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研究生:楊啟宏
研究生(外文):Chi-Hong Yang
論文名稱:淚液樣本中含菌檢體前處理與配合拉曼光譜法之檢測技術開發
論文名稱(外文):Bacteria Test for Tear Samples Combined aPretreating Procedure with Raman Spectroscopy
指導教授:張憲彰
指導教授(外文):HSIEN-CHANG CHANG
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫學工程研究所碩博士班
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:57
中文關鍵詞:角膜潰瘍拉曼光譜細菌指紋淚液
外文關鍵詞:Coffee ringRaman spectroscopyPretreatmentTear
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臨床角膜潰瘍病患檢測流程中,從取得病灶組織再送至病理室進
行感染微生物培養與鑑定常耗時數天至數周。拉曼光譜因具有微量生物樣本檢測與快速分析之特點,近年來於微生物檢測之應用逐漸受到矚目。雖然拉曼光譜可直接針對病灶檢體進行檢測分析,可大幅縮短檢測時間,然其獲得之光譜訊號因相當複雜,致使解析與結果的判斷不易。此研究以角膜潰瘍病患檢體分析後發現,患者淚液及病灶組織刮除後之眼表清洗樣本,常因細菌濃度過低而不易於拉曼光譜之檢測,故本研究藉由「Coffee ring effect」作用,透過控制拉曼基材上的樣本液滴乾燥過程,而達到使細菌濃縮與聚集效果。實驗結果發現,樣本於垂直放置之基材上並配合適當的乾燥時間,可具有較明顯的菌體聚集效果,若再配合光譜mapping 模式可於低濃度細菌樣本中獲得較佳的細菌指紋圖譜和檢測效果。
Traditionally, corneal ulcer is often examined by culture to determine the kind of infected microorganisms. However, it is time-consuming. Recently, there are many reports regarding Raman spectrum analysis to try to promote such a test, because of the rapid detection and analysis under the Raman detection. Although Raman spectra can detect real
sample directly and reduce the examination time. But it’s hard to identify the signal. In this study, we try to use such a method to analyze tears of corneal ulcer patient infected by bacteria. Owing to the rare bacterial
population in the tear sample that may be obtained from patient, a suitable pretreating method based on coffee ring phenomenon is needed. According to coffee ring effect, the latex samples simulated as tear samples with bacteria were dropped on the gold-coated glass slide, and
then we made them to be 90° standing for drying in the air. A lot of latex can be collected at the bottom of edge. From the results, we could compare the effects by the different angles of substrates and the different
periods of time to dry in air. It could achieve concentration and aggregation of the sample. Through improving the aggregation effect at low concentration of the sample, this method may also provide us to detect the Raman spectra of the real tear samples with microorganisms.
圖目錄 ................................ 3
表目錄 ................................ 5
第一章 緒 論 ................ 6
1.1 研究背景 ............. 6
1.2 淚液探討淚液與疾病檢測 ................... 6
1.2.1 淚液介紹 ....... 6
1.2.2 淚液和疾病相關性 .................. 8
1.2.3 角膜潰瘍與淚液檢測方式現況 .......... 10
1.3 光學檢測技術 ...... 13
1.3.1 紅外線吸收光譜 (Infrared absorption
spectrometry) ................. 13
1.3.2 拉曼原理與架構 ............... 14
1.3.3 拉曼應用於微生物的檢測的文獻 ..................... 16
1.4 Coffee ring effect ........................... 17
1.4.1 Coffee ring effect 原理 .............. 17
1.4.2 Coffee ring effect 配合拉曼檢測淚液的文獻 ... 17
第二章 研究方法 .......... 19
2.1 研究設計 .............. 19
2.2 研究設備 .............. 20
2.3 拉曼基材製備 ...... 21
2.4 拉曼儀器架構 ..... 22
2.5 資料收集的程序與分析的方法 ......... 23
2.6 樣本配置 .............. 27
2.6.1 乳鐵蛋白配置 ..... 27
2.6.2 Latex 配置 ... 27
2.6.3 淚液取樣 ..... 27
第三章 結果與討論 ...... 28
3.1 模擬樣本對於Coffee ring effect 之影響 ......... 28
3.1.1 樣本濃度對於Coffee ring effect 之影響 ......... 29
3.1.2 乾燥時間對於Coffee ring effect 之影響 ......... 30
3.1.3 基材放置角度對於Coffee ring effect 之影響 . 31
3.2 模擬樣本對於Coffee ring effect 影響之驗證 ...... 32
3.2.1 拉曼光譜儀驗證 .......... 32
3.2.2 Nano focus 驗證 ................ 36
3.3 模擬樣本測試 ...... 39
3.3.1 不同濃度Latex 拉曼光譜 ....... 39
3.3.2 相同濃度細菌不同角度拉曼光譜 ....... 41
3.4 臨床樣本測試 .......... 42
3.4.1 正常人淚液 ................... 42
3.4.2 角膜潰瘍病人淚液 ....... 44
第四章 結論 .......................... 54
參考文獻.......................... 55
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