腦腫瘤手術切除的完整性對患者手術後復原極為重要，如果無法將腫瘤清除乾淨，可能導致腫瘤復發或轉移。多型性神經膠質母細胞瘤(Glioblastoma Multiforme，GBM)屬於浸潤式，手術上很難完全清除，因此發展一些顯影的方式幫助醫師解決此問題。目前5-ALA螢光分子已被FDA許可運用於手術中，成功提升腫瘤的切除率，但螢光分子的光漂白(Photobleaching)問題限制臨床的實用性，而拉曼標記的光學穩定性佳、擁有指紋般特徵的訊號、低毒性等優點，在生物影像上展現極大的潛力。
先前本實驗室利用50 nm金粒子與20 nm金粒子，已經成功合成出核心－衛星奈米金屬叢集結構，且能穩定地發出拉曼訊號，為了確保拉曼標記結構與其拉曼訊號的穩定性，必須以二氧化矽對此結構進行封裝。本論文第一部份探討如何製厚度薄、表面平滑、完整包覆的二氧化矽殼。
另外，生物標記最重要的是能夠針對腫瘤細胞進行標記，進行病理的檢測，因此利用抗體當作拉曼標記與細胞之間連接的分子，以達到標記的作用。本論文嘗試數種化學物質進行二氧化矽表面改質，也透過EDC與sulfo-NHS反應，將抗體連接到二氧化矽殼表面，完成拉曼標記，且經由多種方式檢驗抗體連接情形。另外也對拉曼標記進行專一性檢驗，確認標記的標靶特性。
在拉曼標記吸附腦腫瘤細胞後，量測細胞上標記的拉曼光譜，並實際以拉曼訊號判別腫瘤細胞與正常細胞，初步模擬未來手術時用此標記分辨腫瘤組織的情形。
整合二氧化矽殼與抗體連接的方法套用至核心－衛星奈米金屬叢集結構，完成拉曼標記，未來希望應用在腦腫瘤的拉曼影像，在手術上協助醫師完整切除多型性神經膠質母細胞瘤。

The completeness of the surgical resection is very important. If the tumor couldn’tbe cleaned, it may lead to tumor recurrence or metastasis. Because it’s difficult to distinguish the boundary of Glioblastoma Multiforme (GBM) which is common and serious brain tumor, completely removing tumor ischallenging. Therefore, the development of imaging methods to help physicians solve this problem.Currently, 5-ALA fluorescence molecule has been used in surgery, andimproved the success rate of tumor excision. However, fluorescence molecules have the problem of photobleaching. It would limit the clinical application. Raman tags show a great potential in biological imaging because of the stable of optical characteristic, non-toxic, allowing multiple marker detection, etc.
Previously, our group has been successfully synthesized core-satellite nanostructures by using 50 nm and 20 nm gold nanoparticles. The nanostructures have stable Raman signals. In order to make the structures stable and keep the signals correct, it is necessary to synthesize silica shell. The shell thickness of 20 nm has been achieved. The silica shellsare smooth and uniform as well.
Moreover, biomarkers must have the ability to target the specific cell, so antibodies were chosen to combine Raman tags and cell. Several chemicals were experimented for modification of silica surface. Through the reaction of EDC and sulfo-NHS, antibodies could conjugated on silica shell. The phenomenon could observed by some checking methods.By specificity test, Raman tags of specific cellular recognition could be confirmed.
After Raman tags labeled on brain tumor cells, their Raman spectra were measured. We tried to identify tumor cells and normal cells by Raman signals.
We have developed a novel Raman tags. We hope that this tags can help physicians discern brain tumor during the surgery in the future.

[1] J. Lazarovits, Y. Y. Chen, E. A. Sykes, and W. C. Chan, "Nanoparticle–blood interactions: the implications on solid tumour targeting," Chemical Communications, vol. 51, no. 14, pp. 2756-2767, 2015.
[2] J. Y. Yhee, S. Lee, and K. Kim, "Advances in targeting strategies for nanoparticles in cancer imaging and therapy," Nanoscale, vol. 6, no. 22, pp. 13383-13390, 2014.
[3] Y. Wang, B. Yan, and L. Chen, "SERS tags: novel optical nanoprobes for bioanalysis," Chemical reviews, vol. 113, no. 3, pp. 1391-1428, 2012.
[4] X. Huang and M. A. El-Sayed, "Plasmonic photo-thermal therapy (PPTT)," Alexandria Journal of Medicine, vol. 47, no. 1, pp. 1-9, 2011.
[5] V. Biju, "Chemical modifications and bioconjugate reactions of nanomaterials for sensing, imaging, drug delivery and therapy," Chemical Society Reviews, vol. 43, no. 3, pp. 744-764, 2014.
[6] T. E. Taylor, F. B. Furnari, and W. K. Cavenee, "Targeting EGFR for treatment of glioblastoma: molecular basis to overcome resistance," Current cancer drug targets, vol. 12, no. 3, pp. 197-209, 2012
[7] M. F. Kircher,A. De La Zerda, J. V. Jokerst, C. L. Zavaleta, P. J. Kempen, E. Mittra, K. Pitter, R. M. Huang, C. Campos, F. Habte, R. Sinclair, C. W. Brennan, I. K. Mellinghoff, E. C. Holland, S. S. Gambhir, "A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle," Nature medicine, vol. 18, no. 5, pp. 829-834, 2012.
[8] FDA官方網站訊息，5-ALA被批准作為膠質細胞瘤手術的顯影劑https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm562645.htm
[9] W. Stummer, A. Novotny, H. Stepp, C. Goetz, K. Bise, and H. J. Reulen, "Fluorescence-guided resection of glioblastoma multiforme utilizing 5-ALA-induced porphyrins: a prospective study in 52 consecutive patients," Journal of neurosurgery, vol. 93, no. 6, pp. 1003-1013, 2000.
[10] D. Ni, Bu, J. Zhang, Bu. W, H. Xing, F. Han, Q. Xiao, Z. Yao, F. Chen, Q. He, J. Liu, S. Zhang, W. Fan, L. Zhou, W. Peng, and J. Shi, "Dual-targeting upconversion nanoprobes across the blood–brain barrier for magnetic resonance/fluorescence imaging of intracranial glioblastoma," ACS nano, vol. 8, no. 2, pp. 1231-1242, 2014.
[11] C. L. Zavaleta,B. R. Smith, I. Walton, W. Doering, G. Davis, B. Sho jaei, M. J. Natan, S. S. Gambhir, "Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy," Proceedings of the National Academy of Sciences, vol. 106, no. 32, pp. 13511-13516, 2009.
[12] J. R. Ferraro, Introductory raman spectroscopy. Academic press, 2003.
[13] 林天心, "以穩定的奈米粒子叢集為基礎的拉曼標記", 碩士論文, 成功大學光電科學與工程學研究所, 2015
[14] S. Y. Chen and A. A. Lazarides, "Quantitative amplification of Cy5 SERS in ‘Warm Spots’ created by plasmonic coupling in nanoparticle assemblies of controlled structure†," The Journal of Physical Chemistry C, vol. 113, no. 28, pp. 12167-12175, 2009.
[15] B. Jankiewicz, D. Jamiola, J. Choma, and M. Jaroniec, "Silica–metal core–shell nanostructures," Advances in colloid and interface science, vol. 170, no. 1, pp. 28-47, 2012.
[16] L. Wang,K. Wang, S. Santra, X. Zhao, L. R. Hilliard, J. E. Smith, Y. Wu,andW. Tan, "Watching silica nanoparticles glow in the biological world," Analytical Chemistry, vol. 78, no. 3, pp. 646-654, 2006.
[17] K.C. Ho, P.J. Tsai, Y.S. Lin, and Y.C. Chen, "Using biofunctionalized nanoparticles to probe pathogenic bacteria," Analytical chemistry, vol. 76, no. 24, pp. 7162-7168, 2004.
[18] D. L. Thorek and A. Tsourkas, "Comparative analysis of nanoparticle-antibody conjugations: carbodiimide versus click chemistry," Molecular imaging, vol. 8, no. 4, pp. 211-229, 2009.
[19] J. Wan, X. Meng, E. Liu, and K. Chen, "Incorporation of magnetite nanoparticle clusters in fluorescent silica nanoparticles for high-performance brain tumor delineation," Nanotechnology, vol. 21, no. 23, p. 235104, 2010.
[20] J. Yu, C.H. Hsu, C.C. Huang, and P.Y. Chang, "Development of Therapeutic Au–Methylene Blue Nanoparticles for Targeted Photodynamic Therapy of Cervical Cancer Cells," ACS applied materials & interfaces, vol. 7, no. 1, pp. 432-441, 2014.
[21] T. Zapf, C. Zafiu, C. Zaba, C.-W. D. Tan, W. Hunziker, and E.-K. Sinner, "Nanoscopic leg irons: harvesting of polymer-stabilized membrane proteins with antibody-functionalized silica nanoparticles," Biomaterials science, vol. 3, no. 9, pp. 1279-1283, 2015.
[22] W. Deng, D. Jin, K. Drozdowicz‐Tomsia, J. Yuan, J. Wu, and E. M. Goldys, "Ultrabright Eu–Doped Plasmonic Ag@ SiO2 Nanostructures: Time‐gated Bioprobes with Single Particle Sensitivity and Negligible Background," Advanced materials, vol. 23, no. 40, pp. 4649-4654, 2011.
[23] Z. Wang, J. Han, H. Gao, C. Li, and Z. Fu, "Protein functionalized titania particle as a nanocarrier in a multiple signal antibody amplification strategy for ultrasensitive chemiluminescent immunoassay," Talanta, vol. 88, pp. 765-768, 2012.
[24] Z. Wang, S. Zong, H. Chen, H. Wu, and Y. Cui, "Silica coated gold nanoaggregates prepared by reverse microemulsion method: Dual mode probes for multiplex immunoassay using SERS and fluorescence," Talanta, vol. 86, pp. 170-177, 2011.
[25] C. Moore, H. Monton, R. O'Kennedy, D. E. Williams, C. Nogues, C. Crean, and V. Gubala, "Controlling colloidal stability of silica nanoparticles during bioconjugation reactions with proteins and improving their longer-term stability, handling and storage," Journal of Materials Chemistry B, vol. 3, no. 10, pp. 2043-2055, 2015.
[26] W. Stöber, A. Fink, and E. Bohn, "Controlled growth of monodisperse silica spheres in the micron size range," Journal of colloid and interface science, vol. 26, no. 1, pp. 62-69, 1968.
[27] S. K. Vashist, B. Zhang, D. Zheng, K. Al-Rubeaan, J. H. Luong, and F.-S. Sheu, "Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay," Analytical biochemistry, vol. 417, no. 1, pp. 156-158, 2011.
[28] R. Huang,S. Harmsen, J. M. Samii, H. Karabeber, K. L. Pitter, E. C. Holland, andM. F. Kircher, "High Precision Imaging of Microscopic Spread of Glioblastoma with a Targeted Ultrasensitive SERRS Molecular Imaging Probe," Theranostics, vol. 6, no. 8, p. 1075, 2016.
[29] M. Jermyn, K. Mok, J. Mercier, J. Desroches, J. Pichette, K. Saint-Arnaud, L. Bernstein, M.-C. Guiot, K. Petrecca , and F. Leblond, "Intraoperative brain cancer detection with Raman spectroscopy in humans," Science translational medicine, vol. 7, no. 274, pp. 274ra19-274ra19, 2015.
[30] A. Indrasekara, B. J. Paladini, D. J. Naczynski, V. Starovoytov, P. V. Moghe, and L. Fabris, "Dimeric Gold Nanoparticle Assemblies as Tags for SERS‐Based Cancer Detection," Advanced healthcare materials, vol. 2, no. 10, pp. 1370-1376, 2013.
[31] L. M. Liz-Marzán, M. Giersig, and P. Mulvaney, "Synthesis of nanosized gold− silica core− shell particles," Langmuir, vol. 12, no. 18, pp. 4329-4335, 1996.
[32] 吳政樑, "二氧化矽/甲基丙烯酸甲酯核殼型複合乳膠粒子之合成與性質", 碩士論文成功大學化學工程學研究所, 2009
[33] C. J. Brinker and G. W. Scherer, Sol-gel science: the physics and chemistry of sol-gel processing. Academic press, 2013.
[34] R. K. Iler, The chemistry of silica: solubility, polymerization, colloid and surface properties, and biochemistry,Canada: John Wiley &Sons Inc, 1979.
[35] G. T. Hermanson, Bioconjugate techniques. Academic press, 2013.
[36] Z. Grabarek and J. Gergely, "Zero-length crosslinking procedure with the use of active esters," Analytical biochemistry, vol. 185, no. 1, pp. 131-135, 1990.
[37] B. Alberts, Essential Cell Biology. Garland Science, 2010.
[38] C. A. Janeway, P. Travers, M. Walport, and M. J. Shlomchik, Immunobiology: the immune system in health and disease. Current Biology, 1997.
[39] 徐榮忠, "具奈米結構之電漿子生物感測器", 碩士論文,成功大學工程科學研究所, 2008
[40] 曾賢德(2010), 金奈米粒子的表面電漿共振特性: 耦合, 應用與樣品製作物理雙月刊,32,2, 126-135
[41] 江宇涵, "奈米粒子應用於癌細胞標定的拉曼光學量測", 碩士論文, 應用化學系分子科學研究所, 2010
[42] 孫逸民, 陳玉舜, 趙敏勳, 謝明學, 劉興鑑, 儀器分析, 全威圖書有限公司,1997
[43] R. J. Hunter, Zeta potential in colloid science: principles and applications. Academic press, 2013.
[44] V. Mohanraj and Y. Chen, "Nanoparticles-a review," Tropical Journal of Pharmaceutical Research, vol. 5, no. 1, pp. 561-573, 2006.
[45] J. M. Walker, The protein protocols handbook. Springer Science & Business Media, 1996.
[46] C. D. Georgiou, K. Grintzalis, G. Zervoudakis, and I. Papapostolou, "Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins," Analytical and bioanalytical chemistry, vol. 391, no. 1, pp. 391-403, 2008.
[47] 付洪蘭, 實用電子顯微鏡技術, 合記圖書發行, 2007
[48] D. C. Bell and N. Erdman, Low voltage electron microscopy: principles and applications. John Wiley & Sons, 2012.
[49] K. K. Chittur, "FTIR/ATR for protein adsorption to biomaterial surfaces," Biomaterials, vol. 19, no. 4, pp. 357-369, 1998.
[50] R. Chao, R. Khanna, and E. Lippincott, "Theoretical and experimental resonance Raman intensities for the manganate ion," Journal of Raman Spectroscopy, vol. 3, no. 2‐3, pp. 121-131, 1975.
[51] Y. Kobayashi,H. Inose, T. Nakagawa, K. Gonda, M. Takeda, N. Ohuchi, and A. Kasuya, "Control of shell thickness in silica-coating of Au nanoparticles and their X-ray imaging properties," Journal of colloid and interface science, vol. 358, no. 2, pp. 329-333, 2011.
[52] C. Wang, Y. Chen, T. Wang, Z. Ma, and Z. Su, "Monodispersed Gold Nanorod‐Embedded Silica Particles as Novel Raman Labels for Biosensing," Advanced Functional Materials, vol. 18, no. 2, pp. 355-361, 2008.
[53] Z. Bai, R. Chen, P. Si, Y. Huang, H. Sun, and D.-H. Kim, "Fluorescent pH sensor based on Ag@ SiO2 core–shell nanoparticle," ACS Applied Materials & Interfaces, vol. 5, no. 12, pp. 5856-5860, 2013.
[54] Y. Kobayashi,R. Nagasu, K. Shibuya, T. Nakagawa, Y. Kubota, K. Gonda, and N. Ohuchi, "Synthesis of a colloid solution of silica-coated gold nanoparticles for X-ray imaging applications," Journal of Nanoparticle Research, vol. 16, no. 8, pp. 1-13, 2014.
[55] N. Sui,V. Monnier, Y. Zakharko, Y. Chevolot, S. Alekseev, J. M. Bluet, V. Lysenko, and E. Souteyrand,., "Plasmon-controlled narrower and blue-shifted fluorescence emission in (Au@ SiO 2) SiC nanohybrids," Journal of Nanoparticle Research, vol. 14, no. 8, pp. 1-10, 2012.
[56] Y. An, M. Chen, Q. Xue, and W. Liu, "Preparation and self-assembly of carboxylic acid-functionalized silica," Journal of Colloid and Interface Science, vol. 311, no. 2, pp. 507-513, 2007.
[57] N. M. Bahadur, T. Furusawa, M. Sato, F. Kurayama, I. A. Siddiquey, and N. Suzuki, "Fast and facile synthesis of silica coated silver nanoparticles by microwave irradiation," Journal of colloid and interface science, vol. 355, no. 2, pp. 312-320, 2011.
[58] W. Ding,J. Cai, Z. Yu, Q. Wang, Z. Xu, Z. Wang, ,and C. Gao, "Fabrication of an aquaporin-based forward osmosis membrane through covalent bonding of a lipid bilayer to a microporous support," Journal of Materials Chemistry A, vol. 3, no. 40, pp. 20118-20126, 2015.
[59] B. Xu,Y. Ju, Y. Cui, G. Song, Y. Iwase, A. Hosoi, and Y. Morita, "tLyP-1–Conjugated Au-Nanorod@ SiO2 Core–Shell Nanoparticles for Tumor-Targeted Drug Delivery and Photothermal Therapy," Langmuir, vol. 30, no. 26, pp. 7789-7797, 2014.
[60] S. Zhan,Y. Yang, Z. Shen, J. Shan, Y. Li, S. Yang, , and D. Zhu, "Efficient removal of pathogenic bacteria and viruses by multifunctional amine-modified magnetic nanoparticles," Journal of hazardous materials, vol. 274, pp. 115-123, 2014.
[61] Q. Chang and H. Tang, "Immobilization of horseradish peroxidase on NH2-modified magnetic Fe3O4/SiO2 particles and its application in removal of 2, 4-dichlorophenol," Molecules, vol. 19, no. 10, pp. 15768-15782, 2014.
[62] E. F. d. Reis,F. S. Campos, A. P. Lage, R. C. Leite, L. G. Heneine, W. L. Vasconcelos, Z. I. P. Lobato , and H. S. Mansur "Synthesis and characterization of poly (vinyl alcohol) hydrogels and hybrids for rMPB70 protein adsorption," Materials Research, vol. 9, no. 2, pp. 185-191, 2006.
[63] V. Zucolotto,M. Ferreira, M. R. Cordeiro, C. J. Constantino, D. T. Balogh, A. R. Zanatta,W. C. Moreira, and O. N. Oliveira,., "Unusual interactions binding iron tetrasulfonated phthalocyanine and poly (allylamine hydrochloride) in layer-by-layer films," The Journal of Physical Chemistry B, vol. 107, no. 16, pp. 3733-3737, 2003.
[64] M. Kahraman, A. I. Zamaleeva, R. F. Fakhrullin, and M. Culha, "Layer-by-layer coating of bacteria with noble metal nanoparticles for surface-enhanced Raman scattering," Analytical and bioanalytical chemistry, vol. 395, no. 8, pp. 2559-2567, 2009.
[65] C. Munro, W. Smith, M. Garner, J. Clarkson, and P. White, "Characterization of the surface of a citrate-reduced colloid optimized for use as a substrate for surface-enhanced resonance Raman scattering," Langmuir, vol. 11, no. 10, pp. 3712-3720, 1995.
[66] M. Mabuchi, T. Takenaka, Y. Fujiyoshi, and N. Uyeda, "Surface enhanced Raman scattering of citrate ions adsorbed on gold sol particles," Surface Science, vol. 119, no. 2-3, pp. 150-158, 1982.
[67] J. S. W. Mak, "Raman Characterization of Colloidal Nanoparticles using Hollow-Core Photonic Crystal Fibers," University of Toronto, 2011.
[68] Y. Zhang, F. Wang, H. Yin, and M. Hong, "Nonuniform Distribution of Capping Ligands Promoting Aggregation of Silver Nanoparticles for Use as a Substrate for SERS," 2013.
[69] 陳俊谷, "微流道流體混合之可視化實驗與分析", 碩士論文, 中興大學機械工程研究所, 2003
[70] J.-W. Park and J. S. Shumaker-Parry, "Structural study of citrate layers on gold nanoparticles: role of intermolecular interactions in stabilizing nanoparticles," Journal of the American Chemical Society, vol. 136, no. 5, pp. 1907-1921, 2014.
[71] M. Schuetz, C. I. Müller, M. Salehi, C. Lambert, and S. Schlücker, "Design and synthesis of Raman reporter molecules for tissue imaging by immuno‐SERS microscopy," Journal of biophotonics, vol. 4, no. 6, pp. 453-463, 2011.
[72] Y. Chen, X. Bai, L. Su, Z. Du, A. Shen, A. Materny, and J. Hu, "Combined labelled and label-free sers probes for triplex three-dimensional cellular imaging," Scientific reports, vol. 6, p. 19173, 2016.
[73] X. Qian, X. H. Peng, D. O. Ansari, Q. Yin-Goen, G. Z. Chen, D. M. Shin, L. Yang, A. N. Young, M. D. Wang, and S. Nie, "In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags," Nature biotechnology, vol. 26, no. 1, p. 83, 2008.
[74] 陳郁淇, "惡性神經膠母細胞瘤所導致之腦水腫產生的影響", 碩士論文,成功大學細胞生物與解剖學研究所, 2012