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Gold nanoparticle colorimetry assay using aptamers is a low cost and a highly effective means for detecting a wide range of biomolecular targets. In this work, this technique is used to detect the protein thrombin as a model system for understanding the relationship between the aptamer-target binding properties and the optical colorimetric response, as well as to gain insight on the secondary structures of the aptamers. The two known aptamers for thrombin, the 15-mer Bock and the 29-mer Tasset aptamer were conjugated to gold nanoparticles to form complexes that bind to thrombin upon contact. The Bock aptamer causes the aggregation of the nanoparticles and the concomitant reduction of the plasmon resonance peak, whereas the 29-mer Tasset aptamer, despite higher affinity, does not cause a spectral change. The data is understood on the basis of the difference in the number of binding sites available on thrombin for the respective aptamers. Additional results on single base substitutions suggest that the G-quadruplex secondary structure in the Bock aptamer is intermolecular and comprises of at least two interacting aptamer molecules. An estimate of the dissociation constant, derived from thrombin titration, is comparable to values reported in the literature.