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研究生： 鄭宜佳
Yi-Chia Cheng
論文名稱： 具高度有序金屬奈米管陣列及網格薄膜用於人類絨毛膜性腺激素表面增強拉曼檢測之研究
Highly-ordered metallic nanotube arrays and metal meshes for SERS detections of human chorionic gonadotropin (hCG)
指導教授： 朱瑾
Jinn Chu
賴志遠
James Lai
口試委員: 朱瑾
Jinn Chu
賴志遠
James Lai
江偉宏
Wei-Hung Chiang
姚栢文
Pak-Man Yiu
林宗宏
Zong-Hong Lin
學位類別： 碩士
Master
系所名稱： 工程學院 - 材料科學與工程系
Department of Materials Science and Engineering
論文出版年： 2023
畢業學年度： 111
語文別： 英文
論文頁數： 93
中文關鍵詞： 金屬玻璃薄膜 、金屬奈米管陣列 、表面增強拉曼散射基板 、人類絨毛膜性腺激素 、癌症檢測
外文關鍵詞： Metallic thin films, metallic nanotube arrays, SERS, human chorionic gonadotropin, disease diagnosis
相關次數： 點閱：194  下載：8 
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    • 表面增強拉曼散射(SERS)光譜相較於拉曼光譜具有微量量測的特性可將訊號增強數倍，不需要對樣品進行螢光或放射標記，從而避免了標記分子可能造成的影響，成功實現無標記的生物分子檢測。
    本研究是以SERS進行人絨毛膜促性腺激素 (hCG)之檢測，hCG主要用於檢測和監測妊娠及妊娠相關疾病，即使在沒有復發的臨床或組織學證據的情況下，復發性絨毛膜癌和睾丸生殖細胞腫瘤的治療通常也是在 hCG 水平升高的基礎上開始的。拉曼應用於此部分的研究目前多為使用抗體進行專一性檢測的免疫分析方式，其檢測樣品準備流程與ELISA檢測使用相同之三明治結構法，為使檢測更加快速及簡易，我們將嘗試透過表面增強拉曼基板進行無標記生物分子之檢測。
    本研究將分為兩大部分，首先透過鎢基金屬玻璃(WNiB)製作成金屬奈米管陣列，並於表面沉積一層純銀薄膜，此金屬奈米管陣列基板相比於其他化學合成之奈米銀顆粒，少去了拉曼 D、G band的背景干擾，使後續發展更多純物質的SERS檢測分析提供了更單純的分析背景，並且經過結構之參數調整，以Rhodamine 6G (R6G)作為檢測SERS之待測物得到最佳純銀薄膜厚度拉曼增強的效果。由此基板進行最低偵測極限濃度檢測(Limit of detection, LOD)可達到10-11 M，增強因子為1.01x108，同時也具備良好之實驗再現性。
    第二部分研究進一步將分別透過傳統拉曼檢測分子探針以及使用SERS基板對hCG直接進行拉曼檢測進行LOD的比較，透過第一部分使用部分實驗得到之最佳拉曼增強效果之基板，於分子探針的免疫分析裝置，我們成功的檢測到標記之抗體且hCG LOD可達20mIU/mL，此濃度已低於市面上驗孕棒快篩的偵測極限25mIU。於無標記純hCG的LOD檢測更可以達到20mIU/mL的程度。雖分子探針可提供檢測上的專一性，但於基板製備上較為繁瑣且須面臨分子探針標記抗體時數量的準確性以及再現性，同時須考慮複雜樣品間拉曼訊號的相互干擾，相較之下，SERS基板直接進行hCG的拉曼檢測更加快速、簡易以及準確。

    Surface enhanced Raman scattering (SERS) spectroscopy is an affordable tool. It offers highly sensitive, timely, accurate, and nondestructive information that demonstrates great potential in clinical diagnosis due to its minor susceptibility to environmental factors[1]. A biomolecule's vibrational and rotational information serves as unique Raman fingerprint for differentiation of its components and molecular structure[2]. It is possible to directly utilize the Raman spectra from biomolecules for multiplexed analysis.
    An important function of human chorionic gonadotropin (hCG) is to detect and monitor pregnancy and pregnancy-related conditions. Recurrent choriocarcinomas and testicular germ cell tumors are often treated based on increased hCG levels even when there is no clinical or histologic evidence of recurrence. There are mainly immunoassay methods for specific detection used in research on Raman applied to this part of this study. In order to detect the samples, the sandwich structure method is used in the same manner as in the ELISA process. To speed up and simplify the detection process, we will attempt to pass through surface-enhanced Raman substrates so that biomolecules can be detected without the need for labeling.
    This research will be divided into two parts. First, metal nanotube arrays are fabricated through tungsten-based metallic glass (WNiB), followed by the deposit of a layer of pure silver thin film on the surface. Compared with other chemically synthesized nano-silver particles, this metal nanotube array substrate reduces the background interference of Raman D and G bands, enabling the subsequent development of pure SERS. The detection analysis provides a simpler analysis background, and after adjusting the parameters of the structure, Rhodamine 6G (R6G) is used as the analyte for SERS detection to obtain the best Raman enhancement effect of pure silver film thickness. There is an excellent reproducibility of results in experiments conducted on this substrate, as well as a limit of detection (LOD) of 10-11 M and an enhancement factor of 1.01x108.
    In the second part of the study, we will compare the LOD through the Raman detection molecular probe and the direct Raman detection of hCG using the SERS substrate. The first part of the experiment involves the use of the substrate with the best Raman enhancement effect obtained in some experiments in conjunction with the immunoassay device of the probe. A labeled antibody was successfully detected, and the hCG LOD reached 20mIU/mL, which is lower than the detection limit of 25mIU for the quick screening of pregnancy test sticks currently available. LOD detection of unlabeled pure hCG is even possible at 20mIU/mL. It is true that molecular probes can provide specificity in detection, but they are considerably more complex to prepare, and they must take into account the accuracy and reproducibility of the number of antibodies labeled with molecular probes. Furthermore, it is necessary to take into account that Raman signals from complex samples can interfere with each other. However, Raman detection of hCG directly on the SERS substrate is significantly faster, more convenient, and more accurate.

    Abstract III Acknowledgements V Content IV List of Figures IV List of Tables VIII Chapter 1 Introduction 1 1.1 Objectives of study 2 Chapter 2 Literature Review 3 2.1 Optical properties of plasmonic nanostructure 3 2.1.1 Surface plasmon resonance (SPR) 4 2.1.2 Localized surface plasmon resonance (LSPR) 4 2.2 Raman spectroscopy 5 2.2.1 Surface Enhancement Raman Scattering (SERS) 7 2.2.2 Chemical enhancement (CM) 8 2.2.3 Electromagnetic enhancement (EM) 9 2.3 Substrate types for SERS enhancement 10 2.3.1 Three-dimensional nanostructure 11 2.3.2 Silver thickness 12 2.3.3 Nanoparticle 13 2.4 Nanotube Arrays 14 2.4.1 Unique properties of Metallic Glass 14 2.4.2 Metallic Nanotube Arrays (MeNTAs) 16 2.4.3 MGNT arrays applied in SERS application 16 2.5 Human chorionic gonadotropin 19 2.5.1 Tertiary structure protein 19 2.6 Liquid SERS application 20 2.7 Dye-labeled immunoassay 22 2.7.1 Polydopamine 22 2.7.2 SERS active substrates 24 2.7.3 PDA application in SERS 25 2.7.4 Rhodamine 6G labeled antibody 25 Chapter 3 Experimental Procedure 27 3.1 Metallic Nanotube arrays (MeNTAs) fabrication 27 3.1.1 Substrate and photoresist preparations 28 3.1.2 Thin-film Metallic Glass (TFMG) deposition 29 3.1.3 Photoresist removal 30 3.2 Fabrication of MeNTAs active substrate 30 3.3 Characterizations of metallic nanotube arrays (MeNTAs) 34 3.3.1 Scanning electron microscope (SEM) 34 3.3.2 Energy Dispersive Spectrometer (EDS) 34 3.3.3 X-ray diffraction (XRD) 34 3.3.4 Micro-Raman spectrometer 35 3.4 Characterizations of Poly dopamine (PDA) thin film 36 3.4.1 Wettability (Water contact angle) 36 3.4.2 X-ray photoelectron spectroscopy (XPS) 37 3.4.3 Surface roughness analysis (AFM) 37 3.5 Characterization of R6G labeled Antibody 38 3.5.1 Conjugation of R6G labeled antibody 38 3.5.2 Desalting and Buffer Exchange 39 3.5.3 UV-visible 39 Chapter 4 Results and Discussion 41 4.1 Triangular MeNTA of Tungsten with Ag coating 41 4.1.1 Surface morphology (SEM) 41 4.1.2 Energy Dispersive Spectrometer (EDS) 47 4.1.3 Wettability (Water contact angle) 48 4.1.4 Crystallographic analysis (XRD) 48 4.1.5 Raman spectra for MeNTAs and film 49 4.1.6 Reproducibility of W-based MeNTAs with Ag coating 53 4.1.7 Limit of detection (LOD) of W-based MeNTAs/ Ag 54 4.2 Polydopamine 57 4.2.1 Surface morphology (SEM) 57 4.2.2 Wettability (Water contact angle) 58 4.2.3 X-ray photoelectron spectroscopy (XPS) 60 4.2.4 Surface roughness analysis (AFM) 61 4.3 Raman spectra of sandwich structure immunoassay 64 4.3.1 Raman spectra of R6G labeled antibody to detect hCG 64 4.4 Raman spectroscopy of liquid immunoassay 67 4.4.1 Raman spectra of Bovine Serum Albumin 67 4.4.2 Raman spectra of Human chorionic gonadotropin 68 4.5 MeNTAs SERS for Selenium detection 70 Chapter 5 Conclusions 73 5.1 Future Work 74 Chapter 6 References 75

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