Fabrication of a Highly Sensitive Glucose Biosensor Based on ...

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Fabrication of a Highly Sensitive Glucose Biosensor Based on Immobilization of Osmium Complex and Glucose Oxidase onto Carbon Nanotubes Modified Electrode Abdollah Salimi,a, b* Begard Kavosi,a, c Rahman Hallaj,a Ali Babaeic a

Department of Chemistry, University of Kurdistan, P. O. Box 416, Sanandaj, Iran Research Center for Nanotechnology, University of Kurdistan, P. O.Box 416, Sanandaj, Iran c Department of Chemistry, Arak University, P. O.Box, 38156-879, Arak, Iran *e-mail: [email protected]; [email protected] b

Received: August 4, 2008 Accepted: December 24, 2008 Abstract In this research a novel osmium complex was used as electrocatalyst for electroreduction of oxygen and H2O2 in physiological pH solutions. Electroless deposition at a short period of time (60 s), was used for strong and irreversible adsorption of 1,4,8,12-tetraazacyclotetradecane osmium(III) chloride (Os(III)LCl2) ClO4 onto single-walled carbon nanotubes (SWCNTs) modified GC electrode. The modified electrode shows a pair of well defined and reversible redox couple, Os(IV)/Os(III) at wide pH range (1 – 8). The glucose biosensor was fabricated by covering a thin film of glucose oxidase onto CNTs/Os-complex modified electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The fabricated biosensor shows high sensitivity, 826.3 nA mM1cm2, low detection limit, 56 nM, fast response time < 3 s and wide calibration range 1.0 mM – 1.0 mM. The biosensor has been successfully applied to determination of glucose in human plasma. Because of relative low applied potential, the interference from electroactive existing species was minimized, which improved the selectivity of the biosensor. The apparent Michaelis-Menten constant of GOx on the nanocomposite, 0.91 mM, exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. Excellent electrochemical reversibility, high stability, technically simple and possibility of preparation at short period of time are of great advantages of this glucose biosensor. Keywords: Carbon nanotubes, Glucose oxidase, Os-complex, Glucose, Biosensor DOI: 10.1002/elan.200804486

1. Introduction Transition elements of ruthenium, iron, platinum, iridium, manganese, cobalt, tungsten, zinc, osmium and vanadium in different forms such as metals or metal oxide, metalocyanides and organometalic complexes have been successfully dispersed on some substrates such as carbon on conductive polymers to make catalyst. Due to electrochemical reversibility and different oxidation states of osmium derivatives [1 – 3], they can be used as electron transfer mediator for modification of different electrode surfaces such as, gold [3], graphite [4], GC [5], Pt [6] and pyrolytic graphite electrode [7]. The electrodes modified with Os-compounds are received interesting interest in the field of electrocatalysis and electroanalysis [8 – 14]. Enzyme based biosensors are being used in an increasing number of clinical, environmental, agricultural and biotechnological applications. In order to achieve high sensitivity and selectivity of biosensors, different electron transfer mediator required. Due to electrochemical reversibility, high electron transfer rate constant, ks, and stability of the Os-complexes, different osmium derivatives have been used  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

as mediator for biosensor fabrication [15]. Benefits of Osmediators compared to other transition metal derivatives are in lower by 300 mV redox potentials and in higher rates of the electron exchange with enzyme active sites [16]. Determination of glucose is one of the most popular and well-known applications of biosensors. Two basic schemes have been used for glucose detection, using glucose oxidase enzyme (GOx) as recognition layer. Measurement of one compound involved in the enzymatic reaction, either the production of hydrogen peroxide or the consumption of oxygen [17]. The indirect glucose measurement through the use of artificial redox mediators is the other protocol [18]. At bare electrodes high overpotentials required for reduction of oxygen or oxidation hydrogen peroxide. The high potential would result the oxidation or reduction of certain electroactive species in the analytical solution. For improving, the biosensor selectivity complex process is needed to remove the interferences. This disadvantageous could be overcome by using electron transfer mediators. A wide variety of osmium derivatives have been successfully used as electron transfer mediators for glucose biosensor fabrications [15, 16, 19 – 23]. The electron transfer mediator can be Electroanalysis 2009, 21, No. 8, 909 – 917

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immobilized on the electrode surface by different methods such as, adsorption [24], self-assembled monolayers [25], simply mixing into carbon paste [26] direct electropolymerization [27] entrapped into conductive polymer [28] covalent attachment [29] and sol-gel techniques [30]. Unfortunately, more of the modified electrodes based on osmium derivatives have certain disadvantageous, such as considerable leaching of electron transfer mediators and poor long term stability. Furthermore, preparations methods are expensive and difficult. Therefore, the immobilization of new osmium-complex derivatives on the surface of various electrode materials is receiving increasing interest in the field of glucose biosensor fabrication. Carbon nanotubes are promising as an immobilization substance because of their significant mechanical strength, high surface area, excellent electrical conductivity, good chemical stability, and high ability for immobilization of different electron transfer mediators [31, 32]. We have recently studied the preparation of modified electrodes with carbon nanotubes and their applications as voltammetric or amperometric biosensor for glucose sensing [33 – 35].The immobilization of osmium compounds onto carbon nanotubes increased the catalytic activity of the modified surfaces [21, 36 – 38]. Lately, we used single-walled carbon nanotubes (SWCNTs) successfully for immobilization of new derivative of Os-complex [39]. Due to chemical stability and high ability of CNTs to immobilize Os-complex, in this paper GC//SWCNTs/Os-complex used for electrocatalytic reduction of hydrogen peroxide and oxygen in physiological pH. In addition by covering a thin film of glucose oxides on the surface of Os-complex/SWCNTs modified GC electrode (using potential cycling) the high sensitive glucose biosensor has been fabricated. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen [40]. These biosensors applied for glucose determinations in human serum without interference effect of cooxidized compound.

2. Experimental 2.1. Chemical and Reagents Single-walled carbon nanotubes (SWCNTs ) from Sigma. The purity of CNTs was 90%, with surface specific area of, 480 m2/g, diameter 1 – 2 nm and length 0.5 – 2 mm. Osmium(III)-complex; 1,4,8,12-tetraazacyclotetradecane osmium(III) chloride, (Os(III)LCl2) ClO4 was synthesized and purified based on the reported procedure [41]. Prepared complex characterized by comparing their IR and UV-vis spectroscopic data with reported values [42]. a-d-glucose (Aldrich anhydrous 96%, Aldrich) and glucose oxidase (Sigma, EC 1.1.3.4. Type II: from Aspergillus niger) were used without further purification. Double distillate water was used to prepare all solutions. Buffer solutions (0.1 M) were prepared from sulfuric acid (H2SO4), phosphoric acid (H3PO4), sodium acetate (CH3COONa), and di-sodium hydrogen phosphate (Na2HPO4). Hydrogen chloride (HCl) Electroanalysis 2009, 21, No. 8, 909 – 917

and sodium hydroxide (NaOH) were used for pH adjustment. Solutions were deaerated by bubbling high purity argon gas (99.99%) through them prior to the experiments. All electrochemical experiments were carried out at room temperature 25  0.1 8C.

2.2. Apparatus Electrochemical measurements were carried out in a conventional three electrodes cell using a m-AUTOLAB-2 PGSTAT computer controlled potentiostat (ECO-Chemie, The Netherlands). A conventional three-electrode cell was used , a Ag/AgCl (sat. KCl) as reference electrode, a Pt wire as counter electrode and GC electrode (˘ ¼ 2 mm) or modified GC as working electrode. All potential were referred to the reference electrode. Voltammograms of GC electrode modified with Os-complex/SWCNTs recorded in buffer solution containing no complex. All of used electrodes were from Metrohm. A draft shaft from Metrohm was used for rotation modified electrode in amperometric measurements. A personal computer was used for data storage and processing.

2.3. Procedure To prepare an osmium/complex-SWCNTs modified electrode a GC electrode was polished with emery paper followed by alumina (1.0 and 0.05 mm) and then thoroughly washed with twice-distilled water. Then electrode was placed in ethanol container and used bath ultrasonic cleaner in order to remove adsorbed particles. The CNTs was immobilized onto the GC electrode, using dimethyl sulfoxide (DMSO) as the dispersing agent. 25 mL of DMSO – CNTs solutions (0.4 mg/mL) was cast on the surface of GC electrode and dried in air to form a CNTs film at electrode surface. By immersing the GC electrode modified with CNTs in acetonitrile solution containing 0.1 mM of Os (III)complex for 60 s, a GC/CNTs/Os-complex modified electrode was prepared. Loosely bound Os-complex was removed by washing carefully with doubly distilled water. For increasing the stability and voltammetric response of the adsorbed redox couple 70 consequent cycles was recorded in buffer solution free of the Os-complex. The effective surface area of the electrodes modified with immobilization of SWCNTs were determined as 0.15 cm2 from cyclic voltammograms of 1 mM K3[Fe(CN)6] in phosphate buffer solution (pH 7).

2.4. Immobilization of Glucose Oxidase at the Surface of SWCNTs/Os-Complex Modified GC Electrode Potential cycling at range 1.0 to  1.0 V was used for immobilization of glucose oxidase film onto glassy carbon electrode modified with SWCNTs and Os-complex. After 40 consequents potential cycling in pH 7 buffer solution

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containing 5 mM GOx, enzyme entrapped into nanocomposite film and stable cyclic voltammogram for adsorbed GOx was obtained. The results indicate during potential cycling the activation of the modified electrode and its surface roughness is increased. Furthermore, surface contaminants and inhibitor layers that hinder electron transfer removed from the electrode surface. The results indicate the activation potential cycling has a great influence on the amount of adsorbed GOx and its electrochemical response. Finally, the modified electrode was removed from glucose oxidase solution, washed with double distilled water and stored in pH 7 buffer solution at refrigerator (4 8C) before using in experiments. Fig. 1. a) Recorded cyclic voltammogram of a SWCNTs/GC modified electrode in PBS (pH 7); b) as (a) for SWCNTs/Oscomplex modified electrode, scan rate 50 mV s1.

3. Results and Discussions 3.1. Electrochemical Properties of Glassy Carbon Electrode Modified with SWCNTs and Os-Complex Cyclic voltammograms of SWCNTs and Os (III)-complex/ SWCNTs modified GC electrodes in buffer solution (pH 7) was recorded (Fig. 1). As shown due to high ability of the CNTs for physical adsorption of Os-complex a well defined redox couple (E8’ ¼  0.07 V) for modified electrode was observed. For GC electrode modified with SWCNTs no recognizable redox couple observed at the same condition (Fig. 1 voltammogram a). The hydrophobic interaction between carbon nanotubes with organic parts of heterocyclic ligand and n – p interaction of Os with carbon nanotubes attached Os-complex on the electrode surface. The electrochemical properties, stability, pH dependent and electron transfer kinetics of the attached redox couple has been investigated in our previous study [39]. Due to reversibility, stability, reproducibility and fast electron transfer kinetics of the modified electrode the electrocatalytic activity of the modified electrode for electroreduction of oxygen and hydrogen peroxide was examined. The ability of the modified electrode in electrocatalysis, electroanalysis, sensors and biosensors fabrication was evaluated.

H2O2. As shown at Os/complex-SWCNTs modified electrode in the presence of H2O2 the cathodic peak current dramatically enhanced while anodic peak current was disappeared. At the surface of the GC electrode modified with SWCNTs, no response was observed for reduction of H2O2 until potential  0.4 V (voltammogram b). The same behavior was observed for electrocatalytic reduction of oxygen. The reduction catalytic current of oxygen starts at 0.05 V and an obvious catalytic reduction peak appears at the potential of  0.10 V (not shown). For GC electrode modified with SWCNTS a small cathodic response for oxygen reduction was observed at more negative potential ( 0.3 V) (not shown). By increasing the concentration of H2O2 and O2, the cathodic peak current of the modified electrode is increased while its anodic peak current decreased (Fig. 3). The

3.2. Electrocatalytic Properties of SWCNTs/Os-Complex Modified Glassy Carbon Electrode for H2O2 and Oxygen Reduction Figure 2 shows cyclic voltammograms of a SWCNTs/GC and SWCNTs/Os-Complex/GC electrodes in the buffer solution (pH 7) in the absence and presence of hydrogen peroxide. It can be seen (Fig. 2 voltammograms a and b) a small response was observed for SWCNTs/GC electrode in the presence and absence of H2O2 at potential range0.1 to  0.4 V. But we found a single and well-defined redox couple E8’ ¼  0.07 V for Os-complex/SWCNTS modified GC electrode in the buffer solution free of H2O2 (Fig. 2c). Figure 2d shows cyclic voltammograms of SWCNTs/Oscomplex modified GC electrode in the presence of 0.5 mM  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Fig. 2. a) Recorded cyclic voltammograms of SWCNTs modified GC electrode in PBS (pH 7) at scan rate 10 mV s1; b) as (a) in the presence 10 mM H2O2. c) and d) as (a and b) for Os-complex/ SWCNTS modified GC electrode.

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catalytic currents increased linearly with the H2O2 concentration in the range of 0.1 mM – 1 mM. The linear regression equation of catalytic currents vs. H2O2 concentration can be obtained from the experimental data, which is Ip (mA) ¼ 2.4 [H2O2] (mM) þ 1.9 mA, R2 ¼ 0.9992. The detection limit is estimated to be 15 mM when the signal to noise ratio is 3. It can be inferred from these results that, the presence Oscomplex and CNTs on the surface of GC electrode facilitates the detection of hydrogen peroxide at low concentration level. Also, as shown in Figure 3B with increasing oxygen bubbling time the cathodic peak current is increased. The ability of the modified electrode for hydrogen peroxide reduction was investigated by recording hydrodynamic chronoamperogram of the modified electrode at applied potential  0.1 V. As shown during successively adding H2O2 to continuously stirred modified electrode a good response was observed (Fig. 4), that demonstrate a stable and efficient catalytic property of the electrocatalyst immobilized on the CNTs film. The response current is linear with the H2O2 concentration in the range 1 mM to 1.6 mM, while for a high concentration of H2O2, the plot of current vs. analyte concentration deviates from linearity (Insets of Fig. 4). The linear least squares calibration curve over the range of 1 – 25 mM (25 points) is I(mA) ¼ 0.0496 [H2O2] þ 0.0063 mA with a correlation coefficient of 0.9998, indicating that the regression line is fitted very well with the experimental data and the regression equation can be applied in the unknown sample determination. The detection limit (signal to noise ratio was 3) and sensitivity were 73 nM and 330 nA mM1 cm2 respectively.

3.3. Evaluation of Biosensor for Glucose Detection Consecutives cyclic voltammograms of Os(III)-complex/ SWCNTs modified GC electrode in buffer solution (pH 7) containing 5 mg mL1 glucose oxidase was recorded. A typical example of cyclic voltammograms obtained during continuous potential cycling (voltage cycling between 1.0 and  1.0 V) is shown in Figure 5. As can be seen a new redox couple (E8’ ¼  0.35 V) for modified electrode was observed due to high ability of the CNTs for entrapping of GOx. The insets of Figure 5 indicate during potential cycling the cathodic and anodic waves of immobilized GOx grew with the number of scans (40 cycles) and then a current plateau and stable voltammetric response was obtained. The enzymatic activity of the biosensor for oxidation of glucose was evaluated by monitoring the changes in the redox peak currents of oxygen or hydrogen peroxide. Since the sensitivity of the modified electrode for oxygen reduction is higher than H2O2 reduction, the amount of glucose is determined by monitoring the decrease in the reduction peak current of GOx/Os-complex/SWCNTs/GC electrode in oxygen saturated solution. The GOx catalyzed the oxidation reaction of glucose, which also consumes the dissolved oxygen. Cyclic voltammograms of biosensor in phosphate buffer solution (pH 7) Electroanalysis 2009, 21, No. 8, 909 – 917

Fig. 3. A) cyclic voltammograms of Os-complex/SWCNTs modified GC electrode in PBS at scan rate 10 mV s1 in different oxygen bubbling time, from inner to outer 10, 20, 30, 40, 50, and 60 s. Inset: plot of cathodic peak current vs. oxygen bubbling time. B) as (A) for different concentrations of H2O2, from inner to outer, 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 mM.

saturated with oxygen in the absence and presence different concentration of glucose were recorded (Fig. 6). As shown in voltammogram b, in oxygen saturated solution the cathodic peak current is dramatically increased and anodic peak current is disappeared. Upon adding the glucose to the O2 saturated solution the reduction peak currents decreased (voltammograms c to h in Fig. 6). Although producing of hydrogen peroxide in enzymatic reaction increasing the cathodic peak current, but it is not comparable with decreasing of cathodic peak current during

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Fig. 4. Amperometric response of rotating Os-complex/SWCNTs modified GC electrode (rotation speed 2000 rpm) held at  0.1 V in PBS (pH 7) for successive addition of A) 0.1 mM and B) 1.0 mM H2O2. a) and b) Plot of currents response vs. H2O2 concentrations.

Fig. 5. Consequent voltammetric response of Os-complex – SWCNTs modified GC electrode in PBS (pH 7) containing 5 mg mL1 glucose oxidase at scan rate 100 mV s1. Insets: enlarged voltammograms for immobilized GOx during repeated potential cycling.

oxygen consumption. The influence of the common interfering substances; ascorbic acid, acetaminophen and uric acid on the glucose biosensor response was investigated. Cyclic voltammograms of a modified electrode in solutions  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

containing 4.0 mM uric acid, ascorbic acid and acetaminophen were recorded (not shown). The biosensor response for glucose, a mixture of glucose and oxidizable interfering compounds (ascorbic acid, uric acid and acetaminophen)

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Fig. 6. CVs of SWCNTs/Os-complex modified electrode in pH 7 buffer solution a) deoxygenated, b) oxygen saturated and c) to h) as (b) in the presence of different concentrations of glucose: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mM, scan rate 10 mV s1. Inset: plot of current decrease vs. glucose concentration.

are similar. The same selectivity for other glucose biosensors has been reported [33, 40, 43]. These results indicate that this biosensor is suitable for practical application of glucose detection in real samples.

3.4. Amperometric Detection of Glucose at GOx/CNTs/ Os-Complex Biosensor Figure 7 shows the steady-state current response of glucose for the constant electrode potential of  0.25 V. As shown, during the successive addition of 0.1 mM (chronoamperogram A) and 1 mM (chronoamperogram B) of glucose, a well defined response is observed. The plot of response current vs. glucose concentration is linear over the wide concentration range of 1 mM to 1 mM. The calibration plot over the concentration range of 1 – 10 mM (10 points) has a slope of 826.3 nA mM1cm2 (sensitivity), correlation coefficient of 0.9994 and the detection limit of 56 nM at signal to noise ratio of 3. An extremely attractive feature of the GOx/Oscomplex/CNTs modified GC electrode, is its fast response time (i.e., < 3 s) and its stable amperometric response toward glucose. These analytical parameters are comparable or even better than results previously reported for electroanalytical determination of glucose with different sensors and biosensors [33 – 35, 40, 44 – 50]. These results indicate that the present biosensor exhibits a wider linear concentration range, low detection limit, fast response time for the determination of glucose. An extremely attractive feature of this biosensor is its stable Electroanalysis 2009, 21, No. 8, 909 – 917

amperometric response toward glucose detection. The amperometric response of 0.40 mM glucose as recorded over a continuous 30 min period (not shown). The response of biosensor remains stable throughout the experiment (only 6% decrease in current is observed after 30 min) indicating no inhibition effect of glucose for modified electrode surface. It was found that the current responses decreased by about 5% and 14% after storage at phosphate buffer solution (pH 7) in 4 8C for one and four weeks, respectively. This result indicates that the biocompatibility of carbon nanotubes for glucose-oxidase enzyme provides substantial improvement in long term stability of the glucose biosensor. For glucose concentration higher than 1 mM, a response plateau was observed, showing the characteristics of the Michaelis – Menten kinetic mechanism. The apparent Michaelis – Menten constant (KM), which gives an indication of the enzyme-substrate kinetics, can be obtained based on the Lineweaver – Burk equation[51]. 1/Iss ¼ 1/Imax þ KM/Imax 1/C

(1)

Here, Iss is the steady-state current after glucose addition, C is the bulk concentration of substrate and Imax is the maximum current measured under saturated substrate(glucose) solution. The Michaelis – Menten constant of the system (KM) in this work is found to be 0.91 mM, implying that the GOx/Os-complex/CNTs modified glassy carbon electrode exhibits a higher affinity for glucose. The value of KM for GOx in this work is much smaller than that obtained

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Fig. 7. Amperometric response of rotating biosensor (rotation speed 1000 rpm) held at  0.25 V in PBS (pH 7) saturated with oxygen during glucose addition of A) 0.1 mM and B) 1 mM: Insets: plots of decreased current vs. glucose concentration.

at glucose biosensors based on Pt nanoparticles/mesoporous carbon matrix, 10.8 mM [52], GOx immobilized at chitosan and gold nanoparticles, 10.5 mM [53], boron doped carbon nanotube modified electrode, 15.19 mM [54], single-walled carbon nanotube/nafion modified electrode, 8.5 mM [55] and carbon nanotube modified carbon film electrode, 2.5 mM [56]. The smaller value of KM validates that the immobilized GOx on CNTs/Os-complex possesses higher enzymatic activity and the proposed electrode exhibits a higher affinity for glucose.

3.6. Application of Biosensor for Determination of Glucose in Human Serum Sample In order to examine the applicability of the glucose biosensor, it was used for glucose determination in the real samples. The standard addition method was used for glucose detection in serum sample. The cyclic voltammograms for buffer solution saturated with oxygen (voltammogram b), saturated oxygen solution and serum sample (voltammogram c) and buffer solutions saturated with oxygen and containing serum sample and different concentration of glucose (voltammograms d to h) were recorded in Figure 8. The glucose concentration in serum was 7.9  0.1 mM, which is in consistent with real value, calculated in clinical laboratory (7.7 mM), indicating that the fabricated glucose biosensor has practical potential. The amperometric technique was also used for detection of glucose in serum  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

sample. The calculated glucose concentration in a serum sample is about 3.1 mM, in agreement with reported value with clinical laboratory (3.2 mM).

4. Conclusions A highly sensitive glucose biosensor has been fabricated based on immobilization of Os-complex and GOx onto glassy carbon electrode modified with SWCNTs. Electroless deposition and consequent potential cycling was used for adsorption of Os(III)-complex and entrapment of glucose oxidase enzyme onto CNTs film. The SWCNTs involved can not only adsorb GOx and Os-complex but also facilitate the electron transfer. The adsorbed Os-complex shows excellent catalytic activity for reduction of oxygen and hydrogen peroxide at reduced overpotential. Furthermore CNTs/Oscomplex nanocomposite can be used successfully for entrapment of GOx, without decreasing its biocatalytic activity. The modified biocomposite electrode exhibited excellent sensitivity and good stability for determination of glucose, based on decreasing cathodic peak current of oxygen upon glucose addition. The biosensor exhibited good performance for electrocatalytic oxidation of glucose, such as high sensitivity, low detection limit, short response time, wide concentration range and high stability. The Oscomplex/CNT/biocomposite modified glassy carbon electrode can be used in designing other dehydrogenase and oxidase biosensors.

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Fig. 8. Cyclic voltammetric response of the biosensor in 20 mL PBS, pH 7 a) deoxygenated, b) oxygen saturated, c) as (b) after adding 0.1 mL serum sample, d) to h) for solutions containing 0.1 mL of serum sample and adding different concentrations of glucose (10 to 50 mM). Inset: plot of decreased current vs. added glucose concentration, scan rate 20 mV s1.

5. Acknowledgements The financial supports of Iranian Nanotechnology Iinitiative and Research Office of Kurdistan University are gratefully acknowledged. The authors are grateful to Dr. S.Soltanian for his valuable discussions.

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