Immobilization, direct electrochemistry and electrocatalysis of hemoglobin on colloidal silver nanoparticles-chitosan film

Electrochimica Acta 55 (2010) 8738–8743

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Immobilization, direct electrochemistry and electrocatalysis of hemoglobin on colloidal silver nanoparticles-chitosan film Chunmei Yu a,b , Xiaohui Zhou b , Haiying Gu b,∗ a b

College of Chemistry and Chemical Engineering and Materials Science, Suzhou University, Suzhou 215123, PR China Institute of Analytical Chemistry for Life Science, School of Public Health, Nantong University, Nantong 226007, PR China

a r t i c l e

i n f o

Article history: Received 16 May 2010 Received in revised form 4 August 2010 Accepted 4 August 2010 Available online 10 August 2010 Keywords: Hemoglobin Colloidal silver nanoparticles Chitosan Direct electrochemistry Electrocatalysis

a b s t r a c t This paper reports on the fabrication and characterization of hemoglobin (Hb)-colloidal silver nanoparticles (CSNs)-chitosan film on the glassy carbon electrode and its application on electrochemical biosensing. CSNs could greatly enhance the electron transfer reactivity of Hb as a bridge. In the phosphate buffer solu tion with pH value of 7.0, Hb showed a pair of well-defined redox peaks with the formal potential (E0 ) of −0.325 V (vs. SCE). The immobilized Hb in the film maintained its biological activity, showing a surfacecontrolled process with the heterogeneous electron transfer rate constant (ks ) of 1.83 s−1 and displayed the same features of a peroxidase in the electrocatalytic reduction of oxygen and hydrogen peroxide (H2 O2 ). The linear range for the determination of H2 O2 was from 0.75 ␮M to 0.216 mM with a detection limit of 0.5 ␮M (S/N = 3). Such a simple assemble method could offer a promising platform for further study on the direct electrochemistry of other redox proteins and the development of the third-generation electrochemical biosensors. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Since Eddowes et al. [1] and Yeh and Kuwana [2] independently reported the reversible electrochemistry of cytochrome c on gold and tin-doped indium oxide electrodes respectively in 1977, the direct electron transfer between redox proteins and the electrode surface has been extensively studied. It is an important means for studying enzyme-catalyzed reactions in biological systems, and for developing bioelectronic systems such as electrochemical biosensors [3] and biofuel cell elements [4]. Hemoglobin (Hb), a typical multi-cofactor protein that has two heme-containing ␣- and ␤dimers (␣␤) [5], is considered to be an ideal model protein for the study of the electron transfer of heme molecules because of its commercial availability, moderate cost as well as well-known and documented structure. However, it has been demonstrated that on conventional solid electrodes, the fast electron transfer between Hb and the electrode is not possible because the redox center of proteins is embedded in polypeptide chain structures and the proteins are easily absorbed on the electrode surface [6]. Therefore, great efforts have been devoted to explore new immobilization methods and supporting materials that accelerate the electron transfer and maintain the enzymatic activity. Various materials such as clay [7],

∗ Corresponding author at: Institute of Analytical Chemistry for Life Science, School of Public Health, 19 Qixiu Road, Nantong, Jiangsu 226001, China. Tel.: +86 513 8501 5417; fax: +86 513 8501 5417. E-mail addresses: [email protected], [email protected] (H. Gu). 0013-4686/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2010.08.010

ion liquid films [8,9], sol–gels films [10], nanomaterials [11] have been used for this purpose. Among these materials, metal nanoparticles are developed rapidly because of their potential uses in microelectronics, optics, catalysis, and chemical/biological sensors. Many nanoparticles such as gold [12,13], platinum [14], copper [15], and metal oxide [16,17] have been widely used in constructing electrochemical biosensors. As a typical noble metal nanomaterial, colloidal silver nanoparticles (CSNs), which are easy to synthesize, have attracted considerable attentions due to their quantum characteristics, such as small granule diameter, large specific surface area and high catalytic activity [18–20]. Based on their biocompatibility and conductivity, CSNs can provide a microenvironment similar to that of the redox proteins in native systems and can be used to immobilize proteins for their direct electrochemistry [21,22]. For example, Hu et al. fabricated a hydrogen peroxide biosensor based on the Hb–Ag sol on the glassy carbon electrode (GCE) [23]. Sun and co-workers developed a biosensor by entrapping Hb/CSNs in titania sol–gel matrix on GCE by a vapor deposition method [24]. In these studies, Hb and CSNs were immobilized on the electrode surface by cast method. As well-known, chitosan (CS) is a natural biopolymer with a large amount of positive charges, which is suitable for film-forming and has been widely used as the electrode material for immobilization proteins [25–27]. Herein the electrodeposition of chitosan [28] was applied to form a positively charged surface on the glassy carbon electrode. Then CSNs and Hb with opposite charges were immobilized respectively on the electrodeposited chitosan to construct Hb-CSNs-CS/GCE through the electrostatic interaction.

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Compared with the cast method, this self-assembled technology could control the film with the precise composition and thickness on the nanometer scale. The fabricated CSNs-CS film could provide a favorable microenvironment for retaining the biological activity of the adsorbed Hb and facilitate its direct electron transfer. The electrocatalysis reduction of oxygen and hydrogen peroxide based on Hb-CSNs-CS film was also studied.

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trophotometer (Shimadzu, Japan). The average particle size and morphology of CSNs were observed by a JEM-2010HR transmission electron microscope (TEM). X-ray photoelectron spectroscopy (XPS) was performed on Perkin-Elmer PHI 550 spectrometer (PHI Co., USA) equipped with the Al K␣ X-ray radiation as the source for excitation at a pressure of less than 10−9 Torr in the chamber. The analyzer pass energy was 50 eV and the step size was 0.1 eV. The scanning electron microscopy (SEM) images were obtained with a HITACHI S4800 SEM (Hitachi, Ltd., Tokyo, Japan).

2. Experimental 2.1. Reagents and apparatus

2.2. Preparation of colloidal silver nanoparticles

Hb (∼90%, bovine blood), silver nitrate and sodium citrate were purchased from Sigma and used without further purification. Chitosan (92.5% deacetylation, Nantong Shuanglin Company), was dissolved in 0.05 M HCl to form 0.2 wt% solution and filtered using a 0.45 mm syringe filter. H2 O2 (30%) was purchased from Shanghai Chemical Reagent Company and was freshly prepared before being used. 0.1 M phosphate buffer solutions (PBS) with various pH values were prepared by mixing stock standard solutions of Na2 HPO4 and NaH2 PO4 and adjusting the pH values with 0.1 M H3 PO4 or NaOH solutions. Other chemicals were of analytical grade. All solutions were prepared with twice-distilled water. Electrochemical measurements were performed with a CHI 660 electrochemical workstation (CH Instruments Co., USA) using the modified glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, a platinum wire as the counter and a saturated calomel electrode (SCE) as reference electrode. Amperometric experiments were carried out in a stirred system by applying a potential of −0.4 V to the working potential. Electrochemical impedance spectroscopy (EIS) experiments were performed at a potential of 0.17 V within the frequency range from 10−2 Hz to 105 Hz in 0.1 M KNO3 containing 5.0 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1). The UV–vis spectra were recorded on an UV-2450 spec-

All glassware used in the following procedures was cleaned in a bath of freshly prepared 3:1 HNO3 –HCl, thoroughly washed with water, and dried prior to use. The CSNs were prepared according to the literature [29]. A solution of 1 mM AgNO3 (100 mL) was added and heated with vigorous stirring. Upon boiling, 1 mL of 10 mg mL−1 sodium citrate solution was added rapidly, a greenish yellow colloid formed almost immediately. Then the solution was heated under reflux for approximately 1 h. Before use, it was stored below 4 ◦ C.

2.3. Electrode modification GCE was first polished with abrasive paper and then with alumina slurry, followed by ultrasonically cleaned in ethanol and water and dried in air. Then the GCE was electrodeposited at −2.0 V for 5 min in chitosan solution. The obtained GCE was washed with twice-distilled water and dried in room temperature. Then the CS modified GCE was dipped into CSNs and the 3 mg mL−1 Hb solution (pH 6.0 PBS) at 4 ◦ C for about 30 min, respectively. The resulting HbCSNs-CS modified electrode was washed with water and stored in pH 7.0 PBS at 4 ◦ C for use.

Fig. 1. (A) UV–vis adsorption spectrum and (B) TEM image of the CSNs. (C, D) SEM images of CSNs-CS film (C) and Hb-CSNs-CS film (D).

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3. Results and discussion 3.1. Characterization of CSNs and morphology of Hb-CSNs film Fig. 1A shows the UV–vis absorption spectrum of the CSNs, which is characterized by the absorption at 425 nm in the visible region. The good symmetric absorption peak implied that the CSNs were relatively uniform. The particle size and shape have been further observed by transmission electron microscope (TEM). The TEM image of CSNs deposited on a carbon-coated copper mesh is shown in Fig. 1B. It can be observed that the CSNs are well-dispersed with spherical shapes and their sizes are around 30–60 nm. Scanning electron microscopy was employed to characterize the morphology of the CSNs-CS and Hb-CSNs-CS film. As shown in Fig. 1C, many particles were decorated across the whole surface, indicating CSNs were adsorbed on the chitosan film. Further immersing the film to Hb solution, a considerable change in morphology has been observed (Fig. 1D). The immobilized Hb molecules were distributed regularly and showed porous structure. This unique porous structure could facilitate access of substrates to the bound proteins.

3.2. Characterization of Hb immobilized on CSNs-CS/GCE In order to further verify if Hb was immobilized on the surface of CSNs-CS film, XPS experiments were performed. Fig. 2 shows the XPS spectra of Hb immobilized on the CSNs-CS film in the N 1s and Fe 2p regions. It can be seen that the characteristic peak of N 1s of N is at 399.5 eV (Fig. 2A), and the peaks Fe 2p3/2 and Fe 2p1/2 of Fe(III) are located at 711.6 and 725.8 eV, respectively (Fig. 2B). Since both Fe and N are from Hb, the results indicate that the Hb molecules are on the surface of CSNs-CS film. The position of the Soret absorption band of heme iron can be applied to study the possible denaturation of the heme protein, especially the conformational change in the heme group region. Fig. 3 exhibits the UV–vis spectra of the Hb solution (curve b) and the Hb-CSNs-CS film on ITO slides (curve c). Hb-CSNs-CS film gives a Soret band at 406 nm, which is the same as the Soret band at 406 nm for native Hb in buffer, indicating that the secondary structure of Hb immobilized on the surface of CSNs is not destroyed and Hb retains its biological activity. EIS was often used to monitor the assembly process. In EIS, the semicircle part at higher frequencies corresponds to the electron transfer limited process. Its diameter equals the electron transfer resistance, Ret , which exhibits the electron transfer kinetics of

Fig. 3. UV–vis adsorption spectra of (a) ITO slide, (b) Hb solution and (c) Hb-CSNs-CS film.

the redox probe at the electrode interface. Fig. 4A displays the EIS observed upon the changes of surface-modified process. A small semicircle can be observed at the bare GCE, indicating a low transfer resistance (curve a). When CS was electrodeposited on the GCE surface (curve b), the semicircle dramatically increased, suggesting that the chitosan film acted as an insulating layer and barriers which made the interfacial charge transfer inaccessible. Next, CSNs were immobilized on the chitosan film, the semicircle obviously decreased (curve c), implying that this material could make the electron transfer easier. After the adsorption of Hb, an obvious increase again in the interfacial resistance was observed (curve d). This demonstrated that Hb had been successfully immobilized on the CSNs-CS film. Generally, Hb is positively charged at pH 6.0 since its isoelectric point (IEP) is 7.4, while CSNs is negative, thus the electrostatic attraction between them will promote Hb to spontaneously adsorb onto the CSNs. Cyclic voltammetry of the ferricyanide system is also a valuable tool for testing the kinetic barrier of the interface. The extent of kinetic hindrance to the electron transfer process increases with the increasing thickness and the decreasing defect density of the barrier. Fig. 4B shows cyclic voltammograms of differently modified electrodes in a 5 mM ferricyanide solution. The electrochemical response for Fe(CN)6 3−/4− is almost reversible with a peak separation of 125 mV at 100 mV s−1 at the bare GCE (curve a). For the GCE deposited by the CS film, an obvious decrease in the redox peak current and increase in the peak–peak separation are observed (curve

Fig. 2. The XPS spectra of Hb immobilized on the CSNs-CS/GCE in (A) N 1s and (B) Fe 2p regions.

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Fig. 4. (A) Electrochemical impedance spectroscopy of (a) GCE, (b) CS/GCE, (c) CSNs-CS/GCE and (d) Hb-CSNs-CS/GCE. (B) Cyclic voltammograms of (a) GCE, (b) CS/GCE, (c) CSNs-CS/GCE and (d) Hb-CSNs-CS/GCE in 0.1 M KNO3 containing 5 mM [Fe(CN)6 ]3−/4− . Scan rate: 50 mV s−1 .

b). After the electrode was assembled with CSNs, the peak current increased and Ep decreased (curve c). CSNs have a large surface area which can increase the active surface area of the modified electrode and may play an important role similarly to a conducting wire or an electroconducting tunnel. When Hb was adsorbed on the electrode surface again, a remarkable current decrease was observed (curve d). These data showed that Hb and CSNs have been successfully attached to the electrode surface, which was consistent with the EIS results. 3.3. Direct electrochemical behavior of Hb on CSNs-CS/GCE In order to investigate the electrochemical properties of HbCSNs-CS film, cyclic voltammograms of different electrodes in PBS (pH 7.0) were recorded, as shown in Fig. 5. No obvious redox peak can be observed at bare GCE (curve a), CS/GCE (curve b) and CSNsCS/GCE (curve c). While for Hb-CSNs-CS/GCE (curve e), a couple of stable and well-defined redox peaks at −0.28 and −0.37 V is clearly discovered, which is the characteristic of heme FeIII/FeII redox couple of the protein. On the other hand, for Hb-CS/GCE (curve d), only a pair of small peaks were observed, which can be explained that only few Hb molecules closest to the electrode could exchange electrons with the electrode and contribute to the observed redox reaction.

Fig. 5. Cyclic voltammograms of (a) GCE, (b) CS/GCE, (c) CSNs-CS/GCE, (d) HbCS/GCE and (e) Hb-CSNs-CS/GCE in 0.1 M pH 7.0 PBS. Scan rate: 50 mV s−1 .

Thus, CSNs must have a great effect on the kinetics of electrode reaction and provide a suitable environment for Hb to transfer electrons  with the underlying electrode. The formal potential E0 (defined as the average of the anodic and cathodic peak potentials) is −0.325 V, which is similar to that reported for Hb entrapped in carbon black [30] and didodecyldimethylammonium bromide (DDAB)-clay film [31]. The separation of the peak potentials (Ep ) is about 100 mV and almost unchanged with the increase of scan rate, implying that the electron transfer rate between Hb and the GCE is relatively fast. Moreover, the anodic and the cathodic peak currents are almost equal. The results indicate that the Hb undergoes a quasi-reversible electrochemical reaction and the immobilized Hb on CSNs is not denatured. The cyclic voltammograms of the Hb-CSNs-CS/GCE at various scan rates were measured as shown in Fig. 6. With an increasing scan rate ranging from 20 mV s−1 to 300 mV s−1 , the anodic and cathodic peak potentials of the Hb showed a small shift and the redox peak currents increased linearly, indicating a surfacecontrolled electrode process. According to Laviron equation [32]: Ip =

n2 F 2 A v nFQ v = 4RT 4RT

(1)

Fig. 6. Cyclic voltammograms of Hb-CSNs-CS/GCE in pH 7.0 PBS with the scan rates from inner to outer as 20, 50, 80, 100, 120, 150, 180, 200, 250 and 300 mV s−1 , respectively. Inset: linear relationship of the peak current vs. the scan rate.

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Fig. 7. Effect of pH on the formal potential (E0 ).

where  is the electroactive Hb amount (mol cm−2 ) and Q is the quantity of charge (C). The symbols n, Ip , F, R and T have their usual meanings. From the slope of the Ip ∝ v, n was calculated to be 0.94. Therefore, the redox of Hb on CSNs is a single electron transfer reaction. The average surface coverage of Hb on the surface of the modified electrode was estimated to be 4.43 × 10−11 mol cm−2 , which is almost two times larger than that of the theoretical monolayer coverage (1.89 × 10−11 mol cm−2 ) [33]. As nEp ≤ 200 mV, the heterogeneous electron transfer rate constant (ks ) can be estimated by Laviron equation [34]: log ks = ˛ log(1 − ˛) + (1 − ˛) log ˛ − log

˛(1 − ˛)nF Ep RT − nF v 2.3RT (2)

Taking the electron transfer coefficient ˛ of 0.5, and a scan rate 250 mV s−1 , Ep = 100 mV, the rate constant can be estimated to be 1.83 s−1 . The estimated value is in the controlled range of surface-controlled quasi-reversible process, and is larger than those obtained for Hb immobilized on polymer-MWNT-modified GCE (0.4 s−1 ) [35], Hb modified CNT powder microelectrodes (0.062 s−1 ) [36]. Such results revealed a reasonably fast electron transfer between the immobilized Hb and the underlying electrode. The direct electrochemistry of Hb immobilized on CSNs-CS film showed a strong dependence on solution pH. With pH increasing from 3.0 to 9.0, both the cathodic and anodic peaks shifted toward negative linearly. The shift in the formal potential as a function of pH, results from the change in the protonation of a water molecule at the sixth coordination position in the heme iron and also protonation of the protolytic groups around the heme with changing pH [37]. Plots of formal potential versus pH were linear with a slope of −40.54 mV/pH (Fig. 7). This value is smaller than the expected theoretical value of 57.6 mV pH−1 at 18 ◦ C for a single-proton coupled to the reversible one-electron transfer [38]. It may be due to the influence of the protonization of ligands to the heme iron and amino acids around the heme [39].

Fig. 8. Cyclic voltammograms of Hb-CSNs-CS/GCE in pH 7.0 PBS containing: (a) 0, (b) 40 ␮L, (c) 100 ␮L and (d) 150 ␮L air. Scan rate: 50 mV s−1 .

(Fig. 8). The reduction peak current increased with the amount of O2 in the solution, which implied that Hb immobilized on CSNsCS/GCE have a satisfactory catalytic activity toward O2 . O2 can bind reversibly to heme to form HbFe(II)-O2 , which can then undergo the electrochemical reduction at the potential of HbFe(III) reduction, producing HbFe(II) again [40]. The electrocatalytic activity of the Hb immobilized on CSNs-CS/GCE to H2 O2 was also observed. Fig. 9 shows cyclic voltammograms of the modified electrodes in the absence and presence of H2 O2 . When H2 O2 was added to a pH 7.0 PBS, the reduction peak currents increased obviously accompanied by the decrease of oxidation peak current, indicating a typical electrocatalytic reduction process, which may be due to the reaction of HbFe(II) with H2 O2 . Furthermore, the reduction peak current increases with increasing concentration of H2 O2 . These results further confirmed that the CSNs-CS film provided a friendly platform for the immobilization of Hb and the bioelectrocatalysis to H2 O2 . The electrocatalytic process can be described as follows [41]: H2 O2 + 2HbHFe(II) → 2HbFe(III) + 2H2 O

(3)

The amperometric response of Hb-CSNs-CS/GCE to H2 O2 at an applied potential of −0.4 V is illustrated in Fig. 10. Upon addition of H2 O2 , the biosensor responded rapidly to the substrates and could achieve 95% of the steady-state current within 4 s, indicating

3.4. Electrocatalysis of Hb immobilized on CSNs-CS/GCE It was reported that heme proteins were able to catalyze the reduction of O2 , NO2 − , H2 O2 and so on. In order to investigate the activity of Hb at GCE modified with CSNs, its response to the reduction of O2 and H2 O2 was studied. The electrocatalytic reduction of O2 by Hb-CSNs-CS/GCE was examined by cyclic voltammetry. When a certain amount of air was injected into the PBS solution by a syringe, a significant increase in reduction peak at about −0.40 V was observed, accompanied by the disappearance of the oxidation peak of HbFeIII/FeII redox couple

Fig. 9. Cyclic voltammograms of Hb-CSNs-CS/GCE in pH 7.0 PBS containing: (a) 0, (b) 1.3 × 10−5 M, (c) 2.7 × 10−5 M, (d) 3.6 × 10−5 M and (e) 4.2 × 10−5 M H2 O2 . Scan rate: 50 mV s−1 .

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Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant numbers: 20675042; 20875051), the Natural Science Foundation of Jiangsu Province (Grant number: BK2009152). References

Fig. 10. Amperometic response of Hb-CSNs-CS/GCE upon successive additions of 0.25 ␮M H2 O2 to 4 mL pH 7.0 PBS with stirring. Inset: amperometric response curve for H2 O2 .

a fast amperometric response to H2 O2 reduction. The amperometric response showed a linear relation with H2 O2 concentration from 0.75 ␮M to 0.216 mM with a correlation coefficient of 0.9997 (n = 15) (inset in Fig. 10). The linear regression equation was I (␮A) = 0.012 + 1.85 [H2 O2 ] (mM). The detection limit was estimated to be 0.5 ␮M (S/N = 3). 3.5. Stability and reproducibility of the modified electrode The stability and reproducibility of the modified electrode was also studied. When the modified electrode was stored in pH 7.0 PBS at 4 ◦ C, the response current retained more than 95% of its original response after 15 days. The relative standard deviation (R.S.D.) was 2.7% for 10 successive measurements at 0.075 mM H2 O2 , indicating a good precision. Moreover, a series of five electrodes prepared in the same manner were also tested and the R.S.D. observed was only 3.4%. 4. Conclusions A simple but effective method was developed for the immobilization of CSNs and Hb to the chitosan modified glassy carbon electrode through the self-assembly method. The presence of CSNs provided the Hb molecules effective adsorption sites that their active centers can be easily accessed so that the direct electron transfer of Hb was significantly promoted. At the same time the immobilized protein retained its biological activity and showed a high electrocatalytic response to O2 and H2 O2 . Due to the troublefree operation, good stability and fast response time of CSNs-CS film, it could provide a good platform for fabricating new generation of biosensors based on other redox proteins without using redox mediators.

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