pubs.acs.org/Langmuir © 2010 American Chemical Society
Combining Host-Guest Systems with Nonfouling Material for the Fabrication of a Biosurface: Toward Nearly Complete and Reversible Resistance of Cytochrome c Pengbo Wan,† Yueyue Chen,‡ Yibo Xing,† Lifeng Chi,‡ and Xi Zhang*,† † Key Lab of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, PR China, and ‡Physikalisches Institut, Westf€ alische Wilhelms Universit€ at, 48149 M€ unster, Germany
Received June 8, 2010. Revised Manuscript Received June 27, 2010 In this letter, a pH-responsive reactivated biointerface is fabricated using an inclusion reaction between an azobenzenecontaining self-assembled monolayer and pH-responsive poly(ethylene glycol)-block-poly(acrylic acid) grafted with cyclodextrins. The pH-responsive interface can be switched between an extended state and a relaxed state for the reversible resistance of cytochrome c adsorption completely in cooperation with protein-resistant poly(ethylene glycol).
Introduction The effective resistance of proteins on surfaces is very desirable for potential applications, including biomedical implants, in vitro diagnostics, biosensors, coatings for ship hulls, carriers for targeted drug delivery, and so on.1 Among various protein-resistant materials, poly(ethylene glycol) (PEG)-based materials have been extensively employed for decades to prevent nonspecific protein adsorption and cell adhesion.2 Recently, numerous attempts have been focused on using PEG-coated surfaces to resist protein adsorption irreversibly.3 For example, Whitesides et al. reported that surfaces modified with PEG-terminated alkanethiol self-assembled monolayer can irreversibly resist cytochrome c (Cyt c), a heme-containing metalloprotein that can be entrapped in the mitochondrial membrane and act as an electron carrier in the respiratory chain.3c However, it still remains a challenge to design reactivated flexible biointerfaces that can reversibly prevent protein adsorption in response to an external stimulus.4 In this letter, we report the fabrication of a pH-responsive reactivated biointerface using the host-guest interaction between an azobenzene-containing selfassembled monolayer (Azo SAM) and a pH-responsive block copolymer. The pH-responsive block copolymer, named PEGPAA-g-CD, was prepared by grafting β-cyclodextrin moieties to poly(ethylene glycol)-block-poly(acrylic acid). Upon pH variation, the reversible resistance of proteins is realized. Smart surfaces that respond to external stimuli can be realized by different approaches.5-7 For example, we have fabricated a dualresponsive interface based on the photocontrolled inclusion and exclusion reaction between azobenzene and the cyclodextrin-grafted *Corresponding author. E-mail:
[email protected]. (1) Jiang, S.; Cao, Z. Adv. Mater. 2010, 22, 920. (2) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (3) (a) Hucknall, A.; Rangarajan, S.; Chilkoti, A. Adv. Mater. 2009, 21, 2441. (b) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359. (c) Chen, X.; Ferrigno, R.; Yang, J.; Whitesides, G. M. Langmuir 2002, 18, 7009. (4) (a) Mohammed, J. S.; Murphy, W. L. Adv. Mater. 2009, 21, 2361. (b) Burkert, S.; Bittrich, E.; Kuntzsch, M.; M€uller, M.; Eichhorn, K. J.; Bellmann, C.; Uhlmann, P.; Stamm, M. Langmuir 2010, 26, 1786. (5) (a) Jiang, Y.; Wan, P.; Smet, M.; Wang, Z.; Zhang, X. Adv. Mater. 2008, 20, 1972. (b) Jiang, Y.; Wang, Z.; Yu, X.; Shi, F.; Xu, H.; Zhang, X.; Smet, M.; Dehaen, W. Langmuir 2005, 21, 1986. (c) Ling, X.; Reinhoudt, D. N.; Huskens, J. Chem. Mater. 2008, 20, 3574. (d) Zhang, L.; Sun, J. Chem. Commun. 2009, 3901. (6) Wang, Y.; Xu, H.; Zhang, X. Adv. Mater. 2009, 21, 2849. (7) Wan, P.; Jiang, Y.; Wang, Y.; Wang, Z.; Zhang, X. Chem. Commun. 2008, 5710.
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pH-responsive poly(acrylic acid) polymer (PAA-g-CD), which can reversibly immobilize large amounts of Cyt c.8 To release Cyt c effectively, PAA is an excellent detaching matrix because it can be switched between an electronegative state at pH 7.0 to adsorb large amounts of positively charged Cyt c (isoelectric point at 10) and an electroneutral state at pH 4.0 to release Cyt c.9 However, only ∼80% of immobilized Cyt c can be released under pH variation in our previous work.8 In the present work, we attempt to combine the pH sensitivity of PAA and the protein resistance of PEG to construct a pH-responsive interface for complete protein release, which reversibly resists Cyt c. First, pH-responsive block copolymer PEGPAA-g-CD can be assembled on Azo SAM by the host-guest interaction between azobenzene and CD (Figure 1).10 Second, the PEG-PAA-g-CD-protecting interface (PEG-PAA-g-CD/Azo SAM) containing protein-resistant PEG can be switched between an electronegative state to immobilize Cyt c (Scheme 1A) and an electroneutral state to resist Cyt c adsorption reversibly (Scheme 1B).
Experimental Section Cytochrome c (from horse heart, purity 98%) and 1-decanethiol (96%) were purchased from Sigma-Aldrich. Other chemicals were analytical-grade reagents and were used as received. All solutions were prepared by using Milli-Q purified water that had been sterilized at high temperature. 10-(4-Phenylazo-phenoxy)decane-1-thiol (AzoSH) was synthesized in our previous work.7 PAA319-g-CD5% and PEG43-PAA153-g-CD5% were synthesized in previous work.8,11 A solution of Cyt c was prepared by dissolving Cyt c powder in 10 mM sodium phosphate buffer solution (PBS) at pH 7.0. An electrochemical measurement was performed using a potentiostat (Autolab PGSTAT12, Netherlands). It was carried out in a conventional three-electrode glass electrochemical cell at ambient temperature. A bare gold electrode (model CHI 101, 2 mm diameter) was used as a working electrode. An auxiliary electrode was platinum, and the reference electrode was a Ag/ AgCl (saturated KCl) electrode. The gold electrode underwent the (8) Wan, P.; Wang, Y.; Jiang, Y.; Xu, H.; Zhang, X. Adv. Mater. 2009, 21, 4362. (9) (a) Zhou, J.; Lu, X.; Hu, J.; Li, J. Chem.;Eur. J. 2007, 13, 2847. (b) Kabanov, A. V.; Vinogradov, S. V. Angew. Chem., Int. Ed. 2009, 48, 5418. (10) (a) Wang, Y.; Ma, N.; Wang, Z.; Zhang, X. Angew. Chem., Int. Ed. 2007, 46, 2823. (b) Willner, I.; Vered, P.; Katz, E.; Ranjit, K. T. J. Electroanal. Chem. 2001, 497, 172. (c) Shipway, A. N.; Willner, I. Acc. Chem. Res. 2001, 34, 421. (11) Wang, Y.; Zhang, M.; Moers, C.; Chen, S.; Xu, H.; Wang, Z.; Zhang, X.; Li, Z. Polymer 2009, 50, 4821.
Published on Web 06/30/2010
DOI: 10.1021/la102336a
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Figure 1. Schematic assembly of pH-responsive block copolymer PEG-PAA-g-CD on an Azo SAM through the host-guest interaction of azobenzene and CD. Scheme 1. pH-Responsive Switching of the PEG-PAA-g-CD-Protecting Biointerface Containing Protein-Resistant PEG between the Stretched State at pH 7.0 To Immobilize Cyt c (A) and the Relaxed State at pH 4.0 To Resist Cyt c Adsorption Reversibly (B)
following pretreatments to get a mirrorlike surface. First, the gold electrode was mechanically polished with 1, 0.3, and 0.05 μm R-Al2O3 and washed ultrasonically with deionized water. Next, it was electrochemically scanned in a 2 mM K3[Fe(CN)6] þ 0.1 M KCl solution (pH 6.2) by potential scanning between -0.05 and 0.55 V until a reproducible cyclic voltammogram was obtained and then completely rinsed with deionized water and tetrahydrofuran. Finally, it was dried with high-purity nitrogen before monolayer adsorption. The solutions for the electrochemical measurements were purged prior to the measurements by continuously bubbling highly purified nitrogen through them for 30 min, and a nitrogen atmosphere was maintained during the electrochemical measurements. The Azo SAM was formed by placing the bare gold electrode in a 10-5 M ethanolic solution of n-C10H21SH and AzoSH (10:1 molar ratio) at room temperature for 90 min and then rinsed extensively with ethanol and dried in a stream of dry, high-purity nitrogen, which has been well established in our previous work. The PEG-PAA-g-CD attached Azo SAM was fabricated by dipping the Azo-SAM-modified gold electrode into a 0.2 mg/mL aqueous solution of PEG-PAA-g-CD at room temperature overnight.8 An in situ quartz crystal microbalance (QCM) measurement was conducted using a Qsense E4 multichannel instrument equipped with gold-coated 5 MHz AT-cut crystals. The same procedures were employed to fabricate Azo SAM and PEG-PAAg-CD-attached Azo SAM on a gold-coated QCM resonant crystal as on an electrochemical gold electrode. For in situ QCM measurements, the PEG-PAA-g-CD-attached Azo SAM on a gold-coated QCM crystal was fixed in the chamber of the QCM instrument, and the crystal was sealed with a silicon rubber O ring in the chamber. Then, the PEG-PAA-g-CD attached Azo SAM 12516 DOI: 10.1021/la102336a
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Figure 2. (a) Cyclic voltammograms of the (A) bare, (B) AzoSAM, and (C) PEG-PAA-g-CD/Azo-SAM modified gold electrodes in the presence of [Fe(CN)6]3-/4-. (b) CV responses of K3[Fe(CN)6] on a PEG-PAA-g-CD/Azo SAM-modified electrode in 2 mM K3[Fe(CN)6] þ 0.1 M KCl solution with different pH values at a scan rate of 0.1 V/s: (A) 3.5, (B) 6.2, (C) 8.3, and (D) 10.2. was stabilized in a flow of pH 7.0 PBS solution until the frequency reached equilibrium. The flow rate is 4 μL/min. Then 5 μM Cyt c-containing pH 7.0 PBS solution was injected over a PEG-PAAg-CD/Azo SAM film at a rate of 4 μL/min. The frequency change of the quartz crystal was monitored throughout the adsorption.
Results and Discussion The Azo SAM that we chose for the immobilization of PEGPAA-g-CD was the mixed SAM of AzoSH and n-C10H21SH. The attachment of PEG-PAA-g-CD on the Azo SAM has been successfully realized. The cyclic voltammetry (CV) responses of the bare, Azo SAM, and PEG-PAA-g-CD/Azo SAM-modified gold electrodes in the presence of [Fe(CN)6]3-/4- are shown in Figure 2a (A-C, respectively). As shown in Figure 2a-C for the PEG-PAAg-CD/Azo SAM-modified gold electrode, the remarkable decrease in peak current, compared with that of the Azo SAM-modified electrode (Figure 2a-B), should be due to the hindrance from the attachment of PEG-PAA-g-CD with ionized electronegative -COO- groups on Azo SAM.7,8 The PEG-PAA-g-CD-coated interface also possesses pH switchability. The CV responses of the PEG-PAA-g-CD-coated interface were obtained in different pH solutions using [Fe(CN)6]3-/4- as the probe, as shown in Figure 2b. A drastic decrease in the current response and an increase in ΔEp (peak-to-peak separation) was observed upon pH variation from 3.5 to 10.2, indicating that the obtained PEG-PAA-g-CD-coated Azo SAM is pH-sensitive. The observed pH switchability can be explained as follows:8,9 at low pH, the carboxylate groups of the PAA segment are hardly ionized and the electroneutral PAA is in a relaxed state; at high pH, the carboxylate groups are fully ionized and ionic repulsion among the -COO- groups expands the PAA chain to form an extended structure with more negative charges on its surface. The PEG-PAAg-CD-coated biointerface can be switched between an electroneutral relaxed state and an ionized stretched state many times, indicating the good stability of the interface. We wondered if the pH-switchable interfaces containing protein-resistant PEG could be used to resist Cyt c reversibly. In situ QCM measurements were employed to provide evidence for the reversible resistance of Cyt c triggered by pH variation.12 The QCM measurement was conducted by fixing the gold-coated quartz crystal with PEG-PAA-g-CD/Azo SAM deposited in a flow chamber, and the solution of the guest molecule candidates was allowed to flow through the chamber. During the flow, the frequency shift was recorded, which represents the number of adsorbed guest molecules on the quartz crystal. Figure 3a shows (12) Niu, J.; Liu, Z.; Fu, L.; Shi, F.; Ma, H.; Ozaki, Y.; Zhang, X. Langmuir 2008, 24, 11988.
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Figure 3. (a) In situ QCM adsorption kinetics of Cyt c on a PEGPAA-g-CD/Azo SAM film in different pH solutions. After flowing pH 7.0 PBS solution to give a baseline (A), 5 μM Cyt c-containing pH 7.0 PBS solution was injected over a PEG-PAA-g-CD/Azo SAM film (B), followed by a pH 7.0 PBS solution to wash physisorpted Cyt c (C). Then pH 4.0 PBS solution was injected to release immobilized Cyt c (D). For comparison with the baseline (A), pH 7.0 PBS solution flowed over the surface again (E). (b) In-situ QCM adsorption kinetics of Cyt c on a PAA-g-CD/Azo SAM film in different pH solutions. After the flow of pH 7.0 PBS solution to give a baseline (A), 5 μM Cyt c-containing pH 7.0 PBS solution was injected over a PAA-g-CD/Azo SAM film (B), followed by pH 7.0 PBS solution to wash physisorbed Cyt c (C). Then pH 4.0 PBS solution was injected to release immobilized Cyt c (D), followed by pH 7.0 PBS.
the in situ QCM adsorption kinetics of Cyt c on a PEG-PAA-gCD/Azo SAM film in different pH solutions. First, after the flow of pH 7.0 PBS solution to give a stable baseline (Figure 3a-A), Cyt c-containing pH 7.0 PBS solution was injected over the PEGPAA-g-CD/Azo SAM film (Figure 3a-B). Second, pH 7.0 PBS solution was allowed to flow over the surface to wash away physisorpted Cyt c (Figure 3a-C). Third, pH 4.0 PBS solution was injected to release adsorbed Cyt c (Figure 3a-D). Last, for comparison with the baseline (Figure 3a-A), pH 7.0 PBS solution was allowed to flow over the surface again (Figure 3a-E). As shown in Figure 3a, the resonance frequency decreases by about 23 Hz after injecting Cyt c-containing pH 7.0 PBS solution (Figure 3a-B), showing that negatively charged PAA in the PEG-PAA-g-CD film adsorbs positively charged Cyt c at pH 7.0. After physisorpted Cyt c was rinsed (Figure 3a-C), the frequency decreases 17 Hz in contrast with the baseline, which corresponds to the amount of immobilized Cyt c, indicating that pH 7.0 PBS solution cannot release immobilized Cyt c. However, upon injecting pH 4.0 PBS solution (Figure 3a-D), the frequency increases immediately, corresponding to the release of immobilized Cyt c. After the flow of pH 7.0 PBS solution (Figure 3a-E), the frequency returns to the baseline state, demonstrating almost 100% of release of immobilized Cyt c. Compared to our previous work where only ∼80% of immobilized Cyt c could be released onto a PAA-g-CD-coated film when changing the pH value from 7.0 to 4.0,8 our present study clearly indicates that the cooperation between protein-resistant PEG and electroneutral PAA can guarantee almost 100% release of the immobilized Cyt c. In addition, it is a reversible process, and the reversible resistance of
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Cyt c adsorption can be repeated several times (Supporting Information, Figure S1). As a control experiment, PAA-g-CD was immobilized on Azo SAM instead of PEG-PAA-g-CD to perform the reversible release of Cyt c at different pH values. Figure 3b shows the in situ QCM adsorption kinetics of Cyt c on the PAA-g-CD/Azo SAM film using the same procedures as for the PEG-PAA-g-CD/ Azo SAM film. As indicated by QCM, the frequency decreases about 35 Hz after injecting Cyt c-containing pH 7.0 PBS solution (Figure 3b-B) and increases 20% to almost 28 Hz after washing the physisorpted Cyt c (Figure 3b-C), which corresponds to the amount of immobilized Cyt c. The frequency decrease for the PAA-g-CD/Azo SAM film (28 Hz) after washing physisorpted Cyt c is about 65% greater than that for the PEG-PAA-g-CD/ Azo SAM film (17 Hz), indicating that PEG resists Cyt c at pH 7.0. As shown in Figure 3b, after the flow of pH 4.0 PBS solution (Figure 3b-D) and pH 7.0 PBS solution (Figure 3b-E) on the PAA-g-CD/Azo SAM film, only 80% of immobilized Cyt c is released compared to almost 100% release of immobilized Cyt c on the PEG-PAA-g-CD/Azo SAM film, which displays the protein-resistance of PEG and is in good agreement with our previous work.8 Therefore, the introduction of PEG is a key point to guaranteeing the complete resistance of Cyt c.
Conclusions We have fabricated a pH-responsive PEG-PAA-g-CD/Azo SAM on gold using the host-guest interaction of azobenzene and CD between Azo SAM and PEG-PAA-g-CD. The pHresponsive interface can be switched between the extended state and the relaxed state, resulting in a nearly 100% release of the immobilized Cyt c in cooperation with protein-resistant PEG. This study represents a successful example of the integration of the pH sensitivity of PAA and the protein-resistance of PEG on a PEGPAA-g-CD coated surface to form pH-responsive reactivated biointerfaces for the reversible resistance of Cyt c adsorption. It is anticipated that this supramolecular biosurface is suitable for the controlled adsorption and release of proteins with an isoelectric point larger than 7.0. Moreover, a similar concept can be further extended to fabricate different functional biosurfaces by combining PEG and other stimuli-responsive materials. Acknowledgment. The work was funded by the National Basic Research Program (2007CB808000), the National Natural Science Foundation of China (50573042), and NSFC-DFG TRR61. We are grateful for the assistance of Prof. Hongwei Ma and Mr. Long Fu (Peking University) with the in situ QCM measurements. Yueyue Chen thanks the China Scholarship Council for financial support. Supporting Information Available: Reversible resistance of Cyt c adsorption on the PEG-PAA-g-CD/Azo SAM-modified surface. This material is available free of charge via the Internet at http://pubs.acs.org.
DOI: 10.1021/la102336a
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