title: 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.
authors.affiliation: Department of Chemistry, National Taiwan University, Taipei, Taiwan.
authors.forename: Chuan-Kuo
authors.initials: CK
authors.lastname: Chen
authors.forename: Yen-Chun
authors.initials: YC
authors.lastname: Shiang
authors.forename: Chih-Ching
authors.initials: CC
authors.lastname: Huang
authors.forename: Huan-Tsung
authors.initials: HT
authors.lastname: Chang
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000032:analysis*; D005340:Fibrinogen / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000032:analysis*
chemical_list: D052157:Aptamers, Nucleotide; C081676:thrombin aptamer; D006046:Gold; D005340:Fibrinogen; D004247:DNA; D013917:Thrombin