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研究生: 黃詩涵
Shih-Han Huang
論文名稱: 超感度電化學生物感測器用於卵巢癌癌細胞偵測
Ultra-sensitive Electrochemical Biosensor for Ovarian Cancer Detection
指導教授: 王復民
Fu-Ming Wang
口試委員: 袁九重
Chiou-Chung Yuan
葉淇臺
Chi-Tai Yeh
高震宇
Chen-Yu Kao
學位類別: 碩士
Master
系所名稱: 應用科技學院 - 應用科技研究所
Graduate Institute of Applied Science and Technology
論文出版年: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 112
中文關鍵詞: 生物感測器石墨烯硫菫卵巢癌CA-125抗原
外文關鍵詞: biosensor, graphene, thionine, ovarian cancer, CA-125
相關次數: 點閱:246下載:2
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  • 本研究之動機即在於希望能降低癌症檢測的濃度,並且提高檢測精確性。此研究開發出一種電化學石墨烯感測器,其氧化石墨烯(rGO)用於接合受容器硫堇(thionine)以及第二抗原(Ab2),而其中thionine的還原反應可以當作與蛋白質接合與否的決定指標。此石墨烯複合材料與另一石墨烯複合材料(第一抗原、金奈米粒子、牛血清蛋白以及抗體)共同結合在玻璃碳電極上,使之成為工作電極。本實驗所採用之參考電極為銀/氯化銀水溶液,相對電極為白金線。


    In this study, an electrochemical immunosensor fabricated on reduced graphene oxide (rGO) sheet has been created. The rGO is used to immobilize the receptor thionine (Thic) and secondary anti-CA125 antibody (Ab2). The resulting rGO/Thic/Ab2, is able to use as the label of the immunosensor. Another graphene composites, primary anti-CA125 antibody (Ab1), gold nanoparticles (AuNPs), bovine serum albumin (BSA), and cancer antigen 125 (CA125) construct on the glassy carbon electrode (GCE) to become working electrode. Ag/AgCl electrode is the reference electrode and platinum wire is the counter electrode in this research.

    摘要 I Abstract II 致謝 III 圖索引 VIII 表索引 XI 第一章 緒論 1 1.1前言 1 1.2研究動機與目的 5 第二章 文獻回顧 6 2.1癌症簡介 6 2.1.1婦科腫瘤總論 6 2.1.2腫瘤免疫學簡介 10 2.1.3腫瘤標誌 13 2.1.4良性及惡性卵巢腫瘤簡介 19 2.1.5綜合診斷 28 2.2生物感測器 31 2.2.1生物感測器簡介 31 2.2.2石墨烯製作與介紹 37 2.2.3金奈米粒子 41 2.2.4生物感測器應用 44 第三章 實驗方法及儀器設備 53 3.1儀器設備 53 3.2實驗藥品 54 3.3實驗步驟 56 3.3.1細胞培養 57 3.3.2細胞繼代 57 3.3.3細胞計數 58 3.3.4蛋白質萃取與定量 58 3.3.5金奈米粒子製備 59 3.3.6石墨烯感測器製備 60 3.4材料鑑定與分析方法 61 3.4.1Western Blot 分析方法 61 3.4.2場發射穿透式電子顯微鏡(Field Emission Electron Microscope,TEM) 64 3.4.3微分脈衝伏安法測試 65 第四章 實驗結果與討論 67 4.1細胞培養與檢測結果 67 4.1.1細胞培養之結果 67 4.1.2蛋白質萃取與定量結果 69 4.2金奈米粒子TEM影像結果分析 73 4.3電化學測試分析 77 4.3.1石墨烯感測器受容器選擇 77 4.3.2石墨烯官能基數量決定 79 4.3.3石墨烯感測器反應時間決定 80 4.3.4石墨烯感測器由電化學分析結果 82 4.3.5石墨烯感測器於不同細胞濃度於電化學分析結果 88 第五章 結論 90 第六章 參考文獻 92

    [1] 衛生福利部統計處103年度死因統計。
    [2] Pujol JL, Molinier O, Ebert W, Daures JP, Barlesi F, Buccheri G,
    Paesmans M, Quoix E, Moro-Sibilot D, Szturmowicz M, Brechot JM,
    Muley T, and Grenier J. CYFRA 21-1 is a prognostic determinant in
    non-small cell lung cancer: results of meta-analysis in 2063 patients.
    Br J Cancer 2004; 91:1-9.
    [3] Nakata B, Takashima T, OGAWA y, Ishikawa T, Hirakawa K. Serum
    CYFRA 21-1 (cytokeratin-19 fragments) is a useful tumor marker for
    detecting disease relapse and assessing treatment efficacy in breast
    cancer. Br J Cancer 2004; 91: 873-8.
    [4] Nakata , Ogawa Y, Ishikawa T, Ikeda K, Kato Y, Nishino H, Hirakawa
    K. Serum CYFRA 21-1 I a one of the most reliable tumor marker for
    breast carcinoma. Cancer 2000; 89:1285-1290.
    [5] Uenishi T, Kubo S, Hirohashi K, Tanaka H, Shuto T, Yamamoto T,
    Nishiguchi S. Cytokeratin 19 fragments in serum (CYFRA 21-1) as a
    marker in primary liver cancer. Br J Cancer 2003; 88:1894-9.
    [6] Hasholzner U, Baumgartner L, Stieber P, Meier W, Hofmann K, Faten-
    Mohadam A. Significance of the tumor markers CA125 II, CA 72.4,
    ASA and CYFRA 21-1 in ovarian carcinoma. Anticancer Res 1994;
    14:2743-6.
    [7] Yuan CC, Yeh JY, Shyong WY, Chang CH, Kao CM, Kang HF. The
    Development and application of MAb Chi-Cx-99 on cancer diagnosis
    and therapy The First Joint Taiwan and Japan Cancer Group
    Conference. Poster presentation, Tokyo, 11/20~23/2009.
    [8] 西滿正, 癌細胞不要來,華文網,pp. 91-94,2000.
    [9] van der Schroeff JG, Evers LM, Boot AJ, Bos JL. Ras oncogene
    mutations in basal cell carcinomas and squamous cell carcinomas of
    human skin. J Invest Dermatol. 1990; 94:423.
    [10] Fettig, O. and Sievers, R. 3H-Index und mittlere Generationszeit des
    menschlichen Portiokarzinoms und seiner Vorstufen. Beitr. Pathol.
    Anat. Allg. Pathol.1966;133,83-100.
    [11] Meyer JS. Growth and cell kinetic measurements in human tumors.
    Pathol Annu 1981; 16: 53–81.
    [12] Fabrikant JI, Cherry J. The kinetics of cellular proliferation in normal
    and malignant tissues. V. Analysis of labeling indices and potential
    tissue doubling times in human tumor cell populations. J Surg Oncol.
    1969;1(1):23-47.
    [13] Klein G. Tumor antigens. Annu Rev Microbiol. 1966; 20:223–252.
    [14] Herberman, RB. Immunogenicity of tumor antigens. Biochim.
    Biophys Acta 1977;473, 93–119.
    [15] Old LJ. Cancer immunology: the search for specificity. Cancer Res.
    1981; 41:361-75.
    [16] Gross L. Intradermal immunization of C3H mice against a sarcoma
    that originated in an animal of the same line. Cancer Res. 1943; 3:326-
    333.
    [17] Foley EJ. Antigenic properties of methylcholanthrene-induced tumors
    in mice of the strain of origin. Cancer Res. 1953; 13:835-7.
    [18] Prehn RT, Main JM. Immunity to methylcholanthrene-induced
    sarcomas. J Natl Cancer Inst. 1957; 18:769-78.
    [19] Hajime Yajima, Tetsuo Noda, Ktliei-Michele de Villiers, Akira
    Yajima, Kaiichiro Yamamoto, Kiichiro Noda, and Yoshiaki Ito.
    Isolation of a New Type of Human Papillomavirus (HPV52b) with a
    Transforming Activity from Cervical Cancer Tissue. cancer res
    1988; 48:7164-7172.
    [20] Cinda M. Boyer, Michael J. Borowitz, Kenneth S. McCarty JR, Robert
    B. Kinney, Lorri Everitt, Deborah V. Dawson, David Ring and Robert
    C. Bast JR. Heterogeneity of antigen expression in benign and
    malignant breast and ovarian epithelial cells. Int J Cancer 1989;
    43:55-60.
    [21] Berchuck, A, Olt, GJ, Soisson, AP et al, Heterogeneity of antigen
    expression in advanced epithelial ovarian cancer. Am J Obstet
    Gynecol. 1990; 162:883–888.
    [22] Rubin SC, Finstad CL, Hoskins WJ, et al. A longitudinal study of
    antigen expression in epithelial ovarian cancer. Gynecol Oncol 1989;
    34:389–394.
    [23] Scoutt LM, Flynn SD, Luthringer DJ, McCauley TR, McCarthy SM.
    Junctional zone of the uterus: correlation of MR imaging and
    histologic examination of hysterectomy specimens. Radiology 1991;
    179:403-407.
    [24] Brown HK, Stoll BS, Nicosia SV, et al. Uterine junctional zone:
    correlation between histologic findings and MR imaging. Radiology
    1991; 179:409–413.
    [25] Ammann AM, Walsh JW. Normal anatomy and technique of
    examination. In: Walsh JW, ed. Computed tomography of the pelvis.
    New York: Churchill Livingstone, 1985:1-28
    [26] Gold P, Freedman SO. Demonstration of tumor-specific antigens in
    human colonic carcinomata by immunological tolerance and
    absorption techniques. J Exp Med. 1965; 121:439–462.
    [27] Bergstrand CG, Czar B. Demonstration of a new protein fraction in
    serum from the human fetus. Scand J Clin Lab Invest. 1956; 8:174.
    [28] Gitlin D, Perricelli A, and Gitlin G.M. Synthesis of a-fetoprotein by
    liver, yolk sac, and gastrointestinal tract of the human conceptus.
    Cancer Res 1972; 32:979-982.
    [29] Huang SC, Chen HC, Kurman RJ, Yang YS, Wen HK, Hsieh CY, Wei
    PY, How SW, Chen TY, Ouyang PC. Secretion of human chorionic
    gonadotropin and alpha-fetoprotein by an ovarian germ cell tumor of
    apparent yolk sac origin. Gynecol Oncol 1984; 18:240-246.
    [30] Kurman RJ, Norris H.J. Malignant germ cell tumors of the ovary. Hum
    Pathol 1977; 8:551–564.
    [31] Teilum G, Albrechtsen, R, Norgaard-Pedersen B: Immunofluorescent
    localization of alpha-fetroprotein synthesis in endodermal sinus tumor
    (yolk sac tumor). Acta Pathol Microbiol Scand 1974;82A:586-588.
    [32] See WA, Cohen MB, Hoxie LD: Alpha-fetoprotein half-life as a
    predictor of residual testicular tumor: Effect of the analytic strategy
    on test sensitivity and specificity. Cancer 1993; 71:2048-2054.
    [33] Scully RE: Tumors of the ovary and maldeveloped gonads. In: Atlas
    of Tumor Pathology Series. Washington, DC, Armed Forces Institute
    of Pathology, 1979.
    [34] Young, R.H., Scully, R.E. Sex cord-stromal, steroid cell, and other
    ovarian tumors with endocrine, paraendocrine, and paraneoplastic
    manifestations. in: R.J. Kurman (Ed.) Blaustein's pathology of the
    female genital tract. 5th edition. Springer-Verlag, New York;
    2002:905–966.
    [35] Piver MS, Baker TR, Piedmonte M, et al. Epidemiology and etiology
    of ovarian cancer. Semin Oncol. 1991; 18:177-185.
    [36] Lynch HT, Watson P, Lynch JF, Conway TA, Fili M Hereditary
    ovarian cancer. Heterogeneity in age at onset. Cancer 1993; 71:573-
    581.
    [37] Schildkraut JM, Collins NK, Dent GA, Tucker JA, Barrett JC,
    Berchuck A, Boyd J. Loss of heterozygosity on chromosome 17q11-
    21 in cancers of women who have both breast and ovarian cancer. Am
    J Obstet Gynecol. 1995; 172:908–913.
    [38] Piver MS, Baker TR, Jishi MF, et al. Familial ovarian cancer—a report
    of 658 families from the Gilda Radner Familial Ovarian Cancer
    Registry. Cancer 1993; 71:582–588.
    [39] P. G. Rose; R. E. Hunter. Advanced ovarian cancer in a woman with
    a family history of ovarian cancer. J Reprod Med 1994; 39:908-910.
    [40] Schottenfeld D, Berg J. Incidence of multiple primary cancers. IV.
    Cancers of the female breast and genital organs. J Natl Cancer Inst
    1971; 46:161–170.
    [41] MF Fathalla. Incessant ovulation--a factor in ovarian neoplasia?
    Lancet 1971; 2:163.
    [42] Daniel L. Clarke-Pearson, MD; Lawrence C. Bandy, MD; Michelle
    Dudzinski, MD; Dennis Heaston, MD; William T. Creasman, MD.
    Computed Tomography in Evaluation of Patients with Ovarian
    Carcinoma in Complete Clinical Remission Correlation With
    Surgical-Pathologic Findings. JAMA 1986; 255:627-630.
    [43] Brenner DE, Shaff MI, Jones HW, Grosh WW, Greco FA, Burnett LS.
    Abdominopelvic computed tomography: evaluation in patients
    undergoing second-look laparotomy for ovarian carcinoma. Obstet
    Gynecol 1985; 65:715-719.
    [44] Megibow AJ, Bosniak MA, Ho AG, et al. Accuracy of CT in
    detection of persistent or recurrent ovarian carcinoma: correlation with
    second-look laparotomy. Radiology 1988; 166:341–345.
    [45] Silverman PM, Osborne M, Dunnick NR, et al. CT prior to second-
    look operation in ovarian cancer. Am J Roentgenol. 1988; 150:829-
    832.
    [46] Miyasaka Y, Hachiya J, Furuya Y, Seki T, Hiromu W. CT evaluation
    of invasive trophoblastic disease. J Comput Assist Tomogr 1985;
    9:459–462.
    [47] Chen CK, Chow SN, Chen RJ, Chen YP, Huang LC, Wang LI,
    LinChanges in definitions of clinical staging for cervix and ovary. Am
    JYH, Huang SC: Primary ovarian cancer at National Taiwan
    UniversityObstet Gynecol 156:263–264, 1987Hospital: 1980–1989.
    Int Surg 1994; 79:48–51,
    [48] Dembo AJ. Epithelial ovarian cancer: The role of radiotherapy. Int J
    Radiation Oncology Biol Phys 1992; 22:835–845.
    [49] Smith, JP, Day, TG. Review of ovarian cancer at the University of
    Texas systems cancer center, M.D. Anderson Hospital and Tumor
    Institute. Am J Obstet Gynecol. 1979; 135:984–993.
    [50] Chow, SN, Chen, YP. Primary ovarian cancer. Int Surg.
    1987; 72:154159.
    [51] 黃炳照、莊睦賢,電化學感測器,化工技術,第七卷,第二期,
    第150-161頁(1999)。
    [52] 陳詩詰,電流式葡萄糖生物感測器之製備及測試,碩士論文,國
    立台灣科技大學化學工程研究所,台北(2009) 。
    [53] Leland C. C., Electrode Systems for Continuous Monitoring in
    Cardiovascular Surgery,Ann. N.Y. Acad. Sci.1962; 102:29.
    [54] Perumal, v., Advances in Biosensors: Principle, Architecture and
    Applications, J.Appl. Biomed. 2014; 12:1.
    [55] Wang, Y., Electrochemical Sensors for Clinic Analysis. Sensors.
    2008; 8:2043.
    [56] Yuqing, M., Ion Sensitive Field Effect Transducer-based Biosensors.
    Biotechnol. Adv. 2003; 21:527.
    [57] Nico J. de Mol. Surface Plasmon Resonance: A General Introduction.
    Methods Mol. Biol. 2010; 627:1.
    [58] Abdulhalim, I., Overview of Optical Biosensing Techniques,
    Handbook of Biosensors and Biochips, John Wiley & Sons, New
    York, U.S.A. (2008).
    [59] Nicu, L., Resonating Piezoelectric Membranes for
    Micro electromechanically Based Bioassay: Detection of
    Streptavidin-Gold Nanoparticles Interaction with Biotinylated DNA,
    Sens. Actuators B Chem. 2005; 110:125.
    [60] Yakovleva, M., The Enzyme Thermistor-A Realistic Biosensor
    Concept, Anal. Chim. Acta. 2013; 766:1.
    [61] Ramanathan, K., Principles and Applications of Thermal Biosensors,
    Biosens. Bioelectron. 2001; 16:417.
    [62] A. K. Geim and P. Kim, “Carbon wonderland,” Scientific American,
    2008; 298(4):90-97.
    [63] G. Eda, G. Fanchini, and M. Chhowalla, "Large-area ultrathin films
    of reduced graphene oxide as a transparent and flexible electronic
    material," Nat Nano. 2008; 3:270-274.
    [64] A. K. Geim, K. S. Novoselov. The rise of graphene. Nature Materials.
    2007; 6:183-191.
    [65] K. S. Novoselov, A. K. Geim1, S. V. Morozov, D. Jiang, Y. Zhang,
    S. V. Dubonos, IV. Grigorieva1, A. A. Firsov. Electric field effect in
    atomically thin carbon films. Science. 2004; 306:666-669.
    [66] C. Lee, X. Wei, J. W. Kysar, and J. Hone, "Measurement of the Elastic
    Properties and Intrinsic Strength of Monolayer Graphene," Science.2008 ;321:385-388.
    [67] A. A. Balandin, S. Ghosh, W. Bao, I. Calizo, D. Teweldebrhan, F.
    Miao, et al., "Superior Thermal Conductivity of Single-Layer
    Graphene," Nano Letters. 2008;8: 902-907.
    [68] K. I. Bolotin, K. J. Sikes, Z. Jiang, M. Klima, G. Fudenberg, J. Hone,
    et al., "Ultrahigh electron mobility in suspended graphene," Solid
    State Communications.2008;146:351-355.
    [69] J. Rafiee, X. Mi, H. Gullapalli, A. V. Thomas, F. Yavari, Y. Shi, et
    al., "Wetting transparency of graphene," Nat Mater.2012;11:217-222.
    [70] A. H. C. Neto and K. Novoselov, "New directions in science and
    technology: two-dimensional crystals," Reports on Progress in
    Physics.2011;74:082501.
    [71] Y. Iyechika, "Application of graphene to high-speed transistors:
    expectations and challenges," Science and Technology Trends –
    Quarterly Review.2010;37:76-92.
    [72] A. Reina, X. T. Jia, J. Ho, D. Nezich, H. B. Son, V. Bulovic, et al.,
    "Large Area, Few-Layer Graphene Films on Arbitrary Substrates by
    Chemical Vapor Deposition," Nano Letters.2009;9:30-35.
    [73] I. Vlassiouk, M. Regmi, P. Fulvio, S. Dai, P. Datskos, G. Eres, et al.,
    "Role of Hydrogen in Chemical Vapor Deposition Growth of Large
    Single-Crystal Graphene," ACS Nano.2011;5:6069-6076.
    [74] Minot Research Group Wiki.
    (http://www.science.oregonstate.edu/~minote/wiki/doku.php).
    [75] H. He, J. Klinowski, M. Forster, and A. Lerf, "A new structural model
    for graphite oxide," Chemical Physics Letters.1998;287:53-56.
    [76] A. Lerf, H. Y. He, M. Forster, and J. Klinowski, "Structure of graphite
    oxide revisited," Journal of Physical Chemistry B.1998;102:4477.
    [77] M. Hirata, T. Gotou, S. Horiuchi, M. Fujiwara, and M. Ohba, "Thin-
    film particles of graphite oxide 1: High-yield synthesis and flexibility
    of the particles," Carbon.2004;42:2929-2937.
    [78] T. Szabo, A. Szeri, and I. Dekany, "Composite graphitic nanolayers
    prepared by self-assembly between finely dispersed graphite oxide
    and a cationic polymer," Carbon.2005;43:87-94.
    [79] D. A. Handley, "Colloidal Gold: Principles, Methods, and
    Applications," Academic Press.1989;1.
    [80] M. Faraday, "The Bakerian Lecture: Experimental Relations of Gold
    (and Other Metals) to Light," Philosophical Transactions of the Royal
    Society of London.1857;147:145-181.
    [81] K. Kneipp, A. S. Haka, H. Kneipp, K. Badizadegan, N. Yoshizawa,
    C. Boone, et al., "Surface-enhanced Raman Spectroscopy in single
    living cells using gold nanoparticles," Applied Spectroscopy.2002;56:
    150-154.
    [82] C. E. Talley, J. B. Jackson, C. Oubre, N. K. Grady, C. W. Hollars, S.
    M. Lane, et al., "Surface-enhanced Raman scattering from individual
    Au nanoparticles and nanoparticle dimer substrates," Nano Letters.
    2005; 5:1569-1574.
    [83] C. J. Orendorff, A. Gole, T. K. Sau, and C. J. Murphy, "Surface-
    Enhanced Raman Spectroscopy of Self-Assembled Monolayers: 
    Sandwich Architecture and Nanoparticle Shape Dependence,"
    Analytical Chemistry.2005;77:3261-3266.
    [84] H. B. Weiser, "Inorganic Colloid Chemistry," Wiley, New York, NY.
    1933; 1:21-57.
    [85] J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, and A. Plech,
    "Turkevich Method for Gold Nanoparticle Synthesis Revisited," The
    Journal of Physical Chemistry B.2006; 110:15700-15707.
    [86] S. Kumar, K. S. Gandhi, and R. Kumar, "Modeling of Formation of
    Gold Nanoparticles by Citrate Method+," Industrial &; Engineering
    Chemistry Research.2006;46:3128-3136.
    [87] Minghui Yang, Alireza Javadib, He Lia, Shaoqin Gongc, Biosensors
    and Bioelectronics. 2010; 26:560-565.
    [88] Zhiyong Guo, Tingting Hao, Shuping Du, Beibei Chen, Zebo Wang,
    Xing Li, Sui Wang, Biosensors and Bioelectronics. 2013; 44:101-107.
    [89] Xia Chen, Xinle Jia, Jingman Han, Jie Ma, Zhanfang Man, Biosensors
    and Bioelectronics. 2013; 50:356-361.
    [90] Xinle Jia, Zhimin Liu, Na Liu, Zhanfang Man, Biosensors and
    Bioelectronics. 2014; 53:160-166.

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