Patterning and Biofunctionalization of Antifouling Hyperbranched

Jun 23, 2014 - Bahman Delalat,. † ... terminal HPG diols to yield reactive aldehyde groups. ... such as carboxylic acids, succinimidyl esters, or ep...
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Patterning and Biofunctionalization of Antifouling Hyperbranched Polyglycerol Coatings Eli Moore,†,‡ Bahman Delalat,† Roshan Vasani,† Helmut Thissen,‡ and Nicolas H. Voelcker*,† †

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Mawson Institute, University of South Australia, GPO Box 2471, Adelaide, South Australia 5001, Australia ‡ CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, Victoria 3168, Australia S Supporting Information *

ABSTRACT: We demonstrate the patterned biofunctionalization of antifouling hyperbranched polyglycerol (HPG) coatings on silicon and glass substrates. The ultralow fouling HPG coatings afforded straightforward chemical handles for rapid bioconjugation of amine containing biomolecular species. This was achieved by sodium periodate oxidation of terminal HPG diols to yield reactive aldehyde groups. Patterned microprinting of sodium periodate and cell adhesion mediating cyclic peptides containing the RGD sequence resulted in an array of covalently immobilized bioactive signals. When incubated with mouse fibroblasts, the HPG background resisted cell attachment whereas high density cell attachment was observed on the peptide spots, resulting in high-contrast cell microarrays. We also demonstrated single-step, in situ functionalization of the HPG coatings by printing periodate and peptide concurrently. Our results demonstrate the effectiveness of antifouling and functionalized HPG graft polymer coatings and establish their use in microarray applications for the first time.

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INTRODUCTION Cell microarrays are high throughput lab-on-a-chip cell capture systems used for a range of applications from fundamental research into cellular responses and cell behavior to biomedical diagnostics.1−3 High parallelism and statistical certainty can be achieved by printing thousands of potential biological factor molecules in microscale spots onto the surface of a single chip.4,5 The key to cell microarrays is the surface coating since this coating needs to (a) enable stable binding of bioactive signals on the spots and (b) provide a low-fouling background that is resistant to protein adsorption and cell attachment in between the spots.6,7 Cell attachment in between the spots complicates the scoring of the cell responses to the spots and may lead to undesired cross-talk of cells from different spots, thereby limiting the density of spots on the array. At the same time, it is desirable that the cell microarray coating facilitates straightforward and reliable bioconjugation. Current techniques for producing cell microarrays often include multiple steps and lengthy reactions to achieve bioconjugation and maintain specificity. Silanization is often used to introduce functionality such as carboxylic acids, succinimidyl esters, or epoxide groups onto glass slides.2,6,8−10 Spots of biomolecules printed onto these surfaces can be conjugated through their amine functional groups. Subsequent backfilling around the printed spots with bovine serum albumin (BSA) or poly(ethylene glycol) (PEG) containing a terminal amine helps to reduce nonspecific binding of proteins and cells in between the spots.9,11 However, © 2014 American Chemical Society

passivation techniques such as these can add up to 2 days to the microarray fabrication time, and the resulting surface may still not be completely protein or cell resistant.12 Other techniques rely on the retention of the studied bioactive signals on hydrogel coatings based on polymers such as PEG and polyacrylamide without any covalent conjugation.13,14 While this negates issues involved with arduous and tedious covalent conjugation reactions, problems can arise with leaching of signals from the surface and these systems may not be suitable for long-term cell culture studies. Here, we introduce hyperbranched polyglycerol (HPG) surface coatings as a novel option for high-contrast cell microarrays. HPG has shown antifouling properties equivalent to PEG with the added benefit of increased stability against oxidation and a high level of functionality.15−18 HPG expresses a high level of functionality on its periphery in the form of diols. These can be rapidly oxidized to yield aldehydes using sodium periodate. Bioconjugation is then achieved via Schiff base formation between the aldehyde on the surface and an amine present on the biomolecule in solution. A fast cyanoborohydride-based reduction step forms a stable secondary amine bond via reductive amination. Aldehyde formation, Schiff base formation, and reductive amination all occur in near neutral aqueous solution at room temperature, which is crucial for Received: April 24, 2014 Revised: June 13, 2014 Published: June 23, 2014 2735

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Contact Angle Measurements. Milli-Q water (18.2 MΩ cm; 3 μL) was placed onto the samples using a 100 μL Hamilton syringe fitted with a hydrophobic sleeve and images were captured using a Panasonic WV-BP550/G CCTV camera. The static contact angle was measured using ImageJ software with the drop analysis plugin. All measurements were repeated a minimum of 3 times and the results averaged. Ellipsometry. The thickness of grafted HPG films were measured with a J.A. Woollam V-Vase ellipsometer using wavelengths between 250 and 1100 nm in 10 nm increments and at 65, 70, and 75° from the surface normal. To obtain the ellipsometric thickness of the grafted HPG films, the VASE spectra were fitted with the multilayer model on the basis of the WVASE32 analysis software, using the optical properties of a generalized Cauchy layer. Atomic Force Microscopy (AFM). AFM imaging was performed using a NanoWizard III BioAFM (JPK Instruments, Berlin). The 5 × 5 μm2 images were recorded in air and pure water at 22 °C in contact mode. The probes used were FORT (AppNano) with a spring constant in the range of 1.2−6.4 N/m (manufacturer’s values). Fluorescence Microscopy. Fluorescence microscopy images were captured using a Nikon Eclipse-TiS microscope fitted with a Nikon Digital Sight DS-2MBWc digital camera head and Nikon NISElements imaging software. ImageJ software was used to overlay different imaging channels. Microprinting. Peptide microarrays were printed using a XactII Compact Microarrayer with a hollow pin (Xtend microarray pin) with a diameter of 350 μm. Preparation of HPG-Grafted Surfaces. Glycidol was polymerized directly from silicon and glass surfaces using procedures first developed by Huck et al.19 In short, silicon wafer and glass microscope slides were cut into 1 cm2 pieces. These were washed with ethanol and dried with a stream of N2. The samples were treated with a mixture of ammonium hydroxide, hydrogen peroxide, and Milli-Q water in a ratio of 1:1:5, also known as RCA, at 70 °C for 30 min. Samples were washed with water and ethanol and dried with a stream of N2. Sodium methoxide (0.15 M) 10 mL was added to the samples under N2 at 70 °C and reacted for 1 h. Sodium methoxide was removed and the samples were washed thoroughly with methanol (anhydrous, 3 × 20 mL) and toluene (anhydrous, 3 × 30 mL) using a syringe. Samples were dried under vacuum at 100 °C for 20 min. Glycidol (distilled the day before and dried over molecular sieves overnight) was added to cover the samples and the polymerization proceeded for 90 min at 100 °C. Polymerization was terminated by removing unreacted monomer and quenching the reaction with water. Samples were washed thoroughly with water and ethanol and dried with a stream of N2. Fluorescent Dye Conjugation to HPG-Grafted Surfaces Microprinted with Sodium Periodate. Sodium periodate (10 mg/mL) was dissolved in Milli-Q water and kept protected from light since it is a light-sensitive reagent.20 Therefore, this and all subsequent reactions involving sodium periodate were conducted away form direct light exposure. HPG-grafted silicon and glass samples were printed with an array of 2 × 2 spots (1.4 mm between the center of each spot and printed in triplicate) of sodium periodate solution and reacted in the dark for 1 h at RT. The microarray samples were then washed with Milli-Q water and submersed in a solution of Lissamine Rhodamine B ethylenediamine (100 μg/mL in carbonate buffer pH 9.6) for 2 h at RT while being protected from light. Sodium cyanoborohydride (5 M in carbonate buffer, 10 μL per 1 mL of reaction solution) was then added to the Lissamine solution and reacted for 30 min.20 Microarray samples were then washed thoroughly with Milli-Q water and dried with a stream of N2. Cyclic RGD Peptide Conjugation to HPG-Grafted Surfaces Microprinted with Sodium Periodate. Sodium periodate (10 mg/ mL) was dissolved in Milli-Q water and kept protected from light. The solution was printed onto HPG-grafted glass and silicon samples in an array of 3 × 3 spots (1.4 mm between the center of each spot and each array printed in duplicate) and reacted in the dark for 1 h at RT. After 1 h, the samples were washed with Milli-Q water and submersed in a solution of either c(RGDfK) or c(RGDfK(PEG−PEG)) peptide (1 mg/mL in carbonate buffer pH 9.6) for 2 h at RT and protected from

successful microarray-based surface immobilization. Furthermore, HPG activation and bioconjugation can be achieved by combining sodium periodate and biomolecule in a one-pot reaction, reducing the number of preparative steps and the time required for microarray assembly. The microarrays are ready for use immediately after a short reductive amination step and do not require further passivation. Our results demonstrate that this approach can produce high-contrast cell microarrays with excellent performance compared to existing microarray formats.9

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MATERIALS AND METHODS

Glycidol (Sigma, 96%) was distilled under vacuum and stored over molecular sieves in the freezer overnight. Glass microscope slides (Menzel-Gläser, 76 × 26 mm2) were cut and washed with ethanol prior to use. p-Type silicon wafers (Siegert Wafer; Boron doped; ⟨100⟩ orientation; ≤0.001 Ω cm; 381 μm thickness) were cut and washed with acetone and dichloromethane and dried under a stream of N2 gas prior to use. Methanol (Chemsupply, 100% undenatured) was dried over molecular sieves before use. Toluene (Sigma, 98%) was distilled over sodium metal and benzophenone (Sigma, 99%) and stored over molecular sieves. Sodium metal (Sigma, ≥99%, stored under mineral oil) was washed in hexane after weighing. Sodium periodate (Sigma, 99.8%), sodium cyanoborohydride (Sigma, 95%), Lissamine rhodamine B ethylenediamine (Invitrogen), cyclo(Arg-Gly-Asp-D-Phe-Lys) (c(RGDfK)), cyclo(Arg-Gly-Asp-D-Phe-Lys(PEG−PEG)) (c(RGDfK(PEG−PEG)); both Peptides International, Inc.) were all used as received. For cell culture, the following reagents were used: 0.01 M phosphate buffered saline pH 7.4 (PBS, Sigma) and paraformaldehyde solution (4%, Electron Microscopy Science). DMEM medium (Invitrogen), fetal bovine serum (FBS, Invitrogen), L-glutamine (Invitrogen), penicillin (Invitrogen), streptomycin (Invitrogen), amphotericin B (Invitrogen), phalloidin-TRITC (Sigma), Hoechst 33342 (Invitrogen), propidium iodide (PI, Sigma), fluorescein diacetate (FDA, Sigma), Fluoro-Gel mounting medium (ProSciTech), and trypsin (0.05%, EDTA 0.53 mM, Life Technologies) were all used as received. All incubation took place at room temperature (RT) unless otherwise stated. All solutions were prepared using ultrapurified water supplied by a Milli-Q system (Millipore). NIH/3T3 mouse embryonic fibroblast cells (ATCC CRL-1658, American Type Culture Collection, Manassas, U.S.A.) were used in the cell culture experiments. X-ray Photoelectron Spectroscopy (XPS). XPS analysis was performed using an AXIS Ultra DLD spectrometer (Kratos Analytical Inc., Manchester, U.K.) with a monochromated Al Kα source at a power of 45 W (15 kV × 3 mA), a hemispherical analyzer operating in the fixed analyzer transmission mode and the standard aperture (1 × 0.5 mm2 slot). The total pressure in the main vacuum chamber during analysis was typically 10−8 mbar. Each specimen was analyzed at an emission angle of 0° as measured from the surface normal. Assuming typical values for the electron attenuation length of relevant photoelectrons in organic compounds, the XPS analysis depth (from which 95% of the detected signal originates) ranged between 5 and 10 nm. An elliptical area with approximate dimensions of 0.3 × 0.7 mm2 was analyzed on each sample. Data processing was performed using CasaXPS processing software version 2.3.15 (Casa Software Ltd., Teignmouth, U.K.). All elements present were identified from survey spectra (acquired at a pass energy of 160 eV). To obtain more detailed information, high resolution spectra were recorded from individual peaks at 40 eV pass energy (yielding a typical peak width for polymers of 1.0−1.1 eV). These data were quantified using a Simplex algorithm in order to calculate optimized curve fits and thus to determine the contributions from specific functional groups. The atomic concentrations of the detected elements were calculated using integral peak intensities and the sensitivity factors supplied by the manufacturer. Binding energies were referenced to the aliphatic hydrocarbon C 1s peak at 285.0 eV. 2736

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light. After 2 h, sodium cyanoborohydride (5 M in carbonate buffer, 10 μL per 1 mL of reaction solution) was added to the peptide solution and reacted for a further 30 min. Microarray samples were then washed thoroughly with Milli-Q water and stored at 4 °C. Microprinting Solutions of Cyclic RGD Peptide and Sodium Periodate onto HPG-Grafted Surfaces. Sodium periodate (10 mg/ mL) and c(RGDfK(PEG−PEG)) (1 mg/mL) were dissolved in carbonate buffer (pH 9.6). The solution was protected from light at all times. Sodium periodate/peptide solution was printed in a 4 × 4 grid (1.4 mm between each spot and each array printed in duplicate) across the surface of HPG-grafted samples and reacted in the dark for 2 h at RT. Microarray samples were removed and lightly washed with Milli-Q water to remove unreacted reagents. They were then submersed in a solution of sodium cyanoborohydride (50 mM in carbonate buffer pH 9.6) for 30 min and washed thoroughly with Milli-Q water.20 Patterned Cell Attachment on Printed RGD Microarrays. Prior to incubation of 3T3 mouse fibroblast cells (American Type Culture Collection) with the microarrays, the arrays were pretreated in sterile PBS containing 200 U/mL penicillin, 200 μg/mL streptomycin, and 500 ng/mL amphotericin B for 2 h. Each HPG-grafted microarray was placed in a 4-well plate (Nunc) and seeded with cells at a density of 1 × 105 cells/cm2. Cells were initially incubated in contact with the array for 60 min at 37 °C and 5% CO2. Loosely attached cells were then removed by washing with prewarmed DMEM. The cells were fixed in 4% paraformaldehyde solution for 30 min and then permeabilised with 0.25% Triton X-100 for 5 min at room temperature. Nuclei of cells were stained with 2 μg/mL Hoechst 33342 dye for 15 min at room temperature. Cells were stained with 100 μM phalloidin-TRITC for 45 min, then mounted with Fluoro-Gel reagent. The fluorescence for Hoechst 33342 and phalloidin-TRITC was detected with an inverted fluorescence microscope (Nikon, Eclipse, Ti−S) equipped with appropriate filters.

Ellipsometry determined that the coating thickness for SiHPG90 and G-HPG90 was 6.7 ± 0.2 and 4.4 ± 0.2 nm, respectively. RMS surface roughness was determined by AFM to be 0.60 and 0.48 nm for Si-HPG90 and G-HPG90, respectively, while sessile drop water contact angle measurements for Si-HPG90 and G-HPG90 were 28.9 ± 1.5° and 32.9 ± 2.4°, respectively (Table 1). Patterned Cell Microarrays on HPG-Grafted Surfaces. One key benefit in using HPG coatings over more linear polymer antifouling coatings such as PEG is the high level of functionality present at the surface of the film.22 Due to the nature of the polymerization of glycidol, a majority of the terminal groups on the periphery of HPGs are diols.23 This presents the opportunity to bioconjugate a plethora of amine containing molecules to the surface after periodate-driven oxidation of terminal diols to aldehydes.20,24,25 The resulting Schiff base can be reduced to a stable secondary amine bond using a mild reducing agent such as sodium cyanoborohydride, without affecting the bioactivity of the attached biomolecule.20 To test the reactivity of the HPG-grafted surfaces, an amine functional fluorescent dye was conjugated via the proposed method. Initially, an aqueous solution of sodium periodate was printed in the dark onto HPG-grafted surfaces in an array of microscale spots of 350 μm diameter and washed off after a short reaction time. A solution of an amino-functional Lissamine dye was then added over the array to drive Schiff base formation, which was subsequently reduced using cyanoborohydride.20 Figure 2 shows that the attachment of Lissamine dye followed the 2 × 2 array of spots where periodate had been printed. In contrast, nonspecific adsorption of the dye on the HPG coating was not detected. This result indicates that Lissamine had indeed reacted with the aldehyde groups generated from the HPG via periodate oxidation. The fact that the spots were not perfectly round is due to the nature of the contact printing using quilled printing pins.26 Inhomogeneous fluorescence intensity across each Lissamine conjugated spot (Figure 2b) indicated inhomogeneous loading of the dye. This could have been a result of incomplete periodate oxidation of available diols at the surface but was more likely to have been caused by steric interference preventing consistent interaction between aldehydes at the surface and the amine on the dye. In the case of the latter, increasing the length of the spacer arm between the bulk of the molecule and the amine to be conjugated should help to overcome issues with steric hindrance. While the Lissamine dye was conjugated for proof of principle and therefore graft density and homogeneity were of no real consequence, the translation of the inhomogeneous loading to bioactive species designed to mediate cell adhesion could adversely affect the ability to uniformly adhere cells across the spots. In the context of microarrays, this could reduce their usefulness in the application of high throughput screening of biomolecules and their respective loading concentrations. After demonstrating the specific conjugation of an aminecontaining dye molecule, we pursued the conjugation of a cell adhesion mediating peptide. RGD peptides are a group of peptides that feature the minimal amino acid sequence (arginine-glycine-aspartic acid) responsible for cell adhesion to a range of extra cellular matrix (ECM) proteins through integrin-ligand binding, and are therefore commonly used as synthetic cell adhesion promoters.27,28 Two cyclic RGD (cRGD) peptides were used to conjugate to sodium-period-

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RESULTS AND DISCUSSION Surface-Initiated HPG Growth. HPG-grafted silicon wafer (Si-HPG90) and glass (G-HPG90) substrates were prepared by the surface-initiated anionic ring-opening polymerization of glycidol from deprotonated silanol groups. After polymerization, the characteristic C−O peak at ∼286.6 eV appeared in the C 1s high-resolution spectra.21 This peak is characteristic for etheric carbon, consistent with the introduction of HPG onto the surface. Figure 1 shows representative high-resolution C 1s spectra obtained for HPG-grafted silicon wafer and glass microscope slide samples. The dominant etheric peak at 286.6 eV accounted for almost 85% of the carbon content detected on the surface. The remaining 15% of carbon on the surface was attributed to adventitious carbon.

Figure 1. High resolution C 1s XPS spectra obtained after the polymerization of glycidol from (a) silicon wafer and (b) glass substrates at 100 °C for 90 min. The dominant etheric carbon peak at 286.6 eV (red line) indicates the presence of HPGs on the surface. Peaks at 285.0 eV (blue line, C−C) and 288.5 eV (green line, CO) were attributed to adventitious carbon. 2737

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Table 1. Chemical and Physical Characteristics of HPG-Grafted Silicon Wafer and Glass Samples Polymerized for 90 mina sample

atomic concn (%) Si

atomic concn (%) O

atomic concn (%) C

film thickness (nm)

surface roughness (nm)

contact angle (deg)

Si-HPG90 G-HPG90

19.1 7.6

33.3 35.8

47.6 55.5

6.7 ± 0.2 4.4 ± 0.2

0.60 0.48

28.9 ± 1.5 32.9 ± 2.4

a

Elemental compositions were determined by XPS in atomic concentration (%), coating thickness was determined using ellipsometry, surface roughness (RMS) was determined by AFM and contact angles were calculated from sessile drop water contact angle analysis.

Figure 2. (A) Fluorescence microscopy image of an array of 2 × 2 spots on HPG-grafted glass after printing of periodate and reaction with Lissamine dye having an amino functional group. Scale bar = 200 μm. (B) The line profile of the fluorescence intensity of Lissamine conjugated spots.

Scheme 1 (at 1 mg/mL) in carbonate buffer (pH 9.6), followed by reaction with sodium cyanoborohydride to perform reductive amination. The peptide microarrays were incubated with 3T3 mouse fibroblasts for 1 h and then gently washed with sterile PBS to remove unattached cells. The remaining cells were fixed, stained, and imaged with a fluorescence microscope (Figure 3). Intermittent bright field microscopy observations of the microarrays during incubation and before cell fixation determined that high cell densities over the printed peptide spots were already achieved after 1 h of incubation. Figure 3 shows that cell attachment was almost exclusively confined to the printed peptide spots. After only 1 h of incubation in a suspension of cells, the attached cell density was high and relatively consistent across all spots and cells had already begun to spread. Comparing the two peptides, the cell density on the c(RGDfK(PEG−PEG)) spots of 108 ± 9 × 103 cells/cm2 (Figure 3a) was considerably higher than on the c(RGDfK) spots with 63 ± 6 × 103 cells/cm2 (Figure 3b). However, cell morphology appeared to be similar for both samples (see higher magnification images). The difference in cell density between the two peptides indicated that the PEG linker arm indeed increased the avidity of the RGD-functionalized surface for integrin binding. For that reason, c(RGDfK(PEG−PEG)) was chosen for all subsequent experiments. As discussed earlier, the extended PEG−PEG linker arm within the c(RGDfK(PEG−PEG)) peptide possibly provided two avenues for the improved cell immobilization seen in Figure 3a. The first avenue being an increased concentration of peptide bound during the conjugation stage due to a reduction in steric hindrance during the reaction as the amine is extended away from the bulk of the molecule, thereby negating the inhomogeneous loading issues observed for the Lissamine dye. The second avenue being an increase in flexibility of the bound peptide, allowing for a greater enthalpy of integrin binding.29

ate-activated HPG surfaces: cyclo(Arg-Gly-Asp-D-Phe-Lys) (c(RGDfK)) was chosen since conjugation can be achieved through the amino group on the lysine residue and cyclo(ArgGly-Asp-D-Phe-Lys(PEG−PEG)) (c(RGDfK(PEG−PEG))) was chosen as the PEG arm spacer extends the amine away from the rest of the molecule, making it potentially more accessible for conjugation (Scheme 1). The PEG spacer also Scheme 1. Chemical Structures of (a) c(RGDfK) and (b) c(RGDfK(PEG−PEG))

helps to extend the conjugated peptide away from the surface, thereby potentially increasing flexibility and improving integrin binding. These factors potentially increase attached cell density within the printed spots in comparison to the c(RGDfK) peptide.29 The peptide microarray fabrication procedure is outlined in Scheme 2. As with the fluorescent dye microarrays, sodium periodate was initially printed onto HPG-grafted glass and silicon surfaces in spots within a 3 × 3 grid and reacted in the dark at RT. After washing off the periodate, samples were incubated with solutions of either of the two peptides shown in 2738

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Scheme 2. Printing of Microscale Spots of Sodium Periodate in 3 × 3 Arrays and Subsequent Reductive Amination for the Covalent Immobilization of c(RGDfK) Peptides Periodate-Activated Spots on the HPG-Grafted Surface

sodium periodate and peptide could be printed together in a single step to facilitate in situ functionalization. If successful, this would allow screening of a large range of conditions such as concentration gradients or biomolecule libraries by printing on a single chip. Here, a mixture of sodium periodate and c(RGDfK(PEG− PEG)) was printed onto HPG-grafted glass and silicon in 4 × 4 grids. Samples were afterward placed in a solution of sodium cyanoborohydride for reductive amination to proceed. 3T3 cells were then once again incubated on the peptide microarrays, fixed and stained. In Figure 5a,b, a 4 × 4 peptide microarray printed on a HPG-grafted glass surface incubated with 3T3 cells is shown. High cell densities within the printed spots along with cell spreading indicated favorable conditions for integrinmediated cell attachment. In contrast, very low levels of nonspecific cell attachment were observed between the spots, demonstrating the low-fouling properties of the HPG background coating. Uniform cell attachment across the spots and a lack of rings of cells around the spot circumference (Figure 5) suggests that the single-step in situ functionalization also produces more homogeneous peptide conjugation than the two-step peptide conjugation procedure. In situ peptide functionalization on HPG-grafted silicon chips gave similar results when compared to the glass substrate in terms of selective cell attachment on the spots (Figure 5c,d).

In several instances, cells appeared to be pinned to the circumference of the spots. This can be explained by a coffee ring effect during periodate activation.6 However, this phenomenon was more noticeable on the microarrays printed with c(RGDfK) due to lower cell densities within the spots. Control samples were tested to determine the extent of nonspecific cell attachment contributed by unreacted aldehyde functionalities remaining on the surface. HPG-grafted surfaces were treated by periodate printing, washed and incubated with 3T3 cells as described above. Very low levels of nonspecific cell attachment were observed across the surface and spots of preferential cell attachment could not be identified (Figure 4). Therefore, we conclude that cell attachment in Figure 3 was attributed to the presence of HPG-immobilized peptides and not the mere presence of aldehyde groups. Importantly, this result also suggested that chemical quenching of the remaining aldehydes within the printed spots is not required for our procedure. In order to broaden the practical applications of HPG-grafted microarray substrates, they must be capable of facilitating the conjugation of a number of different compounds and to screen different conditions across a single microarray chip. However, this aim cannot be achieved using the above-mentioned procedure since the entire chip was incubated in a solution containing a single peptide. We therefore investigated if the 2739

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Figure 3. Fluorescence microscopy images of 3T3 mouse fibroblast cells after 1 h incubation on HPG-grafted glass substrates with (a) depicting a 3 × 3 cell microarray on c(RGDfK(PEG−PEG)) spots with a higher magnification image of a single spot on the right and (b) depicting a 3 × 3 cell microarray on c(RGDfK) spots under the same conditions with a higher magnification image of a single spot on the left.

Figure 4. Fluorescence microscopy images of 3T3 cells attached to HPG-grafted from (a) silicon and (b) glass substrates printed with an array of spots of sodium periodate after 1 h of incubation in a suspension of cells.

spots of sodium periodate without any peptide. Here, fibroblast cells were seeded at a seeding density of 2 × 105 cells/cm2 (double the seeding density used in Figure 5) for 2 h. Figure 6 revealed cell attachment on spots with printed peptide concentrations as low as 100 ng/mL. Typically, printing solutions used for microarray printing consist of biomolecule concentrations between 50 and 1000 μg/mL.4,9,26,30,31 There-

To investigate the dependence of peptide concentration on cell attachment on the chips, a concentration gradient of peptide was printed across HPG-grafted glass substrates. Peptide concentration was reduced stepwise from 1 mg/mL down to 1 ng/mL. The printing procedure followed as previously outlined with mixtures of peptide and sodium periodate being printed onto the surface along with control 2740

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Figure 5. Fluorescence microscopy images of 3T3 cells incubated for 1 h on HPG-grafted glass and silicon chips with a 4 × 4 array of c(RGDfK(PEG−PEG)) spots formed in a single step procedure. (a) Depicts a 4 × 4 array of peptide spots colonized with 3T3 cells on HPG-grafted glass, (b) shows a single peptide spot colonized with 3T3 cells on HPG-grafted glass, and (c, d) representative peptide spots colonized with 3T3 cells on HPG-grafted silicon.

Figure 6. Fluorescence microscopy image of 3T3 cells attached to a peptide concentration gradient. Cells were seeded at 2 × 105 cells/cm2 and incubated for 2 h. Peptide concentrations ranged from 1 mg/mL to 1 ng/mL. Scale bar = 400 μm.

concentration and the 2 h incubation time, which was used to aid cell attachment at lower printed peptide concentrations. There was no evidence of cell attachment to the control spot where only periodate had been printed in the absence of peptide.

fore, our approach demonstrates highly efficient RGD peptide display on the HPG-grafted chips at concentrations orders of magnitude lower than generally used. The high cell density and cell colonization past the boundaries of the printed spots for the highest four peptide concentrations was attributed to the high cell seeding 2741

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Another drawback of currently used coatings for microarrays is the fact that they employ reactive chemistries including epoxide, aldehyde, and N-hydroxysuccinimide (NHS) functionalities that are quenched upon long storage times. Furthermore, backfilling is required after biomolecule printing to quench the remaining reactive functional groups to prevent nonspecific binding, as discussed earlier. These drawbacks are effectively overcome on the HPG-grafted chips which can be activated with periodate on demand. HPG-grafted substrates used throughout this study were stored for up to 3 months prior to microarray printing at room temperature and we did not notice a negative impact of extended storage on the low fouling properties of HPG or the ability to facilitate covalent peptide attachment.

CONCLUSIONS We have studied the prospects of HPG-grafted substrates as novel platforms for cell microarrays. Using cRGD peptides, we have shown that amine-functional molecules were conjugated easily and specifically across sodium periodate activated HPG coatings. Sodium periodate was used as an on-demand activating agent to transform the inert HPG coating into an aldehyde-functionalized coating capable of reacting with amines. Peptide microarrays were generated by microprinting arrays of spots of sodium periodate solution, followed by incubation with cRGD peptides. A short treatment with the mild reducing agent sodium cyanoborohydride resulted in stable bioconjugation. A single-step in situ functionalization procedure was also developed where periodate and peptide were printed concurrently. This approach allowed the generation of microarrays containing different molecular species or of different concentrations of the same species on each spot within the array. Patterned cell microarrays with high cell coverage were obtained following 1 h incubation with 3T3 mouse fibroblasts. Cells were confined to the spots representing conjugated peptide. In contrast, a very low level of nonspecific cell attachment was observed on the background HPG-grafted surface. We also demonstrated that on the HPG-grafted chips, printed peptide concentrations as low as 100 ng/mL elicited fibroblast attachment, whereas higher concentrations between 50 and 1000 μg/mL are generally used with conventional microarray coatings. We therefore conclude that thin coatings produced by grafting of HPG polymers from glass or silicon substrates are a valuable addition to currently available microarray coatings. Improvements include high polymer stability, on-demand surface activation and no backfilling or surface-quenching requirements to obtain an antifouling background. ASSOCIATED CONTENT

S Supporting Information *

XPS survey spectra of bare and HPG-grafted silicon and glass substrates; high magnification fluorescence images of a Lissamine microarray and 3T3 cell microarrays. This material is available free of charge via the Internet at http://pubs.acs.org.

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*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2742

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