荧光微球表面寡核苷酸探针的固定化研究

Study of the Immobilizing of Oligonucleotides Probe onto the Fluorescent Beads Surface

以1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)为偶联剂,将氨基标记的寡核苷酸探针(包含捕获探针、检测偶联效率的Tag序列以及间隔臂三部分)固定在羧基末端荧光微球表面.以生物素标记的cTag与微球表面探针杂交,再加入荧光物质PE标记的亲和素(SA-PE),通过Luminex-100TM荧光微球检测仪对荧光微球表面探针的偶联效果进行了检测.结果表明:不同间隔臂的氨基标记寡核苷酸探针具有不同的偶联效率,C12偶联效率优于C6-5T,C6-5T优于C6;且平均荧光强度(MFI)值最高可达3000以上,为后续核酸杂交检测建立了基础.

Using 1-ethyl-3-（3-Dimethylaminopropyl）-carbodiimide （EDC）, as a kind of coupling reagent, amino-labelled oligonucleotides（including three portions： capture probe, Tag sequence for detecting coupling efficiency and spacing-arm） were immobilized onto the carboxyl functionalized fluorescent beads,surface. Then biotin-labelled cTag hybridized that probes on the fluorescent beads surface, fluorescent substance PE-labeled streptavidin （SA-PE） was added to the hybridization system, and the coupling efficiency of SA-PE with biotin on the fluorescent beads surface was detected using Luminex-100^TM fluorescent microsphere system. The result shows that amino-labelled oligonucleotides with different spacing-arm have different coupling efficiency and C12 precedes C6-5T while C6 5T preceding C6 and that the highest value of mean fluorescent intensity （MFI） is more over 3000, providing the foundation of subsequent detecting hybridization of nucleic acid.