Bisphosphonate Adaptors for Specific Protein Binding on Zirconium Phosphonate-based Microarrays
Bisphosphonate Adaptors for Specific Protein Binding on Zirconium Phosphonate-based Microarrays
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DOI：
10.1021/bc9002597
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发表时间：
2009-12-01
影响因子：
4.7
通讯作者：
Tellier, Charles
中科院分区：
文献类型：
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作者：
Cinier, Mathieu;Petit, Marc;Tellier, Charles
设计了两个双磷酸盐接头，将组氨酸标记的蛋白质固定在涂有锆膦分子层的玻璃基质上，允许在微阵列形式上高效和定向地固定捕获蛋白，即针对溶菌酶的亲和素。这些双功能接头的一端含有两个膦酸链，另一端含有一个或两个NTA基团。如X射线光电子能谱(XPS)显示的那样，膦酸基通过多点连接提供了稳定的键合到锆界面上，并允许连接体的高密度表面覆盖。实时表面等离子体共振增强椭圆偏振法显示了组氨酸标记蛋白的可逆高密度捕获，并以微阵列形式使用AlexaFluor 647标记的目标蛋白的荧光检测。尽管探针是单层排列，但微阵列对目标蛋白的检测灵敏度低于1 nM，这是因为背景染色非常低，这使得荧光信噪比很高。与其他类型的微阵列基板相比，这些Ni-NTA修饰的磷酸锆涂覆载玻片的性能更好，包括具有硝基基质的载玻片、环氧化物载玻片和具有Ni-NTA基团的环氧载玻片。这种固定化策略有很大的潜力将任何组氨酸标记的蛋白质固定在锆离子或钛离子表面。
Two bisphosplionate adaptors were designed to immobilize histidine-tagged proteins onto glass substrates coated with a zirconium phosphonale moriolayer, allowing efficient and oriented immobilization of capture proteins, affitins directed to lysozyme, on a microarray format. These bifunctional adaptors contain two phosphonic acid anchors at one extremity and either one nitrilotriacetic acid (NTA) or two NTA groups at the other. The phosphonate groups provide it stable bond to the zirconium interface by multipoint attachment and allow high density of surface coverage of the linkers as revealed by X-ray photoelectron spectroscopy (XPS). Reversible high-density capture of histidine-tagged proteins is shown by real-time surface plasmon resonance enhanced ellipsometry and in a microarray format using fluorescence detection of AlexaFluor 647-labeled target protein. The detection sensitivity of the microarray for the target protein wits below 1 nM, despite the monolayer arrangement of the probes, due 10 very low background staining, which allows high fluorescent signal-to-noise ratio. The performance of these Ni-NTA-modified zirconium phosphonate coated slides compared favorably to other types of microarray substrates, including slides with a nitrocellulose-based matrix, epoxide slides, and epoxide slides functionalized with Ni-NTA groups. This immobilization strategy has a large potential to fix any histidine-tagged proteins on zirconium or titanium ion surfaces.