1 research outputs found
Molecularly Imprinted Aptamers of Gold Nanoparticles for the Enzymatic Inhibition and Detection of Thrombin
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
(TBA<sub>15</sub>) with different thymidine linkers, and 29-mer thrombin-binding
aptamer (TBA<sub>29</sub>) with different thymidine linkers (Tn) in
the presence of thrombin (Thr) as a template were used to prepare
MP-Thr-TBA<sub>15</sub>/TBA<sub>29</sub>-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-TBA<sub>15</sub>/TBA<sub>29</sub>-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-TBA<sub>15</sub>/TBA<sub>29</sub>-Tn–Au NPs, leading to their stronger binding strength with
thrombin. MP-TBA<sub>15</sub>/TBA<sub>29</sub>-T<sub>15</sub>–Au
NPs (ca. 42 TBA<sub>15</sub> and 42 TBA<sub>29</sub> 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<sup>–11</sup> M. As a result, they had 8 times higher
anticoagulant (inhibitory) potency relative to TBA<sub>15</sub>/TBA<sub>29</sub>-T<sub>15</sub>–Au NPs (prepared in the absence of
thrombin). We further conducted thrombin clotting time (TCT) measurements
in plasma samples and found that MP-TBA<sub>15</sub>/TBA<sub>29</sub>-T<sub>15</sub>–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-TBA<sub>15</sub>-T<sub>15</sub>–Au
NPs and MP-TBA<sub>29</sub>-T<sub>15</sub>–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