ANALELE ŞTIINŢIFICE ALE UNIVERSITĂŢII “AL. I. CUZA” IAŞI Tomul IV, s. Biofizică, Fizică medicală şi Fizica mediului 2008 LACCASE IMMOBILISED ON HYDROTALCITES AS A 3rd GENERATION BIOSENSOR TYPE Alina Manole1, D. Herea2, H. Chiriac2, V. Melnig1 KEYWORDS: Laccase is one of the green enzymes that can directly reduce oxygen into water under ambient conditions, while catalyze the transformation of a large number of phenolic and non-phenolic aromatic compounds. The system laccase intercalated in CoLDH clay with chitosan as enzyme linker seems to be appropriate as direct electron transducer to Pt working electrode. The experimental conditions to obtain current signal in terms of reproducibility and sensitivity have been established as biosensor activity function of pH, temperature and hydroquinone substrate concentration and specific working potential. 1. INTRODUCTION Laccases are cuproteins included in a small group of enzymes called blue copper proteins or blue copper oxidases along with ascorbate oxidase and ceruloplasmin. For the over 60 types of laccases isolated from different sources like insects, plants, fungus and bacteria, differences in thermodynamic and kinetic properties were observed, depending on the source, although the copper centre is similar for all laccases. In general, laccases have, distributed among different binding sites, 4 copper atoms classified in 3 types: types 1 and 2 are involved in electron capture and transfer, while copper types 2 and 3 are involved in binding with oxygen. The direct electron transfer in enzymes was first described for a laccase . Laccases (E.C. 18.104.22.168, benzenediol:oxygen oxidoreductase) are phenol oxidases that catalyses the oxidation of some inorganic and organic compounds (especially phenols) with the concomitant electroreduction of oxygen to water: O 2 4e 4H 2H 2 O Thus, a molecular laccase layer attached at the surface of an electrode forms an oxygen transducer . The electron transfer and reduction of 2 oxygen atoms to water mechanism are not yet fully understand in the case of laccases. In a typical reaction catalysed by laccase, a phenolic substrate is subjected to a one-electron oxidation giving rise to an aryloxyradical. This active species can be converted to a quinone in the second stage of the oxidation. The quinine, as well as the free radical product undergoes non-enzymatic coupling reactions leading to 1 2 “Al.I. Cuza” University, Faculty of Physics, Carol I Blvd., No.11, 700506, Iasi National Institute of Research and Development for Technical Physics 47 Mangeron Blvd., 700050, Iasi 12 Alina Manole, D. Herea, H. Chiriac, V. Melnig polymerization. Simple diphenols such as hydroquinone and catechols are good substrate for the majority of laccases . Fungal laccases, in free form or immobilized, but also in organic solvents, were used in different biotechnological and environment applications like biobleaching, waste water treatment, biosensors, biofuel cells as cathodic biocatalyser, delignification and demethylation. Laccase biosensors are an alternative to phenolic compounds determination besides using peroxidases, for which the background current produced by H2O2 is limiting the biosensor sensitivity . 2. MATERIALS AND METHODS Laccase (EC 22.214.171.124, benzenediol:oxygen oxidoreductase, from trametes versicolor), hydroquinone and chitosan were purchased from Sigma Aldrich. They were used as received, without further purification. The hydrotalcites (CoLDHs) were prepared as follows: an aqueous solution (85 ml) of Mg(NO3)2·6H2O ((0.03-q) mol)/Al(NO3)3·9H2O (0.01mol)/ Co(NO3)2·3H2O (q mol, 0.005 q 0.01) and an aqueous solution of Na2CO3 (1M, 30 ml) were added dropwise together over a period of 2 h at a constant pH value of 8.2. The resulting precipitate was aged at 305 K for 24 h under stirring. The samples were calcined under air at 723 K; the temperature was raised with 8 K·min-1 to 723 K, maintained there for 5 hours and then cooled slowly under nitrogen. All other chemicals are of analytical grade. 0.1M phosphate buffer (PBS) consisted of Na2HPO4 and NaH2PO4 and 0.1M Britton buffer solutions, obtained by mixing the same amount of 0.1M acetic acid, boric acid and phosphoric acid were employed as supporting electrolyte. The desired Britton buffer solutions pH was adjusted by different amounts of NaOH solution. All solutions were made using deionised water. Pt electrode was washed ultrasonically in water and ethanol for a few minutes, respectively. A 1% chitosan solution was prepared by dissolving chitosan in 2% acetic acid in Britton buffer with pH 5 with magnetic stirring for about 2 h. For preparation of laccase/CoLDHs –chitosan/Pt electrode, 3mL of 1% chitosan solution was mixed with 4mg laccase and 8 mg CoLDHs. The laccase is firstly dissolved in 0.5 ml deionized water. Then 10 μL of the laccase/ CoLDHs –chitosan mixture was spread onto the Pt electrode surface with a pipette. Finally, the modified electrode was allowed to dry over night at 50◦C. Chitosan was used for enzyme immobilization because it is an inert, biocompatible and biodegradable amphiphilic polymer  and a special feature of amphiphilic polymers is the formation of association structures in aqueous solution. The unique structural feature of chitosan is the high content of primary amines, and these amines confer important functional properties to chitosan that can be exploited. At low pH, chitosan is a water-soluble cationic polyelectrolyte due to these protonated amines. At high pH, chitosan’s amines become deprotonated and the polymer loses its charge and becomes insoluble. In this case, chitosan’s electrostatic repulsions are reduced allowing the formation of inter-polymer associations (liquid crystalline domains or network junctions). As a result, fibers, films, or hydrogels can be obtained, depending on the conditions used to initiate the soluble-insoluble transition. The soluble-insoluble transition occurs at pH between 6 and 6.5, which is a particularly LACCASE IMMOBILISED ON HYDROTALCITES… 13 convenient range for biological applications . In this case the entrapping of enzyme into the composite film is done without using a cross-linking reagent. The FT-IR spectra were recorded with a 6100 JASCO spectrometer with 4 cm-1 resolution in the range 4000-400 cm-1. Cyclic voltammetry (CV) and chronoamperometry measurements were carried out on a VoltaLab 10 (PGZ 100) potentiometer controled by VoltaMaster 4 software. Hydroquinone solutions in phosphate buffer pH 7 were used as electrochemical probes. A conventional three-electrode setup was used. The modified electrode was applied as the working electrode. Two identical 3 mm diameter platinum electrodes were used as working (WE) and counter (CE) electrodes and Ag/AgCl electrode as reference electrode (REF), respectively. All the voltammograms were performed at 20mV/s scan rate. The chronoamperometry measurements were performed at constant potential (360 mV). Before each electrochemical measurement, the Pt electrodes system was immersed in HNO3 50% v/v solution for 1 min to remove the deposits on the surface electrodes and then it was washed with distillated water and dried thus fresh and clean surfaces could be obtained. All electrochemical measurements were performed at room temperature. For testing the answer of the modified electrode at pH variation the hydroquinone solutions were prepared in Britton buffer. 3. RESULTS AND DISSCUTIONS 3.1 FT-IR spectra The FT-IR spectra show noticeable differences both in the functional group region, and fingerprint region. Inspection of the functional group region and correlation chart reveals characteristic bands of N – H and C – N groups of peptide bond, C = O and N – H bands from amide and O – H from hydroxyl. 895 CH Transmittance (%) 80 2878 CH 60 40 80 2930 Laccase 2885 M 2928 M 3438 LDH 20 3400 3200 Wavenumber (cm-1) 3000 2800 1263 CH 40 0 3600 1262 CH 1156 CH 895 CH 1639 CH 1649 Laccase 1642 CH+Laccase 1800 1600 792 1088 LDH CH 786 804 Laccase Laccase LDH 1156 CH 1089 CH 1400 1200 LDH 555 LDH 667 809 Laccase LDH 60 20 0 3800 Chitosan (CH) Laccase LDH Chitosan, Laccase, LDH (M) 100 Transmittance (%) 100 Chitosan (CH) Laccase LDH Chitosan, Laccase, LDH (M) 657 LDH M 1000 560 LDH 800 600 450 LDH 454 LDH 400 Wavenumber (cm-1) (a) (b) Fig. 1 FTIR spectra a) functional group region and b) fingerprint region, for 1% chitosan solution prepared in 2% acetic acid in britton buffer pH 5, laccase, LDH and 1% chitosan solution mixed with 4mg laccase and 8 mg LDH. 14 Alina Manole, D. Herea, H. Chiriac, V. Melnig Strong water absorption bands are within 3900 – 2800 cm-1 and 1750 – 1550 cm-1, representing the stretching and deformation vibration of O – H group in water, respectively (the fundamental stretching vibrations mode of water occur within the 3900–2800 cm-1 region and the inplane bending mode at around 1640 cm-1. Even tough in the fingerprint region the spectrum is more complex, the general idea is that the result is a physical mixture of laccase, LDH and chitosan in solution. 3.2 Electrochemistry of redox-active system A redox-active system can be characterized by electrochemical measurements. A typical cyclic voltammogram is shown in Figure 2a for the case of an unmodified working electrode immersed in a non-stirred solution containing different hydroquinone concentrations as the electroactive species in phosphate buffer pH 7. The potential axis is versus an Ag/AgCl REF electrode. The switching potential has been set at-300 mV to +500 mV vs. REF etectrode and was selected to be positioned on either side of the main redox peaks. The reduction shows a cathodic peak at +360 mV and an anodic peak at -70 mV resulting from re-oxidation of the reduction product at reverse potential scan, booth dependent of the hydroquinone concentration. (a) (b) Fig. 2 Cyclic voltammograms for hydroquinone solutions with different concentrations using unmodified Pt electrode (a) and cyclic voltammograms for 0.8 mM hydroquinone solutions using unmodified and chitosan modified Pt electrode, respectively (b). The progress of assembling system can be monitored by addition of mixture components on the WE electrode. In Fig. 2b the influence of the chitosan on the double layer of the WE are shown; redox process is increased while the oxidation is not. Differences in the redox processes seen do not appear at normal pH by the presence of laccase and anionic clay in the system (Fig. 3a and 3b). The chronoamperometry measurements demonstrate that in the presence of anionic clay the current densities increase (Fig. 4). Major changes are recorded in redox peaks positions (Fig. 5) and current densities response (Fig. 6) when the experiments are performed as functions of pH and LACCASE IMMOBILISED ON HYDROTALCITES… 15 temperature bath solutions. The optimum working parameter conditions for laccase immobilized CoLDH for hydroquinone biosensor seem to be 70˚ C degree and pH 4. (a) (b) Fig. 3 Cyclic voltammograms for hydroquinone solutions with different concentrations using chitosan and laccase modified Pt electrode (a) and cyclic voltammograms for hydroquinone solutions with different concentrations using chitosan, laccase and anionic clay modified Pt electrode. pH=4 pH=5 pH=6 pH=7 0,5 Current density (mA/cm2) Current density (mA/cm2) Fig. 4 The current answer of the chitosan and laccase and chitosan, laccase and anionic clay modified Pt electrode, respectively, with hydroquinone concentration variation. 0,0 0,0 -0,5 -0,5 -400 pH=4 pH=5 pH=6 pH=7 0,5 -200 0 200 400 Potential (mV) (a) 600 800 -400 -200 0 200 400 Potential (mV) (b) 600 800 16 Alina Manole, D. Herea, H. Chiriac, V. Melnig Fig. 5 Cyclic voltammograms for 0.8 mM hydroquinone solutions at different pH in Britton Buffer solutions using chitosan and laccase modified Pt electrode (a) and chitosan, laccase and anionic clay modified Pt electrode. 0,14 Current density (mA/cm2) 0,12 0,10 0,08 0,06 0,04 0,30 Pt electrode modified with chitosan and laccase Pt electrode modified with chitosan, laccase and anionic clay 0,28 Current density (mA/cm2) Pt electrode modified with chitosan and laccase Pt electrode modified with chitosan, laccase and anionic clay 0,26 0,24 0,22 0,20 0,18 0,16 0,14 0,12 0,02 4 5 6 7 0,10 20 30 pH 40 50 60 70 80 90 Temperature (0C) (a) (b) Fig. 6 Answer of the chitosan and laccase and chitosan, laccase and anionic clay modified Pt electrode, respectively, with pH variation (a) and with temperature variation (b). 4. CONCLUSIONS Free and immobilized laccase/CoLDHs–chitosan/Pt electrode was studied for biosensor activity function of pH, temperature and hydroquinone substrate concentration and specific working potential. The system laccase intercalated in CoLDH clay with chitosan as enzyme linker increase the response in current of the biosensor. The optimal working conditions for this being 70˚ C degree and pH 4. Acknowledgement This research was supported by CEEX 1639/2006 (BIOSNANOMAG) research program. REFERENCES 1. 2. 3. 4. 5. Renato S. Freire, Christiana A. Pessoa, Lucilene D. Mello, Lauro T. Kubota, Direct Electron Transfer: An Approach for Electrochemical Biosensors with Higher Selectivity and Sensitivity, J. Braz. Chem. Soc., Vol. 14, No. 2, p. 230-243, 2003. A. Ghindilis, Direct electron transfer catalysed by enzymes: application for biosensor development, Biochemical Society Transactions, Vol. 28, part 2, p. 84-89, 2000. Nelson Durán, Maria A. 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