Nonbiofouling Polymer Brush with Latent Aldehyde Functionality as

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Biomacromolecules 2010, 11, 284–293

Nonbiofouling Polymer Brush with Latent Aldehyde Functionality as a Template for Protein Micropatterning Yuquan Zou,† Po-Ying J. Yeh,‡ Nicholas A. A. Rossi,† Donald E. Brooks,*,†,§ and Jayachandran N. Kizhakkedathu*,† Centre for Blood Research and Department of Pathology and Laboratory of Medicine, Department of Chemistry, and Department of Mechanical Engineering, 2350 Health Sciences Mall, University of British Columbia, Vancouver, B.C.V6T 1Z3, Canada Received October 9, 2009; Revised Manuscript Received November 23, 2009

A novel, nonfouling polymer brush, poly-N-[(2,3-dihydroxypropyl)acrylamide] (PDHPA), containing latent aldehyde groups, was synthesized by surface initiated atom transfer radical polymerization (SI-ATRP). The synthetic parameters were adjusted to produce brushes with varying graft densities and molecular weights. High-density PDHPA brushes successfully prevented the nonspecific protein adsorption from single protein solutions as well as from human platelet poor plasma. Patterns of nonfouling PDHPA and reactive PDHPA-aldehyde domains on the brush surface were created by a combination of photo and wet chemical lithography from a single homogeneous PDHPA brush. Successful micropatterning of single proteins and multiple proteins were achieved using this novel substrate. The high-density brush prevented the diffusion of large proteins into the brush, while a monolayer of covalently coupled proteins was formed on the PDHPA-aldehyde domains. Atomic force microscopy (AFM) force measurements using a biotin coupled AFM tip showed that covalently coupled streptavidin retained its activity, while PDHPA domains showed little nonspecific adsorption of streptavidin. The current study avoids tedious and complicated synthetic processes employed in conventional approaches by providing a novel approach to protein micropatterning from a single, multifunctional polymer brush.

1. Introduction Polymer brushes form an important class of polymeric material that has many potential applications. When polymer graft density is sufficiently high, strong exclusion forces the polymer chains to adopt a unique stretched conformation, yielding polymer brushes with distinctive properties.1 With advances in synthetic methodology during the past decade, many different approaches have been established to prepare highdensity polymer brushes, including free radical, ionic, and controlled polymerization techniques.2-7 Recent studies of polymer brushes have focused on two main aspects: (1) synthesis of novel polymer brushes containing unique functionality via block/copolymerization or postpolymerization modification, and (2) fabrication of polymer brushes with micro or nanostructures. It is predicted that the combination of these two features will yield some promising advances in the micropatterning of proteins and cells.8,10 Significant attention has been directed toward these developments, mainly due to their potential use in studying protein-protein and protein-cell interactions,11 developing novel biochips and biosensors,12 and in the design of novel templates for tissue engineering.13 To date, a few different approaches have been reported for the fabrication of micropatterned proteins, such as microcontact printing (µCP) and photolithography-assisted patterning.14,15 While the micropatterning of proteins using polymer brushes is an emerging area of research, there are a few important * To whom correspondence should be addressed. Tel.: +1-604-8227085. Fax: +1-604-822-7742. E-mail: [email protected] (J.N.K.); [email protected] (D.E.B.). † Centre for Blood Research and Department of Pathology and Laboratory of Medicine. ‡ Department of Mechanical Engineering. § Department of Chemistry.

prerequisites for the successful micropatterning of proteins via patterned polymer brushes. First, the grafted polymer chains should bear functionalities such as amine, carboxylic acid, hydroxyl, thiol, or aldehyde groups, which can be used for the specific conjugation of proteins onto a surface. Second, to minimize background noise, the underlying polymer layer should resist the nonspecific adsorption of proteins (i.e., nonbiofouling).16 To satisfy these divergent criteria, two types of polymer chains, one representing a functional layer and the other representing a nonfouling layer, have been grafted from the same surface successively.8 For example, Dong et al. utilized poly(acrylic acid) brushes as a coupling segment for patterning bovine serum albumin (BSA) and streptavidin.8a Xu et al. used terminal hydroxyl groups on the side chains of poly(ethylene glycol)methacrylate (PEGMA) as the coupling site for micropatterning human immunoglobulin (IgG).8b In both cases a similar concept was adopted: a nonfouling PEG SAM layer (which resists nonspecific protein adsorption) was deposited prior to the micropatterning of polymerization initiators via photolithography. Surface initiated atom transfer radical polymerization (SI-ATRP) was subsequently employed to generate patterned brushes with appropriate chemical functionality for attaching proteins. As shown by Xu et al., the process is often complicated and requires separate synthetic steps and polymerizations (two independent polymerization steps).8b Another potential problem with this approach is that the chain density, molecular weight, and thickness of the grafted chains in these two layers may vary due to the different polymerization conditions and monomers employed, causing microheterogeneity on the surface, which is rarely noted in the literature. In the present study, we have developed a novel and facile approach for the micropatterning of proteins on a single component, homogeneous, and nonbiofouling polymer brush

10.1021/bm901159d  2010 American Chemical Society Published on Web 12/15/2009

Polymer Brush for Protein Micropatterning

consisting of poly-N-[(2,3-dihydroxypropyl)acrylamide] (PDHPA). The pendant 1,2-diol groups on the PDHPA brush are readily converted to aldehydes via a fast and mild oxidation for protein conjugation. Using a combination of photo- and wet chemical lithography, a patterned, binary polymer brush system containing nonfouling PDHPA and reactive PDHPA-aldehyde zones are obtained and are subsequently used as the template for protein micropatterning.

2. Experimental Section 2.1. Materials and Methods. 2,2-Dimethyl-1,3-dioxolane-4-methanamine (Aldrich, 97%), methyl 2-chloropropionate (Aldrich, 99%), 1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA, Aldrich, 97%), CuCl (Aldrich, 99+%), and CuCl2 (Aldrich, 99.99%) were used as supplied. Tris[2-(dimethylamino)ethyl]amine (Me6TREN) was synthesized according to the literature.9 Rhodamine-labeled streptavidin was purchased from Invitrogen (S6366). All other commercial reagents were purchased from Aldrich of the highest purity and used without further purification. Silicon wafers were purchased from University Wafer (Boston, U.S.A.) with one side polished. Water was purified using a Mili-Q Plus water purification system (Milipore Corp., Bedford, MA) and was used in all experiments. 2.2. Instrumentation. Static water contact angles were determined by dropping a water droplet of 2 µL on the surface before a picture of the droplet was taken using a digital camera (Retiga 1300, Q-imaging Co.). Water contact angles were analyzed using the software Northern Eclipse. Five different sites were tested for each sample and the average value is reported. Dry film thickness of the grafted brushes was measured by ellipsometry. The variable-angle spectroscopic ellipsometry (VASE) spectra were collected on an M-2000 V spectroscopic ellipsometer (J. A. Woollam Co. Inc., Lincoln, NE) at 45, 55, and 65°, at wavelengths from 370 to 1000 nm, with an M-2000 50 W quartz tungsten halogen light source. The VASE spectra were then fitted with the multilayer model on the basis of the WVASE32 analysis software, using the optical properties of a generalized Cauchy layer to obtain the “dry” thickness of the PDMDOMA brush layer. Fluorescence microscopy was performed using a Zeiss Axioskop 2 with an AttoArc2 fluorescence illumination system, an FITC and Rhod filter, and an Axiocam Icc1 digital camera. Absolute molecular weights of the “free” polymers were determined by gel permeation chromatography (GPC) on a Waters 2690 separation module fitted with a DAWN EOS multiangle laser light scattering (MALLS) detector from Wyatt Technology Corp. with 18 detectors placed at different angles (laser wavelength λ ) 690 nm) and a refractive index detector from Viscotek Corp. operated at λ ) 620 nm. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were recorded using a ThermoNicolet Nexus FT-IR spectrometer with a MCT/A liquid nitrogen cooled detector, KBr beamsplitter, and MKII Golden Gate Single Refection attenuated total reflectance accessory (Specac Inc.). Spectra were recorded at 4 cm-1 resolution, and 100 scans were collected for each sample. 2.3. AFM. Atomic force microscopy measurements were performed on a multimode, Nanoscope IIIa controller (Digital Instruments, Santa Barbara, CA) equipped with an atomic head of 130 × 130 µm2 scan range. AFM measurements were performed in air or underwater in contact mode using a commercially manufactured V-shaped silicon nitride (Si3N4) cantilever with gold on the back for laser beam reflection (Veeco, NP-S20). The spring constant of AFM cantilever was measured using thermal equipartition theorem. Force measurements were performed on this instrument using a fluid cell modified to allow temperature adjustment and measurement. The experiments were performed under water in force mode. On tip approach the onset of the region of constant compliance was used to determine the zero distance, and on retraction the region in which force was unchanged was used to determine the zero force. The rate of tip-sample approach speed was typically 1 µm/s but ranged between 0.05 to 5 µm/s. No significant difference was observed between different approach speeds.

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Covalent binding of biotin onto the AFM tip was achieved via a three-step procedure, which includes (1) generation of hydroxyl group on AFM tip via oxygen plasma treatment; (2) surface amination; and (3) covalent coupling of biotin. The detailed experimental condition was described in Figure 4S (Supporting Information). 2.4. Preparation of ATRP Initiator-Deposited Silicon Wafer and Surface Initiated Atom Transfer Radical Polymerization (SI-ATRP). A trichlorosilane-functionalized surface was prepared according to the literature with small modifications.2a Briefly, the silicon wafer was first treated using “piranha” solution (30/70 30% aqueous hydrogen peroxide solution/sulfuric acid) at 90 °C for 1 h. It should be noted that piranha solution is extremely reactiVe and as such should be handled with great care. Ω-Undecylenyl alcohol (4.26 g, 25 mmol) was esterified with an equimolar amount of 2-chloro-propionyl chloride (2.5 mL, 25 mmol) in the presence of triethylamine (4.2 mL, 30 mmol). The crude compound was purified by flash column chromatography and the yield was about 90-95%. The resulting 10-undecen-1-yl 2-chloro-methylpropionate ester (1.10 g, 4.20 mmol) was hydrosilated using 4.2 mL of trichlorosilane (42.6 mmol) and Karstedt’s catalyst (10 µL). The conversion was almost quantitative. The obtained initiator, 11-(2-chloro)-propionyloxyundecenyltrichlorosilane, was deposited onto freshly cleaned wafers by adding 10 µL of initiator into a vial containing 10 mL of anhydrous toluene and the silicon wafer. The typical thickness of the monolayer was 2 nm after 16 h of incubation at ambient temperature. The successful deposition of the ATRP initiator layer was verified by X-ray photoelectron spectroscopy (XPS). Figure 7S (A) (Supporting Information) shows a representative XPS spectrum of silicon wafer modified with the bromine-containing ATRP initiator. The signal at 71 ev belongs to bromine, which verifies the attachment of the initiator. All polymerizations were carried out in a glovebox filled with argon at room temperature. Synthesis of [(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide (DMDOMA) monomer and SI-ATRP of DMDOMA followed the conditions reported earlier by our group.17 For a typical reaction, the initiator-deposited silicon wafer was cut into 1 × 1 cm sizes and placed into a 20 mL glass vial. A two-neck round-bottom flask was loaded with CuCl (15 mg, 0.15 mM), CuCl2 (2 mg, 0.015 mM), HMTETA (80 mg, 0.30 mM), and DMDOMA (1 g, 5 mmol) with 2 mL of DMF as the solvent. The solution was sealed with a rubber septum and cycled three times between argon and vacuum to remove oxygen before addition to the vial containing silicon wafer. Soluble initiator methyl 2-chloropropionate (MCP, 5 µL) was also added. The polymerization proceeded at ambient temperature for a predetermined time, after which silicon wafer was removed and thoroughly rinsed and sonicated (3 min × 3) with DMF and water to remove physically adsorbed polymers. The wafer was then stored in water until use. “Free” polymer formed concomitantly with grafted chains was purified by dialysis against water for 3 days and lyophilized. The obtained PDMDOMA solution was completely hydrolyzed using 10% acetic acid solution at 45 °C for 2 h, dialyzed against water for 3 days and analyzed by GPC-MALLS. The chemical structure of PDMDOMA on silicon wafer was probed using ATR-FTIR and AFM. The successful grafting of the PDMDOMA brush was also verified by XPS (Supporting Information, Figure 7S (B)). A nitrogen signal observed at 400 ev is associated with the grafted PDMDOMA brush. 2.5. Synthesis of Poly-N-[(2,3-dihydroxypropyl)acrylamide] (PDHPA) and PDHPA-Aldehyde Brushes. A Si/SiO2/PDMDOMA polymer brush was prepared as described above. To prepare a PDHPA brush (100%), the polymer brush was placed into a 20 mL glass vial before being filled with 10% acetic acid solution. The reaction proceeded at 45 °C for 2 h, and then the wafer was removed and rinsed thoroughly by water. The resulting PDHPA (100%) brush was put into a solution of sodium periodate (3 mg/mL) for 60 min, removed, and thoroughly washed with water. The cleaned PDHPA-aldehyde (100%) brush was stored in water until use.

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2.6. Nonspecific Protein Adsorption. Silicon wafers of 1 × 1 cm dimensions bearing PDHPA (100%) brushes were incubated with fluorescein isothiocyanate-bovine serum albumin (FITC-BSA; 400 µg/ mL in 50 mM MES buffer, pH ) 5.1) or rhodamine-labeled streptavidin (rhodamine-streptavidin; 100 µg/mL in 50 mM MES buffer, pH ) 5.1) for 1 h. Following the incubation, the substrates were rinsed thoroughly with PBS (pH ) 7.4, >10 times) to remove loosely adsorbed proteins. The images of the protein adsorbed surfaces were taken by a fluorescence microscope after drying the sample and compared with the images before adsorption qualitatively under FITC- and rhodaminefilter, respectively. The power settings for fluorescence microscopy were set constant through the study to provide comparable fluorescence intensity. Silicon wafers of 1 × 1 cm dimensions bearing PDHPA (100%) brushes or ATRP initiator layer were incubated with fibrinogen (1 mg/ mL, PBS buffer, pH ) 7.4) or platelet poor plasma (100%) from a healthy donor for 1 h. Following the incubation, the substrates were rinsed thoroughly with phosphate buffered saline (PBS) to remove loosely adsorbed proteins and dried under argon flow. The thickness changes before and after incubation were measured by ellipsometery. 2.7. Covalent Coupling of Protein. A silicon wafer of 1 × 1 cm size bearing PDHPA-aldehyde (100%) was incubated with FITC-BSA (400 µg/mL) or rhodamine-streptavidin (100 µg/mL) for 1 h. In the case of rhodamine-streptavidin, the surface coupling at 400 and 100 µg/mL protein concentration produced nearly identical results. Therefore, 100 µg/mL was chosen as the final concentration. The washing and qualitative comparison followed the same procedure as the nonspecific protein adsorption. 2.8. Deposition of Photoresist and Photolithography. First, the PDHPA-grafted silicon wafer (1 × 1 cm) was spin-coated with hexamethyldisiloxane (HMDS) as an adhesion layer at 4000 rpm for 30 s to reduce the amount of water present on the surface. The surfaces were left in air for 30 s before the photoresist coating was added. A photosensitive photoresist, SPR 220 (Megaposit SPR 220, Rohm Haas) was spin-coated at 500 rpm for 10 s, followed by 1500 rpm for 30 s, and then 3500 rpm for 10 s. The thickness of coated phoresist was about 10 µm. The surface was then soft baked on a hot plate at 90 °C for 1 min and 110 °C for 1 min before UV exposure. The photoresist coated surface was exposed under a lithography tool (UV 405 nm, Canon mask aligner) for 2 min. The mask for lithography was designed by L-edit (Tanner Research, Inc.) and printed on a transparency with a resolution of 20000 dpi (Output City CAD/Art Services, Inc.). After exposure, the surface was kept under air for 1 h to allow water (which is necessary to complete the photoreaction) to diffuse back into the photoresist film. Then the surface was developed by MF-24A (Rohm Haas) for 3-5 min. After developing, the surface was heated on hot plate at 90 °C for 1 min to increase adhesion strength of the photoresist on the substrate. The surface was then kept in water before use. 2.9. Wet Chemical Lithography and Micropatterning of Rhodamine-Streptavidin. The photoresist-coated PDHPA brushes bearing different patterns were incubated with sodium periodate solution (3 mg/mL) for a given time (0.5-60 min). Following the incubation, the samples were thoroughly washed with water to remove residual sodium periodate. The photoresist was then removed by methanol and acetone via continuous washing and finally rinsed by water. The rinsed sample was immediately incubated with rhodamine-streptavidin solution (100 µg/mL) for 30 min. Sodium cyanoborohydride was then added to a concentration of 1 mg/mL. The sample was incubated for another 15 min, removed, and thoroughly washed with PBS (pH ) 7.4), followed by Milli-Q water to remove loosely adsorbed proteins. The obtained sample was characterized by fluorescence microscopy, SEM and AFM. 2.10. Multiprotein Micropatterning on a Single Substrate. The wafer (1 × 1 cm) bearing the PDHPA-S3 brush was first patterned with a 20 µm-wide stripe of rhodamine-streptavidin as described above. The patterned wafer was incubated in sodium periodate (3 mg/mL) for 5 min, thoroughly rinsed by PBS (pH ) 7.4), and incubated with FITCBSA (400 µg/mL) for 30 min. Sodium cyanoborohydride was then

Zou et al. added to a concentration of 1 mg/mL. The sample was incubated for another 15 min, removed, and thoroughly washed with PBS (pH ) 7.4), followed by Milli-Q water to remove the loosely adsorbed proteins. The obtained sample was characterized by fluorescence microscopy under FITC- and rhodamine-filters.

3. Results and Discussion 3.1. Synthesis of PDMDOMA, PDHPA, and PDHPA-Aldehyde Brushes. Poly-N-[(2,3-dihydroxypropyl)acrylamide] (PDHPA) brushes were obtained via postpolymerization modification of poly-N-[(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide (PDMDOMA) brushes (Figure 1) on silicon wafers. The controlled synthesis of PDMDOMA brushes was achieved by surface-initiated atom transfer radical polymerization (SI-ATRP). Polymerization conditions for SI-ATRP were based on our previously reported ATRP conditions for DMDOMA in solution17 and were optimized for surface initiated polymerization to produce well-controlled grafted brushes with variable molecular weights and thickness (Table 1). Solvent and the ATRP catalyst proved to be critical for controlling polydispersity, molecular weight of the chains (estimated from the solution polymers), and thickness of the grafted layer. By using MeOH/H2O as the solvent and CuCl/Me6TREN (tris[2-(dimethylamino)ethyl]amine) as catalyst, relatively thick PDMDOMA brushes (∼35 nm) were obtained. However, the molecular weight distribution of the “free” solution polymer was relatively high. Therefore, thin and uniform PDMDOMA brushes (∼9 nm) were synthesized in DMF using HMTETA (1,1,4,7,10,10-hexamethyltriethylenetetramine) as the ligand for the current study. It is noted that the graft density of PDMDOMA brushes increased with increased polymerization time indicating a slow initiation process. A similar phenomenon was observed previously for another acrylamide monomer (N,Ndimethylacrylamide),7 suggesting that this unique phenomenon might be typical for acrylamide-based monomers. Further investigations to verify these observations with studies would be worth-while. The graft densities of the PDMDOMA bushes were calculated by the dry polymer thickness (h) determined by ellipsometry utilizing the following equation: σ ) hFNA/Mn, where F is the density of PDMDOMA (1.20 g/cm3), NA is Avogadro’s number, and Mn is the number average molecular weight.20,31 Brush molecular weight characteristics were estimated from the solution polymer formed along with the grafted chains, an approximation that is widely used.18 We supported this assumption by estimating the molecular weight of the grafted S2PDMDOMA polymer via AFM according to our previously reported “pull off” method.19 The number average molecular weights obtained by the two methods were comparable (19300 Da by AFM, 20800 Da from solution GPC).20 Therefore, the molecular weight characteristics obtained in this study should reflect the properties of surface polymers reasonably well. The graft densities of the PDMDOMA brushes varied from 0.12 to 0.47 chains/nm2 (Table 1). The presence of the characteristic amide I peak (1654 cm-1) was verified by ATR-FTIR spectroscopy of the PDMDOMA brushes (Figure 2) on the silicon wafers. The uniformity of the brush layer is evident from the morphology and molecular weight characteristics of the PDMDOMA brush determined by atomic force microscopy (AFM, Supporting Information, Figure 6S (B)) and gel permeation chromatography of the “free” soluble polymer obtained along with the grafted layer (GPC, Supporting Information, Figure 6S (C)), respectively. The controlled nature of the polymerization was also demonstrated by growing block copolymers of



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Figure 1. Synthetic scheme for preparing a micropatterned protein array using a combination of photolithography and chemical lithography from single, homogeneous polymer brush layer. Step 1: Si-ATRP of DMDOMA from ATRP initiator deposited silicon wafer surface; step 2: postpolymerization of PDMDOMA brush via acidic hydrolysis to yield PDHPA brushes; step 3: deposition of photoresist and generation of micropatterns; step 4: wet chemical lithography to generate PDHPA/PDHPA-aldehyde binary brush system; steps 5 and 6: removal of photoresist and selective coupling of steptavidin onto the substrate. PDMDOMA: (poly-N-[(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide); PDHPA: (polyN-[(2,3-dihydroxypropyl)acrylamide]). Table 1. Characteristics of PDMDOMA Brushes Prepared by SI-ATRP of DMDOMA from Silicon Wafersa samples

reaction time

initiator

ligand

solvent

dry thickness (nm)

Mn,SEC and Mw/Mn of “free” polymerb

graft density c (chains/nm2)

S1 S2 S3 S4 S5 S6 S7

8 12 24 24 24 24 24

Cl Cl Cl Br Cl Cl Cl

HMTETA HMTETA HMTETA HMTETA Me6TREN HMTETA Me6TREN

DMF DMF DMF DMF DMF MeOH/H2O (1:1) MeOH/H2O (1:1)

3.1 ( 0.1 6.3 ( 0.3 8.9 ( 0.2 3.4 ( 0.2 7.3 ( 0.3 4.2 ( 0.3 35 ( 2.1

18280, 1.21 20800, 1.18 21800, 1.19 11200, 1.36 15000, 1.15 8900, 1.38 56000, 1.52

0.12 0.22 0.31 0.23 0.37 0.35 0.47

a Molar ratio of [CuCl]/[CuCl2]/[ligand] ) 1:0.1:2; monomer concentration [DMDOMA] ) 2.5 M. b “Free” solution polymer was obtained by addition of sacrificial initiator into the polymerization system. In the case of Cl as initiator, methyl 2-chloropropionate was used as the free initiator; in the case of Br, methyl 2-bromopropionate was used as the sacrificial initiator. c Graft density of PDMDOMA brushes was calculated following the equation: σ ) hFNA/Mn, where F is the density of PDMDOMA (1.20 g/cm3), NA is Avogadro’s number, and Mn is the number average molecular weight.

DMDOMA and N,N-dimethylacrylamide (DMA) from the PDMDOMA brush (Figure 1S, Supporting Information). The linear increase in the thickness of the PDMDOMA brushes with polymerization time (Figure 2S, Supporting Information) also supports the controlled nature of polymerization. The synthesis of PDHPA and PDHPA-aldehyde brushes are highlighted in Figure 1. A simple and efficient acidic hydrolysis (10% acetic acid, 45 °C, 2 h) converted the dioxolane groups on the PDMDOMA brushes to 1,2-dihydroxyl groups to form PDHPA brushes. Subsequent oxidation of 1,2-diols in PDHPA brushes using a mild oxidizing agent, sodium periodate, yielded PDHPA-aldehyde brushes. The current experimental conditions

were chosen based on the relevant reaction conditions for soluble polymers.17 The broad peak around 3400 cm-1 was assigned to hydroxyl groups while the reduced intensity of the peak at 2900 cm-1 (C-H stretching in CH3) in the ATR-FTIR spectrum illustrates cleavage of dioxolane side group (Figure 2A). The conversion of the PDMDOMA brush to a PDHPA brush was also evidenced by the sharp decrease in water contact angle from 58 to 15° (Figure 3S, Supporting Information) and a decrease in the dry thickness of the brush (35 to 32 nm) due to the removal of the pendant group. It has been shown elsewhere that the conversion of dioxolane groups to 1,2-diols groups in soluble polymer is quantitative.17 The presence of aldehyde

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Figure 2. ATR-FTIR spectrum of PDMDOMA (S7, 35 nm), PDHPA (S7, 100% cleaved, 32 nm), and PDHPA-aldehyde (S7, 100% oxidized, 32 nm).

groups in the PDHPA-aldehyde brush was evident from the appearance of a new peak at 1720 cm-1, assigned as -CdO stretching for the aldehyde group (Figure 2A). It is noted that PDHPA brushes can also be synthesized directly via a SI-ATRP of N-(2,3-dihydroxypropyl)acrylamide (DHPA) monomer in DMF using the same catalyst. The PDHPA brushes with comparable thickness and graft density to those obtained via the postpolymerization modification can be controlled by adjusting polymerization time. Extra attention, however, is needed during hydrolysis and column purification to avoid polymerization of the DHPA monomer. Therefore, the postpolymerization modification is preferred and adopted in the current study. 3.2. Nonspecific Protein Adsorption versus Specific Protein Coupling. The most important aspect of the current research is the generation of a multifunctional surface that possesses two essential properties: (1) minimal nonspecific protein adsorption (nonbiofouling) and (2) functionality to aid the covalent coupling of proteins to the surface. In the present case, the nonbiofouling properties of high-density and neutral hydrophilic brushes have been exploited.21 As has recently been reported, PDHPA is a highly hydrophilic and neutral polymer with a “barrier” capacity that prevents hydrophobic interactions, one of the main factors leading to nonspecific protein adsorption, making it a promising nonfouling surface.20 Importantly, the 1,2-diols present on the PDHPA brushes are latent aldehyde functional groups that can be readily converted and used for the covalent conjugation of proteins via reductive amination.22 These properties make the PDHPA brush a unique and novel substrate for protein micropatterning. Verification of the nonfouling properties of the PDHPA brushes, as well as the covalent coupling of proteins on PDHPAaldehyde brushes is presented in Figure 3A using rhodaminestreptavidin and FITC-BSA. The low fluorescent intensity of the protein-incubated PDHPA sample (derivatized from S3; I and III) indicates very low nonspecific protein adsorption. The difference in fluorescence intensity of the sample before and after protein incubation was about 3.9 ( 2.2%. In contrast, the fluorescent intensity of ATRP initiator modified surface shows a 52% increase after FITC-BSA incubation (a 15-fold higher fluorescent intensity compared to PDHPA brush-coated surface). The conclusion was also supported by the absence of any

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significant change in brush thickness as measured by ellipsometry after protein incubation. Further proof of the nonfouling properties of PDHPA brushes is presented in Figure 3B (III), in which S3-PDHPA and a control sample (hydrophobic ATRP-initiator deposited wafer) was incubated with undiluted platelet poor plasma (PPP) and fibrinogen solution (1 mg/mL, PBS buffer, pH 7.4) for 1 h. The control sample shows 3.5 and 6.4 nm increases in thickness after incubation with PPP and fibrinogen, respectively; water contact angle also dramatically decreased from 90 to 46°, suggesting severe nonspecific protein adsorption when PDHPA brush layer was absent. It is noted that PPP with higher fibrinogen concentration (2.6 to 4.5 mg/mL) shows lower increase in thickness compared to fibrinogen solution (1 mg/ mL). The reasons for this observation could be due to the Vroman effect, that is, the maximum protein adsorption normally occurs at intermediate plasma concentration.32a In addition, the coexisting protein in PPP such as kininogen might replace the adsorbed fibrinogen, which could be another factor resulting the lower absorption of fibrinogen from PPP.32b In contrast, S3PDHPA only shows insignificant thickness increase compared to the control sample after incubating with fibrinogen solution and undiluted PPP (ca. 0.1 nm, 35- and 64-fold lower than that of control sample), further verifying that the high-density PDHPA brush can efficiently reduce nonspecific protein adsorption. When PDHPA-aldehyde brushes were used (images II and IV), a significant increase in fluorescence intensity was observed in contrast to the PDHPA brushes, indicating significant covalent binding of proteins on this brush. The clear contrast between the two sets of images reflects the diverse nature of protein interaction with PDHPA and PDHPA-aldehyde homogeneous brushes, guaranteeing the feasibility of using these two brushes as nonbiofouling and coupling zones for further protein micropatterning. A detailed analysis of the protein-resistance property for PDHPA brushes will be presented in a forthcoming publication. The effect of oxidation time on protein coupling was further investigated by using rhodamine-labeled streptavidin. Figure 3B (I and II) shows that the fluorescence intensity and thickness of the coupled streptavidin layer increased sharply in the first 5 min before reaching a plateau. Prolonged oxidation time, which yielded more aldehyde groups, did not lead to further protein coupling. ATR-FTIR on oxidized PDHPA brushes verified that complete oxidation of the PDHPA brush required 45 to 60 min (data not shown). Our previous study using soluble PDHPA polymers also showed that a complete conversion to PDHPAaldehyde took ∼60 min.17 The saturation of protein coupling on partially oxidized PDHPA brushes may be due to the “barrier” effect exerted by the densely grafted hydrophilic brush, which prevents diffusion of protein into the brush.20,23 The ellipsometric and AFM thickness of the grafted streptavidin layer was approximately 4.2 and 4.0 nm, respectively, which was similar to the reported thickness of the surface-adsorbed streptavidin monolayer (4.3 to 4.8 nm) estimated by ellipsometer, X-ray, and neutron reflectometry.24 The formation of a monolayer of streptavidin suggests that the covalent coupling of proteins might only take place on the outer surface of the PDHPA-aldehyde brush, instead of in the interior of the brush (Figure 3C), as the high density of the brush structure prevents the diffusion of large proteins into the brush. This might explain why there is no further increase in protein coupling even though more aldehyde groups were generated. The degree of conversion



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Figure 3. (A) Comparison of nonspecific protein adsorption and covalent protein binding on PDHPA (S3, 100%) and PDHPA-aldehyde (S3, 100%) by fluorescence microscopy. I: Nonspecific adsorption of rhodamine-streptavidin onto S3-PDHPA. II: Covalent binding of rhodamine-streptavidin to PDHPA-aldehyde-S3. III: Nonspecific adsorption of FITC-BSA on PDHPA-S3. IV: Covalent binding of FITC-BSA to PDHPA-aldehyde-S3. (B) Effect of oxidation time on protein binding on PDHPA-S3. I: Relationship of oxidation time with the fluorescence intensity of rhodamine-streptavidin (after incubation with streptavidin, the initiator surface shows 3.2 nm increase in ellipsometric thickness). II: Relationship of oxidation time with ellipsometric thickness change of S3-PDHPA. III: Ellipsometric thickness change after incubation of S3-PDHPA and control (ATRP-initiator deposited wafer) in platelet poor plasma (PPP) and fibrinogen solution (1 mg/mL, PBS buffer, pH ) 7.4). PPP was anticoagulated with 3.2% sodium citrate. The fibrinogen used contains a fraction with 91% clottability. IV: Images of water droplets on control sample before and after the incubation with PPP. (C) Schematic illustration of covalent binding of streptavidin onto the dense PDHPA-aldehyde brushand formation of the monolayer. Due to the steric repulsion, streptavidin can only interact with the outside layer of PDHPA-aldehyde brush, while the interior is nonaccessible.

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Figure 4. Fluorescence images (A, D, G, J, K), scanning electron microscope images (B, E, H), and fluorescence intensity profiles (C, F, I, L) of micropatterned rhodamine-streptavidin on PDHPA-S3. Shape and sizes of different patterns: (A-C) 100 µm wide stripes; (D-F) 20 µm wide stripes; (G-I) 50 µm diameter round dots; (J-L) 50 µm diameter reversed round dots (no clear SEM image was obtained for this pattern). The scale bars in A, D, G, and J were 200, 40, 100, and 100 µm, respectively.

from diols to aldehydes during the short oxidation time (∼5 min) is estimated to be about 15% from an independent study.17 3.3. Protein Micropatterning. The micropatterning of proteins was preceded by the generation of a prepatterned, binary brush system facilitated by the combination of photo- and wet chemical lithography (Figure 1). The photoresist was deposited onto the PDHPA brush prior to patterning by UV irradiation and subsequent incubation in aqueous sodium periodate solution. The exposed region was converted to PDHPA-aldehyde, whereas the covered region remained as a PDHPA brush. Consequently, a unique binary polymer brush system containing both nonbiofouling zones (PDHPA brush) and coupling zones (PDHPA-aldehyde brush) was generated from a single component in a homogeneous brush after washing away photoresist by acetone. During the wash process, PDHPA and PDHPAaldehyde brushes remained intact. The patterning strategy employed in this study is essentially a wet chemical lithography process;25 this allows for selective oxidation of the exposed region on the substrate via a protective photoresist mask prepared by conventional photolithography. Unlike traditional chemical lithography, in which an e-beam is applied to generate reactive amino or carboxylic groups,26 the current approach employs wet chemistry to generate aldehyde functionality via a fast and mild oxidation. Considering the availability and low cost of wet chemistry, the current method provides a facile strategy for preparing micropatterned and multifunctional surfaces from a single substrate. The successful micropatterning of streptavidin with different sizes and patterns after protein conjugation was followed by fluorescence microscopy (Figure 4A,D,G,J,K). Significant contrast of fluorescence intensity between the coupling zone and the nonfouling zone indicates successful fabrication of patterned

PDHPA-aldehyde zones on the PDHPA brush followed by selective immobilization of proteins in the coupling zone. The clear contrast between the coupled protein and noncoupled region also supports the nonfouling nature of PDHPA brushes. Further examination of the patterned surface by SEM and fluorescence intensity profile verified a quite uniform patterning (Figure 4B,E,H,C,F,I,J). The oxidation time proved critical in obtaining successful protein patterning on PDHPA brushes. It was observed that a 5 min oxidation time produced the best results in terms of patterning uniformity and the amount of coupled protein. As shown in Figure 5, no clear patterning was observed at 0.5 min oxidation time. Although optimal patterning was obtained after 3 min oxidation time, the existence of defects (indicated by arrows) made the patterning nonuniform. A 5 min oxidation time led to uniform patterning; no discernible patterning was observed at 30 min oxidation time. Three factors favor use of a shorter oxidation time. First, a prolonged oxidation time can lead to the diffusion of the oxidizing reagent to the masked regions, which neighbor the exposed regions (Figure 5B), as evidenced by the absence of patterning when exposure time was prolonged to 30 min. Second, a prolonged oxidation time can produce a large amount of aldehyde groups that are not available to bind to the proteins due to steric effects of the brush. The remnant of aldehyde groups might affect the property of the grafted layer via possible side reactions such as cross-linking. Third, covalent protein coupling was almost saturated after 5 min oxidation time, as shown in Figure 3B. Thus, a prolonged oxidation time should not lead to the improvement in protein patterning. Atomic force microscopy (AFM) was used to compare the properties of protein patterned brushes. AFM analysis revealed



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Figure 5. (A) Effect of oxidation time on micropatterning of rhodamine-streptavidin on PDHPA/PDHPA-aldehyde binary brush. Oxidation time was 0.5, 3, 5, and 30 min, respectively. Nonuniform patterning was observed at 0.5 and 3 min (as indicated by arrows), while no discernible patterning was observed at 30 min. A total of 5 min of oxidation time yielded successful patterning with much better contrast and uniformity. Scale bars represent 50 µm. (B) Schematic illustration of the oxidation and diffusion process during the oxidation of PDHPA brush.

a ∼4 nm thickness difference (Figure 6A and C) between the coupled zone and nonfouling zone which is in agreement with the ellipsometric thickness change after immobilization of streptavidin onto PDHPA-aldehyde (Figure 3B). To further verify the properties of coupling and nonbiofouling zones, AFM force measurements were conducted using an AFM tip modified by biotin (Supporting Information, Figures 4S and 5S). As shown in Figure 6D and E, the nonfouling zone shows a repulsive interaction during both approach and retraction (6D), which is typical of the interaction of a hydrophilic brush with a hydrophobic Si3N4 tip.20,27 The attachment of biotin did not cause a specific interaction between the nonfouling zone and modified tip, suggesting the absence of coupled or adsorbed streptavidin in the zone. In contrast, a strong adhesive force was observed during pull-off of the biotin-modified tip from the streptavidin coupled zone (Figure 6E). The maximum adhesive force (average of 50 measurements) of the coupling zone was 2.5 nN, which was significantly higher than that of the nonfouling zone (≈ 0 nN; insets in Figure 6D and E). This provides quantitative evidence suggesting that selective protein coupling occurs in the patterned region and the nonfouling zone is highly resistant to nonspecific protein adsorption. In addition, the streptavidin patterned zone shows strong binding to biotin, suggesting the activity of conjugated streptavidin on the brush is preserved. This is significant since it is well-known that the

surface coupling of proteins can lead to conformational changes of the proteins, resulting in a loss of activity.28 The current binary brush system has also been utilized for the micropatterning of multiple proteins. Figure 6F presents the micropatterning of streptavidin and BSA on the S3 substrate. Rhodamine-streptavidin was initially patterned as a 20 µm stripe prior to the second oxidation of the substrate (5 min) and coupling with FITC-BSA. Fluorescence microscopic images under a rhodamine-filter, an FITC-filter, and the merged picture revealed successful patterning of two proteins consecutively. This approach is of great interest considering the importance of micropatterning multiproteins or other biologically relevant molecules onto the same surface.25b,29 It should be noted that current approach for multiprotein patterning may not be applied to proteins containing carbohydrate moieties, which can be oxidized by sodium periodate.

4. Conclusions In summary, a novel binary polymer brush system consisting of nonfouling PDHPA and reactive PDHPA-aldehyde was readily prepared by a combination of photo- and chemical lithography; the latter is simple and facile. The resulting patterned PDHPA-aldehyde/PDHPA substrate proved to be an effective template for protein micropatterning. Successful pat-

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Figure 6. (A) Contact mode AFM image of patterned streptavidin on PDHPA-S3 with 20 µm wide stripes in water. Scanning size: 25 × 25 µm brushes. Z-range was 50 nm. (B) Fluorescence image of 20 µm wide stripe of rhodamine-streptavidin on PDHPA-S3. (C) Section profile of designated area in AFM image. (D) Force measurement used a biotin-tethered AFM tip with nonfouling zone (PDHPA) after protein coupling. Representative force curves (filled circle: approach; open circle: retract) and probability distribution histograms for the maximum adhesive force during retraction. (E) Force measurement using a biotin-tethered AFM tip with a streptavidin-coupled zone (coupling zone) after protein coupling. Representative force curves (filled circle: approach; open circle: retract) and probability distribution histograms for the maximum adhesive force during retraction. (F) Micropatterning rhodamine-streptavidin and FITC-BSA on PDHPA-S3. Rhodamine-streptavidin was first patterned as 20 µm wide stripe. The substrate was then oxidized in sodium periodate solution (3 mg/mL) for 5 min, cleaned, and followed by incubation in FITC-BSA/NaCNBH3 for 30 min. Fluorescence microscope images were recorded under rhodamine- and FITC-filter. The two images were merged by overlapping.

terning of streptavidin with different patterns and sizes were observed. Compared to previously reported approaches for the preparation of patterned binary polymer brush systems using photolithography and SI-ATRP, which vary in terms of mo-

lecular weight, graft density, and thickness,8,30 the method described here provides a facile approach that avoids such heterogeneities. This feature is rarely noted in the literature. Originating from one single, homogeneous brush, the resulting


PDHPA/PDHPA-aldehyde binary brush system shows little variation in thickness, molecular weight, and graft density. Furthermore, this binary brush system provides a straightforward solution for the micropatterning of different biological ligands on a single substrate as a monolayer without losing their activities. Acknowledgment. Financial support from Canadian Institutes of Health Research (CHIR), Canadian Blood Services (CBS), Canada Foundation for Innovation (CFI), Natural Sciences and Engineering Research Council (NSERC) of Canada, and Michael Smith Foundation for Health Research (MSFHR) are gratefully acknowledged. J.N.K. is the recipient of CBS/CIHR New Investigator Award in Transfusion Science. The authors thank the LMB Macromolecular Hub at the UBC Center for Blood Research for the use of their research facilities. These facilities are supported in part by grants from the Canada Foundation for Innovation and the Michael Smith Foundation for Health Research. Supporting Information Available. Figures showing block polymerization of DMA and DMDOMA from PDMDOMA brushes (1S), time-dependent thickness change during SI-ATRP of DMDOMA in DMF (2S), water contact angle pictures (3S), modification of AFM tip by biotin (4S, 5S), AFM morphology and GPC profiles (6S), and XPS spectra (7S). This material is available free of charge via the Internet at http://pubs.acs.org.

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BM901159D

Nonbiofouling Polymer Brush with Latent Aldehyde Functionality as

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