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研究生:楊沛東
研究生(外文):Pei-Tung Yang
論文名稱:高解析掃描式表面電漿共振顯微鏡應用於大腸癌腫瘤標誌陣列晶片之檢測
論文名稱(外文):High-resolution Scanning Surface Plasmon Resonance Microscopy for Imaging of Colorectal Cancer Sensor Array
指導教授:林啟萬林啟萬引用關係
口試委員:林致廷宋孔彬黃念祖
口試日期:2014-05-06
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生醫電子與資訊學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:62
中文關鍵詞:大腸癌預後評估胎盤生長因子金微奈米陣列晶片掃描式表面電漿共振顯微鏡
外文關鍵詞:Colorectal cancerPrognosisPlacental growth factorGold sensor arrayScanning surface plasmon resonance microscopy
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近年藉由多種腫瘤標誌物表現量做為癌症預後評估因子已逐漸受到重視,以腸胃等消化系統來說,隨著生活及飲食方式的改變,大腸癌在世界各國的盛行率不斷的攀升,因超過半數的大腸癌患者大多於第二期或第三期才被診斷接受治療,其中部分患者易有轉移或復發的情形而降低了存活率,因此如何能協助臨床人員針對不同大腸癌的分期以及不同病灶發生的位置,做一個適當的預後評估為一個重要的課題,其中以在腫瘤標誌物檢測的研究領域上,如何能達到多功檢測以及降低檢測成本的情形下,可以有較高的靈敏度以及更快速的檢測,並提供較為直觀的檢測技術也被視為該領域當中主要探討的議題。
本研究以先前對於掃描式表面電漿共振顯微鏡的研究基礎,將此技術全新的應用在微奈米級的蛋白質陣列檢測上,以作為高解析、非標記且於低濃度下有高靈敏度的光學檢測技術之一,嘗試幫助解決目前在蛋白質檢測上所面臨到的問題。本論文在探討檢測應用前,先將過去對於掃描式表面電漿共振顯微鏡的系統研究加以延伸,探討到該顯微鏡之影像品質以及檢測品質,以了解該系統之空間解析度以及檢測極限,量測結果顯示目前所量測到最佳橫向解析度為1 μm左右,證實該技術可以解決因表面電漿共振波傳遞所造成過去影像中橫向解析度失真的問題,而表面電漿共振角解析度於低薄膜樣本量測時可至0.0045度左右,換算成可量測的等效折射率變化可至&;#12310;10&;#12311;^(-4)等級,其檢測極限與過去研究結果相符,顯示若將其作為檢測工具,因表面電漿共振技術具有極佳的靈敏度,高解析掃描式表面電漿共振顯微鏡可作為高檢測品質的影像式檢測技術之一。
本論文的另一主軸,即是將掃描式表面電漿共振顯微鏡應用於腫瘤標誌陣列檢測,以大腸癌預後標誌物之胎盤生長因子做為檢測標的,實驗結果顯示該顯微鏡系統搭配金微奈米陣列晶片的檢測相較於傳統平面金膜晶片的檢測,於低抗原濃度100 pg/mL以下有高出10倍的檢測靈敏度結果,並與陰性對照組比較後其檢測極限可至10 pg/mL,其檢測線性範圍包含胎盤生長因子作為預後評估的濃度閥值20.6 pg/mL,因此將掃描式表面電漿共振顯微鏡應用於微奈米級的胎盤生長因子陣列檢測,可以以金微奈米陣列晶片上修飾少量的檢測試劑的優點達到降低檢測成本外,搭配掃描式表面電漿共振顯微鏡可以於低濃度下,透過金微奈米結構將檢測訊號放大,達到較高的檢測靈敏度和檢測極限的效果,未來期望透過陣列多功檢測的優點,進行多種的大腸癌腫瘤標誌物陣列檢測,以多種腫瘤標誌物之濃度指標針對大腸癌患者進行適當的預後評估,提高大腸癌患者的生存率。


With the changes of living style and diet habit, the occurrence of colorectal cancer (CRC) has steadily increased in recent years. More than half of new CRC patients are initially diagnosed and received treatments beyond stage II or even III and thus have lower 5-years relative survival percentage due to metastasis or relapse. Thus, it is critical to develop a better tool to assist the clinician for appropriate prognosis of recurrence or prediction of survival rate in different cancer stage or in different tumor site or even to deliver a proper personalized treatment. Multiplex detection of prognostic cancer biomarkers has been an important trend in translational medicine and the grand challenge is to achieve higher detection sensitivity at lower sample volume.
In this thesis, our study was based on the foregoing researches of scanning surface plasmon resonance microscopy (sSPRM) and applied this technique to novel detection of submicron scale protein array. It is a label-free optical sensing method of high resolution and sensitivity. Based on the foregoing system researches on scanning surface plasmon resonance microscopy, we firstly characterized the quality of imaging system with fabricated nano-structure. According to measurement results, the lateral resolution of system was 1 μm and the resolution of surface plasmon resonance angle was 0.0045 degrees, which is equal to &;#12310;10&;#12311;^(-4) of effective refractive index changes on thin film measurement. It revealed that this sSPRM can serve as a high sensitivity image-type sensing technique.
At the second part of this thesis, we applied this sSPRM system to cancer biomarker array detection. We used the placental growth factor (PlGF), one of colorectal cancer prognostic biomarkers, as the sensing target. According to the experimental results, compared to the conventional SPR senor chip (gold film), the sensitivity of detection improved 10 folds at low sample concentration (below the 100 pg/mL) by gold array senor chip in submicron scale. The limit of detection was down to the 10 pg/mL in reference to the negative control and the linear range of detection included the prognostic threshold 20.6 pg/mL of placental growth factor. In conclusion, the using of test cost of the sensor array was lower than ELISA method and our detection method can enhance the sensitivity and the detection limit without additional fluorescent label by Au array imaging of SPRM. In the future, we expect that the advantage of sensor array, detecting many prognostic biomarkers by SPRM system, can provide an appropriate prognosis and increase the survival rate of colorectal cancer.


第一章 緒論 1
1.1 大腸癌預後診斷 1
1.2 大腸癌腫瘤標誌物 2
1.3 奈米陣列檢測技術 4
1.3.1. 奈米陣列晶片 4
1.3.2. 高解析影像式檢測技術 5
1.3.2.1. 原子力顯微鏡應用於奈米陣列檢測 6
1.3.2.2. 全反射式螢光顯微鏡應用於奈米陣列檢測 7
1.4 高解析掃描式表面電漿共振顯微鏡 8
1.4.1. 表面電漿共振 8
1.4.2. 表面電漿共振影像 11
1.4.3. 掃描式表面電漿共振顯微鏡 13
1.5 本研究目的與論文架構 17
第二章 掃描式表面電漿共振顯微鏡系統架設 18
2.1 光路架設 18
2.2 造影方法 19
2.3 軟體撰寫 21
2.4 晶片製程 23
第三章 奈米點陣列之表面電漿共振影像 25
3.1 沾筆式奈米微影蝕刻製程 25
3.2 掃描式表面電漿共振顯微鏡之橫向解析度量測 26
3.3 奈米陣列之表面電漿共振影像與製程限制 28
第四章 掃描式表面電漿共振顯微鏡之共振角解析度量測 31
4.1 表面電漿共振角解析度量測 31
4.2 掃描式表面電漿共振顯微鏡之薄膜厚度量測 34
第五章 大腸癌腫瘤標誌陣列晶片檢測 37
5.1 胎盤生長因子之酵素免疫分析 37
5.2 微米球微影製程 40
5.3 掃描式表面電漿共振顯微鏡應用於胎盤生長因子檢測 44
5.3.1. 金微奈米陣列晶片之蛋白質固定化方法 44
5.3.2. 金微奈米陣列晶片之表面電漿共振影像分析方法 46
5.3.3. 蛋白質表面固定化於金微奈米結構之定性量測 48
5.3.4. 蛋白質表面固定化於平面金膜之定性量測 49
5.3.5. 掃描式表面電漿共振顯微鏡應用於胎盤生長因子之定量檢測 50
第六章 結論與未來發展 56
6.1 結論 56
6.2 未來發展 58
參考文獻 59


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