Institute of Materials Research and Engineering, ASTAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602.
Anal Chem. 2009 Aug 1;81(15):6122-9. doi: 10.1021/ac900525k.
We have developed a colorimetric assay for DNA detection based on the aggregation of unmodified metallic nanoparticles. Charge neutral peptide nucleic acids (PNA) are used as a "coagulant" of citrate anion-coated particles and as hybridization probe. In the absence of a complementary target DNA, free PNA molecules in solution induce aggressive particle aggregation because of the removal of charge repulsion as a result of PNA coating on nanoparticles. When a complementary DNA is present and PNA-DNA complexes are formed, the particles remain stable because the negative charges of the DNA strands in the complexes adsorbed on the particle surface ensure sufficient charge repulsions. In this method, no probe immobilization is needed and PNA-DNA hybridization occurs in a homogeneous phase. The assay results are displayed as rapidly as the changes in color and/or in UV-vis adsorption spectra of the colloidal solutions. We have validated the assay principle using gold- and silver-nanoparticles (AuNPs and AgNPs), with the involvement of a shorter (13 mer) and a longer (22 mer) probe sequences. A specific DNA can be detected in the presence of at least 10 times of interference DNA, and the detection limit is at a DNA/PNA ratio of 0.05. When NaCl is added to accelerate the particle aggregation, the selectivity is further improved, and single-base-mismatch discrimination is achieved. A two-component assay using a mixture of AuNPs and AgNPs has also been constituted, aiming to improve the result accuracy by making use of the multiple aggregation signatures from the two types of particles. For single-base-mismatch discrimination, the AgNPs offer a higher sensitivity than AuNPs by showing more obvious spectra and color alternation, and the two-component assay offers three parameters in the UV-vis adsorption spectra.
我们开发了一种基于未修饰的金属纳米粒子聚集的比色法 DNA 检测方法。中性肽核酸（PNA）被用作带负电荷的柠檬酸盐粒子的“凝结剂”和杂交探针。在不存在互补靶 DNA 的情况下，溶液中的游离 PNA 分子由于 PNA 涂层在纳米粒子上的去除而诱导强烈的粒子聚集，因为电荷斥力被去除。当存在互补 DNA 并且形成 PNA-DNA 复合物时，由于吸附在粒子表面上的复合物中的 DNA 链的负电荷确保了足够的电荷斥力，因此粒子保持稳定。在这种方法中，不需要探针固定，并且 PNA-DNA 杂交在均相相中发生。该测定结果与胶体溶液的颜色和/或紫外可见吸收光谱的变化一样迅速显示。我们使用金和银纳米粒子（AuNPs 和 AgNPs）验证了该测定原理，涉及较短（13 个碱基）和较长（22 个碱基）的探针序列。在存在至少 10 倍干扰 DNA 的情况下可以检测到特定的 DNA，并且检测限为 DNA/PNA 比为 0.05。当加入 NaCl 以加速粒子聚集时，选择性进一步提高，并且实现了单碱基错配的区分。还构成了使用 AuNPs 和 AgNPs 混合物的两组件测定，旨在通过利用两种类型粒子的多个聚集特征来提高结果准确性。对于单碱基错配的区分，AgNPs 通过显示更明显的光谱和颜色变化提供比 AuNPs 更高的灵敏度，并且两组件测定在紫外可见吸收光谱中提供三个参数。
