title: Colorimetric detection of DNA by modulation of thrombin activity on gold nanoparticles.
abstract: 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.
authors.affiliation: Institute of Bioscience and Biotechnology and Center for Marine, Bioenvironment and Biotechnology, National Taiwan Ocean University, 2, Beining Road, Keelung, 20224, Taiwan.
authors.forename: Jyun-Wei
authors.initials: JW
authors.lastname: Jian
authors.forename: Chih-Ching
authors.initials: CC
authors.lastname: Huang
mesh_terms: D000755:Anemia, Sickle Cell / Q000235:genetics; D052157:Aptamers, Nucleotide; D003124:Colorimetry / Q000379:methods*; D004247:DNA / Q000032:analysis*; D006046:Gold / Q000737:chemistry*; D053758:Nanoparticles; D009942:Organometallic Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D012689:Sequence Homology, Nucleic Acid; D013917:Thrombin / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D009942:Organometallic Compounds; C081676:thrombin aptamer; D006046:Gold; D004247:DNA; D013917:Thrombin