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Bioconjug Chem. 2009 Dec;20(12):2270-7. doi: 10.1021/bc9002597.

Bisphosphonate adaptors for specific protein binding on zirconium phosphonate-based microarrays.

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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.


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.

[Indexed for MEDLINE]

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