角膜潰瘍病患就診時，醫師須藉由微生物培養以茲判斷，始能調整恰當的藥物進行治療，這需耗費數天或數週的時間。因此，我們想在微少樣本量之分取，且不需複雜之前處理的原則下，發展一種能夠快速鑑定的新方法。相較於傳統檢測，拉曼光譜法配搭予適當波長的雷射光之擇取，並透過主成分分析法(PCA)，即可在短時間內完成檢測。本研究分兩方向探討；(1)針對角膜潰瘍病人與正常人的差異(液滴內含物層積拉曼光譜法(Drop coating deposition Raman spectroscopy, DCDRS))，及(2)使用參考菌株初步測試試樣點滴抽氣過濾法之效用，藉由光譜分析所做的成果報告之。
本研究使用樣本共有三種；(1)合成淚水與(2)其內加入細菌經時所得，及(3)臨床採得的淚水試樣。合成淚水有助於探討臨床淚水中內含物的濃度，利於各光譜波峰歸屬之辨識。其次混予固定量的細菌後，經時觀察比較其差異，可區別因細菌而產生的波峰與否，有利於是細菌本身性的釋出或受感染者之生理應答性分泌的區分。Lactoferrin與Lysozyme為抵禦微生物的生理應答性第一道防線，當調整其濃度混入於蛋白質與鹽類所構成的合成淚水，我們發現其濃度越高，各歸屬的拉曼波峰也越高，PCA分佈也顯示可區別之勢。其次，當合成淚水混合於109 CFU/ml之金黃色葡萄球菌，經不同時間培養作用後，並未發現有其他特徵峰出現。PCA可觀察到作用前與作用12小時後分佈不同，此可歸因細菌本身性的釋出物質影響DCDRS。最後，導入臨床樣本進行比較；發現角膜潰瘍淚水比正常者淚水以及有無加入細菌之合成淚水，在1112、1520與1587 cm-1的位置多出三個明顯波峰，以及958、1307和1390 cm-1三個小的波峰。此外，於855 cm-1波峰強度更強。臨床樣本的變異固然很大，但經PCA分析仍可觀察出正常與患者分布趨勢之不同，顯示此疑似為患者受感染之內生性應答所導致；即TIMP-1、MMP-9及HNP。這也反映出本實驗分析流程實可發揮辨別功能的確據。為此，我們更以ELISA分析套組求證；目前僅發現可能屬於HNP及MMP-9的855及1112 cm-1的波峰，而其他波峰尚無明顯對應關係，雖也顯現出臨床樣本的複雜性，但可確認HNP與MMP-9於淚水中明顯也比正常及非角膜潰瘍眼疾含量還高。最後，另由革蘭氏染色可知淚水中有細菌存在，從檢測菌體角度初步探討，使用試樣點滴抽氣過濾法，評估此法之效用；初步將參考菌株(S. aureus)過濾存留於含銀微粒之濾膜上，其拉曼光譜圖訊息可歸因於該細菌使然。本研究從淚水分成兩方向來探討角膜潰瘍所進行的快速即時診斷評估方法，期望提供另一可靠臨床上應用。

It is a complex and time-consuming process for the detection of microorganism from the clinical specimen of corneal ulcer. A fast and non-invasive spectrum-acquisition tool of specimen, especially in biological samples, is the greatest advantage of Raman spectroscopy. In this study, micro-Raman system is performed for rapidly acquiring Raman spectra of human tears collected from patients and healthy donors. Mimic-tears composed of lactoferrin, lysozyme, albumin, IgA, urea and sodium bicarbonate are also investigated. We adjusted the concentration couple of lactoferrin and lysozyme to simulate the possible differences with or without infection of microorganism. We tried to use the principle component analysis (PCA) to analyze the Raman spectra. Our results show the intensities of Raman spectra would be getting strong with the concentration couple increasing, and were associated with the 1st principal component. Staphylococcus aureus (S. aureus) were added into the mimic-tears to simulate an in vitro disease. As a result, only the signals of S. aureus were presented without apparent difference of signals from different reaction time. Furthermore, clinical samples were introduced to compare. We found that the Raman spectra obtained from the normal participants were similar to those of mimic-tears. The strong peaks at 1112, 1520 and 1587 cm-1 and weak peaks at 958, 1307 and 1390 cm-1 only presented in patients. In addition, the peaks at 855 cm-1 in patients’ tears were stronger than those in other tears. We compared the spectra from normal and corneal ulcer by PCA and could be separated into different groups. Perhaps these peaks in patients of above are endogenous caused. Therefore, we applied three ELISA kits for TIMP-1, MMP-9 and HNP that commonly discussed with ocular diseases and associate with corneal ulcer. Our results showed the concentrations of HNP and MMP-9 were higher in corneal ulcer. The peaks at 855 and 1112 cm-1 in corneal ulcer may be caused by HNP and MMP-9. On the other hand, the bacteria in clinical samples may be filtered, remained on the metal membrane, and their signals were specific for bacterial Raman spectrum. Using S. aureus with the number 109 CFU/ml as a model, silver membrane is proved a good substrate to acquire the Raman spectrum of S. aureus. Finally, decreased number to 105 CFU/ml of S. aureus still could acquire good Raman signals of S. aureus. In this study, micro-Raman system using Au/glass or Ag membrane substrates and PCA techniques may provide a fast and non-invasive platform for tears, giving doctors a diagnostic reference.

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