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 (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

The relationships among the diameter of microfibers, width of observation channel and flow rate of continuous phase.

<p>The relationships among the diameter of microfibers, width of observation channel and flow rate of continuous phase.</p

Microfluidic chip.

<p>Expanded view (A) and a photo (B) of the microfluidic chip: 1, inlets of center channels; 2 and 3, inlets of side channels; 4, cross junction; 5, outlet; 6, observation channel; 7, bottom layer disk; 8, screw orifices; 9, the scale bar = 11 cm. (C) is the geometry of the microfluidic channels.</p

Microfiber formation.

<p>The diagram of the microfluidic system and photographs of observation positions. 1, 2 wt % CaCl₂ solution; 2, deionized water; 3, alginate solution; 4, sunflower seed oil; 5, observation channel; 6, microfibers. The formation of microfibers: A, photograph of the microfiber in the observation channel; B, Photograph of the second cross junction; C, photograph of the first cross junction.</p

Microfiber images.

<p>Microscopic images (A∼B, stained with Rhodamine B) and scanning electron microscopy images (C∼E) of microfibers.</p

Cell culture of microfibers.

<p>Proliferation of GBM cells in microfibers. A. GBM cells; B. microfiber without cells; C. GBM in microfibers at the 1st day; D. GBM in microfibers at the 7th day. Arrows indicate GBM cells.</p

Characteristics of the microfibers.

<p>(A) The hysteresis curve of the microfibers containing MIO nanoparticles. (B) Release profiles of diclofenac from MIO-loaded microfibers without magnetic stimulation as the control (▵), with 2 minutes stimulation at the 10th, 30th and 60th minute (▴), with a 10-minute stimulation after the 20th minutes (•) and with a continuous stimulation from the beginning (○).</p