Tunable adsorption of bovine serum albumin by annealed cationic spherical polyelectrolyte brushes

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OLSUB-5647; No. of Pages 6

Colloids and Surfaces B: Biointerfaces xxx (2013) xxx– xxx

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

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

hort Communication

unable adsorption of bovine serum albumin by annealed cationicpherical polyelectrolyte brushes

iyi Wanga,1, Kaimin Chena,b,1, A. Basak Kayitmazerc, Li Lia,∗, Xuhong Guoa,∗

State Key Laboratory of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, People’s Republic of ChinaMed-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, People’s Republic of ChinaChemistry Department, Bogazici University, Bebek, Istanbul 34342, Turkey

r t i c l e i n f o

rticle history:eceived 20 September 2012eceived in revised form 11 February 2013ccepted 15 February 2013vailable online xxx

a b s t r a c t

By combining turbidimetric titration, dynamic light scattering (DLS), and zeta potential methods, wedemonstrated that the adsorption of bovine serum albumin (BSA) in annealed cationic spherical poly-electrolyte brushes (SPB) can be controlled by modulating the pH, ionic strength, BSA concentration ofthe mixed solution, and SPB thickness. The SPB consist of a polystyrene core with a diameter around80 nm and a dense shell of poly (2-aminoethylmethacrylate hydrochloride) (PAEMH) with a thickness

eywords:anoparticlespherical polyelectrolyte brushrotein adsorptionurbidimetric titrationynamic light scattering

from 10 to 60 nm covalently attached on the core surface. Results revealed the existence of three pHregions, corresponding to (1) adsorption of BSA in SPB, (2) aggregation of SPB induced by BSA adsorption,and (3) desorption of BSA from SPB. The extent of the pH regions can be modulated by ionic strength, BSAconcentration, or SPB thickness. Adsorption measurements demonstrated that the adsorbed amount ofBSA in SPB was affected by pH, ionic strength, and SPB thickness. These findings lay the foundation forprotein separation by SPB.

. Introduction

Direct adsorption of proteins, enzymes, and antibodies withanoparticles has recently attracted many interests in biotech-ology [1,2]. Surface functionalized nanoparticles with adsorbedroteins are employed for applications such as drug deliv-ry, surface enrichment, biological labeling, and antibacterialncapsulation [3–5]. Among various nanoparticles, spherical poly-lectrolyte brushes (SPB) have been reported to be most suitableor immobilization of proteins in aqueous solutions without defor-

ation of native conformation and loss of biological function ofroteins [6,7]. SPB provide a stable environment for a highly effi-ient protein immobilization under controlled ionic strength andH. SPB allow the bound proteins to be released by changing the

onic strength of the medium [7]. Therefore, SPB can serve as tun-ble carriers for protein immobilization [8,9], which have potentialpplication in protein separation [10,11] and biological catalysis

Please cite this article in press as: S. Wang, et al., Tunable adsorption of bbrushes, Colloids Surf. B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.

12].Protein uptake on planar brushes have been observed and sum-

arized by de Vos et al. [13,14], Czeslik et al. [2,15,16], and Kusumo

∗ Corresponding authors.E-mail addresses: [email protected] (L. Li), [email protected]

X. Guo).1 These authors are contributed equally to this work.

927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2013.02.026

© 2013 Elsevier B.V. All rights reserved.

et al. [17] They found that the amount of BSA adsorbed was depend-ent on pH, ionic strength [18], protein concentration, and brushgrafting density. Adsorption of proteins on anionic spherical poly-electrolyte brushes was studied by Ballauff and his coworkers[7,19,20], who observed an unexpected phenomenon: adsorptionof acidic proteins onto anionic SPB took place on the “wrong”side of the isoelectric point of proteins (pI) as evidenced manytimes by Dubin and his co-workers [21–23]. To the best of ourknowledge, a study on the adsorption of acidic proteins by cationicSPB in the full range of pH can hardly be found in literature. Theonly report was from Ballauff et al., who used isothermal titra-tion calorimetry to observe the adsorption isotherm of bovinepancreatic ribonuclease A (RNase A) on cationic SPB as a func-tion of temperature and salt concentration at physiological pH 7.2[24].

In this article, annealed cationic SPB were synthesized by pho-toemulsion polymerization, and used as carriers to immobilizebovine serum albumin (BSA) at various pH values (Scheme 1).The interaction between BSA and SPB at different pH values, ionicstrengths, BSA concentrations, and SPB thicknesses were observedby combining turbidimetric titration, dynamic light scattering(DLS), and zeta potential methods. Adsorbed amount of BSA in SPB

ovine serum albumin by annealed cationic spherical polyelectrolytecolsurfb.2013.02.026

was measured by UV spectroscopy. An investigation on the adsorp-tion of BSA in SPB in the full pH range at various ionic strengths,BSA concentrations, and SPB thicknesses can lay the foundation forprotein separation under optimal conditions.

dx.doi.org/10.1016/j.colsurfb.2013.02.026


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Scheme 1. Structure of cationic SPB and s

. Experimental part

.1. Materials

Bovine serum albumin (BSA), 2-aminoethylmethacrylateydrochloride (AEMH), and standard sodium hydroxide (NaOH)olution (0.1 M) were purchased from Sigma-Aldrich and usedithout further purification. Styrene was distilled under reducedressure and stored in a refrigerator at 4 ◦C before use. Sodiumhloride (NaCl), hydrochloric acid (HCl), standard phosphateolutions, cetyltrimethylammonium bromide (CTAB), 2,2′-zobis(2-methylpropionamidine) dihydrochloride (AIBA), and-(N-morpholino)ethanesulfonic acid (MES) were purchasedrom J & K Chemical and used as received. Cationic SPB witholystyrene (PS) cores and poly(AEMH) (PAEMH) brushes [25,26]ere synthesized by photoemulsion polymerization [27,28]. The

btained cationic SPB consist of a 80 ± 2 nm PS core and a PAEMHrush layer with the thickness of 10 to 60 ± 5 nm. All water used

n this work was purified by reverse osmosis and subsequent ionxchange (Millipore Milli-Q).

.2. Turbidimetric titration

Transmittance (%T) was monitored with a Brinkmann PC 950olorimeter (420 nm filter), connected to a 2 cm path length opticalrobe. The turbidity was reported as 100 − %T, and the fluctuations±0.1%) of transmittance were treated by consistently selectinghe highest transmittance. All solutions were prepared with waternd filtered through 0.45 �m polytetrafluoroethylene PTFE filters25 mm Syringe Filter, PTFE Membrane, Corning Incorporated). Theependence of solution turbidity on pH was obtained by observ-

ng the change of turbidity upon addition of 0.1 M NaOH and/or.1 M HCl standard aqueous solutions to a BSA–SPB mixture by.0 mL microburets at constant ionic strength. Generally, SPB con-entration was 0.004 g/L and protein concentration was 0.020 g/L.itrogen purge was employed during all titrations. SPB-free blanksnd protein-free blanks were subtracted to eliminate the effectf free protein scattering. An Orion pH meter with a combinationlectrode was used to monitor the solution pH.

.3. Dynamic light scattering and zeta potential

The diameter and zeta potential of BSA–SPB complexes andlank SPB were determined by Dynamic Light Scattering (DLS)/Zetaotential/Particle Sizer NICOMP 380 ZLS instrument at variable pH


t 25 ◦C and at a scattering angle of 90◦. Size of BSA–SPB complexesas tested in 1 mM NaCl solution. BSA and SPB concentrations were

.040 and 0.008 mg/mL for DLS, respectively. In the zeta poten-ial measurement, the electric field was 5 V/cm in constant-current

atic representation of the BSA adsorption.

mode. Zeta potential of complexes was measured in 1 mM phos-phate buffer, in which BSA and SPB concentrations were set as0.40 mg/mL and 0.08 mg/mL, respectively. For control experiments,SPB concentration was 0.08 mg/mL and BSA concentration was10 mg/mL to obtain a the high signal-to-noise ratio.

The hydrodynamic radius of the core particles was 40 ± 2 nm,which was measured by dynamic light scattering (DLS) via cumu-lant analysis using the Einstein-Stokes relationship. The shellthickness of SPB can be tuned from 10 to 60 ± 5 nm by changing themonomer (AEMH) amount, which was calculated by subtractingthe hydrodynamic radius of the PS core from the SPB determinedby DLS. For the DLS measurements, the suspensions were filteredthrough a 0.45 �m PTFE filter.

2.4. Adsorption measurement

The ionic strength of all aqueous solutions of SPB (5 mg/mL)in 2-(N-morpholino)ethanesulfonic acid (MES) buffer was tunedthrough the addition of NaCl. The pH of solutions was adjustedwith NaOH or HCl standard aqueous solutions. BSA solutions wereprepared by adding BSA into MES buffer solutions. After mixingof BSA and SPB, the mixture was stirred and incubated in a 4 ◦Crefrigerator for 24 h. The unbound proteins were removed by cen-trifugation (Microfuge 16, Centrifuge, Brinkmann) with 10,000 rpmfor 60 minutes at room temperature. The amount of unboundproteins was determined spectroscopically from the supernatantat 280 nm (UV-2550, UV–visible spectrophotometer, SHIMADZU).The amount of immobilized proteins was calculated by subtractingthe amount of unbound proteins from the total amount of proteins.

3. Results and discussion

3.1. Adsorption and desorption upon changing pH

As shown in Fig. 1a, the turbidity of the mixture of BSA andcationic SPB solutions showed a slight increase from pH 3 to 5 whilea significant jump appeared upon increasing pH from 5 to 6. The pHwhere the turbidity begins to increase corresponds to the onset ofprotein binding to SPB to form soluble complexes between SPB andBSA. This result agrees well with the literature [19,21–23,29,30]that binding is even possible at the “wrong” side of the proteinisoelectric point (the isoelectric point for BSA is 4.9). The particlesize of BSA–SPB remained almost unchanged below pH 5, whilea sharp increase appeared upon increasing pH from 5 to 6. Theturbidity and complex size decreased dramatically at pH near 7,


and returned to the “baseline” at pH >8. The increase in turbidity at5 < pH < 7 was attributed to the aggregation of complexes [21,22,30]formed by SPB and BSA. Since the particle size of blank SPB changedwithin 37 ± 5 nm upon increasing pH (Fig. 1), the dramatic increase


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Fig. 1. (a) Particle size of cationic SPB (with and without BSA) and turbidity as afunction of pH in 1 mM NaCl solution. Symbols denote: ( ) size with BSA (±5 nm,n = 3), ( ) size without BSA (±5 nm, n = 3), and (�) turbidity with BSA (the fluctua-tions (±0.1%) of transmittance). (b) Zeta potential of SPB with and without BSA as afunction of pH in 1 mM phosphate buffer (n = 3, 3% error). Symbols denote: (©) SPBwmM

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ith BSA, ( ) free SPB, and ( ) pure BSA. (c) Adsorbed amount of BSA �ads per unitass SPB particles as a function of pH in SPB with a thickness of 50 ± 5 nm in 10 mMES buffer (n = 3, 2% error).

n turbidity and particle size should be due to the aggregation ofPB induced by BSA adsorption.

In Fig. 1b, the decrease in zeta potential upon addition of NaOHnto BSA–SPB solution below pH 6 implies that the adsorption ofSA in cationic SPB is governed by electrostatic attraction. The netharge of SPB was partly neutralized by adsorbed BSA [19], whichould be predicted by protein–polyelectrolyte interaction theory21–23,30]. The pH region of strong adsorption of BSA in SPB wasocated at pH 3–5.5, where the reduction of complex zeta poten-ial reached the maximum. At pH around 6, the zeta potential waslose to zero, which resulted in formation of the largest aggrega-ion as confirmed by DLS and turbidimetric titration. In the pH rangerom 6 to 7, complex zeta potential increased again where turbid-ty decreased from the maximum. At pH above 7, complex turbidityecreased sharply and the zeta potential of BSA–SPB solution waserged with that of the blank cationic SPB. The zeta potential of

SA–SPB solution was always more positive than that of blank BSA.SA will affect the zeta potential of SPB if adsorption of BSA in SPBccurred, while BSA itself at such low concentration (0.4 mg/mL)


annot give any useful zeta potential signal.Fig. 1c showed that the adsorbed amount of BSA per unit weight

f SPB (�ads) as a function of pH at ionic strength of 10 mM by UV–vispectroscopy, which displayed a peak at pH around 6–7. There was

PRESSBiointerfaces xxx (2013) xxx– xxx 3

almost no adsorption of BSA in SPB when pH was below 3. Adsorbedamount of BSA was increased significantly upon increasing pH from3 to 6, while decreased markedly upon further increasing pH from7 to 9 resulted from desorption of BSA from SPB. Adsorbed amountwas close to zero when pH was above 9, which indicated that almostall BSA were released from SPB and redissolved in aqueous solution.The effect of pH on adsorbed amount was accordant with the resultsby turbidimetric titration, DLS, and zeta potential methods.

The results of turbidity, dynamic light scattering, and zetapotential (Fig. 1) revealed the existence of three pH regions: (1)Adsorption of BSA in SPB at pH <5, (2) aggregation of SPB inducedby BSA adsorption at 5 < pH < 7, and (3) desorption of BSA from SPBat pH >7 (Scheme 2).

Obviously, adsorption of BSA by cationic SPB was tunable by pH(Scheme 2). At low pH (pH <5, around the BSA isoelectric point of4.9), the dynamic equilibrium adsorption of BSA in SPB was pre-dominantly driven by weak electrostatic interaction. By increasingpH over 4.9, the net charge of BSA was turned to negative, which ledto a strong adsorption of proteins into SPB. In this case, the nega-tively charged BSA acted as counterions to diminish the net chargeof SPB. Electrostatic repulsion among SPB with less net charge wasreduced while the hydrophobic force was enhanced. It reduced thecolloidal stability of SPB and led to aggregation of cationic SPBbridged by negatively charged BSA. This BSA induced inter-SPBaggregation led to a huge complex size up to 2 �m (see Fig. 1a).However, the aggregation would be broken upon further increas-ing pH. At pH >7, the reduced charge density of amino groups inSPB led to the shrinkage of polyelectrolyte chains on SPB and thedissolution of aggregates. The shrinkage of SPB chains at high pHresulted in the expulsion of BSA from SPB layers.

3.2. Effect of ionic strength

Fig. 2a showed the effect of ionic strength on the adsorption,aggregation, and desorption of BSA from SPB. Low ionic strengthhad almost no effect on the adsorption, aggregation, and desorp-tion regions, while the aggregation size increased monotonicallywhen the salt concentration increased from 1 to 10 mM. Sincethe net charge of SPB was partly screened by added salt [31], therepulsive force among SPB was reduced which enhanced the aggre-gation among SPB. As shown in Fig. 2a, disaggregation among SPBand desorption of BSA from SPB were delayed to higher pH at theionic strength of 50 and 100 mM. Peak value of complex turbiditydecreased upon increasing ionic strength from 50 mM. The reducedpeak value of turbidity was relative to smaller aggregation size andreduced saturated adsorption amount of BSA in SPB. When the ionicstrength increased to 1 M, there was no adsorption of BSA in SPBat all (Fig. 2b). The small peak in Fig. 2a should be attributed tothe SPB self-aggregation. Therefore, the delay of full desorption tohigher pH values at higher ionic strengths (≥50 mM) was probablyresulted from the aggregation of SPB.

Fig. 2b showed the adsorbed amount of BSA per unit weight ofSPB (�ads) as a function of ionic strength at pH 6. Adsorbed amountof BSA in SPB was decreased upon increasing ionic strength (espe-cially ≥10 mM). The added salt led to screening of the electrostaticinteraction between BSA and SPB [32,33], thus the reduction ofadsorbed amount of BSA in SPB. This result is accordant with theobservation by turbidimetric titration (Fig. 2a).

3.3. Effect of BSA concentration


As shown in Fig. 3, upon increasing BSA concentration, the pHregion of aggregation reduced (especially for BSA concentration≥0.02 mg/mL), the maximum turbidity of aggregation increased,and both aggregation and desorption regions shifted to lower pH.



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4 S. Wang et al. / Colloids and Surfaces B: Biointerfaces xxx (2013) xxx– xxx

Scheme 2. Schematic representation of the interactio

Fig. 2. (a) Turbidimetric titration for BSA–SPB at various ionic strengths with SPBand BSA concentrations of 0.004 and 0.02 mg/mL, respectively (the fluctuations(±0.1%) of transmittance). Symbols denote: ( ) 1 mM, ( ) 5 mM, (�) 10 mM, ( )50 mM, ( )100 mM, (+) 1000 mM. (b) Adsorbed amount of BSA �ads per unit massSPB particles as a function of ionic strength in SPB with a thickness of 50 ± 5 nm atpH 6 in MES buffer (n = 3, 2% error).

n between BSA and SPB in different pH regions.

More adsorbed BSA accelerated the complex neutralization(refer to the peak value of turbidity) [23,30,32], so the pH regionof adsorption was narrowed and aggregation shifted to lower pH.When pH >4.9 (the isoelectric point of BSA), the negative BSA,acted as counterions, reduced the net charge of SPB [19,31]. As dis-cussed above, higher BSA concentration made sharp reduction ofSPB net charge, which led to increased aggregation size and tur-bidity. Meanwhile, SPB with less net charge led to a smaller pHregion to modulate the charge balance and thus to form aggrega-tion. Therefore, the onset of desorption shifted to lower pH, and pHregion of aggregation reduced.

3.4. Effect of SPB thickness


As shown in Fig. 4a, desorption region shifted to higher pH,and peak value of complex turbidity increased upon increasing SPB

Fig. 3. Turbidimetric titration for BSA–SPB as a function of pH at the ionic strengthof 10 mM in 0.004 mg/mL SPB with various BSA concentrations (the fluctuations(±0.1%) of transmittance). Thickness of SPB was 50 ± 5 nm. Symbols denote: (+)0.0004 mg/mL, ( ) 0.002 mg/mL, ( ) 0.004 mg/mL, ( ) 0.002 mg/mL, (�) 0.2 mg/mL,( ) 0.4 mg/mL.


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

Fig. 4. (a) Turbidimetric titration for BSA–SPB as a function of SPB thickness atthe ionic strength of 10 mM in 0.004 mg/mL SPB with 0.02 mg/mL BSA (the fluc-tuations (±0.1%) of transmittance). Symbols denote: ( ) 13 nm, ( ) 36 nm, (�)50 nm. (b) Adsorbed amount of BSA in SPB with various thickness at ionic strengthof 10 mM and pH 6 in MES buffer. Adsorbed amount of BSA �ads per unit massSa5

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PB particles is plotted against SPB thickness (n = 3, 2% error). Linear fitting, withdj. R-square of 0.99923, was performed for the data with thickness less than0 nm.

hickness. Positive net charges on SPB were increased with increas-ng SPB thickness, which contributed to the enlarged pH region to

odulate the charge balance. The increased peak value of complexurbidity was attributed to the increased size of SPB with moreharges.

Fig. 4b showed that adsorbed amount of BSA increased linearlyith SPB thickness smaller than 50 nm. For thickness of ∼60 nm,

he specific amount of BSA (�ads/thickness) decreased. For thick-ess of 10–50 nm, PAEMH chain stretched toward aqueous solutionue to electrostatic and steric repulsion resulted from high graft-

ng density near the PS core. When thickness further increased to0 nm, the PAEMH chain segment away from the PS core curled,esulting buried cations and reducing the adsorbed amount ofSA.

The pH can modulate the adsorption, aggregation, and desorp-ion of BSA by SPB. Ionic strength can change adsorbed amountf BSA in SPB and aggregation size, while protein concentrationan further tune the pH regions of adsorption, aggregation, and


esorption. Furthermore, SPB thickness affects adsorbed amount,ggregation size and the onset of desorption. Interaction betweenrotein and SPB can be applied to protein immobilization, separa-ion, and purification. Through adjusting pH, ionic strength, protein

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PRESSBiointerfaces xxx (2013) xxx– xxx 5

concentration, and SPB thickness, proteins can be separated underoptimal conditions.

4. Conclusions

Annealed cationic spherical polyelectrolyte brushes (SPB) weresynthesized by photoemulsion polymerization. Turbidimetric titra-tion was used to examine the complex formation, aggregation,and desorption of BSA from SPB in a full pH range at fixed ionicstrengths and BSA concentrations. DLS was applied to observe theaggregation of cationic SPB induced by BSA adsorption while zetapotential methods confirmed the adsorption and desorption of BSAby cationic SPB. The pH regions for the interaction between BSAand SPB consist of adsorption, aggregation, and desorption regions.The aggregation region narrowed, and the onset of desorptionshifted to lower pH values with increased protein concentrationand decreased SPB thickness. Adsorption measurements demon-strated that the maximum adsorbed amount of BSA in SPB achievedat moderate pH, low ionic strength, and large SPB thickness. There-fore, the adsorption of BSA by cationic SPB is tunable throughchanging pH, ionic strength, BSA concentration, and SPB thick-ness.

Acknowledgments

We gratefully acknowledge the National Natural ScienceFoundation of China (No. 20774028 and 11076002/A06), theFundamental Research Funds for the Central Universities, thehigher school specialized research fund for the doctoral pro-gram (20110074110003), the Key Basic Research Project ofShanghai Science and Technology Commission (10JC1403800),the China Postdoctoral Science Foundation (2012M511088), andShanghai Synchrotron Radiation Facility for support to thiswork.

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Tunable adsorption of bovine serum albumin by annealed cationic spherical polyelectrolyte brushes

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