Journal of Electroanalytical Chemistry 625 (2009) 33–39
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Electroanalytical properties of haemoglobin in silica-nanocomposite films electrogenerated on pyrolitic graphite electrode T. Rozhanchuk a, O. Tananaiko a,*, I. Mazurenko a, M. Etienne b, A. Walcarius b, V. Zaitsev a a b
Department of Analytical Chemistry, National Taras Shevchenko University, Volodymyrska 64, Kyiv 01601, Ukraine Laboratoire de Chimie Physique et Microbiologie pour l’Environnement, UMR 7564, CNRS-Nancy University, 405 rue de Vandoeuvre, 54600 Villers-les-Nancy, France
a r t i c l e
i n f o
Article history: Received 21 May 2008 Received in revised form 4 September 2008 Accepted 6 October 2008 Available online 17 October 2008 Keywords: Haemoglobin Au nanoparticles Electrogenerated film Sol–gel bioencapsulation Thin-film electrode
a b s t r a c t Haemoglobin (Hb) modified electrochemical devices have been prepared by Hb encapsulation in silica sol–gel films (SiO2), which were generated by electro-assisted deposition onto pyrolitic graphite electrodes (PGEs). The stability and electrocatalytic activity of Hb entrapped into SiO2 network was substantially enhanced in the presence of cationic surfactant (CTAB) and Au nanoparticles (Au-NPs). The composition of sol–gel synthesis medium, i.e., molar ratio of silica precursor to water, contents of Hb, CTAB and Au-NPs, as well as the conditions of electrogeneration had a great influence on the electrocatalytic activity of Hb on PGE surface. The electrochemical response of the PGE modified with the composite SiO2–Hb–CTAB–Au-NPs film was found to vary linearly with the concentration of dissolved oxygen in solution and this was exploited to determine this analyte in the tap water with detection limit 0.12 mg L1. The electrocatalytic current of dissolved oxygen was also found to decrease in the presence of the antivirus drug––amino derivative of adamantane (rimantadine)––which opens the way to the determination of this drug with detection limit 0.3 mg L1 using PGE modified with SiO2–Hb–CTAB– Au-NPs nanocomposite film. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction Amperometric biosensors based on enzymes entrapped in silica matrices coated on solid electrode surfaces become increasingly popular as this way of biomolecule immobilization imparts high stability and good catalytic activity [1–3]. Sol–gel-derived silicabased materials are indeed attractive hosts for enzymes as they are chemically inert towards biomolecules and their three-dimensional structure does not restrict conformation mobility of the latest, so encapsulated biomolecules can retain their catalytic activity [4–6]. On the other hand, it was shown earlier that heme proteins such as haemoglobin (Hb) and myoglobin acquire peroxidase activity while immobilised [7,8] and haemoglobin-based amperometric biosensors were proposed for determination of various analytes, including O2, H2O2 and NO 2 [9–11]. The detection scheme involved the catalytic reduction of these compounds on the Hbmodified electrodes. Another promising application of Hb-modified electrodes is the determination of some pharmaceuticals (e.g., antivirus or anticancer drugs) that are likely to interact with Hb. Examples are available for detection of Taxol and ribavirin using Hb-modified electrodes on the basis of a decrease in the catalytic current relative to O2 reduction upon increasing the drug concentration [12,13]. * Corresponding author. Tel.: +38 0 44 239 34 44; fax: +38 0 44 239 33 45. E-mail address:
[email protected] (O. Tananaiko). 0022-0728/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2008.10.003
Recently the method of electrodeposition was proposed to obtain thin sol–gel coatings on solid electrode surfaces, which was achieved via an electrochemical modulation of pH at the electrode/solution interface to promote gelification of the sol in a controlled way [14,15]. Electrodeposition method shows a few advantages comparing to classic spin-coating and dip-coating methods, particularly the ability to control the thickness of the films, greater porosity and simplicity of procedure [15]. This was notably applied to get organically-functionalized silica thin films on electrodes [16–18] or well-structured and oriented deposits [19,20], which can be applied as sensitive nano-layers with good analytical performance [21,22]. Very recently, we have demonstrated that such an electro-assisted generation approach can be extremely useful for encapsulation of biomolecules, i.e., Hb and glucose oxidase, in silica thin films deposited on glassy carbon electrodes [23]. The encapsulated enzymes retained their catalytic activity without additional use of mediators. Still the problem of the stability of immobilised proteins was not fully solved, giving notably rise to significant decrease in the analytical signal with time, what we have tried to circumvent in the present study by the use of surfactant and/or nanoparticles additives. The addition of cationic surfactant (for example cetyltrimethylammonium bromide, CTAB) at CMC and higher level as template agent into silica sol permits to obtain well-structured porous materials [24,25]. Surfactants indeed improve the properties of sol–gel silica derived materials where encapsulated organic molecules are
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dispersed more uniformly in the silica matrix due to their favourable interaction with the micelles of surfactant formed inside silica cage [26]. It is known that surfactants can form biomembrane-like structures which are disposed to entrap protein molecules like Hb. Such approach was used earlier to construct carbon electrode modified by cationic surfactant and hemoprotein [27]. CTAB prevents leaching of the protein molecules from the silica network and does not influence the catalytic activity of encapsulated enzymes [8,28]. Non-ionic surfactant Tween 20 was also proposed to promote immobilisation of Hb onto the pyrolitic graphite electrode in the form of self-assembled multilayer film [29]. On the other hand, the addition of metal nanoparticles, i.e., gold, into biocomposite materials can greatly enhance the electrochemical properties of film electrodes containing encapsulated enzymes. Nanoparticles act somewhat as wires between the active centre of the enzyme and the electrode surface, thus providing facile electron transport and effective transduction of the biochemical recognition event [30,31]. This approach was successfully applied to the development of mediator-free glucose biosensors based on glucose oxidase absorbed on colloidal gold or encapsulated into a gold nanoparticles–mesoporous silica composite [32,33]. The electrodes modified with gold nanoparticles were also used for further modification with heme-proteins (Hb [34] and peroxidase [35]) and successfully applied for development of biosensors. The fast aggregation of gold nanoparticles in the silica sol can be achieved with the help of various types of stabilizers. Biomolecules themselves can be used as stabilizer of gold nanoparticles in solutions when containing thiol groups in their structure [31,36]. In the present work we have thus examined various approaches to improve the stability of Hb molecules encapsulated into sol–gel silica films which were electrogenerated onto the surface of pyrolitic graphite electrode. The effect of the biocomposite composition, and especially the addition of surfactants molecules and/or gold nanoparticles in the starting silica sol, was thoroughly studied with respect to the electrocatalytic properties of encapsulated Hb. Furthermore, the Hb-modified electrode was tested for the determination of dissolved oxygen and an antivirus drug based on the amino derivative of adamantane (rimantadine).
2. Experimental 2.1. Chemicals and solutions Human haemoglobin (Hb, Mw 64,000) was purchased from Sigma. Tetraethoxysilane (TEOS, 98%) was obtained from Fluka. Cetyltrimethylammonium bromide (CTAB, Sigma) and polyoxyethylene derivative of sorbitan monolaurate polysorbate-20 or Tween 20 (Merck) were used as additives in the sol–gel synthesis procedure as required. All solutions were made up with double-distilled water. All other chemicals were of analytical grade and used without further purification. HCl (0.01 mol L1) was used for precursor hydrolysis. The 0.07 mol L1 phosphate buffer solutions were prepared by mixing stock solutions of KH2PO4 and Na2HPO4 and adjusted at selected pH values using either HCl or NaOH. The suspension of Au nanoparticles was prepared according to the standard procedure [37]. Briefly: 1% HAuCl4 was reduced by 1% aqueous solution of sodium citrate. The dimension of colloidal particles in aqueous suspension and in the silica sol was controlled spectrophotometrically at k = 510–520 nm [38,39]. Water or silica sol was used as blank solution. The mixture of Hb and Au was prepared by addition of Hb solution to Au nanoparticles suspension in various Au:Hb volume ratios (Au:Hb = 4:1; 3:1; 2:1; 0.4:1). The pH of the mixture was adjusted to 10 by NaOH. The solutions were stored at 4 °C when not in use.
The concentration of dissolved oxygen in water samples was determined using the standard Winkler titration method [40]. The solutions with different concentration of dissolved oxygen were prepared by mixing the solution with known oxygen content and oxygen-free solution at different volume ratio. The oxygen free solution was obtained of purging nitrogen through the 15 mL of water for 10 min. All solutions were tightly closed before and during experiments. The solution of rimantadine was prepared by dissolving one pill of Remavir (OlainFarm, Latvia) containing 50 mg of rimantadine hydrochloride in 15 mL of ethanol. Then the solution was diluted with phosphate buffer pH 6.0. The content of rimantadine in solutions was controlled by LC–MS method using as a standard ethanol solution of the substance of the known concentration similar to [41,42] but without previous derivatization of the substance. Before voltammetric measurements the content of dissolved oxygen in rimantadine solutions was measured using the standard Winkler titration method. All solutions were tightly closed during the experiments to avoid contact with air. 2.2. Preparation of Hb-modified electrodes Pyrolitic graphite electrode (PGE, basal plane, ‘‘Burevestnik”, St. Petersburg, Russia) was modified with silica Hb-containing films by electrogeneration technique by adapting a procedure described earlier for glassy carbon electrodes [23]. PGE was first polished with the help of diamond paste, washed with ethanol and water, and dried at 90 °C for 1 h. The thin sol–gel films were electrogenerated on the clean PGE surface from an aqueous TEOS-based sol solution to which the Hb solution was added. A typical silica sol was prepared by dissolving 2.125 g TEOS, 2 mL of water and 2.5 mL of 0.01 mol L1 aqueous HCl, which were mixed for 12 h using a magnetic stirrer. Then 0.07 mol L1 phosphate buffer solution (pH 6.0) was added to silica sol to increase pH and a necessary volume of the protein solution (0.5 mmol L1) was added to the hydrolyzed sol. The mixture was introduced into the electrochemical cell where electro-assisted generation was performed at 1.2 V at room temperature for 7 s (optimized time). After the thin Hb-containing silica film was formed on the surface of PGE (PGE SiO2–Hb), the electrode was rinsed with water, dried for 60 min in air and stored at 4 °C if not in use. To optimize the film composition, selected amounts of CTAB was added into the sol solution just before electro-assisted generation and the so-called Hb–silica-surfactant composite film electrodes were denoted PGE SiO2– Hb–CTAB. Au nanoparticles-doped Hb-containing nanocomposite films were obtained by adding the mixture of Hb and Au solutions (volume ratio 1:2) to the silica sol prior to applying the electrogeneration procedure, in the absence or in the presence of CTAB, leading to the formation of PGE SiO2–Hb–Au and PGE SiO2–Hb– CTAB–Au, respectively. 2.3. Apparatus Electrogeneration and voltammetry experiments have been carried out using analytical voltammeter AVA-2 (‘‘Burevestnik”, St. Petersburg, Russia). Measurements were performed at room temperature in a three-electrode cell, including the modified PGE working electrode, an Ag/AgCl reference and a Pt wire auxiliary electrode. Most often, the electrolyte solutions were deoxygenated by bubbling nitrogen for 10 min prior to the experiments. The ionometer I-130 was used for pH monitoring. Spectrophotometer SF-46 (Severodonetsk, Russia) with quarts cuvette l = 1 cm was used for absorption spectroscopic measurements. The composite electrodes were analyzed by atomic force microscopy (AFM) using a commercial microscope (Thermomicroscope Explorer Ecu+, Veeco Instruments S.A.S.) to get information
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of the film morphology. Scanning electrochemical microscopy (SECM) was further used to visualize the influence of conducting nanoparticles in the thin layers on the interface reactivity. The setup has been built-up in our laboratory (LCPME, Nancy, France) on the base of the apparatus commercialized by Sensolytics (Bochum, Germany). The approach curve experiments have been performed at 0.5 lm s1 with a 25 lm platinum disc electrode in a solution containing 0.1 mol L1 KCl and 1 mmol L1 ferrocene dimethanol. The sample position (z = 0) has been estimated by comparing the experimental approach curve with the corresponding calculated curve (Rg 5) [43]. All approaches have been stopped automatically at a constant distance from the surface by use of a shear-force sensor [44].
catalytic currents, Icat, for oxygen response at PGE SiO2–Hb were observed when applying a deposition potential of 1.2 V and deposition time of 7 s. These optimum values are very close to those reported for similar films deposited on glassy carbon (Edepos = 1.2 V and tdepos = 10 s [23]). The amount of Hb in silica sol was found to influence the value of Icat of the modified electrode. Icat of PGE SiO2–Hb increased linearly with raising of Hb concentration in silica sol from 5 lmol L1 up to 40 lmol L1 and reached saturation at the Hb content of 50 lmol L1. But still the catalytic activity of Hb immobilized on PGE was not stable as Icat values decreased rapidly upon successive analyses and disappeared after 18 potential sweeps.
3. Results and discussion
3.2. Influence of surfactant addition in silica sol on the properties of the Hb-modified PGE
3.1. Voltammetric characteristics of SiO2–Hb modified PGE Fig. 1 shows cyclic voltammogram of PGE and PGE SiO2–Hb in the absence or presence of oxygen, as recorded at 100 mV s1 in phosphate buffer solution (pH 6.0). In the absence of oxygen, the response of PGE SiO2–Hb was characterized by two peaks, a welldefined cathodic signal located at 0.20 V (versus Ag/AgCl) (Ec) and its (less visible) anodic counterpart at 0.06 V (versus Ag/ AgCl) (Ea), as illustrated by curve 2 in Fig. 1 (enlarged in the inset). These peaks were ascribed to reduction and oxidation of iron in porphyrinic complex of Hb molecules. The peak potentials of quasi-reversible Fe(III)/Fe(II) couple on the modified PGE were positively shifted comparing to those measured on GCE [23]. In the presence of oxygen the cathodic peak current dramatically increased, with potential shifting to 0.3 V (versus Ag/AgCl), which refers to catalytic reduction of O2, whereas the anodic part vanished (Fig. 1, curve 3). For the bare PGE no peaks were observed in the mentioned potential range (Fig. 1, curve 1). The obtained data demonstrates that Hb molecules in the SiO2–Hb film on the PGE surface were electroactive and possessed catalytic activity. Electrogeneration parameters such as deposition time and applied potential influence the film thickness, its permeability to external reagents and its mechanical stability and thereby the voltammetric response of the modified electrode [17,18]. The effect of these two parameters was studied here for Hb-containing sol solutions by varying the applied potential between 1.0 and 1.4 V and deposition times ranging from 5 s to 60 s. The highest electro-
Fig. 1. Cyclic voltammetric curves recorded in 0.01 mol L1 phosphate buffer (pH 6.0) and 0.01 mol L1 KNO3 at PGE (1) and PGE SiO2–Hb (2–3) obtained in anaerobic (2) and aerobic (1 and 3) conditions at a scan rate of 100 mV s1. Insetvoltammetric curve 2.
Cationic (CTAB) and non-ionic (Tween 20) surfactants were studied as additives likely to improve the performance of the Hbcontaining electrogenerated silica films. In the absence of oxygen, no detectable difference in cyclic voltammograms of PGE SiO2– Hb–CTAB and PGE SiO2–Hb was observed, indicating that CTAB did not affect the electrochemical response of encapsulated Hb. In the presence of oxygen, however, the electrocatalytic signal was found to increase a little bit and to shift in the positive direction (by ca. 50 mV) when using PGE SiO2–Hb–CTAB instead of PGE SiO2–Hb (compare curves 1 and 2 in Fig. 2). This demonstrates easier reduction of O2 at the electrode modified with biocomposite in the presence of CTAB. On the other hand, this advantage belonging to surfactant was not observed with Tween 20, which resulted in significant decrease in catalytic activity of encapsulated Hb possibly due to fast aggregation of biomolecules in silica sol. It was indeed checked that silica sols containing Hb and Tween 20 were not stable and precipitation of the protein was observed within 30 min so that Tween 20 was not used in further investigation. Addition of CTAB to sol–gel mixtures not only improved the electrocatalytic response of the biocomposite film electrode but also increased it upon successive voltammetric measurements (compare curves 1–3 in Fig. 3 and especially curves 1 and 2 in Fig. 4). For example, the value of Icat for PGE SiO2–Hb–CTAB decreased by 50% after 22 potential sweeps while for PGE SiO2–Hb Icat completely disappeared after 18 sweeps (Fig. 4, curves 1 and 2). The composition of the starting sol, notably the CTAB concentration and TEOS:H2O ratio, was found to affect significantly the per-
Fig. 2. Voltammetric curves recorded under aerobic conditions at PGE SiO2–Hb (1), PGE SiO2–Hb–CTAB (2), PGE SiO2–Hb–Au (3) and PGE SiO2–Hb–CTAB–Au (4). Other conditions as in Fig. 1.
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Fig. 3. Effect of TEOS:H2o molar ratio in silica sol on the catalytic peak current after 2 (1), 10 (2) and 30 (3) potential sweeps for PGE SiO2–Hb–CTAB in the presence of O2. Concentrations in silica sol: CTAB – 0.02 mol L1, Hb – 50 lmol L1. Other conditions as in Fig. 1.
of electroactive Hb, which was equal to (3.5 ± 0.7) 1011 mol cm2. Taking into account the electrode surface area (0.17 cm2) and the surface that would occupy a single protein (5.5 7.0 nm2), the above value would correspond roughly to 7 monolayers, indicating that not only Hb species contacting directly the electrode surface are responsible for the voltammetric signal. From the dependence of cathodic peak potential on scan rates, the apparent heterogeneous electron transfer rate constant was estimated as ks = 0.6 s1 using the method developed by Laviron [45] for a surface-controlled electrochemical system. The obtained results indicate that the electron transfer of Hb entrapped in the SiO2–Hb–CTAB film is a fast process. This ks value is slightly lower than for a SiO2–Hb film electrogenerated on glassy carbon (ks = 1.2 s1 [23]). For the PGE SiO2–Hb–CTAB obtained under the optimal conditions, the influence of scan rate and pH on the voltammetric characteristics was investigated. Peak currents were found to be directly proportional to the potential scan rate with linear regression equation I (lA) = 0.48 + 0.02 v (mV s1) (R = 0.99) in the 10–100 mV s1 range, indicating a thin-layer electrochemical behaviour. Peak potentials were strongly affected by pH, shifting in the negative direction when decreasing acidity, with an average slope of 120 mV/pH (Fig. 5, curve 1), which is higher than the expected value (56 mV/pH at t = 18 °C) [46] for a proton-coupled single electron transfer process probably because of incomplete reversibility of the system. Measuring peak currents as a function of pH in oxygen free solution has revealed an optimal pH value around 6, giving rise to highest signal intensities. 3.3. Interest of Au nanoparticles addition into the biocomposite electrode
Fig. 4. Dependence of catalytic peak currents on the number of potential sweeps at PGE SiO2–Hb (1), PGE SiO2–Hb–CTAB (2), PGE SiO2–Hb–Au (3) and PGE SiO2–Hb– CTAB–Au (4). Other conditions as in Fig. 1.
UV–vis spectroscopy was first used to confirm the presence of the Au particle in silica sol. The absorption spectra of Au, Hb and their mixture in aqueous solution, as well as in silica sol, are presented, respectively, in Fig. 6a and b. The maximum of adsorption spectra of Au colloid observed at k = 520 nm (Fig. 6a, curve 2) is typical for the Au particles with diameter 3–40 nm [38]. Absorption spectra of Hb were essentially changed in the presence of Au (Fig. 6a, curves 1 and 3). Two new maxima at 540 and 570 nm can be attributed to the reduced form of Hb [47,48]. The red shift of absorption maximum to 600 nm observed for Au in silica sol (Fig. 6b, curve 2) indicates aggregation of the particles in the sol [37,38]. No maximum at 600 nm was found in UV–vis absorption
formance of the bioelectrode in terms of electrocatalytic sensitivity and operational stability. The highest catalytic peak current, welldefined even after 30 potential sweeps, was obtained with PGE SiO2–Hb–CTAB electrode prepared in the presence of 0.02 mol L1 CTAB (i.e., higher than its CMC1 [24]). The optimal molar ratio of sol components (TEOS:H2O) was selected as 1:24 as it led to the best operational stability upon successive measurements while maintaining an acceptable sensitivity, even if high sensitivities (but lower long-term stabilities) were also observed in a wider composition range (Fig. 3). We assume that the reason of signal stabilization at PGE SiO2–Hb–CTAB is the interaction of the protein molecules with the micelles of CTAB that entrapped protein molecules and provided more homogenous distribution of encapsulated Hb in the silica film on the PGE surface. As for PGE SiO2–Hb, the electrocatalytic currents sampled at PGE SiO2–Hb–CTAB increased linearly with raising of Hb concentration in the synthesis sol from 5 lmol L1 up to 40 lmol L1 and reached saturation at an Hb content of 50 lmol L1. Integrating the surface area of the cathodic peak obtained in anaerobic conditions enables to estimate the surface concentration
Fig. 5. Variation of peak potentials of cathodic signal for PGE SiO2–Hb–CTAB (1) and PGE SiO2–Hb–CTAB–Au (2) as a function of pH in anaerobic conditions.
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Fig. 7. SECM approach curves recorded in a solution containing 0.1 mol L1 KCl and 1 mmol L1 Fc(MeOH)2 over a flat plastic surface (a), a gold electrode surface (b), the bare carbon electrode surface (a) and the same electrode covered with SiO2– Hb–CTAB (b), SiO2–Hb–Au (e) and SiO2–Hb–CTAB–Au (f). Electrode speed was 0.5 lm s1.
Fig. 6. Absorption spectra of Hb (1), colloidal gold (2) and mixture Hb + Au (3) in aqueous solutions (a) and in a silica sol (b). C HAuCl4 = 88 lmol L1 (a and b curve 2), Hb:Au molar ratio – 1:1 (a and b); pH = 6.0.
spectra of the triple mixture of Hb, Au and silica sol (Fig. 6b, curve 3). Instead, two absorption bands with small intensity at 550 and 580 nm were observed similar to Hb–Au mixture. It can be concluded that Hb prevents aggregation of Au particles in silica sol and particular formation of reduced form of Hb is possible in triple Hb–Au–silica sol mixture. Gold nanoparticles (Au-NPs) were thus added to Hb–silica sols (with or without CTAB) and these media were applied to prepare Au-NPs–containing biocomposite films electrogenerated on PGE (i.e., PGE SiO2–Hb–Au and PGE SiO2–Hb–CTAB–Au). Observation of the surface of these modified electrodes by AFM confirms the good dispersion of Au-NPs within the films and the absence of large aggregates as the morphology of the films did not change significantly in the presence or absence of gold into the biocomposite. Roughness, as given by the root mean square parameter, increased slightly after deposition of the thin sol gel films (2.3 nm on bare electrode and 3.7 nm on SiO2–Hb–CTAB), but the introduction of nanoparticle did affect it in a significant way (4.6 nm for SiO2– Hb–Au and 2.8 for SiO2–Hb–Au–CTAB). On the other hand, X-ray photoelectron spectroscopy (XPS) did not reveal any signal for Au, most probably because of too low Au-NPs content and/or their coverage by an Hb layer, making Au invisible by the surface analysis XPS technique. The presence of gold in the film electrodes was better evidenced by SECM, via approach curves recorded using ferrocene dimethanol as redox probe (Fig. 7). To this end, two reference supports (a conductive gold plate and an insulating plastic
substrate) were used to highlight the positive and negative feedbacks, (respectively for Au and plastic) classically observed when approaching an ultramicroelectrode tip to the surface of conductive and non-conductive supports [49]. As shown, the curve approach to the bare carbon electrode (curve c in Fig. 7) was the very close as that observed with the gold plate (curve b in Fig. 7) because of the conductive nature of the carbon electrode. When covered with a SiO2–Hb–CTAB film, however, a negative feedback was recorded due to the presence of an insulating layer on the carbon surface (curve d in Fig. 7), but it was less pronounced than on plastic (curve a in Fig. 7), indicating that the biocomposite film probably remains porous to the probe. In the presence of Au-NPs, the approach curves turned to positive feedbacks (see curves e and f in Fig. 7), but remained lower than that recorded for the bare electrode (compare to curves b and c in Fig. 7), the lower current values being observed for the CTAB-containing film (curve f in Fig. 7), which can be explained by its more hydrophobic character. Despite this last limitation, the introduction of nanoparticle into the films improves the reactivity of the interface. PGE SiO2–Hb–Au and PGE SiO2–Hb–CTAB–Au bioelectrodes have been characterized by cyclic voltammetry in the presence of oxygen and the results illustrated in Figs. 2 and 4 (curves 3 and 4) indicate clearly a beneficial effect of Au-NPs on the electrocatalytic response of the PGE SiO2–Hb–CTAB–Au in terms of sensitivity and long-term stability. The best electrocatalytic activity appears in the form of lowering overpotentials for PGE SiO2–Hb–Au in comparison to PGE SiO2–Hb, which was even better in the presence of CTAB. Even more interesting is the dramatic increase in operational stability of the Au-NPs-containing bioelectrodes for which only a decrease by about 20% was observed after 30 successive potential sweeps (see Fig. 4, curves 3 and 4). The optimized sol–gel mixture giving rise to the highest catalytic currents and the most stable response to oxygen upon multiple successive measurements with PGE SiO2–Hb–CTAB–Au was the following one (components expressed in molar ratio): TEOS:Au:CTAB = 1:4.9 105:0.02, with a Hb concentration of 40 lmol L1. For PGE SiO2–Hb–CTAB–Au the amount of electroactive Hb molecules on the electrode surface was evaluated from integration of the surface area of the CV peak obtained in anaerobic conditions as (3.8 ± 0.9) 1011 mol cm2 and the apparent electron transfer rate constant ks estimated to be 0.5 s1 from the dependence of cathodic peak potentials on scan rates. Peak currents sampled at PGE SiO2–Hb–CTAB–Au were found to be directly proportional to
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Table 1 Some characteristics of PGE modified with various silica films containing Hb. Type of electrode
Icat/Inoncat
Signal loss/% (after 30 measurements)
Ec/V (versus Ag/AgCl)
Optimal pH range
c Hb/lmol L1
U1/1011 mol cm2
ks/s1
PGE PGE PGE PGE
8.6 11.9 12.0 11.9
100 60 20 20
0.3 0.15 0.22 0.09
6.0 6.0 5.5–6.5 5.5–6.5
50 50 40 40
2.8 ± 0.6 3.5 ± 0.7 2.2 ± 0.6 3.8 ± 0.9
1.2 [23] 0.6 – 0.5
SiO2–Hb SiO2–Hb–CTAB SiO2–Hb–Au SiO2–Hb–CTAB–Au
Icat – electrocatalytic current of dissolved oxygen; Inoncat – maximum of cathodic current in anaerobic conditions; U1 – surface concentration of electroactive Hb; ks – apparent heterogeneous electron transfer rate constant.
the potential scan rate with linear regression equation I (lA) = 0.89 + 0.02 v (mV s1) (R = 0.99) in the 10–100 mV s1 range, indicating again a thin-layer behaviour. Finally, the effect of solution pH on voltammetric signals obtained in anaerobic conditions was less pronounced in the presence of Au-NPs, causing a negative shift of potential values with the slope of 43 mV/pH, which is much less than in the absence of gold (Fig. 5). Peak current depends on pH for both PGE SiO2–Hb–Au and PGE SiO2–Hb–CTAB–Au. Optimal pH range was 5.5–6.5 which is wider than for the electrodes modified without addition of Au-NPs. The main characteristics of the four bioelectrodes studied here are summarized in Table 1. For all modified electrodes fast electron transfer processes were taking place. Simultaneous addition of CTAB and Au nanoparticles into silica sol caused the most positive shift of Ec, improved stability of electrocatalytic signals and increased amount of electroactive proteins on the surface. Combination of all these advantages in a single device has led to choosing PGE SiO2–Hb–CTAB–Au for further investigations. 3.4. Application of PGE SiO2–Hb–CTAB–Au for determination of O2 and antivirus drug For PGE SiO2–Hb–CTAB–Au Icat values were proportional to the concentration of dissolved oxygen (Fig. 8a). In the O2 concentration range from 0.5 mg L1 to 9.0 mg L1, the linear regression equation was DI (lA) = (0.09 ± 0.06) + (1.53 ± 0.02)c (mg L1) (DI = Icat Inoncat) with a correlation coefficient of 0.999. The detection limit was found to be 0.12 mg L1 (3S criteria). The concentration of dissolved O2 in a tap water determined by the present voltammetric method using PGE SiO2–Hb–CTAB–Au was 7.1 ± 2.0 mg L1 (Sr = 0.11). This is in very good agreement with data obtained by the standard Winkler titration method [40], which gave a value equal to 6.8 ± 0.2 mg L1 (Sr = 0.02), pointing out a satisfactory behaviour of the electrochemical method described here (good reproducibility and accuracy). In the presence of antivirus drug amino derivative of adamantine (rimantadine) in phosphate buffer solution (pH 6.0) at constant amount of dissolved oxygen the catalytic reduction current of dissolved O2 noticeably decreased after 3 min of PGE SiO2–Hb– CTAB–Au contact with the drug solution (Fig. 8b). The catalytic activity of the modified electrode was not renewed in supporting electrolyte solution. Non-catalytic current of PGE SiO2–Hb–CTAB– Au in solution contained rimantadine wasn’t changed. Obtained data can be a result of the interaction of the drug’s molecule with the active centre of Hb which caused inhibition of its electrocatalytic activity. Similar results we obtained for different type of amines and phenol which were shown to be the inhibitors of peroxidase [50,51]. The decrease of Icat was proportional to the concentration of rimantadine in solution in the range from 0.5 mg L1 to 2.0 mg L1 in phosphate buffer (pH 6.0). The amount of dissolved oxygen in solution influenced the slope of the calibration graph. But in the presence of buffer and under constant atmospheric pressure and temperature of the solution the linearity of the graph was satisfactory. At O2 content 8 mg L1 the linear regression equation being DI (lA) = (0.45 ± 0.34) + (3.40 ± 0.30)c
Fig. 8. Variation of the cathodic peak currents measured at PGE SiO2–Hb–CTAB–Au as a function of dissolved oxygen concentration (a) and in the presence of antivirus drug rimantadine (amino derivative of adamantine) (b). Concentration: (a) O2/ mg L1 0–4 (a–g), inset represents calibration curve for determination of dissolved O2; (b) rimantadine/mg L1 0–1.5 (a–d), CO2 = 8 mg L1, inset represents calibration curve for determination of rimantadine. Supporting electrolyte solution: 0.01 mol L1 phosphate buffer (pH 6.0) and 0.01 mol L1 KNO3.
(mg L1), R = 0.985, where DI ¼ I0cat I1cat (I0cat and I1cat : catalytic currents of dissolved O2 in the absence and in the presence of rimantadine in solution, respectively). For 0.5 mg L1 of rimantadine in solution the relative standard deviation (Sr) was 0.04. The detection limit for rimantadine using PGE SiO2–Hb–CTAB–Au was 0.3 mg L1 (3S criteria). 4. Conclusions Incorporation of CTAB in the silica sol enhanced electrocatalytic activity, and to improved operational stability of PGE modified
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with electrogenerated SiO2–Hb–CTAB film in comparison to PGE SiO2–Hb. Doping further the sol–gel-derived bioelectrodes with Au-NPs expanded the pH working range for the modified PGE and greatly improved the stability of the oxygen reduction catalytic currents. Simultaneous addition of CTAB and Au nanoparticles in a silica sol containing Hb proteins led to high amount of electroactive Hb, fast electron transfer rates and excellent stability of electrocatalytic currents of the modified PGE. The PGE modified with SiO2–Hb–CTAB–Au film can be used for detection of dissolved oxygen in the mg L1 concentration range. It can be also extended to the determination of antivirus drugs based on amino derivatives of adamantane. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
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