Active Protein-Functionalized Poly(poly(ethylene

Active Protein-Functionalized Poly(poly(ethylene glycol)monomethacrylate)-Si(100)Hybrids from Surface-Initiated Atom Transfer Radical Polymerization for Potential Biological Applications

F.J.Xu,*,?,?L.Y.Liu,?W.T.Yang,*,?E.T.Kang,?and K.

G.Neoh ?

State Key Laboratory of Chemical Resource Engineering College of Materials Science and Engineering,Beijing

University of Chemical Technology,Beijing 100029China,and Department of Chemical and Biomolecular Engineering,National University of Singapore,Kent Ridge,119260Singapore

Received March 17,2009

Revised Manuscript Received April 5,2009

Protein-resistant poly(poly(ethylene glycol)monomethacrylate)-graft -Si(100),or Si-g -P(PEGMA)hybrids,were prepared via surface-initiated atom transfer radical polymerization (ATRP)of the poly(ethylene glycol)monomethacrylate (PEGMA)mac-romonomer from the hydrogen-terminated Si(100)surface (Si -H surface).The resultant robust Si -C bonded P(PEGMA)brushes can be further functionalized by the immobilization of human immunoglobulin (IgG)protein via different strategies,namely,the direct use of the alkyl halide chain ends preserved throughout the ATRP process and the postmodi?cation of the hydroxyl side chains with by 1,1′-carbonyldiimidazole (CDI)or succinic anhydride (SA).The CDI exhibited a higher ef?ciency in activating the hydroxyl groups for coupling proteins.The surface density of the immobilized protein above 2.5μg/cm 2could be readily achieved.The distribution of active protein-docking sites on the Si -C bonded P(PEGMA)brushes can be also controlled by controlling the brush length.The resulting IgG-coupled Si-g -P(PEGMA)hybrid surface interacts only and speci?cally with the anti-IgG protein,while the dense P(PEGMA)brushes effectively prevent nonspeci?c protein binding and fouling.The simple concomitant incorporation of protein-resistant P(PEGMA)brushes and highly speci?c and active protein onto silicon surfaces via robust Si -C bonding should readily endow the silicon substrates with new and interesting properties for applications in silicon-based protein sensors or microarrays.

1.Introduction

Functionalization of oriented single crystal silicon surface with biomacromolecules plays an important role in the develop-ment of new silicon-based biomedical devices,such as biomicro-electromechanical systems (BioMEMS),three-dimensional mi-cro-and nanomemory chips,and DNA-and protein-based biochips and biosensors.1-6Tethering of polymer brushes on a solid substrate is an effective method for modifying the surface properties of the substrate.7,8Stable polymer brushes covalently bonded on silicon surfaces can provide excellent mechanical and chemical protection to the substrate and provide new pathways for the functionalization of silicon surfaces.9Atom

transfer radical polymerization (ATRP)is a recently developed “controlled”radical polymerization method.10-12It allows the preparation of well-de?ned polymer brushes on a variety of substrates via the surface-initiated process.13-18An important application of surface-tethered polymer brushes is in the development of biocompatible surfaces that resist nonspeci?c protein adsorption and minimize cell adhesion.Poly(ethylene glycol)(PEG)brushes are used commonly for the preparation of antifouling surfaces.19Innovative works have been carried out to covalently couple active proteins onto the protein-resistant (“biologically inert”)PEG brushes for protein microarrays and related bioanalytical applications.16,17,20

Pristine silicon surface exhibits strong nonspeci?c protein adsorption,13and the robust Si -C bonded polymer brushes are very attractive for the functionalization of silicon surfaces.9,13In the present work,covalently (Si -C bonded)tethered poly-(poly(ethylene glycol)monomethacrylate)(P(PEGMA))brushes on the Si(100)surface (Si-g -P(PEGMA)surface)are ?rst prepared via surface-initiated ATRP of PEGMA from the 4-vinylbenzyl chloride (VBC)-coupled silicon (Si-VBC)surface (Scheme 1).Our earlier studies indicated the active chloride end groups preserved throughout the ATRP process could be used as leaving groups for further surface functionalization and molecular design.However,the number of halide groups needs to be increased in order for a signi?cant concentration of biomolecules to be immobilized.13Herein,alternative ap-proaches were adopted to activate the hydroxyl groups of the biologically inert P(PEGMA)brushes simply by 1,1′-carbon-yldiimidazole (CDI)or succinic anhydride (SA)for covalent coupling of human immunoglobulin (IgG)to produce the biologically active Si-g -P(PEGMA)-IgG hybrid surface (Scheme 1).The distribution of active protein-docking sites on the Si -C bonded P(PEGMA)brushes can be also controlled by controlling the brush length.The CDI exhibited a higher ef?ciency in activating the hydroxyl groups for coupling proteins.In addition,the resulting IgG-functionalized Si-g -P(PEGMA)hybrid surface interacts only and speci?cally with the anti-IgG and not other proteins.IgG as a model active protein has been widely used for diagnostic assays,environmental testing,and process monitoring in biotechnology and clinical medicine.21For these applications,active proteins are often immobilized on a solid support and employed as a molecular recognition element that binds speci?cally to its antigen with high af?nity.22The present simple approaches to the concomitant incorporation of anti-fouling and Si -C bonded PEG brushes and active proteins onto silicon surfaces should readily endow the silicon substrates with new and interesting properties for applications in silicon-based protein sensors or microarrays.

2.Experimental Section

2.1.Materials.(100)-Oriented single crystal silicon,or Si(100),wafers with a thickness of about 1.5mm and a diameter of 150mm,was purchased from Unisil Co.of Santa Clara,CA.The as-received wafers were polished on both sides and doped lightly with phosphorus as n-type.Details on the preparation and characterization of hydrogen-terminated Si(100)surface (Si -H surface)had been described earlier.9,13Hydro?uoric acid (HF;37wt %),4-vinylbenzyl chloride (VBC,97%),poly(ethylene glycol)monomethacrylate (PEGMA)macromonomer (M n ～360),2,2′-bypridine (Bpy,99%),copper(I)chloride (99%),copper(II)chloride (97%),1,1′-carbonyldiimidazole (CDI,97%),succinic anhy-dride (SA,99%),N -ethyl-N ′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC,99%),and N -hydroxysuccinimide (NHS,98%)

*To whom correspondence should be addressed.E-mail:xufj@https://www.360docs.net/doc/0012291620.html, (F.J.X.);yangwt@https://www.360docs.net/doc/0012291620.html, (W.T.Y.).?

Beijing University of Chemical Technology.?

National University of Singapore.

Biomacromolecules 2009,10,1665–16741665

10.1021/bm900307c CCC:$40.75 2009American Chemical Society

Published on Web 04/30/2009

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E N U N I V o n N o v e m b e r 3, 2009 | h t t p ://p u b s .a c s .o r g P u b l i c a t i o n D a t e (W e b ): A p r i l 30, 2009 | d o i : 10.1021/b m 900307c

were obtained from Aldrich Chemical Co.of Milwaukee,WI.Bovine serum albumin (BSA),?uorescein isothiocynate (FITC)-conjugated BSA (BSA-FITC),human immunoglobulin (IgG),FITC-conjugated IgG (IgG-FITC),and FITC-conjugated antihuman IgG (anti-IgG-FITC)were purchased from Sigma Chemical Co.,St.Louis,MO.Bio-Rad dye reagent for protein assay (Catalog No.500-0006)were purchased from Bio-Rad,Inc.,Hercules,CA.

2.2.Surface-Initiated ATRP of PEGMA.The silicon wafers were sliced into square chips of 2×2cm in size for surface-initiated ATRP.The ATRP initiator was immobilized on the Si(100)surface via 3min of UV-induced hydrosilylation of VBC with the Si -H surface.The process gave rise to a covalently bonded (Si -C bonded)VBC monolayer (the Si-VBC surface).Details on the preparation and characterization of the Si-VBC surface had been described earlier.1For the preparation of PEGMA polymer (P(PEGMA))brushes on the Si-VBC surface,the surface-initiated ATRP was carried out using a [PEGMA (4mL)]/[CuCl]/[CuCl 2]/[Bpy]molar feed ratio of 100:1:0.2:2in 4mL of doubly distilled water at room temperature in a Pyrex tube containing the Si-VBC substrate.The reaction was allowed to proceed for 3-12h to produce the Si-g -P(PEGMA)hybrid.After the reaction,the Si-g -P(PEGMA)hybrid was washed thoroughly by extraction with copious amounts of ethanol and doubly distilled water.The hybrid was subsequently immersed in a large volume of ethanol for about 48h to ensure the complete removal of the adhered and physically adsorbed polymer,if any.13

2.3.Protein Adsorption.The BSA (or BSA-FITC)and IgG (or IgG-FITC)were dissolved separately in phosphate-buffered saline (PBS,pH 7.4)at a concentration of 1mg/mL.The Si -H surface will be quickly oxidized back to the oxide-covered surface,similar to the pristine silicon.5The pristine silicon surface,rather than the Si -H surface,was used for the protein adsorption experiments.The pristine (oxide-covered)silicon and Si-g -P(PEGMA)chips of 1×1cm in area (sliced from the Si-g -P(PEGMA)chips of 2×2cm in size)were used in all protein adsorption experiments.The pristine silicon and Si-g -P(PEGMA)chips were rinsed initially with PBS to rehydrate the surface,prior to being placed in one of the protein solutions.13The adsorption was allowed to proceed at room temperature for 12h.The substrates were then removed from the solution,gently washed four times with PBS or 1%sodium dodecyl sulfate (SDS)from a 5mL Pasteur pipet,and rinsed twice with doubly distilled water to remove the PBS or SDS salt.After drying under reduced pressure,the protein-adsorbed surfaces were analyzed by X-ray photoelectron spectroscopy (XPS).The XPS N 1s core-level signal was used as a maker for the analysis of the relative amount of adsorbed protein on the silicon substrates.13The adsorption of the BSA-FITC and IgG-FITC on the pristine silicon and Si-g -P(PEGMA)chips was imaged with an Olympus BX60?uorescence microscope (Olympus America Inc.,NY),equipped with a 495nm excitation ?lter and a 525nm emission ?lter.

The amount of adsorbed protein on the pristine silicon and Si-g -P(PEGMA)chips was determined by the modi?ed dye-interaction method,using the Bio-Rad protein dye reagent.23-25For the preparation of the dye solution,the Bio-Rad stock dye solution was diluted ?ve times with doubly distilled water.BSA or IgG solution (100μL)of known concentration was added to 5mL of the diluted dye solution and the protein -dye solution was gently stirred for 30min.The absorbance of supernatant at 465nm was used for the standard calibration.For the quantitative determination of adsorbed protein on the silicon surfaces,the dye solution (5mL)was added to a test tube containing 100μL of doubly distilled water and three pieces of the silicon chips of 1×1cm in size.The dye solution was gently stirred for 30min.The chips were removed and the absorbance of the dye solution was measured at 465nm.The amount of protein adsorbed was calculated with reference to the standard calibration curve.

2.4.Covalent Immobilization of IgG on the Si-g -P(PEGMA)Surface.Although earlier studies have indicated that the active chloride groups at the chain ends,preserved throughout the ATRP process,can be used as leaving groups for coupling of biomolecules,the concentra-tion of the halide groups needs to be increased in order to enhance the amount of immobilized biomolecules.13,26In this work,alternative approaches were adopted to activate the hydroxyl groups of the Si -C bonded P(PEGMA)brushes via reactions with 1,1′-carbonyldiimidazole (CDI)27,28or succinic anhydride (SA)29for the immobilization of a high surface density of IgG protein (Scheme 1).Typically,a Si-g -P(PEGMA)hybrid was immersed in 10mL of dried DMSO,containing 2.0g of CDI,in a Pyrex tube for 24h at room temperature to produce the Si-g -P(PEGMA)-CDI hybrid.For the SA activation,a Si-g -P(PEGMA)hybrid was immersed in 10mL of dried THF,containing 2.0g of SA and 1mL of dried pyridine,in a Pyrex tube for 24h at

Scheme 1.Processes of UV-Induced Hydrosilylation of VBC with the Si(100)-H Surface To Give the Si-VBC Surface,Surface-Initiated ATRP of PEGMA from the Si-VBC Surface at Room Temperature,and IgG Immobilization on the Si-g -P(PEGMA)

Surfaces

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room temperature to produce the Si-g -P(PEGMA)-COOH hybrid.After removal from the reaction mixtures,the Si-g -P(PEGMA)-CDI and Si-g -P(PEGMA)-COOH hybrids were washed with copious amounts of THF.The Si-g -P(PEGMA)and Si-g -P(PEGMA)-CDI chips of 1×1cm in size were introduced into 5mL of the PBS solution containing the IgG protein at a concentration of 1mg mL -1.The immobilization reaction was allowed to proceed at room temperature for 48h to produce the corresponding Si-g -P(PEGMA)-IgG1and Si-g -P(PEGMA)-IgG2surfaces (Scheme 1).For the protein immobilization on the Si-g -P(PEGMA)-COOH surface,a Si-g -P(PEGMA)-COOH hybrid was ?rst immersed in 10mL of THF,containing 20mg of N -hydroxysuccinimide (NHS)and 0.1g of N -ethyl-N ′-(3-dimethylaminopropyl)carbodiimide (EDAC),in a Pyrex tube for 4h at room temperature,to activate the carboxylic acid groups.The active Si-g -P(PEGMA)-COOH hybrid was introduced into the PBS solution containing the IgG protein at a concentration of 1mg mL -1for 48h to produce the corresponding Si-g -P(PEGMA)-IgG3surfaces.After the immobilization reaction,the hybrid surfaces were washed sequentially with excess PBS and doubly distilled water.The amount of immobilized IgG on the Si-g -P(PEGMA)-IgG hybdrid surfaces was also determined by the modi?ed dye-interaction methods.For ?uorescence immunostaining,the Si-g -P(PEGMA)-IgG hybrids were incubated in 0.03mg/mL PBS solution of anti-IgG-FITC (or 1mg/mL PBS solution of BSA-FITC)for 4h.The hybrid surfaces were then rinsed with PBS (pH 7.4)and deionized water.The binding of the target protein to the Si-g -P(PEGMA)-IgG hybrid surfaces was also imaged by ?uorescence microscopy.

2.5.Surface Characterization.The chemical composition of the modi?ed silicon surfaces was determined by X-ray photoelectron spectroscopy (XPS).The XPS measurements were performed on a Kratos AXIS HSi spectrometer,using a monochromatized Al K R X-ray source (1486.6eV photons)and procedures similar to those described earlier.9,13The static water contact angles of the pristine and function-alized Si -H surfaces were measured using the sessile drop method with a 3μL water droplet,in a telescopic goniometer (Rame-Hart model 100-00-(230),manufactured by the Rame-Hart,Inc.,Mountain Lakes,NJ).The thickness of the polymer brushes grafted on the silicon substrate was determined by a variable angle spectroscopic ellipsometer (Model VASE,J.A.Woollam Inc.,Lincoln,NE)using procedures similar to those described earlier.13

3.Results and Discussion

Speci?c protein-coupled Si(100)surfaces are prepared ac-cording to the reaction sequence shown in Scheme 1:(i)the P(PEGMA)brushes are ?rst covalently grafted on the Si(100)surface to produce the Si-g -P(PEGMA)hybrid surface via surface-initiated ATRP of PEGMA from the hydrogen-terminated Si(100)surfaces (Si -H surface)modi?ed a prior by hydrosilylation of VBC,(ii)the hydroxyl groups of the protein-resistant Si-g -P(PEGMA)surfaces are activated by 1,1′-carbonyldiimidazole (CDI)and succinic anhydride (SA)to produce the corresponding Si-g -P(PEGMA)-CDI and Si-g -P(PEGMA)-COOH surfaces,and (iii)covalent immobilization of IgG on the Si-g -P(PEGMA),Si-g -P(PEGMA)-CDI,and Si-g -P(PEGMA)-COOH surfaces to produce the Si-g -P(PEGMA)-IgG1,Si-g -P(PEGMA)-IgG2,and Si-g -P(PEGMA)-IgG2hybrid surfaces,respectively.The details of each reaction are discussed below.

3.1.Surface-Initiated ATRP of PEGMA from the Si -H Surfaces.The use of Si -H surface,instead of the as-received Si/SiO 2surface of the Si(100)wafer,allows the preparation of Si-polymer hybrids with robust Si -C linkages.6,9,13Details on the preparation and characterization of the Si -H surface had been described earlier.9,13The Si surface with Si -C bonded ATRP initiator monolayer was prepared via 3min of UV-induced hydrosilylation of VBC with the Si -H surface (the Si-VBC surface).9The C 1s core-level spectrum of the Si-VBC

surface (Figure 1a)can be curve-?tted into three peak compo-nents with binding energies (BE’s)at about 283.8,284.6,and 286.1eV,attributable to the C-Si,C-H,and C-Cl species,respectively.9The corresponding Cl 2p core-level spectrum of the Si-VBC surface with a Cl 2p 3/2BE of about 200eV is consistent with the presence of the alkyl halide species (Figure 1b).The static water contact angle increases from about 72°for the Si -H surface to about 86°for the Si-VBC surface (Table 1).The initiator layer thickness,initiator density,and surface initiator ef?ciency for the Si-VBC surface have been determined previously 9and are about 0.3nm,1.4VBCs/nm 2,and 35%,respectively.

To quickly establish an equilibrium between the dormant and active chains during surface-initiated ATRP,an excess amount of deactivating Cu(II)complex (CuCl 2),rather than “sacri?cial initiators”,was added to control the concentration of the deactivating Cu(II)complex on the Si-VBC surface.9,13This approach allows thicker polymer brushes to be grown at a faster rate in the presence of a higher monomer concentration,because of the absence of accompanying homopolymerization in solution.30,31The molar ratio of [PEGMA (monomer)]/[CuCl (catalyst)]/[CuCl 2(deactivator)]/[Bpy (ligand)]was controlled at 100:1:0.2:2.The presence of grafted PEGMA polymer (P(PEGMA))on the Si-VBC surface was revealed by XPS analysis and ellipsometry measurement after the surface had been subjected to vigorous washing and extraction.The C 1s core-level spectra of the Si-g -P(PEGMA)1(from ATRP time of 3h)and Si-g -P(PEGMA)2(from ATRP time of 12h)surfaces (Figure 1c,e)can be curve-?tted into three peak components with BEs at about 284.6,286.2,and 288.5eV,attributable to the C-H,C-O/C-Cl,and O )C-O species,respec-tively.13The persistence of the Cl 2p core-level signal (Figure 1d,f)is consistent with the presence of alkyl halide chain ends.With the increase in ATRP time from 3to 12h,the [Cl]/[C]ratio (determined from the sensitivity-factor corrected Cl 2p and C 1s core-level spectral area ratio)of the Si-g -P(PEGMA)surface decreases from 1.4×10-3to 6.5×10-4(Figure 1).With the increase in ATRP time,chain termination by bimo-lecular coupling or disproportionation reaction could give rise to an increase in the loss of the active chain ends (the terminal alkyl halides).13,26The thickness (h )values of the corresponding P(PEGMA)?lms on the Si-g -P(PEGMA)1and Si-g -P(PEG-MA)2surfaces are about 52and 136nm (Table 1).Their average static water contact angles decreased from about 86°(for the starting Si-VBC surface)to about 35°(Table 1).Based on the surface initiator density (d ′)of 1.4VBCs/nm 2,an initiator ef?ciency (e )of 35%,P(PEGMA)density (d )of 1.46g/cm 3,and PEGMA molecular weight (M )of 360g/mol,the corre-sponding average degrees of polymerization (DPs)for the Si-g -P(PEGMA)1and Si-g -P(PEGMA)2surfaces [(dh )/(Md ′e )]9,13are thus estimated to be about 240and 620,respectively.An approximately linear increase in P(PEGMA)thickness and DP of the grafted P(PEGMA)chains on the Si-VBC surface with polymerization time is observed in the initial stage of ATRP (Figure 2).With the increase in ATRP time,the increase in surface ?lm thickness starts to deviate from linearity.This phenomenon is probably attributable to chain termination on the surface,followed by bimolecular coupling or dispropor-tionation reactions that consume the active chains.13,26Thus,the chain growth from the Si-VBC surface,at least in the initial stage,is consistent with a “controlled”and well-de?ned process.The above results indicate that PEGMA has been successfully graft polymerized on the Si-VBC surfaces via surface-initiated ATRP.

Notes

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3.2.Protein Adsorption.Protein adsorption studies on the pristine (oxide-covered)Si(100),Si-g -P(PEGMA)1,and Si-g -

P(PEGMA)2surfaces were carried out in phosphate buffered saline (PBS)solutions containing 1mg/mL of bovine serum albumin (BSA)or human immunoglobulin (IgG).Chemical compositions of the protein-adsorbed surfaces were analyzed by XPS.The XPS N 1s core-level signal can be used as a marker for the analysis of the relative amount of adsorbed protein

on

Figure 1.C 1s and Cl 2p core-level spectra of the (a,b)Si-VBC,(c,d)Si-g -P(PEGMA)1,and (e,f)Si-g -P(PEGMA)2surfaces.Table 1.Static Water Contact Angle,[N]/[C]Ratios,and IgG Amount of the IgG-Functionalized Surfaces

sample

reaction time (h)static contact angle h ((3°)

[N]/[C]I

IgG amount (μg/cm 2)

Si-g -P(PEGMA)1a 335Si-g -P(PEGMA)2a

1234Si-g -P(PEGMA)1-IgG1b 4834 1.6×10-4Si-g -P(PEGMA)2-IgG1c 4836 1.3×10-4Si-g -P(PEGMA)1-IgG2d 48380.15 3.2Si-g -P(PEGMA)2-IgG2e 48400.19 4.8Si-g -P(PEGMA)1-IgG3f 48360.10 2.5Si-g -P(PEGMA)2-IgG3g

48

35

0.12

3.3

a

Reaction conditions:[PEGMA]/[CuCl]/[CuCl 2]/[Bpy])100:1:0.2:2in water (1/1,v/v)at room temperature;P(PEGMA)thicknesses for the Si-g -P(PEGMA)1and Si-g -P(PEGMA)2surfaces are 52and 136nm,respectively.b The starting surface is the Si-g -P(PEGMA)1a surface.c The starting surface is the Si-g -P(PEGMA)2a surface.d The starting surface is the Si-g -P(PEGMA)1-CDI surface.e The starting surface is the Si-g -P(PEGMA)2-CDI surface.f The starting surface is the Si-g -P(PEGMA)1-COOH surface.g The starting surface is the Si-g -P(PEGMA)2-COOH surface.h Static water contact angles for the pristine (oxide-covered)Si(100),Si -H,and Si-VBC surfaces are about 20,72,and 86°,respectively.I

Determined from the sensitivity-factor corrected XPS N 1s and C 1s core-level spectral area

ratios.

Figure 2.Dependence of (a)thickness and (b)degree of polymeri-zation (DP)of the grafted P(PEGMA)chains of the Si-g -P(PEGMA)surface on the surface-initiated ATRP time.

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the silicon substrates.13The wide scan (a -d)and N 1s core-level (a ′-d ′)spectra of the pristine Si(100)surfaces after protein adsorption are shown in Figure 3.These Si(100)surfaces were washed with PBS or 1%SDS to remove the weakly adsorbed proteins.A strong N 1s signal at BE of about 400eV has appeared on these surfaces,indicating that a signi?cant amount of proteins has been adsorbed on all the pristine Si(100)surfaces.The [N]/[Si]ratio (determined from the sensitivity-factor corrected N 1s and Si 2p core-level spectral area ratio)of the Si(100)surface washed by 1%SDS (Figure 3b,d)are lower than that of the corresponding protein-adsorbed Si(100)surface washed by PBS (Figure 3a,c),indicating that the SDS solution exhibits a relatively strong protein-removing ability.The amounts of adsorbed BSA and IgG on the Si(100)surfaces,washed by PBS (or 1%SDS solution),are 0.41(or 0.33)and 0.49(or 0.42)μg/cm 2,respectively (Figure 3).

After exposure to the protein solutions and washed with PBS,the N 1s signals are barely discernible in the wide scan spectra of the Si-g -P(PEGMA)1and Si-g -P(PEGMA)2surfaces (Figure 3e -h).In comparison with those of the pristine Si(100)surfaces (Figure 3a -d),the negligible [N]/[C]ratios for the Si-g -P(PEGMA)1and Si-g -P(PEGMA)2surfaces indicate that the graft-modi?ed surfaces exhibit good resistance to protein adsorption.The corresponding [N]/[Si]ratios cannot be obtained,because the P(PEGMA)layer thicknesses of the Si-g -P(PEG-MA)1and Si-g -P(PEGMA)2surfaces (about 50and 136nm,respectively)are larger than the sampling depth (about 7.5nm in an organic matrix 32)of the XPS technique.Subsequent SDS washing did not reduce the intensities of the N 1s signals further.This phenomenon is consistent with the coupling reactions between the active chloride groups (at the chain ends

preserved

Figure 3.Wide scan and N 1s core-level spectra of the (a,a ′,b,b ′,c,c ′,d,d ′)pristine Si(100)surfaces (washed by PBS or 1%SDS solution after protein adsorption)and the (e,e ′,f,f ′,g,g ′,h,h ′)Si-g -P(PEGMA)surfaces (washed by PBS solution after protein adsorption)after being exposed to BSA and IgG solutions for 12h.

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throughout the ATRP process)and protein (see the next section).However,the amounts of BSA and IgG adsorbed on these graft-modi?ed surfaces are too small to be measured quantitatively.Unlike the graft-modi?ed surface,the pristine silicon surface possesses oxygen-containing polar and functional species that can interact directly with proteins through ionic and hydrogen bondings.13,31For the P(PEGMA)-grafted silicon surfaces,the effective reduction in protein adsorption is due to the unique properties of the PEG units.13,16-18The large excluded volume of the PEG units and the highly mobile or ?exible PEG chains in water tend to repel protein molecules approaching the substrate surface.These properties also minimize the ionic interaction and hydrogen bonding of the PEG units with protein molecules.13,33Thus,the immobilized PEG units have the unique ability to reduce the direct contact of proteins with the silicon surface.

Figure 4shows the representative ?uorescence images of the (a -d)pristine Si(100)and (e -h)Si-g -P(PEGMA)surfaces after been exposed to BSA-FITC and IgG-FITC solutions (instead

of the BSA and IgG solutions)under the similar conditions.Prior to protein adsorption,no ?uorescence was observed from the pristine Si(100)surface (Figure 4a ′).After the protein adsorption,the scattered ?uorescence from the silicon surfaces indicates that ?nite amounts of proteins have been adsorbed on the pristine silicon surfaces.For the P(PEGMA)-grafted silicon surfaces,the absence of any signi?cant ?uorescence from the surfaces indicates that these graft-modi?ed surfaces exhibit resistance to protein adsorption.The ?uorescence results are thus consistent with the XPS results of Figure 3.

3.3.Covalent Immobilization of IgG on the Si-g-P(PEGMA)Hybrid Surface.Polymers synthesized by ATRP still retain the terminal alkyl halides (Figure 1d,f).The “dormant”chain ends on the hybrid surfaces offer opportunities for further surface functionalization and molecular design.13,26Our earlier studies indicated the active chloride end groups preserved throughout the ATRP process could be used as leaving groups in the coupling reactions with biomolecules.However,the number of halide groups for the coupling reactions need to be increased in order for a signi?cant concentration of biomolecules to be immobilized.13,26As alternative approaches,the hydroxyl groups of the Si-g -P(PEGMA)1and Si-g -P(PEGMA)2surfaces are activated via the 1,1′-carbonyldiimidazole (CDI)27,28(or succinic anhydride (SA)29)to produce the corresponding Si-g -P(PEG-MA)1-CDI and Si-g -P(PEGMA)2-CDI (or Si-g -P(PEGMA)1-COOH and Si-g -P(PEGMA)2-COOH)surfaces for the immo-bilization of IgG (Scheme 1).The hydroxyl groups of PEGMA can react with CDI to produce the imidazole carbamate groups that react readily with amine-containing biomolecules to form stable carbamate linkages.27,28The hydrolysis of the reactive imidazole carbamate groups is very slow in water at pH 4-8and can last for more than 6days in PBS buffer.28This slow hydrolysis reaction provides a good opportunity for coupling biomolecules in an aqueous buffer solution.The resulting carbamate linkages are stable and uncharged.The absence of charges in the linkages can prevent nonspeci?c adsorption by ion exchange.In addition,the residual imidazole carbamate groups can be readily deactivated in aqueous solutions at higher pH to eliminate potential sites for nonspeci?c binding.27For the protein immobilization on the carboxylic acid group surface,the universal method is to directly couple protein with carboxylic acid groups by using N -ethyl-N ′-(3-dimethylaminopropyl)car-bodiimide (EDAC)as diimide-activation agent.However,the process will lead to undesirable intermolecular conjugation of proteins,because proteins possess rich amine and carboxylic acid groups.In the current work,the carboxylic acid groups of the Si-g -P(PEGMA)-COOH surface were ?rst converted into reactive esters (succinimidyl intermediates)in the presence of EDAC and N -hydroxysuccinimide (NHS).34Then,the activated Si-g -P(PEGMA)-COOH surface with the active intermediates were introduced into the protein solutions.The reactive esters underwent nucleophilic substitution reactions with the amine groups of proteins to form a stable amide linkage without the presence of diimide,thus,avoiding the intermolecular connec-tion of proteins.

The C 1s and N 1s core-level spectra of the (a,a ′)Si-g -P(PEGMA)1-CDI and (b,b ′)Si-g -P(PEGMA)2-CDI surfaces are shown in Figure 5.The C 1s core-level spectra can be curve-?tted into four peak components with BEs at about 284.6,285.2,286.2,and 288.5eV,attributable to the C-H,C-N,C-O,and O )C-O species,respectively.13,23The new N 1s spectra consist of the imine ()N-;at the BE of about 397.5eV)and amine (-N-;at the BE of about 399eV)components,associated with CDI.The above results are consistent with the

successful

Figure 4.Representative ?uorescence images of the (a -d)pristine Si(100)surfaces (washed by PBS or 1%SDS solution after protein adsorption)and the (e -h)Si-g -P(PEGMA)surfaces (washed by PBS solution after protein adsorption)after been exposed to BSA-FITC and IgG-FITC solutions for 12h.The compositions of these surfaces correspond to those of the surfaces shown in Figure 3.Inset (a ′)shows the ?uorescence image of the pristine Si(100)surface before protein adsorption.

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activation of some of the -OH groups of the grafted P(PEGMA)brushes to the imidazole carbamate groups.Based on the surface [N]/[C]ratios (determined from the sensitivity-factor corrected N 1s and C 1s core-level spectral area ratio)and DP of about 34for the P(PEGMA)brushes within the sampling depth (about 7.5nm in an organic matrix 32)of the XPS technique,about 47and 51%of the hydroxyl groups on the respective Si-g -P(PEGMA)1-CDI and Si-g -P(PEGMA)2-CDI surfaces were activated.Figure 5also shows the C 1s core-level spectra of the (c)Si-g -P(PEGMA)1-COOH and (d)Si-g -P(PEGMA)2-COOH surfaces.In comparison with those of the starting Si-g -P(PEGMA)1and Si-g -P(PEGMA)2surfaces (Figure 2),the [O d C -O]/[C -O]ratios of the Si-g -P(PEGMA)1-COOH and Si-g -P(PEGMA)2-COOH surfaces increased substantially,con-sistent with the successful activation of some of the -OH groups to the carboxylic acid groups.Based on the surface [O d C -O]/[C -O]ratios,about 27and 32%of the hydroxyl groups on the respective Si-g -P(PEGMA)1-COOH and Si-g -P(PEGMA)2-COOH surfaces were activated.Prolonging the time of activation reaction (up to 48h)did not appear to result in a higher conversion rate in both Si-g -P(PEGMA)-CDI and Si-g -P(PEGMA)-COOH cases,indicating that the CDI exhibited a higher ef?ciency in activating the hydroxyl groups than SA.Figure 6shows the C 1s and N 1s core-level spectra of the (a,a ′)Si-g -P(PEGMA)1-IgG1(from the Si-g -P(PEGMA)1surface),(b,b ′)Si-g -P(PEGMA)2-IgG1(from the Si-g -P(PEGMA)2surface),(c,c ′)Si-g -P(PEGMA)1-IgG2(from the Si-g -P(PEGMA)1-CDI surface),(d,d ′)Si-g -P(PEGMA)2-IgG2(from the Si-g -P(PEGMA)2-CDI surface),(e,e ′)Si-g -P(PEGMA)1-IgG3(from the Si-g -P(PEGMA)1-COOH surface),and (f,f ′)Si-g -P(PEGMA)2-IgG3(from the Si-g -P(PEGMA)2-COOH surface)surfaces.In comparison with those of the starting Si-g -P(PEGMA)1and Si-g -P(PEGMA)2surfaces (Figure 1c,e),the corresponding C 1s spectral line shapes of the Si-g -P(PEGMA)1-IgG1and Si-g -P(PEGMA)2-IgG1surfaces remain almost unchanged.The corresponding N 1s signals associated with IgG are also very weak (Table 1).These phenomena are consistent with the relatively low concentration of immobilized IgG from the coupling reactions between the chloride groups at the P(PEGMA)chain ends (preserved during ATRP)and the amine groups of IgG.13,26For the Si-g -P(PEGMA)-IgG2and Si-g -P(PEGMA)-IgG3surfaces,the C 1s and N 1s spectral line shapes are signi?cantly different from the corresponding spectral line shapes of the Si-g -P(PEGMA)1-IgG1and Si-g -P(PEGMA)2-IgG1surfaces.The C 1s core-level spectra of the Si-g -P(PEGMA)-IgG2and Si-g -P(PEGMA)-IgG3surfaces can be curve-?tted into ?ve peak components with BE’s at about 284.6,285.5,286.2,287.8,and 288.5eV,attributable to the C-H,C-N,C-O,O )CNH,and O )C-O species,respec-tively.23The C-N and O )CNH peak components are associ-ated with IgG.In comparison with the corresponding Si-g -P(PEGMA)1-IgG1and Si-g -P(PEGMA)2-IgG1surfaces,the [N]/[C]ratio has increased from 1.6×10-4to 0.15(for the Si-g -P(PEGMA)1-IgG2surface)or 0.10(for the Si-g -P(PEGMA)1-IgG3surface)and from 1.3×10-3to 0.19(for the Si-g -P(PEGMA)2-IgG2surface)or 0.12(for the Si-g -P(PEGMA)2-IgG3surface).For the Si-g -P(PEGMA)-IgG2and Si-g -P(PEGMA)-IgG3surfaces,the active chloride groups at the chain ends preserved during the ATRP process,the imidazole carbamate groups and the carboxylic acid groups have been used in the coupling reactions to enhance the amount of the immobilized IgG.The corresponding amounts (determined by the modi?ed dye-interaction meth-ods)of immobilized IgG on the Si-g -P(PEGMA)1-IgG2(or Si-g -P(PEGMA)1-IgG3)(P(PEGMA)thickness ～52nm)

and

Figure 5.C 1s and N 1s core-level spectra of the (a,a ′)Si-g -P(PEGMA)1-CDI and (b,b ′)Si-g -P(PEGMA)2-CDI surfaces and C 1s core-level spectra of the (c)Si-g -P(PEGMA)1-COOH and (d)Si-g -P(PEGMA)2-COOH surfaces.

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Si-g -P(PEGMA)2-IgG2(or Si-g -P(PEGMA)2-IgG3)(P(PEG-MA)thickness ～136nm)surfaces are 3.2(or 2.5)and 4.8(or 3.3)μg/cm 2,respectively (Table 1).The higher IgG loading in the latter hybrid is consistent with the presence of a much thicker P(PEGMA)-CDI (or P(PEGMA)-COOH)layer,indicating that the distribution of active protein-docking sites on the Si -C bonded P(PEGMA)brushes can be controlled by controlling the brush length.The lower IgG loading in the Si-g -P(PEGMA)-IgG3surfaces (than the corresponding in the Si-g -P(PEGMA)-IgG2surfaces)should be attributed to the lower concentration of reactive groups derived from the -OH groups (about 30and 49%,respec-tively,for the Si-g -P(PEGMA)-COOH and Si-g -P(PEGMA)-CDI surfaces)of the P(PEGMA)brushes as described earlier (Figure 5).

After incubation in the PBS solution of anti-IgG-FITC,the representative ?uorescence images of the (a)Si-g -P(PEGMA)1-IgG1,(b)Si-g -P(PEGMA)2-IgG1,(c)Si-g -P(PEGMA)1-IgG2,(d)Si-g -P(PEGMA)2-IgG2,(e)Si-g -P(PEGMA)1-IgG3,and (f)Si-g -P(PEGMA)2-IgG3surfaces are shown in Figure 7.No obvious ?uorescence was observed on the Si-g -P(PEGMA)1-IgG1and Si-g -P(PEGMA)2-IgG1,because of the low concen-tration of immobilized IgG on these surfaces.Upon excitation,uniform and strong ?uorescence is observed across the Si-g -P(PEGMA)-IgG2and Si-g -P(PEGMA)-IgG3surfaces,indicat-ing that signi?cant amounts of anti-IgG-FITC have successfully binded to the uniform and dense IgG immobilized on the well-de?ned PEG brushes.In addition,the ?uorescence intensity is consistent with the corresponding IgG amount on these surfaces.With the increase in the IgG loading from the Si-g -P(PEGMA)1-IgG3surface (IgG amount,～2.5μg/cm 2)to the Si-g -P-(PEGMA)2-IgG2surface (IgG amount,～4.8μg/cm 2),the ?uorescence intensity increased accordingly.Finally,we sought to determine whether the Si-g -P(PEGMA)-IgG2and Si-g -P(PEGMA)-IgG3surfaces exhibit speci?c binding with the target protein.Thus,BSA-FITC,instead of anti-IgG-FITC,was also used in the immunostaining procedure.As shown in Figure 7g,h,no signi?cant ?uorescence can be observed from the

Si-

Figure 6.C 1s and N 1s core-level spectra of the (a,a ′)Si-g -P(PEGMA)1-IgG1,(b,b ′)Si-g -P(PEGMA)2-IgG1,(c,c ′)Si-g -P(PEGMA)1-IgG2,(d,d ′)Si-g -P(PEGMA)2-IgG2,(e,e ′)Si-g -P(PEGMA)1-IgG3,and (f,f ′)Si-g -P(PEGMA)2-IgG3surfaces.

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g -P(PEGMA)2-IgG2and Si-g -P(PEGMA)2-IgG3surfaces after the same period of incubation in the BSA-FITC solution.Furthermore,the control experiments also con?rmed that the (Si-g -P(PEGMA)1and Si-g -P(PEGMA)2surfaces did not exhibit any signi?cant ?uorescence after incubation in the PBS solution of anti-IgG-FITC,consistent with the good protein-resistant properties of the densely grafted P(PEGMA)brushes.The above results suggest that the Si-g -P(PEGMA)-IgG2and Si-g -P(PEGMA)-IgG3surfaces are highly speci?c to the target anti-IgG protein.The good antifouling (“biologically inert”)characters of the well-de?ned and dense PEGMA units must have prevented nonspeci?c protein adsorption on these graft-modi?ed silicon surfaces.

4.Conclusions

Active IgG-coupled Si-g -P(PEGMA)hybrids were suc-cessfully prepared.The P(PEGMA)brushes were ?rst co-valently grafted (via robust Si -C bonds)on the hydrogen-terminated Si(100)surfaces from surface-initiated ATRP of PEGMA.The Si-g -P(PEGMA)hybrids with dense PEGMA

units exhibited good resistance to protein fouling.The hydroxyl groups of the Si -C bonded P(PEGMA)brushes were further activated via CDI or SA for the IgG im-mobilization.The resulting IgG-functionalized Si-g -P(PEG-MA)hybrid surface with a controlled density of IgG (above 2.5μg/cm 2)interacts only and speci?cally with the anti-IgG protein.In addition,the distribution of active protein-docking sites on the Si -C bonded P(PEGMA)brushes can be controlled by controlling the brush length.The CDI exhibited a higher ef?ciency in activating the hydroxyl groups for coupling proteins.With the inherent advantages of the electronic properties of silicon substrates,the good antifouling effects of the well-de?ned P(PEGMA)brushes,the highly speci?c nature of the covalently immobilized protein,and the development of simple approaches to the concomitant incorporation of PEG brushes and active proteins onto silicon surfaces,these multifunctional silicon surfaces are attractive for applications in silicon-based protein sensors or microarrays.Acknowledgment.This work was supported by the Major Project for Polymer Chemistry and Physics Subject Construc-tion from the Beijing Municipal Education Commission (BMEC,XK100100640)and the Program for Changjiang Scholars and Innovative Research Team in University (PC-SIRT,IRT0706).

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