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

Abstract

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.