A novel impedimetric nanobiosensor for low level determination of hydrogen peroxide based on biocatalysis of catalase

Bioelectrochemistry 83 (2012) 31–37

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Bioelectrochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o e l e c h e m

A novel impedimetric nanobiosensor for low level determination of hydrogen peroxide based on biocatalysis of catalase Mojtaba Shamsipur a,⁎, Mehdi Asgari b, Mohammad Ghannadi Maragheh c, Ali Akbar Moosavi-Movahedi d a

Department of Chemistry, Razi University, Kermanshah, Iran Department of Chemistry, Tarbiat Modares University, Tehran, Iran Electrochemistry Laboratory, Chemistry Department, NSTRI, Tehran, Iran d Institute of Biochemistry and Biophysical Chemistry, University of Tehran, Tehran, Iran b c

a r t i c l e

i n f o

Article history: Received 6 April 2011 Received in revised form 27 July 2011 Accepted 8 August 2011 Available online 16 August 2011 Keywords: Impedimetric biosensor Hydrogen peroxide Catalase Ionic liquid Carbon nanotube

a b s t r a c t A robust and effective nanocomposite film-glassy carbon modified electrode based on multi-walled carbon nanotubes and a room temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate was prepared by a layer-by-layer self-assembly method. The fabricated modified electrode was used as a novel impedimetric catalase nanobiosensor for the determination of H2O2. Direct electron transfer and electrocatalysis of catalase were fully investigated. The results suggested that catalase could be firmly adsorbed at the modified electrode. A pair of quasi-reversible redox peaks of catalase was observed in a 0.20 M degassed phosphate buffer solution of pH 7.0. The nanocomposite film showed a pronounced increase in direct electron transfer between catalase and the electrode. The immobilized catalase exhibited an excellent electrocatalytic activity towards the reduction of H2O2. The electrochemical impedance spectroscopy measurements revealed that the charge transfer resistance decreases significantly after enzymatic reaction with hydrogen peroxide, so that the prepared modified electrode can be used for the detection of ultra traces of H2O2 (5–1700 nM). © 2011 Elsevier B.V. All rights reserved.

1. Introduction There is an increasing interest on rapid and accurate detection of hydrogen peroxide (H2O2), not only because it exists as a product of the reactions catalyzed by many highly selective oxidases, but also because it is an essential compound in food, pharmaceutical and environmental analyses [1]. A variety of analytical methods have been developed for the assay of H2O2 including titrimetry [2], spectrophotometry [2,3] spectrofluorometry [4–6], chemiluminescence [7,8] high performance liquid chromatography [9] and, especially, electrochemistry [10–16]. Among different electrochemical methods, the amperometric enzyme-based biosensors have received considerable interest because of their convenience, high sensitivity and selectivity [10,17–22]. Here, the enzymes are immobilized onto different solid surfaces and used to generate signals corresponding to specific analytes in solution [10,17– 19,23]. Retention of the enzymatic activity and prevention of enzyme leaching are two important criteria for constructing successful enzymebased sensors [24,25]. An effective way to avoid enzyme leaching and improve the direct electron transfer of redox enzymes is their incorporating into suitable films modified on electrodes [26–31].

⁎ Corresponding author. Tel.: + 98 21 66908032; fax: + 98 21 66908030. E-mail address: [email protected] (M. Shamsipur). 1567-5394/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2011.08.003

The heme-containing catalases play critical roles in protecting the cell against the toxic effects of hydrogen peroxide by its degradation to water and oxygen (i.e., 2H2O2 = 2H2O + O2) [32]. Thus, in this work, we prepared a robust and effective catalase (CAT)-nanocomposite filmglassy carbon modified electrode based on multi-walled carbon nanotubes (MWCNTs) and a room temperature ionic liquid 1-butyl-3methylimidazolium hexafluorophosphate ([bmim][PF6]) by a layer-bylayer self-assembly method. The fabricated nanocomposite modified electrode, GC/MWCNTs/[bmim][PF6]/CAT, was then used as a novel nanobiosensor for the impedometric determination of nanomolar levels of H2O2. To the best of our knowledge, this is the first enzyme-based impedimetric nanobiosensor reported for the determination of ultra traces of hydrogen peroxide. 2. Experimental procedure 2.1. Chemicals 1-Butyl-3-methylimidazolium hexafluorophosphate ([bmim] [PF6])and catalase (CAT, EC 1.11.1.6) from bovine liver were purchased from Merck and Sigma, respectively, and used as received. MWCNTs with 95% purity (30–50 nm diameters and 3 μm length) were obtained from Timesnano Co. Ltd. (China), and hydrogen peroxide (30%) was obtained from Merck. Other chemicals were of analytical reagents grade from Merck and used without further

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purification. All solutions were prepared with double distilled water. Solutions were deaerated using high-purity argon gas (99.99%, Roham Gas Co., Tehran). 2.2. Apparatus Electrochemical experiments were performed using a computercontrolled Autolab30(2) potentiostat–galvanostat system (Echo Chemie, Utrecht, The Netherlands), driven with GPES and FRA software (Echo Chemie). A conventional three-electrode cell was used with an Ag/AgCl (3 M KCl) as reference electrode, a platinum wire as counter electrode, and the proposed modified electrode as working electrode. Scanning electron microscopy (SEM) was performed on a Zeiss DSM 960A instrument (Zeiss, Germany). 2.3. Purification and activation of MWCNTs Purification and surface activation of MWCNTs were done before use, as follows. The MWCNTs were first treated with reflux in a 1:3 v/v mixture of HNO3 (69%) and H2SO4 (98%) at 85 °C for 12 h. Then, the product was centrifuged, washed with ethanol and dried under vacuum at 50 °C overnight. 2.4. Preparation of modified electrode The glassy carbon (GC) electrode was polished with alumina powder on a polishing cloth and washed with water and ethanol in an ultrasonic bath successively, and allowed to dry at room temperature. Initially, 20 μL of MWCNTs-acetone solution (0.5 mg mL − 1) was cast on the GC electrode surface. After drying, the MWCNTs modified GC electrode was immersed in [bmim][PF6]-ethanol solution for 5 h. Finally, the GC/MWCNTs/[bmim][PF6] electrode was dipped in a 0.04% v/v catalase solution in 0.20 M phosphate buffer solution of pH 7.0 for 4 h to prepare the GC/MWCNTs/[bmim][PF6]/CAT electrode. Prior to electrochemical experiments, the electrode was rinsed thoroughly with water and kept in phosphate buffer solution of pH 7.0 at 5 °C. 2.5. Preparation of milk samples The impedimetric analysis was conducted on the two following milk samples. In sample A, 2.5 mL of milk sample was added to 22.5 mL of 0.2 M phosphate buffer solution of pH 7.0 (i.e., ten times dilution of milk sample). Sample B was prepared by adding 2.5 mL of milk sample together with 0.01 mL of 2.0 × 10 − 3 M H2O2 to 20.49 mL of 0.2 M phosphate buffer solution of pH 7.0. The milk samples were also analyzed by titration with potassium permanganate [33]. It is worth mentioning that, in the cases of both samples A and B, the milk sample was diluted by a factor of 10 to bring their hydrogen contents in the linear range of the prepared biosensor. Before titration, 2.5 mL of 3 M H2SO4 was added to 2.5 mL of milk sample and centrifuged for 10 min, followed by filtration with a micropore membrane filter (0.45 μm).

bundles mediated by local molecular ordering of ionic liquids, which is resulted from the carbon nanotubes interacting with the imidazolium group of bmim with the π–π and/or π-cationic, hydrophobic and electrostatic attractions [34]. The difference between the SEM images of GC/MWCNTs and GC/MWCNTs/[bmim][PF6]/CAT clearly indicates the interaction between MWCNTs with the enzyme and ionic liquid in the composite materials, and shows that the MWCNTs are nicely covered by the ionic liquid, as reported in a previous work [35].

3. Results and discussion

3.2. Cyclic voltammetric studies of direct electron transfer of catalase

3.1. Surface morphologies of modified electrodes

Fig. 2 shows typical cyclic voltammograms of GC/CAT (a), GC/MWCNTs/CAT (b) and GC/MWCNTs/[bmim][PF6]/CAT (c) modified electrodes in deaerated phosphate buffer solutions of pH 7.0 at a scan rate of 10 mV s − 1. At the GC/CAT electrode, almost no redox peak could be observed (Fig. 2a), revealing the difficult electron exchange of catalase with the electrode, because of the deep burying of its electroactive groups [36]. However, the GC electrode modified with an MWCNT film (Fig. 2b) showed a quasi-reversible cyclic voltammogram peak, characteristic of the catalase heme Fe(III)/Fe(II) redox couples [37]. Here, an anodic-cathodic peak potentials separation of ΔEp ≈ 85 mV indicated a relatively fast electron transfer in the

Fig. 1 shows the typical SEM images of the composite materials prepared at the surface of a GCE. From Fig. 1A it can be seen that the MWCNTs are cross-linked with each other to form a highly porous architecture, suitable for the immobilization of catalase. In the case of GC/MWCNTs/[bmim][PF6]/CAT (Fig. 1B), it is clearly seen that a specific untangle and uniform interface appeared with [bmim][PF6] inside the porous structure of MWCNTs. As reported in the literature [34], the interaction mechanism for the formation of a [bmim][PF6]MWCNTs composite is mainly based on the cross-linking of nanotubes

Fig. 1. SEM images of GC/MWCNTs (A) and GC/MWCNTs/[bmim][PF6]/CAT (B).

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Fig. 3A shows the cyclic voltammograms of GC/MWCNTs/[bmim] [PF6]/CAT electrode at different scan rates. The dependences of peak currents and peak potentials on the scan rate are also shown in Fig. 3B and C, respectively. As is obvious from Fig. 3B, the peak currents change linearly with scan rate (υ) over a range of 1 to 250 mV s − 1 (with correlation coefficients of 0.9917 and 0.9907), as prospected for thin layer electrochemistry [13,43]. The slope of corresponding log IP versus log υ linear plot, with a correlation coefficient of 0.9997, was found to be 0.756, relatively close to the theoretical slope 1 for thin layer voltammetry [44]. The surface concentration of electroactive species (Γ) can be estimated from the slope of peak current–scan rate plot based on the following equation [43]:   2 2 IP = n F νAΓ = ð4RTÞ

Fig. 2. Cyclic voltammograms of GC/CAT (a), GC/MWCNTs/CAT (b) and GC/MWCNTs/ [bmim][PF6]/CAT (c) modified electrodes in deaerated phosphate buffer solution of pH 7.0 at a scan rate of 10 mV s− 1.

electrode system. This value is larger than that reported elsewhere [38]. However, the peak currents observed for GC/MWCNTs/CAT (Fig. 2b) are much smaller than those observed at the GC/MWCNTs/ [bmim][PF6]/CAT electrode (Fig. 2c). This is an indication for the fact that the presence of [bmim][PF6] has a significant effect on the electron transfer reaction of catalase via providing a suitable environment for the enzyme to transfer electrons to the modified electrode [39–41]. Although the exact nature of this effect is not clear yet, the synergistic effects of MWCNTs-[bmim][PF6] system brought about via their π–π, π-cationic and/or hydrophobic–hydrophobic interactions [34] can be ascribed to the electron transfer ability of the proposed modified electrode. Meanwhile, as suggested before [42], the enzyme can bind to the ionic liquid through an ionic interaction, resulting in facilitated immobilization of catalase at the electrode's surface.

ð1Þ

where υ is the scan rate, A is the electrode surface area (0.0315 cm 2), and the other symbols have their usual meanings. Thus, the average surface concentration (Γ) of catalase was found to be 3.309 × 10 − 10 mol cm − 2, which indicates that the adsorbed catalase is in the form of an approximate monolayer on the surface of GC/MWCNTs/[bmim][PF6] electrode [45,46]. On the other hand, the peak-to-peak separation at scan a rate of 10 mV s − 1 was approximately 60 mV (Fig. 3C), indicating a quasireversible electron transfer process. However, at higher scan rates, the anodic peak potentials shifted to more positive direction and the anodic peak potentials to more negative direction, which resulted in an increase in the observed peak separations, indicating the limitation arising from charge transfer process. The peak potential separation at higher scan rates could be used to estimate the transfer coefficient and the heterogeneous electron transfer rate constant. According to Laviron model [43], the average values for the transfer coefficient, α, and the heterogeneous electron transfer rate constant, k, were evaluated as 0.472 and 1.95 s − 1, respectively. These values support the quasi-reversible redox process of catalase on the GC/MWCNTs/[bmim][PF6]/CAT modified electrode.

Fig. 3. (A) Cyclic voltammograms of GC/MWCNTs/[bmim][PF6]/CAT electrode in deaerated phosphate buffer solution of pH 7.0 at various scan rates. (B) Relationship between the anodic and cathodic peak currents and scan rates. (C) Relationship between peak potential separation and logarithm of scan rates.

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The cyclic voltammetric studies of the modified GC/MWCNTs/[bmim] [PF6]/CAT electrode at varying solution pHs, from 4.5 to 8.5, revealed that the reduction and oxidation peak potentials of the Fe(II)/Fe(III) redox couple of catalase shifted negatively with an increase in pH, due to the involvement of proton in the electrochemical reaction. The formal potential shifted linearly to negative direction with increasing pH with a slope of −52.5 mV pH− 1, which is close to the theoretical value of −59 mV pH− 1 for a one-electron one-proton electrochemical process [47]. The maximum cathodic current was obtained at a pH of 7.0, which was used as an optimal pH for further experiments. 3.3. Electrocatalytic reduction of H2O2 at GC/MWCNTs/[bmim][PF6]/CAT There are a number of reports which indicate that the enzymes containing the heme groups, such as catalase, hemoglobin and myoglobin, possess the ability to catalyze the H2O2 reduction. [31,32,37,38]. Thus, in this work, cyclic voltammetric studies were carried out to evaluate the bioelectrocatalytic activity of the GC/MWCNTs/[bmim][PF6]/CAT electrode toward H2O2 reduction. Fig. 4 shows the cyclic voltammograms of the GC/MWCNTs/[bmim] [PF6]/CAT electrode in 0.20 M phosphate buffer solution of pH 7.0 in the absence (a) and presence (b) of 6.4 μM H2O2 at a scan rate of 20 mV s − 1. In the absence of H2O2, a quasi-reversible peak of catalase was observed, which was similar to those observed in Figs. 2 and 3. However, in the presence of H2O2, the voltammetric behavior is changed; the appearance of a large cathodic current shows the occurrence of a typical electrocatalytic reduction process. The increased ratio of cathodic current in the presence, relative to that in the absence, of H2O2 can be defined as catalytic efficiency [48]. It was found that the catalytic efficiency decreases with increasing scan rate, and the cathodic current increased with increasing concentration of H2O2, both of which are well-established characteristics of the electrochemical catalysis [48]. As shown in Fig. 5, by increasing concentration of H2O2, the catalase cathodic peak current increased while its anodic peak current diminished significantly. 3.4. Electrochemical impedance spectroscopy In contrast to cyclic voltammetry, EIS uses a small-amplitude perturbation signal, which makes it an excellent tool for obtaining highly resolved kinetic data related to thin films of electroactive

Fig. 5. Cyclic voltammograms of the GC/MWCNTs/[bmim][PF6]/CAT electrode in a phosphate buffer solution of pH 7.0 containing 1.8 (a), 3.6 (b), 4.8 (c) and 7.2 μM H2O2 (d) at a scan rate as 50 mV s− 1.

surfaces [49,50]. Thus, the EIS is frequently used to determine the amount of redox species and probes involved with change in the charge transfer resistance and/or capacitance on the electrode surface. In order to design an impedimetric biosensor, it is necessary to have an appropriate relationship between electrical response of the modified surface in the absence and presence of the species of interest [51]. In this work, the relationship between bulk concentration and charge transfer resistance, Rct, can be described using the following equation [49,52]: 2 2

Rct = RT = n F Akct ½ S 

where, kct is the potential dependent charge transfer rate constant, [S] is the concentration of the redox species and the other symbols have their usual meanings. The EIS behavior of GC/MWCNTs/[bmim][PF6]/CAT electrode, as a biocatalytic interface to produce H2O from H2O2, was examined (Fig. 6A). It can be safely considered that both H2O2/H2O and CAT-Fe (III)/CAT-Fe(IV) = O reactions are fast, and the latter one is also reversible, so that the extent of H2O formation is controlled by the H2O2 concentration. Thus, in Eq. (2) one may replace [S] with k1 [H2O2], where k1 is a constant. If all other parameters are constant, a linear relationship in the form of Eq. (3) is simply found. 1 = Rct = k½H2 O2 

Fig. 4. Cyclic voltammograms of the GC/MWCNTs/[bmim][PF6]/CAT electrode in a phosphate buffer solution of pH 7.0 at a scan rate of 10 mV s− 1 in the absence (a) and presence (b) of 6.4 μM H2O2.

ð2Þ

ð3Þ

where k includes all constants of Eq. (2). According to Eq. (3), a graph of 1/Rct vs. H2O2 concentration should result in a linear plot, the sensitivity of which being dependent on the magnitude of the applied DC potential [53]. The equivalent circuit R1(R2CPE1) (shown in inset of Fig. 6A), consisting of a constant phase element in parallel with a resistance, was found to give the best fit to the experimental data. In this equivalent circuit, R1 corresponds to the resistance of the solution, R2 is the charge transfer resistance between the solution and the electrode surface, and CPE1 is associated with the double layer capacitance. The use of a constant phase element, instead of a capacitor, is required to optimize the fit to the experimental data, and this is due to the non ideal nature of the electrode surface [54]. The chi-square goodness-of-fit, calculated for each fit by the FRA software employed, was systematically checked to validate all of the calculations performed [53]. For all cases studied in this work, the calculated values of chi-square for the mentioned equivalent circuit were in the range of 0.0001–0.01, much lower than the tabulated

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Table 1 Comparison of linear range and limit of detection of different electrochemical sensors reported for hydrogen peroxide determination. Electrode

Linear range

Limit of detection

Ref.

CP/MnO6 GC/sol–gel–peroxidase SWCNH paste Pt/Te Pt/PVA/Ag GC/MB/SiO2/HRP Pg/PEG/HRP Au/Cyst/GNP/Hem CP/Ppy/Pmo12 CP/HRP-GA-BSA CiP/PQQ/GDH GC/MWCNT/Chitosan/HRP PG/NHSS/SWNT/HRP NanoAu/Cyst/PAMAM/GNPs GC/MWCNT/CAT GC/MWCNTs/[bmim] [PF6]/CAT

1 × 10− 4–6.9 × 10− 4 M Up to 3.4 mM 0.5–100 mM 1.7 μM–25 mM 1.25 μM–1 mM 1 × 10− 5–1.2 × 10− 3 M 2.0 × 10− 6–1.0 × 10− 4 M 3.6 × 10− 7–8.6 × 10− 4 2 × 10− 4–3 × 10− 2 M 2 nM–10 μM 1–15 μM 1.67 × 10− 5–7.4 × 10− 4 M 40 nM–1.2 μM 1 × 10− 5–2.5 × 10− 3

2 μM 5 × 10− 7 M – 1.7 μM 1 μM 4 × 10− 6 M 4.0 × 10− 5 M 1.2 × 10− 7 M 5 × 10− 5 M 1 nM 0.17 μM 1.3 × 10− 5 M 40 nM 2 μM

[11] [53] [14] [15] [12] [55] [10] [17] [18] [57] [22] [21] [56] [19]

10–100 μM 5.0 nM–1.7 μM

1 μM 0.25 nM

[31] This work

CP, carbon paste; GC, glassy carbon; SWCNH, single walled carbon nanohorns; PVA, polyvinyl alcohol; MB, methylene blue; HRP, horse radish peroxidase, PG, pyrolytic graphite; PEG, polyethylene glycol; Cyst, cysteine; GNP, gold nano particle; Hem, hemoglobin; Ppy, polypyrrole; Pmo12, phosphomolybdate; GA, glutaraldehyde; BSA, bovine serum albumin; CiP, coprinus cinereus peroxidase; PQQ, pyrroloquinoline quinine; GDH, glucose dehydrogenase; MWCNT, multi walled carbon nanotube; ECD, 1-(3-(dimethylamino) propyl)-3-ethylcarbodiimide hydrochloride; NHSS, N-hydroxysulfosuccinimide; PAMAM, poly(amidoamine); CAT, catalase.

Fig. 6. (A) Nyquist plots obtained on GC/MWCNTs/[bmim][PF6]/CAT electrode for different concentration of H2O2 in a 0.20 M phosphate buffer solution of pH 7.0: (a) buffer without H2O2, (b) 4.89 nM, (c) 58.7 nM, (d) 0.49 μM, (e) 0.98 μM, (f) 1.7 μM. Conditions: EDC = −300 mV, Eac = 10 mV, frequency range of 10 kHz to 100 MHz. Symbols are the experimental data and lines show the approximated results. (B) Calibration graph of 1/Rct as a function of H2O2 concentration.

value for 5 degrees of freedom (i.e., 67.505 at 95% confidence level), thus demonstrating a high significance of the final fit. The variation of 1/Rct versus H2O2 concentration produced a calibration curve (Fig. 6B) over a wide linear range of 5–1700 nM with a regression equation of 1/R c t = 4.0 × 10 − 5 [H 2 O 2 ] + 0.072 (R2 = 0.9923) and a limit of detection of 0.25 nM, as determined from CLOD = 3 Sb/m, where Sb is standard deviation of five replicate blank signals and m is slope of the calibration curve. The reproducibility of five different electrodes prepared was examined at a 1 μM concentration of H2O2, which resulted in a relative standard deviation of less than 7%. Table 1 compares the linear range and detection limit of the proposed GC/MWCNTs/[bmim][PF6]/CAT modified electrode with those of some of the best previously reported voltammetric nonenzymatic [11,12,14,18] and enzymatic [10,18,19,21,31,55,56] hydrogen peroxide biosensors. As is obvious from the summarized data, the proposed impedimetric GC/MWCNTs/[bmim][PF6]/CAT biosensor shows the highest sensitivity and lowest limit of detection among almost all cases. Moreover, in this work, the potential applied for constructing the calibration curve is much less cathodic than that applied in amperometric experiments (i.e., about −500 mV). This behavior reveals the fact that more accurate response data can be obtained by the impedimetric measurements. The stability of GC/MWCNTs/[bmim][PF6]/CAT electrode was also investigated and the results showed that, even after 20 continuous measurements, no measurable change in Nyquist plots can be observed,

suggesting that the enzyme CAT is tightly adsorbed on the surface of GC/MWCNTs/[bmim][PF6] electrode. The modified electrode was also found to keep its activity after keeping in a phosphate buffer solution of pH 7.0 for at least 15 days. 3.5. Analytical application The applicability of the GC/MWCNTs/[bmim][PF6]/CAT modified electrode was evaluated by the determination of hydrogen peroxide concentration in real samples. Due to the fact that hydrogen peroxide is often used as a preservative agent in milk, the electrode was applied to the determination of H2O2 in some milk samples A and B, described in Section 2.5., using the standard addition method. The determination was also carried out by a reported classical titration method [33], for comparison. The results obtained are summarized in Table 2. The results thus obtained clearly revealed that the concentrations of hydrogen peroxide in the sample obtained by the proposed impedimetric sensor are in satisfactory agreement with those determined by the titration method [33]. 4. Conclusions The catalase immobilized on the modified GC/MWCNTs/[bmim] [PF6] electrode exhibited a direct and quasi-reversible electrochemical response. The ionic liquid [bmim][PF6] assembled on the GC/MWCNTs is not only responsible for a better immobilization of the enzyme, but was also found to largely accelerate the electron transfer between catalase and the electrode. Such facilitated electron transfer of catalase in

Table 2 Determination of hydrogen peroxide in milk samples. Milk sample A B

Concentration of hydrogen peroxide (μM) Proposed sensor

Titration [33]

5.67 ± 0.015 6.49 ± 0.011

5.69 ± 0.017 6.51 ± 0.019

Recovery (%) – 103.8

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