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Colloids and Surfaces B: Biointerfaces 110 (2013) 105– 112

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al hom epage: www.elsev ier .com/ locate /co lsur fb

lycosylation of the polypropylene membrane surface via thiol–ynelick chemistry for lectin adsorption

ang Wanga, Yan Fanb, Meng-Xin Huc, Wei Xua, Jian Wua,∗,eng-Fei Renb, Zhi-Kang Xub,∗∗

Department of Chemistry, Zhejiang University, Hangzhou 310027, ChinaMOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou10027, ChinaSchool of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, China

a r t i c l e i n f o

rticle history:eceived 2 February 2013eceived in revised form 18 April 2013ccepted 23 April 2013vailable online 28 April 2013

eywords:lycosylationffinity membrane chromatographyrotein separation/purification

a b s t r a c t

Glycosylated membrane, as one of the most important affinity membranes, permits affinity separa-tion/purification of proteins based on carbohydrate–protein interactions. It is an important scientificissue to screen facile method for fabricating the glycosylated membrane surface with high glycosyl den-sity. Such a surface can be fabricated by the direct covalent immobilization of carbohydrate ligandson the surfaces of microporous polypropylene membrane (MPPM). First, alkyne-functionalized mem-brane surface was fabricated by plasma pretreatment combined with UV-induced graft polymerizationof 3-(trimethylsilyl) propargyl methacrylate. Then, the glycosylated membrane surface was directly fab-ricated with the thiol–yne click reaction to ensure rapid process, improved efficiency, and high glycosyldensity. Chemical and physical properties of the membrane surface were characterized by ATR/FT-IR,

ectinhiol–yne click chemistryicroporous polypropylene membrane

XPS, FESEM and water contact angle measurement. Static lectin adsorption indicates that the glycosy-lated membrane can specifically adsorb lectin concanavalin A (Con A) other than peanut agglutinin (PNA).Break through curves from dynamic Con A adsorption show the membrane has unique properties suchas strong specificity, high adsorption capacity, and reversible binding capability. We suggest that theprepared glycosylated membrane is of great potentials in affinity membrane chromatography for rapid

ration

and high-resolution sepa

. Introduction

Separation/purification of proteins have been fueled for theapid development of biotechnological and pharmaceutical indus-ries. Membrane chromatography is one of the most promisingechniques for high-performance protein separations/purifications1–3]. However, traditional membrane chromatography usuallyas low binding capacity and low selectivity/specificity for pro-eins. It is mainly attributed to the certain interactions whichave been used in the traditional membrane chromatography,uch as electrostatic interaction, hydrophobic interaction and vaner waals interaction. By contrast, the affinity membrane chro-atography is an attractive and competitive method for protein

eparation/purification due to its large binding capacity, high

electivity/specificity, and high reversible binding capability [4,5].sually, the affinity membrane chromatographies achieve their

ervice performance based on the specific interaction between

∗ Corresponding author. Fax: +86 571 8795 1773.∗∗ Corresponding author. Tel.: +86 571 8795 2605; fax: +86 571 8795 1773.

E-mail addresses: jianwu@zju.edu.cn (J. Wu), xuzk@zju.edu.cn (Z.-K. Xu).

927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2013.04.029

/purification of lectins.© 2013 Elsevier B.V. All rights reserved.

immobilized ligands and target proteins. Among various specificinteractions, carbohydrate–protein interactions play importantbiological roles in many cellular processes, such as inflammation,embryogenesis, cellular signal transfer, and immune response [6,7].Based on the carbohydrate–protein interactions, newly developedglycosylated membrane is receiving extensive attentions for poten-tial application in affinity membrane chromatography for rapid andhigh-resolution separation/purification of proteins. Undoubtedly,screening facile method for fabricating the glycosylated membranesurface, with high glycosyl density, tends to be one of the keyscientific issues for developing this kind of affinity membrane chro-matography.

Up to now, much pioneering work was conducted for fabricatingthe glycosylated surfaces [8–18]. In our previous studies, consid-erable efforts were made to fabricate the glycosylated surfaces onmicroporous polypropylene membrane (MPPM) [10–18]. The maingoal of our series work is focused on increasing the surface glyco-syl density. Highly efficient reaction, such as azide–alkyne “click”

chemistry, was recently introduced for fabricating affinity mem-branes from MPPM with high glycosyl density [19]. As we knowwell, azide–alkyne “click” chemistry is a simple, chemoselective,and high-yield reaction. This reaction can be effectively proceeded

106 C. Wang et al. / Colloids and Surfaces B: Biointerfaces 110 (2013) 105– 112

glyco

ucdtitp(ertNtiesit

opwttr“�gtndTsttmr

2

2

p

adsorbed chemicals and dried under reduced pressure at 40 ◦C.The grafting density (GD, �g/cm2) was calculated by the following

Fig. 1. Schematic illustration for the construction of the

nder conventional environment and used in any systems foroupling two compounds. However, we found triazole moietieserived from this reaction unavoidably caused non-specific adsorp-ion of proteins. The thiol–yne “click” chemistry [20,21] was furtherntroduced for preparing the glycosylated surface [22] to overcomehis problem. Similarly, the thiol–yne “click” reaction has simplerocedure with fast reaction rate under mild reaction conditionat normal temperature and pressure). Furthermore, it can yieldven bis-addition products [23–29]. In our preliminary study, thisobust click chemistry showed promising results for fabricatinghe glycosylated membrane surfaces with high glycosyl density.evertheless, the non-quantitative coupling reaction (involving

he carboxyl groups and the alkyne pendants) limited the furthermprovement of the surface glycosyl density due to “bottleneckffect” [22]. Besides, the residue carboxyl groups on the membraneurface also caused non-specific protein adsorption by electrostaticnteractions. Therefore, it is necessary to establish a facile methodo overcome these problems discussed above.

Here, we describe an improved method for the fabricationf the glycosylated membrane surface. First, 3-(trimethylsilyl)ropargyl methacrylate (TMSPA) was synthesized and then itas directly grafted onto the membrane surface by plasma pre-

reatment and UV-induced graft polymerization. Subsequently,he alkyne-functionalized membrane surface was obtained afteremoval of the terminal trimethylsilyl groups. Finally, the thiol–yneclick” reaction was carried out between 2,3,4,6-tetra-O-acetyl--d-glucopyranoside thiol and the alkyne group to fabricate thelycosylated membrane surfaces. Obviously, the improvement ofhe surface glycosyl density will not be influenced by “bottle-eck effect” that seen in the previous studies [19,22], and theensity is easily controllable by changing the grafting degree ofMSPA. In addition, there is no residual of charged compounds,uch as carboxyl groups, on the membrane surface to inducehe undesired protein adsorption. Both the static adsorption andhe filtration experiments show that the newly fabricated affinity

embrane is of great potential for high-performance lectin sepa-ation/purification.

. Experimental

.1. Materials

Unless otherwise specified, analytical grade chemicals wereurchased and used without further purification. MPPM was a

sylated membrane surface by thiol–yne click chemistry.

commercial product from Membrana GmbH (Germany) with anaverage pore size of 0.20 �m and a relatively high porosity ofabout 75%. All membrane samples used were cut into roundswith a diameter of 25 mm and washed by acetone for 2 h toremove impurities adsorbed on the membrane surfaces. Then,they were dried in a vacuum oven at 40 ◦C to constant weight.Trimethylsilyl chlorosilane (TMSCl), �,�-dimethoxy-�-phenyl-acetophenone (DMPA) and 1-propyn-3-ol were purchased fromShanghai ALADDIN Reagent Co., Ltd. (China). Methacryloyl chlo-ride was a commercial product of Nanjing Robiot Co., Ltd. (China)and was purified under reduced pressure. 3-(Trimethylsilyl)propargyl methacrylate (TMSPA) was synthesized according toa procedure described elsewhere (Fig. S1–S4 in SupplementaryInformation for monomer characterization) [30]. 2,3,4,6-Tetra-O-acetyl-�-d-glucopyranoside thiol (TAGT) was synthesized asreported previously [22]. Acetic acid (HAc) was purchased fromSinopharm (China) and used as received without further purifi-cation. Buffer solutions were prepared from analytical-gradechemicals and ultrapure water (18.2 M�) produced from an ELGALab Water system (France). Fluorescence-labeled concanavalin A(FL-Con A) and peanut agglutinin (FL-PNA) were purchased fromVector (USA) and used as received.

2.2. Plasma pretreatment and UV-induced graft polymerization

The experimental procedure is schematically illustrated in Fig. 1.Dielectric barrier discharge (DBD) plasma was used to pretreat thenascent membrane using a plasma apparatus (Nanjing Suman Elec-tronics Co., Ltd. China) at atmospheric pressure [31]. Firstly, thesample was irradiated at 40 V and 10 kHz for a given time. Thetreated membrane was then taken out and exposed in the atmo-spheric air, followed by immersing in a TMSPA solution (5 mmol/mLin THF) which has been degassed for 15 min in Ar atmosphere.Finally, UV-induced graft polymerization on the membrane surfacewas carried out under a high pressure mercury lamp for a pre-determined time. The modified membrane was washed thoroughlywith acetone and THF at 45 ◦C over night to remove physically

equation:

GD = W1 − W0

196A(1)

es B: B

w(c

ptsemt

2

d1saf�Addsedgc

G

R

wnc

2

baedUawcbnttgdCdwrla

2

t

C. Wang et al. / Colloids and Surfac

here W0 and W1 are the mass of nascent and grafted membranesg), respectively. While A represents the area of the membrane (4.91m2).

The trimethylsilyl group (TMS) was removed from the graftedoly(TMSPA) to obtain alkyne-functionalized membrane. This reac-ion was carried out according to previous report. Briefly, theample was immersed in KOH/methanol (0.05 g/mL) at ambi-nt temperature for 1 h. Then, the membrane was washed withethanol, water and dried under reduced pressure at 40 ◦C for 24 h

o constant weight (W2) [29].

.3. Glycosylation of MPPM by thiol–yne click chemistry

TAGT (0.1 g, 0.27 mmol), and 2 wt.% (2 mg, 7.8 �mol) �,�-imethoxy-�-phenyl-acetophenone (DMPA) were dissolved in

mL THF. This formed solution was degassed for 15 min andubsequently transferred into a reaction vessel containing thelkyne-functionalized MPPM sample. The vessel was sealedor thiol–yne click reaction under UV irradiation (∼3 mW/cm2,max = 365 nm) at room temperature for a predetermined time.fter that, the sample was washed extensively with THF andried. Acetyl glucose on the membrane surface was deprotected byipping the membrane in 0.05 g/mL sodium methoxide/methanololution for 1 h at room temperature. Then, the sample was washedxtensively with methanol and deionized water, and subsequentlyried under reduced pressure at 40 ◦C to constant weight (W3). Thelycosyl density (GD′, �mol/cm2) and reaction efficiency (RE) werealculated by the following equations:

D′ = W3 − W2

196A(2)

R = GD′

2GD× 100% (3)

here A represents the area of the membrane (4.91 cm2). Theumber 2 represents the bis-addition process of thiol–yne clickhemistry.

.4. Characterization of the membrane surface

Chemical structure of the membrane surface was characterizedy Fourier transform infrared spectroscopy (FT-IR/Nexus 470) withn ATR accessory (ZnSe crystal, 45◦). Sixteen scans were taken forach spectrum at a resolution of 4 cm−1. XPS analyses were con-ucted on a RBD upgraded PHI-5000 C ESCA system (Perkin Elmer,SA) with Al K� radiation (h� = 1486.6 eV). In general, the X-raynode was run at 250 W and the high voltage was kept at 14.0 kVith a detection angle at 54◦. The base pressure of the analyzer

hamber was about 5 × 10−8 Pa. Binding energies were calibratedy the containment carbon (C1s = 284.6 eV). Field emission scan-ing electron microscopy (FESEM, Sirion-100, FEI, USA) was usedo capture the surface morphologies of membrane at an accelera-ion voltage of 25.0 kV after the samples were sputtered with a thinold layer. Water contact angle (WCA) of the membrane surface wasetermined with a CTS-200 system (Mighty Technology Pvt. Ltd.,hina) at room temperature by sessile drop method. Briefly, a waterrop (2 �L) was carefully dropped onto the top membrane surfaceith a microsyringe, and then images of the water droplet were

ecorded and WCA was calculated with the specific software. Ateast five different surface locations of each sample were measurednd the averaged value was presented.

.5. Lectin adsorption assays

Lectin adsorption assays were performed to evaluate recogni-ion capability of the glycosylated membranes. Briefly, nascent and

iointerfaces 110 (2013) 105– 112 107

modified membranes in round shape (˚d = 3 mm) were immersedinto ethanol for 10 min. Then, the samples were moved into HEPESbuffer solution (pH 7.5) to exchange ethanol at 25 ◦C for 2 h. Subse-quently, the samples were exposed to fluorescence-labeled lectinsolution with a concentration of 20 �g/mL in HEPES buffer solu-tion (pH = 7.5) for a 2 h period at 25 ◦C. Thereafter, each membranewas rinsed in fresh HEPES buffer solution by gentle shaking. Thesample was dried under vacuum at room temperature. Fluorescentimages of the membrane surface were captured by fluorescencemicroscopy (Nikon ECLIPES Ti-U, Japan).

2.6. Affinity microfiltration of lectins by the glycosylatedmembrane

A dead-end microfiltration cell system with an effective mem-brane area of 0.785 cm2 was used to analyze the lectin separationand recognition performance of the nascent and glycosylated mem-branes. The membrane sample was primarily wetted with ethanoland extensively exchanged with HEPES buffer solution before beenplaced in the cell. A microinfusion pump was set to deliver theloading solution at a flow rate of 5 mL/h. Fluorescence-labeledlectin solution of 10 �g/mL was loaded and passed through themembrane. The effluent was collected every 3 min.The lectin con-centration was calculated from the fluorescent intensity at 518 nmby a spectrofluorophotometer (SHIMADZU, RF-5301PC, Japan).Calibration curve was prepared by plotting known standard con-centrations with fluorescent intensities.

After the microfiltration of fluorescence-labeled lectin, the filtercell was washed with HEPES buffer solution until no fluores-cence signal was detected in the effluent. Thereafter, the eluentof 1 mol/L HAc was infused into the filter cell at 5 mL/h. Theelution effect was determined by the lectin concentration ofeffluent.

3. Results and discussion

3.1. Graft polymerization of TMSPA on the membrane surface

As schematically shown in Fig. 1, TMSPA is grafted onto themembrane surface to introduce functional alkyne groups for fur-ther surface glycosylation via thiol–yne “click” chemistry. In thisstudy, we adopted a two-step process to accomplish the immo-bilization of alkyne groups on the membrane surface [32]. Firstly,peroxide/hydroperoxide groups were successfully formed on themembrane surface by plasma pretreatment at atmospheric pres-sure. Then, UV irradiation makes the peroxide/hydroperoxidegroups decomposed into free radicals which initiated the graft poly-merization of TMSPA.

The grafting density of TMSPA is correspondingly varied withplasma pretreatment time and UV irradiation time. As is shownin Fig. 2a, with the increase of plasma pretreatment time, thegrafting density rises obviously at first, and then reaches a max-imum value at about 45 s. Thereafter, it decreases dramaticallywith further increasing time. It is reasonable since prolongedplasma treatment will generate more peroxide/hydroperoxidegroups on the membrane surface and thus produce more free rad-icals by thermal treatment or UV radiation. The produced freeradicals then induce the grafting of vinyl monomers [33–35].Unfortunately, mechanical properties of the membrane will dra-matically decrease as the increase of plasma treatment time [36].

Therefore, 45 s is finally deemed as the optimum pretreatmenttime for the following experiments. Besides, Fig. 2b indicatesthat the grafting density dependents on the UV irradiation time.It increases with increasing UV irradiation time because more

108 C. Wang et al. / Colloids and Surfaces B: Biointerfaces 110 (2013) 105– 112

0 20 40 60 80 10 0 12 0

0

1

2

3

4

5

6

7

8

Gra

ftin

g d

eg

ree

(�� m

ol/

cm

2)

Plasma pre trea tment time (s)

(a)

0 5 10 15 20

0

2

4

6

8

10

Gra

ftin

g d

eg

ree

(��m

ol/

cm

2)

UV irra diation time (min)

(b)

Fig. 2. Dependence of the TMSPA grafting degree on (a) the plasma pretreatmenttp

af

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(TfrrAgc3iwpilr

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%)

Graft ing d ensity (µmol/ cm2)

by FESEM (Fig. S7, Supplementary Information). Compared with the

ime (UV irradiation time is fixed at 15 min) and (b) the UV irradiation time (plasmaretreatment time is fixed at 45 s).

ctive sites are generated and excited on the membrane sur-ace.

.2. Construction of glycosylated surface on the membranes vialick chemistry

To fabricate glycosylated surface, the terminal trimethylsilylTMS) groups on the grafted poly(TMSPA) was removed in advance.hen the thiol–yne “click” chemistry was directly carried out toabricate the glycosylated membrane surface. The thiol–yne “click”eaction has high reaction rate and it nearly reached to equilib-ium after about 1.5 h (Fig. S5 in the supplementary information).s Fig. 3 shows, the “click” efficiency gradually decreases as therafting density ranging from 0.65 to 6.0 �mol/cm2, while the gly-osyl density gradually increases and approximately achieve to

�mol/cm2 as the grafting density up to 6 �mol/cm2. On one hand,t indicates that the increasing steric hindrance of grafting chains

ill block the active reactants diffuse to the active site withinolymer chains on the membrane surface. On the other hand, it

ndicates that the occurred bis-addition process will eventuallyead to high-yields of glycosyl density during the thiol–yne “click”eaction.

Fig. 3. Effect of glycosyl density and conversion of alkyne on the grafting degree ofTMSPA.

3.3. Chemical and physical properties of the prepared membranes

Chemical changes of the membrane surface were clearly char-acterized by FT-IR/ATR spectra (for details, see Fig. S6 in thesupplementary information). Compared with the nascent mem-brane, the poly(TMSPA)-grafted one exhibits an absorption peakat 1738 cm−1, which is assigned to the stretching vibration of C Oin TMSPA unit. In addition, one can also see the absorption peaksinduced by the C≡C and TMS stretching vibrations at 2190 cm−1

and 845 cm−1, respectively. When the terminal TMS groups areremoved, the peaks of C≡C and TMS disappear completely, andthe asymmetrical absorption peaks of C≡C H (2130 cm−1) andacetylenic hydrogen (3290 cm−1) appear immediately. After TGPAwas introduced by thiol–yne “click” reaction, peaks at 1755 cm−1

and 1225 cm−1 can be observed, which are ascribed to the acetyl-protected groups of TAGT. Correspondingly, the absorption peak of

C≡C H at 2130 cm−1 disappears completely. When the glucosepentaacetate was immobilized and the acetyl groups were subse-quently removed from the membrane surface, the absorption peaksat 1755 cm−1 and 1225 cm−1 disappear, while a broad band rangingfrom 3200 cm−1 to 3500 cm−1 appears, which is ascribed to the freehydroxyl groups of glucose pendants.

Quantitative elemental composition was obtained using XPSto gain chemical information of the membrane surface. For thenascent membrane, a major peak at 284.6 eV is ascribed to thebinding energy of C1s (Fig. 4a). For the TMSPA modified membranesurface, additional peaks are obvious at 102.2 eV (Si2p3/2), 151.8 eV(Si1s), and 534 eV (O1s) (Fig. 4b). After the TMS group being depro-tected, the peaks of Si disappear immediately (Fig. 4c). When thethiol–yne click reaction and the subsequent deprotection of acetylstake place, the appearance of S2p3/2 (162.8 eV) and S2s (226.9 eV)is a direct evidence to justify the immobilization of thiol glucosependants on the membrane surface (Fig. 4d). Table 1 summarizesthe changes of chemical components on the membrane surface.There is an obvious increase for the relative content of O1s with theimmobilization of glucose pendants. Furthermore, the efficiency ofthiol–yne “click” reaction can be obtained by calculating the com-ponents ratio of S2p/N1s on the membrane surface. A value of 48.0%is basically accordant with the result by weight method (Fig. S5 inthe supplementary information).

Morphology changes for the membrane surface were evaluated

nascent membrane, less and smaller size pores can be observedon the poly(TMSPA)-grafted membrane due to the grafting poly-merization of TMSPA. As the grafting density increase, the surface

C. Wang et al. / Colloids and Surfaces B: Biointerfaces 110 (2013) 105– 112 109

Table 1Chemical composition of the nascent and modified membrane surfaces by XPS.

Membrane C1s (mol%) O1s (mol%) Si2p (mol%) S2p (mol%) S2p/Si2p

Nascent membrane (a) 100 0 0 0 -Poly(TMSPA)-grafted (b) 90.94 6.80 2.26 0 -TMS deprotected (c) 84.83 15.16 0 0 -Glycosylated (d) 74.34 23.50

0 10 0 20 0 30 0 40 0 50 0 60 0 70 0

Bind ing ener gy (eV)

O1sC

1s

(d)

Si2sSi

2p3/2

(c)

(b)

(a)

S2p3/2

S2s

F(

amp“bs

tiwtFsnatdtSpgsb

3s

fhtmnT

ig. 4. XPS spectra of the membrane surfaces: (a) the nascent, (b) TMSPA modified,c) TMS deprotected, (d) glycosylated.

nd inner pores of the membrane are gradually covered by poly-ers. It leads to the diminishing of both the pore size and the

orosity. When glycosylation has been carried out by thiol–yneclick” chemistry, a lot of pores are still maintained on the mem-rane surfaces, which is benefit for future application in proteineparation/purification.

Water contact angle (WCA) measurements were carried outo evaluate the hydrophilicity of the membrane surfaces. Thenitial contact angle is over 141◦ for the nascent membrane,

hich is ascribed to the intrinsic hydrophobicity and the rela-ively high porosity and roughness of polypropylene membrane.or the poly(TMSPA)-grafted membrane with the grafting den-ity ranging from 0.65 to 2.35 �mol/cm2, the static WCA is almosto obvious decrease (Fig. S8, Supplementary Information). Whilefter glycosylation, the static WCA significantly decreases andhe downtrend becomes notable with the increase of glycosylensity from 0.583 to 3.12 �mol/cm2, which indicate the forma-ion of a stable hydrophilic layer on the membrane surface (Fig.9, Supplementary Information). Consequently, the water dropermeates into the inner pore and gradually disappears on thelycosylated membrane surface. The water contact angle datahows an excellent water permeability of these glycosylated mem-ranes.

.4. Specific adsorption of lectin on the glycosylated membraneurface

Traditionally, the membrane chromatography has been usedor size-based bioseparation with typical characteristics ofigh-throughput but relatively low-resolution requirements. For-

unately, the low-resolution limitation can be resolved with affinity

embrane chromatography, which is based on the specific recog-ition between the immobilized ligands and target proteins.herefore, the specific recognition capability of ligands is one of

0 2.17 0.960

the most important evaluations to affinity membrane chromatog-raphy.

Herein, two fluorescence labeled lectins, FL-Con A and FL-PNA, were used as visual probes to investigate the recognitioncapability of the glycosylated membrane surface. As shown inFig. 5, almost no fluorescence is observable after incubatingthe nascent and poly(TMSPA)-grafted membranes with FL-ConA (Fig. 5a and b). For the membrane with glycosyl density of0.583 �mol/cm2, slight green fluorescence can be detected, whichis attributed to fluorescent probe on FL-Con A adsorbed on themembrane surface (Fig. 5c). When the glycosyl density furtherincreases to 1.50 �mol/cm2, the fluorescent intensity enhancesnotably on the membrane surface and bright green signal canbe observed. It indicates a large amount of Con A has beenadsorbed on the membrane surface. In contrast, the adsorptionof FL-PNA is minor on the membranes surface (Fig. 5e and f).Therefore, this glycosylated membrane has been expected asaffinity membrane, which has high specific recognition capabil-ity to Con A. In addition, the glycosylated membrane is of greatpotential to develop biomaterials for bioengineering because thethiol–yne “click” chemistry employs a promising “metal-free” reac-tion [37,38].

3.5. Microfiltration of lectin by the glycosylated membranes

Affinity membrane chromatography has been proven tobe a promising technology for high-performance protein sep-aration/purification [39]. Usually, the filtration performanceis the direct evidence for evaluating its practice usabil-ity.

The filtration performance of the glycosylated membrane wasinvestigated using FL-Con A and FL-PNA as model proteins. Fig. 6shows the results of specific adsorption and separation of bothlectins on the studied membranes. It can be seen that, the FL-Con A breakthrough curve is similar with that of FL-PNA forthe nascent membrane. The concentration of effluent increasessharply at the first fraction, which means a negligible pro-tein binding on the membrane surfaces. For the glycosylatedmembrane with a glycosyl density of 1.50 �mol/cm2, the break-through curve is definitely different for both lectins. As expected,large amounts of FL-Con A are adsorbed on the glycosylatedmembrane by specific interaction between glucose and Con A.Therefore, the corresponding concentration of effluent is low atthe first fraction. When the effluent time exceeds 80 min, theeffluent concentration rapidly increases and then levels off dueto saturation of the affinity adsorption. Besides, the glycosy-lated membrane adsorbs little amount of FL-PNA on the surface,which is due to the weak affinity between the glucose and PNA[40].

Desorption is a crucial step of affinity membrane chromatog-raphy for extracting the target protein. The efficient recovery ofaffinity membrane is extremely important. Various elution con-

ditions for Con A have been investigated in the previous report[41,42]. Up to now, the glucose solution has been the best com-petitive eluant for Con A. However, it cannot completely elute theadsorbed Con A from the glycosylated membrane. The is mainly

110 C. Wang et al. / Colloids and Surfaces B: Biointerfaces 110 (2013) 105– 112

Fig. 5. Fluorescence images of the membrane surfaces after interaction with FL-Con A (a–c) and FL-PNA (e–g); (a, e) the nascent membrane, (b, f) TMSPA modified(GD = 1.55 �mol/cm2), (c, g) glycosylated (glycosyl density = 0.583 �mol/cm2), (d, h) glycosylated (glycosyl density = 1.50 �mol/cm2).

aaHl

ttributed to the existing glycoside “cluster” effect between Con And the immobilized carbohydrate ligand [16]. In this study, 1 MAc solution has been chosen to elute Con A from the glycosy-

ated membrane. As is reported before, this solution is a desired

hosting solution for proteins [43]. As shown in Fig. 7, the adsorbedCon A can be slightly desorbed at the first fraction. Thereafter,the majority of Con A is gradually eluted from the glycosylatedmembrane surface. It indicates that this glycosylated membrane

C. Wang et al. / Colloids and Surfaces B: B

0 15 30 45 60 75 90 10 5 12 0 13 5 15 0

0.0

0.2

0.4

0.6

0.8

1.0

MPPM -Con A

MPPM-Glu-Con A

MPPM-P NA

MPP M-G lu-PNA

C/C

0

Time (min)

(a)

Fig. 6. Breakthrough curves for lectin binding on the membranes: FL-Con Aadsorbed on the nascent membrane (�) and the glycosylated membrane with gly-cosyl density of 1.50 �mol/cm2 (�); FL-PNA adsorbed on the nascent membrane(♦) and the glycosylated membrane with glycosyl density of 1.50 mol/cm2 (�),respectively.

0 3 6 9 12 15 18 21 24 27 30 33 36

0

2

4

6

8

10

C/C

0

Time (min)

FH

pA

4

scbgchsAtrmtfab

[

[

[

[

[

[

[

[

[

[

[

[

[

ig. 7. Elution curve of Con A adsorbed on the glycosylated membrane with 1 MAc solution.

ossesses excellent separation and regeneration capability for Con.

. Conclusion

A straightforward strategy has been developed for the con-truction of glycosylated membrane surface by thiol–yne “click”hemistry. First, the alkyne groups are introduced on the mem-rane surface by plasma pretreatment combining with UV-inducedrafting. Then, the robust thiol–yne “click” chemistry is directlyarried out to fabricate the glycosylated membrane surface withigh glycosyl density. The static adsorption experiment demon-trates that the glycosylated membrane can specifically adsorb Con

rather than PNA. Meanwhile, the breakthrough curves shows thathe membrane has strong specificity, high adsorption capacity, andeversible binding capability to Con A. In addition, the glycosylatedembrane can be regenerated with 1 M HAc solution. All these jus-

ify that the improved glycosylation strategy is effective and theabricated glycosylated membrane is of great potential for lectinffinity separation/purification based on the specific interactionetween the carbohydrate ligand and the target lectin.

[

[

iointerfaces 110 (2013) 105– 112 111

Acknowledgements

The authors thank a financial support from the National NaturalScience Foundation of China (Grant No. 50933006) and the NationalBasic Research Program of China (2009CB623401).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2013.04.029.

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