Poly(2-hydroxyethyl methacrylate) Brush Surface for Specific andOriented Adsorption of GlycosidasesYan Fang,† Wei Xu,‡ Xiang-Lin Meng,† Xiang-Yu Ye,† Jian Wu,‡ and Zhi-Kang Xu*,†

†MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, and‡Department of Chemistry,Zhejiang University, Hangzhou 310027, China

*S Supporting Information

ABSTRACT: We present a detailed picture to screen generalligands from simple chemicals for fabricating affinity surface toglycosidase enzymes. The surface was constructed by graftingpoly(2-hydroxyethyl methacrylate) (PHEMA) brush on SPRgold chip via surface-initiated atom-transfer radical polymer-ization, after which poly(methoxyethyl methacrylate)(PMEMA) and poly(oligo(ethylene glycol) methacrylate)(POEGMA) brushes were also prepared for comparison.SPR measurements were adopted to monitor the early-stageadsorption of two glycosidases and three other typical proteins.PHEMA resists the adsorption of lysozyme, bovine serumalbumin, and fibrinogen, while it is capable of specificallyadsorbing β-glucosidase (GLU) and β-galactosidase (GAL).These are quite different from the nonspecific adsorption of PMEMA and the anti-nonspecific adsorption of POEGMA to thestudied proteins, because PHEMA is the acceptor substrate of the glycosidases. About 69.6 and 93.7 ng/cm2 of GAL and GLUare adsorbed on the PHEMA brush surface, of which more than 49.6 ng/cm2 is remained after washing with PBS. The specificadsorption process is appropriately described by Freundlich isothermal model rather than Langmuir one, and is also indicated tobe spontaneous, endothermic, and entropy driven through thermodynamic studies. Taking into account all stated results above,we propose that molecular recognition takes place between the hydroxyl groups of PHEMA and the active sites of glycosidases,which subsequently enables the oriented adsorption of glycosidases on the brush surface. The adsorbed enzyme can be effectivelyeluted with 1.0 M aqueous solution of ethanol. Our findings open the door to the further development in the design of novelacceptor substrate−ligand affinity chromatography for enzyme purification.

■ INTRODUCTION

Highly purified enzyme is the foundation of enzymology andenzyme engineering.1,2 Among various methods for enzymepurification, affinity chromatography (AC) is the most powerfulone based on the specific recognition between immobilizedligands and target proteins. Ligand screening, with suitableaffinity and high throughput, tends to be one of the keyscientific issues for developing AC.3 Therefore, severalrequirements on ligands have generally been followed: (1)highly specific affinity between the ligand and the enzymeprotein; (2) appropriate chemical activity of the ligand forcoupling to suitable carrier conveniently; (3) enough hydro-philicity of the immobilized ligand to reduce nonspecificinteractions between the carrier surface and the target protein;(4) search for the ligand particularly providing multipleinteraction sites to enhance the purification efficiency.Following these requirements, enzymes have been reportedto be purified with various ACs depending on different ligands,such as dye−ligand AC,4 metal−ligand AC,5 immuno−ligandAC,6 and inhibitor−ligand AC.7 The former two are developedfrom general ligands, while the latter two are based on specialones. The general ligands are more desirable than the special

ones because they usually show affinity for a broad spectrum ofenzyme. However, major challenges still remain to screengeneral ligands from simple chemicals with high specificity, andto balance the affinity strength as well as the protein resistanceof the ligand-immobilized surface to a family of enzyme or aspecial enzyme.8

We suggest screening general ligands from simple chemicalson the basis of molecular recognition/interaction betweenenzymes and their acceptor substrates, the background of whichlies in the major development in enzymology and enzymeengineering for biotechnology and green chemistry.9 Forexample, glycoenzymes such as dextransucrase,10 alternansu-crase,11 and cellulase12 were found to show high affinity to theiracceptor substrates during the enzymatic synthesis of glycosidicbonds. Therefore, it is to be expected that, as their acceptorsubstrates, dextran and cellulose may be potential ligands forthis family of enzyme. Glycosidase, a kind of glycoenzyme, isclassified into EC 3.2.1 for the linkage and/or breakage of

Received: July 7, 2012Revised: August 23, 2012Published: August 24, 2012

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glycosidic bonds.13,14 To the best of our knowledge, two typicalexamples are β-glucosidase (GLU) from almond (EC 3.2.1.23)and β-galactosidase (GAL) from Aspergillus oryzae (EC3.2.1.23), for which hydroxyl group in organics is theiracceptor. Among various hydroxylated chemicals, poly(2-hydroxyethyl methacrylate) (PHEMA) has attracted muchattention for its hydrophilicity and protein-resistant prop-erty.15−17 To confirm our suggestion, PHEMA brush wasdirectly constructed on a model surface by the surface-initiatedatom-transfer radical polymerization (SI-ATRP) for specificadsorption of glycosidases. Meanwhile, poly(methoxyethylmethacrylate) (PMEMA) and poly(oligo(ethylene glycol)methacrylate) (POEGMA) brushes were also fabricated forcomparison. PMEMA shows nonspecific adsorption, andPOEGMA presents antinonspecific adsorption to the studiedglycosidases and other proteins including lysozyme (LYS),bovine serum albumin (BSA), and fibrinogen (FIB). On theother hand, PHEMA can spontaneously adsorb GLU and GALbut resist the nonspecific adsorption of LYS, BSA, and FIB.Also, the adsorbed GLU and GAL are oriented on the PHEMAbrush surface.

■ EXPERIMENTAL SECTIONMaterials. Sigma-Aldrich (China) provided the following commer-

cial products and they were used as received: 2-hydroxyethylmethacrylate (97%), methoxyethyl methacrylate (99%), oligo(ethyleneglycol) methacrylate (M ≈ 360, 99%), 11-mercapto-1-undecanol(MUD), 2,2′-bipyridyl (99%), 2-bromo-2-methyl-propionyl bromide,chicken egg white LYS, BSA, FIB, GAL from Aspergillus oryzae andGLU from almond. Fluorescence-labeled concanavalin A (FL-Con A)was purchased from Vector (USA). Copper(I) chloride (CuCl) wasstirred in glacial acetic acid overnight, filtered, and washed withabsolute ethanol under an argon blanket. The compound was vacuum-dried at 60 °C overnight.18 Copper(II) bromide, triethylamine, aceticacid, methanol, ethanol, dichloromethane, and all the other chemicalswere purchased from Sinopharm (China) and used as received withoutfurther purification. Water used in all experiments was deionized andultrafiltered to 18 MΩ·cm using an ELGA Lab Water system (France).

SPR chips (12.5 mm × 12.5 mm glass slides coated with 40 nmgold) were purchased from Reicher (America). The chips werecleaned by Pirranha solution (98% H2SO4/30% H2O2 = 7/3, v/v) atambient temperature for 5 min to remove dust particles and organiccontaminants, and then washed with ultrapure water and blew dried byultrapure nitrogen before use.

SI-ATRP on SPR Chips. A two-step process was conducted toprepare ATRP initiator layer for grafting the polymer brushes on thegold chip surface by ATRP (Scheme 1). First, 1.0 mM MUD wasdissolved in ethanol and oxygen was removed from the solution bynitrogen bubbling for 5 min. Then, the precleaned SPR chips weresoaked in the MUD solution thus formed and incubated for 24 h atroom temperature to construct self-assembly monolayer (SAM). Next,the chips were washed sequentially with ethanol and water and thendried in a stream of ultrapure nitrogen. The reaction between 2-bromo-2-methyl-propionyl bromide and hydroxyl groups of the SAMallows the immobilization of the ATRP initiator onto the gold chips.The chips were then put into 6.0 mL of freshly dried dichloromethanecontaining 100 μL triethylamine, and 100 μL of 2-bromo-2-methyl-propionyl bromide was added with 1.5 mL of freshly drieddichloromethane. After reaction for 6 min at room temperature, thechips were taken out and washed with dichloromethane, ethanol, andwater and then dried in a stream of ultrapure nitrogen.

Scheme 2 presents a general strategy to construct PHEMA,POEGMA, and PMEMA brushes onto the gold surface via SI-ATRP.19

One chip was put into 10.0 mL of monomer solution prepared bydissolving CuCl (0.30 mmol), CuCl2 (0.06 mmol), and 2,2′-bipyridyl(0.60 mmol) in 10.0 mL of a proper solvent (pure water for 2-hydroxyethyl methacrylate and oligo(ethylene glycol) methacrylate,methanol for methoxyethyl methacrylate) and then transferring theminto a 50 mL round-bottom flask. The graft polymerization wasproceeded in nitrogen atmosphere at room temperature with a desiredtime to control the brush thickness (20 min for 2-hydroxyethylmethacrylate, 40 min for oligo(ethylene glycol) methacrylate, and 240min for methoxyethyl methacrylate). After that, the chip was washedthree times in the solvent used for polymerization. It was furtherwashed with ethanol followed by water and dried in a stream ofultrapure nitrogen.

Characterization. The variable-angle spectroscopic ellipsometry(VASE) spectra were collected on a MD-2000I spectroscopic

Scheme 1. Two-Step Process for Preparing ATRP Initiator Functionalized Surface

Scheme 2. Construction of PHEMA, POEGMA, and PMEMA Brushes on the Gold Surfaces by SI-ATRP Procedure

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ellipsometer (J. A. Woollam, USA) at an incident angle of 60°, 65°,and 70° in a wavelength range of 800−1000 nm. A refractive index of1.45 was assigned to the studied polymer brushes, the thickness ofwhich was calculated from a two-layer model (Au and Cauchy). Allmeasurements were conducted in dry air at room temperature. Threeseparate spots of each sample were measured to obtain a mean brushthickness and associated standard deviation.FT-IR/MR measurements were performed using a Nicolet FT-IR

spectrometer (Thermo Electron Co., USA) equipped with a mirrorreflection (MR) accessory. Thirty-two scans were taken for eachspectrum at a resolution of 4.0 cm−1.Water contact angles (WCA) were determined using a CTS-200

system (Mighty Technology Pvt. Ltd., China) fitted with a drop shapeanalyzer. Typical experiments were carried out at room temperature bysessile drop method as follows. Briefly, a water drop (2.0 μL) waslowered onto the chip surface from a needle tip. Then, the images ofthe droplet were recorded, from which WCAs were calculated withsoftware. At least five different surface locations of each sample weremeasured and the averaged value was presented.Monitoring of Protein Adsorption by SPR. SPR experiments

were conducted in real time on a SR7000 instrument (Reichert, USA).A peristaltic pump was used to deliver liquids to two independentchannels of the flow cell. A stable baseline signal was established byflowing PBS buffer (50.0 mM, containing 10.0 mM sodium chloride,and pH 7.4) at a rate of 25 μL/min through the sensor. Differentprotein solutions (1.0 mg/mL) of LYS, BSA, GAL, GLU, and FIB,typical parameters of which are summarized in Table 1, were injected

into the channel, respectively. After adsorption for 800 s at 25 °C, PBSbuffer was used for removing the unbound or loosely bound proteinsfrom the chip surface. The adsorbed amounts of proteins per unit ofsurface area (Q, in units of ng/cm2) were calculated from the SPRcurves according to eq 1.20,21

θ= × ΔQ 900(ng/(deg cm )) (deg)2m (1)

For SR7000, 1 μRIU (Response) corresponds to 0.135 mdeg(Δθm). By dividing with the molecular weight of protein, one canobtain the mole concentration (Γ) of the adsorbed protein.Fluorescence Microscopic Observation. Fluorescence micros-

copy was used to confirm the specific and oriented adsorption of GLUon the PHEMA brush surface. The sample was immersed in a PBSsolution of FL-Con A (20.0 μg/mL) for a prescribed time at roomtemperature and was then washed softly after leaving in fresh PBSthree more times. After that, the surface was dried under vacuum atroom temperature. Fluorescence images were taken on an EclipseTE2000 optical microscope (Nikon, Japan) equipped with a highlysensitive CCD camera (ORCA-ER, Hamamatsu Photonics, Japan).The observation was made on at least five spots for each sample.Elution of GLU from the PHEMA Brush Surface. Elution curves

of GLU from the PHEMA brush were measured by SPR with 1.0 Meluant solutions in PBS (pH 7.4). Various solutions, including 2-hydroxyl methacrylate, oligo(ethylene glycol) methacrylate, ethanol,glucose, and acetic acid, were sequentially injected at a rate of 25 μL/min for eluting the adsorbed enzymes.

■ RESULTS AND DISCUSSION

Construction, FT-IR Spectra, and WCAs of PHEMA,POEGMA, and PMEMA Brushes. SI-ATRP has beendeveloped to graft polymer brushes on substrates in the pastyears.22 As demonstrated in Scheme 1, a two-step process wasused to fabricate ATRP initiator layer with 1.3 nm thickness onthe gold surface (glass slide coated with 40 nm gold). Then, weadopted a typical SI-ATRP procedure to construct PHEMA,POEGMA and PMEMA brushes with high density (σ ≈ 0.7chain nm−2, Supporting Information, Table S1).16 The brushthickness is variable and must be identical for comparisonpurposes, which can be simply realized by controlling theATRP time as the monomer concentration is held constant. Itincreases gradually with the increasing ATRP time (SupportingInformation, Figure S1). This increase from hydrophilicmonomers (2-hydroxyethyl methacrylate and oligo(ethyleneglycol) methacrylate) is higher than that from hydrophobicmonomer (methoxyethyl methacrylate). On the basis of theseresults, we synthesized a series of brushes with identicalthickness (20 nm) and high chain density (σ ≈ 0.7 chain nm−2)to ensure protein adsorption in the monolayer.FT-IR/MR spectra were used to characterize the brush

surfaces. Compared to the bare gold surface, PHEMA brushshows an absorption peak at 1720 cm−1 which is assigned to theCO stretching vibration (Supporting Information, FigureS2). Another two bands centered at 1100 and 1037 cm−1

correspond to the stretching vibrations of the ether group (C−O−C). An additional absorption band around 3400 cm−1 isattributed to the OH stretching vibration. Relative results arealso obtained for the POEGMA and PMEMA brushes(Supporting Information, Figure S2).WCAs were measured during the construction procedure of

the brushes (Supporting Information, Figure S3). In contrast tothe bare gold surface, the WCA of the SAM decreases below20° due to the hydroxyl group of MUD.23 The ATRP initiatorlayer is much more hydrophobic (67°) than the SAM. After 20nm of brushes were polymerized on the chip surface, the WCAis 44° for POEGMA, 56° for PHEMA, and 72° for PMEMA,which means the hydrophilicity of the brush surface decreasesas POEGMA < PHEMA < PMEMA. In fact, these results areconsistent with the chemical structures of the brushes.POEGMA has one hydroxyl end-group and six ethylene glycolsegments (−CH2CH2O−) in each monomer unit, both ofwhich are responsible for the hydrophilicity of its brush surface.However, in terms of the PHEMA brush surface, thehydrophilicity is decreased by the reduction in the number ofethylene glycol segments to only one. After methylation of thehydroxyl group, the PMEMA brush surface shows furtherdecrease in hydrophilicity. The decline sequence in hydro-philicity will influence protein adsorption on these brushsurfaces.

Adsorption of Glycosidases and Typical Proteins ontothe Brush Surfaces. SPR was used to monitor in real time theearly-stage adsorption (∼800 s) of glycosidases and other threetypical proteins on the PHEMA, POEGMA, and PMEMAbrush surfaces. Adsorption behaviors were compared in detailfor GLU, GAL, LYS, BSA, and FIB (Supporting Information,Figure S4 and Figure S5). Because the polymer chains aregrafted in high density (σ ≈ 0.7 chain nm−2, SupportingInformation, Table S1), the brush surfaces should adsorbproteins of large three dimensions (Table 1) in themonolayer.16 Therefore, we can calculate and compare the

Table 1. Typical Characteristics of the Proteins Used in ThisWork

protein MW (kD)a pI h × w × l (nm)b

LYS 14.7 10.5 5 × 3 × 3BSA 66.4 4.7 4 × 4 × 11.5GAL 116 4.5 6 × 5 × 11.5GLU 135 5.5 6 × 7 × 12.5FIB 340 5.5 6 × 6 × 45

aMolecular weight of the protein. bApproximate molecular dimensionsof the protein.

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mole concentrations (Γ) of proteins adsorbed on the brushsurfaces (Supporting Information, Table S2). Theoretical valuesare also listed for a complete monolayer of each protein. It canbe seen that the experimental Γ values are far less than thetheoretical ones, which confirms the adsorption of proteins onthe brush surfaces in the monolayer. Figure 1 summarizes the

mass concentration of the adsorbed proteins. It is promisingthat the PHEMA brush surface exhibits expected specificadsorption toward GLU and GAL but high protein resistanceto LYS, BSA, and FIB (the nonspecific adsorption is lower than3.0 ng/cm2). About 69.6 and 93.7 ng/cm2 of GAL and GLU areadsorbed on the PHEMA brush surface, respectively (Support-ing Information, Figure S6). Figure 1 shows more than 49.6ng/cm2 of the adsorbed glycosidases remain on the brush afterwashing with PBS. This specific adsorption can be furtherverified using FT-IR/MR and AFM (Supporting Information,Figure S7 and Figure S8). The POEGMA brush surface, on onehand, is highly protein-resistant and the remaining proteinsafter washing with PBS are less than 3.0 ng/cm2. On the otherhand, the PMEMA brush surface shows nonspecific adsoptionto the studied proteins. More than 93.7 ng/cm2 of proteins areadsorbed on the brush surface and the irreversibly adsorbedamounts are as high as 80.3 ng/cm2. Similar behaviors can beseen from the Γ values (Supporting Information, Table S2).As mentioned above, the PMEMA brush surface is relatively

hydrophobic (WCA = 78°, Supporting Information, Figure S3).Thus, hydrophobic interaction will lead to nonspecificadsorption of proteins on the brush surface. POEGMA iswell-known as a hydrophilic polymer, and its large excludedvolume in aqueous solution, configurational entropy, as well ashydrated chain mobility, endow the POEGMA brush surfacewith outstanding protein resistance.24−26 Nevertheless, theadsorbed amounts of GLU and GAL are higher than those ofLYS, BSA, and FIB, because the hydroxyl end-group ofoligo(ethylene glycol) methacrylate is also a substrate acceptorfor the studied glycosidases. Compared with one hydroxyl end-group, there are six ethylene glycol units (−CH2CH2O−) ineach side chain of POEGMA. This structure enables theformation of a hydration layer on the brush, and finally

contributes predominance of protein resistance to POEGMA.27

For the PHEMA brush, the surface hydrophilicity is highenough to resist the nonspecific adsorption of LYS, BSA, andFIB. However, its hydroxyl groups are directly engaged in themolecular recognition toward the active sites of glycosidases,which can be reasonably proven as 2-hydroxyl methacrylateobviously suppresses the activity of GLU for the hydrolysis ofp-nitrophenyl-β-D-glucopyranoside in aqueous solution (Sup-porting Information, Figure S9). Consequently, the adsorptionof GLU and GAL is specific on the PHEMA brush surface andis much higher than that of the other three proteins.Besides, we propose the specific adsorption of GLU and

GAL oriented on the PHEMA brush surface. This is reasonableon the basis of the following analysis and experimental results.The active site of glycosidase is usually found in a pocket ortunnel lined by two glutamic acid residues (SupportingInformation, Figure S10). There are oligosaccharide chains(carbohydrates) on the outmost surface opposite the active site,which specifically interacts with lectins such as concanavalin A(Con A).28,29 These characteristics enable us to confirm theoriented adsorption of the studied glycosidases on the PHEMAbrush surface. After the specific adsorption of GLU on thebrush surface, the oligosaccharide chains of the enzyme will beexposed to the outermost surface of the brush and adsorb ConA via carbohydrate−lectin interactions.30−32 Figure 2 demon-

strates that the PHEMA brush surface is resistant to theadsorption of BSA and FL-Con A. However, FL-Con A canspecificly interact with GLU adsorbed on the brush surface andno change happens when GLU is replaced by BSA, which inturn confirms the specific adsorption of GLU is extraordinarilyoriented.Furthermore, we checked the effect of temperature on the

specific adsorption of GLU. Temperature will change thehydrated PHEMA chains from order to partial order, followedby further randomization,33,34 which exposes more hydroxylgroups on the brush surface. Figure 3 indicates that theadsorbed amount of GLU increases drastically with temper-ature. These data were analyzed with Langmuir and Freundlichisotherm models to determine which one describes the specificadsorption process more precisely. The Langmuir model isbased on the assumption that there is negligible intermolecular

Figure 1. Comparison of remaining proteins adsorbed on the brushsurfaces after washing with PBS.

Figure 2. SPR curves of BSA and FL-Con A exposure to the PHEMAbrush before and after the adsorption of GLU. Insets are thefluorescence images of FL-Con A adsorbed surfaces (a) with GLUpreadsorption and (b) without GLU preadsorption. The scheme onthe right shows the recognition mechanism between GLU and Con A.

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interaction between adsorbed proteins and there are uniformadsorption sites on the surface. The Freundlich model is a moresophisticated one based on the assumption that there may beintermolecular interactions between the adsorbed molecules,and the surface adsorption sites are energetically heteroge-neous. As illustrated in Table 2, the adsorption behaviors fitsmore satisfactorily to the Freundlich isotherm model(Supporting Information, Figure S11), suggesting that thereare intermolecular interactions between the adsorbed glyco-sidases.Although the Langmuir isotherm model has relatively low

precision in describing the specific adsorption of GLU on thePHEMA brush surface, the equilibrium constant (Ka) obtainedfrom this model can be used to roughly calculate thethermodynamic parameters (change in Gibb’s free energy(ΔG), change in enthalpy (ΔH), and change in entropy (ΔS))of the adsorption process with Van’t Hoff eq 2.

= Δ − Δ × = −ΔK

SR

HR T

GRT

ln1

a (2)

From Table 2, it can be seen that not only Ka but Kf valuesincrease with an increase in temperature, which can beinterpreted by the enhancement of specific adsorption ofGLU with temperature.35 Furthermore, a negative value of ΔGmeans the adsorption process occurs spontaneously on thePHEMA brush surface. ΔG decreases slightly from −31.45 to−33.97 kJ/mol with temperature increases from 288 to 308 K.This spontaneous adsorption results from the highly specificinteraction with high degree of orientation between the activesite of GLU and the hydroxyl group of PHEMA.

The specific adsorption is endothermic demonstrated by apositive value of ΔH,36 and it should increase with an increasein temperature. ΔS of the spontaneous adsorption is only 0.13kJ/mol·K. However, the TΔS value is much higher than that ofΔH, indicating that the influence of enthalpy is minor while theentropy is actually the driving force for the adsorption ofGLU.37 A positive value of entropy also suggests the adsorptioncan be explained by Langmuir replacement reaction.38−41 Thisreaction proposes that water molecules are replaced from eitherGLU or PHEMA or both during the specific adsorption, whichis necessary for enzyme−substrate binding and will influencethe elution of GLU from the PHEMA brush surface.Figure 4 and Figure 5 show the elution characteristics of

GLU adsorbed on the PHEMA brush surface with a sequential

washing. Because 2-hydroxyl methacrylate and oligo(ethyleneglycol) methacrylate are substrate acceptors of GLU, theiraqueous solutions therefore are competitive eluents to theadsorbed GLU. Nevertheless, only 10% of the total adsorbedGLU can be eluted. It may be attributed to the strongpolyvalent interactions between PHEMA and GLU. Glucose isa donor during direct glycosylation for glycoside synthesis bythis kind of enzyme, and its solution can also elute GLU slightlyfrom the brush surface. Ethanol appears more efficient than 2-hydroxyl methacrylate to elute the adsorbed enzyme (>60%),since the water-soluble alcohol can feasibly diffuse to the activesite of GLU in aqueous solution and change the hydration stateof the enzyme surface.14 Furthermore, acetic acid solution isalso a useful eluent because the specific hydrogen bonding

Figure 3. Relationship between the adsorbed amount of GLU on thePHEMA brush surface and the protein concentration at varioustemperatures: (□) 15 °C, (▲) 25 °C, (○) 35 °C.

Table 2. Adsorption Behaviors of GLU on the PHEMA Brush Surface

thermodynamic parameters Langmuir adsorptiona Freundlich adsorptionb

T (K) ΔG (kJ/mol) ΔH (kJ/mol) ΔS (kJ/mol·K) Ka (×105) Qm (ng/cm2) R2 Kf n R2

288 −31.45 5.08 177.92 0.984 5.6 × 103 1.05 0.992298 −32.72 4.53 0.13 5.41 354.61 0.967 4.6 × 105 1.61 0.996308 −33.97 5.71 1139.24 0.939 5.6 × 107 2.98 0.991

aData were caculated by equation [C]/Q = [C]/Qe + 1/Qe × 1/Ka, where Q is the measured amounts of adsorbed GLU per unit of surface area (ng/cm2), Qe is the equilibrium amounts of adsorbed GLU, [C] is the equilibrium concentration of GLU in solution (mol/L), and Ka is the value ofadsorption equilibrium constant. bData were caculated by equation log Qe = log Kf + (1/n)log[C], where Kf and n are the Freundlich characteristicconstants indicating adsorption capacity and adsorption intensity, respectively.

Figure 4. Elution characteristics of GLU adsorbed on the PHEMAbrush surface with different eluant solutions in PBS at 25 °C: (a) 1.0M 2-hydroxyl methacrylate, (b) 1.0 M oligo(ethylene glycol)methacrylate, (c) 1.0 M glucose, (d) 1.0 M ethanol, (e) 1.0 M aceticacid.

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between the enzyme and the hydroxyl group can be broken bythis acid.42,43 We suggest that a 1.0 M aqueous solution ofethanol is effective enough to regenerate the affinity surface forpractical application.

■ CONCLUSIONWe demonstrate an approach to the development of substrateacceptor−ligand AC for enzymology and enzyme engineering.As an example, PHEMA brush was constructed on SPR goldchip to design an affinity surface for glycosidases. The hydroxylgroups of PHEMA are desirable ligands for this family ofenzyme. Glycosidases can be adsorbed on the designed surfacespecifically and orientedly via molecular recognition betweenthe hydroxyl group and the active site of enzyme. Comparedwith Langmuir adsorption isotherm, the specific adsorptionprocess is satisfactorily described by Freundlich adsorptionmodel. Thermodynamic parameters also demonstrate thatadsorption is spontaneous, endothermic, and entropy drivenin nature. In addition, glycosidase−PHEMA interaction may bepolyvalent and can be greatly weakened by appropriate eluents.Therefore, in conclusion, PHEMA should be a promisingaffinity ligand for glycosidases in future.

■ ASSOCIATED CONTENT*S Supporting InformationSI-ATRP on SPR chips and control of the brush thickness;ellipsometry and static WCA measurement of the fabricatedsurface; FT-IR/MR analysis and AFM images of the PHEMAbrushes before and after GLU adsorption; effect of 2-hydroxylmethacrylate on the bioactivity of GLU in aqueous solution;Figures S1−S11; and refs 1−2. This material is available free ofcharge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: xuzk@zju.edu.cn; fax: + 86 571 8795 1592.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank financial support from the National Natural ScienceFoundation of China (Grant No. 50933006) and the NationalBasic Research Program of China (2009CB623401). We also

achnowledge discussion and suggestion from Dr. Xiao-JunHuang and Dr. Ling-Shu Wan in the early stage of this work.

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