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pH-switchable bioelectrocatalysis based on layer-by-layer films assembled through specific boronic acid-diol recognition
Electrochimica Acta 55 (2010) 9185–9192
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
pH-switchable bioelectrocatalysis based on layer-by-layer films assembled through specific boronic acid-diol recognition Huiqin Yao a,b , Fengwei Chang a , Naifei Hu a,∗ a b
Department of Chemistry, Beijing Normal University, Beijing 100875, PR China Department of Chemistry, Ningxia Medical University, Yinchuan 750004, PR China
a r t i c l e
i n f o
Article history: Received 22 July 2010 Received in revised form 1 September 2010 Accepted 4 September 2010 Available online 15 September 2010 Keywords: pH-switchable bioelectrocatalysis Layer-by-layer assembly Boronic acid-diol specific interaction Glucose oxidase Ferrocenedicarboxylic acid
a b s t r a c t Phenylboronic acid (PBA) moieties are grafted onto the backbone of poly(acrylic acid) (PAA), forming the PAA-PBA polyelectrolyte. The PAA-PBA and polysaccharide dextran (Dex) are then assembled layerby-layer (LbL) into {PAA-PBA/Dex}n films on electrode surface through the boronic acid-diol specific recognition between them. The cyclic voltammetric response of ferrocenedicarboxylic acid (Fc(COOH)2 ) at the film electrodes is sensitive to the environmental pH. At pH 5.5, the CV peak currents are quite large, and the films are at the “on” state; at pH 8.0, however, the CV response is significantly suppressed with the films at the “off” state. This pH-sensitive on–off property of the films toward the probe can be used to switch the bioelectrocatalysis of glucose mediated by Fc(COOH)2 in the present of glucose oxidase (GOD) in solution by changing the surrounding pH. Furthermore, PAA-PBA and the glycoenzyme GOD are also assembled into {PAA-PBA/GOD}n LbL films by the boronic acid-diol specific interaction between them, so that the immobilization of the enzyme on electrodes can be realized. The {PAA-PBA/GOD}n films also show pH-sensitive on–off behavior in bioelectrocatalysis of glucose with Fc(COOH)2 as the mediator. This model system may open a new way to establish a foundation for fabricating pH-responsive biosensors based on enzymatic electrocatalysis. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction The bioelectrocatalysis on the basis of various enzymatic reactions nowadays has become an important foundation in the development of biosensors. The high specificity and sensitivity of enzymes in detecting and determining the respective substrates have made this type of electrochemical biosensors more attractive among researchers in recent years [1–3]. In this field, the switchable biosensors based on stimuli-responsive bioelectrocatalysis have aroused increasing interests. In general, the stimuli-controllable bioelectrocatalysis can find its application not only in biosensors but also in bioelectronic devices, biofuel cells, and biocomputing, as well as signal transduction/amplification and information processing [4–8]. Among various external stimuli that activate/deactivate bioelectrocatalysis, pH is the most studied one. By choosing appropriate film-forming materials and selecting proper processing conditions, it is possible to create “smart” interfaces on electrode surface that demonstrate pH-sensitive permeability to electroactive probes, which can be further used to realize the pH-switchable bioelectrocatalysis [9–14]. For example, poly(4-vinyl pyridine) (P4VP) functionalized with Os-complex redox units (P4VP-Os) was
∗ Corresponding author. Tel.: +86 10 5880 5498; fax: +86 10 5880 2075. E-mail address: [email protected] (N. Hu). 0013-4686/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2010.09.012
grafted onto ITO electrodes by Katz and co-workers [9,10]. The formed P4VP-Os brush exhibited the reversible pH-sensitive transition between the swollen and shrunken structures, resulting in pH-dependent switching of the cyclic voltammetric (CV) response of Os-complex units. This property of the brush could be applied to reversibly activate/deactivate the electrocatalytic oxidation of glucose in the presence of glucose oxidase (GOD) by changing environmental pH chemically or biochemically. However, in general, the pH-controllable bioelectrocatalysis has only received a limited attention, and the in-depth understanding of the mechanism is still a great challenge. Among various methods to construct the smart interface, the layer-by-layer (LbL) assembly demonstrates distinguished advantages in its precise control of film thickness at a nanometer scale according to a predesigned architecture and in its extremely simple procedure and high versatility in the assembly [15,16]. The building blocks of LbL films are originally polyelectrolytes, and now extended to proteins, nanoparticles, and other species [17,18]. The driving force of LbL assembly is usually electrostatic interaction between oppositely charged species, but now expanded to a variety of non-electrostatic interactions including hydrogen bonding, hydrophobic interaction, ion-dipole interaction, and biospecific recognition [15,19]. For example, in our previous work, lectinsugar biospecific interaction was used to construct {Con A/Dex}n LbL films by concanavalin A (Con A) and dextran (Dex) on elec-
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trode surface, and the films showed a reversible pH-controlled bioelectrocatalysis in the presence of mediator, GOD, and glucose [12]. Boronic acid-diol recognition is another kind of specific interactions between boronic acid groups and diol units and has attracted more attention in scientific researches, especially in constructing films [20–26]. For example, phenylboronic acid (PBA) and its derivatives can rapidly and reversibly form covalent band with various 1,2- and 1,3-cis-diol compounds such as polyols, carbohydrates and sugar residues located on the surface of glycoenzymes to generate five- or six-membered cyclic boronate ester complexes [27,28]. Since monosaccharides usually have five –OH groups, and one PBA group can react with two –OH groups, the 1:2 sugar–PBA complex is often formed [27,29,30]. Boronic acid-diol interaction has also been used to assemble LbL films [21,24,31–33]. For instance, poly(vinylalcohol) or mannan can be assembled with polyelectrolytes containing PBA moieties into LbL films [21,24]. Dextran (Dex) is a glucose-based polysaccharide and contains many glucose residues on its backbone, which can bind to PBA groups [34–36]. While the specific affinity between Dex and PBA has been studied, the LbL assembly of Dex with polyelectrolytes containing PBA moieties has not been reported until now. In addition, some glycoenzymes, such as horseradish peroxidase (HRP) and GOD, can combine with some compounds containing PBA groups via the boronic acid-diol specific interaction, and form self-assembled monolayer films on electrode surface [20,22,23,26,37–39]. However, no study on the LbL films assembled by glycoenzymes and polymers containing PBA groups has been reported up to now. In the present work, PBA moieties were grafted onto the backbone of poly(acrylic acid) (PAA) through the condensation reaction between aminophenyl-boronic acid and carboxylic acid group of PAA according to the literature [21], designated as PAA-PBA. In addition to a large fraction of PBA moieties in PAA-PBA, a considerable amount of free carboxylic acid groups remained and would be sensitive to surrounding pH. The {PAA-PBA/Dex}n LbL films were then assembled on the surface of pyrolytic graphite (PG) electrodes through the boronic acid-diol specific recognition between PAAPBA and Dex. Electroactive ferrocenedicarboxylic acid (Fc(COOH)2 ) demonstrated pH-sensitive on–off behavior in its electrochemical response at the {PAA-PBA/Dex}n film electrodes. At pH 5.5, the CV response of the probe was quite large at the film electrodes, and the films were at the on state; at pH 8.0, however, the CV response of the probe was greatly suppressed, and the films were at the off state. The mechanism of the pH-dependent on–off property of the films toward the probe was further explored with comparative studies, and believed to be mainly attributed to the electrostatic interaction between the films and probe. The pHswitchable property of the films toward Fc(COOH)2 was further used to activate/deactivate the electrocatalytic oxidation of glucose catalyzed by GOD. In addition, PAA-PBA and GOD were successfully assembled into {PAA-PBA/GOD}n LbL films on PG electrode surface also by the boronic acid-diol specific interaction so that the enzyme could be immobilized. The {PAA-PBA/GOD}n films also demonstrated the pH-sensitive on–off property toward Fc(COOH)2 , which could be used to switch the bioelectrocatalysis of glucose by changing the environmental pH. CV, electrochemical impedance spectroscopy (EIS), quartz crystal microbalance (QCM), scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy were used to characterize the system. To the best of our knowledge, this is the first report on the pH-switchable bioelectrocatalysis based on LbL films assembled through boronic acid-diol specific recognition. This study provided a novel example to construct a smart interface and established a foundation for fabricating pH-controllable biosensors based on the LbL assembly and enzymatic electrocatalysis.
Scheme 1. Synthetic scheme of PAA-PBA.
2. Experimental 2.1. Reagents Poly(acrylic acid) (PAA, MW ≈ 100 000, 35 wt.% solution in water), 3-aminophenyl-boronic acid hemisulfate salt (APBA), 4-(2-hydroxyerhyl) piperazine-1-ethanesulfonic acid (HEPES, 99.5%), N-hydroxysulfosuccinimide sodium salt (NHS), 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), chitosan (CS, the degree of deacetylation is more than 85%, MW ≈ 200 000), dextran from leuconostoc mesenteroides (Dex, MW ≈ 200 000), glucose oxidase (GOD, E.C. 1.1.3.4, type VII, MW ≈ 160 000, 192 000 units g−1 ), 1,1’-ferrocenedicarboxylic acid (Fc(COOH)2 ), hexaammineruthenium(III) chloride (Ru(NH3 )6 Cl3 ), ferrocenemethanol (FcOH), and 3-mercapto-1-propanesulfonate (MPS, 90%) were purchased from Sigma–Aldrich. Potassium ferricyanide (K3 Fe(CN)6 ) and potassium ferrocyanide (K4 Fe(CN)6 ) were obtained from Beijing Chemical Engineering Plant. All other reagents were of analytical grade. Buffers were usually 0.05 mol dm−3 sodium acetate (pH 4–6) or 0.05 mol dm−3 potassium dihydrogen phosphate (pH 6.5–8), all containing 0.1 mol dm−3 NaCl. The pH value of buffers was adjusted to the desired value with dilute HCl or NaOH solutions. The d-glucose stock solutions were allowed to mutarotate at room temperature for 24 h before being used. Solutions were prepared with water purified twice by ion exchange and subsequent distillation. 2.2. Synthesis of PAA-PBA PAA-PBA was synthesized by reaction between PAA and APBA in the presence of NHS and EDC according to the literature [21], and the process is schematically depicted in Scheme 1. In brief, 0.57 g of PAA solution containing 2.77 mmol equiv. of monomer was diluted by HEPES buffers (0.02 dm3 , 50 mmol dm−3 ) and the pH was adjusted to 8.5, APBA (0.02 dm3 , 0.061 mol dm−3 ) in the HEPES buffers were then added. NHS (0.004 dm3 , 0.031 mol dm−3 ) in the HEPES buffers were added dropwise into the solution and stirred for 10 min. EDC (0.004 dm3 , 0.31 mol dm−3 ) in the HEPES buffers were then added to the mixture, followed by stirring of 12 h. The solution was then extensively dialyzed against water for 1 week in order to remove all low-molecular-weight residues, and the white fluffy solid PAA-PBA was obtained after freeze drying. 2.3. Film assembly For electrochemical study, LbL films were grown on basal plane PG (Advanced Ceramics) disks electrodes (geometric area 0.16 cm2 ). Prior to assembly, PG electrodes were abraded on 320grit metallographic sandpaper while flushing with water. After being ultrasonicated in water for 30 s and dried in air, the PG electrodes were first immersed in positively charged CS solutions (1 g dm−3 , pH 5.0) for 20 min to adsorb CS as the precursor layer. The
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PG/CS electrodes were then alternately immersed into negatively charged PAA-PBA (1 g dm−3 , pH 9.0) and neutral Dex (1 g dm−3 , pH 9.0) aqueous solutions for 20 min each with intermediate water rinsing and air stream drying, forming a PAA-PBA/Dex bilayer. This cycle was repeated until the desired number of bilayers (n) was obtained on the PG/CS surface, designated as {PAA-PBA/Dex}n . {PAA-PBA/GOD}n LbL films were assembled on the PG/CS surface with the same way with 1 g dm−3 GOD solution at pH 9.0. For quartz crystal microbalance (QCM) studies, gold-coated quartz crystal resonator electrodes (International Crystal Manufacturing Co.) were first cleaned by a few drops of a freshly prepared piranha solution (3:7 volume ratio of 30% H2 O2 and concentrated H2 SO4 . Caution: the piranha solution reacts violently with most organic materials and must be handled with extreme care, and only a small volume should be prepared at any time) on each side for 10 min, and then washed thoroughly with water and a final rinse with ethanol. The QCM gold electrodes were then immersed in 4 mmol dm−3 MPS ethanol solutions for 24 h to chemisorb an MPS monolayer on gold surface by formation of Au–S bond between Au and MPS, making the Au surface become negatively charged. The CS precursor layer and following {PAA-PBA/Dex}n LbL films were then assembled on the Au/MPS surface in the same way as on the PG electrodes. 2.4. Apparatus and procedures A CHI 660A or CHI 621B electrochemical workstation (CH Instruments) was used for electrochemical measurements. A typical three-electrode cell was used with a saturated calomel electrode (SCE) as the reference, a platinum flake as the counter, and the PG disk electrode with films as the working electrode. The solution was purged with high-purity nitrogen for at least 10 min before electrochemical measurements. The nitrogen atmosphere was then kept above the cell for the entire experiment. Electrochemical impedance spectroscopy (EIS) measurements were performed in 1:1 K4 Fe(CN)6 /K3 Fe(CN)6 mixture solutions with total concentration of 5 mmol dm−3 , and a sinusoidal potential modulation with amplitude of ±5 mV and frequency from 105 to 0.1 Hz was superimposed on the formal potential of Fe(CN)6 3−/4− redox couple at 0.17 V vs SCE. QCM was performed with a CHI 420 electrochemical analyzer (CH Instruments). The quartz crystal resonator (AT-cut) has a fundamental resonance frequency of 8 MHz and is covered by thin gold films on both sides (geometric area 0.196 cm2 per one side). Scanning electron microscopy (SEM) was performed using an S4800 scanning electron microscope (Hitachi) with an acceleration voltage of 3 kV. The CS/{PAA-PBA/Dex}5 films assembled on the PG electrodes were used as the sample. Before the SEM imaging, the surface of samples was coated by thin Pt films with an E-1045 sputtering coater (Hitachi). The Fourier-transform infrared (FTIR) spectra were recorded at a resolution of 4 cm−1 by Nexus 670 Fourier-transform infrared spectrometer (Nicolet). Elemental analysis was performed by a Vario EL CHNOS Elementaranalysator (Elementar Analysesystem GmbH). All experiments were performed at ambient temperature of 20 ± 2 ◦ C. 3. Results and discussion 3.1. Preparation of PAA-PBA Poly(acrylic acid) (PAA) partially grafted by phenyl-boronic acid (PBA) was synthesized by reaction between PAA and APBA in the presence of NHS and EDC according to the literature [21], des-
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Fig. 1. FTIR spectra of (a) PAA, (b) PBA, and (c) PAA-PBA.
ignated as PAA-PBA (Scheme 1). The synthesis of PAA-PBA was confirmed by FTIR spectroscopy (Fig. 1), and the assignment of the observed bands or peaks were discussed as below according to the literatures [40–42]. The characteristic peak at 1712 cm−1 assigned to the C O stretching vibration of the carboxylic acid groups [40–43] of PAA (curve a) was also observed for PAA-PBA (curve c). In addition, the peak at 2950 cm−1 for the stretching mode of CH2 group [42] was observed for both PAA and PAA-PBA. These results indicate that the PAA-PBA keeps the polymer structure similar to that of PAA with some remaining COOH groups. It is noteworthy that the characteristic amide I band centered at 1656 and amide II band at 1550 cm−1 were detected in PAA-PBA (curve c) [40–43], but were not observed in both PAA (curve a) and PBA (curve b). In the meantime, the bending vibration peak of primary amino groups in PBA at 1630 cm−1 [40–42] disappeared in PAAPBA (curve c). These suggest the formation of new amide bond and the successful cross-linking between amino groups of PBA and some carboxylic acid groups of PAA [43]. The typical B–O stretching peak at 1340 cm−1 [42,44], and the characteristic phenyl ring bands at 1490 and 1440 cm−1 [40–42] observed in PBA (curve b) were also detected in PAA-PBA (curve c), demonstrating that the phenyl-boronic acid is successfully grafted onto the PAA polymer. According to the result of elemental analysis of PAA-PBA, the fraction of the carboxylic acid groups in PAA that had been crosslinked with PBA was estimated to be 41%. While this value is only a rough approximation, it is no doubt that a considerable amount of carboxylic acid groups in PAA-PBA keeps “free” after the crosslinking reaction. 3.2. Assembly of {PAA-PBA/Dex}n LbL films CS carries positive charges at pH 5.0 due to its pKa at about 6.5 [45], and was adsorbed on the negatively charged PG surface [46] as the precursor layer. PAA-PBA contained a considerable amount of free –COOH groups in its backbone. With the pKa of PAA at about 6.0 in solution [47–50], almost all these free carboxylic acid groups in PAA-PBA would be ionized and become negatively charged at pH 9.0, and thus could be adsorbed on the oppositely charged PG/CS surface by electrostatic attraction. Dex is a neutral polysaccharide carrying no charge, and could be strongly combined with PAA-PBA through boronic acid-diol specific recognition, forming a PAA-PBA/Dex bilayer. By repeating this cycle, the assembly of {PAAPBA/Dex}n LbL films on the PG/CS surface can thus be realized. The assembly of {PAA-PBA/Dex}n LbL films on PG/CS surface was first confirmed by CV with Fc(COOH)2 as the electroactive probe (Fig. 2A). At bare PG and PG/CS film electrodes, Fc(COOH)2 in pH 7.0 buffers displayed a well-defined and nearly reversible CV oxidation-reduction peak pair at about 0.41 V. However, when
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Fig. 3. QCM frequency shift (−F) with assembly step for assembly of {PAAPBA/Dex}n LbL films on Au/MPS/CS surface: PAA-PBA (䊉) and Dex () adsorption steps.
Fig. 2. (A) CVs of 0.5 mmol dm−3 Fc(COOH)2 at 0.1 V s−1 in pH 7.0 buffers at (a) bare PG electrode, (b) PG/CS films, (c)–(i) PG/CS/{PAA-PBA/Dex}n films with n = 1–7. (B) Dependence of CV oxidation peak current (Ipa ) and CV peak separation (Ep ) of Fc(COOH)2 on the number of bilayers (n) of {PAA-PBA/Dex}n films.
{PAA-PBA/Dex}n LbL films were fabricated on the PG/CS surface, the CV response of probe was severely suppressed, indicating that a barrier was formed on the electrode surface and the probe was hindered from exchanging electrons with underlying PG electrodes. The oxidation peak current (Ipa ) of Fc(COOH)2 decreased with the number of bilayers (n) of {PAA-PBA/Dex}n films, accompanied by the increase of peak separation (Ep = Epa − Epc ) (Fig. 2B), all suggesting that the multilayer films are successfully fabricated on the PG/CS surface. The growth of {PAA-PBA/Dex}n LbL films on PG/CS surface was further confirmed by EIS with Fe(CN)6 3−/4− as the electroactive probe. For bare PG and PG/CS film electrodes, the EIS response of Fe(CN)6 3−/4− in pH 7.0 buffers exhibited a Warburg line in a very wide frequency range (Supplementary materials Fig. S1A, curves a and b), respectively, characteristic of a diffusion-controlled electrochemical process. After the assembly of {PAA-PBA/Dex}n films on the PG/CS surface, semicircles in the high-frequency domain were observed, and the diameter of semicircles became larger with the increase of n values (curves c–i), indicating that the multilayer behaved as a physical barrier and limited the access of the probe to the electrode surface. The diameter of the semicircle usually equals the charge transfer resistance (Rct ) of the probe in electron transfer and the Rct value estimated by using the Randles equivalent circuit model [51–53] showed a nearly linear relationship with n for {PAAPBA/Dex}n films (Supplementary materials Fig. S1B), indicating a roughly uniform growth of the films in the assembly. QCM was also used to characterize the growth of {PAAPBA/Dex}n films on Au/MPS/CS surface (Fig. 3). The QCM resonance frequency decrease (−F) usually reflects the mass increase on QCM gold electrodes. The −F value increased with the adsorption step in a roughly linear mode, suggesting again the successful and uniform assembly of {PAA-PBA/Dex}n LbL films. The stability of {PAA-PBA/Dex}5 films on PG/CS surface was examined by CV with Fe(CN)6 3− as the electroactive probe. For the study of short-term stability, 30 CV cycles were continuously performed in pH 5.5 buffers, and the peak positions and heights of the probe showed no change with the cycle. For the study of long-term
stability, the film electrodes were stored in pH 5.5 blank buffers for most of the storage time, and periodically placed in Fe(CN)6 3− solutions at the same pH for CV testing. After three days of storage, the peak potentials of the probe maintained the same position, and the peak currents decreased by only about 15% of their initial values, suggesting that the films are quite stable. This also indicates that the boronic acid-diol specific recognition between PAA-PBA and Dex is quite strong and can afford long time of immersion in buffers and repeated CV cycling. 3.3. pH-sensitive on–off property of Fc(COOH)2 at {PAA-PBA/Dex}5 film electrodes The solution pH had great influence on the CV response of Fc(COOH)2 for {PAA-PBA/Dex}5 films (Supplementary materials Fig. S2). While Fc(COOH)2 showed relatively large Ipa and quite small Ep in the range of pH 5.0–6.5, the Ipa decreased drastically at pH between 7.0 and 8.0, accompanied by the great increase of Ep . For instance, at pH 5.5, a quite large quasi-reversible CV peak pair was observed (Fig. 4A, curve a), and Ipa showed a linear relationship with square root of scan rates from 0.01 to 1.0 V s−1 (Supplementary materials Fig. S3), suggesting the diffusion-controlled behavior of the probe at the film electrodes. However, when the films were placed in pH 8.0 buffers containing the same amount of Fc(COOH)2 , the CV response was significantly suppressed and could even hardly be observed (Fig. 4A, curve b). This pH-sensitive CV behavior of Fc(COOH)2 should be attributed to the interaction between the films and the probe, since in control experiments, the CV behavior of Fc(COOH)2 at bare PG electrodes was essentially pH-independent (Supplementary materials Fig. S4). By defining the CV response of Fc(COOH)2 at pH 5.5 for the films as the “on” state and that at pH 8.0 as the “off” state, the pH-triggered “on–off” switching property of the films was quite reversible. By switching the film electrode in the probe solutions between pH 5.5 and 8.0, the corresponding CV Ipa could periodically change between the on and off states for many cycles (Fig. 4B). 3.4. Possible mechanism Typically, there are two types of mechanism for pH-sensitive permeation behavior of polyelectrolyte films toward probes. One is mainly controlled by the structure change of films with surrounding pH [9,10,54,55], and the other is mainly tuned by the electrostatic interaction between probes and films, where the surface charge of films can be switched by external pH [11–13,56]. To explore the possible change of film structure induced by environmental pH, the surface morphology of {PAA-PBA/Dex}5 films was examined by SEM after the films were treated with pH 5.5 and 8.0 solutions, respectively (Supplementary materials Fig. S5). With
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Fig. 5. Influence of the number of bilayers (n) for {PAA-PBA/Dex}n films on CV Ipa of 0.5 mmol dm−3 Fc(COOH)2 in buffers at pH 5.5 () and 8.0 () at 0.1 V s−1 .
and demonstrated quite large peak heights at both pH 5.5 and 8.0 (Fig. S6D). All these results suggest that it is the electrostatic interaction between {PAA-PBA/Dex}5 films and probes that controls the pH-sensitive CV on–off property of the system. 3.5. Influencing factors
Fig. 4. (A) CVs of 0.5 mmol dm−3 Fc(COOH)2 at 0.1 V s−1 for {PAA-PBA/Dex}5 films in buffers at pH (a) 5.5 and (b) 8.0. (B) Dependence of CV Ipa of Fc(COOH)2 on the cycle number when the solution pH switched between pH 5.5 () and 8.0 () for the same {PAA-PBA/Dex}5 films.
the same magnification, the films that had been treated with pH 5.5 and 8.0 buffers showed no substantial difference in the surface topography, suggesting that the structure of the films is not very sensitive to the environmental pH at least with the present magnification. Consequently, the pH-sensitive on–off property of {PAA-PBA/Dex}5 films toward Fc(COOH)2 should be most probably attributed to the electrostatic interaction between the films and the probe. Fc(COOH)2 is a weak acid and carries negatively charges at pH ≥ 4.0 due to its ionization [13]. In {PAA-PBA/Dex}5 films, while the Dex component is a neutral polymer carrying no charge, the PAA-PBA constituent is pH-sensitive since it contains a considerable amount of free –COOH groups that is sensitive to environmental pH. At pH 8.0, PAA-PBA carries negative charges since almost all free –COOH groups in the films would be ionized with pKa of PAA at about 6.0 [47–50]. The repulsion between the films and the probe at this pH would hinder the probe to enter the films and block the electron exchange of the probe with underlying electrodes, resulting in the off state of the films. In contrast, at pH 5.5, the films would carry no charge since most of the –COOH groups in the films would be protonated. Without the electrostatic repulsion, the probe would diffuse through the films more easily, leading to the larger CV response. To support this speculation, other electroactive probes with different charges were examined by CV for the films at different pHs. Toward negatively charged Fe(CN)6 3− and Fc(COOH), the {PAAPBA/Dex}5 films demonstrated on state at pH 5.5 and off state at pH 8.0 (Supplementary materials Fig. S6A and B), in good agreement with the pH-dependent behavior of Fc(COOH)2 . However, for positively charged Ru(NH3 )6 3+ , its pH-sensitive property was completely opposite to that of Fc(COOH)2 (Fig. S6C). That is, the films were at the on state at pH 8.0 and at the off state at pH 5.5. For neutral probe FcOH, its CV response was pH-independent
The influence of the film thickness adjusted by the number of bilayers (n) of {PAA-PBA/Dex}n films on the pH-dependent on–off property of the films toward Fc(COOH)2 was studied by CV. For {PAA-PBA/Dex}1 films with n = 1, while the CV response of the probe at pH 5.5 was larger than that at pH 8.0, the CV peak pair of Fc(COOH)2 at pH 8.0 was still clearly observed and quite large (Supplementary materials Fig. S7). This is probably due to relatively small amounts of negative charges for one PAA-PBA/Dex bilayer at pH 8.0, which had weaker electrostatic repulsion with negatively charged Fc(COOH)2 [13]. In addition, the relatively thin films with n = 1 may not completely cover the electrode surface, and the underneath CS layer carrying positive charges may be partially exposed and tend to attract Fc(COOH)2 . With the increase of n, the CV Ipa of the probe gradually decreased at both pH 5.5 and 8.0 (Fig. 5) because the permeability of the films became poorer with thicker films. In pH 8.0 solutions, the Ipa decreased dramatically with n from 1 to 5, and the CV peak almost disappeared when n ≥ 5. This is understandable since the thicker films contain larger amounts of negative charges at this pH, leading to the stronger electrostatic repulsion with the probe. The significant difference in Ipa between pH 5.5 and 8.0 was observed for the films with n = 5, the {PAA-PBA/Dex}5 films were thus usually used in the present work. The influence of outermost layer of PAA-PBA/Dex multilayer films was also investigated (Supplementary materials Fig. S8). At pH 5.5, Fc(COOH)2 demonstrated the nearly identical CVs for {PAAPBA/Dex}4 /PAA-PBA and {PAA-PBA/Dex}5 films with quite large peak heights; at pH 8.0, both films were all at the off state toward the probe. These results suggest that the interpenetration or intermixing of the neighboring layers of the films may happen to a great extent, which is a common phenomenon in LbL assembly [57]. 3.6. pH-controllable bioelectrocatalysis for {PAA-PBA/Dex}5 films The pH-sensitive switching property of {PAA-PBA/Dex}5 films toward Fc(COOH)2 could be utilized to modulate the bioelectrocatalytic oxidation of glucose in the presence of GOD in solution. Herein, Fc(COOH)2 not only acted as the electroactive probe for the films but also acted as the electron transfer mediator to shuttle electrons between GOD and electrodes in bioelectrocatalysis. When the {PAA-PBA/Dex}5 film electrode was placed in the pH 5.5 solution containing glucose, GOD and Fc(COOH)2 , the CV oxidation peak increased dramatically, accompanied by the decrease
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glucose was less than 8 mmol dm−3 , much smaller than that for causing the break of bonding between PAA-PBA and Dex, the films should be quite stable in the bioelectrocatalysis. To support this point of view, {PAA-PBA/Dex}5 films were immersed in pH 5.5 buffers containing 20 mmol dm−3 glucose, and the CV response of Fc(COOH)2 in pH 5.5 solutions at the film electrodes was monitored at different immersing time. During three days of immersion, the CV peak potentials and currents of Fc(COOH)2 maintained nearly the same as their initial values, respectively, suggesting that the films are indeed stable in the presence of glucose in the present work.
3.7. pH-switchable bioelectrocatalysis for {PAA-PBA/GOD}n films
Fig. 6. (A) CVs of {PAA-PBA/Dex}5 films at 0.01 V s−1 in buffers containing 0.5 mmol dm−3 Fc(COOH)2 , 1.0 g dm−3 GOD and 4.0 mmol dm−3 glucose at pH (a) 5.5 and (b) 8.0. (B) Dependence of CV Ipa on the cycle number when the solution pH switched between pH 5.5 () and 8.0 () for the films.
or even disappearance of the reduction peak (Supplementary materials Fig. S9A). The Ipa increased initially with the concentration of glucose in the range of 0.05–4.00 mmol dm−3 , and then tended to level off (Fig. S9B). All these results are characteristic of electrochemical oxidation of glucose catalyzed by GOD and mediated by Fc(COOH)2 [3,58]. At pH 8.0, however, the electrocatalytic response was quite small or even could hardly be observed in the same Fc(COOH)2 GOD-glucose solution system for the films (Fig. 6A). This is because the films are at the off state toward Fc(COOH)2 at this pH, and the catalytic cycle was interrupted. Therefore, the bioelectrocatalytic oxidation of glucose could be switched to the on or off state by tuning the surrounding pH with this system. Moreover, this pHswitchable bioelectrocatalysis was reversible, and the on–off cycle could be repeated for at least several times between pH 5.5 and 8.0 (Fig. 6B). Meanwhile, the CV oxidation peak current ratio of Fc(COOH)2 at pH 5.5 and 8.0, Ipa5.5 /Ipa8.0 , could be amplified by the bioelectrocatalysis from 3 to 10 (Fig. 6A). In control experiments, the electrocatalysis of glucose mediated by Fc(COOH)2 and catalyzed by GOD at the bare PG electrodes could be clearly observed at both pH 5.5 and 8.0 with similar wave heights (Supplementary materials Fig. S10), indicating that the pH-controllable on–off bioelectrocatalysis is not due to the property of Fc(COOH)2 -GODglucose system itself but should be attributed to the interaction between the films and the probe. Theoretically, monosaccharide glucose in solution can also combine PAA-PBA through the boronic acid-diol specific interaction, and thus it is possible for glucose to compete with Dex in the {PAAPBA/Dex}n films and lead to the decomposition of the films. In practice, however, the films possess excellent stability, only the glucose with high concentration (over 20 mmol dm−3 ) and a long period of contacting time can make the films collapse [21] because the higher stability of PBA-Dex complex than that of PBA-glucose [34,36]. In our experiments, since the maximum concentration of
The enzymes dissolved in solution cannot be used efficiently in bioelectrocatalysis, and they are difficult to recover and reuse. The effective immobilization of enzymes on electrode surface without altering their structure and bioactivity is thus highly desirable, especially for the development of biosensors and other biodevices [59,60]. GOD is a kind of glycoenzymes that contain lots of sugar residues on their surface [39,61]. We thus expected that GOD and PAA-PBA could be assembled into {PAA-PBA/GOD}n LbL films by the boronic acid-diol specific recognition between them, so that the GOD could be immobilized on the surface of electrodes. The assembly of {PAA-PBA/GOD}n LbL films on PG/CS surface was confirmed by CV with Fe(CN)6 3− as the electroactive probe (Fig. 7A). When a PAA-PBA/GOD bilayer was assembled on the PG/CS surface, the CV peak currents of the probe was significantly suppressed (curve c), indicating that a bilayer film was formed on the electrode surface and the electron exchange between the probe and the underlying electrodes was hindered. With the increase of n value for {PAA-PBA/GOD}n films, the Ipa of Fe(CN)6 3− continuously decreased, accompanied by the increase of Ep value (Fig. 7A), suggesting that the GOD multilayer films are successfully fabricated on the surface of PG electrodes. The stability of {PAA-PBA/GOD}5 films was examined by CV with Fc(COOH)2 as the probe, and the results demonstrated that the films were quite stable in both pH 5.5 and 8.0 solutions at least for three days. At pH 8.0, GOD carries net negative surface charges with its pI at 4.2 [62] and would repel negatively charged PAA-PBA. However, the {PAA-PBA/GOD}5 films were still quite stable and could not be disintegrated because the main driving force to combine GOD and PAA-PBA is the boronic acid-diol specific interaction, which would overcome the electrostatic repulsion between the similarly charged species. This is one of the advantages of {PAA-PBA/GOD}n films over other electrostatic LbL films, since the net film charge of the {PAA-PBA/GOD}n films can be modulated by surrounding pH without losing the film stability. {PAA-PBA/GOD}5 films also showed pH-sensitive CV on–off behavior toward Fc(COOH)2 , and the films were at the on state at pH 5.5 and at the off state at pH 8.0 (Fig. 7B), similar to the situation of {PAA-PBA/Dex}5 films. The pH-dependent switching property of Fc(COOH)2 at the {PAA-PBA/GOD}5 film electrodes was reversible and could be applied to control the electrocatalytic oxidation of glucose in solution. In pH 5.5 buffers containing Fc(COOH)2 and glucose, the obvious increase of CV oxidation peak current was observed for the films, accompanied by the decrease or disappearance of the reduction peak (Fig. 7C). In pH 8.0 buffers containing the same amount of Fc(COOH)2 and glucose, however, the CV response was too small to be detected. This on–off cycle in bioelectrocatalysis for the system could be repeated for many times by switching the films in the solutions between pH 5.5 and 8.0 (Supplementary materials Fig. S11).
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switch the bioelectrocatalysis of glucose by altering the environmental pH. This is the first report on applying LbL films assembled through boronic acid-diol specific recognition to pH-controllable bioelectrocatalysis. The in-depth understanding of the mechanism of pH-sensitive switching behavior of this model system may guide us to develop new kinds of pH-tunable electrochemical biosensors based on enzymatic electrocatalysis. Acknowledgment The financial support from the National Natural Science Foundation of China (NSFC 20975015 and 20775009) is acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.electacta.2010.09.012. References
Fig. 7. (A) CVs of 1.0 mmol dm−3 K3 Fe(CN)6 at 0.1 V s−1 in pH 7.0 buffers at (a) bare PG electrode, (b) PG/CS films, (c)–(g) {PAA-PBA/GOD}n films assembled on PG/CS electrode surface with n = 1–5. (B) CVs of 0.5 mmol dm−3 Fc(COOH)2 at 0.1 V s−1 for {PAA-PBA/GOD}5 films in buffers at pH (a) 5.5 and (b) 8.0. (C) CVs of {PAAPBA/GOD}5 films at 0.01 V s−1 in solutions containing 0.5 mmol dm−3 Fc(COOH)2 and 5.0 mmol dm−3 glucose at pH (a) 5.5 and (b) 8.0.
4. Conclusions A polyelectrolyte PAA-PBA containing both PBA moieties and carboxylic acid groups is synthesized. PAA-PBA and Dex are then assembled into {PAA-PBA/Dex}n LbL films on electrode surface through boronic acid-diol specific recognition between them. The films exhibit pH-sensitive CV on–off property toward Fc(COOH)2 mainly because the free –COOH groups in PAA-PBA are sensitive to surrounding pH. A series of comparative experiments demonstrate that it is the electrostatic interaction between the films and the probe that plays a key role in deciding the pH-switchable behavior of the system. This pH-responsive property of the system can be used to control or modulate the electrochemical oxidation of glucose catalyzed by GOD and mediated by Fc(COOH)2 through changing the solution pH. Moreover, {PAA-PBA/GOD}n LbL films are also assembled on the surface of electrodes by the boronic acid-diol specific interaction so that the immobilization of enzyme can be realized. The {PAA-PBA/GOD}n films also demonstrate pHtriggered on–off behavior toward Fc(COOH)2 , and can be used to
[1] N.J. Ronkainen, H.B. Halsall, W.R. Heineman, Chem. Soc. Rev. 39 (2010) 1747. [2] A.K. Sarma, P. Vatsyayan, P. Goswami, S.D. Minteer, Biosens. Bioelectron. 24 (2009) 2313. [3] J. Wang, Chem. Rev. 108 (2008) 814. [4] I. Willner, E. Katz, Angew. Chem., Int. Ed. 39 (2000) 1180. [5] E. Katz, V. Privman, Chem. Soc. Rev. 39 (2010) 1835. [6] E. Katz, M. Pita, Chem. Eur. J. 15 (2009) 12554. [7] M. Pita, E. Katz, Electroanalysis 21 (2009) 252. [8] O.A. Loaiza, R. Laocharoensuk, J. Burdick, M.C. Rodriguez, J.M. Pingarron, M. Pedrero, J. Wang, Angew Chem., Int. Ed. 119 (2007) 1530. [9] T.K. Tam, M. Ornatska, M. Pita, S. Minko, E. Katz, J. Phys. Chem. C 112 (2008) 8438. [10] T.K. Tam, J. Zhou, M. Pita, M. Ornatska, S. Minko, E. Katz, J. Am. Chem. Soc. 130 (2008) 10888. [11] H. Yao, N. Hu, J. Phys. Chem. B 114 (2010) 3380. [12] H. Yao, N. Hu, J. Phys. Chem. B 113 (2009) 16021. [13] S. Song, N. Hu, J. Phys. Chem. B 114 (2010) 3648. [14] H. Yao, N. Hu, J. Phys. Chem. B 114 (2010) 9926. [15] G. Decher, J.B. Schneloff (Eds.), Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials, Wiley-VCH, Weinheim, Germany, 2003. [16] Y. Lvov, in: Y. Lvov, H. Möhwald (Eds.), Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology, Marcel Dekker, New York, 2000, p. 125. [17] Y. Lvov, in: R.W. Nalwa (Ed.), Handbook of Surfaces and Interfaces of Materials, vol. 3, Academic Press, San Diego, 2001, p. 170. [18] J.F. Rusling, Z. Zhang, in: R.W. Nalwa (Ed.), Handbook of Surfaces and Interfaces of Materials, vol. 5, Academic Press, San Diego, 2001, p. 33. [19] M. Jiang, A. Eisenberg, G.J. Liu, X. Zhang (Eds.), Macromolecular Self-Assembly, Scientific Press, Beijing, 2008. [20] M. Zayats, E. Katz, I. Willner, J. Am. Chem. Soc. 124 (2002) 14724. [21] T. Levy, C. Dejugnat, G.B. Sukhorukov, Adv. Funct. Mater. 18 (2008) 1586. [22] S. Liu, B. Miller, A. Chen, Electrochem. Commun. 7 (2005) 1232. [23] S. Liu, U. Wollenberger, J. Halamek, E. Leupold, W. Stocklein, A. Warsinke, F.W. Scheller, Chem. Eur. J. 11 (2005) 4239. [24] Y. Ma, L. Qian, H. Huang, X. Yang, J Colloid Interface Sci. 295 (2006) 583. [25] N. Kanayama, H. Kitano, Langmuir 16 (2000) 577. [26] S.Y. Song, H.C. Yoon, Sens. Actuators, B 140 (2009) 233. [27] T.D. James, M.D. Phillips, S. Shinkai (Eds.), Boronic Acids in Saccharide Recognition, RSC Publishing, Cambridge, UK, 2006. [28] D.G. Hall (Ed.), Boronic Acids: Preparation, Applications in Organic Synthesis and Medicine, Wiley-VCH, Weinheim, Germany, 2005. [29] S. Li, E.N. Davis, J. Anderson, Q. Lin, Q. Wang, Biomacromolecules 10 (2009) 113. [30] T. Kawanishi, M.A. Romey, P.C. Zhu, M.Z. Holody, S. Shinkai, J. Fluoresc. 14 (2004) 499. [31] Z. Ding, Y. Guan, Y. Zhang, X.X. Zhu, Soft Matter 5 (2009) 2302. [32] J. Choi, T. Konno, R. Matsuno, M. Takai, K. Ishihara, Colloids Surf. B 67 (2008) 216. [33] J. Choi, T. Konno, M. Takai, K. Ishihara, Biomaterials 30 (2009) 5201. [34] J. Zhang, C.D. Geddes, J.R. Lakowicz, Anal. Biochem. 332 (2004) 253. [35] Y. Miyazaki, K. Yoshimura, Y. Miura, H. Sakashita, K. Ishimaru, Polyhedron 22 (2003) 909. [36] K. Aslan, J. Zhang, J.R. Lakowicz, C.D. Geddes, J. Fluoresc. 14 (2004) 391. [37] A. Kikuchi, K. Suzuki, O. Okabayashi, H. Hoshino, K. Kataoka, Y. Sakurai, T. Okano, Anal. Chem. 68 (1986) 823. [38] J.M. Abad, M. Velez, C. Santamaria, J.M. Guisan, P.R. Matheus, L. Vazquez, I. Gazaryan, L. Gorton, T. Gibson, V.M. Fernandez, J. Am. Chem. Soc. 124 (2002) 12845. [39] S. Liu, L. Bakovic, A. Chen, J. Electroanal. Chem. 591 (2006) 210. [40] BellamyF L.J. (Ed.), The Infra-red Spectra of Complex Molecules, Wiley, New York, 1975.
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H. Yao et al. / Electrochimica Acta 55 (2010) 9185–9192
[41] B.C. Smith (Ed.), Infrared Spectral Interpretation: A Systematic Approach, CRC Press, New York, 1999. [42] B.H. Stuart (Ed.), Infrared Spectroscopy: Fundamentals and Applications, Wiley, New York, 2004. [43] J. Ma, B. Fan, B. Liang, J. Xu, J. Colloid Interface Sci. 341 (2010) 88. [44] S.H. Brewer, A.M. Allen, S.E. Lappi, T.L. Chasse, K.A. Briggman, C.B. Gorman, S. Franzen, Langmuir 20 (2004) 5512. [45] A. Denuziere, D. Ferrier, A. Domard, Carbohydr. Polym. 29 (1996) 317. [46] H.A.O. Hill, Pure Appl. Chem. 59 (1987) 743. [47] K.L. Mittal (Ed.), Acid-Base Interactions: Relevance to Adhesion Science and Technology, Vol.2, VSP, Utrecht, The Netherlands, 2000. [48] H.P. Gregor, L.B. Luttinger, E.M. Loebl, J. Am. Chem. Soc. 76 (1954) 5879. [49] G. Delaittre, J. Nicolas, C. Lefay, M. Save, B. Charleux, Chem. Commun. (2005) 614. [50] A. Laguecir, S. Ulrich, J. Labille, F. Fatin-Rouge, S. Stoll, J. Buffle, Eur. Polym. J. 42 (2006) 1135. [51] E. Katz, I. Willner, Electroanalysis 15 (2003) 913.
[52] A.J. Bard, L.R. Faulkner (Eds.), Electrochemical Methods: Fundamentals and Applications, 2nd ed., Wiley, New York, 2001. [53] J.E.B. Randles, Discuss. Faraday Soc. 1 (1947) 11. [54] M. Motornov, R. Sheparovych, E. Katz, S. Minko, ACS Nano 2 (2008) 41. [55] A.A. Antipova, G.B. Sukhorukov, Adv. Colloid Interface Sci. 111 (2004) 49. [56] A. Calvo, B. Yameen, F.J. Williams, G.J. Soler-Illia, O. Azzaroni, J. Am. Chem. Soc. 131 (2009) 10866. [57] G. Decher, Science 277 (1997) 1232. [58] S.D. Noci, M. Frasconi, G. Favero, M. Tosi, T. Ferri, F. Mazzei, Electroanalysis 20 (2008) 163. [59] M.F. Chaplin, C. Bucke (Eds.), Enzyme Technology, Cambridge University Press, Cambridge, UK, 1990. [60] A.P.F. Turner, I. Karube, G.S. Wilson (Eds.), Biosensors: Fundamentals and Applications, Oxford University Press, New York, 1987. [61] J. Anzai, Y. Kobayashi, Langmuir 16 (2000) 2851. [62] E.J. Calvo, R. Etchenique, L. Pietrasanta, A. Wolosiuk, Anal. Chem. 73 (2001) 1161.
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
pH-switchable bioelectrocatalysis based on layer-by-layer films assembled through specific boronic acid-diol recognition Huiqin Yao a,b , Fengwei Chang a , Naifei Hu a,∗ a b
Department of Chemistry, Beijing Normal University, Beijing 100875, PR China Department of Chemistry, Ningxia Medical University, Yinchuan 750004, PR China
a r t i c l e
i n f o
Article history: Received 22 July 2010 Received in revised form 1 September 2010 Accepted 4 September 2010 Available online 15 September 2010 Keywords: pH-switchable bioelectrocatalysis Layer-by-layer assembly Boronic acid-diol specific interaction Glucose oxidase Ferrocenedicarboxylic acid
a b s t r a c t Phenylboronic acid (PBA) moieties are grafted onto the backbone of poly(acrylic acid) (PAA), forming the PAA-PBA polyelectrolyte. The PAA-PBA and polysaccharide dextran (Dex) are then assembled layerby-layer (LbL) into {PAA-PBA/Dex}n films on electrode surface through the boronic acid-diol specific recognition between them. The cyclic voltammetric response of ferrocenedicarboxylic acid (Fc(COOH)2 ) at the film electrodes is sensitive to the environmental pH. At pH 5.5, the CV peak currents are quite large, and the films are at the “on” state; at pH 8.0, however, the CV response is significantly suppressed with the films at the “off” state. This pH-sensitive on–off property of the films toward the probe can be used to switch the bioelectrocatalysis of glucose mediated by Fc(COOH)2 in the present of glucose oxidase (GOD) in solution by changing the surrounding pH. Furthermore, PAA-PBA and the glycoenzyme GOD are also assembled into {PAA-PBA/GOD}n LbL films by the boronic acid-diol specific interaction between them, so that the immobilization of the enzyme on electrodes can be realized. The {PAA-PBA/GOD}n films also show pH-sensitive on–off behavior in bioelectrocatalysis of glucose with Fc(COOH)2 as the mediator. This model system may open a new way to establish a foundation for fabricating pH-responsive biosensors based on enzymatic electrocatalysis. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction The bioelectrocatalysis on the basis of various enzymatic reactions nowadays has become an important foundation in the development of biosensors. The high specificity and sensitivity of enzymes in detecting and determining the respective substrates have made this type of electrochemical biosensors more attractive among researchers in recent years [1–3]. In this field, the switchable biosensors based on stimuli-responsive bioelectrocatalysis have aroused increasing interests. In general, the stimuli-controllable bioelectrocatalysis can find its application not only in biosensors but also in bioelectronic devices, biofuel cells, and biocomputing, as well as signal transduction/amplification and information processing [4–8]. Among various external stimuli that activate/deactivate bioelectrocatalysis, pH is the most studied one. By choosing appropriate film-forming materials and selecting proper processing conditions, it is possible to create “smart” interfaces on electrode surface that demonstrate pH-sensitive permeability to electroactive probes, which can be further used to realize the pH-switchable bioelectrocatalysis [9–14]. For example, poly(4-vinyl pyridine) (P4VP) functionalized with Os-complex redox units (P4VP-Os) was
∗ Corresponding author. Tel.: +86 10 5880 5498; fax: +86 10 5880 2075. E-mail address: [email protected] (N. Hu). 0013-4686/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2010.09.012
grafted onto ITO electrodes by Katz and co-workers [9,10]. The formed P4VP-Os brush exhibited the reversible pH-sensitive transition between the swollen and shrunken structures, resulting in pH-dependent switching of the cyclic voltammetric (CV) response of Os-complex units. This property of the brush could be applied to reversibly activate/deactivate the electrocatalytic oxidation of glucose in the presence of glucose oxidase (GOD) by changing environmental pH chemically or biochemically. However, in general, the pH-controllable bioelectrocatalysis has only received a limited attention, and the in-depth understanding of the mechanism is still a great challenge. Among various methods to construct the smart interface, the layer-by-layer (LbL) assembly demonstrates distinguished advantages in its precise control of film thickness at a nanometer scale according to a predesigned architecture and in its extremely simple procedure and high versatility in the assembly [15,16]. The building blocks of LbL films are originally polyelectrolytes, and now extended to proteins, nanoparticles, and other species [17,18]. The driving force of LbL assembly is usually electrostatic interaction between oppositely charged species, but now expanded to a variety of non-electrostatic interactions including hydrogen bonding, hydrophobic interaction, ion-dipole interaction, and biospecific recognition [15,19]. For example, in our previous work, lectinsugar biospecific interaction was used to construct {Con A/Dex}n LbL films by concanavalin A (Con A) and dextran (Dex) on elec-
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trode surface, and the films showed a reversible pH-controlled bioelectrocatalysis in the presence of mediator, GOD, and glucose [12]. Boronic acid-diol recognition is another kind of specific interactions between boronic acid groups and diol units and has attracted more attention in scientific researches, especially in constructing films [20–26]. For example, phenylboronic acid (PBA) and its derivatives can rapidly and reversibly form covalent band with various 1,2- and 1,3-cis-diol compounds such as polyols, carbohydrates and sugar residues located on the surface of glycoenzymes to generate five- or six-membered cyclic boronate ester complexes [27,28]. Since monosaccharides usually have five –OH groups, and one PBA group can react with two –OH groups, the 1:2 sugar–PBA complex is often formed [27,29,30]. Boronic acid-diol interaction has also been used to assemble LbL films [21,24,31–33]. For instance, poly(vinylalcohol) or mannan can be assembled with polyelectrolytes containing PBA moieties into LbL films [21,24]. Dextran (Dex) is a glucose-based polysaccharide and contains many glucose residues on its backbone, which can bind to PBA groups [34–36]. While the specific affinity between Dex and PBA has been studied, the LbL assembly of Dex with polyelectrolytes containing PBA moieties has not been reported until now. In addition, some glycoenzymes, such as horseradish peroxidase (HRP) and GOD, can combine with some compounds containing PBA groups via the boronic acid-diol specific interaction, and form self-assembled monolayer films on electrode surface [20,22,23,26,37–39]. However, no study on the LbL films assembled by glycoenzymes and polymers containing PBA groups has been reported up to now. In the present work, PBA moieties were grafted onto the backbone of poly(acrylic acid) (PAA) through the condensation reaction between aminophenyl-boronic acid and carboxylic acid group of PAA according to the literature [21], designated as PAA-PBA. In addition to a large fraction of PBA moieties in PAA-PBA, a considerable amount of free carboxylic acid groups remained and would be sensitive to surrounding pH. The {PAA-PBA/Dex}n LbL films were then assembled on the surface of pyrolytic graphite (PG) electrodes through the boronic acid-diol specific recognition between PAAPBA and Dex. Electroactive ferrocenedicarboxylic acid (Fc(COOH)2 ) demonstrated pH-sensitive on–off behavior in its electrochemical response at the {PAA-PBA/Dex}n film electrodes. At pH 5.5, the CV response of the probe was quite large at the film electrodes, and the films were at the on state; at pH 8.0, however, the CV response of the probe was greatly suppressed, and the films were at the off state. The mechanism of the pH-dependent on–off property of the films toward the probe was further explored with comparative studies, and believed to be mainly attributed to the electrostatic interaction between the films and probe. The pHswitchable property of the films toward Fc(COOH)2 was further used to activate/deactivate the electrocatalytic oxidation of glucose catalyzed by GOD. In addition, PAA-PBA and GOD were successfully assembled into {PAA-PBA/GOD}n LbL films on PG electrode surface also by the boronic acid-diol specific interaction so that the enzyme could be immobilized. The {PAA-PBA/GOD}n films also demonstrated the pH-sensitive on–off property toward Fc(COOH)2 , which could be used to switch the bioelectrocatalysis of glucose by changing the environmental pH. CV, electrochemical impedance spectroscopy (EIS), quartz crystal microbalance (QCM), scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy were used to characterize the system. To the best of our knowledge, this is the first report on the pH-switchable bioelectrocatalysis based on LbL films assembled through boronic acid-diol specific recognition. This study provided a novel example to construct a smart interface and established a foundation for fabricating pH-controllable biosensors based on the LbL assembly and enzymatic electrocatalysis.
Scheme 1. Synthetic scheme of PAA-PBA.
2. Experimental 2.1. Reagents Poly(acrylic acid) (PAA, MW ≈ 100 000, 35 wt.% solution in water), 3-aminophenyl-boronic acid hemisulfate salt (APBA), 4-(2-hydroxyerhyl) piperazine-1-ethanesulfonic acid (HEPES, 99.5%), N-hydroxysulfosuccinimide sodium salt (NHS), 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), chitosan (CS, the degree of deacetylation is more than 85%, MW ≈ 200 000), dextran from leuconostoc mesenteroides (Dex, MW ≈ 200 000), glucose oxidase (GOD, E.C. 1.1.3.4, type VII, MW ≈ 160 000, 192 000 units g−1 ), 1,1’-ferrocenedicarboxylic acid (Fc(COOH)2 ), hexaammineruthenium(III) chloride (Ru(NH3 )6 Cl3 ), ferrocenemethanol (FcOH), and 3-mercapto-1-propanesulfonate (MPS, 90%) were purchased from Sigma–Aldrich. Potassium ferricyanide (K3 Fe(CN)6 ) and potassium ferrocyanide (K4 Fe(CN)6 ) were obtained from Beijing Chemical Engineering Plant. All other reagents were of analytical grade. Buffers were usually 0.05 mol dm−3 sodium acetate (pH 4–6) or 0.05 mol dm−3 potassium dihydrogen phosphate (pH 6.5–8), all containing 0.1 mol dm−3 NaCl. The pH value of buffers was adjusted to the desired value with dilute HCl or NaOH solutions. The d-glucose stock solutions were allowed to mutarotate at room temperature for 24 h before being used. Solutions were prepared with water purified twice by ion exchange and subsequent distillation. 2.2. Synthesis of PAA-PBA PAA-PBA was synthesized by reaction between PAA and APBA in the presence of NHS and EDC according to the literature [21], and the process is schematically depicted in Scheme 1. In brief, 0.57 g of PAA solution containing 2.77 mmol equiv. of monomer was diluted by HEPES buffers (0.02 dm3 , 50 mmol dm−3 ) and the pH was adjusted to 8.5, APBA (0.02 dm3 , 0.061 mol dm−3 ) in the HEPES buffers were then added. NHS (0.004 dm3 , 0.031 mol dm−3 ) in the HEPES buffers were added dropwise into the solution and stirred for 10 min. EDC (0.004 dm3 , 0.31 mol dm−3 ) in the HEPES buffers were then added to the mixture, followed by stirring of 12 h. The solution was then extensively dialyzed against water for 1 week in order to remove all low-molecular-weight residues, and the white fluffy solid PAA-PBA was obtained after freeze drying. 2.3. Film assembly For electrochemical study, LbL films were grown on basal plane PG (Advanced Ceramics) disks electrodes (geometric area 0.16 cm2 ). Prior to assembly, PG electrodes were abraded on 320grit metallographic sandpaper while flushing with water. After being ultrasonicated in water for 30 s and dried in air, the PG electrodes were first immersed in positively charged CS solutions (1 g dm−3 , pH 5.0) for 20 min to adsorb CS as the precursor layer. The
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PG/CS electrodes were then alternately immersed into negatively charged PAA-PBA (1 g dm−3 , pH 9.0) and neutral Dex (1 g dm−3 , pH 9.0) aqueous solutions for 20 min each with intermediate water rinsing and air stream drying, forming a PAA-PBA/Dex bilayer. This cycle was repeated until the desired number of bilayers (n) was obtained on the PG/CS surface, designated as {PAA-PBA/Dex}n . {PAA-PBA/GOD}n LbL films were assembled on the PG/CS surface with the same way with 1 g dm−3 GOD solution at pH 9.0. For quartz crystal microbalance (QCM) studies, gold-coated quartz crystal resonator electrodes (International Crystal Manufacturing Co.) were first cleaned by a few drops of a freshly prepared piranha solution (3:7 volume ratio of 30% H2 O2 and concentrated H2 SO4 . Caution: the piranha solution reacts violently with most organic materials and must be handled with extreme care, and only a small volume should be prepared at any time) on each side for 10 min, and then washed thoroughly with water and a final rinse with ethanol. The QCM gold electrodes were then immersed in 4 mmol dm−3 MPS ethanol solutions for 24 h to chemisorb an MPS monolayer on gold surface by formation of Au–S bond between Au and MPS, making the Au surface become negatively charged. The CS precursor layer and following {PAA-PBA/Dex}n LbL films were then assembled on the Au/MPS surface in the same way as on the PG electrodes. 2.4. Apparatus and procedures A CHI 660A or CHI 621B electrochemical workstation (CH Instruments) was used for electrochemical measurements. A typical three-electrode cell was used with a saturated calomel electrode (SCE) as the reference, a platinum flake as the counter, and the PG disk electrode with films as the working electrode. The solution was purged with high-purity nitrogen for at least 10 min before electrochemical measurements. The nitrogen atmosphere was then kept above the cell for the entire experiment. Electrochemical impedance spectroscopy (EIS) measurements were performed in 1:1 K4 Fe(CN)6 /K3 Fe(CN)6 mixture solutions with total concentration of 5 mmol dm−3 , and a sinusoidal potential modulation with amplitude of ±5 mV and frequency from 105 to 0.1 Hz was superimposed on the formal potential of Fe(CN)6 3−/4− redox couple at 0.17 V vs SCE. QCM was performed with a CHI 420 electrochemical analyzer (CH Instruments). The quartz crystal resonator (AT-cut) has a fundamental resonance frequency of 8 MHz and is covered by thin gold films on both sides (geometric area 0.196 cm2 per one side). Scanning electron microscopy (SEM) was performed using an S4800 scanning electron microscope (Hitachi) with an acceleration voltage of 3 kV. The CS/{PAA-PBA/Dex}5 films assembled on the PG electrodes were used as the sample. Before the SEM imaging, the surface of samples was coated by thin Pt films with an E-1045 sputtering coater (Hitachi). The Fourier-transform infrared (FTIR) spectra were recorded at a resolution of 4 cm−1 by Nexus 670 Fourier-transform infrared spectrometer (Nicolet). Elemental analysis was performed by a Vario EL CHNOS Elementaranalysator (Elementar Analysesystem GmbH). All experiments were performed at ambient temperature of 20 ± 2 ◦ C. 3. Results and discussion 3.1. Preparation of PAA-PBA Poly(acrylic acid) (PAA) partially grafted by phenyl-boronic acid (PBA) was synthesized by reaction between PAA and APBA in the presence of NHS and EDC according to the literature [21], des-
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Fig. 1. FTIR spectra of (a) PAA, (b) PBA, and (c) PAA-PBA.
ignated as PAA-PBA (Scheme 1). The synthesis of PAA-PBA was confirmed by FTIR spectroscopy (Fig. 1), and the assignment of the observed bands or peaks were discussed as below according to the literatures [40–42]. The characteristic peak at 1712 cm−1 assigned to the C O stretching vibration of the carboxylic acid groups [40–43] of PAA (curve a) was also observed for PAA-PBA (curve c). In addition, the peak at 2950 cm−1 for the stretching mode of CH2 group [42] was observed for both PAA and PAA-PBA. These results indicate that the PAA-PBA keeps the polymer structure similar to that of PAA with some remaining COOH groups. It is noteworthy that the characteristic amide I band centered at 1656 and amide II band at 1550 cm−1 were detected in PAA-PBA (curve c) [40–43], but were not observed in both PAA (curve a) and PBA (curve b). In the meantime, the bending vibration peak of primary amino groups in PBA at 1630 cm−1 [40–42] disappeared in PAAPBA (curve c). These suggest the formation of new amide bond and the successful cross-linking between amino groups of PBA and some carboxylic acid groups of PAA [43]. The typical B–O stretching peak at 1340 cm−1 [42,44], and the characteristic phenyl ring bands at 1490 and 1440 cm−1 [40–42] observed in PBA (curve b) were also detected in PAA-PBA (curve c), demonstrating that the phenyl-boronic acid is successfully grafted onto the PAA polymer. According to the result of elemental analysis of PAA-PBA, the fraction of the carboxylic acid groups in PAA that had been crosslinked with PBA was estimated to be 41%. While this value is only a rough approximation, it is no doubt that a considerable amount of carboxylic acid groups in PAA-PBA keeps “free” after the crosslinking reaction. 3.2. Assembly of {PAA-PBA/Dex}n LbL films CS carries positive charges at pH 5.0 due to its pKa at about 6.5 [45], and was adsorbed on the negatively charged PG surface [46] as the precursor layer. PAA-PBA contained a considerable amount of free –COOH groups in its backbone. With the pKa of PAA at about 6.0 in solution [47–50], almost all these free carboxylic acid groups in PAA-PBA would be ionized and become negatively charged at pH 9.0, and thus could be adsorbed on the oppositely charged PG/CS surface by electrostatic attraction. Dex is a neutral polysaccharide carrying no charge, and could be strongly combined with PAA-PBA through boronic acid-diol specific recognition, forming a PAA-PBA/Dex bilayer. By repeating this cycle, the assembly of {PAAPBA/Dex}n LbL films on the PG/CS surface can thus be realized. The assembly of {PAA-PBA/Dex}n LbL films on PG/CS surface was first confirmed by CV with Fc(COOH)2 as the electroactive probe (Fig. 2A). At bare PG and PG/CS film electrodes, Fc(COOH)2 in pH 7.0 buffers displayed a well-defined and nearly reversible CV oxidation-reduction peak pair at about 0.41 V. However, when
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Fig. 3. QCM frequency shift (−F) with assembly step for assembly of {PAAPBA/Dex}n LbL films on Au/MPS/CS surface: PAA-PBA (䊉) and Dex () adsorption steps.
Fig. 2. (A) CVs of 0.5 mmol dm−3 Fc(COOH)2 at 0.1 V s−1 in pH 7.0 buffers at (a) bare PG electrode, (b) PG/CS films, (c)–(i) PG/CS/{PAA-PBA/Dex}n films with n = 1–7. (B) Dependence of CV oxidation peak current (Ipa ) and CV peak separation (Ep ) of Fc(COOH)2 on the number of bilayers (n) of {PAA-PBA/Dex}n films.
{PAA-PBA/Dex}n LbL films were fabricated on the PG/CS surface, the CV response of probe was severely suppressed, indicating that a barrier was formed on the electrode surface and the probe was hindered from exchanging electrons with underlying PG electrodes. The oxidation peak current (Ipa ) of Fc(COOH)2 decreased with the number of bilayers (n) of {PAA-PBA/Dex}n films, accompanied by the increase of peak separation (Ep = Epa − Epc ) (Fig. 2B), all suggesting that the multilayer films are successfully fabricated on the PG/CS surface. The growth of {PAA-PBA/Dex}n LbL films on PG/CS surface was further confirmed by EIS with Fe(CN)6 3−/4− as the electroactive probe. For bare PG and PG/CS film electrodes, the EIS response of Fe(CN)6 3−/4− in pH 7.0 buffers exhibited a Warburg line in a very wide frequency range (Supplementary materials Fig. S1A, curves a and b), respectively, characteristic of a diffusion-controlled electrochemical process. After the assembly of {PAA-PBA/Dex}n films on the PG/CS surface, semicircles in the high-frequency domain were observed, and the diameter of semicircles became larger with the increase of n values (curves c–i), indicating that the multilayer behaved as a physical barrier and limited the access of the probe to the electrode surface. The diameter of the semicircle usually equals the charge transfer resistance (Rct ) of the probe in electron transfer and the Rct value estimated by using the Randles equivalent circuit model [51–53] showed a nearly linear relationship with n for {PAAPBA/Dex}n films (Supplementary materials Fig. S1B), indicating a roughly uniform growth of the films in the assembly. QCM was also used to characterize the growth of {PAAPBA/Dex}n films on Au/MPS/CS surface (Fig. 3). The QCM resonance frequency decrease (−F) usually reflects the mass increase on QCM gold electrodes. The −F value increased with the adsorption step in a roughly linear mode, suggesting again the successful and uniform assembly of {PAA-PBA/Dex}n LbL films. The stability of {PAA-PBA/Dex}5 films on PG/CS surface was examined by CV with Fe(CN)6 3− as the electroactive probe. For the study of short-term stability, 30 CV cycles were continuously performed in pH 5.5 buffers, and the peak positions and heights of the probe showed no change with the cycle. For the study of long-term
stability, the film electrodes were stored in pH 5.5 blank buffers for most of the storage time, and periodically placed in Fe(CN)6 3− solutions at the same pH for CV testing. After three days of storage, the peak potentials of the probe maintained the same position, and the peak currents decreased by only about 15% of their initial values, suggesting that the films are quite stable. This also indicates that the boronic acid-diol specific recognition between PAA-PBA and Dex is quite strong and can afford long time of immersion in buffers and repeated CV cycling. 3.3. pH-sensitive on–off property of Fc(COOH)2 at {PAA-PBA/Dex}5 film electrodes The solution pH had great influence on the CV response of Fc(COOH)2 for {PAA-PBA/Dex}5 films (Supplementary materials Fig. S2). While Fc(COOH)2 showed relatively large Ipa and quite small Ep in the range of pH 5.0–6.5, the Ipa decreased drastically at pH between 7.0 and 8.0, accompanied by the great increase of Ep . For instance, at pH 5.5, a quite large quasi-reversible CV peak pair was observed (Fig. 4A, curve a), and Ipa showed a linear relationship with square root of scan rates from 0.01 to 1.0 V s−1 (Supplementary materials Fig. S3), suggesting the diffusion-controlled behavior of the probe at the film electrodes. However, when the films were placed in pH 8.0 buffers containing the same amount of Fc(COOH)2 , the CV response was significantly suppressed and could even hardly be observed (Fig. 4A, curve b). This pH-sensitive CV behavior of Fc(COOH)2 should be attributed to the interaction between the films and the probe, since in control experiments, the CV behavior of Fc(COOH)2 at bare PG electrodes was essentially pH-independent (Supplementary materials Fig. S4). By defining the CV response of Fc(COOH)2 at pH 5.5 for the films as the “on” state and that at pH 8.0 as the “off” state, the pH-triggered “on–off” switching property of the films was quite reversible. By switching the film electrode in the probe solutions between pH 5.5 and 8.0, the corresponding CV Ipa could periodically change between the on and off states for many cycles (Fig. 4B). 3.4. Possible mechanism Typically, there are two types of mechanism for pH-sensitive permeation behavior of polyelectrolyte films toward probes. One is mainly controlled by the structure change of films with surrounding pH [9,10,54,55], and the other is mainly tuned by the electrostatic interaction between probes and films, where the surface charge of films can be switched by external pH [11–13,56]. To explore the possible change of film structure induced by environmental pH, the surface morphology of {PAA-PBA/Dex}5 films was examined by SEM after the films were treated with pH 5.5 and 8.0 solutions, respectively (Supplementary materials Fig. S5). With
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Fig. 5. Influence of the number of bilayers (n) for {PAA-PBA/Dex}n films on CV Ipa of 0.5 mmol dm−3 Fc(COOH)2 in buffers at pH 5.5 () and 8.0 () at 0.1 V s−1 .
and demonstrated quite large peak heights at both pH 5.5 and 8.0 (Fig. S6D). All these results suggest that it is the electrostatic interaction between {PAA-PBA/Dex}5 films and probes that controls the pH-sensitive CV on–off property of the system. 3.5. Influencing factors
Fig. 4. (A) CVs of 0.5 mmol dm−3 Fc(COOH)2 at 0.1 V s−1 for {PAA-PBA/Dex}5 films in buffers at pH (a) 5.5 and (b) 8.0. (B) Dependence of CV Ipa of Fc(COOH)2 on the cycle number when the solution pH switched between pH 5.5 () and 8.0 () for the same {PAA-PBA/Dex}5 films.
the same magnification, the films that had been treated with pH 5.5 and 8.0 buffers showed no substantial difference in the surface topography, suggesting that the structure of the films is not very sensitive to the environmental pH at least with the present magnification. Consequently, the pH-sensitive on–off property of {PAA-PBA/Dex}5 films toward Fc(COOH)2 should be most probably attributed to the electrostatic interaction between the films and the probe. Fc(COOH)2 is a weak acid and carries negatively charges at pH ≥ 4.0 due to its ionization [13]. In {PAA-PBA/Dex}5 films, while the Dex component is a neutral polymer carrying no charge, the PAA-PBA constituent is pH-sensitive since it contains a considerable amount of free –COOH groups that is sensitive to environmental pH. At pH 8.0, PAA-PBA carries negative charges since almost all free –COOH groups in the films would be ionized with pKa of PAA at about 6.0 [47–50]. The repulsion between the films and the probe at this pH would hinder the probe to enter the films and block the electron exchange of the probe with underlying electrodes, resulting in the off state of the films. In contrast, at pH 5.5, the films would carry no charge since most of the –COOH groups in the films would be protonated. Without the electrostatic repulsion, the probe would diffuse through the films more easily, leading to the larger CV response. To support this speculation, other electroactive probes with different charges were examined by CV for the films at different pHs. Toward negatively charged Fe(CN)6 3− and Fc(COOH), the {PAAPBA/Dex}5 films demonstrated on state at pH 5.5 and off state at pH 8.0 (Supplementary materials Fig. S6A and B), in good agreement with the pH-dependent behavior of Fc(COOH)2 . However, for positively charged Ru(NH3 )6 3+ , its pH-sensitive property was completely opposite to that of Fc(COOH)2 (Fig. S6C). That is, the films were at the on state at pH 8.0 and at the off state at pH 5.5. For neutral probe FcOH, its CV response was pH-independent
The influence of the film thickness adjusted by the number of bilayers (n) of {PAA-PBA/Dex}n films on the pH-dependent on–off property of the films toward Fc(COOH)2 was studied by CV. For {PAA-PBA/Dex}1 films with n = 1, while the CV response of the probe at pH 5.5 was larger than that at pH 8.0, the CV peak pair of Fc(COOH)2 at pH 8.0 was still clearly observed and quite large (Supplementary materials Fig. S7). This is probably due to relatively small amounts of negative charges for one PAA-PBA/Dex bilayer at pH 8.0, which had weaker electrostatic repulsion with negatively charged Fc(COOH)2 [13]. In addition, the relatively thin films with n = 1 may not completely cover the electrode surface, and the underneath CS layer carrying positive charges may be partially exposed and tend to attract Fc(COOH)2 . With the increase of n, the CV Ipa of the probe gradually decreased at both pH 5.5 and 8.0 (Fig. 5) because the permeability of the films became poorer with thicker films. In pH 8.0 solutions, the Ipa decreased dramatically with n from 1 to 5, and the CV peak almost disappeared when n ≥ 5. This is understandable since the thicker films contain larger amounts of negative charges at this pH, leading to the stronger electrostatic repulsion with the probe. The significant difference in Ipa between pH 5.5 and 8.0 was observed for the films with n = 5, the {PAA-PBA/Dex}5 films were thus usually used in the present work. The influence of outermost layer of PAA-PBA/Dex multilayer films was also investigated (Supplementary materials Fig. S8). At pH 5.5, Fc(COOH)2 demonstrated the nearly identical CVs for {PAAPBA/Dex}4 /PAA-PBA and {PAA-PBA/Dex}5 films with quite large peak heights; at pH 8.0, both films were all at the off state toward the probe. These results suggest that the interpenetration or intermixing of the neighboring layers of the films may happen to a great extent, which is a common phenomenon in LbL assembly [57]. 3.6. pH-controllable bioelectrocatalysis for {PAA-PBA/Dex}5 films The pH-sensitive switching property of {PAA-PBA/Dex}5 films toward Fc(COOH)2 could be utilized to modulate the bioelectrocatalytic oxidation of glucose in the presence of GOD in solution. Herein, Fc(COOH)2 not only acted as the electroactive probe for the films but also acted as the electron transfer mediator to shuttle electrons between GOD and electrodes in bioelectrocatalysis. When the {PAA-PBA/Dex}5 film electrode was placed in the pH 5.5 solution containing glucose, GOD and Fc(COOH)2 , the CV oxidation peak increased dramatically, accompanied by the decrease
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glucose was less than 8 mmol dm−3 , much smaller than that for causing the break of bonding between PAA-PBA and Dex, the films should be quite stable in the bioelectrocatalysis. To support this point of view, {PAA-PBA/Dex}5 films were immersed in pH 5.5 buffers containing 20 mmol dm−3 glucose, and the CV response of Fc(COOH)2 in pH 5.5 solutions at the film electrodes was monitored at different immersing time. During three days of immersion, the CV peak potentials and currents of Fc(COOH)2 maintained nearly the same as their initial values, respectively, suggesting that the films are indeed stable in the presence of glucose in the present work.
3.7. pH-switchable bioelectrocatalysis for {PAA-PBA/GOD}n films
Fig. 6. (A) CVs of {PAA-PBA/Dex}5 films at 0.01 V s−1 in buffers containing 0.5 mmol dm−3 Fc(COOH)2 , 1.0 g dm−3 GOD and 4.0 mmol dm−3 glucose at pH (a) 5.5 and (b) 8.0. (B) Dependence of CV Ipa on the cycle number when the solution pH switched between pH 5.5 () and 8.0 () for the films.
or even disappearance of the reduction peak (Supplementary materials Fig. S9A). The Ipa increased initially with the concentration of glucose in the range of 0.05–4.00 mmol dm−3 , and then tended to level off (Fig. S9B). All these results are characteristic of electrochemical oxidation of glucose catalyzed by GOD and mediated by Fc(COOH)2 [3,58]. At pH 8.0, however, the electrocatalytic response was quite small or even could hardly be observed in the same Fc(COOH)2 GOD-glucose solution system for the films (Fig. 6A). This is because the films are at the off state toward Fc(COOH)2 at this pH, and the catalytic cycle was interrupted. Therefore, the bioelectrocatalytic oxidation of glucose could be switched to the on or off state by tuning the surrounding pH with this system. Moreover, this pHswitchable bioelectrocatalysis was reversible, and the on–off cycle could be repeated for at least several times between pH 5.5 and 8.0 (Fig. 6B). Meanwhile, the CV oxidation peak current ratio of Fc(COOH)2 at pH 5.5 and 8.0, Ipa5.5 /Ipa8.0 , could be amplified by the bioelectrocatalysis from 3 to 10 (Fig. 6A). In control experiments, the electrocatalysis of glucose mediated by Fc(COOH)2 and catalyzed by GOD at the bare PG electrodes could be clearly observed at both pH 5.5 and 8.0 with similar wave heights (Supplementary materials Fig. S10), indicating that the pH-controllable on–off bioelectrocatalysis is not due to the property of Fc(COOH)2 -GODglucose system itself but should be attributed to the interaction between the films and the probe. Theoretically, monosaccharide glucose in solution can also combine PAA-PBA through the boronic acid-diol specific interaction, and thus it is possible for glucose to compete with Dex in the {PAAPBA/Dex}n films and lead to the decomposition of the films. In practice, however, the films possess excellent stability, only the glucose with high concentration (over 20 mmol dm−3 ) and a long period of contacting time can make the films collapse [21] because the higher stability of PBA-Dex complex than that of PBA-glucose [34,36]. In our experiments, since the maximum concentration of
The enzymes dissolved in solution cannot be used efficiently in bioelectrocatalysis, and they are difficult to recover and reuse. The effective immobilization of enzymes on electrode surface without altering their structure and bioactivity is thus highly desirable, especially for the development of biosensors and other biodevices [59,60]. GOD is a kind of glycoenzymes that contain lots of sugar residues on their surface [39,61]. We thus expected that GOD and PAA-PBA could be assembled into {PAA-PBA/GOD}n LbL films by the boronic acid-diol specific recognition between them, so that the GOD could be immobilized on the surface of electrodes. The assembly of {PAA-PBA/GOD}n LbL films on PG/CS surface was confirmed by CV with Fe(CN)6 3− as the electroactive probe (Fig. 7A). When a PAA-PBA/GOD bilayer was assembled on the PG/CS surface, the CV peak currents of the probe was significantly suppressed (curve c), indicating that a bilayer film was formed on the electrode surface and the electron exchange between the probe and the underlying electrodes was hindered. With the increase of n value for {PAA-PBA/GOD}n films, the Ipa of Fe(CN)6 3− continuously decreased, accompanied by the increase of Ep value (Fig. 7A), suggesting that the GOD multilayer films are successfully fabricated on the surface of PG electrodes. The stability of {PAA-PBA/GOD}5 films was examined by CV with Fc(COOH)2 as the probe, and the results demonstrated that the films were quite stable in both pH 5.5 and 8.0 solutions at least for three days. At pH 8.0, GOD carries net negative surface charges with its pI at 4.2 [62] and would repel negatively charged PAA-PBA. However, the {PAA-PBA/GOD}5 films were still quite stable and could not be disintegrated because the main driving force to combine GOD and PAA-PBA is the boronic acid-diol specific interaction, which would overcome the electrostatic repulsion between the similarly charged species. This is one of the advantages of {PAA-PBA/GOD}n films over other electrostatic LbL films, since the net film charge of the {PAA-PBA/GOD}n films can be modulated by surrounding pH without losing the film stability. {PAA-PBA/GOD}5 films also showed pH-sensitive CV on–off behavior toward Fc(COOH)2 , and the films were at the on state at pH 5.5 and at the off state at pH 8.0 (Fig. 7B), similar to the situation of {PAA-PBA/Dex}5 films. The pH-dependent switching property of Fc(COOH)2 at the {PAA-PBA/GOD}5 film electrodes was reversible and could be applied to control the electrocatalytic oxidation of glucose in solution. In pH 5.5 buffers containing Fc(COOH)2 and glucose, the obvious increase of CV oxidation peak current was observed for the films, accompanied by the decrease or disappearance of the reduction peak (Fig. 7C). In pH 8.0 buffers containing the same amount of Fc(COOH)2 and glucose, however, the CV response was too small to be detected. This on–off cycle in bioelectrocatalysis for the system could be repeated for many times by switching the films in the solutions between pH 5.5 and 8.0 (Supplementary materials Fig. S11).
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switch the bioelectrocatalysis of glucose by altering the environmental pH. This is the first report on applying LbL films assembled through boronic acid-diol specific recognition to pH-controllable bioelectrocatalysis. The in-depth understanding of the mechanism of pH-sensitive switching behavior of this model system may guide us to develop new kinds of pH-tunable electrochemical biosensors based on enzymatic electrocatalysis. Acknowledgment The financial support from the National Natural Science Foundation of China (NSFC 20975015 and 20775009) is acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.electacta.2010.09.012. References
Fig. 7. (A) CVs of 1.0 mmol dm−3 K3 Fe(CN)6 at 0.1 V s−1 in pH 7.0 buffers at (a) bare PG electrode, (b) PG/CS films, (c)–(g) {PAA-PBA/GOD}n films assembled on PG/CS electrode surface with n = 1–5. (B) CVs of 0.5 mmol dm−3 Fc(COOH)2 at 0.1 V s−1 for {PAA-PBA/GOD}5 films in buffers at pH (a) 5.5 and (b) 8.0. (C) CVs of {PAAPBA/GOD}5 films at 0.01 V s−1 in solutions containing 0.5 mmol dm−3 Fc(COOH)2 and 5.0 mmol dm−3 glucose at pH (a) 5.5 and (b) 8.0.
4. Conclusions A polyelectrolyte PAA-PBA containing both PBA moieties and carboxylic acid groups is synthesized. PAA-PBA and Dex are then assembled into {PAA-PBA/Dex}n LbL films on electrode surface through boronic acid-diol specific recognition between them. The films exhibit pH-sensitive CV on–off property toward Fc(COOH)2 mainly because the free –COOH groups in PAA-PBA are sensitive to surrounding pH. A series of comparative experiments demonstrate that it is the electrostatic interaction between the films and the probe that plays a key role in deciding the pH-switchable behavior of the system. This pH-responsive property of the system can be used to control or modulate the electrochemical oxidation of glucose catalyzed by GOD and mediated by Fc(COOH)2 through changing the solution pH. Moreover, {PAA-PBA/GOD}n LbL films are also assembled on the surface of electrodes by the boronic acid-diol specific interaction so that the immobilization of enzyme can be realized. The {PAA-PBA/GOD}n films also demonstrate pHtriggered on–off behavior toward Fc(COOH)2 , and can be used to
[1] N.J. Ronkainen, H.B. Halsall, W.R. Heineman, Chem. Soc. Rev. 39 (2010) 1747. [2] A.K. Sarma, P. Vatsyayan, P. Goswami, S.D. Minteer, Biosens. Bioelectron. 24 (2009) 2313. [3] J. Wang, Chem. Rev. 108 (2008) 814. [4] I. Willner, E. Katz, Angew. Chem., Int. Ed. 39 (2000) 1180. [5] E. Katz, V. Privman, Chem. Soc. Rev. 39 (2010) 1835. [6] E. Katz, M. Pita, Chem. Eur. J. 15 (2009) 12554. [7] M. Pita, E. Katz, Electroanalysis 21 (2009) 252. [8] O.A. Loaiza, R. Laocharoensuk, J. Burdick, M.C. Rodriguez, J.M. Pingarron, M. Pedrero, J. Wang, Angew Chem., Int. Ed. 119 (2007) 1530. [9] T.K. Tam, M. Ornatska, M. Pita, S. Minko, E. Katz, J. Phys. Chem. C 112 (2008) 8438. [10] T.K. Tam, J. Zhou, M. Pita, M. Ornatska, S. Minko, E. Katz, J. Am. Chem. Soc. 130 (2008) 10888. [11] H. Yao, N. Hu, J. Phys. Chem. B 114 (2010) 3380. [12] H. Yao, N. Hu, J. Phys. Chem. B 113 (2009) 16021. [13] S. Song, N. Hu, J. Phys. Chem. B 114 (2010) 3648. [14] H. Yao, N. Hu, J. Phys. Chem. B 114 (2010) 9926. [15] G. Decher, J.B. Schneloff (Eds.), Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials, Wiley-VCH, Weinheim, Germany, 2003. [16] Y. Lvov, in: Y. Lvov, H. Möhwald (Eds.), Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology, Marcel Dekker, New York, 2000, p. 125. [17] Y. Lvov, in: R.W. Nalwa (Ed.), Handbook of Surfaces and Interfaces of Materials, vol. 3, Academic Press, San Diego, 2001, p. 170. [18] J.F. Rusling, Z. Zhang, in: R.W. Nalwa (Ed.), Handbook of Surfaces and Interfaces of Materials, vol. 5, Academic Press, San Diego, 2001, p. 33. [19] M. Jiang, A. Eisenberg, G.J. Liu, X. Zhang (Eds.), Macromolecular Self-Assembly, Scientific Press, Beijing, 2008. [20] M. Zayats, E. Katz, I. Willner, J. Am. Chem. Soc. 124 (2002) 14724. [21] T. Levy, C. Dejugnat, G.B. Sukhorukov, Adv. Funct. Mater. 18 (2008) 1586. [22] S. Liu, B. Miller, A. Chen, Electrochem. Commun. 7 (2005) 1232. [23] S. Liu, U. Wollenberger, J. Halamek, E. Leupold, W. Stocklein, A. Warsinke, F.W. Scheller, Chem. Eur. J. 11 (2005) 4239. [24] Y. Ma, L. Qian, H. Huang, X. Yang, J Colloid Interface Sci. 295 (2006) 583. [25] N. Kanayama, H. Kitano, Langmuir 16 (2000) 577. [26] S.Y. Song, H.C. Yoon, Sens. Actuators, B 140 (2009) 233. [27] T.D. James, M.D. Phillips, S. Shinkai (Eds.), Boronic Acids in Saccharide Recognition, RSC Publishing, Cambridge, UK, 2006. [28] D.G. Hall (Ed.), Boronic Acids: Preparation, Applications in Organic Synthesis and Medicine, Wiley-VCH, Weinheim, Germany, 2005. [29] S. Li, E.N. Davis, J. Anderson, Q. Lin, Q. Wang, Biomacromolecules 10 (2009) 113. [30] T. Kawanishi, M.A. Romey, P.C. Zhu, M.Z. Holody, S. Shinkai, J. Fluoresc. 14 (2004) 499. [31] Z. Ding, Y. Guan, Y. Zhang, X.X. Zhu, Soft Matter 5 (2009) 2302. [32] J. Choi, T. Konno, R. Matsuno, M. Takai, K. Ishihara, Colloids Surf. B 67 (2008) 216. [33] J. Choi, T. Konno, M. Takai, K. Ishihara, Biomaterials 30 (2009) 5201. [34] J. Zhang, C.D. Geddes, J.R. Lakowicz, Anal. Biochem. 332 (2004) 253. [35] Y. Miyazaki, K. Yoshimura, Y. Miura, H. Sakashita, K. Ishimaru, Polyhedron 22 (2003) 909. [36] K. Aslan, J. Zhang, J.R. Lakowicz, C.D. Geddes, J. Fluoresc. 14 (2004) 391. [37] A. Kikuchi, K. Suzuki, O. Okabayashi, H. Hoshino, K. Kataoka, Y. Sakurai, T. Okano, Anal. Chem. 68 (1986) 823. [38] J.M. Abad, M. Velez, C. Santamaria, J.M. Guisan, P.R. Matheus, L. Vazquez, I. Gazaryan, L. Gorton, T. Gibson, V.M. Fernandez, J. Am. Chem. Soc. 124 (2002) 12845. [39] S. Liu, L. Bakovic, A. Chen, J. Electroanal. Chem. 591 (2006) 210. [40] BellamyF L.J. (Ed.), The Infra-red Spectra of Complex Molecules, Wiley, New York, 1975.
9192
H. Yao et al. / Electrochimica Acta 55 (2010) 9185–9192
[41] B.C. Smith (Ed.), Infrared Spectral Interpretation: A Systematic Approach, CRC Press, New York, 1999. [42] B.H. Stuart (Ed.), Infrared Spectroscopy: Fundamentals and Applications, Wiley, New York, 2004. [43] J. Ma, B. Fan, B. Liang, J. Xu, J. Colloid Interface Sci. 341 (2010) 88. [44] S.H. Brewer, A.M. Allen, S.E. Lappi, T.L. Chasse, K.A. Briggman, C.B. Gorman, S. Franzen, Langmuir 20 (2004) 5512. [45] A. Denuziere, D. Ferrier, A. Domard, Carbohydr. Polym. 29 (1996) 317. [46] H.A.O. Hill, Pure Appl. Chem. 59 (1987) 743. [47] K.L. Mittal (Ed.), Acid-Base Interactions: Relevance to Adhesion Science and Technology, Vol.2, VSP, Utrecht, The Netherlands, 2000. [48] H.P. Gregor, L.B. Luttinger, E.M. Loebl, J. Am. Chem. Soc. 76 (1954) 5879. [49] G. Delaittre, J. Nicolas, C. Lefay, M. Save, B. Charleux, Chem. Commun. (2005) 614. [50] A. Laguecir, S. Ulrich, J. Labille, F. Fatin-Rouge, S. Stoll, J. Buffle, Eur. Polym. J. 42 (2006) 1135. [51] E. Katz, I. Willner, Electroanalysis 15 (2003) 913.
[52] A.J. Bard, L.R. Faulkner (Eds.), Electrochemical Methods: Fundamentals and Applications, 2nd ed., Wiley, New York, 2001. [53] J.E.B. Randles, Discuss. Faraday Soc. 1 (1947) 11. [54] M. Motornov, R. Sheparovych, E. Katz, S. Minko, ACS Nano 2 (2008) 41. [55] A.A. Antipova, G.B. Sukhorukov, Adv. Colloid Interface Sci. 111 (2004) 49. [56] A. Calvo, B. Yameen, F.J. Williams, G.J. Soler-Illia, O. Azzaroni, J. Am. Chem. Soc. 131 (2009) 10866. [57] G. Decher, Science 277 (1997) 1232. [58] S.D. Noci, M. Frasconi, G. Favero, M. Tosi, T. Ferri, F. Mazzei, Electroanalysis 20 (2008) 163. [59] M.F. Chaplin, C. Bucke (Eds.), Enzyme Technology, Cambridge University Press, Cambridge, UK, 1990. [60] A.P.F. Turner, I. Karube, G.S. Wilson (Eds.), Biosensors: Fundamentals and Applications, Oxford University Press, New York, 1987. [61] J. Anzai, Y. Kobayashi, Langmuir 16 (2000) 2851. [62] E.J. Calvo, R. Etchenique, L. Pietrasanta, A. Wolosiuk, Anal. Chem. 73 (2001) 1161.