Choline-sensing carbon paste electrode containing polyaniline (pani)–silicon dioxide composite-modified choline oxidase

Abstract

In this study, a novel carbon paste electrode (CPE) was prepared using the salt form of polyaniline (pani)–silicon dioxide composite that is sensitive to choline. Choline oxidase (ChO) enzyme was immobilized to modified carbon paste electrode (MCPE) by cross-linking with glutaraldehyde. Determination of choline was carried out by the oxidation of enzymatically produced H₂O₂ at 0.4 V vs. Ag/AgCl. The effects of pH and temperature were investigated, and the optimum parameters were found to be 6.0 and 60°C, respectively. The linear working range of the electrode was 5.0 × 10⁻⁷–1.0 × 10⁻⁵ M, R^{2 =} 0.922. The storage stability and operation stability of the enzyme electrode were also studied.

Keywords::

• biosensor
• carbon paste
• choline
• choline oxidase
• Polyaniline (Pani)
• Polyaniline (pani)–silicon dioxide composite

Introduction

Carbon paste electrodes (CPEs) are widely used in electroanalysis owing to their low background current, wide potential window, chemical inertness, and simple and fast preparation from inexpensive materials. CPEs can also be easily modified with electrocatalysts or enzymes by means of simply mixing the modifier and the carbon paste matrix. In addition, the CPE offers a renewable electrode surface (Patia et al. 2007, Arduini et al. 2010).

Choline is a dietary essential nutrient (Zeisel 2000) involved in several body functions, the most relevant being the synthesis of cell membrane's phospholipids. In addition, choline acts as a major source of methyl groups and as a precursor of acetylcholine. Neonates, especially, have a high demand for choline for sustaining growth and brain development (Patia et al. 2007, AAP 1985, Zeisel 1987).

Choline deficiency can affect memory and easily cause liver dysfunction or cancer. Abnormal choline metabolism has been implicated in a number of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases (Zhang et al. 2010, Michel et al. 2006, Zeisel 2004).

Choline determination can be performed using different methods including wet chemical determinations (Martinez 1983), microbiological assays (Lied and Braekkan 1975), gas chromatography (Saucerman et al. 1984), capillary zone electrophoresis (Carter and Trenerry 1996), and high-performance liquid chromatography (HPLC) (Ikarashi and Maruyama 1993).

Thus, choline detection and determination are the basic problems for food and clinical analyses. Generally, analytical methods used up to date are based on the determination of hydrogen peroxide liberated as a result of the enzymatic oxidation of choline by choline oxidase (ChO) (Özdemir et al. 2012).

In this study, a novel CPE was prepared using the salt form of polyaniline (pani)–silicon dioxide composite that is sensitive to choline. ChO enzyme was immobilized to CPE by cross-linking with glutaraldehyde. The optimum working conditions of biosensor were investigated with respect to the substrate concentration, the pH, and temperature. The storage stability and operation stability of the biosensor were investigated.

Material and methods

Equipment and reagents

The electrochemical studies were carried out using an Epsilon EC electrochemical analyzer with a three-electrode cell. The working electrode was a carbon paste (glass tubes with a diameter of 0.8 cm and length of 3 cm) electrode. The auxiliary and reference electrodes were a Pt wire and an Ag/AgCl electrode (3 M KCl), respectively. The pH values of the buffer solutions were measured with an Orion Model 720A pH/ion meter. Temperature control was achieved with a Grant W14 thermostat. ChO (EC 1.1.3.17, purified from the Alcaligenes Species and with an activity of 12.03 unit/ml) and choline were purchased from Sigma. Graphite powder and nujol were supplied by Merck and Sigma, respectively. All other chemicals were obtained from Sigma. All the solutions were prepared using double-distilled water.

Preparation of CPEs

CPE was prepared by thoroughly mixing 160 μL of nujol with 0.15 g of graphite powder in a mortar. For the preparation of CPEs, glass tubes (a diameter of 0.8 cm and length of 3 cm) were filled with the carbon paste. Height of the paste in the tubes was 0.5 cm. Electric contacts were made using platinum wire. The modified carbon paste electrode (MCPE) was prepared with 2 mg of polyaniline–silicon dioxide composite by thoroughly mixing 160 μL of nujol with 0.15 g of graphite powder in a mortar. Polyaniline–silicon dioxide composite was synthesized by Zengin and Erkan (2010). The electrode surface was smoothed on a paper to produce a reproducible working surface.

Preparation of choline oxidase/modified carbon paste electrode

Fifty microliters of ChO enzyme (12.03 Unit/mL), 1 mg of bovine serum albumin, 50 μL of 0.1 M phosphate buffer at a pH of 6.0, and 30 μL of 2.5% glutaraldehyde were dropped upon MCPE. The electrode was dried at room temperature and washed with buffer solution (0.1 M phosphate buffers of pH 6.0) several times in order to remove the excess non-immobilized enzyme and glutaraldehyde. The electrode was kept in a refrigerator at 4°C in phosphate buffer when it was not in use.

Electrochemical measurements

The quantification of choline was achieved via electrochemical detection of the enzymatically released H₂O_2.

ChO/MCPE was immersed in the phosphate buffer (0.1 M) of pH 6.0. The solution contained 0.1 M sodium perchlorate as supporting electrolyte. The electrode was brought to equilibrium by keeping at 0.4 V (vs. Ag/AgCl electrode (3 M KCl)). Steady current (ia) was recorded. Choline solution was added to the cell using a micropipette, and the mixture was stirred. The currents (ib) values obtained at 0.4 V were recorded, and the values (∆i = ib–ia) were plotted against the choline concentration.

Results and discussion

In this study, a novel CPE was prepared using the salt form of polyaniline (pani)–silicon dioxide composite that is sensitive to choline. The parameters affecting the performance of the biosensor and optimum working conditions were investigated.

Amperometric responses of CPE and MCPE to hydrogen peroxide

Amperometric responses of the CP and MCP electrodes to hydrogen peroxide were determined at different hydrogen peroxide concentrations. As shown in Figure 1, when MCP was used, differences in the current were obtained higher than those obtained using CP.

Figure 1. Amperometric responses of carbon paste electrode (CPE) and modified carbon paste(MCPE) to hydrogen peroxide(at 25°C, 0.1 M phosphate buffer of pH = 6.0, 0.4-V operating potential).

The working potential

After preparing MCPE, the hydrogen peroxide oxidation was carried out at different potentials (0.3, 0.4, 0.5, 0.6, and 0.7 V).

Oxidation of hydrogen peroxide was investigated at the mentioned potentials using polyaniline–silicon dioxide composite by Arslan et al. and optimum potential was detected at 0.4 V (Arslan et al. 2012).

The effect of amount of the polyaniline–silicon dioxide composite to selectivity

Arslan et al. investigated the effect of the amount of the polyaniline–silicon dioxide composite on selectivity in another study, which is explained in the following paragraph (Arslan et al. 2012).

The selectivity is an important challenge in the development of a biosensor. In order to obtain the best compromise between sensitivity and selectivity, we evaluated the response of the electrode toward ascorbic and uric acids, and acetaminophen (Luque et al. 2005). Firstly, 5.0 × 10⁻⁴ M hydrogen peroxide was added to the cell. After the addition of hydrogen peroxide; 3.0 × 10⁻⁴ M uric acid, 1.0 × 10⁻⁴ M ascorbic acid and 1.0 × 10⁻⁴ M acetaminophen were also added to the cell. The interference was evaluated at 0.4 V using electrodes prepared with 1.0, 1.5, and 2.0 mg of polyaniline–silicon dioxide composite. It was observed that interference effect decreased when the amount of composite was increased. When the amount of composite was 3 mg, the electrode became very weak. Because of this composite, the amount was limited to 2 mg. The lowest percentage ratio of interference was found when 2 mg composite was used. Therefore, an amount of 2 mg of this composite was used in future studies.

Determination of optimum pH

Since enzyme activity is dependent on the ionization state of the amino acids in the active site, pH plays an important role in maintaining the proper conformation of an enzyme. The effect of pH on the response of the choline biosensor was determined in 0.1 M phosphate buffer of pH range 6.0–10.0. The measurements were taken at a constant choline concentration of 5.0 × 10^{− 3} M. Figure 2 shows that the maximum response was obtained at pH 6.0. Because the isoelectric point of ChO was between pH 4.0 and 5.5, pH values lower than 6.0 were not studied. For choline biosensor, pH values different from 6.0 were employed in the literature (pH 9.0, 7.4, and 8.0) (Özdemir et al. 2012, Razola et al. 2003, Langer et al. 2004). This was attributed to the fact that the used polymer and the type of immobilization were different.

Figure 2. The effect of pH on the response of the biosensor (at 25°C, 5.0 × 10^{− 3} M choline, at 0.4-V operating potential).

Determination of optimum temperature

Enzymes are known to be sensitive to changes in temperature. The relationship between reaction rate of an enzyme and temperature is exponential. Temperature's influence on the response of choline enzyme electrode was tested between 20°C and 65°C at pH 6.0 using constant choline concentration of 5.0 × 10^{− 3} M. As seen in Figure 3, the current difference increases with temperature up to 60°C and decreases afterward. The highest electrode response was obtained at 60°C. For choline biosensor, temperature values were employed in the literature (37 and 30°C) (Razola et al. 2003, Langer et al. 2004).

Figure 3. The effect of temperature on the response of the biosensor (at pH 6.0, 5.0 × 10^{− 3} M choline at 0.4-V operating potential).

The study was carried out at 25°C due to the difficulties involved in working at 60°C. The temperature of 25°C was chosen as a working temperature for all further experiments. ChO enzyme kept its activity even at high temperatures, when it was immobilized to polyaniline–silicon dioxide composite.

When literatures were examined, it was seen that the polyaniline film which was obtained by the electropolymerization of aniline became a good microenvironment around the enzyme. It was observed that the enzyme was stronger even in high temperatures because of the microenvironment (Arslan et al. 2011, Shi et al. 2009, Chen et al. 2006).

Effect of substrate concentration on response of biosensor and calibration curve

The effect of the substrate concentration on the reaction rate, catalyzed by immobilized ChO, was studied using varying concentrations (5.0 × 10^{− 7}–1.0 × 10^{− 3} vM) of choline (Figure 4).

Figure 4. The effect of choline concentration upon the amperometric response of the biosensor (in pH 6.0 phosphate buffer and at a 0.4-V operating potential, 25°C).

The linear working range of the electrode was 5.0 × 10^{− 7}– 1.0 × 10^{− 5} M, R^{2 =} 0.922 (Figure 5). It was shown that the linearity of graphs was highly satisfactory and it could be used for the quantitative determination of choline. The detection limit of the biosensor was 1.0 × 10^{− 7} M and the response time of the biosensor was 200 s. Kinetic parameters Imax(app) and Km(app) for the enzyme biosensor were calculated as 2.39 μA/min and 2.39 × 10^{− 3} mM, respectively, from Lineweaver–Burk plots (Figure 6). Km values for immobilized ChO presented in the literature are 5.58 × 10^{− 4} mM, 0.4 mM, 2.32 mM, and 0.083 mM (Özdemir et al. 2012, Langer et al. 2004, Gülce et al. 2003, Shi et al. 2006). This was attributed to the fact that the polymer used and the type of immobilization were different.

Figure 5. The calibration curve of the choline biosensor (in pH 6.0 phosphate buffer and at a 0.4-V operating potential, 25°C) (n = 3).

Figure 6. The effect of choline concentration upon the amperometric response of the biosensor (Lineweaver–Burk plot, in pH 6.0 phosphate buffer and at a 0.4-V operating potential, 25°C).

The operational stability of the enzyme electrode

The operational stability of the biosensor was studied by performing the activity assay (under optimum conditions) 14 times in the same day (Figure 7). The relative standard deviation obtained after 14 measurements at a constant choline concentration of 5.0 ×10 ^{− 3} M was found to be 7.38%.

Figure 7. Operational stability of the biosensor in pH 6.0 phosphate buffer, at a 0.4-V operating potential, 25°C (n = 3).

The storage stabilization of the enzyme electrode

Storage stability of the biosensor was determined by performing activity assays within 48 days. The activity assay was applied within 48 days to determine the storage stability of the immobilized enzyme. As shown in Figure 8, immobilized enzyme retained 59.09% of its initial activity after 48 days.

Figure 8. Storage stabilization of the biosensor in pH 6.0 phosphate buffer, at a 0.4-V operating potential, 25°C (n = 3).

Conclusion

Choline biosensor prepared in this study is useable in a large concentration range 5.0 × 10^{− 7}–1.0 × 10^{− 5} M (R^{2 =} 0.922). It has a very low detection limit (1.0 × 10^{− 7} M) and an acceptable response time for a biosensor (200 s). Choline biosensor gives perfect reproducible results after 14 measurements with 7.38% relative standard deviation. Also, it has good storage stabilization (gives 59.09% of the initial amperometric response at the end of the 48th day). The optimum pH and temperature parameters for immobilized enzyme were found to be 6.0 and 60°C, respectively. The Km(app) and Imax(app) values of ChO enzyme immobilized in polyaniline–silicon dioxide composite were 2.39 × 10^{− 3} mM and 2.39 μA/min, respectively. Choline biosensor prepared in this study is easy to prepare and is highly cost-effective.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.