博碩士論文 962411001 詳細資訊



以作者查詢圖書館館藏 、以作者查詢臺灣博碩士 、以作者查詢全國書目 、勘誤回報 、線上人數：7 、訪客IP：3.131.13.37
姓名 宋子承(Tzu‐Cheng Sung)  查詢紙本館藏   畢業系所 系統生物與生物資訊研究所
論文名稱 發展微脂粒核適體以應用於高通量生物感測
(Development of liposomal aptamers for the use in high throughput biosensing)
檔案 [Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   [檢視]  [下載]
  1. 本電子論文使用權限為同意立即開放。
  2. 已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
  3. 請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中) 本研究，我們主要開發微脂粒核適體以應用於高通量生物感測。於本研究第一部分中，利用生物素作為主要模式偵測目標，開發微脂粒核適體作為競爭物以應用於置換分析。置換分析較係屬較有利應用於高通量生物檢測之分析形式。我們成功於在96孔盤系統中，開發一高通量樣本數生物感測平台。在第二部分中，我們利用微脂粒核適體與兔子抗體結合之技術，兔子抗體於生物感測中係屬於常見生物識別元件。因此，本研驗中能與兔抗體結合之的”抗兔抗體之微脂粒核適體”可利用於生物感測研究中，針對許多不同的分析物的同時檢測。我們證明於本研究中所使用之兩種微脂粒核適體具有相當潛力可用於最小化分析時間，並簡化了分析步驟與流程，有利於應用於高通量生物感測中。
本研究第一部分，我們開發微脂粒核適體作為生物素的競爭劑，於96孔盤系統中進行置換分析以測定生物素。本研究所使用之ST-21和ST-21M微脂粒核適體之檢測極限分別為1.32微克/ 80微升及0.47微克/ 80微升。該ST-21和ST-21M微脂粒核適體之動態測定範圍分別為七和四個數量級。成功證明了運用微脂粒核適體作為競爭劑於置換分析是可行的。同時也證實去除任何洗滌程序的置換分析法可提供快速並可應用於高通量生物感測之潛力。
在本研究的第二部分，我們應用微脂粒核適體於微陣列分析。我們利用微脂粒核適開發一可用於生物感測之通用試劑，R18微脂粒核適體可與兔抗體結合，吐抗體是在生物感測中常用之生物識別元件。在本研究中，R18微脂粒核適體可成功應用於96孔盤與晶片系統中。R18微脂粒核適體的檢測極限是80毫微克/毫升。動態檢測範圍在96孔盤系統中為三個數量級（80納克/毫升至50微克/毫升）；在晶片上的檢測極限是18.2納克/毫升、在晶片上的動態檢測範圍是三個數量級（18.2毫微克/毫升至10微克/毫升）。我們並R18微脂粒核適體於高密度蛋白質體晶片之檢測，在晶片上可表現出高密度並相當清晰之信號。而R18微脂粒核適體在晶片上之檢測也呈現出明顯的濃度效應，並具有專一性，對其他非兔抗體則不會呈現訊號。因此，本研究成功證實微脂粒核適體可作為高通量生物感測所使用之通用試劑。
綜合本研究之成果，微脂粒核適體不僅能在置換生物感測分析中能作為競爭劑，並能作為免疫分析中之通用試劑。微脂粒核適體具有相當之潛力可應用於高通量生物感測中。
摘要(英) In this research, we developed liposomal aptamers for the use in high throughput biosensing. In the first part, the crucial vitamin biotin was used for model analytes for developing a liposomal aptamer as a competitor for replacement assay, which is an effective assay format for high throughput biosensing. We demonstrated the assay format in the 96-well plate, which provides high sample throughput for biosensing. In the second part, we developed a liposomal aptamer for binding to rabbit antibodies, which is common biorecognition elements in biosensing. Therefore, this anti-rabbit antibody liposomal aptamer can be used for multiplexed biosensing for simultaneous detections of many different analytes. We demonstrate that both liposomal aptamers have the potential to be used to minimize the assay time and simplified the assay protocol to be used in high throughput biosensing
In the first part, we developed a liposomal aptamer as the competitor of biotin for the detection of biotin in 96-wells plate format replacement assay. The limit of detection by using ST-21 and ST-21M liposomal aptamer were 1.32 pg/80 μl and 0.47 pg/80 μl, respectively. The dynamic ranges of our assays using ST-21 and ST-21M aptamers were seven and four orders of magnitude, respectively. This demonstrates the successful use of aptamer as a competitor of analyte in a replacement assay. It also shows the replacements assay without any washing procedure is fast enough and with a potential to use in the high throughput biosensing.
In the second part, we developed a liposomal aptamer as a secondary reagent for immunoassays. The R18 liposomal aptamer can bind with rabbit antibody, which is commonly used biorecognition elements in biosensing. In this research, the R18 liposomal aptamer can be used both on the 96-well plate and chips. LOD for R18 liposomal aptamer was 80 ng/ml, and the dynamic range was three orders of magnitude (80 ng/ml to 50 µg/ml) on the 96-well plate. The LOD for R18 liposomal aptamer on the chip was 18.2 ng/ml. The dynamic range was three orders of magnitude (18.2 ng/ml to 10 µg/ml) on the chip. We further used the R18 liposomal aptamer on high density proteome microarrays. We observed that the R18 liposomal aptamer showed high resolution and clear signals with dose response on the chip and also can R18 showed specific binding to the rabbit antibodies. There results demonstrate the R18 liposomal aptamer can be as a seconadry reagent for the use in high throughput immunoassays.
In summary, liposomal aptamers can be ideal competitors for the replacement biosensing and effective secondary reagents for immunoassays. They are both applicable for high-throughput biosensing.
關鍵字(中) ★ 核適體
★ 微脂粒
★ 高通量生物感測
★ 蛋白質體晶片		 關鍵字(英) ★ aptamer
★ liposomes
★ high throughput biosensing
★ proteome microarray
論文目次 Table of contents
中文摘要 I
Abstract III
Acknowledgements V
I. Introduction 1
1. High-throughput biosensing 1
2. High-throughput biosensing techniques 3
2.1 DNA microarrays 3
2.2 Protein microarrays 3
2.3 Microplate-based assays 4
3. Replacement biosensing assay 4
3.1 Fluorescence polarization-based replacement assay 5
3.2 Fluorescent intercalator replacement assay 5
3.3 Target-induced replacement assay 5
3.4 Fluorescence replacement assay 6
4. Biotin detection 6
4.1 ELISA 6
4.2 Electrochemical immunosensor assay 7
4.3 Electrochemical magneto assay 7
4.4 HPLC/MS 7
4.5 Flow injection liposome immunoanalytical system 8
4.6 Immunoaffinity chromatographic assay 8
4.7 Isotope dilution assay 8
5. Purpose of developing liposomal aptamer for high-throughput biosensing assay 9
Reference 11
II. A replaceable liposomal aptamer for the ultrasensitive and rapid detection of biotin 15
Abstract 16
Introduction 16
EXPERIMENTAL SECTION 19
Results 21
Discussion 27
Conclusions 29
ACKNOWLEDGMENT 29
Reference 29
Supplementary Figure 32
III. Development of an anti-rabbit liposomal aptamer as a secondary reagent for immunoassays 35
Abstract 36
Introduction 36
EXPERIMENTAL SECTION 38
RESULTS 40
DISCUSSION 42
Conclusions 44
Reference 52
IV. Appendix 54
Published and under revision manuscript 55
參考文獻 1. Prével, C., Pellerano, M., Van, T. N. N., and Morris, M. C. (2014) Fluorescent biosensors for high throughput screening of protein kinase inhibitors. Biotechnology Journal 9, 253-265
2. Zhang, Y., Cai, Y., Li, L., Qian, Y., and Lu, L. (2012) High-throughput biosensing of neurotoxic insecticides using polystyrene microplate-immobilized acetylcholinesterase. Analytical Methods 4, 3830-3835
3. Gehring, A. G., and Tu, S.-I. (2011) High-Throughput Biosensors for Multiplexed Food-Borne Pathogen Detection. Annual review of analytical chemistry 4, 151-172
4. Quinn, J. G., O′Neill, S., Doyle, A., McAtamney, C., Diamond, D., MacCraith, B. D., and O′Kennedy, R. (2000) Development and Application of Surface Plasmon Resonance-Based Biosensors for the Detection of Cell–Ligand Interactions. Analytical biochemistry 281, 135-143
5. Boozer, C., Kim, G., Cong, S., Guan, H., and Londergan, T. (2006) Looking towards label-free biomolecular interaction analysis in a high-throughput format: a review of new surface plasmon resonance technologies. Current opinion in biotechnology 17, 400-405
6. Shumaker-Parry, J. S., Aebersold, R., and Campbell, C. T. (2004) Parallel, Quantitative Measurement of Protein Binding to a 120-Element Double-Stranded DNA Array in Real Time Using Surface Plasmon Resonance Microscopy. Analytical chemistry 76, 2071-2082
7. Peterlinz, K. A., Georgiadis, R. M., Herne, T. M., and Tarlov, M. J. (1997) Observation of Hybridization and Dehybridization of Thiol-Tethered DNA Using Two-Color Surface Plasmon Resonance Spectroscopy. Journal of the American Chemical Society 119, 3401-3402
8. Peterson, A. W., Wolf, L. K., and Georgiadis, R. M. (2002) Hybridization of Mismatched or Partially Matched DNA at Surfaces. Journal of the American Chemical Society 124, 14601-14607
9. Gunderson, K. L., Steemers, F. J., Lee, G., Mendoza, L. G., and Chee, M. S. (2005) A genome-wide scalable SNP genotyping assay using microarray technology. Nat Genet 37, 549-554
10. Miller, M. B., and Tang, Y.-W. (2009) Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology. Clinical Microbiology Reviews 22, 611-633
11. Debouck, C., and Metcalf, B. (2000) The Impact of Genomics on Drug Discovery. Annual Review of Pharmacology and Toxicology 40, 193-208
12. Raymond, W., and Sridhar, R. (2005) DNA Microarrays in Clinical Cancer Research. Current Molecular Medicine 5, 111-120
13. Harries, H. M., Fletcher, S. T., Duggan, C. M., and Baker, V. A. (2001) The use of genomics technology to investigate gene expression changes in cultured human liver cells. Toxicology in Vitro 15, 399-405
14. Reymond Sutandy, F. X., Qian, J., Chen, C.-S., and Zhu, H. (2013) Overview of Protein Microarrays. Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 0 27, Unit-27.21
15. Liu, Y., Jia, S., and Guo, L.-H. (2012) Development of microplate-based photoelectrochemical DNA biosensor array for high throughput detection of DNA damage. Sensors and Actuators B: Chemical 161, 334-340
16. Pang, H. L., Kwok, N. Y., Chan, P. H., Yeung, C. H., Lo, W., and Wong, K. Y. (2007) High-throughput determination of biochemical oxygen demand (BOD) by a microplate-based biosensor. Environmental science & technology 41, 4038-4044
17. Schallmey, M., Frunzke, J., Eggeling, L., and Marienhagen, J. (2014) Looking for the pick of the bunch: high-throughput screening of producing microorganisms with biosensors. Current opinion in biotechnology 26, 148-154
18. Cruz-Aguado, J. A., and Penner, G. (2008) Fluorescence Polarization Based Displacement Assay for the Determination of Small Molecules with Aptamers. Analytical chemistry 80, 8853-8855
19. Nygren, J., Svanvik, N., and Kubista, M. (1998) The interactions between the fluorescent dye thiazole orange and DNA. Biopolymers 46, 39-51
20. Tse, W. C., and Boger, D. L. (2004) A Fluorescent Intercalator Displacement Assay for Establishing DNA Binding Selectivity and Affinity. Accounts of chemical research 37, 61-69
21. Zayats, M., Huang, Y., Gill, R., Ma, C.-a., and Willner, I. (2006) Label-Free and Reagentless Aptamer-Based Sensors for Small Molecules. Journal of the American Chemical Society 128, 13666-13667
22. Li, L.-L., Ge, P., Selvin, P. R., and Lu, Y. (2012) Direct Detection of Adenosine in Undiluted Serum Using a Luminescent Aptamer Sensor Attached to a Terbium Complex. Analytical chemistry 84, 7852-7856
23. Xiao, Y., Piorek, B. D., Plaxco, K. W., and Heeger, A. J. (2005) A reagentless signal-on architecture for electronic, aptamer-based sensors via target-induced strand displacement. Journal of the American Chemical Society 127, 17990-17991
24. Tang, Z., Mallikaratchy, P., Yang, R., Kim, Y., Zhu, Z., Wang, H., and Tan, W. (2008) Aptamer switch probe based on intramolecular displacement. Journal of the American Chemical Society 130, 11268-11269
25. Chang, J. C., Tomlinson, I. D., Warnement, M. R., Iwamoto, H., DeFelice, L. J., Blakely, R. D., and Rosenthal, S. J. (2011) A Fluorescence Displacement Assay for Antidepressant Drug Discovery Based on Ligand-Conjugated Quantum Dots. Journal of the American Chemical Society 133, 17528-17531
26. Barthelemy, H., Chouvet, B., and Cambazard, F. (1986) Skin and mucosal manifestations in vitamin deficiency. Journal of the American Academy of Dermatology 15, 1263-1274
27. Mock, D. M., Stadler, D. D., Stratton, S. L., and Mock, N. I. (1997) Biotin status assessed longitudinally in pregnant women. The Journal of Nutrition 127, 710-716
28. Wolf, B., Grier, R. E., Allen, R. J., Goodman, S. I., and Kien, C. L. (1983) Biotinidase deficiency: the enzymatic defect in late-onset multiple carboxylase deficiency. Clinica Chimica Acta 131, 273-281
29. Braun-Falco, O., Plewig, G., Wolff, H., and Burgdorf, W. C. (2000) Vitamin Disorders. Dermatology, pp. 1367-1378, Springer Berlin Heidelberg
30. Coggeshall, J. C., Heggers, J. P., Robson, M. C., and Baker, H. (1985) Biotin status and plasma glucose in diabetics. Annals of the New York Academy of Sciences 447, 389-392
31. Huang, E. Z., and Rogers, Y.-H. (1997) [32] Competitive enzymatic assay of biotin. In: Donald B. McCormick, J. W. S. C. W., ed. Methods in Enzymology, pp. 304-308, Academic Press
32. Ho, J. A., Chiu, J. K., Hong, J. C., Lin, C. C., Hwang, K. C., and Hwu, J. R. (2009) Gold-nanostructured immunosensor for the electrochemical sensing of biotin based on liposomal competitive assay. Journal of nanoscience and nanotechnology 9, 2324-2329
33. Kergaravat, S. V., Gómez, G. A., Fabiano, S. N., Laube Chávez, T. I., Pividori, M. I., and Hernández, S. R. (2012) Biotin determination in food supplements by an electrochemical magneto biosensor. Talanta 97, 484-490
34. Yomota, C., and Ohnishi, Y. (2007) Determination of biotin following derivatization with 2-nitrophenylhydrazine by high-performance liquid chromatography with on-line UV detection and electrospray-ionization mass spectrometry. Journal of Chromatography A 1142, 231-235
35. Ho, J. A., and Huang, M. R. (2005) Application of a liposomal bioluminescent label in the development of a flow injection immunoanalytical system. Analytical chemistry 77, 3431-3436
36. Ho, J. A., and Hung, C. H. (2008) Using liposomal fluorescent biolabels to develop an immunoaffinity chromatographic biosensing system for biotin. Analytical chemistry 80, 6405-6409
37. Hood, R. L. (1979) [49] Isotopic dilution assay for biotin: Use of [14C]biotin. In: Donald B. McCormick, L. D. W., ed. Methods in Enzymology, pp. 279-283, Academic Press
38. Walash, M. I., Rizk, M., Sheribah, Z. A., and Salim, M. M. (2008) Kinetic spectrophotometric determination of biotin in pharmaceutical preparations. International journal of biomedical science : IJBS 4, 238-244
39. Stoltenburg, R., Reinemann, C., and Strehlitz, B. (2007) SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomolecular engineering 24, 381-403
40. Luzi, E., Minunni, M., Tombelli, S., and Mascini, M. (2003) New trends in affinity sensing: aptamers for ligand binding. TrAC Trends in Analytical Chemistry 22, 810-818
41. Bruno, J. G., and Kiel, J. L. (1999) In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection. Biosensors and Bioelectronics 14, 457-464
42. Dwarakanath, S., Bruno, J. G., Shastry, A., Phillips, T., John, A., Kumar, A., and Stephenson, L. D. (2004) Quantum dot-antibody and aptamer conjugates shift fluorescence upon binding bacteria. Biochemical and biophysical research communications 325, 739-743
43. Bitsch, R. (2003) BIOTIN | Properties and Determination. In: Caballero, B., ed. Encyclopedia of Food Sciences and Nutrition, pp. 506-516, Academic Press, Oxford
44. Wagner, D. (2009) Vitamin Analysis for the Health and Food Sciences. Vitamin Analysis for the Health and Food Sciences, pp. 598–599, Elsevier
45. Indyk, H. E., Evans, E. A., Bostrom Caselunghe, M. C., Persson, B. S., Finglas, P. M., Woollard, D. C., and Filonzi, E. L. (2000) Determination of biotin and folate in infant formula and milk by optical biosensor-based immunoassay. Journal of AOAC International 83, 1141-1148
46. Staggs, C. G., Sealey, W. M., McCabe, B. J., Teague, A. M., and Mock, D. M. (2004) Determination of the biotin content of select foods using accurate and sensitive HPLC/avidin binding. Journal of food composition and analysis : an official publication of the United Nations University, International Network of Food Data Systems 17, 767-776
47. Anderson, G. P., Moreira, S. C., Charles, P. T., Medintz, I. L., Goldman, E. R., Zeinali, M., and Taitt, C. R. (2006) TNT detection using multiplexed liquid array displacement immunoassays. Anal Chem 78, 2279-2285
48. Cho, E. J., Lee, J. W., and Ellington, A. D. (2009) Applications of aptamers as sensors. Annual review of analytical chemistry 2, 241-264
49. Ellington, A. D., and Szostak, J. W. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822
50. Tuerk, C., and Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510
51. Chen, C. S., Baeumner, A. J., and Durst, R. A. (2005) Protein G-liposomal nanovesicles as universal reagents for immunoassays. Talanta 67, 205-211
52. Kuo, T. C., Lee, P. C., Tsai, C. W., and Chen, W. Y. (2013) Salt bridge exchange binding mechanism between streptavidin and its DNA aptamer--thermodynamics and spectroscopic evidences. Journal of molecular recognition : JMR 26, 149-159
53. Khor, S. M., Thordarson, P., and Gooding, J. J. (2013) The impact of antibody/epitope affinity strength on the sensitivity of electrochemical immunosensors for detecting small molecules. Analytical and bioanalytical chemistry 405, 3889-3898
54. Ellington, A. D., and Szostak, J. W. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822.
55. Stoltenburg, R., Reinemann, C., and Strehlitz, B. (2007) SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol. Eng. 24, 381-403
56. Edwards, K. A., and Baeumner, A. J. (2006) Liposomes in analyses. Talanta 68, 1421-1431
57. Chen, C.-S., and Durst, R. A. (2006) Simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes with an array-based immunosorbent assay using universal protein G-liposomal nanovesicles. Talanta 69, 232-238
58. Yalow, R. S., and Berson, S. A. (1959) Assay of Plasma Insulin in Human Subjects by Immunological Methods. Nature 184, 1648-1649
59. Loughrey, H., Bally, M. B., and Cullis, P. R. (1987) A non-covalent method of attaching antibodies to liposomes. Biochimica et Biophysica Acta (BBA) - Biomembranes 901, 157-160
60. Zhu, Z., Schmidt, T., Mahrous, M., Guieu, V., Perrier, S., Ravelet, C., and Peyrin, E. (2011) Optimization of the structure-switching aptamer-based fluorescence polarization assay for the sensitive tyrosinamide sensing. Analytica Chimica Acta 707, 191-196
61. Yoshida, Y., Sakai, N., Masuda, H., Furuichi, M., Nishikawa, F., Nishikawa, S., Mizuno, H., and Waga, I. (2008) Rabbit antibody detection with RNA aptamers. Analytical biochemistry 375, 217-222
62. Chen, C. S., Korobkova, E., Chen, H., Zhu, J., Jian, X., Tao, S. C., He, C., and Zhu, H. (2008) A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nature methods 5, 69-74
63. Plant, A. L., Brizgys, M. V., Locasio-Brown, L., and Durst, R. A. (1989) Generic liposome reagent for immunoassays. Analytical biochemistry 176, 420-426
64. Scorilas, A., Bjartell, A., Lilja, H., Moller, C., and P. Diamandis, E. (2000) Streptavidin-Polyvinylamine Conjugates Labeled with a Europium Chelate: Applications in Immunoassay, Immunohistochemistry, and Microarrays. Clinical chemistry 46, 1450-1455
65. Rosenthal, S. J., Chang, J. C., Kovtun, O., McBride, J. R., and Tomlinson, I. D. (2011) Biocompatible Quantum Dots for Biological Applications. Chemistry & biology 18, 10-24
66. Ribble, D., Goldstein, N. B., Norris, D. A., and Shellman, Y. G. (2005) A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnology 5, 12-12
67. Gan, S. D., and Patel, K. R. (2013) Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay. J Invest Dermatol 133, e12
68. Zhang, Q., and Guo, L. H. (2007) Multiple labeling of antibodies with dye/DNA conjugate for sensitivity improvement in fluorescence immunoassay. Bioconjugate chemistry 18, 1668-1672
69. Lueking, A., Horn, M., Eickhoff, H., Bussow, K., Lehrach, H., and Walter, G. (1999) Protein microarrays for gene expression and antibody screening. Analytical biochemistry 270, 103-111
70. Zhu, H., and Snyder, M. (2003) Protein chip technology. Current opinion in chemical biology 7, 55-63
71. Song, K. M., Lee, S., and Ban, C. (2012) Aptamers and their biological applications. Sensors 12, 612-631
72. Toh, S. Y., Citartan, M., Gopinath, S. C. B., and Tang, T.-H. (2015) Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay. Biosensors and Bioelectronics 64, 392-403
73. Brody, E. N., and Gold, L. (2000) Aptamers as therapeutic and diagnostic agents. Journal of biotechnology 74, 5-13
74. Keefe, A. D., Pai, S., and Ellington, A. (2010) Aptamers as therapeutics. Nat Rev Drug Discov 9, 537-550
75. Jayasena, S. D. (1999) Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics. Clinical chemistry 45, 1628-1650
76. Nimjee, S. M., Rusconi, C. P., and Sullenger, B. A. (2005) Aptamers: an emerging class of therapeutics. Annual review of medicine 56, 555-583
77. Mairal, T., Ozalp, V. C., Lozano Sanchez, P., Mir, M., Katakis, I., and O′Sullivan, C. K. (2008) Aptamers: molecular tools for analytical applications. Analytical and bioanalytical chemistry 390, 989-1007
78. Bruno, J. (2015) Predicting the Uncertain Future of Aptamer-Based Diagnostics and Therapeutics. Molecules 20, 6866
79. Baird, G. S. (2010) Where Are All the Aptamers? American Journal of Clinical Pathology 134, 529-531
指導教授 陳建生(Chien‐Sheng Chen) 審核日期 2016-8-31
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   
網路書籤 Google bookmarks   del.icio.us   hemidemi   myshare   

若有論文相關問題，請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407，或E-mail聯絡  - 隱私權政策聲明