials 28(15): 2380-2392.
[63] Martins, M. C. L., E. Naeemi, B. D. Ratner, M. A. Barbosa (2003). "Albumin adsorption on Cibacron Blue F3G-A immobilized onto oligo(ethylene glycol)-terminated self-assembled monolayers." Journal of Materials Science: Materials in Medicine 14(11): 945-954.
[64] Wu, X. Y., Y. Liu, X. Y. Dong, Y. Sun (2006). "Protein Refolding Mediated by Reverse Micelles of Cibacron Blue F-3GA Modified Nonionic Surfactant." Biotechnol. Prog. 22(2): 499-504.
[65] Mitsakakis, K., S. Lousinian, S. Logothetidis (2007). "Early stages of human plasma proteins adsorption probed by Atomic Force Microscope." Biomolecular Engineering 24(1): 119-124.
[66] United States Patent No. US7,153,817; Dec.26.2006
[67] Peter, H. W, S. M. Marshall" Microalbuminuria: Biochemistry, Epidemiology and Clinical Practice ." Cambridge University Press;1998; P8
[68] Koulouris, S., I. Lekatsas, I. Karabinos, G. Ioannidis, T. Katostaras, A. Kranidis, K. Triantafillou, N. Thalassinos, L. Anthopoulos (2005). "Microalbuminuria: A strong predictor of 3-year adverse prognosis in nondiabetic patients with acute myocardial infarction." American Heart Journal 149(5): 840-845.
[69] Higuchi, A., K. Sugiyama, B. O. Yoon, M. Sakurai, M. Hara, M. Sumita, S.-i. Sugawara, T. Shirai (2003). "Serum protein adsorption and platelet adhesion on pluronic(TM)-adsorbed polysulfone membranes." Biomaterials 24(19): 3235-3245.
[70] Nakamura, K., S. Era, Y. Ozaki, M. Sogami, T. Hayashi, M. Murakami (1997). "Conformational changes in seventeen cystine disulfide bridges of bovine serum albumin proved by Raman spectroscopy." FEBS Letters 417(3): 375-378.
[71] Cynthia F. Shuman, M. D. H. U. H. D. (2004). "Kinetic and thermodynamic characterization of HIV-1 protease inhibitors." Journal of Molecular Recognition 17(2): 106-119.
[72] Haidacher, D., A. Vailaya, C. Horvath (1996). "Temperature effects in hydrophobic interaction chromatography." Proceedings of the National Academy of Sciences of the United States of America 93(6): 2290-2295.
[73] Perkins, T. W., D. S. Mak, T. W. Root, E. N. Lightfoot (1997). "Protein retention in hydrophobic interaction chromatography: Modeling variation with buffer ionic strength and column hydrophobicity." Journal of Chromatography A 766(1-2): 1-14.
[74] Buckenmaier, S. M. C., D. V. McCalley, M. R. Euerby (2003). "Determination of pKa values of organic bases in aqueous acetonitrile solutions using capillary electrophoresis." Journal of Chromatography A 1004(1-2): 71-79.
[75] Sosnovsky, G., P. Bell (1998). "In the search for new anticancer drugs. 29. A study on the correlation of lipophilicities, ionization constants and anticancer activities of aminoxyl labeled tepa congeners." Life Sciences 62(7): 639-648.

簡易檢索 / 詳目顯示

相關論文