Counterion-Specific Protein Adsorption on Polyelectrolyte Brushes

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Counterion-Specific Protein Adsorption on Polyelectrolyte Brushes Jun Yang, Zan Hua, Tao Wang, Bo Wu, Guangming Liu, and Guangzhao Zhang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b01145 • Publication Date (Web): 18 May 2015 Downloaded from http://pubs.acs.org on May 20, 2015

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Counterion-Specific Protein Adsorption on Polyelectrolyte Brushes Jun Yang,† Zan Hua,† Tao Wang,† Bo Wu,‡ Guangming Liu*,† and Guangzhao Zhang*‡ †

Hefei National Laboratory for Physical Sciences at the Microscale, Department of Chemical Physics, University of Science and Technology of China, Hefei 230026, P. R. China. ‡ Faculty of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, P. R. China.

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Abstract. Protein adsorption is an important issue in bio-related fields. We have investigated the protein adsorption on the poly(ionic liquid) (PIL) brushes in the presence of different types of counterions. The protein adsorption is driven by a decrease of osmotic pressure within the brushes with an increase of entropy via release of counterions. Our study demonstrates that counterion specificity has a significant influence on protein adsorption on the PIL brushes. There have been two different regimes for the counterion-specific protein adsorption. When the released counterions cannot bind onto the protein surface, the counterion-specific protein adsorption is dominated by the ion-specific counterion condensation within the PIL brushes. If the released counterions can bind onto the protein surface, then the counterion-specific protein adsorption is dominated by the ion-specific re-binding of released counterions onto the protein surface. This work opens up a new opportunity for controlling protein adsorption on polyelectrolyte brushes.

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Introduction Protein adsorption plays a crucial role in a number of medical, biological and physiological processes.1-5 To control protein adsorption at the solid-water interfaces, many types of polymer brushes have been developed to either inhibit or enhance the protein adsorption.6-10 As an important class of polymer brushes, polyelectrolyte brushes have been applied to control protein adsorption in a wide range of fields from drug delivery to enzyme immobilization by tuning pH and salt concentration, because the secondary structures and bioactivities of the adsorbed proteins are largely undisturbed within polyelectrolyte brushes.11-15 In general, protein adsorption on polyelectrolyte brushes is driven by a decrease of osmotic pressure within the brushes with an increase of entropy via release of counterions.16,17 Nevertheless, the counterions have always been treated as non-specific point charges and the ion-specific interactions have never been taken into account during protein adsorption on polyelectrolyte brushes in the conventional theory, regardless of the fact that polyelectrolyte brushes usually possess different types of counterions with distinct structures.17,18 In principle, counterion specificity should strongly influence the protein adsorption on polyelectrolyte brushes. On the one hand, counterion type can affect the osmotic pressure within polyelectrolyte brushes, because the osmotic coefficient of counterions is related to the ion-specific interactions between the counterions and the charged groups associated with the grafted chains.19-21 Therefore, protein adsorption on polyelectrolyte brushes should be counterion-specific as the decrease of osmotic 3


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pressure is one of the driving forces for the protein adsorption. On the other hand, recent studies have shown that ions can bind onto protein surface through ion-specific non-electrostatic interactions even though the protein molecules and ions carry the same charge.22-28 Thus, the released counterions from the polyelectrolyte brushes during protein adsorption may re-bind onto the protein surface, thereby reducing the entropy increase generated by the release of counterions. Such an ion-specific re-binding of the released counterions onto the protein surface which has never been investigated

in

previous

studies

can

also

make

the

protein

adsorption

counterion-specific, as the entropy increase is another driving force for the protein adsorption. However, no studies have been conducted to date to investigate the counterion-specific protein adsorption on polyelectrolyte brushes in spite of the significance of counterion specificity. Due to the large population of cation-anion pairs in ionic liquid chemistry, poly(ionic liquid) (PIL) brushes have recently been recognized as versatile polyelectrolyte brushes.29-33 The incorporation of IL moiety into grafted chains not only broadens the window of physicochemical properties of common polyelectrolyte brushes but also expands the potential applications of polyelectrolyte brushes in a wide range of fields. Herein, we have unraveled the mechanism of counterion-specific protein adsorption on polyelectrolyte brushes by employing PIL brushes as a model system. Our study demonstrates that counterion specificity plays a significant role in the protein adsorption on polyelectrolyte brushes.

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Experimental Section Materials. The p-silicon (100) wafers were purchased from Wafer Works Corporation (Shanghai, China) and were cut into plates with a size of 20 mm × 30 mm. 1-Vinylimidazole (ViIm, 99%) and 4,4'-azobis(4-cyanovaleric acid) (ACVA, 98%) were purchased from Sigma-Aldrich and used as received. Bromoethane (CH3CH2Br),

N,N'-dicyclohexylcarbodiimide

(DCC),

pyridine,

ethyl

acetate,

methanol and ethanol were all analytical reagent (AR) grade (Sinopharm Chemical Reagent Co.) and used as received. (3-Aminopropyl)triethoxysilane (APTES, 99%), sodium tetrafluoroborate (NaBF4, 99.99%) and sodium trifluoromethanesulfonate (CF3SO3Na, 98%) were all purchased from Aladdin Reagent Co. and used as received. Sodium

hexafluorophosphate

(NaPF6,

98%),

bis(trifluoromethane)sulfonimide

sodium [(CF3SO2)2NNa, 98%] and sodium perfluorooctanoate [CF3(CF2)6COONa, 97%] were obtained from Alfa Aesar Chemical Reagent Co. and used as received. Bovine serum albumin (BSA, Mw ~ 68 kDa, pI ~ 4.8) was purchased from Hualvyuan Biotechnology Co. N,N'-Dimethyl formamide (DMF, Sinopharm) was dried in the presence of anhydrous magnesium sulfate (MgSO4) and distilled under reduced pressure prior to use. Dimethylsulfoxide (DMSO, Sinopharm) was dried in the presence of calcium hydride (CaH2) and distilled under reduced pressure prior to use. The water used was purified by filtration through the Millipore gradient system after distillation, giving a resistivity of 18.2 MΩ cm. DMSO-d6 used as a solvent in nuclear magnetic resonance (NMR) measurement was purchased from Sigma-Aldrich.

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Phosphate buffer (PB) was prepared using sodium phosphate dibasic (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4). Synthesis of 1-Vinyl-3-ethylimidazolium Bromide (ViEtImBr). The monomer ViEtImBr was synthesized as follows. 47.1 g ViIm (0.5 M) and 109.0 g CH3CH2Br (1.0 M) were dissolved in 150.0 mL ethyl acetate and stirred at 50 ºC for 24 h, a white-yellow precipitate was formed at the bottom of the glass flask. The precipitate was filtered and dissolved in methanol, and then precipitated again by adding the methanol solution drop wise into ethyl acetate for purification. The powder ViEtimBr was filtered and then dried under vacuum at 40 ºC. The successful synthesis of ViEtImBr was confirmed by the 1H NMR measurement in DMSO-d6. 1H NMR (DMSO-d6): δ = 9.48 (s, N–CH–N, 1H), 8.18 (s, N–CH=CH–N, 1H), 7.94 (s, N–CH=CH–N, 1H), 7.30 (dd, CH2=CH–N, 1H), 5.97 (dd, HCH=CH–N, 1H), 5.41 (dd, HCH=CH–N, 1H), 4.20 (q, N–CH2–CH3, 2H), 1.44 (t, N–CH2–CH3, 3H). Preparation of Poly(Ionic Liquid) (PIL) Brushes. The silicon wafer (or the SiO2-coated resonator) was cleaned by water plasma treatment at a power of 18 W for 15 min. The activated substrate was modified to form APTES monolayer by a vapor deposition of APTES at 60 ºC for 6 h, and then rinsed with ethanol and water, and dried with nitrogen flow. Afterwards, the substrate was immersed in a solution containing 0.2 g ACVA, 2.0 g DCC, 50 µL pyridine and 40 mL DMF in a flask at 25 ºC for 24 h to form the initiator layer, followed by successively rinsing with DMF, ethanol and water, and drying with nitrogen flow. Then, the substrate was immersed in a DMSO solution containing ViEtImBr (2.0 M) in a flask. After bubbling with 6


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argon at room temperature for 2 h, the solution was kept at 80 ºC for 6 h to conduct the surface-initiated free-radical polymerization. After the polymerization, the PViEtImBr brushes grafted substrate was successively rinsed with DMSO, ethanol and water, and dried with a flow of nitrogen. The dry thickness of PViEtImBr brushes was ~ 10.0 nm determined by spectroscopic ellipsometry. The PViEtImX brushes were prepared by counterion exchange of PViEtImBr brushes with relevant salts (0.1 M) in a methanol solution for 10 min, where X represented the different types of counterions. Quartz Crystal Microbalance with Dissipation (QCM-D) Measurements. QCM-D studies of protein adsorption on the PIL brushes were conducted on a Q-sense E1.34 The quartz crystal (AT-cut) with a fundamental resonant frequency of 5 MHz and a diameter of ~ 14 mm was mounted in a fluid cell with one side exposed to the solution.34,35 The resonator had a mass sensitivity constant (C) of ~ 17.7 ng cm−2 Hz−1.36 When a quartz crystal is excited to oscillate in the thickness shear mode at its fundamental resonant frequency (f0) by applying a RF voltage across the electrodes near the resonant frequency, a small layer added to the electrodes induces a decrease in resonant frequency (Δf) that is proportional to the mass change (Δm) of the layer. In vacuum or air, if the added layer is rigid, evenly distributed, and much thinner than the crystal, then Δf is related to Δm and the overtone number (n = 1, 3, 5....) by the Sauerbrey equation37 ∆m =−

ρ q lq ∆f f0

n

=−C

∆f n

7


(1)

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where f0 is the fundamental frequency and ρq and lq are the specific density and thickness of the quartz crystal, respectively. The dissipation factor is defined by34

D=

Ed 2πEs

(2)

where Ed is the energy dissipated during one oscillation and Es is the energy stored in the oscillating system. The measurement of ΔD is based on the fact that the voltage over the crystal decays exponentially as a damped sinusoidal when the driving power of a piezoelectric oscillator is switched off.34 By switching the driving voltage on and off periodically, we can simultaneously obtain a series of changes of the resonant frequency and the dissipation factor. In the present study, all the results obtained were from the measurements of frequency and dissipation changes at the third overtone (n = 3). The protein concentration used in the adsorption experiments was fixed at 1.0 mg mL−1 and all the experiments were performed at 25 ºC. Isothermal Titration Calorimetry (ITC) Measurements. The interactions between anions and BSA were investigated using a MicroCal iTC200 calorimeter (GE Healthcare) at 25 ºC. When studying anion specificity, we employed sodium salts so that the influence of the cation was constant. Both the salts and BSA were dissolved in PB solution (13 mM, pH 7.0). The salt concentration and the BSA concentration were fixed at 10 mM and 30 μM, respectively. Each solution was thoroughly degassed to remove bubbles before loading. Titration was performed by injecting 2 μL aliquots of salt solution in PB buffer into the calorimeter cell containing BSA solution at an interval of 2 min. The data for the titration of salt solution into PB

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buffer as the baseline was subtracted from the raw data to correct the heat of salt dilution. Curve fitting was performed by ITC data analysis module from MicroCal to obtain the binding constant and binding stoichiometry according to the one-type-of-sites model.38-41 Zeta Potential Measurements. SiO2 nanoparticles (~ 70 nm) grafted with PIL brushes were used to determine the surface Zeta potentials (ζ) in the presence of different types of counterions. The PIL brushes on the nanoparticles were prepared in a similar procedure with that on the flat SiO2 surface. The successful preparation of SiO2 nanoparticles grafted with PIL brushes was confirmed by the transmission electron microscopy (TEM) images (Figure S1, Supporting Information). The dry thickness of the PIL brushes on the nanoparticle surface was ~ 10.0 nm. The values of the ζ were measured using a DelsaTM Nano C particle and Zeta potential analyzer in water at 25 ºC. Other Measurements. 1HNMR experiment was conducted on a Bruker AV-400 NMR spectrometer at 400 MHz at room temperature. The chemical shifts (δ) were referred to the solvent peak (δ = 2.5 ppm for DMSO-d6) using tetramethylsilane as an internal standard. The dry thickness of PIL brushes before and after the protein adsorption was determined by a spectroscopic ellipsometer (M-2000V, J. A. Woollam, U.S.A.) by treating the polymer layer as a single Cauchy layer, where the refractive index of the polymer layer was assumed to be 1.450. The water contact angle (WCA) on the surface of PIL brushes was determined using a KSV (Helsinki, Finland) CAM 200 contact angle goniometer at 25 ºC. X-ray photoelectron spectroscopy (XPS) 9


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measurements were conducted on an ESCALAB-250 spectrometer (Thermo Electron, U.K.) with a monochromatic Al Kα X-ray source (hv = 1486.6 eV). TEM measurements of the SiO2 nanoparticles were carried on a JEOL-2010 microscope.

Results and Discussion

Scheme 1. Schematic illustration of the preparation of the poly(ionic liquid) brushes possessing different types of counterions.

Scheme 1 illustrates the preparation of the PIL brushes possessing different types of counterions. Briefly, APTES was anchored on the silicon dioxide surface by a vapor deposition, followed by an immobilization of the initiator ACVA on the surface by an amidation reaction. The PViEtImBr brushes were prepared on the surface by a 10


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surface-initiated free-radical polymerization of ViEtImBr. Then, the PViEtImX brushes were successfully prepared by counterion exchange in methanol, where X represented the different types of counterions. The XPS and WCA results demonstrate that Br− can be completely substituted by other types of anions during counterion exchange (Figures S2 to S4, Supporting Information). The values of the dry thickness (dB) of the PViEtImX brushes were around 10.0 nm (Table 1).

Table 1. Dry thickness of the PViEtImX brushes before (dB) and after (dB-P) the BSA adsorption, the normalized ∆f (∆fN) induced by the BSA adsorption on the PViEtImX brushes, and the normalized change in dry thickness (∆dN) of the PViEtImX brushes induced by the BSA adsorption. Here, X represents the different types of counterions. dB (nm)

dB-P (nm)

ΔfN (Hz)

∆dN (nm)

10.2

52.4

–226

5.3

CF3(CF2)6COO

10.2

63.4

–251

6.3

Br−

9.6

54.7

–288

6.0

9.4

61.0

–291

6.9

9.1

59.0

–305

7.0

8.1

52.0

–337

7.3

X (CF3SO2)2N− −

PF6

−

CF3SO3− BF4−

QCM-D was employed to investigate the protein adsorption on the PIL brushes in water. Figure 1 shows the shifts in frequency (∆f) and dissipation (∆D) as a function of time for the adsorption of BSA on the PViEtImBr brushes. It is known that the frequency shift indicates a mass change on the resonator surface and the dissipation shift reflects the comparative softness or rigidity of the polymeric layer on the resonator surface.36,42,43 After the addition of BSA solution, ∆f rapidly decreases and 11


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then gradually leaves off (Figure 1a), indicating the deposition of BSA molecules on the PViEtImBr brushes. The rinse with water only induces a slight change in ∆f, suggesting that the adsorbed BSA molecules are quite stable on the surface. Interestingly, ∆D also decreases with the adsorption of BSA on the PViEtImBr brushes (Figure 1b), implying that the brushes become stiffer upon the protein adsorption. This result is in contrast with the previous observations that ∆D increases upon the BSA adsorption.6,44

baseline addition of BSA

(a)

0

baseline addition of BSA

∆∆ / 10-6

0

∆f / Hz

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-800

(b)

-20

rinsing

-1600

rinsing

-40 -60

-2400 0

40

80

120

0

Time / min

40

80

120

Time / min

Figure 1. (a) Shift in frequency (∆f) as a function of time for the adsorption of BSA on the PViEtImBr brushes. (b) Shift in dissipation (∆D) as a function of time for the adsorption of BSA on the PViEtImBr brushes. Here, the BSA concentration is 1.0 mg mL−1.

Protein adsorption generally gives rise to an increase in ∆D due to the formation of a soft viscoelastic protein layer on the resonator surface. BSA with a pI of ~ 4.8 is negatively charged in water.45 Thus, the decrease in ∆D in Figure 1b indicates that the BSA molecules penetrate into the inner part of the PViEtImBr brushes and complex 12


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with the grafted positively charged chains, forming a stiffer polymeric layer. This fact suggests that the BSA adsorption on the PIL brushes is driven by a decrease of osmotic pressure with an increase of entropy via the release of counterions induced by the complexation between the BSA molecules and the grafted chains. In addition, the positively charged lysozyme cannot adsorb on the PViEtImBr brushes (Figure S5, Supporting Information), further confirming that the protein adsorption is driven by the decrease of osmotic pressure and the increase of entropy. Since the protein molecules can penetrate into the inner part of PIL brushes, the amount of adsorbed protein should be linearly related to the thickness of the brushes.46-48 To compare the amount of adsorbed protein between the different types of counterions, the ∆f induced by the BSA adsorption on the PViEtImX brushes was normalized by the dry thickness of the relevant brushes (Figure S6, Supporting Information). The normalized ∆f (∆fN) decreases following the series (CF3SO2)2N− > CF3(CF2)6COO− > Br− > PF6− > CF3SO3− > BF4− (Table 1), suggesting that the amount of adsorbed BSA increases following this series. The normalized change in dry thickness (∆dN) of the PViEtImX brushes induced by the BSA adsorption also almost follows this series (Table 1), further suggesting the counterion-specific protein adsorption. Considering that protein adsorption on polyelectrolyte brushes could be influenced by hydrophobic interactions, we have measured the surface wettability of the PViEtImX brushes. In Figure 2, water contact angle (WCA) on the surface of PViEtImX brushes increases following the series Br− < BF4− < PF6− < CF3SO3− < 13


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(CF3SO2)2N− < CF3(CF2)6COO−, indicating the hydrophobicity of the brushes increases following this series due to the distinct hydrophobic characters of the counterions. However, the ordering of anions in this series is quite different from that observed in Table 1. Therefore, the counterion-specific protein adsorption cannot be attributed to the distinct hydrophobic interactions between the BSA molecules and the PViEtImX brushes in the presence of different types of counterions.

WCA / °

120 80 40 0

−

OO ) C6 F2 (3C − CF ) N2 2 SO F3 (C − 3 S3 O CF

−

PF 6

−

4 BF

−

Br

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 2. Change in water contact angle (WCA) on the surface of PViEtImX brushes as a function of counterion type. Here, X represents the different types of counterions.

According to Manning’s theory of counterion condensation, a portion of the confined counterions within the brushes should condense onto the grafted polyelectrolyte chains to reduce the chain charge density to one charge per Bjerrum length.49 These condensed counterions do not contribute to the osmotic pressure (or 14


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Donnan pressure) within the polyelectrolyte brushes. In other words, the osmotic pressure only results from the dissociated free counterions within the brushes.17,50 The condensation of counterions is dependent on counterion type due to the ion-specific interactions between the counterions and the charged groups associated with the grafted chains.21 Thus, the counterions employed in the present work with distinct chemical structures would exhibit different interaction strengths with the positively charged ethylimidazolium groups associated with the grafted chains, leading to an ion-specific counterion condensation.51,52

ζ / mV

66 60 54 48

−

)2 N2 SO F3 (C − − OO 4 BF ) C6 F2 (3C CF − 3 S3 O CF

−

Br

−

PF 6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 3. Change in Zeta potential (ζ) of PViEtImX brushes as a function of counterion type. Here, X represents the different types of counterions.

As a consequence, the disassociation of counterions and the resulting osmotic pressure within the PIL brushes are dependent on counterion type, as reflected by the counterion-specific Zeta potential (ζ) of the PViEtImX brushes (Figure 3). The 15


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surface Zeta potential increases following the series PF6− < Br− < CF3SO3− < CF3(CF2)6COO− < BF4− < (CF3SO2)2N−, implying that the surface charge density and the extent of disassociation of counterions within the brushes increase following this series.53 Consequently, the osmotic pressure within the brushes should also increase following this series. Since the decrease of osmotic pressure via counterion release is one of driving forces for the BSA adsorption, the amount of adsorbed BSA should follow this series as well. However, the ordering of counterions in this series is still different from that observed in Table 1, suggesting that the BSA adsorption on the PIL brushes is not merely determined by the ion-specific counterion condensation. In fact, the released counterions can re-bind onto the protein surface through ion-specific non-electrostatic interactions, e.g., van der Waals and hydrophobic interactions.23-28 The re-binding of released counterions onto protein surface leads to an ion-specific reduction of entropy increase during protein adsorption, thereby resulting in a counterion-specific protein adsorption. Figure 4a shows the ITC data for titration of different salt solutions into the BSA solution at pH 7. Each exothermic peak represents the heat associated with the binding of the anions onto the protein surface. The fact that no obvious exothermic peaks can be observed for Br− and BF4− suggests that both of them do not have obvious interactions with BSA molecules. In contrast, the exothermic peaks observed for other types of anions indicate that these anions can bind onto the BSA surface through the ion-specific non-electrostatic interactions. Note that the influence of cations on the ITC data can be neglected, since all the cations of the salts used in ITC titration experiments are Na+. Figure 4b shows 16


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the titration curves derived from the integrated heats of the exothermic peaks for different types of anions. The binding constants (kB) and binding stoichiometry (nB) for the adsorption of the anions onto the BSA surface can be obtained by fitting the titration curves as reported before.38-41

kcal / mol of Injectant

Br− BF4− CF3SO3− PF6− CF3(CF2)6COO−

(a)

(CF3SO2)2N−

700

1400 2100 2800

0.0

(b) BrBF4-

-0.4

CF3SO3PF6-

-0.8 -1.2

CF3(CF2)6COO(CF3SO2)2N-

0

25

Time / s

50

75

Molar Ratio 100

(c)

(d)

75

1.6

fB / %

kB / 104 M−1

2.4

0.8

50 25

0.0

0 −

−

−

) N2 − 2 SO O O F3 (C ) C6 F2 (3C CF

−

PF 6 − 3 S3 O CF

−

4 BF

Br

−

)2 N2 − SO O O F3 (C ) C6 F2 (3C CF

−

PF 6 − 3 S3 O CF

−

4 BF

Br

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 4. (a) Isothermal titration calorimetry (ITC) data for the titration of salt solutions into the BSA solution as a function of counterion type. (b) ITC titration curves derived from the integrated heats of the exothermic peaks as a function of counterion type. The dashed lines are fits to the ITC titration curves to obtain the binding constant and binding stoichiometry. (c) Binding constants (kB) for the adsorption of anions onto the BSA surface as a function of counterion type. (d) Molar fraction of the bound counterions on protein surface to the totally released counterions (fB) as a function of counterion type. 17


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In Figure 4c. no binding constants can be obtained for Br− and BF4−, indicating the very weak interactions of BSA molecules with these two kinds of anions. For other types of anions, kB increases following the series CF3SO3− < PF6− < CF3(CF2)6COO− < (CF3SO2)2N−, suggesting that the interaction strength between the anions and the protein molecules increases following this series. The average values of nB are 0.4, 5.9, 11.7 and 15.3 for CF3SO3−, PF6−, CF3(CF2)6COO− and (CF3SO2)2N−, respectively, further indicating that the interaction strength between the anions and the BSA molecules increases following the series. Hereafter, we will discuss the counterion-specific BSA adsorption on the brushes separately in terms of the interactions between the anions and the protein molecules. For Br− and BF4−, they do not bind onto BSA surface. That is, the counterion-specific BSA adsorption for these two types of counterions should be dominated by the ion-specific decrease of osmotic pressure within the brushes. Because the Zeta potential of PViEtImBF4 brushes is larger than that of PViEtImBr brushes (Figure 3), the extent of counterion disassociation and the resulting osmotic pressure of the former should be larger than that of the latter. Therefore, the amount of adsorbed BSA on the PViEtImBF4 brushes is larger than that on the PViEtImBr brushes (Figure 5a and 5b).

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9

(a) ∆dN / nm

−∆fN / Hz

420 280 140 0

(b)

6 3 0 4

−

−

3 S3 O − O CF− CO PF 6 F )2 6− (C N 3 CF O )2 2 S F3 (C

−

BF − Br

4

3 S3 O − CF− O CO PF 6 F )2 6− (C N 3 CF O )2 2 S F3 (C

−

BF − Br

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 5. (a) Normalized frequency shift (–∆fN) induced by the BSA adsorption on the PViEtImX brushes as a function of counterion type. (b) Normalized change in dry thickness (∆dN) of the PViEtImX brushes induced by the BSA adsorption as a function of counterion type. Here, X represents the different types of counterions.

For the cases of CF3SO3−, PF6−, CF3(CF2)6COO− and (CF3SO2)2N−, the re-binding of released counterions onto the protein surface can be described as follows: Protein [ P] - x

+

 →

nB Counterion

Protein/(Counterion) n B

[C ] - nB x kB =

x

x ([ P] − x)([C ] − nB x)

(3)

where [P], [C] and x are the protein concentration, the concentration of totally released counterions, and the concentration of protein molecules with bound counterions, respectively. During BSA adsorption, [P] is 15 μM, which should be much larger than x. Thus, kB can be simplified as:

kB =

x [ P ]([C ] − nB x)

(4)

Consequently, the molar fraction of the bound counterions to the totally released counterions (fB) as shown in Figure 4d can be obtained from Eq. 5: 19


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= fB

nB x = [C ]

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1 1 +1 nB k B [ P]

(5)

Obviously, the larger the kB and nB are, the larger the fB will be, thereby resulting in a larger reduction in entropy increase during protein adsorption and a smaller amount of adsorbed BSA. Therefore, the amount of adsorbed BSA decreases following the series CF3SO3− > PF6− > CF3(CF2)6COO− > (CF3SO2)2N− (Figure 5a and 5b). That is, the counterion-specific BSA adsorption is dominated by the ion-specific re-binding of released counterions onto the protein surface for these four types of counterions.

Scheme 2. Schematic illustration of the counterion-specific BSA adsorption on polyelectrolyte brushes.

Above it is demonstrated that the counterion-specific BSA adsorption on polyelectrolyte brushes is not only related to the interactions between the counterions and the charged groups associated with the grafted chains, but also correlated with the interactions between the released counterions and the protein molecules. The 20


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proposed mechanism for the counterion-specific BSA adsorption on polyelectrolyte brushes is schematically illustrated in Scheme 2. Specifically, the counterion-specific BSA adsorption is dominated by the ion-specific counterion condensation within the brushes when the released counterions cannot bind onto the protein surface. If the released counterions can bind onto the protein surface, then the counterion-specific BSA adsorption is dominated by the ion-specific re-binding of the released counterions onto the protein surface. Thus, the counterion specificity can be used to control the protein adsorption on polyelectrolyte brushes. In addition, BSA exhibits a similar counterion-specific adsorption behavior on the PIL brushes with a different thickness, further supporting the proposed mechanism in Scheme 2 (Figure S7, Supporting Information).

Conclusion Our study demonstrates that counterion specificity plays a significant role in the protein adsorption on polyelectrolyte brushes. The protein adsorption is driven by the decrease of osmotic pressure and the increase of entropy, both of which are counterion specific. When the released counterions cannot bind onto the protein surface, the counterion-specific protein adsorption is dominated by the ion-specific counterion condensation within the brushes. If the released counterions can bind onto the protein surface, then the counterion-specific protein adsorption is dominated by the ion-specific re-binding of the released counterions onto the protein surface. The detailed understanding of the mechanism of counterion-specific protein adsorption 21


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elucidated here provides a platform for controlling protein adsorption on polyelectrolyte brushes via specific ion effects.

Associated Content Supporting Information. The data of XPS, surface wettability, protein adsorption and TEM are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding Author *E-mail: [email protected] (G.M.L.). *E-mail: [email protected] (G.Z.Z.).

Acknowledgment The financial support of National Program on Key Basic Research Project (2012CB933802), the National Natural Science Foundation of China (21374110, 91127042 and 21234003) is acknowledged.

References and Notes 1.

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Counterion-Specific Protein Adsorption on Polyelectrolyte Brushes

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