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研究生:魏世忠
研究生(外文):Shih-Chung Wei
論文名稱:區域性激發表面電漿子於金屬螢光增強奈米生物感測器發展之理論驗證及實現
論文名稱(外文):Implementation and verification of the nanobiosensor based on spatially confined surface plasmon and metal enhanced fluorescence
指導教授:林啟萬林啟萬引用關係宋孔彬
口試委員:周家復魏培坤Frank Gu
口試日期:2015-07-24
學位類別:博士
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:2015
畢業學年度:103
語文別:英文
論文頁數:133
中文關鍵詞:針尖增強螢光表面電漿顯微鏡軸向偏振奈米陣列新式圈環型恆溫複製丙型肝炎數值化計數
外文關鍵詞:tip-enhanced fluorescencesurface plasmon microscopynanoarrayloop-mediated isothermal amplificationHepatitis C virusdigitized counting
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在本論文中,我們利用軸向極化光在奈米結構上產生一強而高侷限性的光場,並用之以增強生物感測時之螢光。在奈米針尖增強螢光中,藉由50毫瓦的雷射強度,平均可以將螢光異硫氰酸鹽的光強度提升五倍左右。本研究將此針尖螢光增強技術用於監測丙型肝炎逆轉錄互補核酸的圈環式恆溫複製。針對三種不同模板濃的其結果顯示,反應的偵測時間提早約10-30分鐘。為了進一步提升螢光檢測專一性及多樣本檢測能力,並減少交互污染的可能性,我們以螢光共振轉移的原理,針對乙型和丙型肝炎設計了新型態的圈環式核酸恆溫複製反應。透過此設計,10-3 和 10-4 微克每25微升的核酸模板可以在四十分鐘內透過螢光共振轉一圈環式恆溫複製進行檢測;而一般的圈環式恆溫複製反應則須50到60分鐘的時間檢出10-3 微克每25 微升的核酸模板. 我們更進一步將此設計用於奈米陣列的大量平行化實驗中,並進行數值化的反應點數目計數。透過數值化計數,約可以比使用強度紀錄的螢光共振轉移圈環式恆溫複製增快20分鐘。此外,為了發展非標定式的檢測,我們利用軸向極化光建構出掃描式表面電漿顯微鏡,並將之應用在金模表面疊加分子的折射率變化造影。在本文中,我們針對掃描式表面電漿顯微鏡進行折射率解析以及空間檢析調查後,將之應用在金膜表面修飾丙型肝炎核酸引子、1,2-Dioleoyl-sn-glycero-3-phosphocholine、石墨烯及胎盤生長因子的奈米造影感測器上。

In this manuscript, we used radially-polarized illumination to produce a strong and well-confined optical field at nanostructure to enhance fluorescence signal for biosensing. With the illumination intensity of 50 mW, fluorescein isothiocyanate emission was increased 5 times on average by tip-enhanced fluorescence (TEF). Moreover, we monitored the loop-mediated isothermal amplification (LAMP) on Hepatitis C virus cDNA (complementary DNA) by TEF (tip-LAMP). The increment of amplicon-SYBR at three different template concentrations was found 10-30 minutes earlier than general LAMP. For further increasing of the detection specificity, multiplex detection capability and preventing cross contamination, we have designed a fluorescence resonance energy transfer based loop-mediated isothermal amplification (FRET-LAMP) for Hepatitis C and B virus. The template of 10-3 and 10-4 g per 25l was easily detected in forty minutes by FRET-LAMP, while the reaction performed with SYBR green needs fifty to sixty minutes for 10-3 g per 25 l template. Furthermore, from single point to massive parallel reaction, FRET-LAMP at nanoarray was performed (Digital-LAMP). Digitizing counting was used to monitor the reaction. About 20 min earlier detection time was shown in FRET-LAMP nanoarray comparing to real-time LAMP. In addition, the confined optical field with radially-polarized illumination was also adopted to scanning surface plasmon microscopy (SSPM) to image the refractive index change of stacking layer on Au film. The characteristics of SSPM, lateral resolution and refractive index sensitivity, was inspected. SSPM were then applied to image Hepatitis C virus primer labeled nanoarray, 1, 2-Dioleoyl-sn-glycero-3-phosphocholine array, deposited graphene, placenta growth factor nanobiosensor.

誌謝 I
中文摘要 III
ABSTRACT IV
TABLE OF CONTENTS VI
TABLE OF FIGURE VIII
CHAPTER I. INTRODUCTION 1
A. MOTIVATION 2
B. PURPOSE OF STUDY 2
C. RESEARCH FRAMEWORK 5
CHAPTER II. LITERATURE REVIEW 10
A. NANOBIOSENSOR 11
B. METAL ENHANCED FLUORESCENCE 16
C. SURFACE PLASMON MICROSCOPY 22
D. LOOP-MEDIATED ISOTHERMAL AMPLIFICATION 29
CHAPTER III. MATERIALS AND METHODS 34
A. CHEMICAL AND BIOLOGICAL MATERIALS 35
1. Chemicals for Nanofabrication 35
2. Materials for Biological Experiment 37
B. FABRICATION OF NANOARRAY FOR LAMP AND NANOSTRUCTURE FOR SSPM 39
1. Nanoarray for LAMP 39
2. Nanostructure for SSPM 39
a. Alternative Thin film thickness for SSPM refractive index sensitivity 40
b. Nano-disc array for SSPM 40
c. Nanoarray by Microsphere lithography for SPR nanobiosensor 40
C. OPTICAL EQUIPMENT FOR LOCALLY EXCITING FLUORESCENCE AND SURFACE PLASMON 43
1. Two photon microscope 43
2. Scanning surface plasmon microscope 45
D. SURFACE PLASMON RESONANCE SIMULATION AND MAXWELL-GARNETT THEORY 48
E. LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) 52
F. LAMP AT TIP AND NANOARRAY 53
CHAPTER IV. RESULTS AND DISCUSSION 59
A. LAMP AND FRET-LAMP FOR HEPATITIS B AND C VIRUS 60
B. LOCALLY EXCITED FLUORESCENCE 66
1. Metallic Tip Enhanced Fluorescence (TEF) 66
2. Excitation Power and Excitation Polarization Effect on TEF 71
3. Single Spot LAMP at Metallic Tip 78
4. Parallel FRET-LAMP at Gold Disc Nanoarray 85
a. FRET-LAMP at Nanoarray 85
C. LOCALLY EXCITED SURFACE PLASMON 89
1. Scanning Surface Plasmon Microscopy 89
a. Effective RI sensitivity 89
b. Lateral spatial resolution of SSPM 90
2. SSRM for Non-labeled Sensing and Imaging 94
a. Quality inspection of nanostructure on SPR Chip 94
b. Mapping oligonucleotide functionalized nanoarray 94
c. Other SSPM application 96
CHAPTER V. CONCLUSIONS AND FUTURE DIRECTIONS 102
CHAPTER VI. REFERENCE 108
CHAPTER VII. APPENDIX 127
PUBLICATION LISTS AND PUBLISHED JOURNAL PAPER 128


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