以量子點-適體-染劑複合物發展新穎蛋白質螢光信標試劑

Abstract

本研究發展一種無須進行螢光標記，用於感測蛋白質之新穎螢光量子點信標試劑。本研究策略係利用量子點-適體複合物作為蛋白質之辨識單元，再配合核酸嵌合染劑(intercalating dye)作為訊號單元，最後以螢光共振能量轉移技術(Fluorescence Resonance Energy Transfer, FRET)進行標的蛋白之感測。目前利用量子點-適體複合物進行蛋白質檢測之方法，多建立於藉由目標蛋白與適體之結合所引起之構形變化性質，造成適體尾端標記之螢光熄滅分子與量子點距離變化產生FRET訊號改變。這樣的方法需要額外進行特殊之標記適體設計，且會牽涉適體直接修飾，可能改變適體結構與影響適體之蛋白質辨識能力。本研究利用DNA嵌合式螢光物質作為量子點螢光受體，能提供一不需對目標蛋白質與適體分子進行螢光標定(label-free)且不需進行額外之純化分離的檢測方式，非常適合搭配可攜式光學感測器進行單一或多標的蛋白質標的之臨床或現場快速偵測應用。本研究利用碳二亞酸媒介耦合反應製備量子點-適體複合物，在Sulfo-NHS與EDC比例為0.5的情況反應兩小時能獲得感測靈敏度最高之量子點-適體複合物，在此條件中，適體/量子點比例為3.2，理論螢光轉移效率為88.3 %。在目標蛋白的選擇上則以在凝血機制中扮演關鍵調控蛋白酶角色之凝血蛋白(thrombin)作為標的物，在緩衝溶液中，本研究所提出之量子點-適體複合物在500 nM凝血蛋白中能獲得58.9% ± 1.36的訊號響應，偵測極限為1nM，線性區間為1至20 nM (r=0.95)，同時該複合物對於非專一性標的物牛血清白蛋白並不具有相同訊號響應，並且本研究亦在血清全蛋白干擾下驗證之量子點-適體信標試劑之專一性與靈敏性。在15%血清中，此螢光信標對500 nM凝血蛋白仍具有10.1% ± 0.3的螢光訊號下降。另外在15%血清中此一螢光信標亦能有濃度相依之訊號響應，其線性迴歸之相關係數r可到達0.99979。

This thesis proposes a direct and simple design of fluorescent quantum dot beacon for label-free and separation-free protein detection, and the protein quantification is carried out by measuring the FRET signal reduction between the quantum dot (QD) and the intercalating dyes on the aptamer probe, which is conjugated to the QD. To date, a number of QD-aptamer based sensors have been developed, most of which use displacement strategy of the quencher-carried complementary sequence by target proteins. However, those methods need to design a modified aptamer sequence to enhance the FRET efficiency. Modification of aptamer sequence needs special care on preserving the original protein-binding confirmation because such sequence modification may diminish an aptamer's binding motif and cause malfunction of the aptamer. In this study, quantum dot-aptamer complexes and DNA intercalating dyes are used as the recognition and reporting elements, respectively. Moreover, it also provides a label-free and separation-free diagnostic format. Having these advantages, the proposed method is very suitable for portable optical sensors for single or multiple target proteins detection in clinical or on-site identification. In this study, EDC-mediated coupling strategy was utilized to construct a QD-aptamer complex. By using the ratio of Sulfo-NHS to EDC at 0.5 and conjugation for 2 hours, the highest sensitive performance was observed. In this condition, the ratio of aptamer to quantum dot is 3.2 and the theoretical FRET efficiency is 88.3%. To demonstrate the aforementioned QD-aptamer-intercalating dye-based protein detection, human thrombin is used as a detection target. Thrombin is known as a key protease in blood coagulation mechanism. In phosphate saline buffer condition, the change of fluorescence of the beacon can achieve 58.9% ± 1.36 responding with 500 nM thrombin. The linear range of thrombin detection is between 1 and 20 nM (R=0.95) and limit of detection is as low as 1 nM. In addition, this work also demonstrates that the beacon is compatible with complex matrixes. In 15% serum, the relative fluorescent change of the beacon is 10.1% ± 0.3 against 500 nM thrombin. Furthermore, the dose-dependent response in 15% serum can also be obtained with coefficient of determination, r, approaching 0.99979.