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Potentiometric Glucose Determination in Human Serum Samples with Glucose Oxidase Biosensor Based on Iodide Electrode. E. Karaku a, . Pekyardιmcιb, and ...
ISSN 00036838, Applied Biochemistry and Microbiology, 2013, Vol. 49, No. 2, pp. 194–198. © Pleiades Publishing, Inc., 2013. Original Russian Text © E. Karaku¸s , ¸S . Pekyardιmcι, E. Kiliç, 2013, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2013, Vol. 49, No. 2, pp. 209–214.

Potentiometric Glucose Determination in Human Serum Samples with Glucose Oxidase Biosensor Based on Iodide Electrode E. Karaku¸sa, S ¸. Pekyardιmcιb, and E. Kiliçb a

Yildiz Technical University, 34240, Esenler, Istanbul, Turkey b Ankara University, 06210, Ankara, Turkey email: [email protected] Received February 01, 2012

Abstract—Glucose potentiometric biosensor was prepared by immobilizing glucose oxidase on iodideselec tive electrode. The hydrogen peroxide formed after the oxidation of glucose catalysed by glucose oxidase (GOD) was oxidized by sodium molybdate (SMo) at iodide electrode in the presence of dichlorometane. The glucose concentration was calculated from the decrease of iodide concentration determined by iodideselec tive sensor. The sensitivity of glucose biosensor towards iodide ions and glucose was in the concentration ranges of 1.0 × 10–1–1.0 × 10–6 M and 1.0 × 10–2–1.0 × 10–4 M, respectively. The characterization of pro posed glucose biosensor and glucose assay in human serum were also investigated. DOI: 10.1134/S0003683813020051

Diabetes mellitus is a worldwide problem because many people are diseased. Its main characteristic is the glucose level, which is chronically raised. Rigorous controlling of glucose level can decelerate longterm complications such as microangiopathy, the kidney or nerve damages which are attributed to diabetes [1]. Accurate and rapid measurement of glucose concen tration at low cost is important for industrial and med ical applications [2–4]. Numerous processes and methodologies have been developed for creating new glucose biosensors such as electrochemical methods [5], colorimetry [6], con ductometry [7], optical methods [8] and fluorescent spectroscopy [9]. Among them, the electrochemical glucose sensors have attracted the most attention over the last 40 years because of their unbeaten sensitivity and selectivity. Additionally, electrochemical tech niques show lower detection limit, faster response time, better long term stability and inexpensiveness. The electrochemical glucose sensors are basically cat egorized into 4 major groups depending on the mea surement principles: i.e., potentiometric, amperomet ric, impedemetric or conductometric sensors. Enzymes are important substances since they are used to recognize the target molecule in the complex system very easiliy and accurately. For this reason, researchers have tried to improve the properties of enzyme such as reusability, operational and longterm stabilities by using different immobilization methods. Recently, the use of enzymes such as glucose oxidase has gained popularity in the assay of glucose concentra tions [10–14]. Additionally, there has been consider able interest in the use of enzyme electrodes. Such elec trodes usually consist of a sensor electrode, which mon

itors the change in concentration of a reactant or product of a reaction catalysed by the chosen enzyme; this enzyme is immobilised next to the surface of the sensor [15]. Therefore, numerous sensors were devel oped for fast monitoring of glucose levels in physiologi cal fluids which can be done in vivo or in vitro [15–24]. Enzymatic oxidation of various biological sub strates are often accompanied by the formation of hydrogen peroxide. In particular, glucose oxidation in the presence of glucose oxidase involves the formation of hydrogen peroxide. Classical glucose biosensors were based on monitoring either the consumption of oxygen or the production of hydrogen peroxide. Unfortunately, the amperometric determination of hydrogen peroxide requires high anodic potential [20]. Several potentiometric glucose sensors described in the literature used different preparation methods [25, 26]. Coimmobilization of glucose oxidase and peroxi dase on the electrode permits potentiometric determi nation of glucose on the basis of bioelectrocatalytic detection of hydrogen peroxide formed due to cata lytic oxidation of glucose. The aim of this work was to develop new potentio metric glucose sensor based on glucose oxidase (GOD) and sodium molybdate (SMo) immobilized on an iodide ion selective electrode. Firstly, glucose oxidase enzyme catalyses the oxidation of glucose:

Glucose + 1 2 O 2 + H 2O ⎯⎯⎯⎯ → Gluconic acid + H 2O 2 GOD

(I)

Secondly, formed hydrogen peroxide and iodide ions are reacted by sodium molybdate (SMo) instead

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of peroxidase. Iodine and H2O are formed according to the reaction:

250 mg GOD, 0.1 М ФБ (1 mL, pH 7.0)

МН (II) H 2O 2 + 2H + + 2I −М ⎯⎯⎯ → I 2 + 2H 2O The decrease of iodide concentration will be pro portional to glucose concentration. The optimum working conditions for GOD sensor based on iodide electrode such as lifetime, response time, optimum pH, optimum temperature, optimum working range were investigated and interference studies have been made. Furthermore, we investigated whether the glu cose biosensor can be used to determine glucose level in human serum samples.

MATERIALS AND METHODS Reagents and Apparatus. Glucose oxidase (GOD, E.C. 1.1.3.4) type VII from Aspergillus niger and sodium molybdate (SMo), glucose, sodium iodide, uric acid, ascorbic acid, CuSO4 ⋅ 5H2O, NaF, NaBr, FeCl3 ⋅ 6H2O were obtained from Sigma Chemical (United States). All other chemicals used were of ana lytical reagent grade. Standard solutions and buffer solutions were prepared with deionized water. The blood serum samples were provided by Biochemistry Laboratory at Ibni Sina Hospital at Ankara Univer sity (Turkey). Potential and pH measurements were carried out with an ORION 720A model pHion meter (United States). The iodide electrode (ORION 940011) was used to determine the amount of decresed iodide ions with iodide oxidation. The potential values were given against the Ag/AgCl double junction reference elec trode (ORION 90–02). All measurements were made with a 25 mL glass cell. A magnetic stirrer was used throughout the experiments. Bidestilled water was used for the preparation of solutions. Preparation of Glucose Biosensor. 250 mg of GOD (50kU) was dissolved in 1 mL 0.1M phosphate buffer (pH 7.0). 100 µL of this solution was dropped on the iodide electrodes and left overnight. Electrode surface was covered by dialysis membrane and glucose oxidase biosensor prepared was placed in 0.01 M NaI for 2 h (Fig. 1). First of all, the sensitivity of the GOD biosensor pre pared against iodide was determined by carrying out potential measurement in NaI calibration solution at concentration in the range of 10–1–10–6 M (pH 7.0). To test the sensitivity of the GOD biosensor prepared against glucose the measurements were made in glucose calibra tion solution in 0.1 M phosphate buffer (pH 7.0) con taining 10 mL dichlorometane, 5 mL of 5% SMo (pH 7.0) and 2.5 mL 0.01M sodium iodide (pH 7.0). Measurement of the Biosensor Response to Glu cose. The potential determinations for glucose biosen sor were carried out in 0.1 M phosphate buffer (pH 7.0) by varying glucose concentration in steady state conditions. Measurements were made with the proposed glucose biosensor and Ag/AgCl reference APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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Dialysis membrane GOD Iodide electrode

а

b

Glucose sensor

Fig. 1. Preparation of glucose biosensor. a—iodide sensor; b—glucose biosensor based on GOD and iodide sensor.

electrode. Biosensor was immersed to a depth of 1.5 cm in glucose solution and stirred by a magnetic stir rer. The pH values were determined using an Orion combinated glasspH electrode. All the experimental works were carried out at 25 ± 1°С. The calibration curves were obtained by plotting the potential values of a series of standard glucose solution against the loga rithm of glucose concentration. Procedure for Determination of Glucose in Serum. Human serum samples were collected from diabetes mellitus patients. The glucose level at concentration range of 75–115 mg/L was determined in probes by using the standard addition method [27]. The glucose biosensor was immersed in the 25 mL of 0.1 M phos phate buffer (pH 7.0) containing 0.5 mL of serum. Then stock glucose solution was added to this solution step by step. After each step, potential values against of stock glucose solution taken in different concentra tions were plotted and decrease of iodide amount was determined. Glucose concentration in human serum was calculated according to this plot. The results obtained were compared with data of the analysed blood samples from Ibni Sina Hospital. RESULTS AND DISCUSSION The proposed glucose biosensor was prepared by using GOD catalysed the oxidation of glucose and SMo catalysed the transformation of iodide to iodine. Glucose calibration solutions contain 10 mL of dichlorometane, 2.5 mL of 0.1 M sodium iodide (pH 7.0) and 5 mL of 5% SMo. Reaction mixture was com pleted 25 mL with 0.1 M phosphate buffer (pH 7.0). After the oxidation of glucose at standard glucose calibration solution (10–1–10–6 M, pH 7.0) by using prepared glucose electrode containing GOD, hydro gen peroxide formed from reaction (1), iodide reacted and was oxidized at iodide electrode (reaction 2) in the presence of dichlorometane. Iodine formed passed to dichlorometane phase and SMo catalysed the oxida tion of iodide. Otherwise, iodine formed reacted with

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Fig. 2. The response of glucose electrode against iodide in 0.01 M phosphate buffer (pH 7.0).

E, mV 0 –40 –80 –120 –160 –200 1

2

3

4

5 6 –log[glucose]

Fig. 3. The calibration graph of glucose electrode for glu cose in 0.01 M phosphate buffer (pH 7.0).

mV/[glucose] 80

(а)

60 40 20 0 80

0.05

0.10

0.15

0.20

0.25 М

(b)

60 40 20 0 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 pH Fig. 4. Effect of the phosphate buffer concentration (a) and pH (b) on response of glucose biosensor based on iodide sensor.

iodide being at reaction medium in triiodide form (reaction 3). (3) I 2 + I − → I 3− The decreased amount of iodide ion was deter mined by iodide electrode. The glucose concentration

was calculated from the decrease of iodide concentra tion determined by iodide electrode. The Sensitivity of Glucose Biosensor to Iodide and Glucose. In order to determine responses of the glu cose biosensor to iodide ions and glucose, mV mea surements were carried out with prepared biosensor in a series of standard solutions of every reagent. The calibration curves of glucose biosensor for iodide ions and glucose are shown at Fig. 2 and Fig. 3. The sensitivity of glucose sensor towards iodide ions and glucose is high at the concentration ranges of 10–1–10–6 M and 1.0 10–2–10–4 M, respectively. Therefore, we decided that this type of glucose sensor can be used for glucose determination in biological fluids. The linear range of glucose biosensor extends from 10–2–10–4 M and showed a Nernstian response within this range for glucose (Fig. 3). respectively. The slope of the biosensor (expressed as mV/[glu cose]) was 65.2 ± 0.2 mV/p[glucose]. Effect of Buffer Concentration. To examine the effect of buffer concentration on the response of bio sensor, its slopes (presented as mV/[glucose]) were measured at 5 different phosphate buffer concentra tions varying from 0.05 to 0.15 M. Figure 4a shows the change of potential against glucose concentration. The optimum buffer concentration having the biggest slope was accepted 0.05 M. Effect of pH. Effect of pH on the analytical signal of the glucose biosensor was investigated by measure ments in 0.05 M phosphate buffer at 6 different pH in the range of 5.0–8.0. It was shown that the slopes of glucose biosensor increased up to pH 7.0 and decreased over pH 7.0 (Fig. 4b). Above pH 7.0, GOD was denaturized due to deprotonation at active site. At pH less than 7.0, enzyme could also be denaturized and hence reduced the activity [27, 28]. The response was also diminished with increase of the buffer pH which was common property of the potentiometric systems [27]. Therefore, pH 7.0 was used for all other measurements as optimal value. Effect of Temperature. Effect of temperature on the analytical signal of glucose biosensor based on iodide sensor at 1 mM glucose concentration (pH 7.0) was investigated by measurements for 7 different tempera tures varying from 10 to 50°C (Fig. 5). As seen from Fig. 6, the potential of biosensor increased until 25°C and decreased after 25°C because of decrease in the GOD activity. Because enzyme activity increases with temperature, optimum temperature is very important. On the other hand, high temperatures may lead to thermal deactivation of the enzyme and decrease of O2 concentration [29]. Selectivity Coefficients. The potential interfering species that may influence the glucose measurement can be divided in two groups: (a) interfering with the iodide detection and (b) interfering with the SMo reaction as they competing with iodide, such as ascor

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% 120 100 80 60 40 20

E, mV 300 260 220 180 140 0

10

20

30

40

50

60 °C

0

5

10

15

20

25

30

35 days

Fig. 5. Effect of temperature on on response of glucose biosensor at 1 mM glucose concentration (pH 7.0).

Fig. 6. The lifetime of glucose biosensor based on iodide electrode kept at 4°C.

bic acid and uric acid in the same specific biological samples.

possible changes in the enzyme conformation; there fore, the magnitude of analytical signal found to be lower. For this reason, 250 rpm was determined as the optimal stirring rate. Response Time. The response time of the glucose biosensor was determined by recording the time elapsed to reach a stable potential value after the bio sensor and the reference electrode were immersed in calibration solution. It changed between 1–2 min. Lifetime of the Glucose Biosensor. The lifetime of glucose biosensor kept at 4°С was determined by read ing the potential values of the calibration solution and by plotting the calibration curves for a period of one month. The slopes of the biosensor expressed as mV/[glucose] did not change nearly until 10th day and showed a gradually decrease until 30th day. We can conclude that the lifetime of the prepared glucose bio sensor is rather stable during one month (Fig. 6). Reproducibility of the Glucose Biosensor. The bio sensor was tested for its reproducibility by plotting the calibration curves for five times within one day and determining the slopes of the biosensor presented as mV/[glucose]. The relative standard deviation of the slopes was found to be less than 0.5% for proposed glu cose biosensor. Glucose Determination in Human Serum Samples with the Glucose Biosensor. The glucose level data in human serum measured by glucose sensor were corre lated with Hospital results. Glucose content for two different blood samples is given in Table. The correlation of glucose concentration (range 75–115 mg/L) in human serum determined by the

The prepared glucose biosensor was tested for its sensitivity towards Fe+3, Cu+2 , F–, Br–, Cl–, ascorbic and uric acids (pH 7.0). For determining the interfer ing effects usually observed in biological fluids and several substances such as ascorbic and uric acids oxi dizing with hydrogen peroxide at the same potential, the potentiometric response of biosensor was mea sured in 1 mM glucose (pH 7.0) containing the certain amount of interfering species according to Srinivasan and Rechnitz [30]. The obtained potential values were evaluated and their selectivity coefficients were calcu lated. Interference effects of Fe+3, Cu+2 , F– and Br– on the response of the glucose biosensor were low, 10–5 M ascorbic acid didn’t show interference effect. As uric acid was insoluable in phosphate buffer at pH 7.0, no interference was noted. Effect of Stirring Rate. To study the effect of stir ring rate on the responses of glucose biosensor, the measurements were done in 0.05 M phosphate buffer (pH 7.0) at 3 different stirring rates (100, 250, and 750 rpm) after reaching to stable potential. When stir ring was made at 100 and 750 rpm levels, the time to reach stable potential was longer than when 250 rpm rate was used. An decrease of stirring rate (100 rpm) has not been resulted any influence on the magnitude of the analytical signal, but has caused an increase in the response time of sensor. This situation could be explained by the fact that enzymatic reaction was slow at low stirring rate. At high stirring rate (750 rpm), the rate of enzymatic reactions also decreases because of

The glucose content in human serum samples obtained from the measurement with proposed glucose biosensor and the results of Ibni Sina Hospita Glucose amount, mg/L* Serum samples

Recovery, % Ibni Sina Hospital

iodideselective glucose sensor

86 256

84.7 ± 1.3 255.8 ± 2.1

1 2 * These values are the mean of 4 results.

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