以富含鳥嘌呤核酸適體之矽奈米線場效應電晶體為基礎之鉀離子感應器

Abstract

隨著奈米技術蓬勃發展，矽奈米線場效應電晶體因為具有微量化、高靈敏性、即時回應等優點而受到重視，將矽奈米線場效應電晶體結合特殊生物感測分子形成的生化感測器，對於生化領域也能夠有更深一步的研究。 金屬離子在生理分析中是個很重要的議題，尤其是鉀離子，其在生物系統裡一直扮演著重要的角色，像是神經傳導、或者是血壓的調控等。本篇論文成功的結合矽奈米線場效應電晶體以及富含鳥嘌呤核酸適體，建立一套鉀離子偵測的平台，同時亦透過螢光影像、或電性量測來驗證核酸適體修飾是否成功。當核酸適體與鉀離子作用時，會形成一鳥嘌呤四鏈體結構和小段的雙股脫氧核醣核酸片段，此時矽奈米線場效應電晶體表面電位的變化，造成相對電流值之改變，此電流變化程度即顯示核酸適體與鉀離子間的作用關係。本研究中，實驗結果顯示此偵測系統對於鹼金屬離子的親和力為: (K+ ＞ Rb+ ＞ Na+ ＞ Cs+ ＞ Li+) ，而對鉀離子的偵測下限可以達到10-6 M。此外我們也經由Langmuir adsorption Isotherm計算出此核酸適體與鉀離子的結合常數Ka = (1.0 ± 0.2) × 105 M-1。 我們亦利用此奈米元件，針對腎上腺髓質細胞上受乙醯膽鹼受體調控的離子通道，作一系列的研究探討。當尼古丁刺激該細胞膜上乙醯膽鹼受體調控的離子通道而釋放出鉀離子時，修飾於場效應電晶體表面的核酸適體便能與其作用，而導致元件電流值上升。此外，利用六甲溴銨來調控乙醯膽鹼受體調控的離子通道活性的實驗中發現。當六甲溴銨 (4 mM) 抑制乙醯膽鹼受體調控的離子通道時，腎上腺髓質細胞所釋放的鉀離子濃度只剩原來未抑制的 80%。同時我們也將其應用在血清環境下鉀離子的偵測，在含高鈉鹽 (1.35~1.45 mM) 環境下，我們成功的偵測出鉀離子濃度的變化，其線性操作區間為50 μM~1 mM。

Nanotechnology has been developed vigorously over the past few years. In particular, silicon nanowire field effect transistor (SiNW-FET) has been noticed due to its advantages of size minimization, ultra-sensitivity, and real time response. In this study, a high sensitive SiNW-FET has been integrated with G-rich aptamers for metal ions detection. The detection of metal ions is an important issue for physiological diagnosis, especially K+ analysis. K+ plays an important role in several biological systems, like nerve transmission and the regulation of blood pressure. Here, a K+ detection platform has been successfully demonstrated by a G-rich aptamer modified on the SiNW-FET. the efficiency of G-rich aptamer immobilization has been also demonstrated by fluorescence image and electrical measurement. The conformation of G-rich aptamer changes from random-coil to G-quadruplex and short double-helix stems when binding with K+. The stability of aptamer/metal ion complex was weasured in the following orders K+ ＞ Rb+ ＞ Na+ ＞ Cs+ ＞ Li+, and the association constant of aptamer/K+ was determined to be 105 M-1 in 1 mM Tris buffer at pH 7.4. The detection limit for K+ approached to micromolar level. Furthermore, in order to study the K+ fluxion of nicotine type acetylcholine receptor (nAChR) in neuron cells, chromaffin cells were firstly seeded on the glass and then to place on a G-rich aptamer modified SiNW-FET. Here, nicotine was used as an agonist to activate nAChR. The interaction of nAChR and nicotine controls the net flow of positively-charged ions. The measured conductance change is proportional to the concentration of nicotine added. On the other hand, hexamethonium bromide was used to block the activity of nAChR. The conductance change of treatment with or without hexamethonium bromide, shows a 20 % difference, indicating that K+ efflux could be reduced by hexamethonium bromide. We also detected the K+ flux in serum. Under high Na+ concentration (1.35~1.45 mM) , we successfully detected the different K+ concentration in the linear range of 50 μM ~1 mM.