Colorimetric genotyping using DNA-modified fluorescent nanospheres

点击翻译按钮获取中文摘要

1.Sphere modification by ODNs (oligodeoxynucleotide) and peptidesTo avoid side reactions, modifications were carried out in two steps. First sphere surface was thiolated with cystamine. Maleimide group was modified to the end of DNA or peptide using appropriate bifunctional reagent. Both of them were coupled at ambient temperature to obtain molecular-modified nanospheres in high efficiency.2.Multiplexed multicolor genotyping using selective aggregation of DNA-modified nanospheresThree kinds of differently colored (RGB, red/green/blue) nanospheres (40 nm diameter) bearing unique ODNs on their surface were prepared for detecting the p53 gene. Each ODN is complementary to the different part in the 45-mer sample, which is a part of a conservative region of the p53 gene containing one of the hot spots. The RGB ternary system gave the aggregates with specific colors corresponding to the added ODN samples, wild type or mutant. In addition, in the presence of both samples, all of the spheres formed aggregates with white emission in consequence of mixing three primary colors of lights. Present technique should allow us to conduct an allele analysis.3.Multiplexed multicolor immunoassay using selective aggregation of peptide-modified nanospheresAntigenic Peptides with cationic (KKKKC) or anionic (DDDDC) pentamer tail were immobilized on luminous nanospheres of 40 nm diameter. A mixture of the antigenic peptides of FAK and c-Myc was immobilized to the spheres with red emission, while that of c-Myc and α-catenin was likewise to green spheres. Multiplexed immunoassay was achieved by adding the antibodies to a mixed dispersed solution of these spheres under appropriate conditions. Anti-FAK and anti-α-catenin antibodies formed aggregates with red and green emissions, respectively. On the other hand, the anti-c-Myc antibody formed aggregates emitting a yellow light. This system enabled us to differentiate 3 antibodies in one vessel from the definite differences in aggregate color.