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Carbon nanotube–chitosan modified disposable pencil graphite electrode for Vitamin B12 analysis
Colloids and Surfaces B: Biointerfaces 87 (2011) 18–22
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Carbon nanotube–chitosan modified disposable pencil graphite electrode for Vitamin B12 analysis Filiz Kuralay, Tayfun Vural, Cem Bayram, Emir Baki Denkbas, Serdar Abaci ∗ Department of Chemistry, Faculty of Science, Hacettepe University, 06800 Beytepe-Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 28 July 2010 Received in revised form 14 March 2011 Accepted 22 March 2011 Available online 22 April 2011 Keywords: Single walled carbon nanotubes Chitosan Biosensor Disposable pencil graphite electrode Vitamin B12
a b s t r a c t A single walled carbon nanotube–chitosan (SWCNT–chitosan) modified disposable pencil graphite electrode (PGE) was used in this study for the electrochemical detection of Vitamin B12 . Electrochemical behaviors of SWCNT–chitosan PGE and chitosan modified PGE were compared by using cyclic voltammetry (CV), square-wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) techniques. SWCNT–chitosan modified electrode was also used for the quantification of Vitamin B12 in pharmaceutical products. The results show that this electrode system is suitable for sensitive Vitamin B12 analysis giving good recovery results. The surface morphologies of the SWCNT–chitosan PGE, chitosan modified PGE and unmodified PGE were characterized by using scanning electron microscopy (SEM). © 2011 Elsevier B.V. All rights reserved.
1. Introduction Nano-structured materials have attracted great attention in recent years in many fields and the development of many new technologies [1–3]. Among them, carbon nanotubes (CNTs) play an important role due to their high electrochemically accessible area, good electronic conductance and strong mechanical, structural properties [4]. CNTs are long, thin cylinders of carbon and can be classified as a graphite sheet rolled into a nanoscale-tube (single walled carbon nanotubes, SWCNTs) and an additional graphene tubes around the core of an SWCNT (multi walled CNTs, MWCNTs) [5–7]. As electrode materials, one of the promising applications of CNTs is their use in the design of biosensors and chemical sensors [8–11]. With the advantages of minimizing fouling of electrode surfaces, exhibiting a high ability to promote some type of the electron transfer reactions between electroactive species and electrodes, enhancing electrocatalytic activity, and facilitating the immobilization of molecules on their surface they have superiority to other kinds of carbon based materials used in electrochemistry for developing effective and sensitive biosensors [12]. The incorporation of CNTs into polymer matrices also promises new improvements in biosensing technology [13]. Chitosan is a naturally occurring polymer (a polysaccharide) that is obtained by the partial deacetylation of chitin [13,14]. Chitosan exhibits excellent film-forming ability, nontoxicity, biocompati-
∗ Corresponding author. Tel.: +90 312 297 6080; fax: +90 312 299 2163. E-mail addresses: [email protected], fi[email protected] (S. Abaci). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.03.030
bility, biodegradability, multiple functional groups, pH-dependent solubility in aqueous media, cheapness and a susceptibility to chemical modification [15–18]. All these properties make it widely used as an immobilization matrix for biosensors [19–24]. Li et al. performed DNA detection based on chitosan film doped with carbon nanotubes by using graphite electrode [25]. Arias et al. studied DNA–methylene blue interaction on glassy carbon electrode using carbon nanotubes in chitosan [26]. Carbon nanotube–chitosan system for electrochemical sensing based on dehydrogenase enzymes was developed by Zhang et al. [17]. Glucose biosensor based on carbon nanotubes/chitosan matrix was fabricated by Liu et al. [27]. Tkac et al. used single walled carbon nanotubes dispersed in a chitosan matrix for galactose biosensor by using glassy carbon electrode [23]. Ghica et al. carried out the application of functionalized carbon nanotubes immobilized into chitosan films in amperometric enzyme biosensors [28]. Carbon nanotubes and chitosan composite film for preparation of amperometric hydrogen peroxide biosensor on glassy carbon electrode was studied by Qian and Yang [29]. An amperometric cholesterol biosensor based on multiwalled carbon nanotubes and organically modified sol–gel/chitosan hybrid composite film was performed by Tan et al. [30]. In this study we used carbon nanotubes dispersed in chitosan matrix for electrochemical Vitamin B12 analysis. Vitamin B12 is a corrin based cobalt complex which is important in human physiology. Its deficiency causes pernicious anemia and neuropathy. Monitoring of Vitamin B12 is essential in the quality control of pharmaceuticals, blood plasma serum, milk products for infants and fermentation products [31,32]. There are
F. Kuralay et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 18–22
several methods cited in the literature for the determination of Vitamin B12 including chromatography, spectrophotometry, chemiluminescence and electrochemistry [33]. Electrochemical techniques such as cyclic voltammetry, differential pulse voltammetry, square-wave voltammetry and amperometry have provided sensitive Vitamin B12 detection [34]. Electrochemical reports demonstrated that Vitamin B12 and its derivatives exhibited rich redox chemistry which was centered on the cobalt atom. Vitamin B12a (with Co(III)) can be reduced reversibly to Vitamin B12r (with Co(II)), and be further reduced to Vitamin B12s (with Co(I)), all in aqueous media. However, estimation of standard potentials for the redox couples seems to be difficult [35,36]. This study focused on the development of single walled carbon nanotube–chitosan (SWCNT–chitosan) modified disposable pencil graphite electrode (PGE) for the electrochemical monitoring of Vitamin B12 based on the signal enhancement in comparison to chitosan modified disposable pencil graphite electrode. The incorporation of SWCNTs into the positively charged polymer matrix was carried out easily [25]. Then, electrochemical responses of cobalt redox species in the structure of Vitamin B12 were monitored after successive additions of Vitamin B12 . Electrochemistry of SWCNT–chitosan modified PGE was examined using cyclic voltammetry (CV), square-wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) techniques. Quantification of Vitamin B12 in pharmaceutical products was performed sensitively by using SWCNT–chitosan modified electrode. Scanning electron microscopy (SEM) was also used for the characterization of SWCNT–chitosan modified, chitosan modified and unmodified PGEs.
2. Experimental 2.1. Apparatus Electrochemical studies, cyclic voltammetry and square-wave voltammetry were carried out using CH Instruments (CHI) 660 C (USA). The three-electrode system consisted of a disposable pencil graphite working electrode (PGE), an Ag/AgCl reference electrode (SCE) (BAS, USA) and a Pt wire (Aldrich) counter electrode were used in the experiments. SEM images were obtained by Zeiss Evo 50 EP-SEM Model Scanning Electron Microscope (SEM) (USA).
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2.3. Procedure All the experiments were carried out at room temperature. A new modified electrode surface was used in each electrochemical detection cycle. Each test was repeated three times. 2.3.1. The preparation of SWCNT–chitosan modified PGE PGE was immersed in SWCNT–chitosan mixture and waited for 30 min by stirring. After modification of PGE, modified electrode was immersed in 0.1 M NaOH for 5 min to make film more stable [25]. Modified PGE was then washed with deionized water and allowed to dry. 2.3.2. The preparation of chitosan modified PGE PGE was immersed in chitosan solution and waited for 30 min by stirring. After modification of PGE, modified electrode was immersed in 0.1 M NaOH for 5 min. Modified PGE was then washed with deionized water and allowed to dry. 2.3.3. Voltammetric transduction The electrochemical characterization of SWCNT–chitosan modified, chitosan modified and unmodified PGEs was performed in 5 mM Fe(CN)6 3−/4− containing 0.1 M KCl using CV between −0.2 V and +0.6 V vs. Ag/AgCl. Detection of Vitamin B12 was performed in 0.1 M PBS with successive Vitamin B12 additions to the solution by stirring 1 min using SWV between −0.2 V and −1.0 V at pulse amplitude of 50 mV. 2.3.4. Impedance measurements Electrochemical impedance spectroscopy (EIS) measurements were controlled at the open-circuit value; +0.15 V vs. Ag/AgCl and the frequency was varied over the range 105 –10−2 Hz with amplitude of 5 mV in the presence of 5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) mixture as a redox probe prepared in 0.1 M KCl.
2.3.5. Microscopic characterization of unmodified PGE, chitosan modified PGE and SWCNT–chitosan modified PGE by scanning electron microscopy (SEM) The microscopic characterization of unmodified, chitosan modified and SWCNT–chitosan modified PGEs was performed with SEM in resolution magnitude of 200 nm. 3. Results and discussion
2.2. Reagents and preparation of solutions Chitosan was purchased from Fluka. SWCNT and Vitamin B12 were obtained from Sigma–Aldrich. Pharmaceutical formulations containing Vitamin B12 served as real samples (Sample 1: Solgar; Solgar Inc., USA and Sample 2: Dodex; Deva Ltd., Turkey). Other chemicals were in analytical reagent grade and supplied from Sigma and Merck. 0.1 M phosphate buffer solutions (PBS, pH: 2.0, 5.0) were prepared from NaH2 PO4 (Fluka), Na2 HPO4 ·2H2 O (Fluka) and H3 PO4 (Merck) using deionized water. 5 mM Fe(CN)6 3−/4− containing 0.1 M KCl (Merck) was prepared from K3 Fe(CN)6 (Fisher) and K4 Fe(CN)6 (Fisher). Stock Vitamin B12 solutions (10−3 M) were prepared using PBS freshly. Vitamin B12 pharmaceutical formulations were also dissolved in buffer in PBS and filtered before usage. 0.5% solution of chitosan polymer was prepared in 2 M acetic acid (Sigma–Aldrich). This solution was then filtered. SWCNT was purified according to the early procedure [37]. A dispersion of SWCNT in chitosan was obtained by adding 2.5 mg of SWCNT in 1 mL of chitosan solution and sonicated during 30 min.
The surface morphologies of unmodified, chitosan modified and SWCNT–chitosan modified PGEs were examined using SEM (Fig. 1a–c, respectively). The surface roughness of unmodified PGE was monitored successfully in Fig. 1a. After modification of PGE with chitosan, the electrode surface was covered with chitosan polymer (Fig. 1b). It is clear that some parts of PGE became invisible due to chitosan immobilization onto the electrode surface. In the SEM image of SWCNT–chitosan modified PGE, it was observed that SWCNT was distributed homogenously in the porous structure of chitosan matrix (Fig. 1c) [22]. It can be concluded that SEM images proved different modifications on PGE. Cyclic voltammetric behavior of the Fe(CN)6 3−/4− system is a valuable method to monitor the characteristics of the surface of the modified electrodes [31]. In order to see different modifications on PGE, cyclic voltammetric behavior of the Fe(CN)6 3−/4− system was used in the study. Fig. 2 demonstrates the CVs of unmodified, chitosan modified and SWCNT–chitosan modified PGEs in 5 mM Fe(CN)6 3−/4− redox probe containing 0.1 M KCl. After modification of PGE with chitosan (Fig. 2b), there was no appreciable change at the anodic and cathodic peak currents of Fe(CN)6 3−/4− . However,
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Fig. 2. CVs of (a) unmodified, (b) chitosan modified, (c) SWCNT–chitosan modified PGEs in 5 mM Fe(CN)6 3−/4− redox probe containing 0.1 M KCl. CV measurement: at 100 mV/s scan rate between 0.0 V and +1.4 V.
ity due to SWCNT [6,31,33]. Ep value decreased compared to the result obtained with chitosan modified PGE, suggesting the further enhancement at the electron transfer rate of Fe(CN)6 3−/4− [38]. EIS also used to identify and differentiate the unmodified PGE, chitosan modified PGE and SWCNT–chitosan modified PGE. EIS technique gives information on the impedance changes of the electrode surface during the modification process. Fig. 3 shows the impedance spectra of unmodified PGE, chitosan modified PGE and SWCNT–chitosan modified PGE. In the Nyquist plot of impedance spectra, the diameter of the semicircle represents the charge-transfer resistance (Rct ) at the electrode surface. The Rct of unmodified PGE was calculated as 243.12 (Fig. 3a). Rct value decreased to 120.12 (Fig. 3b) after the modification of PGE with chitosan polymer due to the positively charged amino groups of chitosan which could enhance the electron transfer between negatively charged redox probe and the electrode surface. The decrease in Rct value (85.80 ) was higher for SWCNT–chitosan modified PGE (Fig. 3c) than the decrease in Rct value obtained with chitosan modified PGE indicating the increased charge-transfer in the presence of carbon nanotubes [16]. Carbon nanotubes promoted charge-transfer occurred at the electrode surface. Vitamin B12 analysis was carried out using SWV after the characterization of SWCNT–chitosan modified PGE. The effect of successive additions of Vitamin B12 in 0.1 M PBS was studied. Fig. 4A shows the square-wave voltammograms of SWCNT–chitosan modified PGE in the absence and presence of Vitamin B12 at pH 2.0.
Fig. 1. SEM images of (a) unmodified, (b) chitosan modified, (c) SWCNT–chitosan modified PGEs.
there was a change at the redox peak potentials of Fe(CN)6 3−/4− compared to the redox potentials obtained in the case of unmodified PGE (Fig. 2a). The peak potential difference (Ep ) decreased due to chitosan immobilization. After modification of PGE with SWCNT–chitosan mixture (Fig. 2c), the anodic and cathodic peak currents of Fe(CN)6 3−/4− increased, indicating SWCNT improved the electroactive surface area of the electrode and catalytic abil-
Fig. 3. Impedance spectra of (a) unmodified, (b) chitosan modified, (c) SWCNT–chitosan modified PGEs in 5 mM Fe(CN)6 3−/4− redox probe containing 0.1 M KCl. EIS measurement: at open-circuit value of +0.15 V between 105 and 10−2 Hz range with amplitude of 5 mV.
F. Kuralay et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 18–22
Fig. 4. (A) SWVs for different concentrations of Vitamin B12 at pH 2.0 in 0.1 M PBS: (a) 0 nM, (b) 5 nM, (c) 35 nM, (d) 50 nM, (e) 100 nM. SWV measurement: between −0.2 V and −1.0 V at pulse amplitude of 50 mV. (B) The effect of Vitamin B12 concentration on reduction peak currents of Co(II) to Co(I) using (a) SWCNT–chitosan modified PGE, (b) chitosan modified PGE at pH 2.0 (n = 3).
As seen in Fig. 4A-a, there were no electroactive species between −0.20 V and −0.90 V potential intervals in the voltammograms of SWCNT–chitosan modified PGE. With the addition of Vitamin B12 two peaks existed in the voltammograms. The reduction peak at about −0.32 V vs. Ag/AgCl was attributed to the reduction of Co(III) to Co(II) and the peak at −0.66 V vs. Ag/AgCl was belonged to the reduction of Co(II) to Co(I). There was an electrocatalytic effect using carbon nanotube-polymer modified electrode due to reduction of Co(II) to Co(I) compared to earlier studies [31,35]. The reduction peak currents of cobalt redox couples increased with increasing concentration of Vitamin B12 (Fig. 4A-b to e). In order to test the advantage of SWCNT–chitosan modified electrode over chitosan modified electrode, Vitamin B12 analysis were also carried out using only chitosan modified electrode. The reduction peak currents due to Co(II) to Co(I) were presented in Fig. 4B for SWCNT–chitosan modified and chitosan modified PGEs. As seen from these results enhanced electrochemical response was obtained with SWCNT–chitosan modified PGE (Fig. 4B-a) compared to chitosan modified PGE (Fig. 4B-b). In the presence of carbon nanotubes, the electrode system gave better results indicating high ability to promote the electron transfer reactions between cobalt redox couple and electrode, enhancing electrocatalytic activity and increasing sensitivity of the biosensor [12]. The comparison of SWCNT–chitosan modified electrode over SWCNT modified electrode were also carried out. However, irreproducible results were obtained in the case of SWCNT modified electrode (not shown). The experiments were also carried out at pH 5.0 in 0.1 M PBS. Fig. 5A shows the square-wave voltammograms of SWCNT–chitosan modified PGE in the presence of Vitamin B12 at pH 5.0. With the addition of Vitamin B12 two peaks existed similar to the results obtained at pH 2.0 above. The reduction peak at about −0.40 V vs. Ag/AgCl was attributed to the reduction of Co(III)
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Fig. 5. (A) SWVs for different concentrations of Vitamin B12 at pH 5.0 in 0.1 M PBS. SWV measurement: between −0.2 V and −1.0 V at pulse amplitude of 50 mV: (a) 5 nM, (b) 35 nM, (c) 50 nM, (d) 80 nM. (B) The effect of Vitamin B12 concentration on reduction peak currents of Co(II) to Co(I) using (a) SWCNT–chitosan modified PGE, (b) chitosan modified PGE at pH 5.0 (n = 3).
to Co(II) and the peak at −0.74 V vs. Ag/AgCl was belonged to the reduction of Co(II) to Co(I). The reduction peak currents of cobalt redox couples increased with increasing concentration of Vitamin B12 (Fig. 5A-a to d). However, the magnitude of the peak currents was higher at pH 2.0 than the magnitude of peak currents at pH 5.0. Also, the reduction peak potentials were more positive at pH 2.0 than the reduction peak potentials at pH 5.0. In order to monitor the advantage of SWCNT–chitosan modified electrode over chitosan modified electrode at this pH value, same experiments were carried out using only chitosan modified electrode. The reduction peak currents of Co(II) to Co(I) were presented in Fig. 5B using SWCNT–chitosan modified PGE (Fig. 5B-a) and chitosan modified PGE (Fig. 5B-b). Enhanced electrochemical response was obtained with SWCNT–chitosan modified PGE compared to chitosan modified PGE similar to the results at pH 2.0. The detection limit of the biosensor was found as 0.89 nM with a linear concentration range interval of 5 nM and 100 nM at pH 2.0 (R2 = 0.987) and the detection limit of the biosensor was found as 2.1 nM with a linear concentration range interval of 5 nM and 80 nM at pH 5.0 (R2 = 0.994). The stability of the biosensor was also studied. After 1 week of use, the electrode response decreased about 10%. The pH dependence of the electrode was examined between pH 2 and 5 (Fig. 6A). As seen in this figure, the response of the SWCNT–chitosan modified PGE was maximum at pH 2.0. Vitamin B12 analysis was also carried out using pharmaceutical products at pH 2.0 in 0.1 M PBS. Fig. 6B shows the square-wave voltammograms of SWCNT–chitosan modified PGE after standard additions of pharmaceutical Vitamin B12 product (Solgar). With increasing concentration of pharmaceutical Vitamin B12 product, the peak currents due to Co(II) to Co(I) reduction increased (Fig. 6Ba to c) supporting our earlier results above. The results for two
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response of chitosan modified PGE for Vitamin B12 analysis. A signal enhancement was obtained for the reduction of cobalt redox couples in the structure of Vitamin B12 using SWCNT–chitosan modified PGE at low potentials due to the catalytic activity of SWCNT. The detection limit of the biosensor was found as 0.89 nM with a concentration range interval of 5 nM and 100 nM at pH 2.0 (R2 = 0.987) and the detection limit of the biosensor was found as 2.1 nM with a linear concentration range interval of 5 nM and 80 nM at pH 5.0 (R2 = 0.994). The modified electrode was also provided quantification of Vitamin B12 in pharmaceutical products at pH 2.0 by using two different products with good recovery and reproducibility results. The SWCNT–chitosan modified disposable pencil graphite electrode presented here was prepared in an one step procedure with the advantages of simple, fast, easy and cheap preparation. The selectivity of the electrode was good. The detection method has novelty and comparability with the studies based on Vitamin B12 analysis in the literature [31–35]. The prepared electrodes can also be promising for new biosensing applications based on other real samples. References [1] [2] [3] [4]
Fig. 6. (A) pH dependence of the electrode at 80 nM Vitamin B12 concentration (n = 3). (B) SWVs for different concentrations of Vitamin B12 using one tablet of Solgar at pH 2.0: (a) 35 nM, (b) 50 nM, (c) 80 nM. SWV measurement: between −0.2 V and −1.0 V at pulse amplitude of 50 mV. Table 1 Results for the determination of Vitamin B12 in real samples.
Sample 1
Sample 2
Added (nM)
Found (nM)
RSD (%, n = 3)
Recovery (%)
35.00 50.00 80.00 35.00 50.00 80.00
35.78 51.03 85.52 35.45 50.16 81.11
2.37 2.15 1.18 3.79 3.22 2.89
102.22 102.06 106.90 101.28 100.32 101.38
different pharmaceutical products (Solgar and Dodex) are reported in Table 1. As seen in Table 1, this electrode system is suitable for sensitive Vitamin B12 giving good recovery and reproducibility results. 4. Conclusions In this study, electrochemical Vitamin B12 analysis was carried out using SWCNT–chitosan modified PGE. In the first part of the study, different modifications on PGEs were monitored using SEM, CV and EIS techniques. Then, Vitamin B12 analysis was performed using SWV sensitively. Natural chitosan polymer provided homogeneous dispersion of SWCNT on PGE. The electrochemical response of SWCNT–chitosan modified PGE was compared with the
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Carbon nanotube–chitosan modified disposable pencil graphite electrode for Vitamin B12 analysis Filiz Kuralay, Tayfun Vural, Cem Bayram, Emir Baki Denkbas, Serdar Abaci ∗ Department of Chemistry, Faculty of Science, Hacettepe University, 06800 Beytepe-Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 28 July 2010 Received in revised form 14 March 2011 Accepted 22 March 2011 Available online 22 April 2011 Keywords: Single walled carbon nanotubes Chitosan Biosensor Disposable pencil graphite electrode Vitamin B12
a b s t r a c t A single walled carbon nanotube–chitosan (SWCNT–chitosan) modified disposable pencil graphite electrode (PGE) was used in this study for the electrochemical detection of Vitamin B12 . Electrochemical behaviors of SWCNT–chitosan PGE and chitosan modified PGE were compared by using cyclic voltammetry (CV), square-wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) techniques. SWCNT–chitosan modified electrode was also used for the quantification of Vitamin B12 in pharmaceutical products. The results show that this electrode system is suitable for sensitive Vitamin B12 analysis giving good recovery results. The surface morphologies of the SWCNT–chitosan PGE, chitosan modified PGE and unmodified PGE were characterized by using scanning electron microscopy (SEM). © 2011 Elsevier B.V. All rights reserved.
1. Introduction Nano-structured materials have attracted great attention in recent years in many fields and the development of many new technologies [1–3]. Among them, carbon nanotubes (CNTs) play an important role due to their high electrochemically accessible area, good electronic conductance and strong mechanical, structural properties [4]. CNTs are long, thin cylinders of carbon and can be classified as a graphite sheet rolled into a nanoscale-tube (single walled carbon nanotubes, SWCNTs) and an additional graphene tubes around the core of an SWCNT (multi walled CNTs, MWCNTs) [5–7]. As electrode materials, one of the promising applications of CNTs is their use in the design of biosensors and chemical sensors [8–11]. With the advantages of minimizing fouling of electrode surfaces, exhibiting a high ability to promote some type of the electron transfer reactions between electroactive species and electrodes, enhancing electrocatalytic activity, and facilitating the immobilization of molecules on their surface they have superiority to other kinds of carbon based materials used in electrochemistry for developing effective and sensitive biosensors [12]. The incorporation of CNTs into polymer matrices also promises new improvements in biosensing technology [13]. Chitosan is a naturally occurring polymer (a polysaccharide) that is obtained by the partial deacetylation of chitin [13,14]. Chitosan exhibits excellent film-forming ability, nontoxicity, biocompati-
∗ Corresponding author. Tel.: +90 312 297 6080; fax: +90 312 299 2163. E-mail addresses: [email protected], fi[email protected] (S. Abaci). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.03.030
bility, biodegradability, multiple functional groups, pH-dependent solubility in aqueous media, cheapness and a susceptibility to chemical modification [15–18]. All these properties make it widely used as an immobilization matrix for biosensors [19–24]. Li et al. performed DNA detection based on chitosan film doped with carbon nanotubes by using graphite electrode [25]. Arias et al. studied DNA–methylene blue interaction on glassy carbon electrode using carbon nanotubes in chitosan [26]. Carbon nanotube–chitosan system for electrochemical sensing based on dehydrogenase enzymes was developed by Zhang et al. [17]. Glucose biosensor based on carbon nanotubes/chitosan matrix was fabricated by Liu et al. [27]. Tkac et al. used single walled carbon nanotubes dispersed in a chitosan matrix for galactose biosensor by using glassy carbon electrode [23]. Ghica et al. carried out the application of functionalized carbon nanotubes immobilized into chitosan films in amperometric enzyme biosensors [28]. Carbon nanotubes and chitosan composite film for preparation of amperometric hydrogen peroxide biosensor on glassy carbon electrode was studied by Qian and Yang [29]. An amperometric cholesterol biosensor based on multiwalled carbon nanotubes and organically modified sol–gel/chitosan hybrid composite film was performed by Tan et al. [30]. In this study we used carbon nanotubes dispersed in chitosan matrix for electrochemical Vitamin B12 analysis. Vitamin B12 is a corrin based cobalt complex which is important in human physiology. Its deficiency causes pernicious anemia and neuropathy. Monitoring of Vitamin B12 is essential in the quality control of pharmaceuticals, blood plasma serum, milk products for infants and fermentation products [31,32]. There are
F. Kuralay et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 18–22
several methods cited in the literature for the determination of Vitamin B12 including chromatography, spectrophotometry, chemiluminescence and electrochemistry [33]. Electrochemical techniques such as cyclic voltammetry, differential pulse voltammetry, square-wave voltammetry and amperometry have provided sensitive Vitamin B12 detection [34]. Electrochemical reports demonstrated that Vitamin B12 and its derivatives exhibited rich redox chemistry which was centered on the cobalt atom. Vitamin B12a (with Co(III)) can be reduced reversibly to Vitamin B12r (with Co(II)), and be further reduced to Vitamin B12s (with Co(I)), all in aqueous media. However, estimation of standard potentials for the redox couples seems to be difficult [35,36]. This study focused on the development of single walled carbon nanotube–chitosan (SWCNT–chitosan) modified disposable pencil graphite electrode (PGE) for the electrochemical monitoring of Vitamin B12 based on the signal enhancement in comparison to chitosan modified disposable pencil graphite electrode. The incorporation of SWCNTs into the positively charged polymer matrix was carried out easily [25]. Then, electrochemical responses of cobalt redox species in the structure of Vitamin B12 were monitored after successive additions of Vitamin B12 . Electrochemistry of SWCNT–chitosan modified PGE was examined using cyclic voltammetry (CV), square-wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) techniques. Quantification of Vitamin B12 in pharmaceutical products was performed sensitively by using SWCNT–chitosan modified electrode. Scanning electron microscopy (SEM) was also used for the characterization of SWCNT–chitosan modified, chitosan modified and unmodified PGEs.
2. Experimental 2.1. Apparatus Electrochemical studies, cyclic voltammetry and square-wave voltammetry were carried out using CH Instruments (CHI) 660 C (USA). The three-electrode system consisted of a disposable pencil graphite working electrode (PGE), an Ag/AgCl reference electrode (SCE) (BAS, USA) and a Pt wire (Aldrich) counter electrode were used in the experiments. SEM images were obtained by Zeiss Evo 50 EP-SEM Model Scanning Electron Microscope (SEM) (USA).
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2.3. Procedure All the experiments were carried out at room temperature. A new modified electrode surface was used in each electrochemical detection cycle. Each test was repeated three times. 2.3.1. The preparation of SWCNT–chitosan modified PGE PGE was immersed in SWCNT–chitosan mixture and waited for 30 min by stirring. After modification of PGE, modified electrode was immersed in 0.1 M NaOH for 5 min to make film more stable [25]. Modified PGE was then washed with deionized water and allowed to dry. 2.3.2. The preparation of chitosan modified PGE PGE was immersed in chitosan solution and waited for 30 min by stirring. After modification of PGE, modified electrode was immersed in 0.1 M NaOH for 5 min. Modified PGE was then washed with deionized water and allowed to dry. 2.3.3. Voltammetric transduction The electrochemical characterization of SWCNT–chitosan modified, chitosan modified and unmodified PGEs was performed in 5 mM Fe(CN)6 3−/4− containing 0.1 M KCl using CV between −0.2 V and +0.6 V vs. Ag/AgCl. Detection of Vitamin B12 was performed in 0.1 M PBS with successive Vitamin B12 additions to the solution by stirring 1 min using SWV between −0.2 V and −1.0 V at pulse amplitude of 50 mV. 2.3.4. Impedance measurements Electrochemical impedance spectroscopy (EIS) measurements were controlled at the open-circuit value; +0.15 V vs. Ag/AgCl and the frequency was varied over the range 105 –10−2 Hz with amplitude of 5 mV in the presence of 5 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) mixture as a redox probe prepared in 0.1 M KCl.
2.3.5. Microscopic characterization of unmodified PGE, chitosan modified PGE and SWCNT–chitosan modified PGE by scanning electron microscopy (SEM) The microscopic characterization of unmodified, chitosan modified and SWCNT–chitosan modified PGEs was performed with SEM in resolution magnitude of 200 nm. 3. Results and discussion
2.2. Reagents and preparation of solutions Chitosan was purchased from Fluka. SWCNT and Vitamin B12 were obtained from Sigma–Aldrich. Pharmaceutical formulations containing Vitamin B12 served as real samples (Sample 1: Solgar; Solgar Inc., USA and Sample 2: Dodex; Deva Ltd., Turkey). Other chemicals were in analytical reagent grade and supplied from Sigma and Merck. 0.1 M phosphate buffer solutions (PBS, pH: 2.0, 5.0) were prepared from NaH2 PO4 (Fluka), Na2 HPO4 ·2H2 O (Fluka) and H3 PO4 (Merck) using deionized water. 5 mM Fe(CN)6 3−/4− containing 0.1 M KCl (Merck) was prepared from K3 Fe(CN)6 (Fisher) and K4 Fe(CN)6 (Fisher). Stock Vitamin B12 solutions (10−3 M) were prepared using PBS freshly. Vitamin B12 pharmaceutical formulations were also dissolved in buffer in PBS and filtered before usage. 0.5% solution of chitosan polymer was prepared in 2 M acetic acid (Sigma–Aldrich). This solution was then filtered. SWCNT was purified according to the early procedure [37]. A dispersion of SWCNT in chitosan was obtained by adding 2.5 mg of SWCNT in 1 mL of chitosan solution and sonicated during 30 min.
The surface morphologies of unmodified, chitosan modified and SWCNT–chitosan modified PGEs were examined using SEM (Fig. 1a–c, respectively). The surface roughness of unmodified PGE was monitored successfully in Fig. 1a. After modification of PGE with chitosan, the electrode surface was covered with chitosan polymer (Fig. 1b). It is clear that some parts of PGE became invisible due to chitosan immobilization onto the electrode surface. In the SEM image of SWCNT–chitosan modified PGE, it was observed that SWCNT was distributed homogenously in the porous structure of chitosan matrix (Fig. 1c) [22]. It can be concluded that SEM images proved different modifications on PGE. Cyclic voltammetric behavior of the Fe(CN)6 3−/4− system is a valuable method to monitor the characteristics of the surface of the modified electrodes [31]. In order to see different modifications on PGE, cyclic voltammetric behavior of the Fe(CN)6 3−/4− system was used in the study. Fig. 2 demonstrates the CVs of unmodified, chitosan modified and SWCNT–chitosan modified PGEs in 5 mM Fe(CN)6 3−/4− redox probe containing 0.1 M KCl. After modification of PGE with chitosan (Fig. 2b), there was no appreciable change at the anodic and cathodic peak currents of Fe(CN)6 3−/4− . However,
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Fig. 2. CVs of (a) unmodified, (b) chitosan modified, (c) SWCNT–chitosan modified PGEs in 5 mM Fe(CN)6 3−/4− redox probe containing 0.1 M KCl. CV measurement: at 100 mV/s scan rate between 0.0 V and +1.4 V.
ity due to SWCNT [6,31,33]. Ep value decreased compared to the result obtained with chitosan modified PGE, suggesting the further enhancement at the electron transfer rate of Fe(CN)6 3−/4− [38]. EIS also used to identify and differentiate the unmodified PGE, chitosan modified PGE and SWCNT–chitosan modified PGE. EIS technique gives information on the impedance changes of the electrode surface during the modification process. Fig. 3 shows the impedance spectra of unmodified PGE, chitosan modified PGE and SWCNT–chitosan modified PGE. In the Nyquist plot of impedance spectra, the diameter of the semicircle represents the charge-transfer resistance (Rct ) at the electrode surface. The Rct of unmodified PGE was calculated as 243.12 (Fig. 3a). Rct value decreased to 120.12 (Fig. 3b) after the modification of PGE with chitosan polymer due to the positively charged amino groups of chitosan which could enhance the electron transfer between negatively charged redox probe and the electrode surface. The decrease in Rct value (85.80 ) was higher for SWCNT–chitosan modified PGE (Fig. 3c) than the decrease in Rct value obtained with chitosan modified PGE indicating the increased charge-transfer in the presence of carbon nanotubes [16]. Carbon nanotubes promoted charge-transfer occurred at the electrode surface. Vitamin B12 analysis was carried out using SWV after the characterization of SWCNT–chitosan modified PGE. The effect of successive additions of Vitamin B12 in 0.1 M PBS was studied. Fig. 4A shows the square-wave voltammograms of SWCNT–chitosan modified PGE in the absence and presence of Vitamin B12 at pH 2.0.
Fig. 1. SEM images of (a) unmodified, (b) chitosan modified, (c) SWCNT–chitosan modified PGEs.
there was a change at the redox peak potentials of Fe(CN)6 3−/4− compared to the redox potentials obtained in the case of unmodified PGE (Fig. 2a). The peak potential difference (Ep ) decreased due to chitosan immobilization. After modification of PGE with SWCNT–chitosan mixture (Fig. 2c), the anodic and cathodic peak currents of Fe(CN)6 3−/4− increased, indicating SWCNT improved the electroactive surface area of the electrode and catalytic abil-
Fig. 3. Impedance spectra of (a) unmodified, (b) chitosan modified, (c) SWCNT–chitosan modified PGEs in 5 mM Fe(CN)6 3−/4− redox probe containing 0.1 M KCl. EIS measurement: at open-circuit value of +0.15 V between 105 and 10−2 Hz range with amplitude of 5 mV.
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Fig. 4. (A) SWVs for different concentrations of Vitamin B12 at pH 2.0 in 0.1 M PBS: (a) 0 nM, (b) 5 nM, (c) 35 nM, (d) 50 nM, (e) 100 nM. SWV measurement: between −0.2 V and −1.0 V at pulse amplitude of 50 mV. (B) The effect of Vitamin B12 concentration on reduction peak currents of Co(II) to Co(I) using (a) SWCNT–chitosan modified PGE, (b) chitosan modified PGE at pH 2.0 (n = 3).
As seen in Fig. 4A-a, there were no electroactive species between −0.20 V and −0.90 V potential intervals in the voltammograms of SWCNT–chitosan modified PGE. With the addition of Vitamin B12 two peaks existed in the voltammograms. The reduction peak at about −0.32 V vs. Ag/AgCl was attributed to the reduction of Co(III) to Co(II) and the peak at −0.66 V vs. Ag/AgCl was belonged to the reduction of Co(II) to Co(I). There was an electrocatalytic effect using carbon nanotube-polymer modified electrode due to reduction of Co(II) to Co(I) compared to earlier studies [31,35]. The reduction peak currents of cobalt redox couples increased with increasing concentration of Vitamin B12 (Fig. 4A-b to e). In order to test the advantage of SWCNT–chitosan modified electrode over chitosan modified electrode, Vitamin B12 analysis were also carried out using only chitosan modified electrode. The reduction peak currents due to Co(II) to Co(I) were presented in Fig. 4B for SWCNT–chitosan modified and chitosan modified PGEs. As seen from these results enhanced electrochemical response was obtained with SWCNT–chitosan modified PGE (Fig. 4B-a) compared to chitosan modified PGE (Fig. 4B-b). In the presence of carbon nanotubes, the electrode system gave better results indicating high ability to promote the electron transfer reactions between cobalt redox couple and electrode, enhancing electrocatalytic activity and increasing sensitivity of the biosensor [12]. The comparison of SWCNT–chitosan modified electrode over SWCNT modified electrode were also carried out. However, irreproducible results were obtained in the case of SWCNT modified electrode (not shown). The experiments were also carried out at pH 5.0 in 0.1 M PBS. Fig. 5A shows the square-wave voltammograms of SWCNT–chitosan modified PGE in the presence of Vitamin B12 at pH 5.0. With the addition of Vitamin B12 two peaks existed similar to the results obtained at pH 2.0 above. The reduction peak at about −0.40 V vs. Ag/AgCl was attributed to the reduction of Co(III)
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Fig. 5. (A) SWVs for different concentrations of Vitamin B12 at pH 5.0 in 0.1 M PBS. SWV measurement: between −0.2 V and −1.0 V at pulse amplitude of 50 mV: (a) 5 nM, (b) 35 nM, (c) 50 nM, (d) 80 nM. (B) The effect of Vitamin B12 concentration on reduction peak currents of Co(II) to Co(I) using (a) SWCNT–chitosan modified PGE, (b) chitosan modified PGE at pH 5.0 (n = 3).
to Co(II) and the peak at −0.74 V vs. Ag/AgCl was belonged to the reduction of Co(II) to Co(I). The reduction peak currents of cobalt redox couples increased with increasing concentration of Vitamin B12 (Fig. 5A-a to d). However, the magnitude of the peak currents was higher at pH 2.0 than the magnitude of peak currents at pH 5.0. Also, the reduction peak potentials were more positive at pH 2.0 than the reduction peak potentials at pH 5.0. In order to monitor the advantage of SWCNT–chitosan modified electrode over chitosan modified electrode at this pH value, same experiments were carried out using only chitosan modified electrode. The reduction peak currents of Co(II) to Co(I) were presented in Fig. 5B using SWCNT–chitosan modified PGE (Fig. 5B-a) and chitosan modified PGE (Fig. 5B-b). Enhanced electrochemical response was obtained with SWCNT–chitosan modified PGE compared to chitosan modified PGE similar to the results at pH 2.0. The detection limit of the biosensor was found as 0.89 nM with a linear concentration range interval of 5 nM and 100 nM at pH 2.0 (R2 = 0.987) and the detection limit of the biosensor was found as 2.1 nM with a linear concentration range interval of 5 nM and 80 nM at pH 5.0 (R2 = 0.994). The stability of the biosensor was also studied. After 1 week of use, the electrode response decreased about 10%. The pH dependence of the electrode was examined between pH 2 and 5 (Fig. 6A). As seen in this figure, the response of the SWCNT–chitosan modified PGE was maximum at pH 2.0. Vitamin B12 analysis was also carried out using pharmaceutical products at pH 2.0 in 0.1 M PBS. Fig. 6B shows the square-wave voltammograms of SWCNT–chitosan modified PGE after standard additions of pharmaceutical Vitamin B12 product (Solgar). With increasing concentration of pharmaceutical Vitamin B12 product, the peak currents due to Co(II) to Co(I) reduction increased (Fig. 6Ba to c) supporting our earlier results above. The results for two
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response of chitosan modified PGE for Vitamin B12 analysis. A signal enhancement was obtained for the reduction of cobalt redox couples in the structure of Vitamin B12 using SWCNT–chitosan modified PGE at low potentials due to the catalytic activity of SWCNT. The detection limit of the biosensor was found as 0.89 nM with a concentration range interval of 5 nM and 100 nM at pH 2.0 (R2 = 0.987) and the detection limit of the biosensor was found as 2.1 nM with a linear concentration range interval of 5 nM and 80 nM at pH 5.0 (R2 = 0.994). The modified electrode was also provided quantification of Vitamin B12 in pharmaceutical products at pH 2.0 by using two different products with good recovery and reproducibility results. The SWCNT–chitosan modified disposable pencil graphite electrode presented here was prepared in an one step procedure with the advantages of simple, fast, easy and cheap preparation. The selectivity of the electrode was good. The detection method has novelty and comparability with the studies based on Vitamin B12 analysis in the literature [31–35]. The prepared electrodes can also be promising for new biosensing applications based on other real samples. References [1] [2] [3] [4]
Fig. 6. (A) pH dependence of the electrode at 80 nM Vitamin B12 concentration (n = 3). (B) SWVs for different concentrations of Vitamin B12 using one tablet of Solgar at pH 2.0: (a) 35 nM, (b) 50 nM, (c) 80 nM. SWV measurement: between −0.2 V and −1.0 V at pulse amplitude of 50 mV. Table 1 Results for the determination of Vitamin B12 in real samples.
Sample 1
Sample 2
Added (nM)
Found (nM)
RSD (%, n = 3)
Recovery (%)
35.00 50.00 80.00 35.00 50.00 80.00
35.78 51.03 85.52 35.45 50.16 81.11
2.37 2.15 1.18 3.79 3.22 2.89
102.22 102.06 106.90 101.28 100.32 101.38
different pharmaceutical products (Solgar and Dodex) are reported in Table 1. As seen in Table 1, this electrode system is suitable for sensitive Vitamin B12 giving good recovery and reproducibility results. 4. Conclusions In this study, electrochemical Vitamin B12 analysis was carried out using SWCNT–chitosan modified PGE. In the first part of the study, different modifications on PGEs were monitored using SEM, CV and EIS techniques. Then, Vitamin B12 analysis was performed using SWV sensitively. Natural chitosan polymer provided homogeneous dispersion of SWCNT on PGE. The electrochemical response of SWCNT–chitosan modified PGE was compared with the
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