两种纳米金标记p53抗体探针的制备、表征和性质研究

目的制备IgG-Au-HRP和IgG-Au-DNA-HRP两种纳米金标记p53抗体探针,并对其进行生物学特征表征和性质研究。方法利用纳米金粒子与蛋白质非共价吸附和与巯基修饰的寡核苷酸以Au-S键共价结合的特点,将辣根过氧化物酶(HRP)直接或通过寡核苷酸链间接标记在纳米金表面,制备两种多功能纳米金生物探针(IgG-Au-HRP和IgG-Au-DNA-HRP探针);应用透射电镜、紫外-可见光光谱等对纳米金探针性能进行表征,对纳米金探针表面HRP酶活性进行分析,并通过酶免疫标记技术(enzyme linked immuncsorbent assay,ELISA)优化探针制备条件和比较两种探针标记效率。结果①两种新型探针具有良好的单分散性,大小均一,粒径约10nm;②p53蛋白检测效果对IgG-Au-HRP和IgG-Au-DNA-HRP探针制备条件优化结果表明:制备IgG-Au-HRP探针时,HRP分子和p53抗体的最佳比例为10:1;制备IgG-Au-DNA-HRP探针时,DNA和p53抗体的最佳比例为2.5:1;③HRP分子定量检测显示:一个IgG-Au-HRP探针可标记HRP数量约11个,IgG-Au-DNA-HRP探针可标记HRP数量为20个;④两种探针结合ELISA法初步检测p53蛋白判定IgG-Au-DNA-HRP探针比IgG-Au-HRP探针检测蛋白灵敏度高。结论两种新型纳米金探针制备简单,可作为一种新型探针应用于微量蛋白的高灵敏检测。

Preparation and characterization of two kinds of gold nanoparticle labled p53 antibody probes

Objective Two novel p53 antibody gold nanoparticle probes （IgG-Au-HRP probes and IgG-Au-DNA-HRP probes） were synthetized,the biological characteristics of which were studied.
Methods Two kinds of novel multifunctional gold nanoparticle probes （IgG-Au-HRP probes and IgG-Au-DNA-HRP probes） were synthesized,the gold nanoparticles were functioned with both antibodies and Horseradish-peroxidase （HRP）,which can be immobilized onto gold nanoparticles directly （IgG-Au-HRP probes） or through the thiol-terminated oligonucleotides strand （IgG-Au-DNA-HRP probes）.The morphology and spectroscic properties of these probes were evaluated by transmission electron microscopy （TEM）,UV-vis spectra analyses and ELISA.
Results ①New gold nanoparticles probes were monodisperse,good morphology and dispersed evenly,the average diameter was 10 nm;②Experimental parameters involved in preparation of IgG-Au-HRP and IgG-Au-DNA-HRP probes were optimized.The ideal ratio of HRP： detecting antibody p53 was 10：1 （IgG-Au-HRP）,and the ratio of DNA： detecting antibody p53 was 2.5：1 （IgG-Au-DNA-HRP）;③Based on quantification of HRP immobilized on Au NP probe,the number of HRP molecules was 11 in IgG-Au-HRP probe and was 20 in IgG-Au-DNA-HRP probe;④From initial detection through ELISA,the sensitivity in protein detection of IgG-Au-DNA-HRP probes was higher than IgG-Au-HRP probes.
Conclusion The preparation of the new nanoparticle gold probes was simple,which can be used as a new type of probe used in high sensitivity detection of protein.