A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and te...A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.展开更多
Direct mixture of Au^3＋ with glutathione （GSH）, which act as both reduction agents and stabilizers, in aqueous solution gave rise to production of gold nanoparticles （Au NPs） with uniform sizes of around 21 nm. T...Direct mixture of Au^3＋ with glutathione （GSH）, which act as both reduction agents and stabilizers, in aqueous solution gave rise to production of gold nanoparticles （Au NPs） with uniform sizes of around 21 nm. The GSH stabilizer Au NPs in solution show immediate aggregation after addition of 1 mol/L NaCI aqueous solution containing Pb^2＋ ions. The Pb^2＋-induced aggregation in Au NP solution is monitored by both colorimetric response and UV-vis spectroscopy. A rather broad linear range （from 0.1 to 30 pmol/L） and low detection limit （0.1 pmol/L） are explored for Au NP sensors used for detection of Pb^2＋ ions. Furthermore, the response of GSH-stabilized Au NPs toward Pb^2＋ ions is specific compared with other possible interferants （Hg^2＋, Mg^2＋, Zn^2＋, Ni^2＋, Cu^2＋, Co^2＋, Ca^2＋, Mn^2＋, Cd^2＋, and Ba^2＋）.展开更多
基金supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (20070075003, 20091301120003)the Natural Science Foundation of Hebei Province (B2009000170)
文摘A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards, the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.
文摘Direct mixture of Au^3＋ with glutathione （GSH）, which act as both reduction agents and stabilizers, in aqueous solution gave rise to production of gold nanoparticles （Au NPs） with uniform sizes of around 21 nm. The GSH stabilizer Au NPs in solution show immediate aggregation after addition of 1 mol/L NaCI aqueous solution containing Pb^2＋ ions. The Pb^2＋-induced aggregation in Au NP solution is monitored by both colorimetric response and UV-vis spectroscopy. A rather broad linear range （from 0.1 to 30 pmol/L） and low detection limit （0.1 pmol/L） are explored for Au NP sensors used for detection of Pb^2＋ ions. Furthermore, the response of GSH-stabilized Au NPs toward Pb^2＋ ions is specific compared with other possible interferants （Hg^2＋, Mg^2＋, Zn^2＋, Ni^2＋, Cu^2＋, Co^2＋, Ca^2＋, Mn^2＋, Cd^2＋, and Ba^2＋）.
