- 學位論文
以金奈米微粒偵測口腔癌症基因
Oral Cancer Gene Detection Based on Gold Nanoparticles
摘要
奈米科技的進步，對於醫療領域的轉變有很大的影響。目前全球已有許多研究團隊積極將金奈米顆粒應用在各種生醫檢測的發展上面，希望在疾病檢測上能夠更為準確、快速與靈敏。金奈米顆粒的檢測是利用生物探針與待測物的交互作用，例如與特定的基因序列鍵結，產生不同的螢光強度作為偵測結果。 我們本論文是利用金奈米顆粒優異的螢光共振能量轉移特性，製作出一高靈敏度及高專一性之偵測平台。我們將有硫醇修飾與螢光標記的單股去氧核糖核酸(DNA)探針藉由金硫鍵結固定在金奈米顆粒的表面。而藉由DNA序列的設計，探針DNA會形成環狀結構，使得螢光標記端反摺，進而使螢光分子與金奈米顆粒的距離靠近，產生螢光共振能量轉移現象，將螢光分子的能量轉移到金奈米顆粒上，形成消光。當加入完全互補的目標DNA，因與探針DNA雜交，使原本的彎曲結構拉直伸展開來，螢光分子與金奈米顆粒之間之距離增加，螢光分子再度恢復螢光。 本論文以上述之平台發展癌症基因複合型檢測方法，應用於口腔癌指標性致癌因子。我們選擇數種與口腔癌相關的癌症因子作為偵測標的，探討金奈米顆粒表面修飾與靈敏度之關係，以及不同環境條件下此檢測系統的穩定性，並探討目標分析物濃度對於消光效應之成效。研究結果顯示，我們製作出的金奈米顆粒，在高濃度探針的表面修飾下，對於環境介質的穩定度較高，與目標分析物鍵結後，螢光恢復量也較多，當目標分析物濃度高達500pM時，螢光變化有達到飽和的現象。另外，此檢測系統可辨識完全互補序列與單一鹼基不互補之序列。完全互補序列目前可檢測至1pM。 藉由完成上述工作，未來我們將進一步製作複合型的口腔癌相關因子檢測，探討多種癌症因子之間的關係。藉由複合型口腔癌症基因金奈米顆粒檢測系統的建立，期待能提供一個簡易且操作方便的低成本篩檢方式，以提高口腔癌的早期篩出率，提升存活率。
關鍵字
金奈米顆粒；口腔癌基因檢測並列摘要
The development of nanotechnology provides great benefits for the medical field.Currently, many research groups in the world have studiedthe applications of gold nanoparticles in biomedical detectionfor a rapid,simple and sensitive method to monitor diseases. The principle of gold nanoparticlesin biomedical detection usesthe biological interaction between probe DNA and analyte. For example, a specific gene sequence binding to the probe DNAcauses a recovered fluorescence, and one can observe their interactions by measuring the fluorescence intensities. A highly-sensitive and highly-selective genes detection platform based on fluorescence resonance energy transfer(FRET) of gold nanoparticles has been developed. The probe DNAwith a thiol-modified and fluorescence-labeled on each terminals was conjugated to the gold nanoparticlessurfaceusing gold-sulfur binding.Through design of the sequence, the probe DNA formed a stem-loop structure, and hence the fluorescence on DNA terminal was close to the gold nanoparticle.The fluorescence was quenched due to FRET occurred. When a complementary target DNA was added, the target DNA hybridized to the probe DNA, and the fluorescence was then recovered due to the stem-loop structure was released. In this thesis, a multi-functional oral cancer genes detection system using foregoing technique was developed. We chose various oral cancer-related genes as the detecting targets, and examined the dependence between surface modification and sensitivity, as well as stability at different environments. The DNA-concentration related quench efficiency was also studied. Our experiment results demonstrate that more stable gold nanoparticles and greater recovered fluorescence can be obtained when higher concentration probe DNA modified on the surface.The detection limit of target DNA can be down to 1 pM, and saturated as the target DNA is up to 500 pM. In addition, the complementary and one base mismatchtargetDNA can be distinguish using this gold nanoparticles detection system. Through this study, we will fabricate a multiple oral cancer detection system to explore the relationship of cancers factors.This system is expected to provide an simple, easy-operation, and low-cost detection method to improve the early screen-out and cure rate of oral cancer.