Bisphosphonate Adaptors for Specific Protein Binding on Zirconium

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Bisphosphonate Adaptors for Specific Protein Binding on Zirconium Phosphonate-based Microarrays Mathieu Cinier,† Marc Petit,‡ Monique N. Williams,§ Roxane M. Fabre,§ Fre´de´ric Pecorari,† Daniel R. Talham,§ Bruno Bujoli,‡,* and Charles Tellier†,* Laboratoire de Biotechnologie, Biocatalyse et Biore´gulation, UFR Sciences et Techniques, Universite´ de Nantes, CNRS, UMR 6204, 2, rue de la Houssinie`re, BP 92208, 44322 NANTES Cedex 3, France, Chimie Et Interdisciplinarite´: Synthe`se Analyse Mode´lisation (CEISAM), Universite´ de Nantes, CNRS, UMR 6230, 2 Rue de la Houssinie`re, BP92208, 44322 Nantes Cedex 03, France, and Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200. Received June 16, 2009; Revised Manuscript Received September 29, 2009

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.

INTRODUCTION Proteins play a key role in cellular mechanisms, but most of their functions remain unknown (1). Proteomics, which is the study of protein function, expression, and localization on a cellular-wide scale, needs more high-throughput analysis tools to achieve this goal. By analogy to work in genomics with DNA microarrays (2, 3), protein capture microarrays have been used successfully to identify differentially expressed biomarker proteins (4-8). However, it is still an emerging technology, which needs selective methods for surface immobilization (1, 9) and alternative strategies to antibodies for use as capture agents in protein microarrays (10, 11). A significant challenge for protein microarrays is attachment on the chip surface. Immobilization of the protein by nonspecific adsorption is often associated with problems such as a high background signal and significant loss of the protein probes during stringent washes (12). Furthermore, random orientations of the immobilized proteins affect their activities, which decrease the sensivity of detection (13, 14). Therefore, specific protein attachment through covalent coupling (15-22) or affinity interaction (23-27) is considered to be a better strategy (13). In the present work and in other studies (28-31), specific orientation of capture proteins is achieved by affinity interaction between a histidine-tag protein and a transition metal chelated * To whom correspondence should be addressed. E-mail addresses: [email protected]; [email protected] † Laboratoire de Biotechnologie, Biocatalyse et Biore´gulation, Universite´ de Nantes. ‡ Chimie Et Interdisciplinarite´: Synthe`se Analyse Mode´lisation, Universite´ de Nantes. § University of Florida.

to a nitrilotriacetic acid (NTA) moiety. Ni-NTA resins (32-34) are routinely used for the purification of oligo-histidine-tagged proteins, which can be easily prepared by genetic fusion of an oligohistidine tag to the C- or N-terminus. Despite the moderate affinity (Kd ≈ 1-10 µM) (35-37) and stability of individual Ni-NTA/oligohistidine complexes, this strategy has been successfully used in a microarray format for the immobilization of about 6000 histidine-tag-labeled yeast proteins, of which more than 80% were shown to retain their biological activity after immobilization (38). However, most NTA surfaces used for the design of protein microarrays are obtained by spreading a functionalized polymer matrix on a glass surface, leading to inhomogeneous surface coatings without rigorous control of the NTA density. In previous work on DNA microarrays, our laboratories described a new ultraflat support with which the biological probes are bound to a monolayer-coated surface through an “inorganic” linkage, in contrast to existing systems based on the covalent attachment of the DNA probes via covalent organic bonds. We demonstrated the spontaneous linkage of oligonucleotide probes, phosphorylated at their 5′ end, onto a zirconium phosphonate modified surface (39). More recently, we demonstrated that the phosphate/zirconium bond is strong enough for dsDNA attachment and for studying DNA/protein interactions (40). The zirconium-phosphonate linkage has been shown to be very robust between pH 1 and pH 10 and stable to displacement by competing bases (39). On the other hand, unmodified proteins are poorly adsorbed on such zirconium phosphonate surfaces and oligo-histidine-tagged proteins do not provide specific binding on these surfaces. In order to provide specific anchoring of oligo-histidinetagged proteins to the zirconium phosphonate surface, we

10.1021/bc9002597  2009 American Chemical Society Published on Web 11/23/2009

Protein Binding on Zr/Phosphonate-Based Arrays

designed a bifunctional adaptor containing a multivalent phosphonic acid anchor at one extremity and a NTA group at the other. The phosphonate groups provide a stable bond to the zirconium interface by multipoint attachment. This affinity tag strategy provides a uniform orientation of proteins on the surface and high-density coverage without the need to perform complicated chemistry on the protein targets or on the solid support. Stable binding of the bifunctional adaptor is demonstrated, allowing reversible capture of histidine-tagged proteins. This technology is applied to a new class of small and stable capture proteins, the affitins (41, 42), which are shown to keep their binding properties when immobilized on the zirconium phosphonate surface and to exhibit a high signal-to-noise ratio relative to arrays prepared from other NTA-functionalized supports.

EXPERIMENTAL PROCEDURES Materials. Glass slides were purchased from Gold Seal Products (cat no. 3013, 3 × 1 in.2, thickness 0.93-1.05 mm). Reagents were of analytical grade and used as received from commercial sources, unless indicated otherwise. Hydrophobic glass slides were made using octadecyltrichlorosilane (OTS) following a method by Sagiv (43). The zirconium octadecylphosphonate Langmuir-Blodgett monolayers were prepared on the hydrophobic slides as described previously (44, 45). Nexterion Slides E were obtained from Schott. FASTslide coated with nitrocellulose were obtained from Whatman/Schleicher & Schuell.NR,NR-bis(carboxymethyl)-L-lysinehydrateandlyzozyme were purchased from Sigma Aldrich. Synthesis of the Mono-NTA and Bis-NTA Bisphosphonate Adaptors. The synthesis of the monovalent and multivalent linkers is outlined in Scheme 1; the details are given in Supporting Information. Protein Expression and Purification. H4 and B3 are recombinant proteins derived from Sac7d from Sulfolobus acidocaldarius and evolved by ribosome display to bind lysozyme (41, 42). H4 was overexpressed in a recombinant Escherichia coli BL21 strain carrying the corresponding plasmid. The culture was grown in Luria-Bertoni medium containing ampicillin (50 µg/mL) at 37 °C until an OD600 of 0.8-1. Induction using isopropyl-1-β-D-thio-1-galactopyranoside (IPTG) (0.5 mM) was continued overnight. The protein was purified on nickel-nitrilotriacetic acid (Ni-NTA) columns according to the manufacturer’s recommendations (Qiagen, Courtaboeuf, France), eluting with a TBS 250 buffer (20 mM TrisHCl, pH 7.4, 150 mM NaCl, 250 mM imidazole). The protein concentration was measured using a Nanodrop 1000 spectrophotometer (Thermoscientific). The same procedure was applied to B3. Microoarray Spotting and Incubation Conditions. To prepare the LB Ni-NTA substrate, the zirconium phosphonate modified slides were incubated overnight with a 1 mM monoNTA (13) or bis-NTA (18) adaptor aqueous solution. For the Nexterion Slide E Ni-NTA substrate, Nexterion Slides E were incubated overnight with a 3 mM solution of NR,NR-bis(carboxymethyl)-L-lysine hydrate in PBS (12 mM phosphate, pH 8.3, 150 mM NaCl). Slides were washed 3 times with ultrapure water and then incubated for 1 h with a 100 mmol L-1 NiCl2 aqueous solution. The slides were washed again 3 times with ultrapure water and dried by centrifugation (1500 rpm, 1 min). The slides were then spotted with a microcaster TM 8-pin system (Whatman/Schleicher & Schuell). The spotted slides were placed overnight in an incubation chamber at 4 °C and 75% humidity. To passivate the unspotted areas, slides were treated after spotting with a solution of 0.3% R-casein (Sigma) in a TBS (Tris-buffered saline) solution of TrisHCl (20 mM) and NaCl (150 mM) at pH 7.4. Incubation was performed by applying an Alexa 647-labeled lysozyme solution (1 µM in

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TBS-0.3% R-casein) to the substrate for 1 h at room temperature. Microarrays were washed 3 times with TBS-0.05% Tween 20 for 5 min, then once with TBS and ultrapure water. Finally, the slides were spun dry centrifuging at 1500 rpm for 1 min. All washes and incubations were performed in small staining jars at room temperature on an oscillating shaker. Microarray Analysis. All microarrays were scanned on a Scanarray Gx apparatus (Perkin-Elmer) with a laser power and gain value of 60. Suitable excitation wavelength and emission filter were used to detect Alexa 647: 650 nm (excitation), 665 nm (emission). The location of each analyte spot on the array was outlined using the mapping software Genepix (Axon laboratories, Palo Alto, CA). X-ray Photoelectron Spectroscopy (XPS). XPS was performed using a UHV XPS/ESCA PHI 5100 system. Survey scans and multiplex scans (Zr 3d, P 2p, N 1s, Ni 2p3) were taken with an Al KR X-ray source (using a power setting of 300 W and a takeoff angle of 45° with respect to the surface). Survey scans were taken for all samples with a pass energy of 89.4 eV and multiplex scans were taken with a pass energy of 22.36 eV. The peak areas were determined using commercial XPS analysis software with Shirley background subtraction. Elemental ratios reported in the text are averages of at least three experiments. Slides for XPS analysis containing the NTA linkers were prepared using a Whatman Fast-Frame slide plate and a twopad incubation chamber. One pad of the zirconium phosphonate modified slide was immersed in 1 mL of 1 mM biphosphonate adapter, while the other pad was immersed in 1 mL of nanopure water as an experimental control. The slide was kept at 4 °C overnight, rinsed with water, and then deposited in a solution of 100 mM NiCl2 for 1 h. Finally, the slide was then rinsed with nanopure water and spin-dried in air for analysis. Surface Plasmon Resonance (SPR) Spectroscopy. Surface plasmon resonance enhanced ellipsometry measurements were performed on a commercial EP3-SW imaging system (Nanofilm Surface Analysis, Germany). The ellipsometer employed a frequency-doubled Nd:YAG laser (adjustable power up to 20 mW) at 532 nm. Linearly p-polarized light was directed through a 60° equilateral SF10 prism coupled to a gold-coated SF10 slide via diiodomethane index matching oil in the Kretschmann configuration. The angle of incidence was kept at 64° for all kinetics experiments because this condition provided the highest sensitivity. Curve fitting of the kinetics data used the AnalysR software from Nanofilm, using a 1:1 Langmuir binding model. SPR slides were prepared by rendering the gold-coated slides hydrophobic with octadecylmercaptan before transferring the zirconium phosphonate layer. The slides were then immersed overnight in 1 mM aqueous solutions of either the mono-NTA linker or the bis-NTA linker, followed by rinsing with ultrapure water and drying under a stream of nitrogen. The NTA-modified slides were placed in the SPR flow cell and treated with a 100 mM solution of nickel chloride. Protein binding onto the NiNTA surface was carried out by injecting 5 µM protein solutions in TBS. The surface was regenerated using 250 mM imidazole in TBS buffer.

RESULTS AND DISCUSSION Design of the Anchoring Linker. In previous studies, we showed that oligonucleotides and dsDNA can be immobilized on zirconium phosphonate surfaces and that phosphateterminated oligonucleotides are selectively adsorbed over those without terminal phosphates (39, 40). It was also shown that dsDNA phosphorylated at both ends binds better to the zirconium surface than monophosphorylated DNA. Based on these results, in order to make the zirconated phosphonate film suitable for oligohistidine-tagged protein

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Scheme 1. Synthesis of the Mono-NTA and Bis-NTA Bisphosphonate Adaptors

immobilization, surface modification with either mono-NTA or bis-NTA end groups was performed using adaptors 13 and 18, respectively, which contained a similar scaffold bearing two phosphonic acid functions for binding to the zirconated surface (Scheme 1A,B). These two adaptors were synthesized in around 15 steps in good yield. First benzylamine was alkylated twice using compound 1 synthesized by reacting 4-chloro-1-butanol with 3,4-dihydro-2H-pyran to yield compound 2. After hydrogenolysis, compound 3 was reacted with succinimide to produce the acid compound 4. Reaction with p-toluene sulfonic acid in ethanol resulted in the esterification of the carboxylic acid function and removal of the THP protective groups. The two alcohol groups were then activated (mesylate) and substituted by bromine atoms, which were subsequently replaced by diethyl phosphonate groups using sodium salt of diethylphosphite to

yield compound 8. Finally, saponification of the ester function led to compound 9, which was then coupled to the mono-NTA or bis-NTA precursors 11 and 16, respectively, as described in Scheme 1B, to obtain the two expected bisphosphonate monoand bis-NTA adaptors 13 and 18. Compounds 11 and 16 were derived from L-lysine, as previously reported by Lata et al. (46). Similar adaptors with only one phosphonic acid moiety were found to bind poorly to the zirconated surface and were inadequate for the present application. Zr/phosphonate Surfaces and Linker Anchoring. Zirconium phosphonate modified surfaces are generated by adsorbing Zr4+ ions to surface phosphonate or phosphate groups. An active metal layer results when the phosphorylated groups are closely organized into a monolayer so that the Zr4+ ions bind to the surface groups to form a layer while retaining some coordination



Figure 1. Peaks of Ni(2p3), Zr(3d), and P(2p3) obtained from XPS analysis for (a) zirconium phosphonate monolayer surface, (b) mono-NTA modified surface, and (c) bis-NTA modified surface. No Ni(2p3) is seen on the bare zirconium phosphonate surface.

sites that are free of the surface phosphate or phosphonate ligands. The starting phosphorylated surfaces can be prepared in different ways, including covalent attachment of the phosphorylated groups to silica (47-54) or gold (49, 50, 53, 55, 56) and by Langmuir-Blodgett deposition of an organophosphonic acid (44, 45, 57-60). We have long utilized LB methods to prepare the zirconium phosphonate monolayers because the surface films formed in this way are of high quality, stable, and highly reproducible, and the surface chemistry can be applied to nearly any substrate material, permitting application of a wide range of surface analytical techniques on the same surface (61) Modification of the zirconium phosphonate surface was thus investigated by incubating the substrate in a 1 mM aqueous solution of the desired bisphosphonate adaptor (13 or 18), the binding of which was then monitored by XPS. XPS is a powerful surface analytical technique, which provides qualitative and quantitative information about the elements on a surface, and was recently shown to be an efficient, label-free method for studying DNA surface coverage on zirconium phosphonate surfaces (62, 63). The P/Zr ratio of 1.1 for the naked zirconated phosphonate monolayer indicates that it is almost fully reticulated by the zirconium ions (44). Upon reaction with adaptor 13, a significant increase of this ratio was observed (P/Zr ) 1.4), which confirms binding of the bisphosphonate on the surface (Figure 1). Knowing the surface density of the zirconium ions within the monolayer, 4.2 × 1014 atoms/ cm2 (44), the mono-NTA adaptor coverage can be estimated at 6.3 × 1013 molecules/cm2, which corresponds to one NTA moiety every 1.58 nm2. With the bis-NTA adaptor (18), a P/Zr ratio of 1.18 was determined, which indicates half the surface coverage observed for the mono-NTA molecule. However, because this adaptor presents two NTAs per molecule, the NTA density on the surface is roughly similar to that obtained with the mono-NTA adaptor. Nickel complexation to the NTA end groups was then performed to make them capable of covalent binding to histidine-tagged proteins. In Figure 1, XPS spectra of monoNTA and bis-NTA coated slides are compared with the bare zirconium phosphonate surface, after exposure to Ni2+. Binding to the NTA-coated slides is evident, with more Ni2+ complexed to the bis-NTA coated slide. Protein Immobilization. The octadecylphosphonic acid (OPDA)-Zr surfaces modified with mono-NTA and bis-NTA end groups were used to study the specificity and reversibility of their interaction with histidine-tagged proteins as well as the

proteins’ activity when immobilized. For this purpose, monomeric proteins (affitins) with a single N-terminal oligohistidine tag were used. These affitins were derived from the Sac7d scaffold and selected by ribosome display to selectively recognize lysozyme (41, 42). Binding of the H4 affitin on the zirconium phosphonate monolayer functionalized with nickelloaded NTA groups was first studied using SPR (Figure 2). As a control experiment, low and totally reversible binding of the protein was observed in the absence of nickel complexation to the NTA group (Figure 2a). In the case of both adaptors 13 and 18, the surface exhibits a high and specific binding of the histidine-tagged-affitin (Figure 2b,c). A higher phase shift amplitude was observed for the binding of the affitin to the mono-NTA adaptor (∆∆ψ ≈ 0.22°) than for the bis-NTA (∆∆ψ ≈ 0.17°), reflecting a higher amount of bound protein using the mono-NTA adaptor. This result can be correlated to the higher surface coverage with this adaptor. However, a significant dissociation of the affitin was observed with the mono-NTA linker upon prolonged washing (∼1 h) (Figure 2b), while washing out of the protein was reduced in the case of the bisNTA linker (Figure 2c). This observation reflects the intrinsically low affinity of mono-NTA toward a polyhistidine sequence (Kd ≈ 10-5 to 10-6 M), which can be improved when bis-NTA functional groups are used (29). After washing, the amount of the proteins retained on the surface was similar for both adaptors. The specificity of the interaction is confirmed by the full reversibility of binding upon injection of a high concentration of imidazole (Figure 3). The protein loading followed by imidazole washing cycle can be repeated without a decrease in binding amplitude, as shown in Figure 3 for two cycles. Microarray Experiments. Having shown that both linkers 13 and 18 can be efficiently immobilized on the zirconium phosphonate surface, making it suitable for histidine-tagged protein immobilization, the next step was to verify that the immobilized proteins are still functional and capable of binding lysozyme. Affitin immobilization on a microarray format was carried out according to Scheme 2. The nickel-loaded NTA functionalized slides were spotted with affitin at fixed concentrations prior to a blocking step with R-casein, which due to its high phosphate content provides efficient saturation of the nonspotted areas, hindering nonspecific protein binding (40). The activity of the immobilized affitin was investigated upon incubation of the surface with AlexaFluor 647 labeled lysozyme overnight. To verify that immobilization of the affitin proceeds via the expected Ni-NTA/histidine-tag interaction, the same


Figure 2. Affitin immobilization on the zirconium phosphonate surface functionalized with mono- and bis-NTA, probed in real time by surface plasmon resonance enhanced ellipsometry (SPREE). Kinetic adsorption curves of affitin on (a) mono-NTA without nickel, (b) mono-NTA with nickel, or (c) bis-NTA with nickel. Experimental data (dotted line) were fitted (solid line) using a 1:1 Langmuir binding model for each step. Curves b and c were shifted by 0.05° and 0.2°, respectively.

Figure 3. Sensorgram for the interaction of affitin with a mono-NTA functionalized surface that shows binding of Ni2+, adsorption and desorption of affitin, and regeneration of the surface by 250 mM imidazole. This procedure was then repeated to show the reproducibility of the surface. Experimental data (dotted line) were fitted (solid line) using a 1:1 Langmuir binding model for each step.

experiment was performed on the naked zirconium octadecylphosphonate surface. To avoid interslide variation, each slide had only half of its surface functionalized with Ni-NTA groups, and incubation chambers with two compartments were used. As an additional control, the affitins were spotted under two different conditions, one in a 25 mM imidazole-containing buffer to reduce nonspecific bonding to the Ni-NTA surface and the second in a 250 mM imidazole containing buffer to prevent histidine-tagged protein binding by competition (Figure 4). A negative control experiment was also performed with an affitin

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that does not bind lysozyme. At low imidazole concentration, only spots present on the surface functionalized with the NiNTA groups gave high signal intensities, which indicated that affitins were still functional after immobilization. As expected, a high concentration of imidazole (250 mM) prevented affitin binding on the Ni-NTA surface and subsequent lysozyme capture. Interestingly, a very low background value was observed in the absence of affitin, confirming the low nonspecific interaction of lysozyme with the surface when saturated with R-casein. However, nonspecific binding of the affitin on the naked zirconated surface was not completely avoided by R-casein saturation since a low intensity signal was detected in that control. Different spotting concentrations of the affitins were explored, incubating at a fixed concentration of the lysozyme target. An increase in signal intensity was detected, with no saturation of the lysozyme capture, using affitin concentrations of 1.25-10 µM (Figure 5), confirming the high density of Ni-NTA groups available on the surface of the slides. Both bisphosphonates 13 and 18 led to similar fluorescence intensities at a given affitin concentration, in agreement with SPR data, which had shown that the amount of bound protein after washing is similar with the two linkers. However, the spot shape remains better defined on the bis-NTA modified surface. On the mono-NTA functionalized surface, the spot shape was irregular if affitin was spotted at high concentration, likely as a result of significant dissociation occurring during the washing steps. In the case of the bis-NTA functionalized surface, stronger binding of the histidine tag by a bivalent interaction strongly reduces this effect (Figure 5). The sensitivity of the microarrays was explored by spotting with a constant affitin concentration and incubating with decreasing concentrations of AlexaFluor 647-labeled lysozyme, using the same slide divided into several independent incubation areas. For this experiment, two affitins differing in their affinity for lysozyme were chosen (H4, Kd ) 5 × 10-9 M, and B3, Kd ) 2.9 × 10-7 M) (see Supporting Information). For the H4 affitin, the observed limit of detection was below 1 nM (Figure 6). As expected, the slide spotted with B3 affitin, which has a lower affinity for lysozyme, exhibited a detection limit around 0.1 µM, with low sensitivity even at high lysozyme concentration, likely because the weakly associating B3 lysozyme is lost during the washing process. Comparison with Other Microarray Surfaces. The performance of our Ni-NTA-modified zirconium phosphonate surface was evaluated relative to other types of microarray substrates, including slides coated with a nitrocellulose-based matrix (FAST slides, Schleicher & Schuell), epoxide slides (Nexterion, Schott), and epoxide slides functionalized with NiNTA groups using NR,NR-bis(carboxymethyl)-L-lysine hydrate. All slides were spotted with the same affitin concentrations and incubated with a fixed concentration of the labeled lysozyme. The resulting fluorescence intensities are reported in Figure 7.

Scheme 2. Description of the Different Steps Employed To Test Affitin Activity on a Microarray Platform


Figure 4. Detection of lysozyme captured by affitin spotted at 1 µM on a Ni-NTA-modified zirconium phosphonate microarray: white bar, buffer spotted only; light gray bar, affitin spotted with 250 mM imidazole; dark gray bar, affitin spotted with 25 mM imidazole. Fluorescence intensities were measured at 90% laser power and 50% photomultiplier gain.

Figure 5. Fluorescence intensity versus concentration of spotted affitin on zirconium phosphonate slides functionalized with mono-Ni-NTA (dark gray) and bis-Ni-NTA (light gray) groups. Fluorescence data values correspond to the mean and the ecart type range for three replicates within one slide (inset) image obtained at 60% laser power and 60% photomultiplier gain.

Slides coated with nitrocellulose membrane gave the highest fluorescence signal, making it necessary to use a lower laser power in this case to avoid excessive background. However, even at lower laser power, the background signal is still high, resulting in a low signal-to-noise ratio (S/N ) 1.5). It is known that this polymeric material with defined microporosity binds large amounts of protein in a noncovalent and nonspecific way (13). The poor signal-to-noise ratio could be related to the nature of the affitin, which is very small, and possibly easily washed out during the incubation process. Slides modified with a chemical reactive group, such as an epoxide, provide a covalent yet random immobilization of the proteins via reaction with amino groups present on the protein surface. At the highest concentration of spotted protein (10 µM), the fluorescence intensity using the epoxide E slide is about 50% lower than that obtained using the zirconium phosphonate slides functionalized with Ni-NTA. Somewhat surprisingly, when the epoxide E slides were functionalized with Ni-NTA groups using NR,NR-bis(carboxymethyl)-L-lysine hydrate, the observed fluorescence intensities were comparable to those obtained for the unmodified E slides. Although modifying the epoxide slides with Ni-NTA groups should provide an oriented immobilization of the affitins, similar to the Ni-NTA-functionalized zirconium phosphonate surface, the observed fluorescence intensity was lower throughout the 1.25-10 µM spotting concentration range. A possible reason might be that the


Figure 6. Fluorescence intensity versus concentration of AlexaFluor 647-labeled lysozyme in the incubation solution. Affitins H4 (black line) and B3 (gray line) were spotted (5 µM) on a mono-Ni-NTAfunctionalized ODPA/Zr surface. The gray dotted line corresponds to spotting of buffer.

Figure 7. Compared performances for lysozyme capture of H4 affitin immobilized on different substrates: slide E, epoxide surface from Nexterion; slide E-NTA, Ni-NTA functionalized epoxide surface; monoNTA, mono Ni-NTA functionalized ODPA/Zr surface; FAST slide. The / indicates the fluorescence intensities were measured at lower laser power (30) and gain value (40) with the FAST slide.

zirconium phosphonate surface provides a higher density of NTA groups compared with the epoxide surface. Alternatively, the long spacer separating the NTA group from the phosphonic acid groups attached to the zirconium phosphonate surface may provide better accessibility to the proteins.

CONCLUSION In this paper, we demonstrate that a zirconium phosphonate modified surface can be efficiently functionalized with metal NTA groups in order to specifically bind histidine-tagged proteins in an oriented way. The NTA linkers bind the zirconated surface via a bis-phosphonic acid functional group with high avidity for the surface. These results further extend to the protein microarray field the applications of mixed organic/inorganic zirconium phosphonate surfaces, on which simple and irreversible modifications can be introduced without the need for chemical activation steps. By use of these bifunctional adaptors, a high density of accessible NTA groups was obtained, allowing high efficiency of protein binding. Proteins immobilized in this way retain their ability to capture protein target with high sensitivity, and the modified zirconium phosphonate surfaces were shown to compete favorably with commercially available substrates designed for protein microarrays. Finally, we also show that the affitin scaffold (41, 42) can be used as a capture agent on different types of microarray slides. The small size of this scaffold (66 aa), which has been evaluated as an alternative to antibodies, allows a high density of spotting and consequently a high level of specific activity of the surfaces.


ACKNOWLEDGMENT This work was partially supported by grants (Nos. 04.04.025 and 2008.34.0010) from the De´le´gation Ge´ne´rale a` l’Armement (DGA). M.C. was supported by a grant from the DGA. Partial support for this work was provided by the US National Science Foundation through Grant Number CHE-0514437 (D.R.T.). Supporting Information Available: Detailed description of the synthesis, purification, and spectroscopic characterization of the mono- and bis-NTA bisphosphonate adaptors (13 and 18) and determination of the affitin dissociation constants by SPR measurement. This material is available free of charge via the Internet at http://pubs.acs.org.

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Bisphosphonate Adaptors for Specific Protein Binding on Zirconium

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