石英晶體微天平基因感測系統之開發及其應用

Abstract

本研究以壓電性石英晶體微天平 (QCM) 為基礎，建構基因型生物感測器，用以檢測核酸雜合效率。以氣相 QCM 偵測系統作模組，分別建構兩類修飾核酸作為探針：一類為抓取探針 (capture probe)，利用金與硫氫基間良好的親合性作基礎，將探針修飾硫氫基直接固定於石英晶體之金電極上，或利用 11-Mercaptoundecanoic acid (11-MUA) 及 6-Mercapto-1-hexanol (MCH) 連接系統 (Linker system) 將探針與前述有機碳鏈長分子黏連而固定於石英晶體之金電極上，再與目標核酸雜合 (Hybridization) 偵測頻率變化；另一類探針則作為回報探針 (Reporter probe)，在抓取探針與目標核酸雜合後，與目標核酸二度雜合，此回報探針接有生物素 (Biotin)，利用生物素與卵白素 (Streptavidin) 間高親合之特性，在卵白素接上酵素或螢光物質而呈色，進而檢測雜合效率並增進系統靈敏度，作為QCM 頻率檢測外的佐證工具。除基礎偵測條件之探討，本研究主要著重於增進雜合效率之探討。本系統可在兩小時內檢測到 200 pg/μL 之目標核酸，並應用呈色系統確認雜合結果，成功建立一靈敏度高之壓電性石英晶體微天平核酸偵測系統。

We developed a quartz crystal microbalance (QCM) based genosensor to detect the DNA hybridization efficiency. We used a gas QCM system model to construct two kinds of DNA probes. One were capture probe, which was used the high efficiency between the Au electron and thiol group to immobilize a thiolated probes on the Au electron surface of quartz chip, or used 11-mercaptoundecanoic acid (11-MUA) and 6-Mercapto-1-hexanol (MCH) linker system to condense the capture probe on the Au electron surface of quartz chip, then the immbilized capture probe will hybridize with target DNA and detected the QCM frequency shift; the other kind of probe as a reporter probe were used for secondary hybridization with target DNA. The biotinylated reporter probes provide a high affinity with streptavidin. The biotinylated reporter probes active with streptavidin-coated enzyme or fluorescent to have color or fluorescent signal, enhance the genosensor sensitivity and provide stronger evidence.The QCM based genosensor system was not only used to establish the basic condition versus frequency shift, but also used to promote the hybridization efficiency. The system can detect 200 pg/μL target DNA in two hours. We established a colorful system to confirm the correction of the genosensor system.