Laccase (polyphenoloxidase; EC 18.104.22.168) is a well-known enzyme for the oxidization of a wide range of compounds such as polyphenols, methoxy-substituted phenols and diamines. This enzyme, which belongs to the blue multi-copper-oxidase family undergoes typical reactions where a phenolic group is often oxidised via one electron transfer to form a phenoxyl free radical. This results in an active oxygen species that could yield a quinone or further undergo polymerisation of the free radical. The ability of laccase to react with phenolic compounds opens up many opportunities of applications in agricultural, food, industrial, medical and environmental sectors [1
Laccase based voltammetric/amperometric biosensors have been developed for food and beverages analyses. These biosensors have found wide applications in the analysis of phenol, polyphenol, guaiacol, gallic acid, caffeic acid, catechin, catechol, hydroquinone and resorcinol to name a few in various food and beverage products [2
]. However, such biosensors have not been explored for food colour additives such as tartrazine. The selection of a suitable matrix for laccase enzyme immobilization in the construction of a biosensor for food analysis is a crucial part of the fabrication process [5
]. The enzyme laccase immobilisation methods for the construction of biosensors reported for food analysis were based on several procedures involving laccase adsorptions (e.g., in carbon/graphite materials, nafion nanocomposite and screen-printed gold); enzyme entrapments (e.g., in polyazetidine prepolymer, chitosan, MWCNTs, PVA photopolymer and sonogel), enzyme cross-linkings (e.g., with cyanuric chloride/chitosan, polyvinylpropylidone gel, glutaradehyde on glassy carbon) and laccase covalent attachments (e.g., on nickel nanoparticles/MWCNTs/PANI, DEAE cellulose, ITO-APTES monolayer, glutaradehyde-cysteamine monolayer, magnetic nanoparticles or copper nanoparticles composite with MWCNTs and PANI) [6
Polymeric microspheres are favourable materials for enzyme immobilization due to their high surface area in 3D shape, chemical stability, porosity and functional group density that can easily be tailored in accordance to their specific needs [7
]. The poly(glycidyl methacrylate) (PGMA) containing epoxy group has been widely applied due to its attractive properties and discovered as an ideal support for enzyme immobilization [8
]. They have the ability to form strong linkages with amino, hydroxyl, and thiols group under mild conditions [9
]. The modifications of the epoxy ring with amine groups endow PGMA with excellent affinity to a variety of proteins, which makes it applicable in many areas, and easily available for immobilization of the enzyme [11
]. The use of methacrylic based polymers in the forms of microspheres or cryogels for the immobilization of laccase have been reported with the aim of application to bioremediation and waste water treatment [12
This study highlights the development of a new tartrazine biosensor based on laccase. To the best of our knowledge, detection of tartrazine catalyzed by laccase using the electrochemical biosensor technique has not yet to be reported. Tartrazine (Figure S1
) is a synthetic organic food dye found in common food products such as beverages, candies, dairy products and bakery products [2
]. However, the content of tartrazine must be controlled due to its potential harm to human beings, contributed from the azo groups (N=N) and aromatic ring structures [14
]. In China, the permitted maximum limit of tartrazine additive in foods is 0.1 g/kg (individually or in combination [2
]. Tartrazine will cause many adverse health effects such as allergies, migraines, eczema, anxiety, diarrhoea and childhood hyperactivity if they are excessively consumed [15
]. Therefore, convenient, rapid and reliable methods for rapid determination of tartrazine are essential for the food safety assurance. Traditionally, tartrazine has been analysed using spectrophotometry [3
], chromatography [4
], mass spectrometry-chromatography [5
], capillary electrophoresis [16
] and electrochemical methods using various chemically modified electrodes [17
In this work, laccase enzyme was immobilized on poly(glycidyl methacrylate-co
-n-butyl acrylate) (poly(GMA-co
-nBA)) microspheres, which we have reported recently [18
]. Poly (n-butyl acrylate) has a hydrophobic property and thus the microspheres are hydrophobic where the laccase immobilization will be confined to the surface of the spheres; this allows the enzymatic reaction with tartrazine to occur at the surface and diffusion limitation within the polymer matrix is eliminated [7
]. Thus, we have attempted for the first time to utilize laccase enzyme for the successful construction of a biosensor for tartrazine analysis.
2. Materials and Methods
Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were performed with AutolabPGSTAT 12 (Autolab, Metrohm, Zofingen, Switzerland) potentiostat. The parameters used for CV were 0.007 V for step potential and 0.05 V/s of scan rate from −1.25 to 0.75 V. For DPV, the parameters used were 0.02 V step potential in the scan range of −1.0 to −0.1 V. A screen printed electrode (SPE) supplied by Scrint Technology (M) Sdn. Bhd. coated with methacrylate-acrylate microspheres in the presence of gold nanoparticles (AuNPs) was used as working electrode. A rod-shaped glassy carbon electrode and Ag/AgCl electrode were used as auxiliary and reference electrodes, respectively, and the KCl solution of 3.0 M was used as the internal solution of the Ag/AgCl electrode. All potentials measured in this study were referred to Ag/AgCl electrode and homogeneous mixture of material solutions was prepared using sonicator bath Elma S30H.
The following chemicals were obtained from commercial sources: glycidyl methacrylate, GMA (Sigma-Aldrich, St. Louis, MO, USA), n-butyl acrylate, nBA (Merck, Kenilworth, NJ, USA), ethylene glycol dimethacrylate, EGDMA (Sigma-Aldrich, St. Louis, MO, USA), sodium dodecyl sulphate, SDS (Systerm), 2,2-dimethoxy-2-phenylacetophenone, DMPP (Sigma-Aldrich, St. Louis, MO, USA), glutaric aldehyde, GA (Sigma-Aldrich, St. Louis, MO, USA), Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA) and bovine serum albumin, BSA (Sigma-Aldrich, St. Louis, MO, USA). Deionized water was used for preparing aqueous solution during experiments.
2.2. Fabrication of Functionalized Microspheres and Electrochemical Characterization
Colloidal AuNPs (<100 nm particle size) was purchased commercially. About 1 g of methacrylate-acrylate microspheres were added to 10 mL enzyme solution and kept at 4 °C for 24 h in order to immobilize the enzyme onto the microspheres. The SPE working electrode (AuNPs/SPE) was prepared by depositing AuNPs onto SPE and dried at room temperature. Then, methacrylate-acrylate microspheres immobilized with laccase enzyme was suspended in 90:10 of ethanol to water before being deposited onto the AuNPs/SPE. It refers as microspheres-laccase/AuNPs/SPE and dried at room temperature. The response of tartrazine biosensor was later examined with CV and DPV in 0.05 μM phosphate buffer pH 5.0. Four SPEs were selected and fabricated including (a) bare SPE, (b) Laccase/SPE, (c) microspheres-laccase/SPE and (d) microspheres-laccase/AuNPs/SPE respectively. Electrochemical investigation of tartrazine was carried out in an electrochemical cell containing 4 mL of 0.1 M PBS (pH = 5.0) and 0.5 μM of TT. The potential range was 0.50 to 1.30 V using microspheres-laccase/AuNPs/SPE as working electrode, a glassy carbon counter electrode and Ag/AgCl reference electrode.
2.3. Optimization and Evaluation of Electrochemical Tartrazine Biosensor
The response of tartrazine biosensor was examined based on the effect of various parameters on the immobilized laccase. The influence of pH was analysed by varying pH of tartrazine solution that was prepared in 0.05 M sodium phosphate in the pH range 2.0–8.0 [19
]. The exposure time, which is the duration of the exposure of the electrode to tartrazine, could affect the optimum biosensor response. For this study, the exposure time was performed from 1–15 min before measurement. The procedure was carried using a different amount of AuNPs between 0.02 mg and 0.10 mg. The microspheres amounts were also optimized. Effect of laccase concentration on biosensor response was also examined by varying laccase concentration over the range of 1.0–3.0 mg/mL. The voltammograms were recorded from +0.60 to +1.20 V at 0.07 V·s−1
scan rate. Under optimum conditions, DPV measurements were recorded from +0.60 to +1.20 V to obtain the linear range and low detection limit of tartrazine biosensor. Furthermore, to evaluate the interference that may interfere on the tartrazine determination, tartrazine concentration is fixed at 0.5 μM. Then, the interferences were added with various ratios of concentration. Other performances of biosensor including reproducibility and repeatability were also examined using DPV. Stability studies were carried out by storing electrode at 4 °C for 90 days. The electrode was immersed in 0.1 M PBS (pH = 5.0) when it was not in use. The electrode performance was checked daily using DPV [20
2.4. Determination of Tartrazine in Food Samples
The samples were purchased from a local market. All simultaneous determinations of each sample were performed by standard addition method and have been repeated 3 times under the same conditions to examine and to improve the results’ accuracy. For electrochemical analysis, 20.0 mL commercial mango juice or 20.0 g candy coated chocolate was taken and dissolved in 20 mL 0.1 M PBS (pH 5.0). The synthetic dye content in commercial mango juice was determined by the standard addition method to prevent any matrix influence.
2.5. Validation and Recovery Studies of Tartrazine in Food Samples
Samples were also validated and analysed by high performance liquid chromatography (HPLC). HPLC system consisted of a binary pump, a degasser, an automated injector, a column oven and an UV–vis detector (Agilent 1100 Series HPLC, Agilent Technologies, Massachusetts, USA). HPLC was also introduced to detect tartrazine in drinks according to Alves et al. [22
]. The column was a C18 analytical column (4.6 mm × 250 mm × 5 μm). One mobile phase system was employed to accomplish a quick separation of the analysed dyes in samples. It contained methanol (solution A) and 0.08 mol·L−1
aqueous ammonium acetate (solution B). Prior to usage, the aqueous solution and methanol were further filtered through 0.45 μm membranes. A constant flow rate was 0.7 mL·min−1
and the injection column was 20 mL. After completing the chromatographic elution, the mobile phase was programmed to its initial condition within 5 min, while 10 min reconditioning time was set before next injection. The detection was performed at a wavelength of 417 nm for both sunset yellow and tartrazine with UV-vis spectra detector. Statistical analysis (Student t
-test) was performed by Microsoft Excel datasheets for comparison of both techniques.