Bisphosphonate adaptors for specific protein binding on zirconium phosphonate-based microarrays.
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.
Cinier
Mathieu
M
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.
Petit
Marc
M
Williams
Monique N
MN
Fabre
Roxane M
RM
Pecorari
Frédéric
F
Talham
Daniel R
DR
Bujoli
Bruno
B
Tellier
Charles
C
D019843:Archaeal Proteins / Q000737:chemistry*; D001665:Binding Sites; D004800:Enzymes, Immobilized / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008567:Membranes, Artificial; D009113:Muramidase / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry*; D063065:Organophosphonates / Q000737:chemistry*; D010316:Particle Size; D040081:Protein Array Analysis; D011994:Recombinant Proteins / Q000737:chemistry; D016945:Sulfolobus acidocaldarius / Q000737:chemistry; D013499:Surface Properties; D015040:Zirconium / Q000737:chemistry*
D019843:Archaeal Proteins; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D063065:Organophosphonates; D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel; D015040:Zirconium; D009113:Muramidase; D009571:Nitrilotriacetic Acid