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研究生： 曾子清
Tseng, Zi-Qing
論文名稱： 開發一種利用抗體化磁珠以及表面增強拉曼散射進行牙齦卟啉單胞菌簡易且準確快速之定性及定量方法
Development of a Simple, Accurate, and Rapid Method for Qualitative and Quantitative Detection of Porphyromonas gingivalis Using Antibody-Conjugated Magnetic Beads and Surface-Enhanced Raman Scattering
指導教授： 吳炳慶
Wu, Ping-Ching
學位類別： 碩士
Master
系所名稱： 工學院 - 生物醫學工程學系
Department of BioMedical Engineering
論文出版年： 2024
畢業學年度： 112
語文別： 英文
論文頁數： 94
中文關鍵詞： 慢性牙周病 、牙齦卟啉單胞菌 、表面增強拉曼散射 、奈米金棒 、抗體化磁珠
外文關鍵詞： Chronic periodontitis, Porphyromonas gingivalis (P. gingivalis), Surface-Enhanced Raman Scattering (SERS), gold nanorods (GNRs), antibody-conjugated magnetic beads
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    • 牙齦卟啉單胞菌（Porphyromonas gingivalis, P. gingivalis）是慢性牙周炎的主要病原菌，其存在和數量與牙周病的嚴重程度密切相關。慢性牙周炎是一種常見的口腔疾病，會導致牙齦炎症、牙槽骨吸收和最終的牙齒脫落，並且與多種全身性疾病（如心血管疾病、糖尿病等）有密切關聯。傳統檢測方法如細菌培養和聚合酶鏈反應（PCR）由於處理時間長、操作複雜和成本高，難以滿足快速臨床診斷的需求。因此，開發一種快速且準確的檢測方法對於提高臨床診斷效率和患者治療效果具有重要意義。
    本研究開發了一種結合抗體化磁珠和表面增強拉曼散射（Surface-Enhanced Raman Scattering, SERS）技術的P. gingivalis檢測方法。SERS技術利用奈米金棒作為基底材料，通過其局域表面電漿共振（Localized Surface Plasmon Resonance, LSPR）效應來顯著增強拉曼信號。此外，奈米金棒通過表面電荷相互作用有效吸附細菌，進一步提高檢測靈敏度。同時，抗體化磁珠技術利用磁珠表面修飾上抗體，對P. gingivalis進行高效分離和聚集，提升檢測的特異性和準確性。
    在實驗過程中，首先合成了奈米金棒，並通過調控反應條件來優化其尺寸和形狀，以獲得最佳的LSPR效應。隨後將奈米金棒與細菌混和吸附，同時製備抗體化磁珠，並將其與含有奈米金棒的細菌再次混合，利用磁性分離技術實現目標細菌的聚集和分離，最終進行SERS檢測。結果顯示，該方法能夠在60分鐘內實現P. gingivalis的高靈敏度和高特異性檢測，並提供準確的定量結果，最低檢測值能達到10^4 CFU/mL。
    本研究結果顯示。透過抗體化磁珠與SERS技術的結合，我們成功開發出一種滿足臨床需求，簡便、準確且快速的細菌檢測方式。特別是針對P. gingivalis這種與多種全身性疾病相關的病原菌，本技術的應用將提升牙周病的診療效率，改善患者的口腔健康和生活品質，並為未來其他病原菌的快速檢測提供新的方法和思路。

    Porphyromonas gingivalis (P. gingivalis) is the primary pathogen of chronic periodontitis, with its presence and quantity closely related to the severity of periodontal disease. Chronic periodontitis is a common oral disease that leads to gingival inflammation, alveolar bone resorption, and ultimately tooth loss. It is also associated with various systemic diseases, such as cardiovascular diseases and diabetes. Traditional detection methods, such as bacterial culture and polymerase chain reaction (PCR), are limited by long processing times, complex operations, and high costs, making them unsuitable for rapid clinical diagnosis. Therefore, developing a rapid and accurate detection method is crucial for improving clinical diagnostic efficiency and patient treatment outcomes.
    This study developed a rapid and accurate detection method for P. gingivalis by combining antibody-conjugated magnetic beads (MBs) with Surface-Enhanced Raman Scattering (SERS) technology. SERS technology utilizes GNRs as the substrate material, leveraging their Localized Surface Plasmon Resonance (LSPR) effect to significantly enhance Raman signals. Additionally, the GNRs effectively adsorb bacteria through surface charge interactions, further improving detection sensitivity. The antibody-conjugated MBs technology efficiently separates and enriches P. gingivalis, enhancing detection specificity and accuracy.
    During the experimental process, GNRs were first synthesized and their size and shape were optimized by adjusting reaction conditions to achieve the best LSPR effect. The GNRs were then mixed with and adsorbed onto the bacteria. Antibody-conjugated MBs were prepared and mixed with the bacteria-containing GNRs, utilizing magnetic separation technology to achieve the aggregation and separation of the target bacteria, followed by SERS detection. The results show that this method can achieve high sensitivity and specificity in the detection of P. gingivalis within 60 minutes and provide accurate quantitative results, with a detection limit as low as 10^4 CFU/mL.
    The findings of this study demonstrate that by combining antibody-conjugated MBs with SERS technology, we have successfully developed a simple, accurate, and rapid bacterial detection method that meets clinical needs. Specifically, for P. gingivalis, a pathogen associated with various systemic diseases, the application of this technology will improve the diagnostic efficiency of periodontal disease, enhance patients’ oral health and quality of life, and provide new methods and approaches for the rapid detection of other pathogens in the future.

    摘要 I Abstract II 誌謝 IV Contents VI List of Table X List of Figures XI Abbreviation List XIII Chapter 1 Introduction 1 1.1 Chronic Periodontitis 2 1.1.1 Definition and Clinical Features of Chronic Periodontitis 2 1.1.2 Epidemiology of Chronic Periodontitis 3 1.1.3 Chronic Periodontitis 3 1.2 Periodontal Pathogen- P. gingivalis 4 1.2.1 Microbiological Characteristics of P. gingivalis. 4 1.2.2 Mechanism of Colonization of P. gingivalis in Periodontal Tissues 6 1.2.3 Mechanisms by which P. gingivalis Triggers and Exacerbates Periodontal Inflammation 6 1.2.4 The Pathological Relationship of P. gingivalis to Non-Periodontal Diseases 7 1.2.5 The Importance of Qualitative and Quantitative Analysis of P. gingivalis10 1.3 Surface-enhanced Raman Scattering (SERS) 12 1.3.1 History of Raman Scattering 12 1.3.2 Basic Principles of Raman Scattering 12 1.3.3 History of SERS 15 1.3.4 Basic Principles of SERS 17 1.3.5 The Application of Metals in SERS for Bacterial Analysis 18 1.3.6 Morphologies of Gold Nanoparticles 19 1.3.7 Synthesis of GNRs 20 1.4 Application of Antibody-Coated Magnetic Beads (MBs) in the Separation of P. gingivalis 21 1.5 Specific Aim 21 Chapter 2 Materials & Methods 23 2.1 Materials 24 2.2 Oral Pathogens Culture 25 2.2.1 Bacterial Strain Selection 25 2.2.2 P. gingivalis Culture 25 2.2.3 S. mitis Culture 25 2.2.4 Establishment of a Standard Curve for Bacterial Count 25 2.3 SERS Substrate Preparation 26 2.3.1 Preparation of GNRs 26 2.3.2 Characterization of Gold Nanoparticles and Bacteria 28 2.4.1 Antibody Modification on MBs 28 2.4.2 Detection of Antibody Residuals Using Bradford Protein Assay 29 2.4.3 Confirmation of Functional Groups on the Surface of antibody-conjugated MBs 30 2.4.4 Capturing Bacteria Using Antibody-Conjugated MBs was Performed 30 2.4.5 Analysis of Morphological Characteristics of Metals and Bacteria 30 2.5 Acquisition of Raman Spectra 31 2.5.1 Creation of Liquid SERS wells 31 2.5.2 Spectral Data Acquisition 32 2.5.3 Quantification of Bacteria 32 Chapter 3 Results 33 3.1 Correlation Between P. gingivalis OD Values and Plate Count 34 3.2 Characterization of Gold Nanoparticles and Bacteria 37 3.2.1 The Absorption Spectrum of Gold Nanoparticles 37 3.2.2 Surface Charge of GNRs and Bacteria 38 3.2.3 The TEM Images of Bacteria and GNRs 41 3.2.4 The SEM Images and of Bacteria and GNRs 43 3.3 Characterization of MBs after Antibody Modification 48 3.3.1 Modification Verification of MBs 48 3.3.2 Characterization of MBs after Bacterial Capture 51 3.4 Acquisition of Raman Spectra 55 3.4.1 Comparison of P. gingivalis Raman Spectra With and Without the GNRs 55 3.4.2 Comparison of Enhancement Effects of Different GNRs Wavelengths 57 3.4.3 The Standard Curve of P. gingivalis OD value and Raman Intensity 58 3.4.4 Raman Spectra and Specificity Test of the MBs 60 Chapter 4 Discussion 63 4.1 Challenges Encountered in the Synthesis of NRs 64 4.2 The Selection of MBs Size 65 4.3 Comparison of Different GNRs for Raman Signal 67 4.4 Evaluation of Raman Shift Peaks in Bacterial Raman Spectroscopy 67 4.5 Limit of Detection 69 4.6 The Aggregation of MBS Affects the Raman Signal 70 Chapter 5 Conclusion 71 References 75

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