A novel supporting electrolyte – tris(hydroxymethyl)aminomethane hydrochloride (TRIS) buffer solution was used to

obtain polypyrrole (PPy) layers based on the anodic oxidation of pyrrole. Influence of some parameters: temperature,

monomer and supporting electrolyte concentration, polarization potential and hydrodynamic conditions on electrodeposition process were investigated. Obtained layers were preliminary tested in construction of an amperometric biosensor

based on enzyme tyrosinase, which response was tested using catechol as a substrate

Adamski, Juliusz

Kochana, Jolanta

Acta Chimica Slovenica

English

Jagiellonian Univeristy Repository

623Acta Chim. Slov. 2008, 55, 623–626Adamski and Kochana:   Polypyrrole Layers Electrodeposited from TRIS Buffer  ...Short communicationPolypyrrole Layers Electrodeposited from TRIS Buffer Solution as Matrix of Tyrosinase BiosensorJuliusz Adamski* and Jolanta KochanaDepartment of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University, 30-060 Cracow, Ingardena 3, Poland* Corresponding author: E-mail: adamskij@chemia.uj.edu.plReceived: 04-02-2008AbstractA novel supporting electrolyte – tris(hydroxymethyl)aminomethane hydrochloride (TRIS) buffer solution was used toobtain polypyrrole (PPy) layers based on the anodic oxidation of pyrrole. Influence of some parameters: temperature,monomer and supporting electrolyte concentration, polarization potential and hydrodynamic conditions on electrodepo-sition process were investigated. Obtained layers were preliminary tested in construction of an amperometric biosensorbased on enzyme tyrosinase, which response was tested using catechol as a substrate.Keywords: Biosensor, tyrosinase, polypyrrole, electropolymerization, catechol determination1. IntroductionAccording to the IUPAC recommendation biosensoris defined as an integrated receptor-transducer device,which is capable of providing selective quantitative or se-mi-quantitative analytical information using a biologicalrecognition element.1 Immobilization of bioreceptor ontomatrix plays an important role for biosensor applicationand stability. Recently, the immobilization of biocatalysisin electropolymerized conducting polymers has madeconsiderable progress.2 Conducting polymers can be defi-ned as polymers with long-range conjugation. Polypyrrole(PPy) seems to be potentially very attractive for the bioe-lement immobilization, it is biologically compatible andcan be easily deposited onto electrodes of different shapeand size at room temperature from the aqueous solutions.The electrochemical approach for preparing PPy layers isvery versatile and provides facile vary the film propertiesby a simple modification of the electrolysis conditions.In this work application of TRIS buffer solution asthe supporting electrolyte for electrodeposition of PPy la-yers is proposed. Influence of some critical parameters onelectrodeposition process was investigated. Obtained un-der the optimized conditions PPy layers were preliminarytested as matrix for immobilization of tyrosinase in ampe-rometric biosensor for determination of phenolic com-pounds. Tyrosinase catalyses the oxidation of monophe-nols and o-diphenols to corresponding o-quinones. Qui-nones can be electrochemically reduced to allow low po-tential detection of phenolic compounds.32. Experimental2. 1. Chemicals and ReagentsMushroom tyrosinase (5370 U/mg) and pyrrole(98%), were purchased from Sigma (USA); buffer TRIS –tris(hydroxymethyl)aminomethane hydrochloride- wasfrom Fluka (Switzerland); ammonium chloride (Suprapur)was bought from Merck (Germany); potassium chloride,disodium tetraborate and catechol (all analytical grade) we-re from POCh (Poland). Pyrrole was cleaned prior to use bypassing through a neutral Al2O3-column to remove any co-lored components. 0.3 µm alumina (Buehler Micropolish,USA) was used for polishing surface of working electrode.Solutions were prepared using double-distilled water and, ifnecessary, deareated using high purity nitrogen (99.996%).2. 2. ApparatusEMU (WUT, Poland) was employed for electroche-mical measurements. Potential of working electrode, WE,(vs. reference electrode, RE) was additionally controlledusing Digital Multimeter V553 (Meratronik, Poland). Athree electrode system was used consisted of platinum orglassy carbon disc WE, saturated silver/silver chloride RE(Mtm-anko, Poland) and platinum auxiliary electrode624 Acta Chim. Slov. 2008, 55, 623–626Adamski and Kochana:   Polypyrrole Layers Electrodeposited from TRIS Buffer  ...(AE). Before each experiment the WE was polished ontoglass plate using alumina suspension, rinsed with doubledistilled water and methanol.2. 3. Electropolymerization of the MonomerAnodic oxidative polymerization of pyrrole was car-ried out in mixtures of monomer and TRIS that weredeoxygenated using high purity nitrogen. Polymer layerswere deposited using two techniques: potentiostatic or po-tentiodynamic. In potentiostatic experiments, two stepswere applied: 300 mV for 120 s and 800 mV for 400 s.The polymerization process occurred only in the secondstep. However, it was noticed (based on charge used) thatthe employment of the first step improved the repeatabi-lity of electrodeposition process. During this step, impuri-ties presented on electrode surface or in buffer solutionwere probably oxidized. In potentiodynamic method,cyclic voltammetry technique was used. The WE potentialwas changed between 0 and 1000 mV.2. 4. Construction of the Tyrosinase BiosensorImmobilization of tyrosinase was performed by aphysical adsorption of enzyme onto electropolymerizedpolypyrrole layer (Electrode I) and by an incorporation ofenzyme into growing polymer layer (Electrode II). Forphysical adsorption 10 µL of enzyme solution (6.7 mgml–1) was dipped onto a polymer-modified electrode for-med from unstirred 0.1 mol L–1 solution of Py using thepotentiostatic technique. In order to entrap tyrosinase intogrowing polypyrrole layers the cyclic voltammetry or apulse technique were employed. For the pulse method theWE potential was maintained for 10 s at 300 mV and thenchanged to 800 mV for 1 s. These changes were repeated20 times. In both cases of potentiodynamic experiments,electrolyzed solutions were 0.1 mol L–1 pyrrole in 0.05mol L–1 TRIS to which 50 µL of the enzyme solution (thesame as for Electrode I) was added.2. 5. Evaluation of Biosensor ResponseChronoamperometric curves (WE potential set at 50mV) were recorded for different catechol concentrationusing 0.1 mol L–1 phosphate buffer solution (pH = 6) asthe supporting electrolyte.3. Results and Discussion3. 1. Electrodeposition Process3. 1. 1. Effect of Monomer ConcentrationMonomer concentration influence was examinatedby potentiodynamic technique using Pt electrode. Foreach monomer concentration (0.01, 0.1, 0.2, 0.3 and 0.4mol L–1), ten successive voltammograms were recordedwith scan rate 0.5 V s–1. It was noticed that for the fixedWE potential, current corresponding to the pyrrole elec-trooxidation was linearly proportional to the monomerconcentration (Fig. 1).Fig 1:  Effect of monomer concentration on electrodeposition pro-cess. Voltammograms (first cycle, 0.5 V s–1) obtained on Pt electro-de in 0.05 mol L–1 TRIS. Inset: relationship between current (for1000 mV) and Py concentration.This linear dependence suggested that at employedscan rate the oxidation process was diffusion-limited. Forthe lower scan rates the dependence current vs. squareroot of scan rate was non-linear. Moreover, after each cy-cle the increase of current corresponding to the oxidationof previously reduced polymer layer as well as the increa-se of peak current for oxidation of the monomer were ob-served (data not shown). This proves that in each cycle anadditional amount of polymer was deposited onto theelectrode.3. 1. 2. Effect of the Supporting Electrolyte ConcentrationFormation of the polypyrrole layer is associatedwith incorporation of supporting electrolyte anions (A–)into growing positive charged polymer. In order to studythe influence of electrolyte concentration on electropoly-merization, potentiostatic electrodeposition of polymeronto Pt electrode was performed. Pyrrole concentrationwas set at 0.1 mol L–1 and TRIS concentration was chan-ged from 0.001 mol L–1 to 0.1 mol L–1. Because the enzy-me’s denaturation probability rose with electrolyte con-centration, 0.1 mol L–1 was chosen as its highest value.Good linear relationship between the consumed charge(obtained after integration of area under chronoampero-metric curves) and the supporting electrolyte concentra-tion was obtained (Q = 1002.2 c + 11.0; R2 = 0.9927 for Qin mC and c in mol L–1). Since the WE potential for eachTRIS concentration was the same (ohmic drop in elec-trolyte was compensated) it could be concluded that the625Acta Chim. Slov. 2008, 55, 623–626Adamski and Kochana:   Polypyrrole Layers Electrodeposited from TRIS Buffer  ...incorporation of anions into growing layer was easier insolution with higher amount of anions and the whole pro-cess ran faster.3. 1. 3. Effect of the Polarization PotentialPotentiostatic technique (see 2.3) was used to studyinfluence of the WE potential on electrodeposition pro-cess. Results of experiments (charge vs. WE potential) arepresented in Fig. 2. It is seen that increasing the potentialof WE caused the rise of the electrodeposition rate, butthis dependence is non-linear. This phenomenon could beexplained as follows: the higher the WE potential was, thehigher was the anodic reaction rate constant, but in the sa-me time the higher were the rate of overoxidation processand probability of nucleophilic attack of water moleculesand anions on formed radical cations.4 These side reac-tions disturb the main process of the electrodeposition ofpolymer layers. It can be observed (data not shown) thatfor the lower potentials of WE (ex. 800 mV), where theoveroxidation process seems to be negligible, there exi-sted linear dependence between the consumed charge andthe polarization time.Fig. 2: Charge used during electrodeposition of PPy layer from 0.1mol L–1 Py in 0.05 mol L–1 TRIS as a function of platinum WE po-tential. Polarization time was 400 s.3. 1. 4. Effect of the TemperatureThe effect of temperature on electrodeposition pro-cess was evaluated using potentiodynamic electrodeposi-tion technique. The relationship of current vs. temperature(Fig. 3) exhibits increase of the rate of the electrooxida-tion process when temperature was enlarged. This couldbe explained by rise of two factors: monomer diffusioncoefficient and increasing reaction rate constants.3. 1. 5. Effect of StirringAccording to the mechanism described by Diaz atal.5 pyrrole oxidative deposition begins by electron trans-fer (E) (with formation of radical cation) followed by asuccession of chemical reaction (C) and electron transferreactions, which can be described as E(CE)n. Because theoverall process takes place near the electrode surface, stir-ring of solution has great influence on it. This effect isshown in Fig. 4, where the amperometric curves obtainedfor different stirring rate were presented. It was found thatelectropolymerization process was possible only in non-stirred solutions or in solution stirred with low rate. Thisfact could be explained by formation of very reactive radi-cal cations. Intensive stirring could reduce their concen-tration near the electrode surface and decrease the chanceof coupling reaction between two radicals. In non-stirredsolution, the high radical concentration near the electrodecould be maintained by the continual diffusion of mono-mer towards the electrode.3. 2. Biosensor Response3. 2. 1. Electrode I (Physical Adsorption of Enzyme)A typical amperometric electrode response is pre-sented in Fig. 5. It was found that successive additions ofcatechol solution caused increase of current correspon-ding to quinone reduction.Estimated sensitivity of the proposed biosensor forcatechol was equal to 35 nA µmol–1 L–1 and the responsetime (t95%) was about 20 s. The obtained sensitivity appea-red higher then is offered by other biosensors: constructedby covalent tyrosinase immobilization onto copolymerpoly(N-3-aminopropyl pyrrole-co-pyrrole) film (3.46 nAµmol–1 L–1),6 based on 1,6-heksanedithiol and nano-Auself-assembled monolayers (3.94 nA µmol–1 L–1),7 and ba-sed on Os-complex functionalized polymer (6.10 nAµmol–1 L–1).8The constructed biosensor exhibited poor stability.Most of biomolecules were lost from the electrode surfaceFig. 3: Effect of temperature on electrodeposition process. Voltam-mograms for first cycle obtained in solution contains 0.1 mol L–1Py and 0.05 mol L–1 TRIS; scan rate 0.5 V s–1. Inset: relationshipbetween current (at 1000 mV) and temperature.626 Acta Chim. Slov. 2008, 55, 623–626Adamski and Kochana:   Polypyrrole Layers Electrodeposited from TRIS Buffer  ...after few measurements. It was detected as decrease of theelectrode response and color changes of tested solutions(from colorless to deep brown) caused by the enzymaticreaction products.3. 2. 2. Electrode II (the enzyme entrapped in polypyrrole layer)In this case entrapment of the enzyme in polypyrro-le layer was performed but obtained electrode gave no sig-nificant response in the catechol solutions of differentconcentrations irrespective of method of polymer deposi-tion: pulse or cyclic voltammetry technique. This sugge-sted that either the enzyme was not incorporated onto gro-wing layer due to their high hydrophobicity or the enzymewas incorporated in denaturated form. It was also pro-bably that analyte (catechol) transport to enzyme active si-te was disturbed by the structure of polymer film.4. ConclusionsElectrochemical deposition of the PPy layers fromTRIS buffer solution was performed. The influence of so-me critical parameters (temperature, monomer and sup-porting electrolyte concentration, polarization potentialand hydrodynamic conditions) on electrodeposition pro-cess was investigated. It was found that mechanicallystable polymer layers could be obtained on electrochemi-cal way using this supporting electrolyte. Based on promi-sing results of preliminary experiments, especially the va-lue of biosensor sensitivity, polypyrrole layers electrode-posited from TRIS could be employed for construction oftyrosinase biosensor. Further research will be focused onan improvement of biosensor performance.5. References1. D. R. Thévenot, K. Toth, R. A. Durst, G. S. Wilson, Pure Appl.Chem. 1999, 71, 2333–23482. S. Cosnier, Biosens. Bioelectron. 1999, 14, 443–4563. L. G. Fenoll, J. N. Rodriguez-Lopez, F. Garcia-Sevilla, J. Tu-dela, P. A. Garcia-Ruiz, R. Varon, F. Garcia-Canovas, Eur. J.Biochem. 2000, 267, 5865–56784. S. Ghost, G. A. Bowmaker, R. P. Cooney, J. M. Seakins,Synth. Met. 1998, 95, 63–675. B. L. Funt, A. F. Diaz, Organic Electrochemistry: an Introduc-tion and a Guide, Marcel Dekker, New York, 19916. Rajesh, W. Takashima, K. Kaneto, Sens Actuators B 2004,102, 271–2777. Z. Liu, J. Liu, G. Shen, R. Yu, Electronalysis 2006, 16,1572–15778. H. B. Yildiz. J. Castillo, D. A. Gushin. L. Toppare, W. Schu-mann, Micrchim. Acta 2007, 159, 27–34Fig. 4: Influence of stirring on electrodeposition process. Chro-noamperograms at the Pt working electrode recorded for 0.1 molL–1 pyrrole solution in 0.05 mol L–1 TRIS; polarization potential:800 mV.Fig. 5: Electrode I response for catechol in stirred 0.1 mol L–1phosphate buffer solution (pH = 6); polarization potential: 50 mV.PovzetekDa bi z anodno oksidacijo pirola iz raztopine dobili polipirol (PPy) smo uporabili elektrolit tris(hidroksime-til)aminometan hidroklorid (TRIS). Preu~ili smo vpliv temperature, koncentracije monomera in elektrolita, polarizaci-jskega potenciala in hidrodinamskih pogojev na proces elektrodepozicije. Dobljene sloje PPy smo preizkusili priizdelavi amperometri~nega biosenzorja temelje~ega na encimu tirozinazi. Odziv dobljenega senzorja smo preizkusili zuporabo katehola kot substrata.

Polypyrrole layers electrodeposited from TRIS buffer solution as matrix of tyrosinase biosensor

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Polypyrrole layers electrodeposited from TRIS buffer solution as matrix of tyrosinase biosensor

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