Electrochemical Impedance Spectroscopy Based ...

Electrochimica Acta 146 (2014) 659–665

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Electrochemical Impedance Spectroscopy Based Evaluation of 1,10-Phenanthroline-5,6-dione and Glucose Oxidase Modified Graphite Electrode Arunas Ramanavicius a,b,∗ , Povilas Genys a , Almira Ramanaviciene c a

Department of Physical Chemistry, Faculty of Chemistry, Vilnius University, Vilnius, Lithuania Laboratory of NanoBioTechnology, Department ofMaterials Science andElectronics, Institute of Semiconductor Physics, State Scientific Research Institute Centre for Physical Sciences and Technology, Vilnius, Lithuania c NanoTechnas - Center of Nanotechnology and Materials Science, Department of Analytical and Environmental Chemistry, Faculty of Chemistry, Vilnius University, Vilnius, Lithuania b

a r t i c l e

i n f o

Article history: Received 12 May 2014 Received in revised form 24 August 2014 Accepted 26 August 2014 Available online 18 September 2014 Keywords: Biosensor electrochemical impedance spectroscopy redox mediator 1,10-phenanthroline-5,6-dione glucose oxidase.

a b s t r a c t 1,10-Phenanthroline-5,6-dione and glucose oxidase modified and unmodified graphite electrodes were analysed in buffer/glucose media by the electrochemical impedance spectroscopy (EIS) method. The EIS analysis was carried out under potentiostatic conditions. The gathered impedimetric data was evaluated applying estimated equivalent circuits. It was determined that equivalent circuit R (RF2 C2 )(C[RF W]) most optimally describes this electrochemical system. The study revealed redox mediating properties of 1,10phenanthroline-5,6-dione deposited on graphite electrodes. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Glucose oxidase (GOx) based electrodes are used in design of glucose biosensors [1] or biofuel cells [2]. GOx-based electrodes are suitable for the generation of electrical current, which is proportional to glucose concentration in the sample [1]. The reaction rate of GOx depends on electrolyte concentration, pH, temperature and presence and nature of redox mediators or other electron acceptors [3,4]. If GOx is used in biosensor design an efficient redox mediator, which is capable to transfer electrons efficiently from the active site of GOx to the electrode is required [5]. For example one natural electron acceptor of GOx is oxygen, but some other redox mediators can be used for the same purpose. Moreover there are several redox mediators that can compete with oxygen in electron transfer efficiency [6]. Various methods including optical microscopy [7], atomic force microscopy [8], and electrochemical methods, [9,10] can be applied to investigate properties of layers that are used for bioanalytical purposes. Electrochemical impedance spectroscopy (EIS) is one of the most informative out of many recently available

∗ Corresponding author. E-mail address: [email protected] (A. Ramanavicius). http://dx.doi.org/10.1016/j.electacta.2014.08.130 0013-4686/© 2014 Elsevier Ltd. All rights reserved.

electrochemical methods [10]. The EIS is based on electrochemical response of a system towards perturbations applied at different frequencies [10,11]. Using other electrochemical methods, such as amperometry or potentiometry there is always a problem of the measured system being non-linear because of changing electrochemical conditions, due to relatively high variation of electric current or voltage, and in this way influencing measured results. In potentiostatic EIS, which is based on constant electrode polarization voltage, a small sinusoidal perturbation of potential is usually applied. The amplitude of such perturbation usually is in the range of 5-20 mV. Such perturbation does not disturb the system’s linearity and in this way it enables to acquire results, which are also linear in time and suitable for further analysis [12,13]. The EIS is mostly used in order to determine the double electric layer capacity and resistance of various modifiers, which carry the charge from electrolyte to electrode and also for the estimation of ion diffusion in solution towards the studied electrode [12]. Because of these abilities the EIS provides detailed information on characteristics of the electrochemical system. There are also some studies on the application of EIS for the characterization of enzyme-modified surfaces [13,14]. It was demonstrated that a phenanthroline derivative and glucose oxidase can be used together for the modification of electrodes,

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after which they can serve as glucose sensors or fuel cells [15]. The 1,10-phenanthroline-5,6-dione (PD), which was chosen for the research presented here, is known as an efficient redox mediator [16] and as a versatile bidentatic ligand in complexes with metal ions [17]. Formation of an enzyme-based layer on top of the electrode modified by this redox mediator provides unique opportunity to study redox properties of PD layer, when it accepts electrons from the enzyme and shuttles them to the electrode [18]. Because of this property, the 1,10-phenanthroline-5,6-dione based layer is expected to act as a conductor rather than an insulator [19]. On the other hand it is known that the transfer rate of charges, which are moving through the enzyme (e.g. GOx), is ‘slowed down’ due to relatively slow enzymatic electron exchange processes. It indicates that the enzyme is a poor conductor but a fairly good charge holder [6,20]. During preliminary evaluation of 1,10-phenanthroline-5,6-dione and glucose oxidase modified electrode basic R (RA CA ) circuit was applied [20], however the circuit well fitted with experimental results only in very narrow frequency range from 1390 Hz to 14.37 mHz. Therefore the aim of this research was advanced EIS-based evaluation of 1,10-phenanthroline-5,6-dione and glucose oxidase modified electrodes, in order to adjust most suitable equivalent circuit, which is describing this electrochemical system. 2. Experimental 2.1. Chemicals and equipment Glucose oxidase (GOx) from a fungus Aspergillus niger (E.C.1.1.3.4.) 295 Umg-1 and 25% glutaraldehyde were purchased from AppliChem GmbH (Darmstadt, Germany). D-(+)-Glucose was obtained from Carl Roth GmbH&Co (Karlsruhe, Germany). The 0.04 M solution of glucose was prepared in a phosphate – acetate buffer, pH 6.0, (A-PBS) at least 24 hours before use, in order to reach equilibrium of ␣- and ␤-forms glucose was allowed to mutarotate. The 10.0 mgmL-1 glucose oxidase solution was prepared by mixing the enzyme in A-PBS, pH 6.0, containing 50.0 mM sodium acetate and 50.0 mM sodium phosphate with 100.0 mM KCl. The 10.0 mgmL-1 solution of 1,10-phenanthroline-5,6-dione in acetonitrile was prepared. All other chemicals were commercially available and were purchased from global suppliers of ‘analytical grade’ purity. The electrochemical impedance spectroscopy measurements were performed in a three-electrode electrochemical cell inside of a Faraday-cage with AUTOLAB PGSTAT30 at ambient temperature (25 ◦ C). Potentiostat/Galvanostat was controlled by the frequency response analyser (FRA) software Eco Chemie (Utrecht, Netherlands). Ag/AgCl electrode in saturated KCl (Ag/AgCl/KClsat ) from Metrohm was used as reference electrode. Handcrafted graphite rod (GR) auxiliary electrode was constructed from 3 mm diameter×40 mm length, 99.999% graphite rod, which was purchased from Sigma–Aldrich (Berlin, Germany). Bare or modified graphite rod electrodes were used as working electrodes, depending on requirements of the experiment. The cell’s electrochemical impedance spectrum was recorded under potentiostatic conditions at 0.0 V vs Ag/AgCl/KClsat . Although the selected potential of 0.0 V is not the best for the charge transfer process [19], the 0.0 V potential was chosen as the most informative for EIS evaluation, because at this potential the initial spectrum of a graphite electrode modified with 1,10-phenanthroline-5,6-dione and glucose oxidase, in buffer solution possesses a more informative characteristic shape (Fig. 1), which is suitable for the evaluation of layered electrochemical system. Also the 0.0 V potential allows to avoid additional influence of electrode polarisation on formation of complicated ion layers on the electrode surface. The EIS spectra for further data analysis are presented as Nyquist plots.

Fig. 1. Initial EIS spectrum for potential selection of graphite electrode modified with 1,10-phenanthroline-5,6-dione and in buffer solution. Plus (+) symbolized data series was gathered at the potential of 0.0 V, triangle () symbolized data series at 0.1 V and rhomb (♦) symbolized data series at 0.5 V vs Ag/AgCl/KClsat.. The impedance spectra were recorded in the logarithmic frequency range from 40 kHz to 8 mHz while applying 10 mV sinusoidal perturbation amplitude.

2.2. Electrode preparation, modification and characterization In order to obtain a clean electrode surface, the surface of the graphite electrode was polished with fine emery paper. Then the electrodes for 10 minutes were boiled in a 4:1 mixture according to volume of 25% ammonia and 30% H2 O2 . After this they were washed with acetone, ethanol and distilled water prior to use, and dried at room temperature. To achieve the best results the edge of the graphite rod surface was limited to only a circular disk of 0.071 cm2 of geometrical surface, which was polished to mirror smoothness in order to minimize the charge holding double electric layer disturbance and to have a similar roughness of all electrodes. Preparation of enzyme-modified electrodes was performed as previously described in other researches [21,22]. In order to obtain a 1,10phenanthroline-5,6-dione and glucose oxidase–modified graphite (GR/PD/GOx) electrode surface: (i) firstly the GR electrode was covered by 1,10-phenanthroline-5,6-dione, for this 3.0 ␮L of 1,10phenanthroline-5,6-dione 10.0 mgmL-1 solution was distributed on the surface of GR electrode; (ii) then, the immobilization of glucose oxidase onto the 1,10-phenanthroline-5,6-dione modified graphite (GR/PD) electrode was performed by procedure described previously [22]. The electrodes were evaluated by EIS during different steps of preparation: (i) bare GR electrode, then (ii) developed GR/PD electrode, after this (iii) formed GR/PD/GOx in buffer and (iv) in buffer/glucose solution was investigated. Then the EIS spectra were evaluated by adjusting several equivalent circuits and their parameters were calculated in order to achieve the best fits between measured results and theoretical values [23]. 3. Results and discussion 3.1. Electrochemical impedance spectroscopy characterizations of electrode at different modification steps Glucose oxidase alone cannot transfer electrons to the electrode directly and for this reason a charge transferring agent is necessary. The 1,10-phenanthroline-5,6-dione is quite compatible with GOx [15,18]. Active site of glucose oxidase is oxidizing


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Fig. 2. Bare graphite electrode in buffer solution. A – (i) principle scheme of GR electrode and (ii) equivalent circuit applied for evaluation; B, C – EIS results represented in a Nyquist plot (circles) and fitting results (solid line) to equivalent circuit model. B – EIS results for the whole range, C – initial part of the same EIS spectra; Equivalent circuit element values: R= 148 , RF = 59.1 k, CCPE = 650 ␮F (n = 0.8811), CW = 1.20 mF. The impedance spectra were recorded in the logarithmic frequency range from 40 kHz to 8 mHz while applying 10 mV sinusoidal perturbation amplitude. The EIS measurement was performed at 0 V potential vs Ag/AgCl/KClsat.

glucose molecule, then it initiates electron ‘flow’ from this active site via the 1,10-phenanthroline-5,6-dione based layer [6]. In order to evaluate this process, in this study the GR/PD/GOx system was examined by means of electrochemical impedance spectroscopy to determine the 1,10-phenanthroline-5,6-dione/glucose oxidase system‘s charge carrier capacity and transfer properties. For the comparison an EIS analysis of the bare graphite electrode was performed. The surface of the electrode consisting of a conductive material such as graphite, when immersed in an ionic solution, gains a double electric layer, which under specific conditions can exhibit capacitive and conductive properties. After achieving steady-state conditions, the double electric layer capacitance and charge transfer through the phase limit resistance in equivalent circuits are interpreted as a capacitor and a resistor respectively. The evaluated system supports both states in parallel, therefore the electrode‘s surface in equivalent circuit is represented as a simple RF C circuit that could only be used in representation of solid state electronics or a electrochemical cell with extremely small dimensions. Keeping in mind, that the working and auxiliary electrodes are much further away from each other than a nanometer, the diffusion and solution ohmic resistance both should be taken into account as important elements of equivalent circuit, therefore the equivalent circuit is altered into R (C[RF W]) (Fig. 2A). A classic expression of this circuit in a Nyquist plot would be an incomplete semicircle with a line, which straightens out with decreasing frequency. Such a model under right selected circuit element values fit well with gathered measurement data as seen in Fig. 2B and 2C. The EIS spectra of bare graphite electrode were registered in A-PBS buffer, pH 6.0, without any additives. After this the composition of this solution was changed by adding glucose up to a concentration of 0.04 M in order to investigate possible influence of glucose on the EIS spectra. The evaluation of such system showed that the addition of glucose to the solution does not change the shape of the Niquist plot (Fig. 3B and 3C) and therefore the circuitry remains the

same (Fig. 3A), only some changes in R (C[RF W]) circuit values are observed. Comparing the system with the previous bare electrode in buffer solution system, the solution resistance R has increased by 8.1%, charge transfer resistance RF – by 0.17%, the Warburg element capacitance CW – by 117%, and the double electric layer capacitance CCPE – by 0.3%. However the decrease of the capacity degree n by 0.56% was observed as well. Comparing the magnitudes of changes of corresponding elements by the addition of glucose to the solution, the most significant influence was noticed in changes of element R and CW values. These changes are mostly dependent on ionic properties of the solution and that were the most affected by addition of glucose, which is a nonionogenic agent. There are also some changes in circuit element CCPE and RF values that represent the surface properties of the electrode, which were not affected by glucose addition, indicating just insignificant glucose molecule adsorption on the graphite surface. During the next step, after the modification of the electrode surface with 1,10-phenanthroline-5,6-dione, the recorded EIS shape differs from that of the bare graphite electrode in buffer solution. In this case, the previously applied equivalent circuit does not fit with experimental results. Therefore the circuit was supplemented with an additional R2 C2 part that corresponds to an additional electrochemically active layer (Fig. 4A), which in the Nyquist plane appears as a new semicircle that merges with the previously registered one (Fig. 4B). Due to a small difference between the capacities of the elements CA and CB the curvature separating the two semicircles is also relatively small, but in this case the difference is significant enough to distinguish the two parts of the circuit and to determine their element values (Fig. 4C). Comparing the bare GR electrode and the 1,10-phenanthroline-5,6-dione modified GR electrode (GR/PD) in buffer solution a great decrease of equivalent circuit parameters was observed: RF – by 71%, C – by 67.7%, n – by 23%. These changes in the GR/PD electrode properties are mostly related to 1,10-phenanthroline-5,6-dione layer

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Fig. 3. Bare graphite electrode in buffer solution containing 0.04 mol/L of glucose. A – (i) principle scheme of GR electrode and (ii) equivalent circuit applied for evaluation; B, C – EIS results represented in a Nyquist plot (circles) and fitting results (solid line) to equivalent circuit model. B – EIS results for the whole range, C – initial part of the same EIS spectra; Equivalent circuit element values: R= 160 , RF = 59.2 k, CCPE = 652 ␮F (n = 0.8762), CW = 2.60 mF. The impedance spectra were recorded in the logarithmic frequency range from 40 kHz to 8 mHz while applying 10 mV sinusoidal perturbation amplitude. The EIS measurement was performed at 0 V potential vs Ag/AgCl/KClsat.

properties, which show that the PD is a poor charge holder, but a good conductor, what is a useful characteristic for electron transfer mediator. The decrease of parameters R – by 58% and CW – by 71% indicate that the composition of the buffer solution has

changed, the most likely explanation for this is that the ionic form of 1,10-phenanthroline-5,6-dione diffuses out from the layer into the solution. The parameters of the newly added R2 C2 circuit part are also of a relatively small magnitude (Table 1). Such

Fig. 4. Graphite electrode modified with 1,10-phenanthroline-5,6-dione and in buffer solution. A – (i) principle scheme of GR/PD electrode and (ii) equivalent circuit applied for evaluation; B, C – EIS results represented in a Nyquist plot (circles) and fitting results (solid line) to equivalent circuit model. B – EIS results for the whole range, C – initial part of the same EIS spectra; Equivalent circuit element values: R = 62 , RF = 17 k, CCPE = 210 ␮F (n = 0.6766), CW = 350 ␮F, RF2 = 24.2 , CCPE2 = 113.7 ␮F (n = 0.7300). The impedance spectra were recorded in the logarithmic frequency range from 40 kHz to 8 mHz while applying 10 mV sinusoidal perturbation amplitude. The EIS measurement was performed at 0 V potential vs Ag/AgCl/KClsat .


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Fig. 5. Principle scheme and complete, theoretical equivalent circuit for graphite electrode modified with 1,10-phenanthroline-5,6-dione and glucose oxidase in A – buffer solution and B – buffer solution containing 0.04 mol/L of glucose.

Fig. 6. Graphite electrode modified with 1,10-phenanthroline-5,6-dione and glucose oxidase, in buffer solution. A – (i) principle scheme of GR/PD/GOx electrode and (ii) equivalent circuit applied for evaluation; B, C – EIS results represented in a Nyquist plot (circles) and fitting results (solid line) to equivalent circuit model. B – EIS results for the whole range, C – initial part of the same EIS spectra; Equivalent circuit element values: R = 140 , RF = 27 k, CCPE = 505 ␮F (n = 0.7500), CW = 1000 ␮F, RF2 = 100 , CCPE2 = 252 ␮F (n = 0.9289). The impedance spectra were recorded in the logarithmic frequency range from 40 kHz to 8 mHz while applying 10 mV sinusoidal perturbation amplitude. The EIS measurement was performed at 0 V potential vs Ag/AgCl/KClsat .

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Fig. 7. Graphite electrode modified with 1,10-phenanthroline-5,6-dione and glucose oxidase, in buffer solution containing 0.04 mol/L of glucose; A – (i) principle scheme of GR/PD/GOx electrode and redox reaction on the surface of this electrode, and (ii) equivalent circuit applied for evaluation; B, C – EIS results represented in a Nyquist plot (circles) and fitting results (solid line) to equivalent circuit model. B – EIS results for the whole range, C – initial part of the same EIS spectra; Equivalent circuit element values: R = 90 , RF = 19 k, CCPE = 46 ␮F (n = 0.8100), CW = 290 ␮F, RF2 = 200 , CCPE2 = 652 ␮F (n = 0.9289). The impedance spectra were recorded in the logarithmic frequency range from 40 kHz to 8 mHz while applying 10 mV sinusoidal perturbation amplitude. The EIS measurement was performed at 0 V potential vs Ag/AgCl/KClsat .

Table 1 Calculated EIS parameters for estimated equivalent circuits. Conditions

R( ,

RF ,

CCPE , ␮F

n

CW , ␮F

RF2 ,

CCPE2 , ␮F

n

C C, + glucose C\PD C\PD\GOx C\PD\GOx, + glucose

148 160 62 140 90

59.1k 59.2k 17k 27k 19k

650 652 210 505 46

0.8811 0.8762 0.6766 0.7500 0.8100

1200 2600 350 1000 290

24.2 100 200

113.7 252 652

0.7300 0.9289 0.9289

results were registered because the R2 C2 circuit corresponds to the 1,10-phenanthroline-5,6-dione interface with solution and/or graphite and it confirms the 1,10-phenanthroline-5,6-dione surface electrochemical properties mentioned previously, when comparing the parameters RF , C and n before and after the addition of the PD layer on the graphite surface. After the final modification, when the 1,10-phenanthroline5,6-dione modified electrode was additionally modified by immobilized glucose oxidase in order to achieve a fully developed GR/PD/GOx electrode, the impedance spectrum was then recorded in A-PBS buffer without any additives. In this case results were compared with those of the bare graphite electrode under the same conditions, and the Nyquist plot shape (Fig. 6B and 6C) was found to be similar to that of the 1,10-phenanthroline-5,6-dione modified electrode, therefore the equivalent circuit of GR/PD/GOx electrode based system is the same as that for the GR/PD electrode (Fig. 4A). In the developed system three clear interfaces are observed that are characterised by different redox and electrochemical properties (Fig. 5A). These three interfaces theoretically could correspond to a three partial circuit chain, but due to the very similar surface capacity some parts of the circuit practically merge into one without leaving any clear trace observable in corresponding Niquist plots (Fig. 6B and 6C). Hence, equivalent circuit R (RF2 C2 )(C[RF W]) (Fig. 6A) most optimally describes this electrochemical system. The optimisation of circuit element values was

performed in such way that the whole circuit theoretical model data would correspond to experimental data. Compared to the bare electrode, the values of equivalent circuit, which correspond to electrochemical properties of the solution, in contrast to the previously studied GR/PD system, have changed only very slightly: R decreased by 5.4% and CW – by 16.7%. Most likely this effect was observed due to relatively fast electron transfer from glucose oxidase to the 1,10-phenanthroline-5,6-dione. The surface parameters of GR/PD/GOx in contrast to the bare graphite electrode changed more significantly: RF decreased by 54.3%, C – by 22.3%, n – by 15%. But the observed decrease is relatively low in contrast with that observed for GR/PD electrode. This means that the layer of glucose oxidase acts as an electrically insulating layer, which increases the capability to accumulate and hold the charge. Very interesting results were obtained when comparing the R2 C2 circuits of the 1,10-phenanthroline-5,6-dione and 1,10-phenanthroline-5,6dione/glucose oxidase systems: RF2 increased by 313% and C2 – by 122% (n – by 27.2%), meaning that the layer of 1,10-phenanthroline5,6-dione is being strongly influenced by glucose oxidase and this can only be possible at the 1,10-phenanthroline-5,6-dione – glucose oxidase interface. Therefore the R2 C2 circuit part now also corresponds to the 1,10-phenanthroline-5,6-dione/glucose oxidase interface, which is responsible for distinct changes in the analysed experimental data, which are presented in Nyquist plot, at higher frequencies (Fig. 6C).


In the final evaluation step EIS analysis was carried out for GR/PD/GOx electrode in buffer/glucose solution. At first, the most noticeable observation is the uneven distribution of experimental points at lower frequencies (Fig. 7B), which suggests that the electrochemical system is changing in time. This effect is observed because the enzyme glucose oxidase reacts with the glucose and coverts it into glucono lactone, which is quickly hydrolysed to gluconic acid. Formed gluconic acid is important for the change of electrochemical properties of the solution. But the most important influence is from the glucose oxidase itself, because during the catalytic reaction of enzyme with glucose, which is present in the solution, electrons from glucose are accepted by active sites of the enzyme. During the next stage these electrons are accepted by PD and transferred towards the electrode. This process results in electron flow, which interferes with the electrical current used by the measuring equipment and in this way it influences the data point distribution and equivalent element values of the system. Moreover, comparing properties of the bare GR electrode and the GR/PD/GOx electrode in pH 6.0 buffer solution, containing 0.04 M of glucose, it can be noticed, that the equivalent circuit parameters have changed more significantly than that of the same electrode in buffer solution without any glucose (Table 1), but this does not reveal the hidden part of the complete circuit (Fig. 5B). Hence, R (RF2 C2 )(C[RF W]) equivalent circuit (Fig. 7A) is the most suitable for the description of here evaluated electrochemical system. After the addition of glucose the parameters of circuit represented in Fig. 7A have been changed: R has decreased by 39% and CW decreased by 76%, indicating that ionic products, in this particular case – gluconic acid, are being generated by enzymatic reaction and further hydrolysis of formed gluconolactone. Simultaneously some other parameters of the GR/PD/GOx electrode have also decreased (RF – by 68%, CCPE – 93%, n – by 8%) showing significant increase in charge transfer rate towards the GR electrode and decrease in capacitance of the GOx-based layer. The R2 C2 circuit parameters in comparison to the same electrode in buffer solution have increased: the RF2 by 200% and the C2 – by 159%, however no changes of the n value were observed. The explanation for this effect is related to the involvement of the 1,10-phenanthroline-5,6-dione based layer into redox reactions by catalytic action of GOx (Fig. 7A). Besides the analysed data there is a very small but significant part of the EIS spectrum in higher frequencies, which deviates from the well fitted part. The deviation at high frequencies can be explained by the non-ideal experiment conditions, even though the electrochemical system was held in a Faraday cage some experiment elements were not so well optimised, such as the shape of electrochemical cell, electrode position and form and other conditions. Despite the fact that this part cannot be well fitted, as it is observed, the deviating points remain in a constant shape in all analysed systems’ spectra, such characteristic shows that the non-ideal conditions, which possibly lead to the deviation, are also constant what enables to compare here studied electrochemical systems. 4. Conclusion Electrochemical impedance spectroscopy based study of 1,10-phenanthroline-5,6-dione and glucose oxidase modified

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electrodes clearly demonstrated redox mediating properties of 1,10-phenanthroline-5,6-dione. Addition of glucose to the solution causes disturbances in ion diffusion. Modification of the electrode with 1,10-phenanthroline-5,6-dione, creates a redox mediator layer that exhibits good electron transfer properties. If glucose is absent in the solution the GOx layer in GR/PD/GOx electrode acts as a usual dielectric insulator and it increases the ability of this layer to hold a charge what increases charge capacitance properties of the system. In addition the GOx layer is acting as an external barrier, which is holding the 1,10-phenanthroline-5,6-dione layer fixed on the electrode. The modification of the electrode with both glucose oxidase, and 1,10-phenanthroline-5,6-dione creates a sensitive system where even low concentrations of glucose are resulting in a significant change of interface properties. These characteristics reveal that a simple layered system composed of an electrode modified with 1,10-phenanthroline-5,6-dione and GOx is capable to serve as an electrochemical glucose biosensor. Acknowledgements The work was supported by Research Council of Lithuania, Support to research of scientists and other researchers (Global Grant), Enzymes functionalized by polymers and biorecognition unit for selective treatment of target cells (NanoZim’s), Project Nr. VP1-3.1ˇ SMM-07-K-02-042. References [1] H. Liu, H. Li, T. Ying, K. Sun, Y. Qin, D. Qi, Anal. Chim. Acta 358 (1998) 137–144. [2] T. Kuwahara, H. Ohta, M. Kondo, M. Shimomura, Bioelectrochem. 74 (2008) 66–72. [3] C.G. Whiteley, D.J. Lee, Enzym. and Microb. Technolog. 38 (2006) 291–316. [4] A. Schmid, F. Hollmann, J.B. Park, B. Buhler, Curr. Opin. in Biotechnol. 13 (2002) 359–366. [5] J. Raba, H.A. Mottola, Crit. Rev. in Anal. Chem. 25 (1995) 1–42. [6] V. Leskovac, S. Trivic, G. Wohlfahrt, J. Kandrac, D. Pericin, The Int. J. of Biochem. & Cell Biol. 37 (2005) 731–750. [7] B. Ballarin, M.C. Cassani, R. Mazzoni, E. Scavetta, D. Tonelli, Biosens. and Bioelectron. 22 (2007) 1317–1322. [8] A.M. Chiorcea, A.M.O. Brett, Bioelectrochem. 63 (2004) 229–232. [9] A.J. Bard, L.R. Faulkner, Electrochem. Methods: Fundam. and Appl., Wiley, New York, 2001. [10] A. Ramanavicius, A. Finkelsteinas, H. Cesiulis, A. Ramanaviciene, Bioelectrochem. 79 (2010) 11–16. [11] E. Katz, I. Willner, Electroanal. 15 (2003) 913–947. [12] D.D. Macdonald, Electrochim. Acta 51 (2006) 1376–1388. [13] R.K. Shervedani, A.H. Mehrjardi, N. Zamiri, Bioelectrochem. 69 (2006) 201–208. [14] T. Hoshino, S. Sekiguchi, H. Muguruma, Bioelectrochem. 84 (2012) 1–5. [15] Y. Oztekin, A. Ramanaviciene, Z. Yazicigil, A.O. Solak, A. Ramanavicius, Biosens. and Bioelectron. 26 (2011) 2541–2546. [16] M.V. Mirifico, E.L. Svartman, J.A. Caram, E.J. Vasini, J. of Electroanal, Chem. 566 (2004) 7–13. [17] K. Yokoyama, A. Wakabayashi, K. Noguchi, N. Nakamura, H. Ohno, Inorg. Chim. Acta 359 (2006) 807–814. [18] A. Chaubey, B.D. Malhotra, Biosens. and Bioelectron. 17 (2002) 441–456. [19] D.M. Murphy, K. McNamara, P. Richardson, V.S. Romaguera, R.E.P. Winpenny, L.J. Yellowlees, Inorg. Chim. Acta 374 (2011) 435–441. [20] A. Ramanavicius, P. Genys, Y. Oztekin, A. Ramanaviciene, J. Electrochem. Soc 161 (2014) 31–33. [21] N. German, A. Ramanavicius, J. Voronovic, Y. Oztekin, A. Ramanaviciene, Microchim. Acta 172 (2011) 185–191. [22] Y. Oztekin, V. Krikstolaityte, A. Ramanaviciene, Z. Yazicigil, A. Ramanavicius, Biosens. Bioelectron. 26 (2010) 267–270. [23] D.A. Harrington, P. Driessche, Electrochim. Acta 56 (2011) 8005–8013.