發展以官能化氧化鐵磁性奈米粒子為基材之磷酸化生物分子之偵測方法

Abstract

氧化鐵磁性奈米粒子具有超順磁性、表面積大和表面易改質等優點所以在分析化學上有很廣泛的應用，是一種極具潛力的生物感測器材料。本論文的兩個主題主要是以表面修飾有螢光染料分子的官能化氧化鐵磁性奈米粒子為基材發展出可快速感測磷酸化生物分子的方法。 本論文的第一個主題是以官能化磁性奈米粒子做為DNA萃取探針和偵測探針，首先使用DNA萃取探針進行目標DNA之濃縮萃取並利用氧化鐵磁性奈米粒子的超順磁特性將目標DNA透過外加磁鐵分離出目標DNA；而再進一步利用升溫方法將目標DNA重新回溶至溶液中並與經由螢光染料核黃素磷酸鈉修飾的氧化鋁磁性奈米粒子偵測探針作用，透過目標DNA結構上5端的磷酸根與偵測探針表面的核黃素磷酸鈉螢光分子發生競爭取代作用，造成核黃素磷酸鈉解離至溶液中，根據溶液中的黃綠色之螢光放光，達到偵測目標DNA的目的。整個分析流程可在數十分中內結束，偵測極限約為40 pM左右，並且可以用於辨識與目標DNA僅單一鹼基突變的DNA序列。 本論文的第二個主題則以相同染料分子修飾的官能化氧化鐵磁性奈米粒子為磷酸腺苷小分子的探針，可用於間接定量溶液中磷酸化小分子，即透過目標物上的磷酸根可與偵測探針上的核黃素磷酸鈉發生競爭取代反應，取代出核黃素磷酸鈉至溶液中並產生黃綠色螢光放光，可達到定量偵測目標物之目的，此偵測方法針對ATP及ADP標準溶液的偵測極限分別為70 nM及500 nM。實驗中也已證實此方法對複雜樣品如細胞溶解物中的磷酸化小分子進行定量，且在複雜樣品中ATP的偵測極限為500 nM。此外，實驗中也證明此偵測方法可成功應用於觀察活細胞樣品中磷酸根分子存在與分佈。

Iron oxide magnetic nanoparticles (Fe3O4 MNPs) have been widely used in the field of analytical chemistry because of the features including superparamagnetic property, high surface-to-volume ratio, and ease of surface modification. These unique properties also lead Fe3O4 MNPs to be as suitable materials for being employed in biosensing researches. In this thesis, functional Fe3O4 MNPs were explored as sensing materials for phosphorylated biomolecules. In the first part of this thesis, two types of functional Fe3O4 MNPs were designed as trapping probes and detection probes for sensing DNA. The trapping probe immobilized with DNA was used to concentrate their complementary DNA from sample solutions. Owing to the magnetic property, the probe-target DNA conjugates can be readily isolated from the sample solution by employing an external magnetic field. The DNA released from the conjugates by temperature elevation then interacted with the detection probes, which were core/shell Fe3O4@Al2O3 MNPs immobilized with fluorescent dye, i.e. riboflavin 5’-monophosphate (RFMP- Fe3O4@Al2O3) through Al-phosphate chelating. RFMP molecules on the detection probes were then replaced by the DNA and released to the solution. As a result, the solution had enhanced fluorescence as the concentration of the DNA in the sample solution increased. The results also demonstrated that this sensing method can be used to distinguish single-base mismatched DNA. The analysis time is as short as 1 h. The detection limit is ~40 pM. In the second part of this thesis, functional RFMP-Fe3O4@Al2O3 MNPs were used as concentration and detection probes for nucleoside polyphosphates. When using the probes to interact with target species from a sample solution, the level of nucleoside polyphosphates in the sample solution can be estimated based on the fluorescence intensity derived from the RFMP molecules released from the probes. Cell lysate and living cells have been used as model samples. The detection limits for ATP and ADP are 70 and 500 nM, respectively.