- 學位論文
金/氧化鋅奈米薄膜之表面電漿共振生物感測器於疾病分子診斷與環境檢測之應用
A Gold/Zinc Oxide Thin Film-Based Surface Plasmon Resonance Biosensor for Disease and Environmental Applications
摘要
本論文主要利用金/氧化鋅奈米薄膜之表面電漿共振(surface plasmon resonance, SPR)晶片發展高選擇性與高靈敏度腫瘤標記分子(carbohydrate antigen 15-3, CA15-3)、汞離子(Hg2+)、銀離子(Ag+)、半胱氨酸(cysteine)與丙型干擾素( interferon-gamma, INF-γ)之光學感測器。首先，利用透明導電金屬氧化物-氧化鋅(zinc oxide, ZnO)作為中介層以改善表面電漿共振特性，並以乳癌腫瘤標記分子- CA15-3為標的進行實驗之量測。其生物檢測結果顯示，金/氧化鋅晶片的強度變化至少為傳統結構(金/鉻)晶片的兩倍，此外，當濃度低至於檢測極限時，金/氧化鋅晶片比一般傳統表面電漿共振晶片可將檢測極限再增進四倍，因此可利用此種超靈敏之晶片結構應用於其他分子檢驗或環境醫學之應用上。在環境檢測方面，利用Hg2+與核酸鹼基(thymine, T)產生T- Hg2+-T之配位結構原理，可將髮夾（hairpin）形式之核酸探針打開，並藉由DNA標記之奈米金粒子(DNA conjugated nanoparticle)與探針進行雜交反應進而放大反應之訊號。藉由此項技術，可檢測至1nM的Hg2+。另外，亦利用核酸探針進行銀離子與半胱氨酸之光學感測技術。其原理為Ag+誘導富含胞嘧啶鹼基(cytosine, C)之單股直鏈寡核苷酸藉由C-Ag+-C 之特殊配位結構產生構型變化；然而，當半胱氨酸存在時會與C-C核酸配對錯誤(C-C mismatch)之位置競爭結合銀離子，因此抑制了此特殊配位結構(C-Ag+-C)形成，而直鏈寡核苷酸探針亦不會產生摺疊變化。此外，核酸嵌入劑-daunorubicin則被用來增加檢測的靈敏度，基於此檢測原理，Ag+和cysteine的檢測極限將可分別達到10 nM和50 nM。最後，使用核酸適合體(DNA aptamer)在金/氧化鋅晶片上進行丙型干擾素量測。將丙型干擾素與卵白素（streptavidin）之適合體合併形成一髮夾型之適合體核酸探針。加入丙型干擾素分子可使適合體探針的結構重新排列，因此核酸探針之卵白素適合體將會形成可被其蛋白質辨識之二級結構；若無丙型干擾素的存在，因需辨認之卵白素二級結構被限制住，卵白素分子將無法辨識此一適合體探針，所以卵白素分子具有加強丙型干擾素感測反應之功能。經過最佳感測參數測試，檢測極限可達33pM線性範圍將至300 nM。此核酸適合體感測器具有高靈敏度、簡單、可重複性以及不須外加螢光標記之優點。利用金/氧化鋅奈米薄膜之表面電漿共振檢測平台結合不同之辨識分子(抗體、核酸、適合體)，已成功應用於生物醫學分析和重金屬汙染之環境檢測，因此此一檢測晶片將可作為日後發展生物感測領域之重要平台。
並列摘要
The purpose of this dissertation is to develop highly sensitive and selective assays for carbohydrate antigen 15-3 (CA15-3), mercury ion (Hg2+), silver ion (Ag+), cysteine (Cys) and interferon-gamma (INF-γ) using gold/zinc oxide (Au/ZnO) thin film-based surface plasmon resonance (SPR) sensors. First, Au/ZnO nanocomposite films were effectively employed to enhance the performance of SPR for the detection of CA15-3. Compared with the degree of the shift in SPR intensity induced by the specific binding event between antibody and antigen, the change of intensity on the Au/ZnO layers was increased by at least 2 fold over that on the gold/chromium (Au/Cr) layers. In addition, the Au/ZnO layers allowed for a detection limit 4 times lower than the Au/Cr layers, which are in widespread use as the sensing interfaces in current SPR-based detectors. Next, the “turn-on” reaction of a hairpin probe via coordination of Hg2+ by the thymine–thymine base pair results in a substantial increase in the SPR response, followed by specific hybridization with a gold nanoparticle probe to amplify the sensor performance. Meanwhile, the limit of detection is 1nM, which is lower than other recently developed techniques. An SPR sensor was then developed for the detection of Ag+ and Cys in aqueous solutions. This assay was based on the Ag+-induced conformational change of a cytosine-rich, single-stranded DNA. In the free state, single-stranded oligonucleotides fold into double-helical structures through the addition of Ag+ to cytosine-cytosine (C-C) mismatches. However, in the presence of Cys, which competitively binds to Ag+, the formation of the C-Ag+-C assembly is inhibited, resulting in free-state, single-stranded oligonucleotides. To enhance sensitivity, the DNA intercalator, daunorubicin, was employed to achieve signal enhancement. The detection limit for Ag+ was 10 nM with a measurement range of 50–2,000 nM, and the detection limit for Cys was 50 nM with a measurement range of 50–2,000 nM. Finally, the DNA aptamer with the hairpin structure was developed for the detection of IFN-γ with high sensitivity and selectivity. The streptavidin DNA aptamer was incorporated into the aptamer prober for the amplified detection of target molecules. Initially, the probe remains essentially in the inactive configuration. Addition of IFN-γ induced rearrangement of aptamer structure, allowing the self-assembly of the active streptavidin aptamer conformation for streptavidin molecular recognition. Under optimized conditions, the detection limit was determined as 33 pM and a dynamic range was up to 333 nM, which are even lower than those of the corresponding optical sensors. Since combined aptamers are composed of nucleic acids, this optical aptasensor provide advantages of high sensitivity, simplicity, reusability, and no further label or sample pretreatment. Given the disease and environmental applications, such biosensors have great potential to provide the targeted information in a direct, rapid, and simple manner. Thus, a gold/zinc oxide thin film surface plasmon resonance–based sensor will be an useful system for sensing applications.
參考文獻
延伸閱讀
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