建構高敏感免標定波導共振核酸適合體式生物感測器並於生物分析應用之研究

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林聖富(Sheng-fu Lin) 查詢紙本館藏

畢業系所

光電科學與工程學系

論文名稱

建構高敏感免標定波導共振核酸適合體式生物感測器並於生物分析應用之研究
(Development of high sensitivity label-free guided mode resonance aptasensor and its bioanalytical application)

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摘要(中)

本研究主要在於發展一低成本、免標定與高敏感度的波導共振生物感測器。波導共振元件是一種光學的濾波器，它藉由其特殊的次波長波導光柵結構來反射特定的光波。當波導共振元件的邊界條件被改變的時候，如，生物分子附著於結構表面，結果會造成其共振波長的偏移。本研究藉由此特性將波導共振元件做為一個體外的生物感測器，用以檢測生物分子。本文內容包括：此共振元件的敏感度與結構關係之理論探討、一個高敏感度的型變波導共振結構（金屬輔助波導共振）與建立其相對應的檢測平台，並實際展示此系統於生物分子檢測應用的效能。在實驗部分，我們發展出一套適用於製作低成本高均勻性波導共振元件的奈米壓印技術與一個對凝血酶具有專一性結合能力的核酸適合體式波導共振晶片並具有抗汙的能力。最後，本文亦建立適用於金屬輔助波導共振元件的波長解析檢測平台與適用於一般波導共振的光強解析的檢測平台。
於敏感度理論分析中，我們提出一個簡化的波導模型來預測表面淺蝕刻式波導共振生物感測器的敏感度。這個簡化的模型相對於現有常見的模擬方法，如：嚴格耦合波理論或是有限時域差分法來說，是個便利且快速的計算方法，並且可以更加深入探討共振機制。所以本研究藉由此提出的模型，有系統的分析與探討波導共振元件之結構與其對應敏感度關係。其中，我們亦將此模型之預測結果與嚴格耦合波理論的計算結果相互比較，我們發現，此簡化模型所提出的預測結果與嚴格耦合波的結果具有高度的吻合性。
在高敏感型變波導共振的單元中，為了使波導共振感測器更能接近一般生醫檢測應用的範疇，我們提出高敏感度金屬輔助波導共振的結構，根據實驗結果，其敏感度相較於傳統波導共振結構提升一倍。其中，金屬輔助波導共振元件的反射光譜展現出一個特殊的反轉型光譜，這個共振的機制亦在本單元討論。另一方面，由數值模擬結果可知，此元件之高敏感度來自於其強烈的非對稱共振場分布型態與波導內的低傳播角共振。數值計算結果顯示，在藉由此方法，晶片表面的消逝波強度被增強了一倍，進而達到提升感測器之靈敏度。此外，本研究中亦開發奈米壓印方法並應用於晶片製造，用以達到低成本的高均勻性晶片。經模擬與實驗證實，壓印型金屬波導共振元件可再次明增加感測器之靈敏度。
接著，本研究分別建立適用於金屬輔助波導共振與波導共振之檢測平台，其分別屬於光譜解析與光強解析的架構。在光譜解析的設置中，我們使用低輸出（éntendue）設置法，此法大幅減小建構系統與實驗量測的困難度。此外，此設置法也減少因為基板所造成的干涉條紋。在另一方面，在強度解析的設置中，透過調制通過波導共振元件的偏振光，將特徵橢圓偏振轉換成線性偏振，接著使用檢偏器遮蔽，最後可得到近乎全暗的背景訊號，使任何的折射率變化皆會造成光強增加，而達到檢測的目的。
最後呈現的是一個以核酸適合體為固定化分子的免標定、即時檢測之金屬輔助波導共振生物感測器。實驗中，兩條不同序列的抗凝血酶核酸適合體被固定於金屬輔助波導共振晶片的表面，用以作為生物辨識分子而進行凝血酶之檢測。其中此檢測器亦通過非專一性的抗污測試。於凝血酶之檢測中，本感測器使用15與29鹼基抗凝血酶核酸適合體檢測凝血酶分別可達到0.55與0.44 nm/μM的靈敏度，其檢測極限分別為190與230 nM。

摘要(英)

The guided mode resonance (GMR) device is developed to be a low-cost, label-free and high sensitivity biosensor. Typically, the GMR is an optical filter which reflects a specific light wave according to its sub-wavelength waveguide grating structures. As biomolecules attach on GMR surface, it changes the boundary conditions of GMR and results in a shift of characteristic spectrum. This presented work includes the theoretical study of GMR sensitivity for biosensing, a mutated high sensitive GMR structure (metal-assisted guided mode resonance, MaGMR), constructions of the related sensing platform and in vitro bioanalytical application. In experimental part, a nano-imprint method is developed for low-cost and high uniformity chip processes. A surface modification for antifouling and aptamer immobilized chip which specific binds to thrombin is also demonstrated. Finally, spectrum-resolved and intensity-resolved sensor platforms based on MaGMR and GMR are individually demonstrated.
In theoretical study of the GMR sensitivity, a simplified waveguide model for predicting the sensitivity of surface-relief GMR biosensors is presented. The proposed model is a convenient and rapid calculation method compared to those current optical simulation tools such as the rigorous couple wave analysis (RCWA) or finite-difference time domain (FDTD). Thus, by using this proposed model, a systematic and theoretical discussion on the GMR geometric structure and its sensitivity is also presented. Calculations of sensitivity between the proposed model and the RCWA method show a good agreement.
For the high sensitive mutation type of GMR section, a novel metal layer assisted guide mode resonance (MaGMR) device is proposed for bioanalytical applications and its performances are experimentally proved. Compare to the typical GMR, the sensitivity is one fold enhanced. We find the reflection spectra show a unique inversed response and the resonance mechanism is discussed as well. Numerical calculation results indicate that the high sensitivity performance of MaGMR comes from the strongly asymmetric resonance modal profile and low propagation angle inside the waveguide. There is a one-fold enhancement of the evanescent wave in the analytes region compared to typical GMR. In addition, a nano-imprint method for high uniformity and low cost MaGMR chip is developed. Furthermore, the surface to bulk sensitivity is also enhanced through the nano-imprint method.
In construction of sensing platforms, two sensing systems for MaGMR and GMR are individually demonstrated. One is a spectrum-resolved method for MaGMR sensors, and the other is a polarization-control setup for intensity-resolved GMR sensors. The spectrum-resolved system is performed under a low éntendue design which makes the measurement much easier and totally reduces the spectrum fringe noises from substrate. On the other hand, the intensity-resolved setup transfers polarization ellipses into linear polarization state and then suppresses the signals to a dark signal by an analyzer. Hence, any changes in the refractive index results in an increase in the intensity signals.
Finally, an aptamer based MaGMR sensor (aptasensor) for label-free and real-time detection is demonstrated. Two strains of thrombin binding aptamer (TBA) are immobilized on the surface of MaGMR chips as a recognizing ligand for thrombin. The MaGMR aptasensors also passed the nonspecific antifouling tests. The sensitivity of 15 and 29-mer TBA MaGMR aptasensor is 0.55 and 0.44 nm/μM and the limit of detection (LOD) is achieved 190 and 230 nM, respectively.

關鍵字(中)

★ 波導共振
★ 生物感測器
★ 金屬
★ 核酸適合體

關鍵字(英)

★ Guided mode resonance
★ Biosensor
★ Metal
★ Aptamer

論文目次

中文摘要 I
ABSTRACT III
ACKNOWLEDGEMENT V
CONTENTS VI
LIST OF FIGURES VIII
LIST OF TABLES XIII
I. INTRODUCTION 1
I- 1 FUNDAMENTALS OF GUIDED MODE RESONANCE 2
I- 2 GMR BASED SENSORS 2
I- 3 MOTIVATION AND ARRANGEMENT OF THESIS 5
II. THEORY AND NUMERICAL METHOD 7
II- 1 GUIDED MODE RESONANCE 7
II- 2 RIGOROUS COUPLED-WAVE ANALYSIS 16
II- 3 FINITE-DIFFERENCE TIME-DOMAIN 18
III. SIMPLIFIED MODEL FOR CALCULATING GMR PHENOMENON 22
III- 1 THE CONCEPT 22
III- 2 GMR ON A SUBSTRATE 24
III- 2.a The resonance, sensitivity and geometric conditions 24
III- 2.b Compare to the RCWA 28
III- 3 IMPROVEMENT OF THE SENSITIVITY- SUSPENDING GMR 30
IV. METAL-ASSISTED GUIDED MODE RESONANCE 32
IV- 1 THE CONCEPT 33
IV- 2 PHYSICS OF THE MAGMR 34
IV- 2.a Resonance mechanism 34
IV- 2.b Sensitivity 42
IV- 2.c Experimental results 43
IV- 3 NANO-IMPRINT TYPE MAGMR 45
IV- 3.a Optimization 45
IV- 3.b Chip processes 52
IV- 3.c Experimental results 54
V. POLARIZATION CONTROLLED GMR SYSTEM 57
V- 1 THE CONCEPT 57
V- 2 EXPERIMENTAL RESULTS 60
VI. DEMONSTRATION ON BIOANALYTICAL APPLICATION- APTASENSOR 63
VI- 1 THE MULTI-CHANNEL DETECTION SYSTEM 63
VI- 2 MAGMR APTASENSOR FOR THROMBIN DETECTION 68
VI- 2.a Method 69
VI- 2.b Experimental results 70
VII. CONCLUSIONS 75
VIII. REFERENCES 77
IX. OPTICAL CONSTANTS 86
X. PUBLICATION LIST 87

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指導教授

張正陽(Jenq-yang Chang)

審核日期

2013-11-5

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