Diazo Coupling Method for Covalent Attachment of Proteins to Solid

Bioconjugate Chem. 2006, 17, 359−365

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Diazo Coupling Method for Covalent Attachment of Proteins to Solid Substrates Yang Wu,† Tione Buranda,‡ Robert L. Metzenberg,§ Larry A. Sklar,‡ and Gabriel P. Lopez*,† Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico 87131, Department of Pathology and Cancer Research Facility, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, and Department of Biological Sciences, Stanford University, Stanford, California 94305. Received September 16, 2005; Revised Manuscript Received February 6, 2006

We describe a process for covalently linking proteins to glass microscope slides and microbeads in a manner that optimizes the reactivity of the immobilized proteins and that is suitable for high-throughput microarray and flow cytometry analysis. The method involves the diazo coupling of proteins onto activated self-assembled monolayers formed from p-aminophenyl trimethoxysilane. Proteins immobilized by this method maintained bioactivity and produced enhanced levels of protein-protein interaction, low background fluorescence, and high selectivity. The binding of immobilized proteins to their specific binding partner was analyzed quantitatively and successfully correlated with solution concentrations. Diazotized surfaces bound more efficiently to proteins containing a hexahistidine tag than those without a his-tag. Moreover, significantly higher reactivity of the immobilized histagged proteins was observed on diazotized surfaces than on amine-terminated surfaces. Results suggest that his-tagged proteins are immobilized by reaction of the his-tag with the diazotized surface, thus offering the possibility for preferential orientation of covalently bound proteins.

INTRODUCTION Understanding of the functions, modification and regulation of proteins will continue to improve our knowledge of complex biological systems (1-4). Because each biological system can include thousands of different proteins, the ability to simultaneously analyze proteins by a cost-effective and reproducible methodology has become extremely attractive (5). Protein arrays that provide high-throughput, quantitative measurement of receptor-ligand complexation are finding applications broadly in proteomics (6, 7), drug development (8, 9), and diagnostics (10, 11). Two popular array formats, planar substrates used in high-throughput microarray technology (12-15), and microparticle-based assays that can be used for high-throughput flow cytometry (16-18), have been developed for these applications. Both type of arrays are fabricated by immobilizing receptor proteins onto a solid substrate. The reactivity of the receptor proteins, which can directly affect the sensitivity and specificity of the array, is largely dependent on the immobilization chemistry (1, 19). There are several substrates commonly used in the fabrication of protein arrays. These include aldehyde-containing silane surfaces (i.e. SuperAldehyde substrate from TeleChem, International Inc.) that react with primary amines on the proteins, amine-terminated surfaces (i.e. GAPS-coated slides from Corning) that carry a positive charge at a neutral pH and will form ionic bonds with negatively charged amino acid residues, Nicoated surfaces that will specifically bind proteins engineered with histidine tags (4, 6, 20), BSA-N-hydroxysuccinimide modified substrates (3, 12), and glyoxylyl-modified surfaces (13). A detailed comparison of several methods for protein immobilization is found in Seong’s work (21). However, * To whom correspondence should be addressed. Tel: (505) 2774939; Fax: (505) 277-5433; E-mail: [email protected]. † Department of Chemical and Nuclear Engineering, University of New Mexico. ‡ Department of Pathology and Cancer Research Facility, University of New Mexico Health Sciences Center. § Stanford University.

because proteins have a heterogeneous structure, and are very likely to denature or lose activity after immobilization, the search for a suitable solid support for protein arrays remains active (1, 22). The optimization of the orientation and stability of immobilized receptor proteins, which will likely affect binding efficiency and specificity of the immobilized proteins, is one of the issues that remains to be addressed. We describe a chemical process for covalently linking proteins onto ordinary microscope slides and glass microbeads in a manner that preserves their activity. We have previously used diazo coupling chemistry to covalently immobilize nucleic acids to activated self-assembled monolayers (SAMs)1 formed from p-aminophenyl trimethoxysilane (ATMS) in the manufacture of DNA microarrays(23). Our goal in this study is to extend the application of this method to covalently couple proteins to glass substrata. Although the application of diazo coupling reaction for biomolecular immobilization can be traced back to the late 1970s when Stark et al. coupled single-standed DNA on diazotized Whatman paper (23), and has been extended to coupling proteins with cellulose (to be used in affinity chromatography) (24) as well as polymer supports (25), in-depth, quantitative analysis of immobilized proteins over glass substrata has not been performed. To this end, we have characterized the binding efficiency of the substrata and the binding specificity and reactivity of the immobilized proteins. Moreover, because randomly oriented proteins may exhibit severely reduced reactivity, the possibility of directed orientation of the immobilized proteins on the surfaces is also studied. To achieve preferential orientation, hexahistidine-tagged (his-tagged) proteins were used because (a) histidine is one of two common 1 Abbreviations: ATMS, p-aminophenyl trimethoxysilane; GFP, green fluorescent protein; His6GFP, hexahistidine-tagged wild-type GFP; HRP, horseradish peroxidase; HisProbe-HRP, HisProbe horserasish peroxidase; FITC, fluorescein-5-isothiocyanate; FITC-antiHRP, FITC-conjugated affinity purified anti-horseradish peroxidase; SAMs, self-assembled monolayers; EtOH, ethanol; MeOH, methanol; BSA, bovine serum albumin; SSC, sodium citrate buffer (0.3 M sodium citrate/3 M sodium chloride); SDS, sodium dodecyl sulfate; FLAG peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys.

10.1021/bc050278m CCC: $33.50 © 2006 American Chemical Society Published on Web 02/24/2006

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amino acids that can be covalently coupled with diazotized surfaces (25, 26), (b) the attachment of a hexahistidine tag is commonly used in the production of recombinant proteins, and (c) currently there are many commercially available his-tagged proteins (27-29). Our data suggest that the process described results in preferential reaction of the his-tags on proteins with surface diazonium groups, thus enabling covalent immobilization of oriented protein layers.

EXPERIMENTAL PROCEDURES Materials. p-Aminophenyl trimethoxysilane (ATMS) (90%; Gelest Inc., PA), sodium nitrite (Sigma, St. Louis, MO), sodium acetate (Sigma), ethanol (EtOH, absolute; Aaper Alcohols and Chemical Company, KY), glycine (Aldrich, WI), 20× SSC (0.3M sodium citrate/3M sodium chloride, Sigma), and SDS (sodium dodecyl sulfate, Sigma) were used as obtained. Glass beads (4.9 µm diameter) were obtained from Duke Scientific Corp. (Palo Alto, CA). Hexahistidine-tagged wildtype green fluorescent protein (His6GFP, λex≈395 nm) and HisProbe horseradish peroxidase (HisProbe-HRP) were obtained from Pierce Biotechnology Inc. (Rockford, IL) and used without further purification. FITC-conjugated, affinitypurified anti-horseradish peroxidase (FITC-antiHRP) was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). M1 anti-FLAG antibody and FITC-FLAG (FITCDYKDDDDK) were obtained from Sigma. Unless otherwise indicated, all antibodies are monoclonal to ensure high binding specificity. All other biological reagents were obtained from Molecular Probes Inc. (Eugene, OR) and used without further purification unless specified. Protein solutions were made in Tris buffer (100 mM Tris, 150 mM NaCl, pH 7.4 to 8.0, all from Sigma) or Tris-BSA buffer (Tris buffer containing 0.1% bovine serum albumin (BSA), pH 7.5). Cleaning of Substrata. The covalent attachment of organosilanes to glass surfaces is partially dependent on the number of hydroxyl groups exposed on the substrate (30). There are several commonly used methods for cleaning silica substrata, including washes with acid or base solutions (e.g. concentrated HCl (31), NH4OH/H2O2(32), piranha(31, 33-36)), and UV/ ozone treatment (37-39). Eight different chemical cleaning methods for glass were compared by Cras et al. (30), who found that 30 min incubation in 1:1 MeOH:HCl followed by an additional 30 min incubation in concentrated H2SO4 yielded the highest concentration of surface OH groups after treatment and provided the best surface for subsequent silanization without the requirement of heating or other extreme procedures. This method was chosen as our treatment procedure. Formation of Silane Layers. After the cleaning procedure, glass slides were rinsed with deionized water (dH2O) and EtOH, dried in a stream of N2, and immersed into 3 mM ATMS in toluene for 3 h. ATMS-functionalized glass slides were then rinsed exhaustively in EtOH and dH2O, dried in a stream of N2, and stored in an airtight container at 4 °C until further use. After the cleaning procedure, glass beads were rinsed with dH2O and EtOH, centrifuged briefly at 135 000g to remove the rinse solutions, and then incubated with 3mM ATMS in toluene for 3 h with mild vortexing. ATMS-coated glass beads were then washed with EtOH and dH2O, recentrifuged, and used within 24 h. Diazotization of ATMS-Functionalized Surfaces. Before reacting with biomolecules, ATMS-coated surfaces were converted to the diazobenzyl form by treatment with a solution containing 40 mL of water, 80 mL of 400 mM HCl, and 3.2 mL of freshly prepared solution of NaNO2 (200 mM) for 30 min at 4 °C (40). The diazotized surfaces were then washed three times, each for 3 min, with ice-cold sodium acetate buffer (50 mM, pH 4.7) followed by washing with ice-cold deionized

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water and then EtOH two times each (5 min washes). The diazotized glass slides were air-dried and gently blotted with Kimwipes (Kimberly-Clark, GA) and kept at 4 °C. Diazotized glass beads were also maintained at 4 °C after discarding the wash solution. Diazotized glass slides or glass beads were used immediately for protein immobilization. Uibel and Harris have carried out quantitative studies of this diazo coupling reaction on porous silica gel, and concluded that a surface coverage of 0.36 µmol/m2 of amine group resulted in approximately 80% diazotization (41-44). We estimate a surface coverage of ≈ 2 × 107 amines per 4.9 µm diameter bead prior to diazotization (43, 44). Assuming an 80% rate of diazotization, we expect at least 1 × 107 diazonium salts per bead to be formed. Formation of Protein Microarrays. Aliquots (10 µL) of different types and concentrations of sample proteins were loaded into a 96-well microtiter plate prior to each set of experiments. These proteins were transferred onto the diazotized microscope slides using a microarrayer (Packard Bioscience, Meriden CT) at room temperature and > 40% humidity. This array-printing process was computer driven, and took ∼ 15min/ slide when employing four printing pins. Printed slides were then transferred from the microarrayer to a humid chamber and incubated for 1-4 h at room temperature before use. Printed glass slides were blocked with 1% BSA in Tris (pH 7.5) for 1-2 h and incubated with up to 1 µM solution of complementary antigens or antibodies under a glass coverslip in a humid chamber at room temperature for 2 h, followed by sequential washes in 1 × SSC + 0.1% SDS, 0.1 × SSC + 0.1% SDS, 0.1 × SSC, and dH2O. Protein-Bearing Microbeads. Suspensions containing ∼100 000 ATMS-functionalized glass microbeads in 400 µL aliquots of pH 8.0 Tris buffer were reacted with nM to 3 µM protein (streptavidin, M1 anti-FLAG antibody, or His6GFP) for 1 h at room temperature under mild vortexing. The samples were then rinsed with deionized water and resuspended in Tris buffer. The respective site densities of streptavidin, M1 antiFLAG antibody, and His6GFP were analyzed on the basis of their binding to fluorescent ligands. It is worth noting that the wild-type His6GFP obtained from Pierce has an excitation wavelength maximum of 395 nm, which is not suitable for routine flow cytometric detection. Thus a fluorescently labeled anti-GFP was employed to detect the presence of His6GFP. The experimental procedures used have been described elsewhere (45). The binding characteristics of protein-coated beads to fluorescein biotin (streptavidin beads), FITC-FLAG peptides (M1 beads), and Alexa488-antiGFP were generally determined by incubation of 40 000 protein-coated beads in 400 µL of fluorescently labeled ligand solutions (10-9 to 10-6 M) for 1-2 h, unless otherwise specified. Protein-bearing beads were exposed to 1% BSA in Tris buffer for 1 h before ligand incubation in order to reduce nonspecific binding. Measurements of fluorescence associated with samples blocked with large excesses of nonfluorescent ligands (>5-fold) before exposure to fluorescently labeled ligands were preformed in parallel to estimate nonspecific binding. We determined the amount of specific binding by subtracting signals of blocked samples from unblocked samples and then quantified the fluorescence intensity (488 nm excitation) by comparison to calibrated fluorescent beads (45, 46). For diazotized substrates, nonspecific binding was less then 30% of total binding in all experiments. Data Collection and Analysis. Protein microarrays were visualized with a confocal laser scanner, GenePix 4000B from Axon Instruments, Inc., and data were processed using the associated image-processing software (GenePix 3.0) (40). The surface density of immobilized proteins on beads was determined by centrifugation assays and flow cytometry (45, 46). The flow cytometric analysis used a Becton-Dickinson FACScan

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Figure 1. A protein microarray formed on an ATMS/diazotized glass slide. Spot sizes are about 100 µm in diameter. A. Scanned image before FITC-streptavidin incubation. Different proteins were arrayed onto the slide in duplicates of five different concentrations: Cy3-labeled anti mIgG (0.06, 0.6, 6, 60, 600 pM), biotinylated rIgG (0.06, 0.6, 6, 60, 600 pM), and biotinylated BSA (0.3, 3, 30, 300, 3000 pM). B. Scanned image of the same protein microarray after incubation with FITCstreptavidin.

flow cytometer (Sunnyvale, CA) interfaced to a Power PC Macintosh running CellQuest software package. The FACScan is equipped with a 15 mW air-cooled argon ion laser. It has been shown elsewhere (46) that the mean of the histogram is the quantity relevant to binding capacity. The average fluorescence of a single bead is converted to the number of fluorophores per bead on the basis of flow cytometric calibration beads (46). Data were subsequently analyzed using Microsoft Excel or GraphPad Prism software (GraphPad Software, Inc. San Diego, CA).

RESULTS AND DISCUSSION Protein Microarrays. In a previous report, we demonstrated that the ATMS-based diazo coupling method can produce reusable DNA microarrays that provide uniform DNA spots with high spot intensity (40). These microarrays have been used to successfully quantify differential gene expression in different RNA libraries (Dolan P. L., Nelson M. A., unpublished data). Similar experimental procedures were used as a starting point for the fabrication of protein microarrays. Scanned images and quantitative analyses of protein microarrays are presented in Figure 1 and Figures 2 and 3, respectively. Figure 1 shows the scanned images of a typical protein microarray before (Figure 1A) and after 2 h incubation of 1 µM FITC-labeled streptavidin in Tris-BSA (Figure 1B). Three

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Figure 2. Quantitative intensity analysis of protein microarrays imaged in Figure 1 before and after incubation with FITC-streptavidin. A. Intensity of Cy3-labeled anti mIgG spots (λem ) 530 nm). B. Intensity of biotinylated BSA spots. The microarrays were prepared by spotting the proteins at the concentrations indicated.

different proteins, including a fluorescently labeled “control protein”, Cy3-anti mouse IgG (Cy3-anti mIgG), and two biotinylated proteins, biotinylated BSA and biotinylated rabbit IgG (rIgG), that bind specifically to FITC-labeled streptavidin, were arrayed on diazotized glass microscope slides at five different concentrations, each in duplicates. The scanned images in Figure 1 show that (1) signals representing the control protein, i.e., Cy3-anti mIgG, appear clearly on both images with similar spot intensity, (2) signals from biotinylated areas are visible only after the incubation in FITC-labeled streptavidin, and (3) the signal intensity of all three types of proteins reflect protein concentration in the spotted samples. Results of spot intensity analysis are shown in Figure 2. Figure 2A shows the background-subtracted mean spot intensity of Cy3-anti mIgG before and after incubation with FITC-streptavidin. Although some signal decrease is observed (likely due to photobleaching occurring during scanning), neither the mean spot intensity nor the proportional relationship between different concentrations of the immobilized control protein is significantly influenced by the incubation of FITC-streptavidin. In contrast, spot intensities of biotinylated BSA shown in Figure 2B increase dramatically after incubation with FITC-streptavidin, which binds specifically with the biotinylated protein. The spot intensity after FITC-streptavidin incubation reflects the concentration of biotinylated BSA in the spotted samples. Similar results were obtained on the spots of biotinylated rIgG (data not shown). These observations indicate the possibility of applying the diazo coupling method to produce protein microarrays for quantitative analysis.

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Figure 4. Equilibrium binding curve of Alexa488-antiGFP binding to His6GFP. Samples for binding curve analysis were prepared by incubating diazotized beads in 400 µL of 200 nM His6GFP for 1 h at room temperature under mild vortexing, and then the beads were reacted with nM to µM range of Alexa488-antiGFP for 1 h.

Figure 3. Responses of different proteins arrayed on diazotized surface to their specific binding partner. The microarrays were prepared by spotting the proteins at the concentrations indicated. Intensity of antimIgG spots (A), biotinylated rIgG spots (B), and His6GFP spots (C) before and after the incubation of 1 µM Cy5-mIgG, FITC-streptavidin, or FITC-antiGFP.

An important criterion for the success of covalent protein immobilization strategies is the preservation of bioactivity of immobilized proteins, i.e., their ability to recognize specific binding partners. We demonstrated binding specificity of proteins immobilized on diazotized substrates by first forming microarrays consisted of five different concentrations of three different types of proteins, an antibody (anti-mIgG), an antigen (GFP), and a conjugated protein (biotinylated rIgG). The printed slides were then incubated with Cy5-labeled mIgG, FITClabeled antiGFP, or FITC-labeled streptavidin at room temperature for 1 h, to compare the reactivity of the arrayed proteins. Intensity analysis results in Figure 3 show all three immobilized proteins bound specifically to their binding partners, and the signal intensities are proportional to the protein concentration in spotted samples. These observations indicate that protein microarrays formed on the ATMS-based diazonium surfaces maintain relatively high binding specificity, and that it is also possible to obtain quantitative data using this protein immobilization method. Protein-Bearing Micro Particle Assays. Among the 20 common amino acids, only four contain an aromatic ring, and

of these, only histidine and tyrosine readily bind covalently to diazonium salts (25, 26). Our expectation is that the rapid reaction between the diazonium group and tyrosine or histidine residues in proteins is the basis for the immobilization described here. Polyhistidine tags that consist of five or six contiguous histidine residues, attached to either the N or the C terminus of the protein and exposed at protein surfaces, are widely employed in the purification of recombinant proteins (28, 47). Therefore, we compared the reactivity of diazotized surfaces with proteins bearing and lacking histidine tags. We investigated the binding of His6GFP, streptavidin, and M1 anti-FLAG antibody to diazotized glass microbeads (4.9 µm in diameter) and performed quantitative analysis by flow cytometry. We used fluorescein biotin to measure the surface concentration of streptavidin (MW ∼ 66 kDa; Kd ) 10-13 M (46, 48)), FITC-FLAG to detect M1 anti-FLAG antibody (MW ∼ 150 kDa; Kd ∼ 8 nM (45)), and Alexa488-antiGFP to detect His6GFP (MW ∼ 27 kDa). High concentrations of fluorescently labeled ligands (at least 10 times higher than Kd) were used in order to ensure characterization of the amount of proteins bound to the diazotized surface at saturation. To determine the affinity of Alexa488-antiGFP to His6GFP bound to the diazotized bead surface, a binding curve (Figure 4) was measured. A one-site binding hyperbola was used to analyze the experimental data in Prism software, and the best-fit values gave the equilibrium dissociation constant, Kd, after background subtraction, as ∼40 nM (Figure 4). On the basis of this result, 400 nM antiGFP (i.e., 10 × Kd of the antiGFP/GFP reaction) was chosen for the characterization of the reaction of His6GFP with the diazo beads, while 100 nM of FITC-FLAG and 200 nM of fluorescein biotin were chosen to characterize the reaction of M1 and streptavidin, respectively, with the diazotized beads. Figure 5 compares the binding efficiency of His6GFP (Figure 5A), streptavidin, and M1 anti-FLAG antibody (Figure 5B). These data indicate that each of the tested proteins was immobilized to the diazotized surface consistently, and that the diazotized surface showed especially high binding of the histagged protein. To ascertain whether this is due to high binding reactivity of diazotized surfaces for his-tagged proteins or to the size of the immobilized proteins, the maximum possible protein coverage over diazotized microbead surfaces was estimated. As described in the Experimental Procedures, approximately 1 × 107 active sites/bead are expected after diazotization; however, the degree of protein binding may also depend on characteristics of the protein molecule, e.g., tertiary structures. GFP is an 11-stranded β-barrel threaded by an R-helix running through the axis of the cylinder (49). These cylinders have a diameter of about 30 Å and a length of 40 Å (50). The

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Figure 6. A. Experimental design for studying the reactivity of histagged protein immobilized on diazotized surfaces. B. Results of experiments in part A detected by flow cytometry. Diazo: represents response of immobilized His6GFP on diazotized beads coupled to HisProbe-HRP detected by FITC-antiHRP. Amine: response of immobilized His6GFP on primary amine-terminated beads coupled to HisProbe HRP detected by FITC-antiHRP. Inserts schematically depict possible protein orientations on each surface. Figure 5. Comparison of diazo coupling reaction for proteins with and without histidine tag. A. Comparison of binding efficiency of His6GFP with diazotized and amine terminated surface. B. Binding efficiency of proteins without a histidine tag (streptavidin, and M1 antiFLAG antibody) with diazotized surfaces.

minimum surface coverage for a single GFP molecule is thus ∼780 Å2. Because the surface area of a 4.9 µm diameter bead is ∼7.5 × 109 Å2, the maximum number of His6GFP molecules that can be attached to a 4.9 µm bead is ∼1 × 107. On the basis of a similar estimation, there should be a maximum of about 4 million streptavidin molecules [dimension ∼ 40 Å × 50 Å × 50 Å] (51, 52) and 0.4 million M1 molecules [∼15 nm in diameter] that can be packed onto a single 4.9 µm bead. Results in Figure 5 show that when incubated in 300 nM protein solution, approximately 900 000 His6GFP molecules per bead were detected, while less than 5000 protein/bead (or less than 0.5% of the bound His6GFP) were detected for both nonhis-tagged proteins (streptavidin and M1 anti-FLAG antibody). Moreover, as illustrated in the Figure, detectable His6GFP molecules/bead increased to approximately 2.2 million (when the His6GFP concentration in solution was 3 µM), which is about 20% of the estimated maximum His6GFP binding sites/ bead. Geometrically, the maximum number of His6GFP that can pack on a single glass bead should only be about twice that of streptavidin and twenty times more than that of M1 anti-FLAG antibody. Thus, these results indicate that the his-tagged proteins are substantially more reactive with the diazotized surface than non-his-tagged proteins. The identification of an immobilization chemistry that can preserve the reactivity of the bound proteins has always been a challenge for high-throughput protein analysis (21, 53). Reactivity of the surface bound proteins can be a function of the structure, orientation, and packing of the protein. We compared the reactivity of his-tagged proteins for activated diazotized surfaces with their reactivity to unactivated primary amineterminated surfaces. Figure 5A presents the amount of reactive His6GFP bound to diazotized and unactivated SAMs formed from ATMS as detected by the binding of Alexa488-antiGFP.

The amount of reactive his-tagged protein measured on the diazotized surface is considerably higher than that observed on the amine surface, reaching a maximum of 1000% higher at a solution concentration of 3 µM of His6GFP. Similar experiments with proteins without a his-tag did not show any significant difference between diazotized surfaces and amine-terminated surfaces. These observations indicate that when compared to amine surfaces, the diazo-coupling method can result in much higher levels of adsorbed his-tagged proteins. Because the ligand binding site(s) over a protein surface is generally asymmetric, the geometric orientation of the immobilized protein may determine whether it retains its binding activity (22, 54). Figure 5A indicates that the coupling reaction of His6GFP with diazotized beads yields a much greater surface density of the reporter Alexa488-antiGFP antibodies than the primary amine (i.e. unactivated ATMS) surfaces. The reporter antibody (Alexa488-antiGFP) can be considered as an indirect measure of surface His6GFP coverage. However, it is not possible to definitively distinguish whether the higher surface coverage of the reporter antibody at diazotized surface is due to improved coupling efficiency to His6GFP molecules, or to proper orientation of GFP that allows optimum antibody capture, or both. To assess whether the results shown in Figure 5 were related to the geometric orientation of the immobilized histagged proteins, we further investigated the diazo coupling method as a means of covalent immobilization of oriented proteins on surfaces. An assay specifically targeting surfaceaccessible his tags using HisProbe-HRP was employed to explore this issue. HisProbe-HRP is a Ni2+ activated HRP derivative, that can be used to detect surface-exposed his-tags (http:// www.piercenet.com/). Figure 6A shows a typical experimental scheme that was followed. The results are shown in Figure 6B. The results indicate that a higher amount of HisProbe-HRP is bound to GFP treated primary amine surfaces than to GFPtreated diazotized SAMs. Because HisProbe-HRP is only able to bind to surface-exposed histidine residues, this indicates that

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few his-tags are available for binding on the GFP-treated diazotized SAMs. Taken together with the data presented in Figure 5A, these results suggest that high levels of His6GFP were bound to the diazotized SAMs, and that a large percentage of the immobilized His6GFP was bound in an oriented fashion on these surfaces (see inserts of Figure 6B). These proteins were likely coupled to the diazotized surfaces via a covalent linkage to the his-tags. The results further suggest that amine-terminated surfaces do not react as efficiently with his-tagged proteins as diazotized surfaces, and that they may react nonspecifically with other negatively charged amino acid residues scattered on the protein surface, as discussed previously (21), to result in a nonpreferential orientation of the adsorbed His6GFP molecules.

SUMMARY A diazo coupling method was used to covalently immobilize proteins onto solid substrates. When compared with commonly used microarray supports, e.g., amine-terminated surfaces, the diazotized surfaces provide low background fluorescence and high signal/noise ratio. Proteins containing a hexahistidine tag exhibited over 10× higher reactivity compared to non-his-tagged proteins. Results indicate that immobilized his-tagged proteins are likely coupled to diazotized surfaces through the histidine tag, which allows for preferential orientation of the immobilized proteins and thereby can be used to optimize the reactivity of the protein to interact with specific binding partners. Because of the wide availability of his-tagged proteins, the described surface immobilization described herein may be useful in the fabrication of protein arrays and protein-coated microbeads for high throughput analyses.

ACKNOWLEDGMENT This project was funded by the National Science Foundation (Grant CTS-0332315), the National Institutes of Health (Grant GM60799/EB00264), and the Department of Energy through the US/Mexico Materials Corridor Initiative.

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