Colloids and Surfaces B: Biointerfaces 109 (2013) 176– 182
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Colloids and Surfaces B: Biointerfaces
jou rn al hom epage: www.elsev ier .com/ locate /co lsur fb
ioinspired surface modification of poly(2-hydroxyethylethacrylate) based microbeads via oxidative polymerization of
opamine
atma Yilmaza, Kazim Kosea, Mufrettin Murat Sari a,b, Gokhan Demirel c, Lokman Uzuna,∗,dil Denizli a
Hacettepe University, Department of Chemistry, Biochemistry Division, Ankara, TurkeyTurkish Military Academy, Department of Basic Sciences, Ankara, TurkeyGazi University, Department of Chemistry, Bio-inspired Materials Research Laboratory (BIMREL), Ankara, Turkey
a r t i c l e i n f o
rticle history:eceived 17 July 2012eceived in revised form 14 March 2013ccepted 19 March 2013vailable online 3 April 2013
Surface modification of support materials is crucial for improving their selectivities and biocompatibil-ities in bioaffinity applications. However, conventional modification techniques including chemical orphysical conjugations mostly suffer from limitations of their multistep and complicated procedures, sur-face denaturations, batch-to-batch inconsistencies, and insufficient surface conjugations. In this study,we demonstrate a simple yet effective bioinspired approach for the surface modification of poly(2-hydroxyethyl methacrylate) [PHEMA] based bioaffinity adsorbents through oxidative polymerization ofdopamine. The magnetic (mPHEMA) and non-magnetic (PHEMA) polymeric microbeads were fabricatedby suspension polymerization technique. Surface modification of obtained microbeads was then car-ried out by using dopamine molecules under alkaline conditions. The polydopamine (PDOPA) coated
opamineatechol
microbeads were further employed as a bioaffinity absorbent targeted for immunoglobulin G (IgG)molecules. The effects of pH, temperature, protein concentration and ionic strength on the IgG adsorptionprocess have been investigated. We found that PDOPA coated microbeads display dramatically higher IgGadsorption capacities when compared with their un-modified forms. Adsorption capacities also increasedwith increasing temperature. Monolayer Langmuir adsorption model can be thought more applicable for
these adsorbent systems.
. Introduction
Development of novel strategies for biomaterial surface modi-cations is essential for a variety of applications including medicalevices, biosensors, tissue engineering scaffolds and bioaffin-
ty platforms due not only to prevent undesirable non-specificnteractions between biomolecules and biomaterials but alsoor improving their mechanical and thermal stabilities [1–3].ecently, various innovative strategies based on environmentalreatment, covalent chemical modification, and physical adsorp-ion, have been attempted for the modification of biomaterialurfaces [4–6]. However, most of these methods have severalrawbacks such as requiring multistep and complicated pro-edures or prior functionalization via chemical treatments or
lasma. For example, Liu et al. reported a controllable modificationrocess for polyamide surfaces by photo-induced graft polymer-
zation [4]. In another study, poly(tetrafluoroethylene) films were
modified via plasma treatment and following acrylic acid graft-ing [5]. Hu et al. also demonstrated the surface modification ofpoly(dimethylsiloxane) microfluidic devices by ultraviolet poly-mer grafting technique [6]. Although surface modifications weresucceeded above mentioned works, treatments or chemicals usedfor surface activation can induce surface denaturation or lead tolow surface conjugation. Therefore, new strategies for the effectivesurface modification of biomaterials are still needed. The poly-dopamine (PDOPA) based surface modification inspired by marinemussels is a simple but versatile material-independent approachand has recently attracted considerable attention for a diversearray of applications including catalysis, biomolecule immobi-lization, tissue engineering, metallization, and bio-mineralization[7–10]. This material-independency is mainly due to the uniquechemical structure of dopamine (DA) molecule. The DA has ortho-dihydroxyphenyl, catechol, and amine functional groups, whichmay polymerize under alkaline condition. During polymeriza-
tion, catechol groups oxidize to quinone. The catechols/quininesgroups also exhibit capability of covalently binding to nucleo-philes without using any activator or further functionalization[9,10].
Human serum contains numerous proteins along with theirleaved or modified forms and plays a crucial role in clinicalreatments and biotechnological processes [11]. One of the mostbundant proteins in human serum is the human immunoglob-lins, especially G isotype (IgG), which are a group of functionallycoproteins. They can be used both at low dose as a substitu-ive therapy for patients with primary [12,13] or secondary [14]mmunodeficiencies, and at high dose as an immunomodulatorygent [15] for treatment of autoimmune [16], hematologic [17],nd neurologic diseases [18]. The growing need of human IgG atufficient purity for therapeutic, diagnosis, or immunochemicalpplications, has been the basis for the development of variety ofethods including chromatographic and adsorption based separa-
ion, ion-exchange membranes, immobilized metal-chelate affinitylatforms, and molecular sieving techniques [19]. Among them,ffinity chromatography using protein A or protein G molecules ishe most commonly method used for separation of immunoglobu-ins, including human IgG and their subclasses [20]. In spite of theirigh purity, good selectivity, and recovery, protein A carrying affin-
ty adsorbents have some drawbacks including high cost of process,ossible contamination from leached protein A, and drastic dena-uring condition required for elution of IgG. Furthermore, it is stillhallenging to immobilize protein A in the proper orientation ontohe solid substrates [21]. These restrictions have stimulated theevelopment of other separation techniques and new adsorbentsith affinity, specificity, and selectivity under mild conditions toreserve its integrity and activity.
Herein, we reported a novel bioaffinity adsorbent for efficientgG adsorption based on bioinspired PDOPA coating onto poly-
eric microbeads. The polymeric supports both magnetic andon-magnetic forms have been synthesized through suspensionolymerization technique using HEMA as a monomer and ethylenelycol dimethacrylate (EGDMA) as a cross-linker and subsequenturface functionalization of their surfaces with PDOPA coating. Theerformance of prepared adsorbents were then evaluated for selec-ive IgG adsorption investigating the effects of time, initial IgGoncentration, pH and temperature. We demonstrated that PDOPAoated microbeads may be utilized as a new generation of bioaffin-ty platforms for enhanced efficiency of biomolecule separation.
. Experimental
.1. Materials
Human-immunoglobulin G (IgG), dopamine hydrochloride,8H11NO2.HCl, l-ascorbic acid, C6H8O6, 2,2-azobisisobutyronitrileAIBN), poly(vinyl alcohol) (PVAL) and magnetite nanopow-er (Fe3O4, average diameter: 20–50 nm) were obtained fromigma–Aldrich (St. Louis, MO, USA). Hydroxyethyl methacrylateHEMA) and ethylene glycol dimethacrylate (EGDMA) were alsoupplied from Fluka A.G. (Buchs, Switzerland) and distilled undereduced pressure in the presence of hydroquinone inhibitor beforese. All other chemicals were of reagent grade and purchased fromerck A.G. (Darmstadt, Germany). Double distilled deionized wateras used to prepare all the solutions. All the water used in the
xperiments was purified using a Barnstead (Dubuque, IA, USA)Opure LP® reverse osmosis unit with a high flow cellulose acetateembrane (Barnstead D2731) followed by a Barnstead D3804ANOpure® organic/colloid removal and ion-exchange packed-ed system.
.2. Methods
.2.1. Preparation of PHEMA based microbeadsThe polymeric microbeads were synthesized via suspension
olymerization technique using HEMA, AIBN, toluene, EGDMA and
iointerfaces 109 (2013) 176– 182 177
PVAL as the monomer, initiator, pore former, cross-linker and thestabilizer, respectively [22]. Briefly, the dispersion medium wasfirst prepared by dissolving 200 mg of PVAL within 50 mL of dis-tilled water and 50 mg of AIBN was dissolved in a second phaseincluding monomers EGDMA/HEMA/toluene as volumetric ratio as8:4:12. The monomer solution was then transferred into the dis-persion medium placed in a glass polymerization reactor (100 mL)that was placed in a thermostatic water bath and mixed at 600 rpmstirring rate. In the case of the preparation of magnetic microbeads,1.0 g of magnetite was added into the polymerization mixture. Thereactor was flushed by bubbling nitrogen and then was sealed. Thepolymerization was allowed to proceed under nitrogen atmosphereat 65 ◦C for 4 h and termination was occured at 90 ◦C for 2 h. Finally,soluble residuals were removed from the polymer by repeateddecantation with water and ethyl alcohol. Synthesized microbeadswere washed several times with ethyl alcohol and water to removeany unreacted monomer or diluents and stored in distilled waterat 4 ◦C.
2.2.2. PDOPA coating onto microbeadsThe PDOPA modification of the microbeads was carried out
through oxidative self-polymerization of dopamine molecules[2,23]. In a typical experiment, the microbeads (2 g) were placedinto a dopamine solution (2 mg/mL, 10 mM, pH 8.5) for 30 min withgenerous shaking. Afterwards, the PDOPA coated microbeads werecentrifuged (5000 rpm, 5 min) and washed with fresh buffer solu-tion to remove the colored solution until the supernatant remainedcolorless.
2.2.3. Characterization of the microbeadsThe chemical and physical properties of the microbeads were
characterized by using various techniques. The average sizes andsize distributions of the microbeads were determined by screenanalysis through Tyler Standard Sieves. Water uptake ratios of themicrobeads were also determined in distilled water. The exper-iments were conducted as follows: the dried microbeads werecarefully weighed (Wo) before being placed in a 10 mL vial con-taining distilled water. The vial was put into an isothermal waterbath with a fixed temperature (25 ± 0.5 ◦C) for 2 h. The microbeadswere then removed from the water, wiped using a filter paper, andweighed. The water contents of the microbeads were calculated byusing the following expression:
Water uptake ratio % =[
(Ws − Wo)Wo
]× 100 (1)
where Wo and Ws are the weights of microbeads before and afteruptake of water (g), respectively. The surface morphologies of themicrobeads were examined using a scanning electron microscopy(SEM, JEOL, JM5 5600, Japan). Prior to analysis, the samples wereinitially dried in air at 25 ◦C for 7 days. A small amount of the driedmicrobeads was then mounted on a SEM holder and coated witha gold layer using sputter coater for 2 min. To analyze the chemi-cal characteristics of the microbeads, a Fourier transform infraredspectrophotometer (FTIR, PerkinElmer, Spectrum One, USA) wasalso used. Thermogravimetric analysis (TGA, Shimadzu DTG-60,Tokyo, Japan) was performed in a nitrogen environment using a10 mg of dried beads and heated from room temperature to 500 ◦C.
2.2.4. Bioaffinity experimentsIn this part of study, the microbeads were interacted with aque-
ous IgG solutions by means of rotator. Briefly, a 50 mg of themicrobeads were incubated with a 10 mL of IgG solution having
varied concentration (0.1–2.0 mg/mL) under stirring at 150 rpm for2 h. Effects of interaction time, initial IgG concentration, pH andtemperature on the adsorption capacity were then evaluated. ThepH of the medium adjusted by using acetate and phosphate buffer
178 F. Yilmaz et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 176– 182
EMA;
sTboTatiifdtbT
Fig. 1. SEM micrographs of (a) PHEMA; (b) mPH
ystems in own buffering ranges was varied in the range of 4.0–8.0.he adsorbed IgG concentration was spectroscopically determinedy measuring the absorbance of solutions at 280 nm. The amountf adsorbed IgG was calculated using appropriate mass balances.he desorption of IgG molecules from microbeads were also evalu-ted using 0.1 M glycine-HCl buffer (pH 3.5) to show reusability ofhe microbeads. In order to evaluate the efficiency of the desorb-ng agent and whether any denaturation problems was occurredn biomolecule, we performed spectrofluorimetic measurementsor the solutions including native IgG molecules in loading and
esorbing buffers; desorbed solution; denatured with tempera-ure or chemical treatment. The measurements were performedy using Shimadzu RF53010 spectrophotometer (Tokyo, Japan).he emission spectra were recorded in a range of 300–900 nm
(c) PDOPA@PHEMA; and (d) PDOPA@mPHEMA.
when excitations were applied at 293 nm that was optimal valuefor IgG molecules. Other experimental parameters were appliedas slit width was 5.0 nm for both excitation and emission, scanspeed was super, sensitivity was high and response time andshutter were automatically controlled. In addition, the desorptionefficiency were evaluated by measuring amount of IgG in desorp-tion medium and calculating the ratio according to the equation,�R% = [(Cdesorbed × V/Qadsorbed × m) × 100%], where �R, desorptionratio (%); Cdesorbed, amount of IgG in medium (mg/mL); V, volumeof desorbing agent (mL); Qadsorbed, adsorption capacity (mg/g); m,
weight of microbeads (g). For each set of data, standard statisti-cal methods were used to determine the mean value and standarddeviations. Confidence intervals of 95% were calculated for each setof samples in order to determine the margin error.
es B: Biointerfaces 109 (2013) 176– 182 179
3
3
asaaaimpaasssmofm4ictmiFps1gh
bgts
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Fig. 3, there were relatively faster adsorption rates observed at thebeginning, and then saturation was observed gradually in about60 min. This may be due to a high driving force, which is the IgG
F. Yilmaz et al. / Colloids and Surfac
. Results and discussion
.1. Characterization of microbeads
In this study, we mainly focused on to prepare a novel bioaffinitydsorbents combining with HEMA based microbeads and bioin-pired PDOPA coating for the affinity adsorption of IgG fromqueous solutions. The PDOPA coating on the microbeads werechieved by oxidative polymerization of dopamine molecules inlkaline medium. After treatment, the surface coating can be eas-ly distinguished by color change of the microbeads (Fig. 1). As
entioned before, the microbeads were prepared by suspensionolymerization which may be caused by a size distribution. Theverage size of microbeads, both PHEMA and mPHEMA, wereround 100–150 �m (Fig. 1a–d). The microbeads, which wereynthesized, have crosslinked polymeric chains. Therefore, theywell when interacting with solvent instead of dissolution. Thewelling abilities of the microbeads (PHEMA, PDOPA@PHEMA,PHEMA, PDOPA@mPHEMA) were also determined under aque-
us conditions. The swelling ratios were determined as 46%or PHEMA, 49% for mPHEMA. After PDOPA coating onto the
icrobeads, these values decreased to 42% for PDOPA@PHEMA,3% for PDOPA@mPHEMA, respectively. Although PDOPA coat-
ng improves the hydrophilicities of microbeads, which would beaused an increment into the swelling ratios, however, it is clearhat, the pores into the microbeads were closed during the poly-
erization of dopamine. Thus, water molecules cannot penetratento the microbeads which causing a decrease in the swelling ratios.TIR spectra of microbeads were also collected and given in Sup-lementary informations (Fig. SI-1). They showed common bandstemmed from HEMA groups around 3440 cm−1, 2930 cm−1 and730 cm−1 related to OH, aliphatic C H and C O functionalroups, respectively. Additionally, PDOPA modified microbeadsave vibration bands at 1635 cm−1 and ∼1390 cm−1 (aromaticC C stretching), 1197 cm−1 (aromatic C OH out-of-planeending), and 963 cm−1 (aliphatic C C N stretching of amineroups of PDOPA), respectively, as reported in the related litera-ure [24]. The FTIR spectra confirm that PDOPA accumulation wasucceeded onto the microbeads.
The SEM images of the microbeads were shown in Fig. 1. Fig. 1and b show that the microbeads were almost spherical and had
smooth surface. Their average diameters were also calculatedo be 100–150 �m. After PDOPA coating, the surface rough-esses increased meanwhile spherical form of the microbeadsas retained. The increase in surface roughness causes increase
n external surface of the microbeads. PDOPA coating onto theicrobeads was also confirmed with thermal gravimetric analy-
is (TGA). As seen in Fig. 2, decomposition of the microbeads havetarted at around 300 ◦C and completed at about 500 ◦C. It is clearhat PDOPA coating onto both magnetic and non-magnetic PHEMA
icrobeads increased the thermal stability and resistance. In allases, decomposition temperature increases about 50 ◦C.
.2. IgG Adsorption studies
.2.1. Adsorption rateFor the determination of IgG adsorption kinetics and adsorp-
ion efficiencies of synthesized beads, various incubation durationhanged from 5 to 120 min have been investigated in 0.5 mg/mLnitial IgG concentration at room temperature. It is known thathe factors, which influence the interactions between biological
aterials and solid supports such as adsorption rate, pH, initial
oncentration and temperature, play an important role for possiblesage in biotechnological applications. Adsorption characteristicsnd kinetic behaviours are also dependent on the solid and liquid-hase diffusivity, boundary layer mass transfer and internal mass
Fig. 2. Thermal gravimetric analysis profiles (TGA) of the microbeads.
transfer effect [25]. Mainly, three general steps are defined for theexplanation of adsorption process: (i) diffusion of protein from thesolution to the surface of the media, (ii) adsorption at the surfaceof the media, and (iii) diffusion inside the media. In principle, anyof these steps may be controlled via manipulating of the abovementioned factors [26]. Equilibrium adsorption time curves, whichobtained by following the decrease of the concentration of IgGwithin the samples with desired time intervals, have been plottedin Fig. 3. It is apparent that the non-specific adsorption, whether onPHEMA or mPHEMA microbeads, was relatively low due to the factthat both microbeads do not contain functional group for specificIgG binding. Conversely, PDOPA@PHEMA and PDOPA@mPHEMAhave exhibited higher IgG adsorption capacities. Although the non-specific adsorption may be only due to the diffusion of IgG tothe pores and weak interactions between IgG molecules and themicrobeads, specific IgG adsorption on PDOPA coated microbeadswas occurred through specific interaction between cathecol groupsand glycoprotein moieties of IgG molecules. As also seen from
Fig. 3. Adsorption rates of IgG on microbeads. IgG concentration: 0.5 mg/mL; pH:6.0; T: 25 ◦C. Each result is the average calculated in 95% confidence interval of threeparallel studies.
180 F. Yilmaz et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 176– 182
F09
ctI
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ismsmoPcwbivpihtIv
3
mcmwivbAmtsatwc
Fig. 5. Effect of initial IgG concentration on IgG adsorption capacities of themicrobeads. Contact time: 120 min; pH: 6.0; T: 25 ◦C. Each result is the average
ig. 4. Effect of pH on the IgG adsorption on the microbeads. IgG concentration:
.5 mg/mL; contact time: 120 min; T: 25 ◦C. Each result is the average calculated in5% confidence interval of three parallel studies.
oncentration difference between the liquid (i.e., the aqueous solu-ion) and the solid (i.e., the microbeads) phases, in the case of highgG concentration.
.2.2. Influence of pH on adsorptionAcidity of the medium generally affects the protein-ligand
nteractions because of the total charge, solubility, chemicaltability and spatial arrangement of which is a function of pri-ary, secondary, tertiary and quaternary structure of proteins are
trongly depend on it. The pH-dependency of IgG adsorption ontoicrobeads was performed in the pH range of 4.0–8.0 (Fig. 4). We
bserved that maximum IgG adsorption onto PDOPA@PHEMA andDOPA@mPHEMA microbeads was observed at pH 6.0 for bothases, which is near to the isoelectric point of IgG (pH 6.2). Theell-known explanation for this fact is the zero net charge of
iomolecules at their isoelectric point. In this respect, unfavorablenteractions depending on opposite charges were limited at this pHalues; so on, the maximum adsorption is mostly observed at thisoint. Another explanation is that the cis-diols of cathecol groups
s most favorable at this point. At higher and lower pH values, theyave some electrostatic repulsion forces depending on their depro-onation and redox activities. It should be noted that non-specificgG adsorption is independent of pH and it can be occured at all pHalues.
.2.3. Effect of initial IgG concentration and adsorption isothermThe concentration dependency of IgG adsorption behavior of the
icrobeads was investigated to determine the optimal IgG con-entration range to be studied. The IgG adsorption capacities of theicrobeads were given as a function of the initial IgG concentrationithin the aqueous phase in Fig. 5. The initial IgG concentration was
ncreased up to 2.0 mg/mL in order to clearly observe the plateaualues, which represent saturation of the active sites on the adsor-ent, in other terms to obtain the maximum adsorption capacity.s presented in the Fig. 5, the amount of IgG adsorbed per unitass of the microbeads linearly increased first (up to 0.5 mg/mL);
hen started to decline and reached to platue value that repre-ented saturation of the active binding sites which are available
nd accessible for IgG on the microbeads. From starting concen-ration, adsorption capacity showed a significant increase, whichas indicative of high affinity between the IgG molecules and
athecol groups of dopamine molecules. Thus, IgG adsorption is
calculated in 95% confidence interval of three parallel studies.
favored at a higher initial concentration. This was obvious becausemore efficient utilization of the binding capacities of the beadswas expected on account of a greater driving force with a higherconcentration gradient. The binding of IgG reached a saturationlevel at a bulk concentration, that is, higher than the concentra-tion value of 1.0 mg/L. This phenomenon means that the all bindingsites on the PDOPA modified microbead surfaces have been occu-pied. This increase in the IgG coupling capacity may have resultedfrom especially specific interaction between cathecol group on thePDOPA and IgG molecules. The data also shows that compared withthe negligible amount of non specific IgG adsorption only resultedfrom the diffusion of IgG molecules into the swollen PHEMA basedmicrobeads.
During the batch experiments, one of the main requirements forthe explanation of protein-ligand binding and for the evaluation ofadsorption isotherms is the determination of suitability of theo-retical isotherm which fits the experimental data. This informationprovides a relationship between the concentration of protein inthe solution and the amount of protein adsorbed on the solid-phasewhen the two phases are at equilibrium [27]. The Langmuir adsorp-tion isotherm is expressed by Eq. (2). Langmuir adsorption modelassumes that the molecules are adsorbed at a fixed number of well-defined sites each of which can only hold one molecule. Thesesites are also assumed to be energetically equivalent and distantto each other so that there are no interactions between moleculesadsorbed to adjacent sites [28,29]. The corresponding transforma-tions of the equilibrium adsorption data for IgG gave rise to a linearplot, indicating that the Langmuir model could be applied in thissystem.
Qeq = QmaxCeq
b + Ceq(2)
where Qeq is the adsorbed amount of IgG (mg/g), Ceq is the equi-librium IgG concentration in solution at equilibrium (mg/mL), b isLangmuir constant (mL/mg), and Qmax is the theoretical maximumIgG capacity (mg/g). This equation can be linearized into the formexpressed by Eq. (3).
Ceq[
1] [
1]
Qeq=
Qmaxb+
Qmax.Ceq (3)
Another isotherm generally used for explaining the adsorp-tion processes is Freundlich model. The isotherm assumes that the
F. Yilmaz et al. / Colloids and Surfaces B: B
Fig. 6. Effect of temperature on IgG adsorption onto the microbeads. Contact time:1i
aiii
l
1t
wpclstfiaawsiPactmsashim
3
tttsta
emission spectra because of unfolding and irreversible denatur-
20 min; IgG concentration: 0.5 mg/mL; pH: 6.0. Each result is the average calculatedn 95% confidence interval of three parallel studies.
dsorption process occurred by forming multiple adsorption layersncluding interaction between biomolecules adsorbed and retainedn solution [28]. The linearized forms of the equations for bothsotherms can be given as:
n Qeq = ln KF + 1n
. ln Ceq (4)
Additional parameters, KF and 1/n, are Freundlich constants and/n shows the heterogeneous distribution of interaction points onhe surface.
Linear representations of Langmuir and Freundlich equationsere shown in Supplementary Informations (Fig. SI-2). Some modelarameters (Langmuir and Freundlich adsorption constants andorrelation coefficients) for IgG adsorption process were calcu-ated from the curves with commercially available software andummarized in Table 1. In the light of the comparison of theheoretical approaches, Langmuir isotherm model was the bestt to the experimental data and hence, was found to be morepplicable for IgG adsorption onto, especially, PDOPA@PHEMAnd PDOPA@mPHEMA microbeads. The plot of Ceq versus Ceq/Qeq
as employed to generate the intercept of 1/Qmax·b and thelope of 1/Qmax. The theoretical maximum IgG adsorption capac-ty (Qmax) was obtained from experimental data as 4.37 mg/g forHEMA, 63.08 mg/g for PDOPA@PHEMA, 6.31 mg/g for mPHEMA,nd 75.19 mg/g for PDOPA@mPHEMA, respectively. The correlationoefficient (R2) for Langmuir type isotherm model is much higherhan Freundlich type and indicating that the Langmuir adsorption
odel is better fitted to these systems. Moreover, Table 1 alsohows the Freundlich adsorption isotherm constants, 1/n and KFnd the correlation coefficients. The magnitude of KF and 1/n valueshowed easy uptake of IgG molecules from aqueous medium with aigh affinity. The 1/n values are lower than 1, closer to zero, which
ndicates monolayer adsorption is dominant similar to Langmuirodel.
.2.4. Effect of temperature on IgG adsorptionTo evaluate the effect of temperature on IgG adsorption onto
he microbeads, IgG adsorption studies were performed at variousemperatures (5–37 ◦C) and results were given as a function of the
emperature in Fig. 6. As expected, adsorption process was quiteensitive to incubation temperature. Theoretically, increasing theemperature enhances protein retention and lowering the temper-ture generally promotes the protein elution [30]. Experimental
iointerfaces 109 (2013) 176– 182 181
results correspond to this prevision. With an increment in temper-ature from 4 to 37 ◦C, IgG adsorption capacities, especially PDOPAmodified microbeads, significantly enhanced. The probable rea-sons for the increasing of adsorption capacity in the presenceof hydrophobic interaction were reported as: (i) At higher tem-perature during unfolding process, the proteins expose buriedhydrophobic amino acid residues on the surface. Thus, the con-tact area between the protein and the ligand on the matrix shouldincrease, resulting in an increase in affinity of proteins for the adsor-bent at higher temperature; (ii) it is interesting to note that the vander Waals attraction forces, which operate in hydrophobic interac-tions, also increase with increase in temperature [31,32]. As a result,adsorption capacity increased due to the hydrophobic interactionforces. The data also indicate that the interactions between IgGmolecules and cathecol groups of PDOPA have mainly hydropho-bic nature besides the interaction between cis-diol of cathecol andglycoprotein moieties of IgG.
3.2.5. Desorption and repeated useThe recycling possibility of the affinity adsorbents is an
important criteria in the protein adsorption processes. Repeatedusability provides an unique property because it makes theadsorbent cost-friendly alternative for benchmark competitors. Inaffinity bioseparation, the desorption of adsorbed biomoleculesshould be achieved in the shortest time to prevent denatur-ation problem and as highest amount as possible. To verify therecycling efficiency of the microbeads synthesized in this study,adsorption–desorption cycles were repeated ten times using thesame microbeads. For this purpose, 0.1 M glycine-HCl buffer (pH3.5) was used for the desorption of IgG molecules adsorbed ontothe microbeads. The microbeads were placed within the desorp-tion buffer at room temperature for 1 h (Fig. 7a). We found that theadsorption capacities of PDOPA@PHEMA and PDOPA@mPHEMAmicrobeads did not noticably decreased. After tenth cycle,they decreased only 15.8% for PDOPA@PHEMA and 11.3% forPDOPA@mPHEMA, respectively. In all cycle, desorbing rate washigher than 97.0%. It could be emphasized that PDOPA modifiedmicrobeads can be used for repeated IgG adsorption–desorptionprocesses by using glycine-HCl buffer (pH 3.5) as desorbingagent.
Evalution of that whetether there were any denaturationproblems during desorption process was performed via spec-trophotometry. For this aim, we carried out the measurementsfor seven different solutions: native IgG solution in loading buffer(PBS, pH: 6.0), native IgG solution in desorbing agent (glycine-HCl, pH: 3.5), desorption solution, IgG solution treated at 40 ◦C for1 h, IgG solution treated at 80 ◦C for 1 h, denaturated IgG solutiontreated with ethanol (1:1, v:v), denaturated IgG solution treatedwith sodium azide. As seen in Fig. 7b, native IgG molecules havesimilar fluorescent emission spectra. In addition, the desorbed IgGsolution has similar spectrum with low intensity shift depend-ing on conformational changes of side residues in IgG molecules.For heat denaturated (@40 ◦C) IgG solution, the intensity shiftwas higher than the shift for desorption solution. The increase intemperature of heat denaturation process caused more intensityshift in emission spectrum. The chemical treatment caused muchmore increased in emmision peak intensity. Two possible changescould be occurred in the emisison spectra of biomolecules dur-ing denaturation process: (i) the lost of emission band because ofquenching of emitting groups and (ii) much more increase in the
ation of biomolecules [29]. The results indicated that the desorptionagent did not cause any irreversible denaturation problem in IgGmolecules and appropriate for masking/distributing the interac-tions between PDOPA and IgG molecules.
182 F. Yilmaz et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 176– 182
Table 1Adsorption isotherm parameters of the microbeads for IgG adsorption.
Fig. 7. Desorption of IgG from PDOPA modified microbeads. (a) Repeated use ofPDOPA modifed microbeads. Contact time: 120 min; IgG concentration: 0.5 mg/mL;pEs
4
tfbadabpsi
[[[[[
[
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937–944.
H: 6.0; T: 25 ◦C. (b) Spectrofluorimetry characterization of desorbed IgG molecules.ach result is the average calculated in 95% confidence interval of three paralleltudies.
. Conclusion
The time consumption, high cost and extensive purifica-ion steps of well-known purification methods for separation ofunctional molecules such as enzymes, coenzymes, cofactors, anti-odies, amino acid derivatives, oligopeptides, proteins, nucleiccids, and oligonucleotides have inspired researchers for theevelopment of alternative and suitable methods including rapidpplicable and low-cost adsorbents. In this study, a novel
ioinspired surface modification technique based on oxidativeolymerization of dopamine molecules was reported to modify theurface of plain microbeads and their potential usage as an bioaffin-ty adsorbent for specific IgG adsorption. The most important
advantage of using the PDOPA modified microbeads is eliminationof surface activation and ligand immobilization steps, which makethe adsorbent more cost-friendly. In the light of the experimentalresults, we found that the PDOPA modified microbeads provide arapid but efficient separation for IgG molecules via cathecolic adhe-sion. The results also demonstrate the existence of good adsorptiveproperties for the microbeads such as cost effectiveness, enoughmechanical rigidity, thermal stability, effective separation abilityfrom reaction medium after adsorption, and provide a workingopportunity at wider pH and temperature range. The experimentaldata were well-fitted to the Langmuir model.
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.2013.03.041.
References
[1] H. Lee, K.D. Lee, K.B. Byo, S.Y. Park, H. Lee, Langmuir 26 (2010) 3790–3793.[2] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Science 318 (2007)
426–430.[3] J.H. Waite, Integr. Comp. Biol. 42 (2002) 1172–1180.[4] N. Liu, G. Sun, S. Gaan, P. Rupper, J. Appl. Polym. Sci. 116 (2010) 3629–3637.[5] H.X. Sun, L. Zhang, H. Chai, H.L. Chen, Desalination 192 (2006) 271–279.[6] S. Hu, X. Ren, M. Bachman, C.E. Sims, G.P. Li, N. Allbritton, Anal. Chem. 74 (2002)
4117–4123.[7] A. Zhang, J.L. Neumeyer, R.J. Baldessarini, Chem. Rev. 107 (2007) 274–302.[8] Y. Lin, M. Yin, F. Pu, J. Ren, X. Qu, Small 7 (2011) 1557–1561.[9] H. Lee, Y. Lee, A.R. Statz, J. Rho, T.G. Park, P.B. Messersmith, Adv. Mater. 20 (2008)
1619–1623.10] H. Lee, J. Rho, P.B. Messersmith, Adv. Mater. 21 (2009) 431–434.11] G. Francisco, C. Hernandez, R. Simo, Clin. Chim. Acta 369 (2006) 1–16.12] A. Elkak, S. Ismail, L. Uzun, A. Denizli, Chromatographia 69 (2009) 1161–1167.13] P. Mathew, M.E. Conley, Pediatr. Allergy Immunol. 6 (1995) 91–94.14] H.M. Chapel, M. Lee, R. Hargreaves, D.H. Pamphilon, A.G. Prentice, Lancet 343
(1994) 1059–1063.15] C. Ibanez, P. Sune, A. Fierro, S. Rodriguez, M. Lopez, A. Alvarez, J. De Gracia, J.B.
Montoro, BioDrugs 19 (2005) 59–65.16] C. Larroche, Y. Chanseaud, P. Garcia de la Pena-Lefebvre, L. Mouthon, BioDrugs
16 (2002) 47–55.17] A. Otten, P.M. Bossuyt, M. Vermeulen, A. Brand, Eur. J. Haematol. 60 (1998)
73–85.18] B.M. Koffman, M.C. Dalakas, Muscle Nevre 20 (1997) 1102–1107.19] G. Tishchenko, J. Dybal, K. Meszarosova, Z. Sedlakova, M. Bleha, J. Chromatogr.
A 954 (2002) 115–126.20] S.P. Bottomley, B.J. Sutton, M.G. Gore, J. Immunol. Methods 182 (1995) 185–192.21] L. Uzun, D. Turkmen, V. Karakoc, H. Yavuz, A. Denizli, J. Biomater. Sci. 22 (2011)
2335–2341.22] N. Basar, L. Uzun, A. Guner, A. Denizli, Int. J. Biol. Macromol. 41 (2007) 234–242.23] A. Postma, Y. Yen, Y. Wang, A.N. Zelikin, E. Tjipto, F. Caruso, Chem. Mater. 21
(2009) 3042–3044.24] A. Lagutschenkov, J. Langer, G. Berden, J. Oomens, O. Dopfer, Phys. Chem. Chem.
Phys. 13 (2011) 2815–2823.25] P.R. Wright, F.J. Muzzio, B.J. Glasser, Biotechnol. Prog. 14 (1998) 913–921.26] S. Zhang, A. Tanioka, K. Saito, H. Matsumoto, Biotechnol. Prog. 25 (2009)
1115–1121.27] M.M. Sari, C. Armutcu, N. Bereli, L. Uzun, A. Denizli, Colloids Surf. B 84 (2011)
140–147.28] B. Akkaya, L. Uzun, F. Candan, A. Denizli, Mater. Sci. Eng. C 27 (2007) 180–187.29] Y. Saylan, M.M. Sari, S. Ozkara, L. Uzun, A. Denizli, Mater. Sci. Eng. C 32 (2012)
30] S. Hjerten, K. Yao, K.O. Eriksson, B. Johansson, J. Chromatogr. 359 (1986) 99–109.31] S. Oncel, L. Uzun, B. Garipcan, A. Denizli, Ind. Eng. Chem. Res. 44 (2005)
7049–7056.32] L. Uzun, R. Say, S. Unal, A. Denizli, J. Chromatogr. B 877 (2009) 181–188.
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