Improvement of the thermal stability of streptavidin immobilized on magnetic beads by the construction of a mixed poly(ethylene glycol) tethered-chain layer
Improvement of the thermal stability of streptavidin immobilized on magnetic beads by the construction of a mixed poly(ethylene glycol) tethered-chain layer
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DOI：
10.1038/pj.2011.5
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发表时间：
2011-05-01
期刊：
影响因子：
2.8
通讯作者：
Nagasaki, Yukio
中科院分区：
文献类型：
--
作者：
Kubota, Masaki;Yoshimoto, Keitaro;Nagasaki, Yukio
在开发高性能的生物传感和生物分离材料中，有效的封闭处理对于减少蛋白质固定化底物表面的非特异性吸附和提高底物颗粒的分散稳定性具有重要意义。聚乙二醇(PEG)是一种优良的封闭剂。由于聚乙二醇的非离子性质、亲水性和大的空间排斥效应，5种聚乙二醇化表面和纳米和微米级粒子分别表现出优异的无污垢性能和高分散稳定性，分子数分别为6和7-9。此外，在我们最近的研究中，我们发现聚乙二醇化改善了固定在固体表面和颗粒上的蛋白质的功能。例如，抗C反应蛋白抗体固定的金传感器表面10和抗铁蛋白抗体固定的胶乳颗粒11的抗原结合效率通过共固定致密的聚乙二醇层而得到改善，聚乙二醇层由端羟基和端寡胺的聚乙二醇层组成。通过共固定化聚乙二醇多胺嵌段共聚物8和脂肪酶/聚乙二醇多胺/葡萄糖脱氢酶固定化金纳米粒子杂化材料，在581C下重复5次热处理10min后，固定化金纳米粒子的底物反应活性几乎相同。12这些结果表明，将聚乙二醇衍生物共固定在蛋白质固定化的底物上，有可能大大提高生物传感和生物分离材料的性能。蛋白质的热诱导失活是限制其应用领域的一个缺点。人们认为，热诱导失活是由于分子内蛋白质相互作用的解离造成的。13在这里，我们报告了致密填充的聚乙二醇层对链霉亲和素固定的磁珠(SA-MB)热诱导灭活的预防作用。通过在SA-MB上共固定端寡胺基团，构建了SA-MB与聚乙二醇系链层共固定化的SA-MB，并通过测定修饰后SA-MB捕获的生物素化单链DNA(SsDNA)的量来评价其与生物素的结合效率。我们发现，在751C重复热处理5min后，聚乙二醇/SA-MB对生物素化单链DNA的捕获率是未经修饰的SA-MB的两倍。
In the development of high-performance materials for biosensing and bioseparation, an effective blocking treatment is important to decrease nonspecific adsorption onto the surfaces of protein-immobilized substrates and to increase the dispersion stability of substrate particles. Poly (ethylene glycol)(PEG) is known as an excellent blocking agent. Because of the nonionic properties, hydrophilicity and large steric-exclusion effect of PEG, 5 PEGylated surfaces and nano-and microscale particles show excellent non-fouling properties with various molecules 6 and high-dispersion stabilities, 7–9 respectively. Furthermore, in our recent studies, we discovered that PEGylation improves the functioning of immobilized proteins on solid surfaces and particles. For example, the antigen-binding efficiencies of an anti-C-reactive protein antibody-immobilized gold sensor surface 10 and anti-ferritin antibody-immobilized latex particles 11 were improved by the co-immobilization of densely packed PEG layers, consisting of sulfhydryl-terminated PEGs and oligoamine-terminated PEGs. The substrate reactivity of glucose dehydrogenase-immobilized gold nanoparticles was improved by the co-immobilization of PEG/polyamine block copolymer, 8 and lipase/PEG-polyamine/glucose dehydrogenaseimmobilized gold nanoparticle hybrids showed almost the same initial enzymatic activity after five repeated thermal treatments at 581C for 10 min. 12 These results indicate that the co-immobilization of PEG derivatives onto protein-immobilized substrates has potential as a treatment to strongly improve the performance of biosensing and bioseparation materials.The heat-induced inactivation of proteins is a disadvantage that often restricts their field of application. It is thought that heat-induced inactivation occurs because of the dissociation of intramolecular protein interactions. 13 Here, we report the preventative effect of a densely packed PEG layer against the heat-induced inactivation of streptavidin-immobilized magnetic beads (SA-MB). SA-MB co-immobilized with a PEG tethered-chain layer (PEG/SA-MB) were constructed by the co-immobilization of oligoamine-terminated PEGs onto SA-MB, and their biotin-binding efficiency was evaluated by measuring the amount of biotinylated single-stranded DNA (ssDNA) captured by the modified SA-MB. We found that the capture efficiency of biotinylated ssDNA by PEG/SA-MB was twice that of unmodified SA-MB after repeated thermal treatments for 5min at 751C.