Site-Specific and Covalent Attachment of His-Tagged Proteins by

Sep 5, 2013 - This is a reminder that also when immobilizing ligands via residues far from the binding site, there are many other factors influencing ...
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Site-Specific and Covalent Attachment of His-Tagged Proteins by Chelation Assisted Photoimmobilization: A Strategy for Microarraying of Protein Ligands Emma M. Ericsson,†,# Karin Enander,*,†,# Lan Bui,‡,∥ Ingemar Lundström,§ Peter Konradsson,‡ and Bo Liedberg†,⊥ †

Division of Molecular Physics, ‡Division of Organic Chemistry, and §Division of Applied Physics; Department of Physics, Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden S Supporting Information *

ABSTRACT: A novel strategy for site-specific and covalent attachment of proteins has been developed, intended for robust and controllable immobilization of histidine (His)-tagged ligands in protein microarrays. The method is termed chelation assisted photoimmobilization (CAP) and was demonstrated using human IgG-Fc modified with C-terminal hexahistidines (His-IgGFc) as the ligand and protein A as the analyte. Alkanethiols terminated with either nitrilotriacetic acid (NTA), benzophenone (BP), or oligo(ethylene glycol) were synthesized and mixed self-assembled monolayers (SAMs) were prepared on gold and thoroughly characterized by infrared reflection absorption spectroscopy (IRAS), ellipsometry, and contact angle goniometry. In the process of CAP, NTA chelates Ni2+ and the complex coordinates the His-tagged ligand in an oriented assembly. The ligand is then photoimmobilized via BP, which forms covalent bonds upon UV light activation. In the development of affinity biosensors and protein microarrays, site-specific attachment of ligands in a fashion where analyte binding sites are available is often preferred to random coupling. Analyte binding performance of ligands immobilized either by CAP or by standard amine coupling was characterized by surface plasmon resonance in combination with IRAS. The relative analyte response with randomly coupled ligand was 2.5 times higher than when site-specific attachment was used. This is a reminder that also when immobilizing ligands via residues far from the binding site, there are many other factors influencing availability and activity. Still, CAP provides a valuable expansion of protein immobilization techniques since it offers attractive microarraying possibilities amenable to applications within proteomics.

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optimizing the His-tag length,8,9 but upon addition of a competing molecule such as imidazole or by changing the pH to below 6, the His-tagged protein is displaced. In order to overcome this limitation, amine coupling to carboxylic acid groups of the chelator has been suggested.10 However, this results in a less well-defined orientation of the ligand due to the carboxylic acid groups of NTA losing their coordinating ability upon activation. Allowing the His-tagged protein to coordinate to Ni2+-NTA prior to activation may lead to a nonfunctional ligand.10 Retention of binding function in this situation has indeed been reported with certain ligands,11 but the approach cannot be recommended in the general case since many proteins would be sensitive to cross-linking and carboxylic acid derivatization associated with activation. An attractive alternative to covalently tethering sitespecifically captured ligands by means of amine coupling would be to combine Ni2+−NTA coordination with a

INTRODUCTION Numerous strategies are available for immobilization of proteins on sensing (transducer) substrates and in microarray formats.1,2 The majority of these, e.g., spontaneous adsorption or amine coupling based on conventional carbodiimide chemistry, typically result in random orientation of the attached recognition molecules (ligands), causing a fraction of the interaction sites to be unavailable to analyte binding. To increase accessibility, site-specific attachment of the ligand distantly from the binding site can be realized via the interaction between a region or tag (e.g., a histidine (His)tag) and its surface-immobilized affinity partner (e.g., nitrilotriacetic acid (NTA) chelating Ni2+ and other bivalent metal ions).1,2 In the context of protein microarray fabrication, piezodispensing of His-tagged ligands onto substrates coated with Ni2+NTA is an established method, although it does not provide a stable attachment.3,4 His-tagged ligands bind with moderate (micromolar) affinity to the Ni2+−NTA complex at neutral pH.5 The interaction can be stabilized by continuous rebinding to NTA at a sufficiently high surface concentration5−7 and by © XXXX American Chemical Society

Received: March 28, 2013 Revised: August 22, 2013

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amine coupling resulted in a higher normalized analyte response than CAP. Still, considering the wide range of Histagged ligands relevant for covalent arraying, CAP provides a valuable expansion of currently available protein immobilization strategies.

photochemistry approach, which would have the added benefit of allowing for convenient arraying of ligands in protein chip formats. For this purpose, several photolabile agents such as arylazides, benzophenones, diazirines, and nitrobenzyl groups are available.12 Benzophenone (BP) is activated by UV light (350−360 nm, a range that does not damage proteins or cells13) and attacks C−H, O−H, or N−H bonds to form a new covalent bond by insertion. It has been frequently used for photoimmobilization of proteins14−16 and even cells17 to various substrates, and its combination with affinity capture was recently demonstrated when a recombinant protein containing an amino acid analogue with a BP side chain was captured by surface bound β-cyclodextrin followed by photoimmobilization.18 In this contribution we introduce chelation assisted photoimmobilization (CAP), a new strategy for site-specific capturing followed by covalent attachment of His-tagged proteins (Figure 1) that is amenable to photopatterning applications. Gold

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EXPERIMENTAL SECTION

Synthesis and Characterization of Alkanethiols and Alkane Disulfides. The synthetic strategies for modification of ethylene glycols have been reported earlier by Svedhem et al.23 The three alkanethiols and two alkane disulfides shown in Figure 2 were synthesized as described in the Supporting Information, Schemes S1− S4. Triethylene glycol was used to build the EG3 alkanethiol, the EG3 disulfide, and the NTA alkanethiol. The BP alkanethiol was built from two tetraethylene glycol units. Finally, the NTA and BP alkanethiols were combined into the asymmetric so-called BPNTA alkane disulfide. The structures of the five synthesized molecules were confirmed with IR transmission spectroscopy as described in the Supporting Information. Preparation and Characterization of SAMs. Experimental details of preparation and characterization of SAMs are described in the Supporting Information. In brief, alkanethiolate SAMs were prepared by incubating gold-coated silicon or glass substrates in ethanolic solutions of the compounds shown in Figure 2. For basic characterization, pure SAMs of each compound were prepared from 100 μM solutions. SAMs for ligand immobilization experiments were prepared from three different incubation solutions, each of a total concentration of 100 μM, in the following compositions: EG3 and BPNTA disulfides (80:20), EG3 and NTA thiols (90:10), EG3 and BP thiols (90:10). These mixed SAMs are referred to as 20% BPNTA, 10% NTA, and 10% BP, respectively. The thickness, wettability, and structure of the formed SAMs were observed by null ellipsometry, contact angle goniometry, and infrared reflection absorption spectroscopy (IRAS), respectively. Biomolecular Model System. The ligand was the hexahistidinetagged Fc dimer of human IgG4 (Mw 57.6 kDa), kindly provided by D. Kanmert,24 and the analyte was staphylococcal protein A (Mw 42 kDa) from Nordic BioLabs. Analyte Binding on 10% NTA and 20% BPNTA Following Ligand Immobilization by Amine Coupling or Ni2+-NTA Chelation. Analyte binding to ligand, either amine coupled or noncovalently bound via the His-tag to NTA groups on 10% NTA or 20% BPNTA, was studied with a Biacore 3000 instrument at a flow rate of 10 μL/min. For studies of ligand immobilized via amine coupling, HBS-EP (Hepes-buffered saline (10 mM Hepes, 150 mM NaCl, pH 7.4) with an addition of 0.005% (v/v) surfactant P20 and 3 mM EDTA; GE Healthcare) was used as running buffer. Carboxylate groups were activated with a mixture of ethyl(dimethylaminopropyl)carbodiimide (EDC, 0.2 M (aq); GE Healthcare) and N-hydroxysuccinimide (NHS; 0.05 M (aq); GE Healthcare) for 5 min. His-IgGFc (4 μM, 5 min) in phosphate-buffered saline (PBS: 0.14 M NaCl, 2.7 mM KCl, 10 mM phosphate, pH 7.4) was then immobilized, and residual activated groups were deactivated by ethanolamine-HCl (1 M, pH 8.5; GE Healthcare) for 5 min. The ligand was conditioned with two 0.5 min pulses of conditioning solution (50 mM NaOH, 0.5 M NaCl(aq)) before injecting protein A (0.5 μM in HBS-EP, 1 min). As a reference, analyte interaction with a SAM was observed where activation and prompt deactivation, followed by injection of conditioning solution, had been performed. For studies of ligand coordinated to Ni2+−NTA, both ligand and analyte were diluted in HBS-N (10 mM Hepes, 150 mM NaCl, pH 7.4; GE Healthcare) which was also used as running buffer. Injection of NiCl2 (0.5 mM (aq); Fluka) for 5 min was followed by injection of His-IgGFc (4 μM, 5 min). The surface was then blocked with two consecutive 2 min injections of 10% (w/v, aq) trehalose dihydrate (Fluka) before injection of protein A (0.5 μM, 1 min). As a reference, analyte interaction with an untreated SAM was observed.

Figure 1. Schematic picture of a protein immobilized to an alkanethiolate SAM by the CAP principle. The SAM is represented by the tilting bars and only NTA and BP functionalities are shown in detail. The ligand has a hexahistidine tag, located distantly from the analyte binding site, which first coordinates to Ni2+−NTA. Photoimmobilization to BP then tethers the ligand permanently to the surface. The length of the OEG linker chain of the BP alkanethiol was chosen to allow for covalent attachment of BP to the His-tag or another ligand region far from the binding site.

substrates were modified with self-assembled monolayers (SAMs) of alkanethiolates,19−22 exposing NTA groups for capturing and BP groups for photoimmobilization. All alkanethiolates contained an oligo(ethylene glycol) (OEG) portion for reduction of nonspecific binding. The CAP concept was demonstrated using His-tagged human IgG-Fc (HisIgGFc) as the ligand, where the His-tag was located far from the binding site, and protein A as the analyte. In order to evaluate CAP in terms of analyte binding capacity, His-IgGFc was immobilized either by CAP or by conventional amine coupling, and binding of protein A was monitored in relation to the ligand immobilization level. Amine coupling targets primary amines and results in a randomly attached ligand, as positively charged amino acid residues, including lysines, are fairly homogeneously distributed on the surface of His-IgGFc. With this biomolecular model system, B

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Figure 2. The five molecules for surface modification used in this work, referred to as (I) EG3 thiol, (II) NTA thiol, (III) BP thiol, (IV) EG3 disulfide, and (V) BPNTA disulfide. Analyte responses were reference subtracted. Assuming two analyte binding sites in His-IgGFc, the binding efficiency E (%) was then calculated as

E=

R(analyte)M w (ligand) × 100 2I(ligand)M w (analyte)

where R(analyte) is the analyte response recorded 15 s after end of injection and I(ligand) is the immobilization level of the ligand. Mw(ligand) and Mw(analyte) are the molecular weights of ligand and analyte. This model is a simplification of reality since there is a possibility that some analyte molecules bind two ligand molecules, but it is still useful when comparing binding efficiency on different twodimensional surfaces where ligand immobilization levels are similar. Demonstration of CAP and Analysis of Analyte Binding Efficiency. Ligand Immobilization and Analyte Interaction. For CAP, a 20% BPNTA surface was incubated in a drop of 0.5 mM NiCl2 for 30 min, then rinsed briefly in water, and dried with N2 gas. A drop (10 μL) of 1 μM ligand solution in PBS was added to the surface, which was then brought in contact with a clean quartz disk so that a thin film of protein solution was spread over the surface (0.069 μL/ mm2). This was followed by 30 min incubation in darkness to allow time for ligand orientation by NTA. The surface−quartz disk assembly was then placed under a UV lamp and irradiated for 1 min to activate BP and covalently attach the ligand to the surface (Figure 3). A 100 W Hg lamp (Newport) was used in conjunction with a dichroic mirror (280−400 nm, Newport) and a 360 nm bandpass filter (Edmund Optics) to select the wavelengths that activate BP without damaging the protein or the SAM. The UV box was purged with N2 gas before and during the irradiation to reduce the effect of harmful oxygen radicals formed in UV light. The surface was then removed from the quartz disk by rinsing in PBS. The excess of unbound protein was

Figure 3. Schematic drawing of the optical setup used for photoimmobilization of a preoriented His-tagged protein. The surface was attached to a UV transparent quartz disk by the capillary forces of a thin film of protein solution. removed by immersions twice in PBS and three times in imidazole (0.3 M (aq), pH 7.4; Merck). Finally, the surface was incubated 2 min in 3% (w/v, aq) trehalose dihydrate to protect the protein from denaturation upon drying of the surface in flowing N2 gas. Control surfaces (20% BPNTA) were irradiated with UV light in the absence of ligand. Amine coupling was performed on 10% NTA surfaces. These were activated by 30 min incubation in 1 μL/mm2 EDC/NHS (0.2 M/0.05 M), followed by ligand incubation during 30 min (as above) and rinsing in PBS and Milli-Q water. Residual activated groups were deactivated by 30 min incubation in 1 μL/mm2 ethanolamine-HCl (1 M) followed by rinsing in Milli-Q water. Finally, the surface was incubated during 2 min in 3% (w/v) trehalose dihydrate. In this experiment, 3 mM EDTA (Merck) was added to the PBS buffer to exclude any possibility of metal ion mediated coordination of ligand. Also, immobilization was performed at neutral pH to keep the His-tag C

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uncharged in order to avoid an orientation effect by electrostatic interaction with the negatively charged surface. Control surfaces (10% NTA) were activated and promptly deactivated. The dried surface, where ligand had been immobilized either by CAP or by amine coupling, was mounted on a chip holder and docked into a Biacore 3000 SPR instrument for studies of analyte interaction. The running buffer was HBS-EP, and the flow rate was 10 μL/min. The surface was conditioned by two 0.5 min pulses of conditioning solution (0.5 M NaCl and 50 mM NaOH). After baseline stabilization, protein A (0.5 μM in HBS-EP) was injected for 1 min. The interaction between the ligand and the analyte was quantified as the response 15 s after the end of injection. Ligand Quantification. The ligand immobilization procedures described above were analogously performed on surfaces with a thicker gold layer for IRAS and null ellipsometry measurements. Here, not only CAP (20% BPNTA) and amine coupling (10% NTA) were studied but also the performance of photoimmobilization alone (10% BP) and noncovalent binding to NTA via metal ion chelation (10% NTA). The ellipsometric film thickness was measured, and the infrared reflection absorption (RA) spectrum was recorded on untreated SAMs and after each treatment step as described in Table S1. An imidazole rinsing step was performed to study detachment of ligand. The surface was rinsed in Milli-Q water and dried in N2 gas after each treatment step before ellipsometry and IRAS measurements. Any protein conformational changes resulting from this treatment were not expected to affect the quantification of the amount of surface bound ligand. RA spectra were recorded as described in the Supporting Information. They were corrected for water interference, cut, and baseline corrected using the OPUS software (Bruker), and thereafter the peak corresponding to the amide I bond (CO stretch) was integrated in the intervals 1810−1610 cm−1 (for 20% BPNTA and 10% BP surfaces) and 1800−1600 cm−1 (for 10% NTA surfaces). These large intervals were chosen to take into account the protonation changes in the carboxylate peak overlapping the amide I peak as well as the general baseline rise in this interval due to the presence of protein. The integral value of the peak in the untreated SAM was then subtracted from the integral values of the peaks in subsequent spectra, and the resulting value (referred to as the corrected integral) was used as a measure of ligand quantity and compared to the ellipsometric value of the protein film. Normalization of Analyte Response. The normalized analyte response with 10% NTA and 20% BPNTA surfaces, where ligand had been immobilized by amine coupling and CAP, respectively, was calculated by dividing the analyte response (RU) measured with SPR by the corrected integral (au) from the corresponding IRAS spectrum. CAP-Based Photopatterning. Photopatterning was demonstrated using a custom-built imaging surface plasmon resonance (iSPR) instrument.25 A UV lamp (100 W Hg lamp, Newport) with a 360 nm bandpass filter (Edmund Optics Ltd.) was mounted on top of the instrument so that the surface could be irradiated while mounted in the Teflon sample cell. The sample cell, with a circular hole (8 mm diameter), held the gold surface in place on the SPR prism, leaving it exposed so that buffer could be changed manually. All measurements were performed in PBS prepared from tablets (Medicago). The illumination wavelength was scanned between 650 and 760 nm, and the SPR wavelength was recorded with lateral resolution. The SPR wavelength shift (ΔλSPR) between the sample and an untreated surface was used to create images. A 20% BPNTA surface was loaded with Ni2+ by 15 min incubation in 100 μL of NiCl2 (0.5 mM) followed by PBS rinsing. During ligand incubation (100 μL of His-IgGFc, 1 μM in PBS, 30 min), a quartz mask with a chromium pattern of 300 μm diameter circles with 200 μm spacing was placed on top of the drop of ligand solution, creating a liquid film of about 1 mm thickness on the surface. The UV lamp shutter was opened for 1 min, and the protein solution and surface were exposed to UV light through the patterned mask. After PBS rinsing, noncovalently bound ligand was eluted by rinsing in imidazole (0.3 M, pH 7.4) and PBS. The surface was then incubated with 100 μL of protein A (5 μM) for 30 min followed by PBS rinsing.

Article

RESULTS AND DISCUSSION Design, Synthesis, and Characterization of Alkanethiols and Alkane Disulfides. Three alkanethiols and two alkane disulfides (Figure 2) were synthesized as described in the Supporting Information (Schemes S1−S4) to provide a controllable surface composition for the demonstration of CAP. A high surface content of OEG-terminated alkanethiolates reduces nonspecific binding of proteins. The length of the OEG linker in the BP alkanethiol was chosen so that the BP moiety could protrude from the surface and insert into a covalent bond of the protein upon UV light activation. The OEG linker lengths were not optimized but can be directly controlled during synthesis. The structures of the five synthesized molecules were confirmed with IR transmission spectroscopy (Figure S1). Characterization of SAMs. The synthesized molecules were used to prepare five single component SAMs and three mixed SAMs. These were characterized as described in Supporting Information with ellipsometry (Figure S2), contact angle goniometry (Figure S3), and infrared reflection absorption spectroscopy (Figure S4). Surfaces with mixed monolayers assembled from solutions of 20% BPNTA, 10% NTA, or 10% BP (referred to as 20% BPNTA, 10% NTA, and 10% BP, respectively) were used in subsequent experiments. Biomolecular Model System. The ligand used in the demonstration of CAP was the Fc dimer of human IgG, where both heavy chains had been modified with a hexahistidine sequence at the C terminus (His-IgGFc). The analyte was protein A, a staphylococcal cell surface protein that binds to the Fc region of mammal immunoglobulins. Whether protein A has four or five binding sites has been discussed,26 but it is agreed upon that it can bind two IgG molecules simultaneously and that one IgG molecule can bind two protein A molecules. The binding sites for protein A are located in the junctions between the CH2 and CH3 domains of IgG-Fc, distantly from the Histags. Analyte Binding on 10% NTA and 20% BPNTA Following Ligand Immobilization by Amine Coupling or Ni2+−NTA Chelation. With two-dimensional surfaces there is a risk that molecular crowding reduces analyte binding efficiency of immobilized ligands, defined as the fraction of theoretically available binding sites being occupied by analyte. An SPR analysis was performed in order to investigate any differences in this efficiency when the ligand was covalently immobilized via lysines and when it was noncovalently captured by Ni2+−NTA. Results are summarized in Table 1. Table 1. Comparison of Binding Efficiency of His-IgGFc on Different Surfaces upon Exposure to 0.5 μM Protein A 10% NTA 20% BPNTA CM5 a

amine coupling (%)

Ni2+−NTA coordination (%)

5 5 10

10 5a not determined

Less reliable value due to nonspecific adsorption of analyte (see text).

When similar amounts (about 500 RU, which is less than 10% of a monolayer) of His-IgGFc were covalently immobilized via amine coupling to carboxylic acid groups on 10% NTA or 20% BPNTA followed by introduction of 0.5 μM protein A, binding efficiency was around 5% in both cases (it should be noted that at this concentration of analyte ligands do D

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not get saturated). This demonstrates that when the ligand is covalently immobilized via its different lysine side chains, the composition of the underlying monolayer does not influence analyte binding. No nonspecific binding of analyte to the SAMs was observed in these cases. When performing amine coupling of His-IgGFc on a commercially available dextran-coated sensor chip (CM5; GE Healthcare) in an identical manner, binding efficiency was 10%, in line with previous observations that binding efficiency is higher when ligands are immobilized to CM5 chips compared to when they are attached to twodimensional surfaces.27 When the ligand was site-specifically and noncovalently attached (about 750 RU) via coordination of the His-tag to Ni2+-NTA, binding efficiency was 10% with 10% NTA and 5% with 20% BPNTA. In these cases, where surface carboxylic acid groups were highly charged, nonspecific binding of analyte to the SAMs was observed, but in the case of 10% NTA, this could be completely eliminated by introducing a trehalose solution after ligand immobilization. Increased binding efficiency on 10% NTA when the ligand was site-specifically attached, compared to when it was randomly immobilized, may in part be explained by increased accessibility, as NTA groups are expected to be well-dispersed in the background of EG3 thiolates due to charge repulsion between carboxylic acid groups, allowing for protein A interaction at both His-IgGFc binding sites in some cases. Also, covalent bond formation in the case of amine coupling may have impaired the binding sites in some ligand molecules due to structural changes. Data for 20% BPNTA is less reliable, since almost half of the obtained response on this surface was due to nonspecific binding of the analyte to the underlying SAM. In this case, the problem could not be eliminated despite the use of trehalose (a tween detergent and a highly concentrated NaCl solution were also tried, without success). There is an obvious risk that nonspecifically adsorbed molecules hindered specific interactions, giving a less than 10% binding efficiency with this surface. However, the important (and expected) result from these experiments was that noncovalent, His-tag mediated attachment of ligand at 20% BPNTA is associated with an analyte binding efficiency that is at least as good as that obtained with conventional amine coupling. Demonstration of CAP. In order to demonstrate CAP, a series of experiments with IRAS, ellipsometry, and SPR were performed. Amine coupling was performed in parallel and used in a subsequent analysis of analyte binding efficiency. The integrity of the formed SAMs was not disturbed by UV illumination when a 360 nm bandpass filter was used (not shown). NTA and BP chemistries were evaluated separately in order to find a set of suitable experimental conditions for CAP, including UV irradiation time and UV wavelength interval. Maximal ligand photoimmobilization on 20% BPNTA was achieved after 1 min irradiation (not shown). There was no nonspecific binding of ligand to 100% EG3 surfaces, but some occurred on surfaces containing BP or BPNTA. The ligand was immobilized on three types of surfaces in four different ways, as described in detail in Table S1. Photoimmobilization was performed on 20% BPNTA and 10% BP, while 10% NTA was used for amine coupling and noncovalent ligand attachment via Ni2+−NTA chelation. The amount of immobilized ligand was determined by null ellipsometry (Figure 4a) and IRAS (Figure 4b), where the increase in the amide I peak due to presence of protein was used for quantification (n = 3 for each immobilization method). The

Figure 4. Ligand immobilization levels evaluated by (a) null ellipsometry and (b) IRAS, based on values of integration of amide I peaks. The ellipsometer has a sensitivity of 1 Å, and the amount of ligand attached by photoimmobilization on 10% BP or by amine coupling (AC) on 10% NTA was too low to be detected by this method. Negative values were interpreted as no change compared to the untreated SAM.

results show that a Ni2+-loaded 10% NTA surface was able to bind the His-tagged protein (1.6 Å), but after imidazole treatment, the ligand was eluted. In contrast, most of the ligand bound to 20% BPNTA was still present after imidazole treatment (4.7 Å before, 2.9 Å after), indicating that photoimmobilization had permanently locked 60% of the ligands coordinated to Ni2+−NTA. There were not enough photolabile groups present on the surface to immobilize all ligand molecules coordinated to NTA, and further optimization of the BP/NTA surface ratio would improve the immobilization yield. The reason for the lower immobilization level on 10% NTA (Ni2+) compared to 20% BPNTA before imidazole treatment is that the noncovalent bond is sensitive to the rinsing and drying which was necessary for the ligand quantification measurements. There was a small tendency of His-IgGFc to associate noncovalently with BP without assistance from NTA, evident from the fact that it was possible to photoimmobilize some protein onto 10% BP, as determined by IRAS. However, the great majority of photoimmobilized ligands on 20% BPNTA were site-specifically attached via NTA interaction before UV illumination. E

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Amine coupling to 10% NTA resulted in low immobilization levels, detectable by IRAS but not by ellipsometry. Here, as the ligand was covalently bound, it could not be eluted by imidazole. The lower immobilization level was mainly due to less ligand preconcentration on activated 10% NTA than on nonactivated 10% NTA (Ni2+) and 20% BPNTA at the pH used for coupling. Interaction with analyte was confirmed with both 20% BPNTA (CAP) and 10% NTA (amine coupling) (Figure 5a,b). The SPR instrument available for these experiments was not amenable to integration with a UV lamp for photochemistry, which is why ligand immobilization and analyte interaction monitoring had to be performed in separate steps. The ligand was protected from drying during immobilization on the lab bench and incubated in trehalose prior to drying and docking into the SPR instrument. This treatment resulted in very low nonspecific binding of analyte to surfaces without ligand, also with 20% BPNTA. The SPR postinjection phase (Figure 5a) indicated the presence of both fast and slow dissociation events. This was very reproducibly observed with both CAP and amine coupling, and the fast dissociation of protein A is probably due to weak interactions with ligand molecules that were disturbed by the drying procedure during immobilization. This was supported by the fact that interaction sensorgrams at identical conditions where His-IgGFc had been amine coupled online demonstrated slow dissociation only (not shown). In the further analysis of binding efficiency, response values were sampled 15 s after the end of injection, to include only high affinity binding events. Analyte Binding Efficiency Following Ligand Immobilization by CAP. In order to monitor the binding performance of ligands immobilized by CAP, analyte levels in relation to amount of immobilized ligand were analyzed in the case of CAP (20% BPNTA) and amine coupling (10% NTA). Analyte responses (Figure 5b) were normalized using ligand amounts quantified from the amide I peak in the IRAS spectrum (Figure 4b). The normalized response was 2.5 times higher when the ligand was immobilized by amine coupling compared to when it was site-specifically attached by CAP (Figure 5c). Site-specific attachment is often described as superior to random immobilization with respect to increased accessibility, but systematic studies show that depending on the nature of the interacting molecules (e.g., binding valency), the surface chemistry, and the method of immobilization, sitespecific ligand attachment can either improve analyte binding efficiency,28,29 leave it virtually unchanged,30,31 or decrease it slightly32 compared to random immobilization. As discussed above, analyte binding efficiency was not decreased when noncovalently immobilizing the ligand via its His-tag on 20% BPNTA compared to when amine coupling was used. UV-mediated tethering to BP thus reduced binding efficiency, probably by decreasing the flexibility of the ligand and/or by impairing some of the binding sites, either upon covalent bond formation per se or as a result of UV illumination. Also, although submonolayer coverage of ligand was obtained with both CAP and amine coupling, the fact that the absolute ligand immobilization level was 3 times higher with CAP (Figure 4b) may have contributed to ligands being less available to the analyte in this case. Several previous studies have reported such steric hindrance in the case of immobilized Fab fragments, similar in size to His-IgGFc, covering fractions of two-dimensional surfaces.28,32,33

Figure 5. (a) Typical SPR sensorgram obtained from protein A interacting with His-IgGFc immobilized by CAP on a 20% BPNTA surface (solid line). The control refers to an identical surface where no ligand was present (dotted line). Very similar binding curves were obtained when the ligand was amine coupled to 10% NTA surface (not shown). (b) Absolute SPR responses of analyte interactions with ligand immobilized by amine coupling (AC) on 10% NTA and by CAP on 20% BPNTA. Values were sampled 15 s after the end of injection. Standard deviations are based on n = 4 surfaces per group. (c) The normalized analyte response with CAP was lower than that with amine coupling by a factor 2.5. Normalization was performed with respect to ligand amount on the different surfaces.

It is also possible that island formation34,35 in the threecomponent SAM used for CAP (indicated by the hysteresis in the advancing and receding contact angle measurements, Figure S3) caused local crowding of ligand molecules, resulting in steric hindrance in analyte binding. However, lacking detailed F

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Figure 6. SPR images demonstrating photopatterning based on CAP on a 20% BPNTA surface. The area shown is 1 mm2, and each unit on the yaxis corresponds to a shift of 2 nm in the SPR wavelength (ΔλSPR), which in turn corresponds to approximately 10 Å of immobilized protein. (a) After Ni2+ loading. (b) After ligand incubation and UV light irradiation. (c) After imidazole rinsing. (d) After analyte incubation.

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insights into the chemical heterogeneities on these surfaces, this remains a speculation. Demonstration of CAP-Based Photopatterning. The major motivation for the development of a new protein immobilization strategy was to provide a means of robust, flexible, and controllable immobilization of His-tagged ligands in microarray patterns. In order to demonstrate how CAP can be used for photopatterning, a custom-built imaging SPR instrument was equipped with a UV lamp. The NTA groups of 20% BPNTA were loaded with Ni2+ (Figure 6a) followed by ligand incubation and UV light irradiation through a mask with 300 μm sized circular holes. After rinsing in PBS, there was a homogeneous layer of ligand on the surface (Figure 6b), but when eluting noncovalently bound His-IgGFc a pattern appeared, indicating that photoimmobilization had occurred in the irradiated areas and permanently tethered the ligand molecules to the surface (Figure 6c). The covalently immobilized ligand was then allowed to interact with analyte, and binding was observed in a qualitative manner (Figure 6d). Control experiments with 10% NTA surfaces resulted in no patterns after imidazole treatment, while some photoimmobilization of protein was observed with 10% BP (not shown), in full agreement with results obtained with nonpatterned surfaces by null ellipsometry and IRAS (Figure 4).

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and characterization of alkanethiols and alkane disulfides; surface preparation and characterization of SAMs; overview of ligand immobilization steps. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Ph +46-13-282359 (K.E.). Present Addresses ∥

L.B.: Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, 52074 Aachen, Germany. ⊥ B.L.: Centre for Biomimetic Sensor Science, Nanyang Technological University, 50 Nanyang Drive, 637553 Singapore. Author Contributions #

E.M.E. and K.E. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Swedish Research Council (VR), Sweden. E.E. and L.B. were enrolled in the multidisciplinary graduate school Forum Scientium at Linköping University. The authors thank D. Kanmert for kindly providing the ligand protein and R. Selegård for help with NMR measurements.

CONCLUSION

We have designed a novel surface chemistry for attachment of proteins to sensing surfaces, relying on two well-established methods for anchoring of biomolecules: chelation chemistry based on NTA and photochemistry based on BP. Further, we have successfully demonstrated how a model ligand is immobilized in its active analyte-binding form by initial capturing followed by covalent tethering, and indicated how this chemistry can be used to create protein microarrays. With the biomolecular model system used here, the ligand was sensitive to covalent attachment via BP, reducing binding efficiency when the ligand was tethered by CAP compared to when it was immobilized by amine coupling. Still, in the choice of immobilization strategy for systematic attachment of multiple ligands in a protein microarray format, CAP is attractive for many reasons. In contrast to amine coupling, the photo induced immobilization lends itself to patterning in a system for sequential injection of ligands, interspersed by imidazole rinsing, which are homogeneously and covalently attached for reproducible analyte responses with low nonspecific binding. Also, capturing is realized via interactions with His-tags, the most frequent amino acid modification in proteins produced by recombinant methods, ensuring availability of this immobilization strategy to a wide range of protein ligands.

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