Biosensor based on a glassy carbon electrode modified with tyrosinase immmobilized on multiwalled carbon nanotubes
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We describe a biosensor for phenolic compounds that is based on a glassy carbon electrode modified with tyrosinase immobilized on multiwalled carbon nanotubes (MWNTs). The MWNTs possess excellent inherent electrical conductivity which enhances the electron transfer rate and results in good electrochemical catalytic activity towards the reduction of benzoquinone produced by enzymatic reaction. The biosensor was characterized by cyclic voltammetry, and the experimental conditions were optimized. The cathodíc current is linearly related to the concentration of the phenols between 0.4 μM and 10 μM, and the detection limit is 0.2 μM. The method was applied to the determination of phenol in water samples.
KeywordsTyrosinase Multiwalled carbon nanotubes Electrochemical biosensors Phenols
Phenolic compounds often exist in the wastewaters of many industries. Many of them are very toxic, showing adverse effects on animal and plants . High levels of phenols have detrimental effects on animal health. Prolonged oral or subcutaneous exposure causes damage to the lungs, liver, kidney and genitourinary tract . Therefore, the determination of phenolic compounds is of great importance. Spectrophotometry and chromatography are commonly used to determine phenols [3, 4]. However, these methods are complicated in sample pretreatment and unsuitable for in situ monitoring. Biosensor based on tyrosinase (Tyr) have been proven to be a promising method for a simple, fast and sensitive detection of phenolic compounds [5, 6, 7, 8, 9, 10].
Recently many tyrosinase biosensors for phenols have been presented. Various materials such as magnetic nanoparticles , metal nanoparticles [15, 16] and polymers [12, 17] have been used to fabricate tyrosinase biosensors. Many methods have been employed for stabilizing and immobilizing the enzyme. Polymer entrapment [18, 19], electropolymerization [20, 21], sol–gels [22, 23, 24], self-assembled monolayers [25, 26], covalent linking [2, 27] and incorporation within carbon paste [11, 16, 28] have been used to construct tyrosinase biosensors. Although these materials have their own advantages in enzyme immobilization, they also have some drawbacks. Take silica sol–gel films as a example, it can retain the catalytic activities of enzymes to a large extent. But the fabrication of silica sol–gel films is time consuming and the silica sol–gel matrix is fragile, it easily shrinks, cracks and delaminates from the electrode surface . Many methods which have been reported about biosensor fabrication are time consuming and consist of many steps, which result in difficulties in obtaining sensors with comparable sensitivity. Thus, more efforts are needed to develop a simple and reliable method to fabricate biosensors.
Nanomaterials possess extreme small size, a high specific surface area, a high surface-to-volume ratio and unique physicochemical characteristics. The use of nanomaterials superstructures for the creation of electrochemical devices is an extremely promising prospect. They hold important applications as catalysts. Carbon nanotubes (CNTs), as a new form of carbon, are of great interest for many applications in, for example, batteries and chemical sensors [30, 31, 32, 33]. Furthermore, high accessible surface area, low resistance and high stability suggest that CNTs are suitable for the potential catalyst supports [34, 35, 36]. After acid treatment, CNTs possess more active sites. CNTs can promote the electron transfer of the active site of biological molecules, and improve relative activity of the enzyme.
Recently, there has been growing interest in using CNTs to fabricate biosensors. Korkut et al. reported an amperometric biosensor based on horseradish peroxidase and CNTs/polypyrrole nanobiocomposite film on a gold surface for determination of phenol derivatives . Yin et al. fabricated an amperometric biosensor based on tyrosinase immobilized onto multiwalled carbon nanotubes-cobalt phthalocyanine (MWNTs-CoPc) silk fibroin film to determine bisphenol A .
We describe sensitive biosensors for phenols here which based on tyrosinase immobilized onto MWNTs modified glassy carbon electrodes (Tyr/MWNTs/GCE). The novel characteristics of the biosensors are simple fabrication, excellent stability and high sensitivity for the determination of phonlic compounds. The characterization and performance of this tyrosinase biosensor was studied. Additionally, the biosensor has been successfully used for the detection of phenol in real samples.
Tyrosinase (EC 232-653-4, 3933 unit·mg−1 from mushroom) was purchased from Aldrich (http://www.sigmaaldrich.com, USA). Multiwalled carbon nanotubes (95%) were purchased from Shenzhen Nanotech Port Co. Ltd. (http://www.seasunnano.com, China). Pretreatment of MWNTs was performed by refluxing in HCl + HNO3 solution (3:1) at 110 °C for 8 h, followed by filtrating and washing with doubly distilled water till pH 7.0, dried at 110 °C for 2 h. After then, 1.0 mg MWNTs was dispersed in 10 mL DMF with the aid of ultrasonic agitation. 0.1 mg ·mL−1 treated MWNTs suspension was therefore obtained . Phosphate buffer solution of pH 7.5, 0.1 M was prepared using K2HPO4 and KH2PO4 in redistilled water. All of the chemicals were of analytical reagent grade and all the solutions were prepared with redistilled deionized water.
All electrochemical measurements were performed with CHI842B electrochemical analyzer (Shanghai Chenhua Co., China) with a conventional three-electrode cell. A glassy carbon electrode (GCE) with diameter of 3 mm was used as working electrode, saturated calomel electrode (SCE) as reference electrode, and a platinum wire was used as auxiliary electrode.
Preparation of the electrodes and amperometric measurements
The bare GCE was polished successively with 0.5 μm Al2O3 slurry to a mirror, and ultrasonically cleaned in ethanol and water for 5 min, respectively, then washed with redistilled deionized water and dried in air before use. A 2.0 μL aliquot of the treated MWNTs solution was cast on the GCE surface, dried in air. The chemically modified electrode was denoted as MWNTs/GCE.
To dissolve 1.2 mg of tyrosinase (3,933 units·mg−1) and 3.6 mg of bovine serum albinum (BSA) in 0.2 mL phosphate buffer solution, 10 μL of this solution was mixed with 2 μL of 2.5% glutaraldehyde. 4 μL of this mixture was then deposited upon the MWNTs/GCE surface and allowed to cross-link to dryness at room temperature. This enzyme activity was calculated and it approximately equal to 80 units on each electrode surface. The electrode was denoted as Tyr/MWNTs/GCE. For comparison, Tyr/GCE was fabricated with the same method. When not in use, the biosensor was stored at 4 °C in a refrigerator.
Amperometric experiments were carried out in an electrochemical cell containing 10 mL of 0.1 M pH 7.5 phosphate buffer solution. All measurements were performed at room temperature.
Results and discussion
Electrocatalysis of MWNTs modified electrodes to quinone
The effect of the amounts of immobilizing tyrosinase and MWNTs on the response current
The effect of pH and temperature of the solution on the response current
Linear range and detection limit
Linear range and detection limit
Linear range (M)
Detection limit (M)
4.0 × 10−7 to 1.0 × 10−5
2.0 × 10−7
2.0 × 10−7 to 6.0 × 10−5
2.0 × 10−7
2.0 × 10−7 to 1.0 × 10−5
2.0 × 10−7
2.0 × 10−6 to 1.0 × 10−4
5.0 × 10−7
Reproducibility, stability and interference
The repeatability of the current response of one enzyme electrode to 50 μM phenol was examined. The relative standard deviation (RSD) was 3.4% for eight successive assays. The electrode-to-electrode reproducibility was determined from the response to 50 μM phenol at five different enzyme electrodes, an acceptable reproducibility was obtained with a variation coefficient of 4.0%.
The enzyme electrode was stored in a dry state at 4 °C in a refrigerator when not in use. The enzyme electrode retained 72% of its original response after 1 month testing.
In order to evaluate the selectivity of the biosensor, the influence of some possible interfering substances was examined in phosphate buffer solution (pH 7.5) containing 20 μM phenol. The results suggested that 100-fold concentration of K+, Ca2+, Mg2+, Zn2+, Pb2+, Cu2+, Cl−, Br−, I−, SO42−, PO43−, CO32−, NO3−, 100-fold concentration of glucose, 50-fold concentration of L-cysteine and uric acid, 20-fold concentration of aminoacetic acid, 5-fold concentration of benzoic acid did not interfere with the determination of phenol.
Recovery of tyrosinase biosensor (n = 6)
Phenol added (M)
3.98 × 10−7
4.00 × 10−7
4.14 × 10−7
3.70 × 10−7
4.00 × 10−7
4.22 × 10−7
3.55 × 10−7
4.00 × 10−7
3.77 × 10−7
Comparison of the biosensor for determination of phenol with others
Comparison of the biosensor fabricated by this paper for determination of phenol with others (GCE, glassy carbon electrode; CPE, carbon paste electrode; MWNTs, multiwalled carbon nanotubes; PDDA, poly(dimethyldiallylammonium) chloride; Tyr, tyrosinase)
70% after 30 days
Covalently immobilized to MgFe2O4-SiO2
1.0 × 10−6–2.5 × 10−4
100% after 20 days
5.0 × 10−6–2.2 × 10−5
Loss 100% after 3 weeks
Mix Tyr and sol–gel solution
5.0 × 10−7–3.0 × 10−5
Sol–gel silicate/Nafion composite
74% after 2 weeks
MixTyr and sol–gel silicate/Nafion
5.0 × 10−6–1.0 × 10−4
Layer by layer self-assembled
2.0 × 10−6–1.0 × 10−4
Mix Tyr, MWNTs and Nation
1.0 × 10−6–1.9 × 10−5
80% after 60 days
Mix Tyr and palygorskite
5.0 × 10−8–1.0 × 10−4
72% after 30 days
Cross-linking with glutaraldehyde
4.0 × 10−7–1.0 × 10−5
We describe an amperometric biosensor here which was fabricated based on tyrosinase immobilized onto MWNTs film for the determination of phenolic compounds. The MWNTs film enhanced the direct electron transfer rate of benzoquinone produced by enzymatic reaction at the electrode. Compared with those biosensors reported in other literatures, the biosensors reported in this work possess main merits are simple fabrication method, good performances in terms of response rate, high sensitivity and operational stability.
This work was supported by the National Natural Science Foundation of China (No. 20247002), Beijing Natural Science Foundation (No.8102009), the Beijing Municipal Education Commission Scientific Technological Project Foundation (No. KZ201110005006) and Beijing Municipal institution of higher learning academic innovating group projects (No. PHR 201007105).