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

Abstract

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 TBA 27 functionality, a T 5 linker, and an 11-base sequence for hybridization) and TBA 15′ (comprising a 15-base sequence providing TBA 15 functionality, a T 5 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 (50 pM thrombin, 2 nM TBA 15′, 2 nM TBA 27′, and 38 pM Fib-Au NPs), the linear relationship of the response of the probe toward DNA pm extended from 0.1 to 2 nM, with a correlation coefficient of 0.97. The limit of detection (LOD) for DNA pm was 20 pM, 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 32 pM. Relative to conventional molecular beacon-based and crosslinking aggregation-based Au NP probes, our new approach offers higher sensitivity and higher selectivity toward DNA.