Colloids and Surfaces B: Biointerfaces 129 (2015) 169–174
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Layer by layer assembled films between hemoglobin and multiwall carbon nanotubes for pH-switchable biosensing Zhongqin Pan a,b,1 , Xiaojun Liu a,b,1 , Jing Xie a,b,c , Ning Bao a,b , Hong He c , Xiaodong Li a,b , Jiang Zeng a,b , Haiying Gu a,b,∗ a
Institute of Analytical Chemistry for Life Science, Nantong University, Nantong 226019, China School of Public Health, Nantong University, Nantong 226019, China c Affiliated Hospital, Nantong University, Nantong 226021, China b
a r t i c l e
i n f o
Article history: Received 27 September 2014 Received in revised form 3 March 2015 Accepted 19 March 2015 Available online 27 March 2015 Keywords: Multiwall carbon nanotubes Hemoglobin Layer by layer assembly pH switchable Biosensing
a b s t r a c t Although pH-switchable behaviors have been reported based on multilayer films modified electrodes, their pH-switchable biosensing is still difficult due to the existence of the electroactive mediator. In this study, we report the pH-dependable determination of hydrogen peroxide (H2 O2 ) based on a fourbilayer film fabricated through layer by layer assembly between hemoglobin (Hb) and multiwall carbon nanotubes (MWCNTs). We observed that response of electroactive probe Fe(CN)6 3− at the multilayer films was very sensitive and reversible to pH values of phosphate buffer solutions phosphate buffer solution with cyclic voltammetry. The reduction peak height of Fe(CN)6 3− at the multilayer film could reach ∼221 A at pH 3.0 while 0 A at pH 9.0. The linear range for the detection of H2 O2 at pH 3.0 was from 12.5 M to 10.4 mM, which was much wider than that at pH 9.0. Our results demonstrated that the detection of H2 O2 with the proposed modified electrode is dependent on pH values of phosphate buffer solution. Moreover, the component of multilayer films has impacts on the performance of biosensors with pH-switchable behaviors. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The study of molecular switches and memories has attracted much attention during the past decade owing to their potential function as the key component for manufacturing materials at molecular and electronics levels [1–6]. The development of the devices was mainly based on their controllable responses to external stimuli [7], such as stress [8], temperature [9], pH value [10], moisture [11], light [12], and ionic strength [13]. Since pH value can be adjusted conveniently, it had been utilized to control the process of biological reaction in aqueous solutions [10,14]. To date, most of the pH-controlled switchable electrochemical devices were constructed based on the combination of enzyme or proteins, such as concanavalin A (Con A) and glucose
Abbreviations: MWCNTs, multiwall carbon nanotubes; GCE, glassy carbon electrode. ∗ Corresponding author at: Institute of Analytical Chemistry for Life Science, Nantong University, Nantong 226019, China. Tel.: +86 513 8501 2916; fax: +86 513 8501 2916. E-mail addresses: [email protected], [email protected] (H. Gu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2015.03.044 0927-7765/© 2015 Elsevier B.V. All rights reserved.
oxidase (GOD), Con A and horseradish peroxide (HRP), Con A/Dextran (Con A/Dex) and myoglobin (Mb) [15–18]. An advantage for those enzyme or protein based devices is that pH-controllable bioelectrocatalysis could be performed on the basis of direct electrochemistry of enzyme or protein. However, with the existence of electroactive mediators (such as Fe(CN)6 3− ), direct electrochemistry of most enzymes or proteins could not be fully realized. As a result, their bioelectrocatalysis and the biosensing functions were significantly influenced due to the electroactive mediator. For the same reason, the pH-switchable determination has appeared in a few previous reports [10,19–22]. Most recently, based on chitosan modified glassy carbon electrode (GCE), our group reported that instead of Con A or Con A/Dex, silver (Ag) nanoparticles (NPs) could be coupled with hemoglobin (Hb) with layer by layer assembly (LBL) method for the pH-switchable behavior and pH-controllable bioelectrocatalysis of H2 O2 [10]. The introduction of NPs with LBL assembly technique could enhance the pH-dependent devices on biosensing functions with the additional advantages of nanomaterials including large surface areas, controllable sizes [23,24], etc. By comparison, LBL assembly demonstrates distinguished advantages over other film preparation methods in the precise control of film composition and thickness at a molecular or nanometer level [15]. In constructing
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LBL films, the driving force is usually electrostatic interaction, while nonelectrostatic interactions such as hydrogen bonding and biospecific recognition [18]. Herein, the LBL assembly process is mainly based on electrostatic interaction between NPs of negative charge and Hb of positive charge. Hb in the LBL film may provide a new interface for the study of electrochemical biosensors and other molecular biodevices (e.g., pH-dependent switch). Moreover, it is believed that such pH-sensitive “on–off” function benefited from porphyrin structure in the Hb molecules and the derived switchable actions at different pH values in the existence of mediator (Fe(CN)6 3− ) [25]. As is well-known, multiwalled carbon nanotubes (MWCNTs) play important role in scientific and industrial fields and serve as an excellent matrix for promoting the electron transfer rate between solution and electrode owing to their great chemical stability, outstanding electrical conductivity and high mechanical strength [26,27]. Therefore, in this study, we used MWCNTs instead of AgNPs to fabricate the multilayer films for the study of pH-switchable functions. Our study indicated that with the multilayer films modified glassy carbon electrode (GCE), the bioelectrocatalysis of H2 O2 strongly depends on pH values of phosphate buffer solution phosphate buffer solution in cyclic voltammograms (CVs). In addition, our investigations show that it is valuable to fabricate Hb-based films with both switchable and biosensing functions. Fig. 1. The TEM image of MWCNTs dispersion.
2. Experimental 2.1. Materials and methods Bovine Hb (Sigma–Aldrich Co., St. Louis, MO, http://www. sigmaaldrich.com) was used without further purification. MWCNTs (95%, diameter ∼20–40 nm) powders were prepared for better dispersibility according to our previous work [26] (Shenzhen Nanotech. Port. Co. Ltd., http://www.nanotubes.com). Other chemicals were of analytical reagent grade. 0.10 M phosphate buffer solution with different pH values were prepared by mixing the stocking standard solutions of Na2 HPO4 and NaH2 PO4 , and the pH was adjusted by 0.10 M H3 PO4 or 0.10 M NaOH solution. Twice-distilled water was used in all experiments. Electrochemical impedance spectroscopy (EIS) was performed in 0.10 M KCl solution including 1.0 mM Fe(CN)6 3− /Fe(CN)4− (1:1) as a supporting electrolyte at its open circuit potential with the AUTOLAB PGSTAT 302 N electrochemical working station (Metrohm Co. Ltd., Switzerland) accompanying with the frequency from 1.0 × 10−3 to 1.0 × 105 Hz. All electrochemical measurements were performed with a CHI 660 C electrochemical working station (CH Instruments Co., USA) equipped with conventional threeelectrode system, in which the modified GCE (diameter 4 mm) was as the working electrode, a platinum wire and a saturated calomel electrode (SCE) were employed as the counter electrode and the reference electrode, respectively. As shown in Fig. 1, the optimal MWCNT dispersion was characterized by transmission electron microscopy (TEM) (Tecnai 12, Netherland). Scanning electron microscope (SEM) was used to characterize the morphology of the modified GCE (S-4800, Japan). UV–Vis absorption spectra based on indium tin oxides were recorded using a UV-2450 spectrophotometer (Shimadzu Co., Japan).
was incubated in 3.0 mg mL−1 Hb solution (dissolved in pH 6.0 phosphate buffer solution) at 4 ◦ C for 20 min, washed with double-distilled water and dried in air. Then the first bilayer ({MWCNTs/Hb})–Chitosan modified GCE was obtained. The procedure was repeated for the optimal number of {MWCNTs/Hb} bilayer (The detailed assembly process was shown in Scheme 1). By comparison, the four-bilayer film modified electrode was found best for the study, denoted as {MWCNTs/Hb}4 –Chitosan–GCE. 3. Results and discussion 3.1. Characterization of the assembly of {MWCNTs/Hb}4 multilayer film The assembly of the monolayer ({MWCNTs/Hb}1 ) film was characterized by SEM. As shown in Fig. 2, the SEM images of the bare GCE (A), the Chitosan–GCE (B), the MWCNTs–Chitosan–GCE (C) and the Hb-MWCNTs–Chitosan–GCE (D) were taken respectively. The differences demonstrated that Hb had been successfully assembled on the surface of the MWCNTs–Chitosan–GCE. Moreover, well dispersed MWCNTs nanoparticles confirmed that MWCNTs had been successfully entrapped in the chitosan film on the GCE. Moreover, CV was employed to investigate the assembly of {MWCNTs/Hb}n films on the Chitosan–GCE in phosphate
2.2. Preparation of {MWCNTs/Hb}4 multilayer films The chitosan modified GCE was prepared according to our previous work [26]. Then the dry chitosan film modified GCE was dipped into MWCNTs dispersion at 4 ◦ C for 20 min, washed with double-distilled water and dried at room temperature, simplified as MWCNTs–Chitosan–GCE. Afterwards, the modified GCE
Scheme 1. The assembly process of the {MWCNTs/Hb}4 on the modified GCE.
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Fig. 2. The SEM images of (A) bare GCE, (B) Chitosan–GCE, (C) MWCNTs–Chitosan–GCE and (D) Hb–MWCNTs–Chitosan–GCE.
Fig. 3. (A) Cyclic voltammograms of {MWCNTs/Hb}n films at 100 mV s−1 in pH 7.0 phosphate buffer solution phosphate buffer solution (n = 1–5). (B) The redox peak currents varied with the number of bilayers n (from 1 to 5). (C) Electrochemical impedance spectroscopy of the {MWCNTs/Hb}n films (n = 1–4). (D) Ultraviolet-visible absorption spectra of the {MWCNTs/Hb}6 film on the ITO substrates with bilayer number (n) from 1 to 4.
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Fig. 4. (A) Cyclic voltammograms of Fe(CN)6 3− at the {MWCNTs/Hb}4 –Chitosan film at 100 mV s−1 in different phosphate buffer solution (pH 3.0–9.0). (B) The reduction peak current (䊉) and the peak separation () of Fe(CN)6 3− on the surface of the {MWCNTs/Hb}4 –Chitosan modified GCE at 100 mV s−1 in phosphate buffer solution with different pH levels (pH 3.0–9.0). (C) Dependence of the CV reduction peak current of Fe(CN)6 3− on the cycle number when the solution pH switched between 3.0 and 9.0 for the same {MWCNTs/Hb}4 –Chitosan film.
buffer solution. Fig. 3A shows that redox peak currents of the {MWCNTs/Hb}n film increased gradually with the bilayer number increased from 1 to 4. Fig. 3B shows the redox peak currents with the increase of the bilayer number (n < 5). It could be easily found that the redox peak curves of {MWCNTs/Hb}5 films were almost the same as the peak curves of {MWCNTs/Hb}4 films. Therefore, in the following experiments, the {MWCNTs/Hb}4 films were studied in detail. EIS and UV–Vis spectra were then used to investigate the LBL assembly process of {MWCNTs/Hb}4 films. In EIS, the electron transfer limited process could be observed using the semicircle part at higher frequencies. In this case, the electron transfer resistance, Ret , is equal to the semicircle diameter in EIS. Ret is the key parameter for the electron transfer kinetics of probe Fe(CN)6 3− /Fe(CN)6 4− at the electrode interface. On the other hand, the diffusion process could be characterized with the linear part at lower frequencies in EIS. As shown in Fig. 3C, only a very small semicircle could be observed at bare GCE (curve a), after chitosan was deposited on the GCE, the diameter of the semicircle increased (curve), implying that the chitosan film obstructs the direct electron transfer of probe due to poor conductivity of the chitosan film. With the assembly of the MWCNTs NPs, the semicircle Ret decreased obviously, showing excellent conductivity of the nanocomposite (curve c), which also indicates that MWCNTs NPs can enhance the direct transfer. When Hb was immobilized, Ret increased again (curve d), confirming successful immobilization of Hb. Afterwards, with
the increase number (n < 5) of the bilayer of {MWCNTs/Hb}n , Ret increased sequentially (curve d–g). Such results illustrate successful assembly of the multilayer films. As shown in Fig. 3D, the Soret band of Hb in pH 6.0 phosphate buffer solution was 406 nm [10], and in this work the multilayer films modified on ITO substrates all showed Soret bands at 412 nm, which were very close to 406 nm. In addition, the spectra also displayed that the characteristic absorption of Hb increased with the increase of bilayer number. All the results demonstrate successful assembly of {MWCNTs/Hb}4 films. 3.2. pH-switchable behavior of {MWCNTs/Hb}4 multilayer film toward Fe(CN)6 3− Fig. 4A illustrates responses of the {MWCNTs/Hb}4 –Chitosan film modified GCE in phosphate buffer solution of different pH values with Fe(CN)6 3− as the mediator. It could be observed that the redox peak currents (Ip ) and the peak potential difference (Ep ) were substantially influenced by the pH level. The reduction peak current of Fe(CN)6 3− at the multilayer film could reach ∼221 A at pH 3.0 but only 0 at pH 9.0. However, the reduction peak current of Fe(CN)6 3− at the {Hb/AgNPs}4 film was only about ∼80 A at pH 3.0 [10]. Fig. 4B shows that Ep gradually increased with the increase of the pH value. The results of six successive measurements were almost the same: the reduction peak currents are all around ∼221 A at pH 3.0
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Fig. 5. (A) Plots of cathodic and anodic peak current vs. scan rate in 0.10 M pH 3.0 phosphate buffer solution at various scan rates: 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, and 100 mV s−1 . (B) Cyclic voltammograms of Fe(CN)6 3− at the {MWCNTs/Hb}4 film in 0.10 M pH 3.0 phosphate buffer solution at 10 mV s−1 with the presence of 0 (a), 1.0 (b), 2.0 (c), and 4.0 mM (d) H2 O2 .
(the “on” state) but nearly 0 at pH 9.0 (the “off” state) (Fig. 4C). Such results revealed that the {MWCNTs/Hb}4 –Chitosan film modified GCE could perform reversible pH-sensitive “on–off” functions. Similar pH switchable behaviors could be observed on the four-bilayer MWCNTs/Hb film and the four-layer
AgNPs/Hb film. By comparison, the redox peak currents of the {MWCNTs/Hb}4 –Chitosan film were larger than that of the {AgNPs/Hb}4 –Chitosan film. We are assuming that this is because of smaller sizes and larger surface area of MWCNTs compared with AgNPs.
Fig. 6. (A) Amperometric responses of the {MWCNTs/Hb}4 –Chitosan film by successive additions of H2 O2 to 8 mL 0.10 M pH 3.0 phosphate buffer solution containing 1.0 mM Fe(CN)6 3− with stir. (B) Amperometric responses of the {MWCNTs/Hb}4 –Chitosan film upon successive additions of H2 O2 to 8 mL 0.10 M pH 9.0 phosphate buffer solution containing 1.0 mM Fe(CN)6 3− with stir. (C) Amperometric response curve for H2 O2 at pH 3.0 () and pH 9.0 ().
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3.3. Scan rate and bioelectrocatalysis study of the {MWCNTs/Hb}4 multilayer film In the following we investigated the influence of the scan rates on the bioelectrocatalysis of H2 O2 with the proposed pHswitchable biosensor. Fig. 5A illustrates CVs of Fe(CN)6 3− at the {MWCNTs/Hb}4 –Chitosan film in 0.10 M pH 3.0 phosphate buffer solution at various scan rates. It could be found that redox peak currents increased linearly with the increase of the square root of the potential scan rate, indicating that the reaction at the modified electrode is controlled by diffusion process. As the determining reaction step, the diffusion process could improve the bioelectrocatalysis of H2 O2 at the modified electrode. Fig. 5B shows pH-controllable bioelectrocatalysis of H2 O2 toward Fe(CN)6 3− at 10 mV s−1 . The results demonstrated that the reduction peak height increased with continuous addition of H2 O2 .
with MWCNTs NPs can largely enhance the performance of the pHswitchable biosensors on detection of H2 O2 . Our study indicated that it is valuable to continue the investigation of Hb-based films with both switchable and biosensing functions. Further fabrication of Hb-based films with switchable and biosensing functions will focus on using those devices for more practical applications. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant numbers: 21175075; 21475070; 21075070), the Natural Science Foundation of Jiangsu Province (Grant number: BK2011047, BK2012651, BK2012652), the Postgraduate Technology Innovation Project of Nantong University (YKC13043), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
3.4. Effect of pH value on the bioelectrocatalysis
References
Fig. 6 illustrates the amperometric current–time response at pH 3.0 and pH 9.0 by adding H2 O2 continuously at an applied potential of −0.30 V. It could be observed from Fig. 6C that amperometric current grew linearly with the increase of H2 O2 concentration ranging from 12.5 M to 10.4 mM, and the detection limit was 6.25 M (S/N = 3) in pH 3.0 phosphate buffer solution containing Fe(CN)6 3− (curve a). By comparison, the amperometric currents in pH 9.0 phosphate buffer solution with the same H2 O2 concentration were much lower than those in pH 3.0 phosphate buffer solution although there was a linear relationship between the currents and the H2 O2 concentration from 25 M to 2.3 mM. With the increase of the H2 O2 concentration, the difference of amperometric currents in both phosphate buffer solution solutions increased. The peak current in pH 9.0 phosphate buffer solution could not be over 50 A even the H2 O2 concentration reach 10 mM while that in pH 3.0 could reach ∼270 A. Such relationship demonstrated that the proposed biosensors could be applied for the pH-switchable determination. The linear range with the {MWCNTs/Hb}4 films in pH 3.0 phosphate buffer solution is larger than the previous reports using the {Con A/HRP/Con A/GOD}3 films and the {Con A/Dex}4 –Mb films [15,16]. Such differences suggested that the nano-material enhanced LBL films have far-reaching influences on the construction of pH switches and biosensors in molecular devices. And the mechanism of pH-controllable bioelectrocatalysis could also be attributed to the characteristics of Hb at different pH values [10].
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4. Conclusions We report the pH-controllable bioelectrocatalysis of H2 O2 at the {MWCNTs/Hb}4 –Chitosan film modified GCE. The detection of H2 O2 could be controlled by pH values of the electrolyte solution with Fe(CN)6 3− as the mediator. The replacement of AgNPs
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Layer by layer assembled films between hemoglobin and multiwall carbon nanotubes for pH-switchable biosensing Zhongqin Pan a,b,1 , Xiaojun Liu a,b,1 , Jing Xie a,b,c , Ning Bao a,b , Hong He c , Xiaodong Li a,b , Jiang Zeng a,b , Haiying Gu a,b,∗ a
Institute of Analytical Chemistry for Life Science, Nantong University, Nantong 226019, China School of Public Health, Nantong University, Nantong 226019, China c Affiliated Hospital, Nantong University, Nantong 226021, China b
a r t i c l e
i n f o
Article history: Received 27 September 2014 Received in revised form 3 March 2015 Accepted 19 March 2015 Available online 27 March 2015 Keywords: Multiwall carbon nanotubes Hemoglobin Layer by layer assembly pH switchable Biosensing
a b s t r a c t Although pH-switchable behaviors have been reported based on multilayer films modified electrodes, their pH-switchable biosensing is still difficult due to the existence of the electroactive mediator. In this study, we report the pH-dependable determination of hydrogen peroxide (H2 O2 ) based on a fourbilayer film fabricated through layer by layer assembly between hemoglobin (Hb) and multiwall carbon nanotubes (MWCNTs). We observed that response of electroactive probe Fe(CN)6 3− at the multilayer films was very sensitive and reversible to pH values of phosphate buffer solutions phosphate buffer solution with cyclic voltammetry. The reduction peak height of Fe(CN)6 3− at the multilayer film could reach ∼221 A at pH 3.0 while 0 A at pH 9.0. The linear range for the detection of H2 O2 at pH 3.0 was from 12.5 M to 10.4 mM, which was much wider than that at pH 9.0. Our results demonstrated that the detection of H2 O2 with the proposed modified electrode is dependent on pH values of phosphate buffer solution. Moreover, the component of multilayer films has impacts on the performance of biosensors with pH-switchable behaviors. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The study of molecular switches and memories has attracted much attention during the past decade owing to their potential function as the key component for manufacturing materials at molecular and electronics levels [1–6]. The development of the devices was mainly based on their controllable responses to external stimuli [7], such as stress [8], temperature [9], pH value [10], moisture [11], light [12], and ionic strength [13]. Since pH value can be adjusted conveniently, it had been utilized to control the process of biological reaction in aqueous solutions [10,14]. To date, most of the pH-controlled switchable electrochemical devices were constructed based on the combination of enzyme or proteins, such as concanavalin A (Con A) and glucose
Abbreviations: MWCNTs, multiwall carbon nanotubes; GCE, glassy carbon electrode. ∗ Corresponding author at: Institute of Analytical Chemistry for Life Science, Nantong University, Nantong 226019, China. Tel.: +86 513 8501 2916; fax: +86 513 8501 2916. E-mail addresses: [email protected], [email protected] (H. Gu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2015.03.044 0927-7765/© 2015 Elsevier B.V. All rights reserved.
oxidase (GOD), Con A and horseradish peroxide (HRP), Con A/Dextran (Con A/Dex) and myoglobin (Mb) [15–18]. An advantage for those enzyme or protein based devices is that pH-controllable bioelectrocatalysis could be performed on the basis of direct electrochemistry of enzyme or protein. However, with the existence of electroactive mediators (such as Fe(CN)6 3− ), direct electrochemistry of most enzymes or proteins could not be fully realized. As a result, their bioelectrocatalysis and the biosensing functions were significantly influenced due to the electroactive mediator. For the same reason, the pH-switchable determination has appeared in a few previous reports [10,19–22]. Most recently, based on chitosan modified glassy carbon electrode (GCE), our group reported that instead of Con A or Con A/Dex, silver (Ag) nanoparticles (NPs) could be coupled with hemoglobin (Hb) with layer by layer assembly (LBL) method for the pH-switchable behavior and pH-controllable bioelectrocatalysis of H2 O2 [10]. The introduction of NPs with LBL assembly technique could enhance the pH-dependent devices on biosensing functions with the additional advantages of nanomaterials including large surface areas, controllable sizes [23,24], etc. By comparison, LBL assembly demonstrates distinguished advantages over other film preparation methods in the precise control of film composition and thickness at a molecular or nanometer level [15]. In constructing
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LBL films, the driving force is usually electrostatic interaction, while nonelectrostatic interactions such as hydrogen bonding and biospecific recognition [18]. Herein, the LBL assembly process is mainly based on electrostatic interaction between NPs of negative charge and Hb of positive charge. Hb in the LBL film may provide a new interface for the study of electrochemical biosensors and other molecular biodevices (e.g., pH-dependent switch). Moreover, it is believed that such pH-sensitive “on–off” function benefited from porphyrin structure in the Hb molecules and the derived switchable actions at different pH values in the existence of mediator (Fe(CN)6 3− ) [25]. As is well-known, multiwalled carbon nanotubes (MWCNTs) play important role in scientific and industrial fields and serve as an excellent matrix for promoting the electron transfer rate between solution and electrode owing to their great chemical stability, outstanding electrical conductivity and high mechanical strength [26,27]. Therefore, in this study, we used MWCNTs instead of AgNPs to fabricate the multilayer films for the study of pH-switchable functions. Our study indicated that with the multilayer films modified glassy carbon electrode (GCE), the bioelectrocatalysis of H2 O2 strongly depends on pH values of phosphate buffer solution phosphate buffer solution in cyclic voltammograms (CVs). In addition, our investigations show that it is valuable to fabricate Hb-based films with both switchable and biosensing functions. Fig. 1. The TEM image of MWCNTs dispersion.
2. Experimental 2.1. Materials and methods Bovine Hb (Sigma–Aldrich Co., St. Louis, MO, http://www. sigmaaldrich.com) was used without further purification. MWCNTs (95%, diameter ∼20–40 nm) powders were prepared for better dispersibility according to our previous work [26] (Shenzhen Nanotech. Port. Co. Ltd., http://www.nanotubes.com). Other chemicals were of analytical reagent grade. 0.10 M phosphate buffer solution with different pH values were prepared by mixing the stocking standard solutions of Na2 HPO4 and NaH2 PO4 , and the pH was adjusted by 0.10 M H3 PO4 or 0.10 M NaOH solution. Twice-distilled water was used in all experiments. Electrochemical impedance spectroscopy (EIS) was performed in 0.10 M KCl solution including 1.0 mM Fe(CN)6 3− /Fe(CN)4− (1:1) as a supporting electrolyte at its open circuit potential with the AUTOLAB PGSTAT 302 N electrochemical working station (Metrohm Co. Ltd., Switzerland) accompanying with the frequency from 1.0 × 10−3 to 1.0 × 105 Hz. All electrochemical measurements were performed with a CHI 660 C electrochemical working station (CH Instruments Co., USA) equipped with conventional threeelectrode system, in which the modified GCE (diameter 4 mm) was as the working electrode, a platinum wire and a saturated calomel electrode (SCE) were employed as the counter electrode and the reference electrode, respectively. As shown in Fig. 1, the optimal MWCNT dispersion was characterized by transmission electron microscopy (TEM) (Tecnai 12, Netherland). Scanning electron microscope (SEM) was used to characterize the morphology of the modified GCE (S-4800, Japan). UV–Vis absorption spectra based on indium tin oxides were recorded using a UV-2450 spectrophotometer (Shimadzu Co., Japan).
was incubated in 3.0 mg mL−1 Hb solution (dissolved in pH 6.0 phosphate buffer solution) at 4 ◦ C for 20 min, washed with double-distilled water and dried in air. Then the first bilayer ({MWCNTs/Hb})–Chitosan modified GCE was obtained. The procedure was repeated for the optimal number of {MWCNTs/Hb} bilayer (The detailed assembly process was shown in Scheme 1). By comparison, the four-bilayer film modified electrode was found best for the study, denoted as {MWCNTs/Hb}4 –Chitosan–GCE. 3. Results and discussion 3.1. Characterization of the assembly of {MWCNTs/Hb}4 multilayer film The assembly of the monolayer ({MWCNTs/Hb}1 ) film was characterized by SEM. As shown in Fig. 2, the SEM images of the bare GCE (A), the Chitosan–GCE (B), the MWCNTs–Chitosan–GCE (C) and the Hb-MWCNTs–Chitosan–GCE (D) were taken respectively. The differences demonstrated that Hb had been successfully assembled on the surface of the MWCNTs–Chitosan–GCE. Moreover, well dispersed MWCNTs nanoparticles confirmed that MWCNTs had been successfully entrapped in the chitosan film on the GCE. Moreover, CV was employed to investigate the assembly of {MWCNTs/Hb}n films on the Chitosan–GCE in phosphate
2.2. Preparation of {MWCNTs/Hb}4 multilayer films The chitosan modified GCE was prepared according to our previous work [26]. Then the dry chitosan film modified GCE was dipped into MWCNTs dispersion at 4 ◦ C for 20 min, washed with double-distilled water and dried at room temperature, simplified as MWCNTs–Chitosan–GCE. Afterwards, the modified GCE
Scheme 1. The assembly process of the {MWCNTs/Hb}4 on the modified GCE.
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Fig. 2. The SEM images of (A) bare GCE, (B) Chitosan–GCE, (C) MWCNTs–Chitosan–GCE and (D) Hb–MWCNTs–Chitosan–GCE.
Fig. 3. (A) Cyclic voltammograms of {MWCNTs/Hb}n films at 100 mV s−1 in pH 7.0 phosphate buffer solution phosphate buffer solution (n = 1–5). (B) The redox peak currents varied with the number of bilayers n (from 1 to 5). (C) Electrochemical impedance spectroscopy of the {MWCNTs/Hb}n films (n = 1–4). (D) Ultraviolet-visible absorption spectra of the {MWCNTs/Hb}6 film on the ITO substrates with bilayer number (n) from 1 to 4.
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Fig. 4. (A) Cyclic voltammograms of Fe(CN)6 3− at the {MWCNTs/Hb}4 –Chitosan film at 100 mV s−1 in different phosphate buffer solution (pH 3.0–9.0). (B) The reduction peak current (䊉) and the peak separation () of Fe(CN)6 3− on the surface of the {MWCNTs/Hb}4 –Chitosan modified GCE at 100 mV s−1 in phosphate buffer solution with different pH levels (pH 3.0–9.0). (C) Dependence of the CV reduction peak current of Fe(CN)6 3− on the cycle number when the solution pH switched between 3.0 and 9.0 for the same {MWCNTs/Hb}4 –Chitosan film.
buffer solution. Fig. 3A shows that redox peak currents of the {MWCNTs/Hb}n film increased gradually with the bilayer number increased from 1 to 4. Fig. 3B shows the redox peak currents with the increase of the bilayer number (n < 5). It could be easily found that the redox peak curves of {MWCNTs/Hb}5 films were almost the same as the peak curves of {MWCNTs/Hb}4 films. Therefore, in the following experiments, the {MWCNTs/Hb}4 films were studied in detail. EIS and UV–Vis spectra were then used to investigate the LBL assembly process of {MWCNTs/Hb}4 films. In EIS, the electron transfer limited process could be observed using the semicircle part at higher frequencies. In this case, the electron transfer resistance, Ret , is equal to the semicircle diameter in EIS. Ret is the key parameter for the electron transfer kinetics of probe Fe(CN)6 3− /Fe(CN)6 4− at the electrode interface. On the other hand, the diffusion process could be characterized with the linear part at lower frequencies in EIS. As shown in Fig. 3C, only a very small semicircle could be observed at bare GCE (curve a), after chitosan was deposited on the GCE, the diameter of the semicircle increased (curve), implying that the chitosan film obstructs the direct electron transfer of probe due to poor conductivity of the chitosan film. With the assembly of the MWCNTs NPs, the semicircle Ret decreased obviously, showing excellent conductivity of the nanocomposite (curve c), which also indicates that MWCNTs NPs can enhance the direct transfer. When Hb was immobilized, Ret increased again (curve d), confirming successful immobilization of Hb. Afterwards, with
the increase number (n < 5) of the bilayer of {MWCNTs/Hb}n , Ret increased sequentially (curve d–g). Such results illustrate successful assembly of the multilayer films. As shown in Fig. 3D, the Soret band of Hb in pH 6.0 phosphate buffer solution was 406 nm [10], and in this work the multilayer films modified on ITO substrates all showed Soret bands at 412 nm, which were very close to 406 nm. In addition, the spectra also displayed that the characteristic absorption of Hb increased with the increase of bilayer number. All the results demonstrate successful assembly of {MWCNTs/Hb}4 films. 3.2. pH-switchable behavior of {MWCNTs/Hb}4 multilayer film toward Fe(CN)6 3− Fig. 4A illustrates responses of the {MWCNTs/Hb}4 –Chitosan film modified GCE in phosphate buffer solution of different pH values with Fe(CN)6 3− as the mediator. It could be observed that the redox peak currents (Ip ) and the peak potential difference (Ep ) were substantially influenced by the pH level. The reduction peak current of Fe(CN)6 3− at the multilayer film could reach ∼221 A at pH 3.0 but only 0 at pH 9.0. However, the reduction peak current of Fe(CN)6 3− at the {Hb/AgNPs}4 film was only about ∼80 A at pH 3.0 [10]. Fig. 4B shows that Ep gradually increased with the increase of the pH value. The results of six successive measurements were almost the same: the reduction peak currents are all around ∼221 A at pH 3.0
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Fig. 5. (A) Plots of cathodic and anodic peak current vs. scan rate in 0.10 M pH 3.0 phosphate buffer solution at various scan rates: 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, and 100 mV s−1 . (B) Cyclic voltammograms of Fe(CN)6 3− at the {MWCNTs/Hb}4 film in 0.10 M pH 3.0 phosphate buffer solution at 10 mV s−1 with the presence of 0 (a), 1.0 (b), 2.0 (c), and 4.0 mM (d) H2 O2 .
(the “on” state) but nearly 0 at pH 9.0 (the “off” state) (Fig. 4C). Such results revealed that the {MWCNTs/Hb}4 –Chitosan film modified GCE could perform reversible pH-sensitive “on–off” functions. Similar pH switchable behaviors could be observed on the four-bilayer MWCNTs/Hb film and the four-layer
AgNPs/Hb film. By comparison, the redox peak currents of the {MWCNTs/Hb}4 –Chitosan film were larger than that of the {AgNPs/Hb}4 –Chitosan film. We are assuming that this is because of smaller sizes and larger surface area of MWCNTs compared with AgNPs.
Fig. 6. (A) Amperometric responses of the {MWCNTs/Hb}4 –Chitosan film by successive additions of H2 O2 to 8 mL 0.10 M pH 3.0 phosphate buffer solution containing 1.0 mM Fe(CN)6 3− with stir. (B) Amperometric responses of the {MWCNTs/Hb}4 –Chitosan film upon successive additions of H2 O2 to 8 mL 0.10 M pH 9.0 phosphate buffer solution containing 1.0 mM Fe(CN)6 3− with stir. (C) Amperometric response curve for H2 O2 at pH 3.0 () and pH 9.0 ().
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3.3. Scan rate and bioelectrocatalysis study of the {MWCNTs/Hb}4 multilayer film In the following we investigated the influence of the scan rates on the bioelectrocatalysis of H2 O2 with the proposed pHswitchable biosensor. Fig. 5A illustrates CVs of Fe(CN)6 3− at the {MWCNTs/Hb}4 –Chitosan film in 0.10 M pH 3.0 phosphate buffer solution at various scan rates. It could be found that redox peak currents increased linearly with the increase of the square root of the potential scan rate, indicating that the reaction at the modified electrode is controlled by diffusion process. As the determining reaction step, the diffusion process could improve the bioelectrocatalysis of H2 O2 at the modified electrode. Fig. 5B shows pH-controllable bioelectrocatalysis of H2 O2 toward Fe(CN)6 3− at 10 mV s−1 . The results demonstrated that the reduction peak height increased with continuous addition of H2 O2 .
with MWCNTs NPs can largely enhance the performance of the pHswitchable biosensors on detection of H2 O2 . Our study indicated that it is valuable to continue the investigation of Hb-based films with both switchable and biosensing functions. Further fabrication of Hb-based films with switchable and biosensing functions will focus on using those devices for more practical applications. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant numbers: 21175075; 21475070; 21075070), the Natural Science Foundation of Jiangsu Province (Grant number: BK2011047, BK2012651, BK2012652), the Postgraduate Technology Innovation Project of Nantong University (YKC13043), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
3.4. Effect of pH value on the bioelectrocatalysis
References
Fig. 6 illustrates the amperometric current–time response at pH 3.0 and pH 9.0 by adding H2 O2 continuously at an applied potential of −0.30 V. It could be observed from Fig. 6C that amperometric current grew linearly with the increase of H2 O2 concentration ranging from 12.5 M to 10.4 mM, and the detection limit was 6.25 M (S/N = 3) in pH 3.0 phosphate buffer solution containing Fe(CN)6 3− (curve a). By comparison, the amperometric currents in pH 9.0 phosphate buffer solution with the same H2 O2 concentration were much lower than those in pH 3.0 phosphate buffer solution although there was a linear relationship between the currents and the H2 O2 concentration from 25 M to 2.3 mM. With the increase of the H2 O2 concentration, the difference of amperometric currents in both phosphate buffer solution solutions increased. The peak current in pH 9.0 phosphate buffer solution could not be over 50 A even the H2 O2 concentration reach 10 mM while that in pH 3.0 could reach ∼270 A. Such relationship demonstrated that the proposed biosensors could be applied for the pH-switchable determination. The linear range with the {MWCNTs/Hb}4 films in pH 3.0 phosphate buffer solution is larger than the previous reports using the {Con A/HRP/Con A/GOD}3 films and the {Con A/Dex}4 –Mb films [15,16]. Such differences suggested that the nano-material enhanced LBL films have far-reaching influences on the construction of pH switches and biosensors in molecular devices. And the mechanism of pH-controllable bioelectrocatalysis could also be attributed to the characteristics of Hb at different pH values [10].
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4. Conclusions We report the pH-controllable bioelectrocatalysis of H2 O2 at the {MWCNTs/Hb}4 –Chitosan film modified GCE. The detection of H2 O2 could be controlled by pH values of the electrolyte solution with Fe(CN)6 3− as the mediator. The replacement of AgNPs
