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Colorimetric Detection of DNA by Modulation of Thrombin Activity on Gold Nanoparticles
Author(s) -
Jian JyunWei,
Huang ChihChing
Publication year - 2011
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201002825
Subject(s) - thrombin , colloidal gold , aptamer , chemistry , detection limit , dna , oligonucleotide , nanoparticle , analytical chemistry (journal) , biophysics , nuclear chemistry , chromatography , microbiology and biotechnology , biochemistry , nanotechnology , materials science , biology , platelet , immunology
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 m M , pH 7.4), KCl (5 m m) , MgCl 2 (1 m m) , CaCl 2 (1 m M ), NaCl (150 m m) , thrombin (10 p m) , and TBA 29 –AuNPs (20 p M )], the new TBA 29 –AuNP/Fib–AuNP probe shows linear sensitivity to DNA pm in the concentration range 20–500 p M with a correlation coefficient of 0.96. The limit of detection for DNA pm was experimentally determined to be 12 p M , 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.