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Adlayer-Mediated Antibody Immobilization to Stainless Steel for Potential Application to Endothelial Progenitor Cell Capture Pasquale Benvenuto,† Miguel A. D. Neves,† Christophe Blaszykowski,‡ Alexander Romaschin,§ Timothy Chung,† Sa Rang Kim,† and Michael Thompson*,†,‡ †

Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada Econous Systems Inc., Toronto, Ontario M5S 3H6, Canada § Clinical Biochemistry, St. Michael’s Hospital, Toronto, Ontario M5B 1W8, Canada ‡

S Supporting Information *

ABSTRACT: This work describes the straightforward surface modification of 316L stainless steel with BTS, S-(11trichlorosilylundecanyl)-benzenethiosulfonate, a thiol-reactive trichlorosilane cross-linker molecule designed to form intermediary coatings with subsequent biofunctionalization capability. The strategy is more specifically exemplified with the immobilization of intact antibodies and their Fab′ fragments. Both surface derivatization steps are thoroughly characterized by means of X-ray photoelectron spectroscopy. The antigen binding capability of both types of biofunctionalized surfaces is subsequently assessed by fluorescence microscopy. It was determined that BTS adlayers achieve robust immobilization of both intact and fragmented antibodies, while preserving antigen binding activity. Another key finding was the observation that the Fab′ fragment immobilization strategy would constitute a preferential option over that involving intact antibodies in the context of in vivo capture of endothelial progenitor cells in stent applications.

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INTRODUCTION Stainless steels (SS) have been extensively used in the fabrication of a variety of biomedical devices such as coronary stents, which are employed for lumen expansion within arteries constricted by atherosclerosis. The prominence of SS is largely due to their desirable mechanical properties and resistance against corrosion. Implantable SS possess the nonmagnetic austenite crystal lattice structure and contain chromium as an alloying element, which is vital to impart SS with their necessary resistance to corrosion through the formation of a passivating oxide layer.1 Despite their beneficial properties and widespread use, SS do not constitute the perfect material, or biomaterial for that matter, as they do suffer from biocompatibility-associated challenges such as protein surface fouling with conformational restructuration resulting in the exposure of neoepitopes capable of triggering deleterious cell processes.2,3 As such, surface functionalization strategies (i.e., biofunctionalization) become important as a means of addressing specific problems without having to substitute the SS base material and thus sacrificing its desirable properties. For example, Yuan et al.4 modified SS with lysozyme-coupled polymer brushes in the development of a fouling- (protein) and bacterial-resistant coating, which is relevant to food processing and biomedical fields. In the context of coronary stent technology, in-stent renarrowing of arteries (restenosis) as a © XXXX American Chemical Society

result of neointima formation remains a significant hurdle that may require the patient to undergo additional medical interventions.5 Neointima arises from the proliferation of smooth muscle cells and extracellular matrix production within the intima (the layer of endothelial cells in contact with the circulating blood), which may lead to its thickening and a reduction in the luminal space.6 Several factors can influence neointima formation such as blood vessel damage during implantation,7 stent design and material,8 and inflammation.5 Efforts toward the reduction of in-stent restenosis have led to the development of numerous surface modification strategies for bare metal stents. Among these, the clinically approved drug-eluting stents (DES) have arguably made the most significant contribution to reduce restenosis, and to decrease the necessity for repeated surgical interventions by over 50% when contrasted to bare metal stents.9 DES reduce restenosis through the localized delivery of immunosuppressants or antiproliferative drugs, which interfere with vascular smooth muscle cell proliferation.10 While successful, long-term safety trepidations concerning incidents of very late stent thrombosis in certain first-generation DES have arisen.11 There is some Received: March 4, 2015 Revised: April 13, 2015

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single step without the need for preactivation and coupling agents. In an attempt to exploit the preferred reactivity of the thiosulfonate moiety for thiols, and ideally compel paratopes to face outward through oriented immobilization (Scheme 1, panel C), Fab′ antibody fragments bearing a freed thiol group(s) were generated through enzymatic digestion followed by selective chemical reduction (Scheme 1, panel B). While presented in the context of coronary stents, this BTS surface modification strategy is readily amenable to other technologies (i.e., sensors), hydroxylated/oxidized substrates (quartz, aluminum nitride, indium−tin oxide, etc.), and capture biomolecular receptors (aptamers, binding peptides, etc.). As a matter of fact, BTS has already been implemented by Neves et al. for the tethering of thiol-terminated aptamers to quartz in the successful construction of a label-free acoustic wave biosensor dedicated to the detection of cocaine.22 The following proof-of-concept study characterizes the immobilization of whole antibodies and their Fab′ fragments to BTS-functionalized 316L steel surfaces. X-ray photoelectron spectroscopy (XPS) was used to analyze bare, BTS-modified, and antibody-functionalized SS surfaces. Antigen binding to biofunctionalized surfaces (with whole or fragmented antibodies) was assessed by means of fluorescence microscopy.

evidence that these complications may be linked to delayed healing of the injured vasculature at the stented site.11,12 An interesting alternative approach is centered around the restoration of healthy endothelium at the site of implantation, as opposed to solely preventing smooth muscle cell proliferation. It was hypothesized that the promotion of a normally-functioning, antithrombotic endothelium could help mitigate restenosis and reduce the risk of late stent thrombosis. This approach has been made feasible through the discovery and use of endothelial progenitor cells (EPC), which are naturally present in the bloodstream13 and participate in the regeneration of damaged endothelium.14,15 There has been interest in recruiting and capturing these cells for the endothelialization of implant surfaces, as excellently reviewed by Avci-Adali et al.16 OrbusNeich Medical Technologies has developed a coating for the in vivo capture of EPC on SS stents and evaluated its potential in a 2005 human study.17 The coating featured immobilized antibodies directed against an EPC surface receptor. Other antibodies, as well as other capture biomolecules (such as aptamers and binding peptides), have since been used to tackle EPC capture.18,19 Herein, we describe a new coating for the surface immobilization of antibodies on 316L SS that could be easily implemented for EPC capture. SS surface modification first involves a derivatization step with an adlayer built from the previously described BTS cross-linker molecule depicted in Scheme 1 (panel A).20 In the process, the trichlorosilyl function condenses with surface hydroxyl groups to form a robust polysiloxane network, as shown in Scheme 1 (panel A). The distal benzenethiosulfonate moiety can next be functionalized,21 notably with thiol molecules to form disulfide bridges,20 in a

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MATERIALS AND METHODS

Materials. Octadecyltrichlorosilane (≥90%), anti-goat IgG (whole molecule) produced in rabbit, pepsin (from porcine gastric mucosa), bovine serum albumin (BSA, essentially fatty acid- and globulin-free, ≥98%), 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s Reagent, 99%), DL-dithiothreitol (DTT, 99.5%), anhydrous N,N-dimethylformamide (DMF, 99.8%), and toluene (ACS grade, ≥99.5%) were all purchased from Sigma-Aldrich. Methanol (ACS grade, ≥99.9%), Slide-A-Lyzer dialysis cassettes (2000 Da MWCO), EZ-Run Pre-Stained Rec Protein Ladder, and the Pierce BCA Protein Assay Kit were purchased from Thermo Fisher Scientific Inc. Reagent-grade concentrated sulfuric and glacial acetic acids, as well as pentane (spectrograde, ≥98%), were purchased from Caledon Laboratories Ltd. Chloroform (ACS, ≥99.8%), Amicon Ultra centrifugal filters (3, 30, and 50 kDa), and Millex-HA syringe-driven filter units (0.45 μm) were purchased from EMD Millipore. Hydrogen peroxide (30% solution, ACS) was purchased from ACP Chemicals Inc. Rabbit F(ab′)2 IgG (pepsin digest of rabbit IgG) was purchased from SouthernBiotech. Alexa Fluor 488 goat anti-mouse IgG (H+L) and BenchMark Pre-Stained Protein Ladder were purchased from Life Technologies. The Precision Plus Protein All Blue standards were purchased from Bio-Rad. Technical grade acetone (≥96.5%) and 95% ethanol were always used. DI water herein refers to deionized water with a resistance ≥18.0 MΩ· cm. All chemicals were used as purchased, unless otherwise noted. Anhydrous DMF was freshly distilled (from CaH2, under high vacuum) prior to use. Phosphate buffered saline (PBS) contained 154 mM NaCl and 10 mM sodium phosphate dibasic in DI water and was adjusted to pH 7.2, unless otherwise noted. The acetate buffer contained 200 mM of sodium acetate in DI water and was adjusted to pH 4. The DTT reduction buffer contained 3 mM EDTA, 0.1 M NaCl, 0.05 M sodium citrate tribasic dehydrate, and 0.1 M sodium tetraborate decahydrate in DI water and was adjusted to pH 5.5.23 The stop reaction buffer contained 10 mM sodium phosphate dibasic and 154 mM NaCl in DI water and was adjusted to pH 11.9. The 2 M TRIS base was made in DI water and had a pH of approximately 11. BTS Synthesis. BTS 3 was synthesized in two steps from 11bromoundecene 1 with a 95% overall yield (Scheme S1 of Supporting Information section X): S-(10-Undecenyl)-benzenethiosulfonate 2. To a stirred solution of 11-bromoundecene 1 (4.6 mL, 20.1 mmol, 1.0 equiv) in anhydrous acetonitrile (100 mL) was added benzenethionosulfonic acid sodium salt (85%, 9.2 g, 39.9 mmol, 2.0 equiv) at room temperature. The reaction was refluxed overnight and then submitted to an ethyl

Scheme 1. Illustration of (A) BTS Cross-Linker Condensing with Steel Surface Hydroxyl Groups, (B) Production of Fab′ Fragments, and (C) Ideal Reaction between the Fab′ Fragment and the BTS Adlayer

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Langmuir acetate/water extraction. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and then evaporated under reduced pressure. Purification by column chromatography (hexanes/ ethyl acetate: 100/0 to 95/5) finally provided thiosulfonate 2 as a pale yellow oil (6.15 g, 93% yield). 1H NMR (300 MHz, CDCl3) δ: 7.96 (m, 2H), 7.63 (m, 1H), 7.57 (m, 2H), 5.81 (m, 1H), 4.96 (m, 2H), 3.00 (t, J = 7.2 Hz, 2H), 2.03 (m, 2H), 1.60 (m, 2H), 1.29 (m, 12H). 13 C NMR (75 MHz, CDCl3) δ: 145.2, 139.4, 133.7, 129.4, 127.2, 114.4, 36.3, 34.0, 29.5, 29.4, 29.2, 29.1, 29.0, 28.8, 28.7. IR (neat): 3070, 1640, 1328, 1147 cm−1. HRMS (ESI, m/z) calcd for C17H27S2O2 (MH+) 327.1446; found 327.1447. S-(11-Trichlorosilylundecanyl)-benzenethiosulfonate 3 (BTS). In a heavy-walled tube equipped with a magnetic stirring bar, thiosulfonate 2 (411 mg, 1.3 mmol, 1.0 equiv) and chloroplatinic acid hexahydrate (6.5 mg, 0.013 mmol, 1.0 mol %) were loaded. The tube was transferred into a glovebox, and trichlorosilane (0.26 mL, 2.6 mmol, 2.0 equiv) was added to the solution. The tube was tightly fastened and then removed from the glovebox. The resulting solution was stirred at room temperature for 43 h behind a protecting shield. The excess of trichlorosilane was finally removed under vacuum to provide BTS 3 as a viscous orange oil (578 mg, 99% yield). 1H NMR (400 MHz, CDCl3, see Supporting Information section X) δ: 7.95 (m, 2H), 7.64 (m, 1H), 7.56 (m, 2H), 3.01 (t, J = 7.3 Hz, 2H), 1.59 (m, 4H), 1.41 (m, 2H), 1.25 (m, 14H). 13C NMR (100 MHz, CDCl3) δ: 145.2, 133.7, 129.4, 127.2, 36.3, 32.0, 29.6, 29.5 (2 13C), 29.2, 29.1, 28.8, 28.7, 24.5, 22.5. Surface Polishing. AISI 316L stainless steel foils (2 mm thick) were purchased from Goodfellow Cambridge Ltd. (Huntingdon, England). The steel is composed of 69% Fe, 18% Cr, 10% Ni, and 3% Mo. The foils were cut into approximately 5 × 5 mm coupons (for a micropolished surface finish) or 1 × 1 cm coupons (for an electropolished surface finish). Micropolishing was performed on a Leica EM TXP target sectioning system using successively finer abrasive grit papers (15, 9, 6, and finally 1 μm grit paper). An ∼100 μm slice was also removed from the face of some coupons in order to better level the Leica polishing head on the coupon surface. Electropolishing was performed by Electro-Kleen Alloy Polishing Ltd. (Mississauga, Ontario, Canada), who are experienced with polishing 300 series steels. While some electropolishing details are proprietary, the general procedure begins with degreasing the steel coupons in a heated alkaline solution. The coupons are then rinsed with tap water and electropolished in a heated (∼52 °C) solution containing phosphoric acid (75%), sulfuric acid (25%), and an additive. The coupons are then degreased again and rinsed in tap water followed by deionized water. Lastly, the coupons are passivated in a nitric acid solution (20−40%). Stainless Steel Cleaning Protocol. Individual polished steel coupons were first successively sonicated for 20 min in pentane, acetone, and 95% ethanol in order to remove weakly adsorbed nonpolar and polar contaminants. The coupons were then rinsed copiously with 95% ethanol, followed by distilled water. Once rinsed, the coupons were individually soaked for 30 min in approximately 5 mL of piranha solution (2.7:1 v/v mixture of concentrated H2SO4 and 30% H2O2) that was preheated to approximately 90 °C using a water bath. (Caution: piranha solutions are corrosive. Handle with extreme care.) The coupons were next rinsed three times with distilled water followed by methanol (×3). The coupons were then baked for 2 h in an oven maintained at 150 °C. Once dried, the coupons were nitrogen plasma-cleaned for 5 min, then immediately placed in a humiditycontrolled chamber (70% RH, room temperature), and left overnight. From here on in, a “bare” steel coupon refers to a steel coupon that was cleaned according to the aforementioned protocol. Silanization Protocol. Because of the high moisture sensitivity of trichlorosilanes, silanization of the stainless steel coupons were always performed in a glovebox under an inert (N2) and anhydrous (P2O5) atmosphere using anhydrous toluene as the solvent. In addition, glassware used for silanization was pretreated with an octadecyltrichlorosilane solution in anhydrous toluene (1/20 v/v) overnight, in order to prevent unwanted reactions between trichlorosilane molecules and any exposed hydroxyl groups from glassware walls.

Silanization was always conducted in a glovebox the day after the steel coupons were left in the humidity chamber overnight. BTS adlayers were produced from a 1 μL/mL BTS solution prepared by diluting neat BTS cross-linker in anhydrous toluene. 1 mL of BTS solution (2 mL for the 1 × 1 cm coupons) was then added to test tubes containing individual steel coupons. The test tubes were then sealed with rubber stoppers, removed from the glovebox, and then placed on a spin plate for various, increasing periods of time. Once the desired time elapsed, the coupons were rinsed three times with toluene followed by a 5 min sonication in toluene on the third rinse. After sonication, the coupons were rinsed one final time with toluene. The same rinsing procedure was then repeated with chloroform, before the coupons were finally dried under a gentle N2 stream. Silanization times of 3, 5, 10, 15, 20, 25, 30, 60, 90, 120, and 240 min were performed, in triplicate. Fab′ Fragment Generation. See Supporting Information section III. SDS PAGE Protocols. See Supporting Information section IV. Ellman’s Test for Thiols. See Supporting Information sections VI and VII. Antibody Immobilization. Whole IgG molecules or their Fab′ fragments were immobilized on BTS-modified or bare steel coupons by soaking individual coupons in a 0.1−0.2 mg/mL antibody solution (made in PBS) overnight (14−17 h). The glass test tubes used for antibody immobilization were stoppered, covered in tin foil, and then placed on a spin plate at room temperature. Following immobilization, three different rinsing protocols were evaluated. The first method (protocol A) included three rinses with approximately 5 mL of filtered PBS, followed by three rinses with copious amounts of DI water, and then drying under a gentle stream of N2. The second (protocol B) rinsing protocol began with three rinses with approximately 5 mL of filtered PBS, with a 5 min sonication step occurring on the third rinse. Once sonicated, a final rinse with ∼5 mL of filtered PBS was performed. This rinsing−sonication combination was then repeated using copious amounts of DI water. Once rinsed, the coupons were dried under a gentle N2 stream. The third rinsing procedure (protocol C) started with three rinses in copious amounts of DI water. The coupons were then individually soaked in 3 mL of a 2% SDS solution, and then placed on a spin plate at room temperature for 50 min, after which they were immediately sonicated for 10 min. The coupons were subsequently rinsed three times using copious amounts of DI water, and then dried under a gentle stream of N2. All coupons were then analyzed by CAG and XPS. As neither protocol was found to provide any advantage in terms of removing nonspecifically bound antibodies, protocol A was thus implemented for all subsequent immobilization studies because of its simplicity. Antigen Binding. Alexa Fluor 488 goat anti-mouse IgG (H+L) was used as the labeled antigen to the anti-goat IgG. Antigen binding studies were performed by individually soaking bare steel coupons, BTS adlayer only-modified coupons, and BTS-modified coupons biofunctionalized either with whole IgG or their fragmentswith or without BSA blocking. Three different concentrations of fluorescentlylabeled antigen (30, 3000, and 300 000 ng/mL prepared through serial dilution in filtered PBS) were used in this study. Experiments were performed in glass test tubes that were sealed, tin-foiled, and then set on a spin plate for 1 h at room temperature. After 1 h, the incubation was stopped by removing the labeled antibody solution and then rinsing each slide three times in approximately 5 mL of filtered PBS followed by copious amounts of DI water (×3). The slides were then gently dried under N2 and stored in sealed scintillation vials that were shielded from light until analysis by fluorescence microscopy. Blocking with sacrificial BSA was always performed prior to antigen binding by soaking individual bare or treated steel coupons in a 1% BSA solution in filtered PBS, for 1 h on a spin plate at room temperature (with tinfoil-covered glassware). Each slide was then rinsed three times in approximately 5 mL of filtered PBS, followed by copious amounts of DI water (×3). XPS Analysis. XPS analysis was performed with two instruments: the Thermo Scientific K-Alpha XPS spectrometer (ThermoFisher Scientific) or the Thermo Scientific Theta Probe (ThermoFisher Scientific) located at Surface Interface Ontario (University of Toronto, C

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Langmuir Toronto, Ontario, Canada). The latter is capable of angle-resolved analysis (multiple takeoff angles), whereas the former is generally restricted to 20° and 90° takeoff angles relative to the sample surface. In either case, both instruments utilize monochromatized Al Kα Xrays. The binding energy scale was always calibrated to the main C1s signal at 285.0 eV. The Avantage Data system software package (ThermoFisher Scientific) was used for data analysis. Peak fitting and integration were performed using the “Smart” method with a background at start and end set to 0.8 eV. Fluorescence Microscopy. Fluorescence microscopy was performed at the Advanced Optical Microscopy Facility (AOMF) in the MaRS Center (Toronto, Ontario, Canada) using a Zeiss AxioImager Z1 upright fluorescence microscope equipped with a Hamamatsu Flash4 camera. Excitation was performed with the X-Cite eXacte fluorescence lamp set to 100%. The 10×/0.3 NA objective lens was used with the FITC filterset and 500 ms exposure times. Data acquisition was performed with Metamorph software. The Metamorph software output grayscale TIFF images that were processed and their fluorescence intensities calculated using the ImageJ software. Applying a batch processing macro (provided in the Supporting Information section IX) to the outputted TIFF files, the stainless steel blank images (controls) were subtracted from each image, the maximum and minimum brightness adjusted, and the images tinted with the “green fire blue” fluorescence color profile, and finally converted to JPGs. Lastly, an ∼1 MP area (1380 × 777 pixels) was manually cropped from each image, and then, using the built-in “measure” batch processing function, the fluorescence intensity of the ∼1 MP crop was calculated.

coverage of BTS atop the steel surface across our three replicate time trials.26,27 At face value, the data from the curve of Figure S4 do reflect oscillatory behavior that is first dominated by a fast adsorption followed by a slight desorption at 5 min, and then a slow readsorption from 10 min onward. However, the size of the standard deviation at each point precludes any definitive conclusions to be drawn about the adsorption behavior of BTS since the converse argument of monotonic adsorption (where BTS deposition simply increases over time) could also be made within the stated error range. As such, more replication would be needed to try and reduce the standard deviation so that the adsorption behavior of BTS onto SS could be elucidated, or at least known with better accuracy. Despite its elusive adsorption behavior, BTS successfully depositing onto the steel surface was further strongly evidenced through comparative analysis of S2p XPS spectra. For bare SS, only one S2p signal at ∼169 eV (Figure 1A) is present that

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RESULTS AND DISCUSSION BTS Adlayer Formation on Stainless Steel. The deposition of BTS on micropolished stainless steel was assessed through low-resolution angle-resolved XPS (25°−75°) by tracking the Si2p signal as a function of coupon incubation time in BTS solution (a process referred to as “silanization”). The substrate signals, Fe2p and Cr2p, were also monitored along with those for C1s and O1s. Description/discussion of XPS data will be focused herein on the 55° takeoff angle relative to the normal as this simultaneously provides a representative elemental composition of both the BTS adlayer and underlying SS substrate. The data for all other measured takeoff angles and scanned elements are provided in section I of the Supporting Information. As shown in Figure S1, the silicon atomic percentage rises sharply within the first few minutes of silanization from ∼2% at time zero (control coupon that has been cleaned but not silanized) to just under 6% for 3 and 5 min of incubation in the BTS solution. It continues to rise to ∼7.5% after 15 min and then slightly dips before peaking and plateauing to ∼8.5% after 60 min incubation. The C1s curve (Figure S2) generally follows a similar trend, which is not surprising since both carbon and silicon mainly come from the BTS cross-linking molecules. The O1s (Figure S2), Cr2p (Figure S3) and Fe2p (Figure S3) signals, on the other hand, all steadily decrease as silanization proceedsas anticipated for a now buried, underlying SS substrate. Overall this indicates, as expected, that a carbon-rich, silicon-containing species is depositing onto and thus covering the metal oxide surface of the steel coupon. The apparent oscillation in the silicon curve could be the result of an adsorption−desorption−readsorption mechanism,24 a phenomenon already observed for organosilane SAMs (both trichloro- and trialkoxy-based) on various metallic substrates.24−26 In light of this, the Si2p data of Figure S1 were re-expressed through the following Si2p:(Si2p + Cr2p + Fe2p) ratio, which takes into account the XPS signals of elements from the substrate. This ratio attempts to normalize the

Figure 1. S2p spectra showing the appearance of a sulfur oxide peak (∼169 eV) after piranha treatment (A) and the appearance of a sulfide peak (∼164 eV) after silanization with BTS (B).

corresponds to sulfur oxide species,28 which likely are sulfate salts formed between iron and sulfuric acid from the piranha treatment used to clean the steel coupons. After exposing the cleaned steel coupons to BTS, a clear and distinct second peak appears at ∼164 eV, as shown in Figure 1B. Given that this peak is indicative of a sulfur sulfide moiety,28 and that it only and always appears after exposure of the steel to BTS, its presence was thus confidently attributed to the sulfide D

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Figure 2. Nitrogen relative atomic percentage for various antibody immobilization treatments (left) and the corresponding N1s XPS spectra (right).

When the surface of cleaned SS coupons is incubated in a BTS solution, this signal diminishes to ∼0.7%, which is consistent with a BTS adlayer depositing on the surface and partly attenuating the signal of the nitrogen-containing species located beneath. The nitrogen relative atomic percentages after individually incubating BTS adlayer-free steel coupons in solutions of Fab′ fragment or whole antibody (with equivalent total protein concentrations) were 2.7% and 5.1%, respectively. The nitrogen percentage after Fab′ incubation was slightly more elevated relative to bare clean steel, indicating very little fouling of SS by Fab′. Fouling by whole IgG was otherwise more substantial (∼100% increase compared to bare SS). The more pronounced adsorption of whole antibodies compared to their Fab′ fragments onto bare steel could be attributed to increased physicochemical interactions, either directly between the Fc region of the whole antibody (which Fab′ fragments lack) and the steel surface, or through glycan residues present on the Fc region. It may also be the result of a concerted effect between several regions of the intact antibody, which enables a greater number of interactions with the surface to take place. When steel coupons are functionalized with BTS, and then incubated in solutions of Fab′ fragment or whole antibody as was done with the bare clean coupons, the nitrogen percentage dramatically rises to 10.4% and 11.7%, respectively (Figure 2). The nitrogen peak after incubation of BTS-coated SS coupons in the antibody solutions is centered at ∼400 eV for both intact IgG and its Fab′ fragment. This peak may be attributed to protein-borne amines, which is in close agreement with a study conducted by Iwata et al., wherein a nitrogen peak at 399.8 eV after the site-directed immobilization of Fab′ fragments onto activated polymer brushes was observed.31 While the significant increase in nitrogen suggests successful antibody immobilization, the similar percentages achieved for both the intact antibody and its Fab′ fragment were surprising as a higher nitrogen signal would have been expected for the latter given the presence of the freed thiol moiety and lack of free thiols in the intact antibody as determined by Ellman’s test for thiols (protocol and data are presented in sections VI and VII, respectively, of the Supporting Information). This result questions whether the Fab′ fragments were in fact covalently bound through their freed thiol, or through amino acid side-

component in the benzenethiosulfonate function of BTS surface-modifying residues (Figure 1B). These results are well in line with those observed in our own previous research with BTS on quartz,22 aluminum nitride,29 and steel substrates.20 Lastly, when the steel coupons are exposed to a solution of BTS synthesis precursor (the corresponding alkene molecule lacking the SiCl3 anchoring function, Scheme S1 of Supporting Information section X), the sulfide peak at ∼164 eV does not appear in the S2p spectra, indicating that it is indeed through this trichlorosilyl moiety that BTS molecules attach to the surface of SS coupons. For the following experiments of antibody immobilization, we used BTS adlayers formed after 90 min, as this reaction time belongs where XPS data plateau, far enough away from the hypothetical initial adsorption−desorption phenomenon. We acknowledge however that this time of adlayer formation may not necessarily coincide with that of optimal antibody attachment (loading amount, distribution, etc.). Whole Antibody and Fab′ Fragment Immobilization. With successful BTS adlayer formation, antibody immobilization was performed next for the whole intact structure, as well as its Fab′ fragments. Briefly, the latter were generated by digesting whole antibodies with pepsin, an endopeptidase. This enzyme cleaves IgGs at the C-terminal side of the disulfide bonds in the hinge region to yield the F(ab′)2 fragment (Scheme 1, panel B).30 The F(ab′)2 fragment was then chemically reduced with dithiothreitol (DTT) to obtain two Fab′ fragments (Scheme 1, panel B). The full cleavage protocol and data work-up for the production of Fab′ fragments are provided in sections III and V of the Supporting Information, respectively. Because of the significant nitrogen content in proteins arising from both peptide bonds (−CO−NH−) and amino acid sidechains (e.g., lysine and arginine), the nitrogen relative atomic percentage was utilized as an indicator of antibody deposition. The XPS results are presented in Figure 2 for a 20° takeoff angle relative to the sample surface. The bare steel control surface has a N1s peak centered at ∼399 eV with a relative atomic percentage of ∼2.5%, which likely results from the exposure and reaction of steel coupons with nitrogen charged/ radical species during plasma cleaning prior to silanization. E

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Figure 3. S2p XPS spectra after antibody immobilization (recorded at a 20° takeoff angle relative to the surface).

chain amines, or even through physisorption. The phenomenon of protein physisorption is actually quite general and frequently encountered in many fields of biotechnology and is better known as “fouling”. As observed from a previous study by Blaszykowski et al., the reaction of BTS with a thiol-bearing, small molecule probe resulted in the loss of the sulfur oxide XPS peak as a result of the removal of the benzenesulfonyl leaving group (Ph−SO2−) of BTS (as benzenesulfinic acid) upon nucleophilic displacement.20 When the sulfur XPS narrow scan is consulted before and after exposure of BTS to any of the antibody solutions, we do not observe the same reduction/loss of the sulfur oxide peak (Figure 3). Instead, both the sulfur oxide and sulfide peaks are simply attenuated as a result of an overlying antibody layer. Hence, the single analysis of XPS data did not allow us to conclude whether antibodies, whole or fragmented, were covalently attached or not. To investigate the strength of antibody immobilization, we used harsher rinsing conditions than PBS and deionized water, that is, sonication and/or rinsing with a 2% SDS surfactant solutionthe latter being used during SDS PAGE protocols to disrupt noncovalent interactions and denature antibody samples. As shown in Figure 4, even when submitted to those harsh conditions, both intact and fragmented antibodies were barely removed from the adlayer surface (≤1% drop in the N1s percentage after rinsing). If not a proof of chemisorption (i.e., covalent attachment), this clearly demonstrated the robustness of antibody immobilization on the BTS adlayer. Even though the nature of the antibody linkage to the surface could not be definitively ascertained, we performed antigen binding studies as an indirect means of assessing whether there existed differences in paratope orientation between intact and fragmented antibodies. The ideal, covalent attachment of Fab′ fragments through their targeted freed thiol function of the hinge region would warrant proper paratope orientation (Scheme 1, panel C), and hence optimal antigen binding affinity. A much contrasted result would be displayed by randomly physisorbed whole antibodies. This investigation is the objective of the following section.

Figure 4. Change in nitrogen XPS atomic percentage (at 20° take-off angle relative to the surface) after rinsing antibody-immobilized surfaces with either PBS and deionized water, with sonication, or rinsing with SDS followed by sonication.

Antigen Binding to Immobilized Whole Antibodies and Fab′ Fragments. Antigen binding was assessed through fluorescence microscopy using fluorescently-labeled goat IgG (antigen). In order to obtain a greater surface area for fluorescence measurements, these experiments were performed on 10 × 10 mm electropolished rather than 5 × 5 mm micropolished coupons. For rigor purposes, antigen binding experiments and subsequent XPS analysis with cleaned bare SS, BTS-derivatized, and antibody-functionalized surfaces were carried out for both micropolishing and electropolishing treatments of the substrates. Gratifyingly, the observation was made that both polishing methods provided comparable results, albeit a smoother surface finish for electropolished coupons (see section VIII of the Supporting Information). As noted earlier, these binding experiments were performed not only to determine whether the immobilized antibodies remained active toward their antigen, but also to try and indirectly determine whether the immobilized Fab′ fragments were properly, or at least better oriented than whole antibodies. This was hypothesized to be expressed through enhanced antigen binding capability, that is, brighter fluorescence for F

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exposure to the labeled antigen (Figure 7B). It is safe to say that sacrificial BSA32 successfully prevented the labeled IgG antigen from interacting with the BTS adlayer. All measured fluorescence intensity values can be found in Table S3 of section IX in the Supporting Information. Antigen binding was also assessed for both whole antibodyand Fab′ fragment-functionalized surfaces, both with and without the use of sacrificial BSA. When BSA was omitted, there appeared to be no advantage in utilizing fragmented over intact antibodies. Both surfaces indeed produced very similar images and mean fluorescence intensities (Figure 7C and D). It is worth noting that several different surface fluorescence features can be seen on both of these images. For instance, the very bright spots are believed to be the aforementioned aggregates, whether deposited from solution or self-assembled through surface seeding. The streaks are artifacts likely revealing surface scratches, while the lightly illuminated background fluorescence, on the other hand, is believed to be the fluorescence signal of interest that arises from antibodyantigen interactions. Interesting results were obtained when BSA was used in combination with the capture antibody (whole or Fab′ fragment) and the resulting surfaces then exposed to the fluorescently-labeled antigen. As expected, the fluorescence signal was attenuated, but not totally lost, when Fab′-modified BTS adlayers were precoated with BSA (Figure 7F), in comparison to no BSA treatment (Figure 7D). Surface coverage was still quite homogeneous as well. Unexpectedly, however, whole antibody-modified BTS adlayers precoated with BSA produced a (much) higher fluorescence than without BSA treatment (Figure 7E vs Figure 7C)with a much heterogeneous, uncontrolled surface coverage. This observation, while surprising, may hypothetically reflect the conformational restructuration of BSA with concomitant formation of neoepitopes with high antigen affinity.33 Regardless, given that whole antibody-modified BTS adlayers without BSA pretreatment would constitute, overall, a better option for antigen capture, there seems to be no practical advantage in going through the hassle of producing and purifying antibody Fab′ fragments, and implementing an extra blocking step with sacrificial BSA. This may be true in vitro but certainly would not be in vivo, where naturally plentiful albumin (albumin is the most abundant blood plasma protein at 30−50 g/L3) would fill in interstitial areas of Fab′-modified BTS adlayers to produce surfaces alike that observed in Figure 7F. In this scenario, the use of Fab′ fragments over intact IgG could actually be favorable. Another benefit of using the body’s own naturallyoccurring albumin would reside in the minimization of fouling, and possibly the prevention of unwanted adverse biological processes such as surface-induced thrombosis and even neointimal hyperplasia. Overall, the BTS adlayer enabled the robust immobilization of functional antibodies and their fragments, as shown through XPS and fluorescence microscopy experimental data. The BTS strategy offers a simple, one-step modification of oxidized/ naturally-hydroxylated surfaces as compared to more complicated methods such as that based on atom transfer radical polymerization (ATRP), which has been used to couple Fab′ fragments onto polymer brush-coated surfaces.31

fragmented vs whole antibody-immobilized BTS adlayers. Antigen binding was assessed at low (30 ng/mL), medium (3000 ng/mL), and high (300 000 ng/mL) concentrations of fluorescently-labeled antigen. While the lowest concentration value rarely produced any detectable fluorescence (see section IX, Table S3 of the Supporting Information), the highest concentration value (although successful in displaying antigen binding) resulted in an increased number of aggregates relative to the midrange concentration, as observed by large areas of extremely intense fluorescence (Figure 5A). We believe these

Figure 5. Fluorescence images of (A) a BTS-modified surface with immobilized intact IgG and incubated in a solution of the highest test concentration (300 000 ng/mL) of labeled antigen, and (B) a bare steel surface exposed to the midrange concentration (3000 ng/mL) of labeled antigen. The fluorescence intensity color scale, which also applies to Figures 6 and 7, is also provided.

results of uncontrolled antibody−antigen binding to be deposited aggregates formed in solution, and/or aggregates built on-surface through self-assembly. As a result, the following results and discussion only pertain to the midrange concentration of labeled antigen, unless otherwise noted. BTS adlayer-only, antibody-functionalized BTS, and BSAcoated BTS surfaces did not exhibit any significant intrinsic fluorescence relative to cleaned bare SS (Figure 6). Bare steel

Figure 6. Background fluorescence image for a cleaned bare steel surface. The fluorescence intensity color scale is provided in Figure 5.

exposed to the antigen exhibited limited fluorescence too (Figure 5B vs Figure 6), i.e., was not fouled. This result was consistent with the low nitrogen XPS atomic percentage observed in the previous section (Figure 2) when bare steel was exposed to whole antibodies (or their fragments). When the BTS-modified surface was exposed to the antigen, the fluorescence intensity dramatically increased as evident from Figure 7A. The substantially increased and widespread fluorescence over the entire surface area indicates not only that BTS was heavily and homogeneously fouled by the IgG antigen, but also that it exhibited good coverage over the steel substrate. When the BTS adlayer was treated with BSA, a well-known and commonly used blocking agent in many assay protocols such as ELISA, the fluorescence was significantly attenuated after

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CONCLUSION It was demonstrated by this study that both whole and Fab′ fragment antibodies could be successfully tethered to the G

DOI: 10.1021/acs.langmuir.5b00812 Langmuir XXXX, XXX, XXX−XXX

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Langmuir

Figure 7. Fluorescence images of BTS-modified surfaces incubated in the midrange concentration (3000 ng/mL) of labeled antigen (A) without and (B) with BSA blocking. Second row fluorescence images are those of BTS adlayers functionalized with (C) intact antibodies or (D) Fab′ fragments, and then exposed to the midrange concentration of labeled antigen, without BSA blocking. Third row fluorescence images are those of BTS adlayers functionalized with (E) intact antibodies or (F) Fab′ fragments, and then exposed to the midrange concentration of labeled antigen, with BSA blocking. Mean intensities are reported in the bottom left corner for images C and D. The fluorescence intensity color scale is provided in Figure 5.

measured fluorescence intensities, and synthesis and proton NMR spectrum for BTS. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b00812.

surface of 316L stainless steel through a precoated BTS conditioning adlayer. While the mode of attachment, either through chemi- or physisorption, could not be determined for Fab′ fragments, it was observed that the immobilized antibodies were strongly bound to the BTS-derivatized surfaces, as evidenced by the lack of protein detachment upon exposure to harsh rinsing conditions. Fluorescently-labeled antigen binding experiments without the use of BSA blocking revealed that intact antibody- and Fab′ fragment-functionalized coatings display similar antigen binding capability. However, in the context of in vivo EPC capture, an environment where albumin would be naturally abundant, the Fab′ fragment approach was concluded to be a more appealing option. Currently, the implementation of the BTS adlayer/capture antibody strategy to the directed and controlled proliferation of EPC on SS surfaces for application in stent technology is under investigation.

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AUTHOR INFORMATION

Corresponding Author

*Tel +1 416 978 3575; e-mail [email protected] (M.T.). Present Address

S.R.K.: Yale School of Medicine, New Haven, CT 06510. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for support, Drs. Peter Brodersen and Rana Sodhi from Surface Interface Ontario for XPS analysis, James Jonkman from the Advanced Optical Microscopy Facility at the MaRS Center for fluorescence analysis, and Isaac Li and Duncan Smith-Halverson of the Walker group at the University of Toronto for the AFM measurements.

ASSOCIATED CONTENT

S Supporting Information *

XPS data of BTS-modified steel surfaces, XPS data of selected elements in the silanization of steel with BTS, protocol for Fab′ fragment production and SDS PAGE, sample work-up data for antibody fragment production, protocol and results for Ellman’s test for thiols, electropolished versus micropolished steel, H

DOI: 10.1021/acs.langmuir.5b00812 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.5b00812 Langmuir XXXX, XXX, XXX−XXX