Colorimetric genotyping using DNA-modified fluorescent nanospheres

1.寡核苷酸和多肽修饰球体为避免副反应，修饰分两步进行。首先，用半胱胺对球体表面进行硫醇化。用适当的双功能试剂将马来酰亚胺基团修饰到DNA或多肽的末端。2.DNA修饰纳米球的多重多色基因分型制备了3种不同颜色(RGB，红/绿/蓝)的纳米球(直径40 nm)，其表面带有独特的ODN，用于检测p53基因。每个ODN都与45聚体样本中的不同部分互补，45聚体样本中的不同部分是包含其中一个热点的p53基因保守区的一部分。RGB三元体系给出的聚集体具有与添加的ODN样本、野生型或突变体相对应的特定颜色。此外，在两个样品的存在下，由于混合了三种原色的光，所有的球体都形成了具有白色发射的聚集体。目前的技术应该允许我们进行等位基因分析。3.利用多肽修饰的纳米球选择性聚集的多重多色免疫分析-将带有阳离子(KKKKC)或阴离子(DDDDC)五聚体的抗原肽固定在直径为40 nm的发光纳米球上。将FAK和c-Myc抗原肽的混合物固定在红色发光的球上，c-Myc和α-catenin的抗原肽也固定在绿色发光的球上。在适当的条件下，将抗体加入到这些球体的混合分散溶液中，实现了多重免疫分析。抗粘着斑块抗体和抗α-连环蛋白抗体分别形成红色和绿色发光聚集体。另一方面，抗c-Myc抗体形成了发出黄色光的聚集体。该系统使我们能够根据聚集颜色的明确差异来区分同一血管中的三种抗体。

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.