混合型自主單層膜修飾之石英晶體微量天平DNA感測器製備與應用於小分子交互作用之研究

Development of a Quartz Crystal Microbalance DNA Sensor Based on Mixed Self-Assembled Monolayer and Application for Small-Molecule Interaction Study

葉政穎(Cheng-Ying Yeh) ； 羅敏瑞(Min-Ruei Luo) ； 蔡文琦(Wen-Chi Tsai)

《臺灣農業化學與食品科學》 51卷1期 (2013/02) Pp. 43-49

https://doi.org/10.6578/TJACFS.2013.006

摘要

本研究的目標為製備石英晶體微量天平DNA感測器並應用於互補股DNA的雜交分析以及以DNA為作用標的的小分子交互作用之研究。採用硫醇修飾的探針與短碳鏈硫醇（6-mercapto-1-hexanol；6-MCH）所形成的混合式自組單層膜修飾石英晶體金表面以提高感測器的敏感度。結果顯示，在高離子強度的溶液中，有利於探針固定化的進行。DNA探針與6-MCH兩者之間的莫耳比例為1：5時，有最佳的雜交反應。此結果可歸因於短碳鏈硫醇的存在有助於探針方向性的排列，並降低感測表面的空間及靜電障礙。與互補DNA雜交試驗的結果顯示，在互補股DNA濃度為0.5－5.0 μg/mL之間呈線性，並以非互補股證實此感測表面的專一性。至於DNA感測表面與小分子交互作用之可行性研究，則以一個已知能與DNA結合的小分子－小蘗鹼為樣品，分析計算其與DNA之間的解離常數。結果顯示兩者間的KD為1.023 × 10^(-3) M（25℃）。此數值顯示在所測試的條件下，小蘗鹼與DNA感測表面之間的結合並不強。

關鍵字

石英晶體微量天平 ； 硫醇修飾的探針 ； DNA雜交 ； DNA/小分子化合物交互作用 ； 小蘗鹼 ； 混合式自主單層膜

並列摘要

In this paper we present a QCM-based sensor for measuring DNA hybridization and DNA/small-molecule interactions. A mixed self-assembled monolayer (SAM) was used to optimize the biosensor sensitivity. The result showed that the DNA probe immobilization was favorable under high ionic strength. In addition, it was observed that the mixed SAM formed by mixing thiolated-DNA probe and 6-mercapto-1-hexanol (6-MCH) at a 1:5 molar ratio showed the best results. This could be ascribed to the oriented assembly of the DNA probe and the decrease of steric and electrostatic hindrances in the mixed SAM. The ssDNA-coated sensor was first evaluated as a nucleic acid biosensor through a DNA-DNA hybridization assay. A linear calibration curve was observed in the range of 0.5~5.0 μg/mL for the complementary strand. Non-complementary DNA was used to verify the specificity of the assay. Berberine was used as a model molecule in the study of DNA/smallmolecule interactions. The dissociation constant between the immobilized DNA and berberine was determined to be 1.023 × 10^(-3) M at 25℃. This data seems to suggest that the binding between berberine and the DNA sensing surface was not strong under the studied situation.

並列關鍵字

Quartz crystal microbalance ； Thiolated probe ； DNA hybridization ； DNA/small-molecule interaction ； Berberine ； Mixed self-assembled monolayer