利用B型抗諧振反射光波導表面電漿子共振生物感測元件進行α-凝血蛋白酵素對單股DNA適體鍵結與登革熱病毒DNA雜交之即時檢測

Abstract

本論文研究適用於水中環境的B型抗諧振反射光波導表面電漿子共振生化感測元件。由於此生化感測元件具有免標定、高效率以及對於金表面所固定的生物分子層之折射率變化具有高靈敏度等特性，因此能應用在即時感測表面生物分子之交互作用。感測元件的設計以及製作過程在論文中有詳盡的介紹與討論，生物感測實驗部分則根據生物分子不同鍵結特性分為兩方面。（一）進行α-凝血蛋白酵素對單股DNA適體鍵結特性之即時檢測，並且在兩者鍵結之後，通入表面修飾抗α-凝血蛋白酵素抗體的奈米金粒子對α-凝血蛋白酵素進行抗體抗原親合力鍵結以作為SPR訊號放大之用途。此生化感測器對α-凝血蛋白酵素之偵測極限可達到1 pM，與商品化的Biacore 3000之偵測極限 (1 pM)相同，但是本研究所建構之量測系統成本較低。最後，由實驗結果驗證了此生化感測元件能定性且定量地即時檢測DNA適體與α-凝血蛋白酵素分子間具高度專一性的鍵結反應。（二）進行對於登革熱病毒單股DNA雜交反應之即時檢測，登革熱單股DNA分子DENV2-P利用末端修飾的硫醇基先行固定化於金表面，之後登革熱單股DNA分子DENV2-T再利用互補DNA序列與DENV2-P進行專一性鍵結。最後，由實驗結果顯示此生化感測元件能精確地定性且即時檢測此雜交反應。

In this thesis, an antiresonant reflecting optical waveguide of type B (ARROW-B) surface plasmon resonance (SPR) biosensor operating in the aqueous environment has been investigated. The ARROW-B SPR biosensor is proposed to provide a label-free, high-throughput and highly surface-sensitive platform to detect the bimolecular interactions in real time. The design and fabrication process of the ARROW-B SPR sensor chips are described and discussed. Besides, the primary analytes for the bioassay experiments are divided into two categories based on different binding characteristics. First, the real-time detection of α-thrombin binding to ssDNA aptamers was under in-depth investigation. The gold nanoparticles modified with anti-thrombin antibodies were employed to bind to the α-thrombins for signal amplification. The detection limit of this biosensor to α-thrombin was measured at 1 pM level, which was comparable to that of the Biacore 3000 system but at much lower cost. Second, the real-time detection of dengue virus ssDNA hybridization was studied. The dengue virus DNA probe was modified with a thiol group at one end to achieve effective immobilization on the Au surface, while the DNA target utilized the complementary sequence to bind to the immobilized probe. In summary, the measurement results have shown that the ARROW-B SPR biosensors can be applied to detect the ssDNA aptamer/α-thrombin interaction and dengue virus ssDNA hybridization both quantitatively and qualitatively in real time.