Laboratoire de Biotechnologie, Biocatalyse et Bioregulation, UFR Sciences et Techniques, Universite de Nantes, CNRS, UMR 6204, 2 Rue de la Houssiniere, BP 92208, 44322 NANTES Cedex 3, France.
Bioconjug Chem. 2009 Dec;20(12):2270-7. doi: 10.1021/bc9002597.
Two bisphosphonate adaptors were designed to immobilize histidine-tagged proteins onto glass substrates coated with a zirconium phosphonate monolayer, 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 a 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 was below 1 nM, despite the monolayer arrangement of the probes, due to 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.
设计了两种双膦酸盐接头，将组氨酸标记的蛋白质固定在涂有锆膦酸盐单层的玻璃基底上，允许亲和素（针对溶菌酶的亲和素）在微阵列形式上有效地定向固定在捕获蛋白上。这些双功能接头在一端包含两个磷酸基团锚，在另一端包含一个或两个亚氨基二乙酸（NTA）基团。磷酸基团通过多点附着提供与锆界面的稳定键合，并允许通过 X 射线光电子能谱（XPS）揭示的接头的高表面覆盖率。通过实时表面等离子体共振增强椭圆光度法和使用 AlexaFluor 647 标记的靶蛋白荧光检测，在微阵列形式上显示出组氨酸标记的蛋白质的可逆高密度捕获。尽管探针呈单层排列，但由于背景染色非常低，微阵列对靶蛋白的检测灵敏度低于 1 nM，这允许高荧光信号与噪声比。这些 Ni-NTA 修饰的锆膦酸盐涂层载玻片的性能与其他类型的微阵列基底相比具有很大的优势，包括基于硝酸纤维素的基质载玻片、环氧化物载玻片和用 Ni-NTA 基团功能化的环氧化物载玻片。这种固定化策略具有将任何组氨酸标记的蛋白质固定在锆或钛离子表面上的巨大潜力。
