Using self-assembled aptamers and fibrinogen-conjugated gold nanoparticles to detect DNA based on controlled thrombin activity

We have developed a colorimetric probe, based on the aggregation of gold nanoparticles (Au NPs), for the detection of DNA and for the analysis of single-nucleotide polymorphism (SNP); this probe functions through the modulation of the activity of thrombin (Thr) in the presence of bivalent thrombin-binding aptamers (TBAs). The bivalent TBAs were formed from TBA_27' (comprising a 27-base sequence providing TBA₂₇ functionality, a T₅ linker, and an 11-base sequence for hybridization) and TBA_15' (comprising a 15-base sequence providing TBA₁₅ functionality, a T₅ linker, and a 12-base sequence for hybridization) through their hybridization with perfectly matched DNA (DNA_pm). The bivalent TBAs interacted specifically with thrombin, suppressing its activity toward fibrinogen-modified Au NPs (Fib-Au NPs). The potency of the inhibitory effect of TBA_15'-TBA_27'/DNA_pm toward thrombin - and, thus, the degree of aggregation of the Fib-Au NPs - was highly dependent on the concentration of DNA_pm. Under the optimal conditions (50pM thrombin, 2nM TBA_15', 2nM TBA_27', and 38pM Fib-Au NPs), the linear relationship of the response of the probe toward DNA_pm extended from 0.1 to 2nM, with a correlation coefficient of 0.97. The limit of detection (LOD) for DNA_pm was 20pM, based on a signal-to-noise ratio of 3. We also applied a corresponding TBA_15″-TBA_27″/Thr/Fib-Au NP probe to the detection of the SNP of the Arg249Ser unit in the TP53 gene, with an LOD of 32pM. Relative to conventional molecular beacon-based and crosslinking aggregation-based Au NP probes, our new approach offers higher sensitivity and higher selectivity toward DNA.