Pii: s0013-4686(02)00777-6

Electrochimica Acta 48 (2003) 855 /865 Tris (2,2?-bipyridil) copper (II) chloride complex: a biomimetic tyrosinase catalyst in the amperometric sensor construction Maria Del Pilar Taboada Sotomayor a, Auro Atsushi Tanaka b, Lauro Tatsuo Kubota a, a Instituto de Quı´mica, UNICAMP, P.O. Box 6154, 13083-970 Campinas, SP, Brazil b Departamento de Quı´mica, UFM, 65070-270 Sa˜o Luı´s, MA, Brazil Received 25 September 2002; received in revised form 21 November 2002 The use of tris (2,2?-bipyridil) copper (II) chloride complex, [Cu(bipy)3]Cl2×/6H2O, as a biomimetic catalyst, is reported in the construction of an amperometric sensor for dopamine. The sensor was prepared modifying a glassy carbon electrode with aNafion† membrane doped with the complex. The optimized conditions for the sensor response were obtained in 0.25 mol dm3Pipes buffer (pH 7.0) containing 150 mmol dm3 H2O2, with an applied potential of /50 mV versus saturated calomel electrode(SCE). In these conditions, a linear response range between 9 and 230 mmol dm3, with a sensitivity of 1.439/0.01 nA dm3 mmol1cm2 and a detection limit of 4.8 mmol dm3 were observed for dopamine. The response time for this sensor was about 1 s,presenting the same response for at least 150 successive measurements, with a good repeatability (4.8%) expressed as relativestandard deviation for n /13. After its construction, this sensor can be used after 180 days without loss of sensitivity, kept at roomtemperature. The difference of the sensor response between four preparations was 4.2%. A detailed investigation about the sensorresponse for other eighteen phenolic compounds and five interfering species was performed. The sensor was applied for dopaminedetermination in pharmaceutical preparation with success.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Copper complexes; Biomimetic catalyst; Tyrosinase; Catecholamines; Amperometric sensors trocatalytic amplification cycle . When the quinoneis reduced quantitatively and reversibly on an electrode In the last years many biosensors for phenols detec- surface, the catalytic recycling gives a significant in- tion have been described in the literature. Most of them crease in the reduction current that is proportional to using extracted enzymes from natural sources, purified the concentration of the phenolic substrate . How- or lyophilized . The principle of bioelectrocataly- ever, still exist a necessity to improve the performance of tic conversion of phenols to quinone by the Tyr these biosensors, principally due to the short lifetime, immobilised in these biosensors is well known .
poor stability and relatively high detection limits. These The phenolic substrate diffuses through the sensitive problems suggest that the electrochemical reduction step layer, where it is enzymatically oxidized to quinone must be improved. An alternative for this is the species by tyrosinase under the O2 consumption. The improvement of the catalytic efficiency in the sensitive substrate is regenerated through the electrochemical layer. In this sense, Hedenmo et al. demonstrated reduction of these quinones, which is the critical step that immobilized an osmium complex (mediator) and in the response of these biosensor based on tyrosinase Tyr together, improved the catalytic performance as a (Tyr). This regeneration leads to a significant amplifica- consequence of a more efficient mediated electroreduc- tion of quinone species production, forming a bioelec- tion, recycling the produced quinones enzymatically.
Another alternative to improve the performance of thesebiosensors for phenols detection is making the electron * Corresponding author. Tel.: /55-19-3788-3127; fax: /55-19- transfer easier between the active site of the enzyme and E-mail address: (L.T. Kubota).
the electrode surface, reducing or removing the protein 0013-4686/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 1 3 - 4 6 8 6 ( 0 2 ) 0 0 7 7 7 - 6 M.D.P.T. Sotomayor et al. / Electrochimica Acta 48 (2003) 855 /865 (protective shell) around the active site. In this sense, the 4-Aminophenol, 4-nitrophenol and p -phenylenediamine use of biomimetic chemistry synthesizing artificially dihydrochloride were obtained from Riedel, De Hae¨n, active sites of Tyr , trying to imitate the natural Germany. Tetrahydrofuran and methanol were acquired enzyme with the same efficiency and selectivity, is very from J.T. Baker, Xalostoc, Mexico.
attractive. Based on this context, the term ‘enzymelessbiosensors' can be used For the other hand, the 2.2. Preparation of tris(2,2?-bipyridil) copper(II) use of biomimetic catalysts will be able to infer more robustness to the sensors, overcoming the low stabilitypresented by the enzyme.
The complex was synthesized according to the proce- Various biomimetic catalysts of tyrosinase have been dure described by Meyer , with some modifications.
described in literature and demonstrating For 25.0 cm3 of a 0.04 mol dm3 CuCl2×/2H2O aqueous efficient catalyst to oxidize phenolic and catecholic solution (1 mmol), was used 25.0 cm3 of a 0.12 mol substrates to quinones. The redox behavior of all these dm3 2,2?-bipyridine ethanol solution (3 mmol), and compounds is very interesting, however, they have rarely then they were slowly mixed. The solvent in the resulting been studied in detail, mainly for sensor development.
blue solution was removed under vacuum, until to With the aim to develop amperometric sensors based on obtain a volume of 20 cm3. Then, this solution was biomimetic catalyst of Tyr, an approximation of its stand at 4 8C, in order to allow slow evaporation of the active site can be made using molecules containing remaining solvent, such as recommended by Palmer and copper atoms surrounded by nitrogen. This approach Piper After about 30 days, blue color crystals were mimics the Tyr deoxy form , that is able to obtained, corresponding to the tris(2,2?-bipyridil) cop- catalyze the catecholamines oxidation, in the same form per(II) chloride complex, with formulae [Cu(bipy)3]Cl2×/ that the Tyr. On this context, this work investigates the 6H2O, which was confirmed through elementary analy- use of tris(2,2?-bipyridil) copper(II) chloride complex as sis (C, H, N). The obtained data were: 49.0% C; 4.8% H a biomimetic catalyst of Tyr, in the construction of an and 11.3% N versus 49.4% C; 4.9% H and 11.5% N, for amperometric sensor for dopamine determination.
theoretical ones. The Cu amount determined by atomicabsorption spectrometry was 9.1 against 8.7% fortheoretical one.
2.3. Sensor construction In a first step, solutions containing 1, 2, 3 and 5 g All chemicals were analytical grade reagent. Buffer dm3 of the complex in DMF were prepared, in order and phenol solutions were prepared using de-ionized to test the influence of the amount of copper (II) water (Milli-Q Millipore system), and the actual pH of complex on the sensor construction.
the buffers was determined employing a pH electrode A glassy carbon (GC) electrode, acquired from connected to a pH-meter (Corning pH/Ion analyzer 350, Metrohm†, Switzerland with a geometrical area of New York, USA).
0.071 cm2, was used for the sensor construction.
Phenol, 30% (w/v) hydrogen peroxide, monopotas- Initially, the surface of the GC electrode was cleaned sium phosphate (KH2PO4), sodium hydroxide, sodium according to the procedure described in the literature chloride, sodium bisulfite and N,N -dimethyformamide (DMF) were acquired from Synth, Sa˜o Paulo, Brazil.
After cleaning the GC electrode, 200 mm3 of copper Catechol, uric acid, 3-nitrophenol, epinephrine, (9/) complex solution was mixed with 100 mm3 of 5% (w/v) norepinephrine L-bitartrate hydrate, tetrachloro-1,2- Nafion† solution, and an aliquot of 150 mm3 of this benzoquinone, guaiacol and 5% (w/v) Nafion† solution mixture was put on the GC electrode surface. Finally the were purchased from Aldrich, Milwaukee, USA. Ascor- solvent was evaporated at room temperature during 4 h, bic acid, o -phenylenediamine, 2-aminophenol, 3-amino- forming a film of 23 mm thickness on the electrode phenol, 2-nitrophenol and sodium salicylate were from surface, estimated as described by Langmaier et al. Merck, Darmstadt-Germany. 3,4-Dihydroxyphenethy-lamine (dopamine), 5-hydroxytryptamine (serotonin), 2.4. Electrochemical measurements N? -bis[2-ethanesulfonic acid] (Pipes) and N -[2-hydro- The voltammetric and amperometric measurements xyethyl]piperazine-N ?-[2-ethanesulfonic acid] (Hepes) were carried out in a potentiostat from Echo Chemie were acquired from Sigma, St. Louis, USA. Copper (Autolab PGSTAT10 model), Utrecht, Netherlands and (II) chloride dihydrate was acquired from Vetec, Rio de using an electrochemical cell with three electrodes. A Janeiro, Brazil. Acetaminophen and 2,2?-bipyridyl saturated calomel electrode (SCE) was used as reference, (bipy) were purchased from Acros, New Jersey, USA.
a Pt wire as counter and the GC electrode, modified M.D.P.T. Sotomayor et al. / Electrochimica Acta 48 (2003) 855 /865 with Nafion† membrane doped with copper (II) com-plex (sensor), as working electrode.
Before the electrochemical measurements the elec- trode was kept in buffer solution for 20 min, formembrane hydration. The measurements were carriedout in 5.00 cm3 of buffer solution, applying an adequatepotential. Initially the current was continuously mon-itored until it reaches the steady state (from 3 to 5 min).
After that, an addition of hydrogen peroxide solutionwas made into the buffer solution, which it was stirredfor a few seconds (30 s), in order to homogenize thesolution before current monitoring. Then, the currentwas monitored until reach a new steady state. Successiveadditions of phenol standard solutions were performedevery 30 s, always stirring the solution for a few secondsbefore current monitoring.
2.5. Determination of the apparent Michaelis /Mentenconstants (Kapp The apparent Michaelis  were determined from the electrochemical Eadie /Hofs-tee form of the Michaelis /Menten equation plot-ting Djs/C versus Djs, where js is the current density inthe steady state and C is the concentration of phenoliccompound. In these graphs the Kapp obtained from their respective slopes. The curves wereplotted considering concentrations of phenolic com-pounds in the saturated range to assure that the sensorresponse is being kinetically controlled by the biomi-metic reaction.
3. Results and discussion 3.1. Copper complex and membrane thickness effects onthe sensor response In a first step, an experiment carried out in order to test the response of the unmodified (andmodified GC electrode with a Nafion† membrane(and a Nafion† membrane doped with coppercomplex (indicated clearly the role of thecomplex in sensor preparation. For the redox coupleof copper complex the formal potential was /140 mVversus SCE. The shape of the voltammogram suggeststhat the process is not fully reversible, as the peakseparation is about 105 mV and is almost constant for Fig. 1. Cyclic voltammograms obtained with (a) a bare GC electrode; different scan rates (v ). The peak current was propor- (b) a GC electrode covered with a Nafion† membrane; and (c) a GCelectrode tional to the v1/2, suggesting that the process is similar to [Cu(bipy)3]Cl2×/6H2O. Scan rate 10 mV s1.
those controlled by diffusion. Since the copper complexis adsorbed on the electrode surface, the supporting copper complex, amperometric measurements were electrolyte should be responsible to keep the electro carried out at /50 mV in presence of H2O2 and, no satisfactory response was observed with this electrode, After that, in order to verify the response of the GC as can be observed in the curve a of . When the GC electrode coated with a Nafion† membrane without electrode was coated with a Nafion† membrane doped M.D.P.T. Sotomayor et al. / Electrochimica Acta 48 (2003) 855 /865 the sensing membrane, since the complex has animportant role in the film reticulation. Thus, when thecopper concentration increases on the electrode surface,the analyte/product diffusion through the membranebecomes more difficult and the sensor response de-creases. In order to minimize the diffusion effect,membranes prepared using a small amount of complexare more adequate, but should be considered the sensorstability.
For concentrations higher than 3 g dm3 the obtained membrane was not uniform and the responsewas unstable. Based on this aspect, the membraneprepared with 2 g dm3 copper complex solution waschosen as giving the best sensor performance, assuringthat the complex is homogenously dispersed on the film.
Fig. 2. Amperometric responses for dopamine obtained with GC The sensor prepared in this way allowed to obtain electrodes coated with a Nafion† membrane (a) without coppercomplex; (b) with [Cu(bipy)3]Cl2×/6H2O. Applied potential of /50 uniform films, good signals, reproducibility and stabi- mV vs. SCE, in 0.1 mol dm3 phosphate buffer (pH 7.0).
The influence of the membrane thickness on the sensor response was also investigated. The results in with [Cu(bipy)3]Cl2×/6H2O a good reduction current indicate that there is no significant influence of density (j ) was observed at /50 mV in the presence of the membrane thickness on the sensor response, in the H2O2 (curve b in All these studies suggest that range between 15 and 46 mm. This behavior suggests the copper complex is working as a catalyst to the that the Nafion† film is not influencing in the interac- reaction between H2O2 and dopamine (or other phenolic tion of the analyte/biomimetic catalyst (copper com- compounds) on the sensor surface, and then this is plex). This aspect is important to assure the same electrochemically reduced. Based on these characteris- response in a wide range of thickness (15 /46 mm) giving tics, the amount of copper complex in the Nafion† a high reproducibility.
membrane is very important to get larger response.
For thicker membranes the response decreases, prob- The effect of the copper (II) complex concentration in ably in this case the Nafion† film generates a barrier the membrane preparation was investigated. between the complex and analyte. On the other hand, its shows the results obtained for the sensor prepared with adherence on the electrode surface was poor. Thus, different concentrations of the copper complex in DMF membranes with 23 mm thickness were chosen, because solution. It can be observed that, the best response was allowed to construct sensors with a good sensitivity and obtained for a copper complex concentration of 1 g mainly an optimal repeatability in the sensors prepara- dm3. However, using lower concentrations, lower than 2 g dm3, the sensor was not stable suggesting thatcopper complex aid in the film reticulation. Although, 3.2. Hydrogen peroxide influence the sensor prepared using a solution of 2 g dm3presents a lower sensitivity, it was much more stable.
shows the results obtained in the experiments The sensitivity decreases as complex concentration is carried out in the presence and absence of hydrogen increased. This might be probably explained, consider- peroxide. In the absence of H2O2 (curve a) no signal was ing a compromise between the available amount of observed, while in the presence of H2O2 (curve b) a good cupric centers and the diffusion of analyte/product in response for dopamine was obtained, suggesting that the Influence of complex concentration used for membrane preparation on Influence of the membrane thickness on the sensor response in the sensor sensitivity, in the presence of 150 mmol dm3 H2O2 presence of 150 mmol dm3 H2O2 [Cu(bipy)3]Cl2×/6H2O (g dm3) Sensitivity (nA dm3 mmol1 cm2) Membrane thickness (mm) Sensitivity (nA dm3 mmol1 cm2 l) *Standard deviation for three replicates.
*Standard deviation for three replicates.
M.D.P.T. Sotomayor et al. / Electrochimica Acta 48 (2003) 855 /865 curve a), and the second is the H2O2 reduction catalyzedby the copper complex (c). Thus, initially areduction current is always observed before the phenolsaddition.
In the proposed sensor, the prior addition of hydro- gen peroxide, before the additions of phenolic com-pounds, can be explained considering the catalyticmechanism of Tyr from Cu4 In the monophenolase cycle (ortho -hydroxylation ofmonophenols), the active site must be oxygenated togenerate the oxy species. In the presence of the substratea series of proton exchanges occur resulting in anoxygen insertion into the phenolic ring and electrondonation from the copper atoms yielding the o -quinone, Fig. 3. Current density (j ) obtained with the proposed sensor in the water and the deoxy reduced form, which is oxidized in absence (a) and presence (b) of H2O2. Applied potential of /50 mV vs.
order to regenerate the Cu4 deoxy form, with the aid SCE, 0.1 mol dm3 phosphate buffer (pH 7.0).
of an oxidizing agent or by applying an adequate peroxide is very important in the sensor response. The potential, in order to bind new H2O2. In the cycle signal observed after H2O2 addition is due to mainly two involving catechol oxidation (o -diphenols to o -qui- contributions; the first one, is the inherent electroche- nones), the coordination of the catecholic substrate to mical reduction of H2O2 on the sensor surface , the oxy-cupric form results in the breakdown of the Fig. 4. Mechanism for Tyr in which the oxy species is formed from Cu4 and H2O2, in order to oxidize phenol and catechol compounds. T: tyrosine and D: DOPA bound forms.
M.D.P.T. Sotomayor et al. / Electrochimica Acta 48 (2003) 855 /865 complex with electron transfer from the catechol to dence of the current obtained on the measurement oxygen, giving the corresponding o -quinone and water.
carried out in anaerobic or aerobic conditions This leaves the copper atoms at the active site in the The factors that can be affecting the signal are: (1) the oxidized state (Cu2). In this state, it cannot bind O2 or possible O2 reduction catalyzed by the [Cu(bipy)3]Cl2×/ H2O2; therefore it is inactive to phenolic substrates. On 6H2O complex; (2) the efficiency of copper-oxy species the other hand, this form is able to coordinate another formation; and (3) possibly the spontaneous dopamine molecule of catecholic substrate and oxidizes it to the o - oxidation in presence of oxygen at neutral media quinone by donation of electrons to copper atoms, but not significant. In order to evaluate the influence of generating the deoxy-reduced form (Cu2 the measurement's conditions, studies were carried out In the sensor is expected that [Cu(bipy)3]Cl2×/6H2O in N2 saturated solutions (absence of O2), air saturated perform the same role of the Tyr active site, according to solution (bubbling air) and air equilibrated solution the mechanism proposed in This mechanism is (without bubbling any gas).
based on the Tyr enzyme mechanism, which catalyses shows clearly that in aerobic conditions (air the oxidation of phenolic compounds through copper- equilibrated and air saturated solutions) the sensitivities oxy species generation , and considering that the are lower than the responses obtained in the anaerobic copper complexes are dispersed in the Nafion† mem- condition. This behavior can be explained due to O2 brane in a favorable way to the formation of the Cu4 reduction catalyzed by the [Cu(bipy)3]Cl2×/6H2O, as sites. In a first step, the hydrogen peroxide clearly observed in . When the sensor is used in (or eventually O2) could react directly with two Cu2 aerobic conditions the O2 reduction is catalyzed by the ions, forming the oxy species, necessary for phenol complex, competing for the copper active centers with oxidation to o -quinone species. In this stage the copper the H2O2. Thus, the copper-oxy species generation is centers are reduced to Cu. However, an adequate affected resulting in lower sensitivity observed when potential should be applied in order to allow the copper compared with those obtained in anaerobic condition.
oxidation to complete the catalytic cycle, and at the Although, during the O2 reduction by the complex could same time the o -quinone might be reduced to catechol form copper-oxy site a high potential is necessary to electrochemically. The two electrons left in the copper reduce O2. The higher sensitivity obtained in anaerobic oxidation could be used for oxygen reduction in the oxy condition indicates the importance of hydrogen perox- species to form H2O and o-quinone. In a following step, ide in the sensor response mechanism, indicating that the catechol electrochemically produced, can react with the intermediate copper-oxy species should be the another oxy species, forming the o -quinone and pseudo- responsible for the dopamine oxidation. Thus, the meta tyrosinase. At this stage, the most important step sensitivity obtained is a compromise between; (1) the for phenol quantification, the electrochemical reduction facility of oxy species formation in O2 presence; and (2) of the o -quinone could occur on the electrode surface, the O2 reduction by the complex, diminishing the and a signal amplification may be obtained due to the amount of the active cupric centers that could react to cycle formed, making an analogy with the enzymatic form the oxy species.
systems for catecholamines detection The same In the present work, the measurements were carried mechanism is reported for many amperometric biosen- out in air equilibrated solution, which is more practical sors based on the Tyr enzyme Nevertheless, the for applications, because the sensitivity in this condition cathodic current observed will be proportional to the is enough to detect dopamine in the desired samples.
phenol concentration. In order to complete the catalyticcycle, another catechol species could reacts with the 3.4. Influence of the applied potential pseudo-meta form, generating o -quinone and the Cu.
Copper is electrochemically oxidized to complete the In is clearly seen that the sensor presents best catalytic cycle, and these two electrons are used to response at /50 mV versus SCE. This result is a reduce the o -quinone formed. This last cycle always compromise between the optimum potential for electro- allow the conversion of pseudo-meta species to reduced chemical reduction of quinone species to dopamine, and copper in order to complete the catalytic cycle.
the peroxide consumption at more negative potentials, Considering this mechanism and the obtained results through its electrochemical reduction Thus, in in the evaluation of the influence of peroxide concentra- more negative potentials the amount of H2O2 available tion on the sensor response the concentration of to form the reactive oxy species diminishes, due to the 150 mmol dm3, was used in all further experiments.
inherent electrochemical reduction of H2O2; conse-quently the sensitivity is lower. It is also important 3.3. Measurements conditions that the copper be in oxidized form to present catalyticproperty, thus the potential cannot be much negative to In the development of this amperometric sensor for avoid the copper reduction. For higher potentials the phenolic compounds is observed that there is a depen- possibility of o -quinone species reduction is minimized.

M.D.P.T. Sotomayor et al. / Electrochimica Acta 48 (2003) 855 /865 Fig. 5. Proposed mechanism for dopamine and other phenolic compound oxidation for the sensor modified with [Cu(bipy)3]Cl2×/6H2O.
Fig. 7. Profile of the sensor response, in aerobic and anaerobic media, Fig. 6. Dependence of H2O2 concentration on the current density (Dj) for dopamine. Applied potential of /50 mV vs. SCE, in 0.25 mol obtained with the proposed sensor. Applied potential of /50 mV vs.
dm3 Pipes buffer (pH 7.0) containing 150 mmol dm3 H2O2.
SCE, in 0.1 mol dm3 phosphate buffer (pH 7.0) containing 100 mmoldm3 of dopamine.
influence of the Pipes buffer concentration on the sensorresponse showed that the best result was obtained in a 3.5. Influence of the pH, buffer and buffer concentration concentration of 0.25 mol dm3 The investigation to evaluate the pH effect on the 3.6. Sensor characteristics sensor response showed an optimum pH at 7.0 (in 0.1 mol dm3 phosphate buffer solution. Experi- In the optimized conditions the proposed sensor ments carried out in three different buffer solutions showed a linear response range from 9.5 up to 230 (in a concentration of 0.10 mol dm3, indicated mmol dm3 (which can be expressed according that Pipes buffer gives the best response. Finally, the to the following equation: M.D.P.T. Sotomayor et al. / Electrochimica Acta 48 (2003) 855 /865  37(92)1:43(90:01) [DA]=mmol dm3 with a correlation coefficient of 0.999, for n /22. Theprofile of the full curve showed that the sensor responseis linear at low concentrations of dopamine and becomesindependent at higher dopamine concentration (data notshown). This result provides a mechanistic indicator thatthe dopamine oxidation indeed follows Michaelis /Menten kinetics such as occurs in enzymaticbiosensors.
The detection limit of 4.8 mmol dm3 was calculated according to Serra et al. The response time, Fig. 8. Cyclic voltammograms obtained with the proposed sensor in considering the time to reach 100% of the signal, was 0.25 mol dm3 Pipes buffer (pH 7.0) in (a) N2 saturated solution and approximately 1 s. The response time showed by this (b) after the saturation with air (dot line). Scan rate 10 mV s1.
Table 3Influence of the buffer on the current density (Dj ) obtained for theproposed sensor for 110 mmol dm3 dopamine, in presence of 150mmol dm3 H2O2 Buffer (0.1 mol dm3) *Standard deviation for three replicates.
Table 4Influence of the pipes concentration on current density (Dj ) for 100mmol dm3 dopamine, in presence of 150 mmol dm3 H2O2 [Pipes] (mol dm3) Fig. 9. Influence of the applied potential on the sensor response.
Measurements carried out in 0.1 mol dm3 phosphate buffer (pH 7.0) containing 100 mmol dm3 of dopamine and 150 mmol dm3 of H2O2.
*Standard deviation for three replicates.
Fig. 10. Response profile for the sensor in phosphate buffer solutionswith different solution pH. Applied potential of /50 mV vs. SCE, in Fig. 11. A typical profile of the sensor response in the optimized 0.1 mol dm3 phosphate buffer containing 100 mmol dm3 of conditions. Applied potential /50 mV vs. SCE, in 0.25 mol dm3 dopamine and 150 mmol dm3 of H2O2.
Pipes buffer solution (pH 7.0), containing 150 mmol dm3 of H2O2.
M.D.P.T. Sotomayor et al. / Electrochimica Acta 48 (2003) 855 /865 Table 5Analytical and kinetic parameters of the proposed sensor for dopamine and analogous compounds, in the optimised conditions sensor was better than the other sensors presented in the as R.S.D. was lower than 5%. This result indicates a literature for phenolic compounds .
good repeatability in the sensor construction.
The repeatability in the measurements was evaluated This sensor can be kept at room temperature and through thirteen successive experiments carried out with used, without loosing the sensitivity, after several 30 mmol dm3 dopamine solutions. The repeatability months. The good stability is probably due to the was evaluated as the relative standard deviation (R.S.D) elimination of enzyme denaturation problems caused giving a value of 4.8%.
by time and temperature.
Repeatability in the sensors construction was evalu- Under operational conditions the proposed sensor ated preparing four sensors and determined the sensi- presented good stability during more than 150 determi- tivity obtained for each one. The repeatability expressed nations, as shown in Fig. 12. Relative response (%) as a function of the number ofdeterminations. The parameter was calculated considered the sensorresponse in the first determination as 100%. Applied potential of /50 Fig. 13. Eadie /Hofstee plot, obtained for the dopamine using the mV vs. SCE, in 0.1 mol l1 phosphate buffer (pH 7.0) containing 100 proposed sensor, in which the [Cu(bipy)3]Cl2×/6H2O is a biomimetic mmol l1 of dopamine and 150 mmol l1 of H2O2.
catalyst of the Tyr enzyme.
M.D.P.T. Sotomayor et al. / Electrochimica Acta 48 (2003) 855 /865 3.7. Effect of the phenolic substrates Table 7Determination of dopamine in pharmaceutical preparations In this work a detailed study was carried out, in order Dopamine containing in 10.0 ml of sample (mg) to investigate the sensor response for other phenoliccompounds besides dopamine, including: aromatic amines, nitro-phenols, catecholic compounds and neu- rotransmitters. Among the eighteen compounds evalu- ated, the proposed sensor presented response only for Standard deviation for three replicates.
b Standard deviation for five replicates.
five of them. These compounds are; dopamine (DA),catechol (CAT), norepinephrine (NE), 4-aminophenol the high responses observed for 4-APh and p -PhA, and (4-APh) and p -phenylenediamine (p -PhA). The last two lower response obtained for DA, CAT and NE.
compounds presented responses 8.4 and 5.4 times,respectively, higher than that observed for dopamine.
In is shown the analytical and kinetics parameters obtained for the compounds with structure 3.8. Effect of interfering compounds very similar to dopamine. It can be observed that the The sensor response was tested in presence of 130 dopamine presented best sensitivity and linear response mmol dm3 dopamine and compounds such as uric acid, range, but slightly smaller affinity than catechol; this ascorbic acid and acetaminophen, and other compounds last parameter was evaluated through the value of the commonly contained in the pharmaceutical formula- apparent Michaelis /Menten constant. Although, this tions, such as sodium chloride and sodium bisulfite, in constant is commonly defined for enzymatic systems, different levels of molar ratios. The results obtained with this kinetic parameter is being used evidently by the proposed sensor () showed no significant analogy. The used of the Eadie /Hofstee form is quite interference in presence of the investigated compounds.
efficient in the kinetics analysis because, it could provide Ascorbic acid gave some small interference at a molar an instrument to discriminate kinetics from diffusion ratio of 1:1, presumably due to reaction with hydrogen process. The linear plot in (correlation coeffi- peroxide. However, as the sensor response is indepen- cient 0.992 for n /17) shows that apparently it is dent for concentrations of H2O2 higher than 150 mmol kinetically controlled, and the biomimetic reaction is dm3, it could be overcome using more H2O2.
the rate controlling process. Thus, this result is otherevidence that the mechanism proposed in iscoherent.
The sensitivity and selectivity order observed for the studied compounds can be explained based on their The applicability of the amperometric sensor in the redox potential, as it stands with respect to the use of dopamine determination was evaluated, on pharmaceu- metal complex. It is know that hydroquinone and other tical samples. In the analyzed sample containing in its analogous compounds such as 4-APh and p -PhA, are formulation preservatives and salts, such as the anti- easily and rapidly oxidized Their oxidation poten- oxidant sodium bisulfite no significant interference tials are lower than the ortho -analogous compounds, was observed. The results (were compared such as catechol. The facility for oxidation diminished, favorably with those obtained with the USP official respectively, for resorcinol, 2-nitrophenol, 3-nitrophe- method showing that the result is statistically the nol, 4-nitrophenol and phenol This could explain same with 95% of confidence.
Table 6Recovery (%) obtained for 130 mmol dm3 dopamine in presence of different interfering compounds in various levels of molar ratios Molar ratio interfering: dopamine *Standard deviation for three replicates; n.e., non evaluated.
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