Molecularly Imprinted Aptamers of Gold Nanoparticles for the Enzymatic Inhibition and Detection of Thrombin

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• 作者：Yu-Ju Liao ; Yen-Chun Shiang ; Chih-Ching Huang ; Huan-Tsung Chang
• 刊名：Langmuir
• 出版年：2012
• 出版时间：June 19, 2012
• 年：2012
• 卷：28
• 期：24
• 页码：8944-8951
• 全文大小：318K
• 年卷期：v.28,no.24(June 19, 2012)
• ISSN：1520-5827

文摘

We prepared thrombin-binding aptamer-conjugated gold nanoparticles (TBA鈥揂u NPs) through a molecularly imprinted (MP) approach, which provide highly efficient inhibition activity toward the polymerization of fibrinogen. Au NPs (diameter, 13 nm), 15-mer thrombin-binding aptamer (TBA₁₅) with different thymidine linkers, and 29-mer thrombin-binding aptamer (TBA₂₉) with different thymidine linkers (Tn) in the presence of thrombin (Thr) as a template were used to prepare MP-Thr-TBA₁₅/TBA₂₉-Tn鈥揂u NPs. Thrombin molecules were then removed from Au NPs surfaces by treating with 100 mM Tris-NaOH (pH ca. 13.0) to form MP-TBA₁₅/TBA₂₉-Tn鈥揂u NPs. The length of the thymidine linkers and TBA density on Au NPs surfaces have strong impact on the orientation, flexibility, and stability of MP-TBA₁₅/TBA₂₉-Tn鈥揂u NPs, leading to their stronger binding strength with thrombin. MP-TBA₁₅/TBA₂₉-T₁₅鈥揂u NPs (ca. 42 TBA₁₅ and 42 TBA₂₉ molecules per Au NP; 15-mer thymidine on aptamer terminal) provided the highest binding affinity toward thrombin with a dissociation constant of 5.2 脳 10^鈥?1 M. As a result, they had 8 times higher anticoagulant (inhibitory) potency relative to TBA₁₅/TBA₂₉-T₁₅鈥揂u NPs (prepared in the absence of thrombin). We further conducted thrombin clotting time (TCT) measurements in plasma samples and found that MP-TBA₁₅/TBA₂₉-T₁₅鈥揂u NPs had greater anticoagulation activity relative to four commercial drugs (heparin, argatroban, hirudin, and warfarin). In addition, we demonstrated that thrombin induced the formation of aggregates from MP-TBA₁₅-T₁₅鈥揂u NPs and MP-TBA₂₉-T₁₅鈥揂u NPs, thereby allowing the colorimetric detection of thrombin at the nanomolar level in serum samples. Our result demonstrates that our simple molecularly imprinted approach can be applied for preparing various functional nanomaterials to control enzyme activity and targeting important proteins.