Bisphosphonate Adaptors for Specific Protein Binding on Zirconium Phosphonate-based Microarrays

Abstract

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.

Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold

“…40 A lysozyme-binding protein with a K D of 5 nM has been isolated from a library of ∼ 10 12 mutants derived from the nearly identical Sac7d scaffold, using ribosome display. 41 By contrast, the lysozyme-binding Sso7d mutant isolated in this study using yeast surface display has a K D of ∼ 350 nM. Nevertheless, the yeast display system provides certain key advantages, such as the use of flow cytometric sorting, the ability to discriminate between clones with small differences in binding affinity and ease of quantitative determination of binding affinities (K D ) through cell surface titrations.…”

Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

“…In this case, the MO6 are octahedrally coordinated by oxygen atoms, with the three oxygens of each phosphonate bound to three different zirconium ions 31 . Notably, the unique coordination of Zr 4 + or Ti 4 + with the phosphate/phosphonate group has been shown to have a richness of applications, including DNA microarrays 32 , protein microarrays 33 and self-assembled monolayers 34 . We recently developed a new generation of IMAC for phosphopeptide enrichment [35][36][37][38][39] by using the unique property of Zr(IV) or Ti(IV) phosphate/ phosphonate chemistry.…”

Straightforward Micropatterning of Oligonucleotides in Microfluidics by Novel Spin-On ZrO2 Surfaces

“…On the other hand, some of the main limitations of widespread technologies for oligonucleotide positioning and micropattern fabrication, such as automated microspotting, microcontact printing 21,22 or Dip-Pen lithography 23 , are overcame with the presented strategy, with particular regard to their high cost, low accuracy of positioning, small area covering and low robustness in the case of microcontact printing. 24,25 A second issue concerns the characteristics of the engineered photosensitive ZrO 2 material, which allows to exploit a functionalization protocol based on phosphate groups and the Zr surface chemistry, already described in literature 4,7,26,27 ; generally the reported materials, sometimes made by sol-gel route, are no photosensitive or require multistep depositions. Our approach offers a straightforward functionalization method with respect to some of the current bio-grating procedures.…”

“…This outcome was achieved thanks to the naturally occurring terminal 5'-phosphate groups in DNA strands ( Figure 1c) and exploiting the surface specificity and affinity between the Zr atoms and phosphate groups. 4,7,26,27 To the best of our knowledge, the use of a spin-on ZrO 2 material which can be both patterned and functionalized in a direct and straightforward process with different oligonucleotides and is 5 easily integrated in microfluidic platform has been investigated and demonstrated for the first time. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 6 Synthesis of ZrO 2 …”

“…The anchoring of phosphate compounds on the zirconium oxide surface first proceeds by a reversible noncovalent interaction with the adsorbed H 2 O, OH, and oxo groups covering the surface, followed by formation of strong highly covalent bonds resulting from the displacement of oxo and hydroxyl groups by the phosphate oxygen atoms. 27 This mechanism can explain the stability of interaction between the phosphorylated groups and the ZrO 2 surface.…”