Molecularly Imprinted Aptamers of Gold Nanoparticles for the Enzymatic Inhibition and Detection of Thrombin
journal contributionposted on 19.06.2012, 00:00 by Yu-Ju Liao, Yen-Chun Shiang, Chih-Ching Huang, Huan-Tsung Chang
We prepared thrombin-binding aptamer-conjugated gold nanoparticles (TBA–Au 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–Au 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–Au 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–Au NPs, leading to their stronger binding strength with thrombin. MP-TBA15/TBA29-T15–Au 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 × 10–11 M. As a result, they had 8 times higher anticoagulant (inhibitory) potency relative to TBA15/TBA29-T15–Au 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–Au 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–Au NPs and MP-TBA29-T15–Au 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.