Colorimetric genotyping using DNA-modified fluorescent nanospheres

1.用寡核苷酸和肽修饰球为避免副作用，修饰分两步进行。第一个球体表面用半胺硫化。用合适的双功能试剂将马来酰亚胺基团修饰到DNA或肽的末端。在室温下，将两者耦合，得到了高效的分子修饰纳米球。采用dna修饰纳米球选择性聚集技术进行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），其表面具有独特的odn，用于检测p53基因。每个ODN与45-mer样本中的不同部分是互补的，该样本是p53基因保守区域的一部分，包含一个热点。RGB三元体系给出了与添加的ODN样品、野生型或突变型相对应的特定颜色的聚集体。此外，在两种样品存在的情况下，由于混合了三种原色的光，所有的球体都形成了白色发射的聚集体。目前的技术应该允许我们进行等位基因分析。将带有阳离子（KKKKC）或阴离子（DDDDC）五聚体尾部的抗原肽固定在直径为40 nm的发光纳米球上。FAK和c-Myc抗原肽的混合物固定在红色发射球上，c-Myc和α-catenin的混合物同样固定在绿色发射球上。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，实现了多重免疫测定。抗fak抗体和抗α-catenin抗体分别形成红色和绿色辐射的聚集体。另一方面，抗c- myc抗体形成聚集体，发出黄光。该系统使我们能够从聚集颜色的明确差异中区分出一个血管中的3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.