Document Type : Research Paper
1 Faculty of Chemistry, Razi University, Kermanshah, Iran
2 Faculty of Chemistry, Sensor and Biosensor Research Center (SBRC) & Nanoscience and Nanotechnology Research Center (NNRC), Razi University, Kermanshah, Iran
Biosensors make a bridge between various fields of medicine, agriculture, biotechnology, materials science, optics, electronics, physics, and chemistry . Nowadays, biosensors are one of the developed domains to introduce novel developments. Biosensors are substitutes of classical analytical systems in the real world that are annoying, costly, and complex and cannot be used for in situ controlling . They include three essential components; transducer, bioreceptor, and electronic circuit . Bioreceptor is a biomolecule surrounded by the transducer such as DNA, enzyme, protein, antibodies, and whole-cell . Biosensors are an interesting strategy for working at neutral pHs and low temperatures, that they are used as enzymes and catalysts . The enzymes with high stability are very expensive . Enzymes are active biocatalysts and one of the most used methods to enhance the selectivity of the biosensor, which catalyze specific reactions . In addition, the enzymes are usually applied in bioassays, diversified biosensors, and biofuel cells. The limited stability of enzymes is the main obstacle in extensive biological application of enzymes .
To make biosensors, using enzymes on solid electrode surfaces, often need immobilization steps. Various strategies involve adsorption, membranes, chemical binding or encapsulation have been used to immobilize enzymes on the surface of solid electrodes successfully .
The stabilization of enzymes is of great interest in many applications, and the purpose is to offer prolonged active lifetime at normal environments with common substrates in buffered solutions. To make and store the devices without losing activity, the enzyme should be kept in a dry state, since it is very stable in this state, and is not stable in the long term or in the solution process .
There are two ways for electron transfer between the electrode and enzymes; mediated electron transfer (MET) and direct electron transfer (DET) approaches. This capability depends on various factors such as the location of the redox center, the structure of the enzyme, the distance of electron transfer and the orientation of the enzyme on the electrode surface . Modifying the electrode surface and immobilization of enzymes on the electrode surface can easily improve the electron transfer .
Laccases are phenoloxidases which are in the category of multi-copper enzymes. Furthermore, laccases have the ability to accelerate the oxidation of phenols and its derivatives including catechol, benzenethiols, and anilines [12, 13]. Laccase biosensors can accelerate the catechol oxidation to o-quinone for calculable recognition .
Novel nanomaterials, such as carbon nanotube (CNT)  and gold nanoparticles (AuNPs)  are the most common promoters used to improve the electron transfer rate and can be utilized as a support medium for the laccase immobilization . They cause to an appropriate moieties orientation onto the electrode surface and diminish the distance between the transducer surface and electro-active species improving the rate of electron transfer . Amongst all nanomaterials for laccase immobilization, CNT received great attention due to its excellent conductivity, robustness, well-ordered pore structure, higher sensitivity, better selectivity, and high surface area . Nano-sized CNTs can get closer to the active centers of biomolecules and perform better electron transfer . By immobilizing enzymes on functionalized CNTs, the enzyme molecules seem to approach the electrode surface. In addition, enzyme molecules on the electrode surface may be directed by functionalized CNTs, causing the electron transfer process becomes easier and the oxidation potential is reduced .
Catechol (1,2-dihydroxybenze) is one of the phenolic compounds broadly utilized in industry . The European Union and the US Environmental Protection Agency considered catechol as “an environmental pollutant” owing to its toxicity and partial degradation in the ecological system [22, 23]. Of the major conducting polymers, poly (3, 4-ethylenedioxythiophene) (PEDOT) heteroaromatic thiophene has a poor solubility, and therefore its substituted derivatives were synthesized . PEDOT demonstrates some motivating properties, such as optical transparency in its conducting state, high conductivity and stability, and low redox potential and band gap. Polymerization of PEDOT can be conducted by aqueous solutions, and it can be utilized in applications of a biosensor as well [25, 26]. Silva et al.  stabilized AuNPs in poly (allylamine hydrochloride) and applied them as a support for the laccase immobilization and were successfully applied in the expansion of a new biosensor for the determination of dopamine.
The purpose of this research was to fabricate a sensitive laccase-based biosensor for catechol recognition. Here, PEDOT, AuNPs, and MWCNTCOOH were mixed, and then the mixture was electrodeposited on a glassy carbon electrode since their combination effect ensures the quality of biosensor working.
Materials and reagents
Laccase (EC=22.214.171.124, from Trametes Versicolor, 21.8 U/mg), N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were acquired from Sigma-Aldrich. Laccase was used without further purification. Catechol was purchased from Merck, and its stock solutions were provided in twice distilled water. HAuCl4 and 3, 4-ethylenedioxythiophene were purchased from PubChem and Tokyo chemical industry companies, respectively. The other compounds were purchased from Merck.
A Sama 500 electro analyzer system and a three-electrode cell were applied for recording the cyclic voltammetry (CV) and DPV graphs. Glassy carbon electrode, Ag/AgCl electrode (saturated KCl) and a Pt rod were utilized as working, reference and counter electrodes, respectively. To investigate the suitable working electrode or laccase-based biosensor, electrochemical experiments of catechol on the working electrode were studied by CV. FTIR was used for characterization of the electrode surface and enzyme immobilization at the wave-number range of 4000-400 cm-1.
Preparation of a modified electrode
Alumina slurries and fine emery paper were used for cleaning the glassy carbon electrode, thereafter was rinsed carefully with distilled water, and permitted to dry at room temperature. Next, a solution containing 0.01 M 3, 4-ethylenedioxythiophene, 5.0×10-4 M SDBS, and 1.0×10-4 M HAuCl4 was confected in 0.1 M KNO3 solution. MWCNTCOOH powder was dispersed in twice distilled water and sonicated for 4 hours to form a dark and homogeneous suspension. MWCNTCOOH suspension was added to the KNO3 solution and stirred and sonicated for 30 min, then electrochemically treated onto cleaned electrode under the CV from -0.6 to 1.2 V (vs. Ag/AgCl) for 10 cycles (at a scan rate of 0.05 V/s) to obtain the MWCNTCOOH, AuNPs, SDBS, and PEDOT/GCE. Thereafter, the modified electrode was separated from the cell and eluted with distilled water because some molecules may adsorb physically. Then 8 µL of EDC (0.5% w/v), NHS (0.5% w/v), and Laccase (3 mg/mL, pH 3) were dropped on the electrode surface respectively, thereafter kept to dry in the fridge at 4 ºC overnight. After drying, the modified electrode was characterized by FTIR and CV techniques to examine the electrochemical behaviors of catechol at biosensor.
Characterization of laccase biosensor
Voltammetry techniques were applied to study the biosensing systems for catechol detection. The electrochemical experiments were achieved with a computer-controlled electro-analyzer SAMA 500, conventional cell, and three electrodes; glassy carbon electrode, Ag/AgCl (saturated) (Azar electrode), and platinum auxiliary electrode. The potential range of -0.5 to 0.8 V was selected for recording the cyclic voltammograms in the presence of K3Fe(CN)6 and catechol separately.
To investigate the proposed biosensor for catechol detection, its different concentrations were applied in phosphate buffer (0.1 M, pH 3), and DPV technique was used to monitor the effects of catechol concentration onto cathodic currents.
For the better investigation, the proposed biosensor was applied in probable interferences with the concentration 100 times higher than catechol. Hydroquinone is an isomer molecule for catechol; also, catechol was investigated in the presence of hydroquinone.
RESULTS AND DISCUSSION
Morphology study of electrodes
In the FTIR, each type of bond has unlike vibrational frequency, therefore FTIR is considered as a useful tool  to check the bonds of the materials. The covalent binding between laccase and MWCNTCOOH was confirmed using FTIR spectroscopy. FTIR spectroscopy was performed for two modified electrodes, with and without laccase. Fig. 1 demonstrates the model FTIR spectra of modified electrodes. Lower intensity bands appear at 1165, 1079, and 983 cm-1, which are responsible for the stretching of ethylenedioxy ring and to the C-S bond. The functional groups of carboxylic acid appeared at 1631 cm-1 (strong band), and 3428 cm-1 (stretch, broad) which are related to the C=O and O–H, respectively. 816 cm-1 is related to N-H bond, and C-N bond is in the range of 1000-1350 cm-1 .
The CV of the electrodepositing mixture is shown in Fig. 2.
Effect of pH
The best pH was determined for the optimization of laccase biosensor, which included the pH from 3.0 to 8.0. It is known that the activity of the enzymes is affected by the change in the medium acidity , hence the performance of biosensor is affected as well. There are reasons responsible for selecting the optimal pH. In this case, laccase has the ability to reduce molecular oxygen to water associated with the oxidation of numerous organic substrates such as catechol  subsequently, back to the substrate type . Also, the optimal activity of laccase in the pH buffer solution is ranged from 3.0-7.0, when the selected substrate is a phenolic compound  and the activity of fungal laccase enhances as the pH is decreased . In addition, the ionic interaction between a charged surface of the support and enzyme has control on shifting the optimum pH for an immobilized enzyme to a lower or higher pH .
The biosensor response was considered in phosphate buffer (0.1 M) at the pH range of 3-8 containing 1 × 10-3 M catechol. The results (Table 1) for laccase-based biosensor indicated that the highest sensitivity is obtained at pH 3 due to enhancing the laccase activity in acidic media. At a pH of more than 3, the anodic and cathodic currents were decreased perhaps due to reducing the activity of laccase as shown in Table 1. This is why, pH 3 buffer solution was selected for further experiments. According to Table 1, anodic and cathodic potentials shifted to negative amounts by increasing pH. The optimum pH for the proposed laccase biosensor was obtained in acidic media which is in accordance with the previous experiments [36, 37].
Fig. 3 shows clearly that the anodic (Epa) and cathodic (Epc) peak potentials of a laccase redox couple are highly affected by pH and the formal potential (E0′) of laccase has a linear correlation with the pHs 3–8, where E0’ = (Epa + Epc)/2. The linear regression equation was found as E0′ = − 0.0808 pH + 0.8979 with the correlation coefficient of 0.9988.
The various modified electrodes were characterized by CV graphs to examine the changes in their electrochemical behavior.
Some electrochemical parameters such as Epc and Epa, as well as the corresponding cathodic (ipc) and anodic (ipa) peak currents were calculated.
Fig. 4 A displays the CVs of the modified electrodes in an electrochemical solution including 0.1 M KCl and K3Fe(CN)6 at a constant scan rate (0.1 V/s). The data are shown in Table 2. MWCNTCOOH, AuNPs, SDBS, and PEDOT\GCE exhibit high activity for the redox reactions of K4[Fe(CN)6]/K3[Fe(CN)6]. The AuNPs and MWCNTCOOH have a good effect on the modified electrode due to reducing the difference between redox potentials and increment of the redox currents; they increase the electrode surface area and play a significant role; e.g., a conducting wire that makes the electron transfer easier to occur.
Fig. 4 B shows CVs of two modified electrodes with and without laccase in the presence of catechol (the data are shown in Table 3). Laccase\MWCNTCOOH,AuNPs,SDBS,PEDOT\GCE has higher anodic and cathodic currents and also lower different potential peak (ΔE) as comparable with MWCNTCOOH,AuNPs,SDBS,PEDOT\GCE, so the laccase biosensor is more suitable.
Investigation of scan rate effect
The effect of scan rate was considered for Laccase/MWCNTCOOH, AuNPs, SDBS, and PEDOT/GCE at diverse scan rates in phosphate buffer (0.1 M, pH 3) containing 1 × 10-3 M catechol (Fig. 5). Fig. 6 A demonstrates the relation between anodic and cathodic peak currents with the scan rate, correlation coefficients of 0.96 and 0.98 for anodic and cathodic currents, respectively; this refers to the process controlled by laccase. Furthermore, it determines the mechanism of adsorption.
Fig. 6 B displays the changes of anodic and cathodic potentials versus the logarithm of scan rates. The Potentials of an anodic peak proceed in the positive potential direction, while the potentials of a cathodic peak slightly shifts to the negative potential direction in the case of increasing scan rates. The practical behavior depends on α and ks when the charge-transfer rate is adequately slow [38, 39]. The cathodic peak potential (Epc) altered linearly versus log υ with an equation of Epc = -0.0909 Log ʋ + 0.2134; R2 = 0.92, in the range from 0.02 to 0.2 V/s scan rate. The peak potentials can be denoted by Laviron  equations number (1) and (2):
where k is heterogeneous electron transfer rate constant, n is the number of electrons, α is the electron transfer coefficient, R, F, and T are gas constant, Faraday constant, and temperature, respectively. Correspondent to the slope of a cathodic process, it was considered that (1-α) n = 0.64. Given 0.3<αs was estimated to be 0.083 s-1. The redox reaction of catechol with the proposed biosensor was pH dependent to 2 electron numbers. Therefore, two electrons have exchanged in the transfer process and ks could be estimated to be 0.083 s-1 that is near to ks of the other literature ; this indicates that the biosensor has been engaged in electron transfer step.
Calibration curve and detection limit
Fig. 7A demonstrates DPVs that is obtained in different concentrations for the detection of catechol in phosphate buffer (0.1 M, pH 3) with -0.5 to 0.8 V. Fig. 7B demonstrates the relationship between the cathodic current of the biosensor and the catechol concentrations (calibration curve) obtained by DPV techniques. It was found a linear correlation with a detection limit of 0.35 μM in the concentration range of 1 to 4 μM. Table 5 illustrates the performance of the proposed biosensor which has a lower limit of detection (LOD), and better sensitivity than some other biosensors [42-45].
According to Table 4, the presence of probable interferences was studied by CV, using the concentrations of 100 times higher than that of catechol, which indicated no interferences with catechol response. Hydroquinone is the structural isomer of catechol. When the concentration of hydroquinone was 2 or less than 2 times of catechol, two isolated redox peaks were observed, related to catechol and hydroquinone.
The proposed biosensor incorporates the favorable electrocatalytic properties of AuNPs, SDBS, and MWCNTCOOH with the PEDOT properties to decrease the oxidation potential and improve electron transfer rate. FTIR records confirm the presence of amide bonds between laccase and the electrode indicating the immobilization process of laccase. This study shows that the modification of laccase-based biosensor employing PEDOT, SDBS, AuNPs, and MWCNTCOOH on the glassy carbon electrode improves sensitivity, detection limit, and selectivity for the detection of catechol.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of interest.