基于戊二醛的氨基微球上DNA编码和抗体蛋白固定

DNA-barcode and antibody protein immobilization on amino-microparticles based on glutaraldehyde

用戊二醛和EDC/sulfo-NHS分别活化表面修饰了氨基和羧基的微球,在2种微球上分别固定相等摩尔浓度的氨基标记的发卡DNA和未经修饰的人TNF α捕获抗体,结果表明戊二醛的交联效果优于EDC/sulfo-NHS。从而选择戊二醛为交联剂将Cy3标记的发卡DNA和未经修饰的藻红蛋白(RPE)分别固定到微米球表面并进行了荧光观察,再通过调整蛋白和DNA的比例将FAM标记的发卡DNA和RPE同时固定到微米球上并观察荧光,为实现用DNA序列编码的微米球上多种生物标志物酶联免疫吸附检测奠定了基础。用RPE为荧光标记物采用双抗体夹心法在微球上进行荧光免疫反应,为商品化诊断试剂的研究提供一定的参考。

Amino-and carboxyl-modified microspheres are activated with glutaraldehyde and EDC/sulfoNHS,respectively.An equal mole of amino-labeled hairpin DNA and human TNF αcapture antibody are immobilized onto both microspheres.The results show that the cross-linking effect with glutaraldehyde is better than that with EDC/sulfo-NHS.Therefore,glutaraldehyde is used to immobilize Cy3-labeled hairpin DNA and R-PE onto the surface of microspheres,respectively,and the conjugates are investigated under the fluorescence microscope.Then,adjusted ratio of FAM-labeled hairpin DNA to R-PE are co-immobilized on the surface of microbeads using glutaraldehyde,and the resulting conjugates investigated under the fluorescence microscope.This would lay a foundation for high-throughput immunoassay by encoding various biomarkers with DNA sequences.The solid-phase fluorescence immunoreaction of microparticles was carried out by using double antibody sandwich method with R-PE labled fluorescent,which provides reference for the research of commercial diagnostic reagents.