利用功能性金奈米材料抑制凝血酶活性及偵測三磷酸腺苷

Abstract

本論文分兩部分，第一部分是利用分子模板技術(Molecularly Imprinted Technology，MP)將凝血酶適合體(Thrombin-Binding Aptamer，TBA)修飾在金奈米粒子(Gold Nanoparticles，Au NPs)表面後，形成具有抗凝血酶與偵測凝血酶活性的適合體─金奈米粒子複合物(MP-TBA−Au NPs)；第二部份是利用金銀奈米棒(Gold-Silver Nanorods，Au/Ag NRs)修飾上肌動蛋白(Actin)，製備肌動蛋白─金銀奈米棒(Ac−Au/Ag NRs)」感測器於三磷酸腺苷(Adenosine-5'-triphosphate, ATP)的偵測。經由凝血酶凝集時間(thrombin clotting time，TCT)的測試，在最佳化的條件下，MP-TBA15/TBA29-T15−Au NPs可使TCT值延遲至160秒，優於其他抑制劑(如Argatroban (61 秒)、Hirudin (44秒)、Warfarin (40秒)、Heparin (34秒))。MP-TBA−Au NPs亦可應用於凝血酶的偵測，利用凝血酶分子同時與MP-TBA15−Au NPs和MP-TBA29−Au NPs作用產生聚集現象，在模擬的生理環境偵測凝血酶分子，其線性範圍為1.0−10 nM (R2 = 0.98)。MP-TBA−Au NPs對於凝血酶有極高的選擇性與高穩定性，可應用於真實樣品(例如:人類血清以及血漿)中凝血酶的抑制及偵測。第二部分是利用Ac−Au/Ag NRs偵測樣品中ATP濃度，因ATP的存在會使肌動蛋白單體(G-Actin)產生聚合作用(polymerization)形成絲狀肌動蛋白聚合體(F-actin)進而造成Au/Ag NRs的聚集，藉由測量金銀奈米棒的吸收值比(Ex900/730；吸收值波長900 nm與730 nm比值)即可得知金銀奈米棒聚集的程度，進而定量出樣品中ATP濃度。Ac−Au/Ag NRs在模擬的生理環境中對於ATP偵測的線性範圍為50 nM−1.0 μM (R2 = 0.99)。Ac−Au/Ag NRs對ATP、ADP、AMP以和Adenosine有很好的選擇性，可應用於複雜的樣品(如人類血漿)中偵測ATP濃度。

There are two parts in this thesis: the first part is focused on that we prepared thrombin-binding aptamer-conjugated gold nanoparticles (TBA−Au NPs) through a molecularly imprinted (MP) approach, which provided highly efficient inhibition activity toward the polymerization of fibrinogen; another is to prepared actin-conjugated gold-silver nanorods (Ac−Au/Ag NRs) to measure Adenosine-5'-triphosphate (ATP) concentration in the aqueous solution by colorimetric method. MP-TBA15/TBA29-T15−Au NPs provided the highest binding affinity toward thrombin with a dissociation constant of 5.2 × 10−11 M. Finally, we tested the thrombin clotting time (TCT) measurements in plasma samples and found that MP-TBA15/TBA29-T15−Au NPs had greater anticoagulation activity than heparin, argatroban, hirudin and warfarin. In addition, we demonstrated that thrombin induced the formation of aggregates from MP-TBA15-T15−Au NPs and MP-TBA29-T15−Au NPs, thereby allowing the colorimetric detection of thrombin at the nanomolar level in serum samples. The second part is to demonstrated a colorimetric sensing approach for the determination of the concentration of adenosine-5'-triphosphate (ATP) using actin-conjugated gold nanorods (Ac−Au/Ag NRs). In the presence of the ATP, the color of the Ac−Au/Ag NRs solution changed from red to purple as a result of actin polymerization. For mixtures of ATP, polymerization buffer (40.0 mM NaCl and 2.0 mM MgCl2) and physiological buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5.0 mM KCl, 1.0 mM MgCl2, 1.0 mM CaCl2), a linear correlation (R2=0.99) existed between the ratio of the extinctions of the Ac−Au NRs at 900 and 730 nm (Ex900/730) and the concentration of ATP.