Son Ahjeong, Dosev Dosi, Nichkova Mikaela, Ma Zhiya, Kennedy Ian M, Scow Kate M, Hristova Krassimira R
Department of Land, Air, and Water Resources, University of California Davis, One Shields Avenue, Davis, CA 95616, USA.
Anal Biochem. 2007 Nov 15;370(2):186-94. doi: 10.1016/j.ab.2007.08.001. Epub 2007 Aug 9.
Nanoscale magnetic/luminescent core-shell particles were used for DNA quantification in a hybridization-in-solution approach. We demonstrated a rapid, simple, and non-polymerase chain reaction-based DNA hybridization-in-solution assay for quantifying bacteria capable of biodegrading methyl tertiary-butyl ether. Fe3O4/Eu:Gd2O3 core-shell nanoparticles synthesized by spray pyrolysis were biofunctionalized with NeutrAvidin. Following immobilization of a biotinylated probe DNA on the particles' surfaces via passive adsorption, target DNA labeled with fluorescein isothiocyanate was hybridized with probe DNA. The hybridized DNA complex was separated from solution with a magnet, while nonhybridized DNA remained in solution. The normalized fluorescence (fluorescein isothiocyanate/nanoparticles) measured with a spectrofluorometer indicated a linear quantification (R(2)=0.98) of the target bacterial 16 S rDNA. The rate of hybridization increased concurrently with the target DNA concentration. In addition, this approach differentiated between the signal outputs from perfectly complementary target and two-base mismatched target DNA in a range of concentrations, showing the specificity of the assay and the possibility for environmental applications.
纳米级磁性/发光核壳颗粒被用于溶液杂交法中的DNA定量分析。我们展示了一种快速、简单且基于非聚合酶链反应的溶液杂交DNA分析法，用于定量能够生物降解甲基叔丁基醚的细菌。通过喷雾热解法合成的Fe3O4/Eu:Gd2O3核壳纳米颗粒用中性抗生物素蛋白进行了生物功能化修饰。在通过被动吸附将生物素化的探针DNA固定在颗粒表面后，用异硫氰酸荧光素标记的靶DNA与探针DNA杂交。杂交后的DNA复合物用磁铁从溶液中分离出来，而未杂交的DNA则保留在溶液中。用荧光分光光度计测量的归一化荧光（异硫氰酸荧光素/纳米颗粒）表明对目标细菌16S rDNA进行了线性定量分析（R(2)=0.98）。杂交速率随靶DNA浓度的增加而同时增加。此外，该方法在一系列浓度范围内区分了完全互补靶标和两个碱基错配靶标DNA的信号输出，显示了该分析方法的特异性以及在环境应用中的可能性。
