Effect of film-forming solution pH on the properties of chitosan-ferrocene film electrodes

Journal of Electroanalytical Chemistry 767 (2016) 160–166

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Effect of film-forming solution pH on the properties of chitosan-ferrocene film electrodes Weiwei Yang a, Jinlong Fan a, Yongsheng Yu a,⁎, Geping Yin a, Haibo Li b a b

School of Chemical Engineering and Technology, Harbin Institute of Technology, Harbin, Heilongjiang 150001, China Key Laboratory of Functional Materials Physics and Chemistry of the Ministry of Education, Jilin Normal University, Changchun 130103, China

a r t i c l e

i n f o

Article history: Received 29 January 2016 Received in revised form 16 February 2016 Accepted 19 February 2016 Available online 22 February 2016 Keywords: pH Glucose Chitosan Ferrocene Biosensor pH Glucose oxidase

a b s t r a c t Ferrocene-branched chitosan (CHIT-Fc) film electrodes were prepared from CHIT-Fc solution of variable pH to investigate the effects of film-forming solution pH on charge transport of the ferrocene. The redox behavior of CHIT-Fc polymer film electrodes was studied by cyclic voltammetry. The increase in both C⁎D1/2 ct and film permeability with decreasing film-forming solution pH was observed. Incorporation of glucose oxidase (GOD) into these films demonstrated efficient electrical communication between the enzyme molecules and the electrodes for the oxidation of glucose. Optimal electrocatalytic response to glucose based on these films occurred at filmforming solution pH 2.8, which exhibited a high sensitivity of 77.5 μA·mM−1·cm−2. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The development of novel redox polymers has been the focus of considerable research in the areas of electrocatalysis and biosensing applications [1–8]. Among various polymer backbones [9], chitosan attracts our attention due to its intrinsic excellent properties. Chitosan (CHIT) is a linear polysaccharide that is obtained by the partial deacetylation of chitin. Because of incomplete deacetylation, chitosan is mainly composed of the (1-4)-2-amino-2-deoxy-D-glucopyranose (D-glucosamine, GlcN) repeating unit and includes a small amount (b 20%) of N-acetyl-Dglucosamine (GlcNAc) residues. The unique structural feature of chitosan is the presence of the primary amine at the C-2 position of the glucosamine residues. Few biological polymers have such a high content of primary amines that confer important functional properties to chitosan that can be exploited for bio-fabrication [10]. In addition, chitosan displays a rare combination of physicochemical properties including an excellent membrane-forming ability, good biocompatibility, high permeability toward water, good adhesion, nontoxicity, and high mechanical strength [11–15]. These properties make it a promising matrix for enzyme immobilization [16,17]. On the other hand, chitosan's amines are reactive allowing a range of chemists to be employed to graft substituents to functionalize chitosan. When modified properly, chitosan might be expected to develop favorable ⁎ Corresponding author. E-mail address: [email protected] (Y. Yu).

http://dx.doi.org/10.1016/j.jelechem.2016.02.031 1572-6657/© 2016 Elsevier B.V. All rights reserved.

physical and/or chemical properties [18–21]. The attachment of electroactive groups to chitosan that can be cast on the electrode surface is an attractive route to obtain an efficient electron-transfer system for biosensing [22,23]. It is important to seek for simple, inexpensive, effective and stable methods to immobilize biomolecules on electrodes for their applications in biosensors. Many kinds of methods have been used for construction of biosensor interfaces such as Langmuir–Blodgett, selfassembled monolayers, layer-by-layer electrostatic adsorption of alternate multilayers, avidin–biotin interaction etc. [24–27]. The sol–gel technique provides a unique approach to prepare a three-dimensional network suited for the encapsulation of a variety of biomolecules because of their good mechanical and chemical stability. Worth of note, one of feasible methods to construct stable enzyme construction by forming covalent bond, Schiff bases bond, has been proposed [28]. It is well known that the reaction between amino-group and aldehydegroup easily proceeds in a moderate condition [29]. This is very vital for the fabrication of the enzyme biosensors, as it is not necessary to introduce other material or energy to the system and avoid the contamination and deactivation of the enzyme. In this work, ferrocenebranched chitosan (CHIT-Fc) was synthesized by the previously reported method [30]. Using chitosan's susceptibility to chemical modifications, we attempt to entrap IO− 4 -oxidized glucose oxidase in CHIT-Fc polymer by forming Schiff base bonds and construct reagentless glucose biosensors. The superiority of using CHIT-Fc is that it ensures a stable immobilization of enzyme on the electrodes as well as an efficient

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electrical communication between the enzyme and electrodes. In addition, the GOD/CHIT-Fc film is easy to form in one step, which avoids using cross-linking agents which are necessary for other redox hydrogels. In order to design and optimize the performance of the biosensors, we investigated the effect of film-forming solution pH on charge transport, permeability, and catalytic oxidation for glucose of the GOD/CHIT-Fc compound films. A systematic change in the pH of the CHIT-Fc solution leads to a variation in the charge transport carried by the redox polyelectrolyte and strongly affects the polyelectrolyteenzyme composite structure and catalytic properties. The aim of the present work is to report the film-forming solution pH effects on the catalytic current, concentration of electroactive sites, diffusion coefficient due to electron hopping, and their influence on the glucose biosensor response. The integration of chitosan's biocompatibility and susceptibility to chemical modifications will open new routes to design bioelectrochemical devices. The study of the effects of film-forming solution pH on charge transport of the ferrocene-branched chitosan may make chitosan and its derivatives more powerful as functionalized matrix in biosensors for food safety detection, environmental monitoring and clinical diagnosis. 2. Experimental section 2.1. Materials Chitosan (CHIT) with a degree of deacetylation (DDA) of 92% was purchased from Sanland-chem International Inc. (Xiamen, China). Glucose Oxidase (EC 1.1.3.4, from Aspergillus niger, 100 units·mg−1, GOD) was purchased from Sangon Company (Shanghai, China). Sodium cyanoborohydride (NaCNBH3, 95%) and ferrocenecarboxaldehyde (FcCHO, 98%) were obtained from Acros and Fluka, respectively. All other chemicals were of analytical grade and all the solutions were prepared using doubly distilled water. Chitosan-ferrocene polymer (CHIT-Fc) was synthesized according to our reported work (Fig. 1). The degree of substitution for ferrocene groups was 0.17. CHIT-Fc solutions (0.10 wt.%) were prepared by dissolving CHIT-Fc in 0.1 M acetate buffer solution with different pH from 2.8 to 4.6. The solutions were filtered using a 0.45-μm Miller-HA syringe filter unit (Millipore). Carbohydrate groups on the peripheral surface of the glucose oxidase molecules were oxidized to carbaldehydes with periodate according to the established procedure, and the concentration of the enzyme solution was about 40 μM [31]. 2.2. Preparation of film electrodes Glassy carbon electrodes (GCE, 3 mm diameter) were used as substrates to prepare chitosan film electrodes. Prior to use, the electrodes were wet-polished with alumina slurry and then cleaned by ultrasonication in ethanol and deionized water. CHIT-Fc film electrodes were prepared by the casting technique. In a typical procedure, 10 μL of 0.10 wt.% CHIT-Fc solutions with different pH was placed on the surface

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of GCE and allowed to evaporate at 35 °C. By evaporating water from the polyelectrolyte solutions, stable CHIT-Fc films are easily formed because of the presence of hydrogen bonds between the hydroxy groups, amino groups, and hydroxy and amino groups. This procedure resulted in a transfer of ~10 μg CHIT-Fc, which corresponds to 8.6 × 10−9 mol of ferrocene groups. CHIT-Fc/GOD film electrodes were prepared as follows: A casting solution was first prepared by mixing 100.0 μL of 0.10% CHIT-Fc solution with 10.0 μL of IO− 4 -oxidized GOD solution. After being incubated for 2 h, 11.0 μL of above casting solution was placed on the surface of GCE and allowed to dry at 35 °C. This procedure resulted in a transfer of 0.74 units of GOD and the same amount of ferrocene groups as on the CHIT-Fc film electrodes. All the chitosan film electrodes were thoroughly rinsed with water and then stored in air at 4 °C when not used. 2.3. Electrochemical measurements Cyclic voltammetric (CV) and amperometric measurements were performed with a CHI660A electrochemical workstation (CH Instruments, USA). The electrochemical cell consisted of a conventional three-electrode system with the CHIT-Fc and/or CHIT-Fc/GOD film electrodes as working electrodes, a platinum wire as a counter electrode, and a saturated calomel electrode (SCE) as a reference electrode. The pH 7.40 phosphate buffer solution (0.10 M, PBS) served as a background electrolyte in all experiments. A glucose stock solution was prepared in pH 7.40 PBS and was allowed to mutarotate overnight before use, and oxygen removal from the working solutions was achieved by purging with nitrogen. Constant temperature (25 ± 1 °C) was maintained during the experiments. 3. Results and discussion 3.1. Electrochemistry of CHIT-Fc film electrodes The effect of film-forming solution pH on the electrochemical characteristics of CHIT-Fc film electrodes was examined using cyclic voltammetry (CV). Prior to each set of experiments, the CHIT-Fc film electrodes were immersed in aqueous electrolyte for 30 min to swell. Fig. 2 shows the typical CVs in background electrolyte for CHIT-Fc film electrodes obtained from CHIT-Fc solutions with different pH. All the electrodes exhibited well-defined redox couples for one-electron redox reactions of CHIT-Fc+/CHIT-Fc. It should be noted that a significant increase in the redox voltammetric response is obtained, when the film-forming solution pH decreases. This indicates that there are more redox sites on the CHIT-Fc film electrode obtained from the lower film-forming pH solution, despite the equal amount of ferrocene groups on all CHIT-Fc film electrodes. Table 1 summarizes the effect of film-forming solution pH on the electrochemical properties of CHIT-Fc film electrodes. First, a slight positive shift of the formal potential (E0′), calculated from the average values of respective cathodic and anodic peak potentials, is observed as the film-forming solution pH

Fig. 1. Synthesis of ferrocene-branched chitosan derivatives (CHIT-Fc).

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Fig. 2. Cyclic voltammetry of CHIT-Fc film electrodes obtained from different film-forming pH solution: pH 2.8 (black); pH 3.4 (red); pH 4.0 (green); pH 4.6 (blue) Sweep rate 50 mV s−1; 0.1 M PBS (pH 7.4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

decreases, which may be a result of the microenvironments effects within the films [32]. The direction of the potential shift is consistent with enhanced stabilization of the ferricinium ion by electrostatic interaction with anions [33]. On the other hand, direct integration of charge Q at low scan rate shows that the redox charge increases as film-forming solution pH decreases. From calculation of the coverage Γ of redox sites on the CHITFc film electrodes, ratios of electroactive ferrocene amount to total ferrocene amount increase from 6.4% to 18% when the film-forming solution pH changes from 4.6–2.8, which indicates that more redox sites in the polymer participate in the electron transfer reaction as filmforming solution pH decreases. The analysis of the CHIT film electrode behaviors shows a diffusionlike process for charge-transfer within redox sites in the CHIT-Fc films, when evaluating peak currents as a function of scan rate. A linear regression of Ip vs. v1/2 suggests the charge-transfer model of Randles–Sevcik for semi-infinite diffusion in a reversible system (Eq. (1) [34,35]. Ip ¼ 0:4463ðnF Þ3=2 ADct 1=2 C  v1=2 =ðRT Þ1=2

ð1Þ

monolayer of redox reactants undergoes a surface redox process. In the present system, where redox molecules are covalently attached to polymers, diffusion of the redox polymer and chain reptation are not possible, so charge must propagate through the electron-hopping between neighboring ferrocene redox sites in a concentration gradient and/or counterion motion. Diffusion of counterions inside the films to compensate charge when the CHIT-Fc film electrodes are oxidized can be ruled out as the rate-determining step of charge propagation, since redox anions in the external electrolyte can easily diffuse through the films and their electrochemistry can be recorded, as will be shown later in Fig. 3. There has yet been no quantitative theoretical results, because C*, and Dct are not independent parameters in fixed site redox polymers, and the relationship between them is not well defined in the face of the polymeric structural uncertainties. However, there have been several reports of the dependence of electron-hopping rate on the concentration of redox sites (C*) within fixed site redox polymers [36–39]. In each, the qualitative expectation that electron transport rate increases with site concentration has been observed. Values of C⁎D1/2 ct for each CHIT-Fc film electrodes, calculated from respective slopes of Ip vs. v1/2 plots (Eq. (1)) are estimated in Table 1. Thus, if C⁎, or, equivalently, the film thickness is known, Dct may be calculated. Since the film thickness under experimental conditions is usually not known precisely, the value of C⁎Dct1/2 is often reported [40,41]. We observed that the values of C⁎D1/2 ct become larger as the film-forming solution pH decreases, which can be explained by an increase in concentration of redox sites in the CHIT-Fc films as pH decreases, since a larger Dct could be expected for higher redox site concentration as discussed above. The strong effect of the film-forming solution pH on the behavior of CHIT-Fc film electrodes should be related to the configuration of polymer in solution. Chitosan is a weak polyelectrolyte so that a systematic variation of pH resulted in a change of degree of protonation and charge density of polycation [42]. The fraction of positive charge that CHIT-Fc carries in solution was increased as film-forming pH value decreased, which leads to a rod-like structure of polymer that corresponds to a fully extended configuration. As the solution pH increased, a rod-coil transition is believed to occur in CHIT-Fc solution. On the other hand, as the pH of chitosan solution increased, we favor the formation of intramolecular hydrogen bond which also leads to a conformational arrangement of loops and tails to some extent. At lower pH, the CHIT-Fc film is formed mainly by intermolecular interaction as a result of lateral arrangement of segments of different macromolecules chains, since the polymer chains are strongly stretched because of their high degree of

The terms are defined as follows: Ip is the peak anodic current, n is the number of electrons in the oxidation, F is Faraday's constant, Dct is the diffusion coefficient of charge-transfer through the polymer film, C* is the concentration of redox sites in the film, v is the scan rate, R is the universal gas constant, and T is temperature (Fig. S1 Supporting information). This result suggests three-dimensional charge propagation in the CHIT-Fc films, unlike other redox hydrogel electrodes in which a

Table 1 Electrochemical data for CHIT-Fc film electrodes obtained from solutions with different film-forming pH. pH

E0 ′ (mV)

Qa (C)

Γb (mol cm−2)

%c

C*D1/2d ct (mol·cm−2·s–1/2)

2.8 3.4 4.0 4.6

312.5 310 301.5 294

1.5 × 10−4 8.4 × 10−5 7.0 × 10−5 5.3 × 10−5

2.2 × 10−8 1.2 × 10−8 1.0 × 10−8 7.7 × 10−9

18% 10% 8.4% 6.4%

1.2 × 10−8 8.9 × 10−9 7.4 × 10−9 5.4 × 10−9

a b c d

Integration at a scan rate of 5 mV·s−1. Γ = Q/nAF. Ratio of electroactive ferrocene to total ferrocene amount on the film electrodes. Calculated from the respective Randles–Sevcik equation.

Fig. 3. Cyclic voltammetry of Fe(CN)4− probe at bare GCE and CHIT film electrodes 6 obtained from different film-forming solution pH: from pH 2.8 to pH 4.6. Sweep rate in 0.1 M KCl. 50 mV s−1; 2 mM Fe(CN)4− 6

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ionization and weak intramolecular hydrogen bond. At higher pH, the CHIT-Fc adopts a coiled conformational arrangement, and the formation of CHIT-Fc film may accompany the intramolecular aggregation of a fraction of polymer chains. It is thus expected that a more compact form of CHIT-Fc film from lower pH solution in comparison with that from higher pH solution, which resulted in a thickness transition from a thick film to a thin film as CHIT-Fc solution pH decreases. This thickness transition would bring the ferrocene moieties closer to each other, resulting in the apparent increase in redox sites concentration of ferrocene groups at lower pH.

so the probe is transported predominantly by the pore mechanism in the CHIT film [1]. These porous channels may allow counterions in aqueous buffers to move into or out of the films more easily and greatly so that it can enhance the charge transport mobility within the films. More porous films would also allow the enzyme substrates to enter into the films more easily and have more chance to react with all of the proteins in the films, thus significantly improving the catalytic efficiency, as will be shown later.

3.2. Transport of probe in chitosan films

Having established that we can control the concentration of redox sites and the film structure by adjusting the film-forming solution pH, we now examine the extent of glucose oxidation catalyzed by the GOD covalently attached to the CHIT-Fc and mediated by this redox polymer molecular wire. When the GOD/CHIT-Fc redox polymer compound is immobilized on a GCE, the electrochemistry of GOD/CHIT-Fc films in PBS shows similar redox potentials with the CHIT-Fc film (Fig. S2 Supporting information), which indicates the presence of enzyme does not affect the free energy of the redox sites in this system. Note that the presence of the enzyme produces a decreased peak current, which can be explained in two aspects: GOD entrapping in the polymer films reduces concentration of electroactive groups, and the presence of enzyme molecules reduces the collision chances between the adjacent ferrocene redox sites. Upon addition of glucose to the background electrolyte, obvious catalytic reaction appears, accompanied by a dramatic increase in the oxidation current and a complete disappearance of the reduction current for the couple of CHIT-Fc+/CHIT-Fc. Fig. 5 shows the typical CVs in 300 mM glucose for GOD/CHIT-Fc film electrodes obtained from different film-forming solution pH. The absence of reduction waves shows that, at low scan rate, these films are approximately maintained in the reduced state by the transfer of electrons from GOD (FADH2) to the CHIT-Fc (Eq. (6)). This is also evidenced by the almost complete lack of hysteresis between the forward and the reverse scans for the films from film-forming solution pH 4.0 and 4.6. However, at pH 2.8 and 3.4, hysteresis appears, and thus the films are no longer completely reduced by enzyme-mediated electron transfer. This indicates film-forming solution pH have an effect on the film thickness since all these catalytic waves are obtained under the same other conditions, i.e. same substrate concentration, scan rate, and ionic strength. On the other hand, the catalytic current grows with the decreased film-forming solution pH following a similar trend to the redox charge and the concentration of redox sites in the films. We have also tried to fabricate the sensor with even lower pH (pH 2.0). The activity of GOD, however, has degraded at this severe condition (Data not shown). These typical enzyme-dependent catalytic processes can be expressed as follows (Eqs. (4)–(6)):

The ion transport and permeability of chitosan films were probed using electrochemical techniques. Fig. 3 shows cyclic with Fe(CN)4− 6 voltammograms for the control bare GCE and CHIT film electrodes obtained from unmodified chitosan solutions with different pH in equilibrium with solutions containing Fe(CN)46 − probe ions. The electron , is not inhibited transfer in the one-electron redox system, Fe(CN)4−/3− 6 by the chitosan coatings. This conclusion is supported by a fact that the anodic and cathodic peak potentials of the probe are practically the same at both bare electrode and CHIT film electrodes [43]. Further, electrochemical impedance spectroscopy measurements reveals that electron transfer resistances of probe molecule at each CHIT film electrode are all lower than 400 Ω (data not shown), which indicates that the effect of chitosan coatings on the kinetics of the electrode process due to the uncompensated film resistance is weak and neglectable. From linear Ip vs. v1/2 plots, apparent diffusion coefficient (Dapp) of the Fe(CN)4− 6 probe in the film electrodes was calculated from Randles–Sevcik equation (Table 2). These values of apparent diffusion coefficient are the at same order of magnitude as the diffusion constant of the Fe(CN)4− 6 bare electrode, which shows that the CHIT film electrode has an open structure and small molecules have high mobility in water-containing channels of the films. The increase in Dapp with decreasing filmforming solution pH was also observed. This can be explained by more probe diffusion in the CHIT film elecefficient channels for Fe(CN)4− 6 trode obtained from lower pH solution then that from higher pH solution, which results in a preferential partitioning of probe from the solution into the CHIT films. The permeability of the films to the Fe(CN)46 − probe is also investigated. The permeability, P, of a film to the redox probes was calculated from the following expression (Eq. (3)), where Ifilm and Ibare are the anodic voltammetric peak currents recorded at the CHIT film electrodes and the bare electrode, respectively.
P ¼ I film =Ibare  100%

ð3Þ

Table 2 shows that all the CHIT film electrodes have good permeability (P = 145–280%) to the probe. It should be noted that the P of probe molecule in the films increases with decreasing film-forming solution pH. The variation of permeability of chitosan films to probe can be explained by considering the structural differences between these CHIT films. Fig. 4 shows the SEM of CHIT films obtained from different filmforming solution pH. It can been seen that the CHIT film at low pH possesses more pore and larger pore size than that at high pH, which may increase the diffusion coefficient of redox probe molecule in the film, Table 2 at the CHIT film elecApparent diffusion coefficients Dapp and permeability of Fe(CN)4− 6 trodes obtained from different film-forming solution pH. pH 2.8 3.4 4.0 4.6 Bare electrode

Dapp(cm2 s−1) −6

34 × 10 30 × 10−6 26 × 10−6 13 × 10−6 7.6 × 10−6

P 281 225 191 145 100

3.3. Catalytic oxidation of glucose on the GOD/CHIT-Fc film electrodes

k

CHIT−Fc → CHIT−Fcþ þ e−

ð4Þ

k

kc

GODðFADÞ þ G ⇌ GODðFAD−GÞ → GODðFADH2 Þ þ GL k

‐1

k

GODðFADH 2 Þ þ 2CHIT− Fcþ → GODðFADÞ þ 2CHIT−Fc þ 2H þ

ð5Þ

ð6Þ

where CHIT-Fc and CHIT-Fc+ represent reduced and oxidized forms of the redox polysaccharide, GOD (FAD) and GOD(FADH2) the oxidized and reduced forms of glucose oxidase, GOD(FAD-G) the enzymeglucose complex, and G and GL are the glucose and glucose-lactone. In this redox-enzyme double catalytic cycle, electrons should diffuse to the electrode surface by electron hopping between adjacent ferrocene redox sites, as discussed above. In the presence of sufficient excess of substrate the enzyme is completely reduced, and thus reaction (Eq. (6)) is practically pseudo-first. For charge propagation in the

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Fig. 4. SEM CHIT-Fc films obtained from different film-forming solution pH: A pH 2.8, B pH 3.4, C pH 4.0 and D pH 4.6.

hydrogel under semi-infinite diffusion conditions and reaction (5) under steady state, the catalytic wave can be described by case II of Pratt and Bartlett kinetic analysis [44,45]: 1=2

I lim ¼ 2FAð2k DCT C E Þ

C

ð7Þ

The activity of immobilized enzyme, CE, was measured by the limiting catalytic current for the anaerobic oxidation of glucose in the presence of soluble ferrocene (Fc-CH2OH) and excess of substrate. The process of enzyme FADH2 oxidation by the artificial redox mediator undergoes three main steps: 1) diffusion of the redox mediator to the enzyme surface, 2) suitable positioning with the respect to the prosthetic group, 3) electron transfer from the FADH2 to the mediator. In the present case, however, the ferrocene groups are covalently attached to the

polymer backbone, and the later two steps are the decisive steps. Due to the anisotropy for the electron-transfer process in the FADH2 oxidation by the CHIT-Fc redox polymer, only a fraction of redox mediator that approaches the enzyme surface at an optimum orientation leads to efficient electron transfer. When GOD is immobilized on the surface of the GCE by unmodified chitosan at different film-forming solution pH, nearly equal limiting catalytic currents confirms the comparable activity of GOD, CE, in all four of the films (Not shown). Thus, assuming a same constant k for the mediation process, the decaying catalytic current with increasing pH must be the result of a decrease in the concentration of ferrocene redox sites in the films that are electrically connected to the electrode. This has been confirmed by the linear dependence of the catalytic current with C*D1/2 CT , shown in Fig. S3 (Supporting information) and expected by Eq. (7). The total concentration of active GOD is thus always larger than the concentration of “wired enzyme” on the electrodes, which decreases as the redox charge drops. Fig. S4 (Supporting information) depicts the catalytic currents for the anaerobic oxidation of glucose as a function of substrate concentration for E ≫ E0′. The glucose response curves measured under N2 are consistent with those expected for Michaelis–Menten kinetics. In this case, the rate-determining step is the enzymatic reaction (Eq. (5)) with fast reoxidation of FADH2 by the redox mediator. The dependence of the catalytic current with substrate concentration, Cs, can be described for these films by case VII of Pratt and Bartlett [46]: 1=2

I cat ¼ 2FAð2kcat C  DCT C E Þ

Fig. 5. GOD catalyzed oxidation of glucose mediated by CHIT-Fc at GOD/CHIT-Fc film electrodes obtained from different film-forming pH solution: pH 2.8 (black); pH 3.4 (red); pH 4.0 (green); pH 4.6 (blue). Nitrogen atmosphere; Sweep rate 5 mV s−1; 300 mM glucose, 0.1 M PBS (pH 7.4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

ð1 þ K M 0=C S Þ−1=2

ð8Þ

Best fits of Eq. (8) with data in Fig. S4 (Supporting information) yield apparent Michaelis constant, KM′ = (k−1 + kcat)/k1. The values of KM′ measured for the four electrodes ranged from 10.0–15.4 mM and varied slightly. The KM′ characterizes the enzyme electrodes, not the enzyme itself. Up to now, we can conclude that film-forming solution pH of 2.8 not only make a higher ferrocene redox concentration in the films but also ensure effective enzyme catalytic oxidation for glucose. KM′ is a measure of the substrate concentration range over which the electrode response is approximately linear [1]. Thus, the CHIT-Fc-GOD film electrodes can be used as an amperometric reagentless glucose biosensor due to its

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excellent bioelectrocatalytic response to the oxidation of glucose. Fig. 6 illustrates a typical current-time plot for the GOD/CHIT-Fc film electrode (film-forming solution pH 2.8) on successive step additions of glucose to a stirred and degassed solution at a working potential of 0.42 V. When an aliquot of glucose is added into the buffer solution, the oxidation current rises steeply and the steady state is achieved within 50 s. The sensitivity calculated from the linear region of calibration curves is 77.5 μA·mM−1·cm−2. The substantially high sensitivity is obtained with this polysaccharide redox film as compared with other glucose sensors [47–49], which is due to following aspects: 1) CHIT-Fc provides a very suitable microenvironment for enzyme entrapment and retains its activity; 2) this three-dimensional network structure of the GOD/ CHIT-Fc films and optimal film-forming solution pH can make more active enzyme effectively wired; 3) The electron-transfer process is actually efficient between the GOD and ferrocene redox sites for the oxidation of glucose. 4. Conclusions In this paper, we successfully prepared CHIT-Fc and GOD/CHIT-Fc film electrodes at film-forming solution pH from 2.8 to 4.6. The higher ferrocene coverage and higher C*D1/2 ct on the CHIT-Fc film electrode obtained from lower film-forming pH solution demonstrated apparent increase in efficient redox sites of electroactive ferrocene groups at lower pH. The electron transport of Fe(CN)4− 6 probe ions on the CHIT film electrodes demonstrated that the CHIT film obtained from lower pH solution possessed higher apparent diffusion coefficients and higher permeability for Fe(CN)46 − probe. These suggested that there were more efficient channels for electron transfer through the CHIT film obtained from lower pH than that from higher pH, which allows counterions in aqueous buffers to move into or out of the films more easily. The incorporating the GOD into the CHIT-Fc film can quantitatively detect the target-glucose by the reagentless electrochemical method. The catalytic response of the CHIT-Fc film electrode to glucose grew with the decreased film-forming solution pH, and the highest sensitivity of 77.5 μA·mM−1·cm−2 was obtained at pH 2.8. This strategy not only maintains GOD enzyme activity, but also retains the CHIT-Fc electrochemical activity. The study of the effects of film-forming solution pH on charge transport of the ferrocene branched on chitosan will help in designing new chitosan-based biosensing platform, which may provide enormous potential for applications in food safety detection, environmental monitoring and clinical diagnosis.

Fig. 6. Amperometric-time curve obtained at GOD/CHIT-Fc film electrode (film-forming solution pH 2.8) upon successive addition of aliquots of 1.0 M glucose solution to a 10 mL stirred 0.1 M PBS (pH 7.4) with an applied potential of 0.42 V under nitrogen atmosphere. Inset: Calibration curve of glucose.

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Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21305025 and 51571072), China Postdoctoral Science Foundation (No. 2015M81436) and Heilongjiang Postdoctoral Science Foundation (No. LBH-Z15065). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jelechem.2016.02.031. References [1] B.A. Gregg, A. Heller, Cross-linked redox gels containing glucose oxidase for amperometric biosensor applications, Anal. Chem. 62 (1990) 258–263. [2] S.C. Barton, H.H. Kim, G. Binyamin, Y.C. Zhang, A. Heller, The “wired” laccase cathode: high current density electroreduction of O2 to water at + 0.7 V (NHE) at pH 5, J. Am. Chem. Soc. 123 (2001) 5802–5803. [3] H. Wang, Sh. Li, Y.M. Si, Platinum nanocatalysts loaded on graphene oxide-dispersed carbon nanotubes with greatly enhanced peroxidase-like catalysis and electrocatalysis activities, Nanoscale 6 (2014) 8107–8116. [4] K. Sirkar, M.V. Pishko, Amperometric biosensors based on oxidoreductases immobilized in photopolymerized poly (ethylene glycol) redox polymer hydrogels, Anal. Chem. 70 (1998) 2888–2894. [5] E.J. Calvo, A. Wolosiuk, Wiring enzymes in nanostructures built with electrostatically self-assembled thin films, ChemPhysChem 6 (2005) 43–47. [6] E.J. Calvo, C. Danilowicz, L. Diaz, Enzyme catalysis at hydrogel-modified electrodes with redox polymer mediator, J. Chem. Soc. Faraday Trans. 89 (1993) 377–384. [7] P.P. Joshi, S.A. Merchant, Y.D. Wang, D.W. Schmidtke, Amperometric biosensors based on redox polymer-carbon nanotube-enzyme composites, Anal. Chem. 77 (2005) 3183–3188. [8] K.W. Lin, Sh.L. Ming, Sh.J. Zhen, Molecular design of DBT/DBF hybrid thiophenes πconjugated systems and comparative study of their electropolymerization and optoelectronic properties: from comonomers to electrochromic polymers, Polym. Chem. 6 (2015) 4575–4587. [9] B.Y. Lu, Sh.J. Zhen, Sh.M. Zhang, Highly stable hybrid selenophene-3, 4ethylenedioxythiophene as electrically conducting and electrochromic polymers, Polym. Chem. 5 (2014) 4896–4908. [10] H. Yi, L.Q. Wu, W.E. Bentley, R. Ghodssi, G.W. Rubloff, J.N. Culver, Biofabrication with chitosan, Biomacromolecules 6 (2005) 2881–2894. [11] H.Y. Chen, H.Q. Chen, H.B. Yang, A dual-targeting nanocarrier based on modified chitosan micelles for tumor imaging and therapy, Polym. Chem. 5 (2014) 4734–4746. [12] H. Chen, M.W. Chen, X.H. Wang, Self-assembled conjugated polymer/ carboxymethyl chitosan grafted poly(p-dioxanone) nanomicelles and their use in functionalized indicator paper for fast and visual detection of a banned food dye, Polym. Chem. 5 (2014) 4251–4258. [13] R. Jin, Ch. Lin, A.N. Cao, Enzyme-mediated fast injectable hydrogels based on chitosan–glycolic acid/tyrosine: preparation, characterization, and chondrocyte culture, Polym. Chem. 5 (2014) 391–398. [14] D. Lee, K. Singha, M.K. Jang, Chitosan: a novel platform in proton-driven DNA strand rearrangement actuation, Mol. BioSyst. 5 (2009) 391–396. [15] Y.F. Wang, X. Wang, Z.H. Geng, Electrodeposition of a carbon dots/chitosan composite produced by a simple in situ method and electrically controlled release of carbon dots, J. Mater. Chem. B 3 (2015) 7511–7517. [16] X. Wei, M.G. Zhang, W. Gorski, Coupling the lactate oxidase to electrodes by ionotropic gelation of biopolymer, Anal. Chem. 75 (2003) 2060–2064. [17] X. Chen, J.B. Jia, S.J. Dong, Organically modified sol–gel/chitosan composite based glucose biosensor, Electroanalysis 15 (2003) 608–612. [18] M. Yalpani, L.D. Hall, Some chemical and analytical aspects of polysaccharide modifications. III. Formation of branched-chain, soluble chitosan derivatives, Macromolecules 17 (1984) 272–281. [19] H. Sashiwa, Y. Makimura, Y. Shigemasa, R. Roy, Chemical modification of chitosan: preparation of chitosan–sialic acid branched polysaccharide hybrids, Chem. Commun. (2000) 909–910. [20] S.S. Silva, R.A.S. Ferreira, L. Fu, L.D. Carlos, J.F. Mano, R.L. Reis, J. Rocha, Functional nanostructured chitosan–siloxane hybrids, J. Mater. Chem. 15 (2005) 3952–3961. [21] M. Bodnar, J.F. Hartmann, J. Borbely, Preparation and characterization of chitosanbased nanoparticles, Biomacromolecules 6 (2005) 2521–2527. [22] M.G. Zhang, W. Gorski, Electrochemical sensing platform based on the carbon nanotubes/redox mediators-biopolymer system, J. Am. Chem. Soc. 127 (2005) 2058–2059. [23] M.G. Zhang, W. Gorski, Electrochemical sensing based on redox mediation at carbon nanotubes, Anal. Chem. 77 (2005) 3960–3965. [24] S. Sun, P.H. Ho-Si, D.J. Harrison, Preparation of active Langmuir–Blodgett films of glucose oxidase, Langmuir 7 (1991) 727–737. [25] A.J. Guiomar, J.T. Guthrie, S.D. Evans, Use of mixed self-assembled monolayers in a study of the effect of the microenvironment on immobilized glucose oxidase, Langmuir 15 (1999) 1198–1207. [26] L. Deng, Y. Liu, G. Yang, L. Shang, D. Wen, F. Wang, Z. Xu, S. Dong, Molecular “wiring” glucose oxidase in supramolecular architecture, Biomacromolecules 8 (2007) 2063–2071.

166

W. Yang et al. / Journal of Electroanalytical Chemistry 767 (2016) 160–166

[27] N. Anicet, C. Bourdillon, J. Moiroux, J.-M. Savéant, Electron transfer in organized assemblies of biomolecules. Step-by-step avidin/biotin construction and dynamic characteristics of a spatially ordered multilayer enzyme electrode, J. Phys. Chem. B 102 (1998) 9844–9849. [28] W.W. Yang, J.X. Wang, S. Zhao, Y. Sun, C.Q. Sun, Multilayered construction of glucose oxidase and gold nanoparticles on Au electrodes based on layer-by-layer covalent attachment, Electrochem. Commun. 8 (2006) 665–672. [29] W.W. Huang, X. Yang, S. Zhao, Fast and selective recognizes polysaccharide by surface molecularly imprinted film coated onto aldehyde-modified magnetic nanoparticles, Analyst 138 (2013) 6653–6661. [30] W.W. Yang, H. Zhou, C.Q. Sun, Synthesis of ferrocene-branched chitosan derivatives: redox polysaccharides and their application to reagentless enzyme-based biosensors, Macromol. Rapid Commun. 28 (2007) 265–270. [31] O.R. Zaborsky, J. Ogletree, The immobilization of glucose oxidase via activation of its carbohydrate residues, Biochem. Biophys. Res. Commun. 61 (1974) 210–216. [32] F. Battaglini, E.J. Calvo, C. Danilowicz, A. Wolosiuk, Effect of ionic strength on the behavior of amperometric enzyme electrodes mediated by redox hydrogels, Anal. Chem. 71 (1999) 1062–1067. [33] G.K. Rowe, S.E. Creager, Redox and ion-pairing thermodynamics in self-assembled monolayers, Langmuir 7 (1991) 2307–2312. [34] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamental and Applications, New York, John Wiley & Sons, 1980 (Chapter 6). [35] R.J. Forster, J.G. Vos, Charge transport properties of poly (N-vinylimidazole) containing [Os (N) 6] 2+/3+ moieties, J. Inorg. Organomet. Polym. 1 (1991) 67–86. [36] E.F. Dalton, N.A. Surridge, J.C. Jernigan, K.O. Wilbourn, J.S. Facci, R.W. Murray, Charge transport in electroactive polymers consisting of fixed molecular redox sites, Chem. Phys. 141 (1990) 143–157. [37] T. Ohsaka, H. Yamamoto, N. Oyama, Thermodynamic parameters for charge-transfer reactions in pendant viologen polymers coated on graphite electrodes and at electrode/pendant viologen polymer film interfaces, J. Phys. Chem. 91 (1987) 3775–3779. [38] F.C. Anson, D.N. Blauch, J.M. Saveant, C.F. Shu, Ion association and electric field effects on electron hopping in redox polymers. Application to the tris(2,2′-

[39]

[40]

[41]

[42]

[43] [44]

[45] [46]

[47]

[48] [49]

bipyridine)osmium(3+)/tris(2,2′-bipyridine)osmium(2+) couple in nafion, J. Am. Chem. Soc. 113 (1991) 1922–1932. M. Sharp, B. Lindholm, E.L. Lind, Aspects of charge propagation through nafion/Os films on glassy-carbon electrodes, J. Electroanal. Chem. 274 (1989) (bipy)2+/3+ 3 35–60. R.J. Russell, A.C. Axel, K.L. Shields, M.V. Pishko, Mass transfer in rapidly photopolymerized poly (ethylene glycol) hydrogels used for chemical sensing, Polymer. 42 (2001) 4893–4901. E.J. Calvo, R. Etchenique, C. Danilowicz, L. Diaz, Electrical communication between electrodes and enzymes mediated by redox hydrogels, Anal. Chem. 68 (1996) 4186–4193. P. Sorlier, A. Denuziere, C. Viton, Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan, A. Domard, Biomacromolecules 2 (2001) 765–776. J. Cruz, M. Kawasaki, W. Gorski, Electrode coatings based on chitosan scaffolds, Anal. Chem. 72 (2000) 680–686. P.N. Bartlett, K.F.E. Pratt, Theoretical treatment of diffusion and kinetics in amperometric immobilized enzyme electrodes part I: redox mediator entrapped within the film, J. Electroanal. Chem. 397 (1997) 61–78. F. Battaglini, E.J. Calvo, Enzyme catalysis at hydrogel-modified electrodes with soluble redox mediato, J. Chem. Soc. 90 (1994) 987–995. P.N. Bartlett, K.F.E. Pratt, Theoretical treatment of diffusion and kinetics in amperometric immobilized enzyme electrodes part I: redox mediator entrapped within the film, J. Electroanal. Chem. 397 (1995) 61–78. X. Wei, J. Cruz, W. Gorski, Integration of enzymes and electrodes: spectroscopic and electrochemical studies of chitosan-enzyme films, Anal. Chem. 74 (2002) 5039–5046. W.G. Koh, A. Revzin, M.V. Pishko, Poly (ethylene glycol) hydrogel microstructures encapsulating living cells, Langmuir. 18 (2002) 2459–2462. H.C. Yoon, M.Y. Hong, H.S. Kim, Functionalization of a poly (amidoamine) dendrimer with ferrocenyls and its application to the construction of a reagentless enzyme electrode, Anal. Chem. 72 (2000) 4420–4427.