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

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Archaeal Proteins / chemistry*
  • Binding Sites
  • Enzymes, Immobilized / chemistry
  • Histidine / chemistry
  • Membranes, Artificial
  • Muramidase / chemistry
  • Nickel / chemistry
  • Nitrilotriacetic Acid / analogs & derivatives
  • Nitrilotriacetic Acid / chemical synthesis
  • Nitrilotriacetic Acid / chemistry*
  • Organophosphonates / chemistry*
  • Particle Size
  • Protein Array Analysis*
  • Recombinant Proteins / chemistry
  • Sulfolobus acidocaldarius / chemistry
  • Surface Properties
  • Zirconium / chemistry*


  • Archaeal Proteins
  • Enzymes, Immobilized
  • Membranes, Artificial
  • Organophosphonates
  • Recombinant Proteins
  • Histidine
  • Nickel
  • Zirconium
  • Muramidase
  • Nitrilotriacetic Acid