Colorimetric detection of DNA by modulation of thrombin activity on gold nanoparticles

A colorimetric, non-cross-linking aggregation-based gold-nanoparticle (AuNP) probe has been developed for the detection of DNA and the analysis of single-nucleotide polymorphism (SNP). The probe acts by modulating the enzyme activity of thrombin relative to fibrinogen. A thrombin-binding aptamer with a 29-base-long oligonucleotide (TBA(29)) assembled on the nanoparticles (TBA(29)-AuNPs) through sandwich DNA hybridization was found to possess ultra-high anticoagulant potency. The enzyme inhibition of thrombin was determined by thrombin-induced aggregation of fibrinogen-functionalized 56 nm AuNPs (Fib-AuNPs). The potency of the inhibition of TBA(29)-AuNPs relative to thrombin--and thus the degree of aggregation of the Fib-AuNPs--is highly dependent on the concentration of perfectly matched DNA (DNA(pm)). Under optimal conditions [Tris-HCl (20 mM, pH 7.4), KCl (5 mM), MgCl(2) (1 mM), CaCl(2) (1 mM), NaCl (150 mM), thrombin (10 pM), and TBA(29)-AuNPs (20 pM)], the new TBA(29)-AuNP/Fib-AuNP probe shows linear sensitivity to DNA(pm) in the concentration range 20-500 pM with a correlation coefficient of 0.96. The limit of detection for DNA(pm) was experimentally determined to be 12 pM, based on a signal-to-noise ratio (S/N) of 3. The new probe was successfully applied to the analysis of an SNP that is responsible for sickle cell anemia. Relative to conventional molecular-beacon-based probes, the new probe offers the advantages of higher sensitivity and selectivity towards DNA and lower cost, showing its great potential for practical studies of SNPs.