Biosensor based on platinum nanoparticles dispersed in ionic liquid and laccase for determination of adrenaline

Sensors and Actuators B 140 (2009) 252–259

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Biosensor based on platinum nanoparticles dispersed in ionic liquid and laccase for determination of adrenaline Daniela Brondani a , Carla Weber Scheeren b , Jairton Dupont b , Iolanda Cruz Vieira a,∗ a b

Departamento de Química, Laboratório de Biossensores, Universidade Federal de Santa Catarina, 88040-970, Florianópolis, SC, Brazil Departamento de Química Orgânica, Laboratório de Catálise Molecular, Universidade Federal do Rio Grande do Sul, 91501-970, Porto Alegre, RS, Brazil

a r t i c l e

i n f o

Article history: Received 25 February 2009 Received in revised form 3 April 2009 Accepted 16 April 2009 Available online 3 May 2009 Keywords: Biosensor Platinum nanoparticles Ionic liquid Laccase Adrenaline

a b s t r a c t The platinum nanoparticles dispersed in ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (Pt-BMI.PF6 ) were used in the construction of a novel biosensor for the determination of adrenaline in pharmaceutical formulations by square-wave voltammetry. This biosensor was constructed based on laccase from Aspergillus oryzae and the responses under optimized conditions were obtained for a composition of 50:20:15:15% (w/w/w/w) graphite powder:laccase:Nujol:Pt-BMI.PF6 , phosphate buffer solution (0.1 mol L−1 , pH 6.5), with frequency, pulse amplitude, and scan increment at 20 Hz, 80 mV, and 5.0 mV, respectively. The analytical curve was linear for adrenaline concentrations from 9.99 × 10−7 to 2.13 × 10−4 mol L−1 (r = 0.9998) with a detection limit of 2.93 × 10−7 mol L−1 . This biosensor demonstrated suitable stability (ca. 90 days; 300 determinations) and good repeatability and reproducibility, with a relative standard deviation of 3.7% and 4.8%, respectively. The recovery study of adrenaline in pharmaceutical samples gave values from 95.5% to 104.2% and the concentrations determined are in agreement with those using the standard method at the 95% confidence level. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Adrenaline belongs to the catecholamine or biogenic amines group exhibiting the properties of a chemical neurotransmitter and a hormone. It is biosynthesized in the adrenal medulla and sympathetic nerve terminals and acts in the renal, hormonal, cardiovascular and central nervous systems of animals and humans [1–3]. It is a potent vasoconstrictor and cardiac stimulant [4]. It is a drug widely used in medicine in the treatment of allergic emergencies, heart attack, bronchial asthma and cardiac surgery [5–7]. Owing to their importance in pharmaceuticals, the determination of adrenaline has attracted much attention from researchers. A great number of methods have been developed for the determination of this drug, mainly employing spectrophotometry [4,6], liquid chromatography [8], capillary electrophoresis [9], chemiluminescence [5] and electrochemical detection with various sensors and biosensors [1–3,10,11]. The development of biosensors was driven by the need for faster and more versatile analytical methods for application in important areas including clinical, biomedical, environmental, industrial and pharmaceutical analysis [12]. The biosensors are an attractive alternative to conventional analytical methods, such as liquid

∗ Corresponding author. Tel.: + 55 48 3721 6844; fax: +55 48 3721 6850. E-mail address: [email protected] (I.C. Vieira). 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.04.037

chromatography, used for determination of phenolic compounds in several types of the samples. Some advantages of biosensors, relative to chromatographic techniques, are their fast response, cost-effectiveness, simplicity of operation and manufacturing. It is recognized that a judicious choice of the biological receptor and applied potential much improves their selectivity and sensitivity [13]. Enzyme electrodes are one of the most intensively researched biosensors because enzymes are highly selective and respond quickly to a specific substrate [13,14]. Laccase (EC 1.10.3.1, p-benzenediol: oxygen oxidoreductase) belongs to a family of multicopper oxidases that catalyze the oxidation of a range of inorganic and aromatic compounds (particularly phenols) with the concomitant reduction of molecular oxygen to water [15–17]. A few laccases are isolated from plant sources (e.g., lacquer, sycamore and tobacco), however, most known laccases have fungal origins (e.g., white rot fungi). Recently, it has been reported that laccases are widespread in bacteria [15,16,18]. Laccases are mainly used in paper and textile industries, for wastewater treatment, fiber modification, delignification and dye bleaching. [15,16]. In addition, many electrodes combined with laccase have been developed for detection of phenolic compounds [13,14,18,19]. Recent advances in science and technology have led to the development of electrochemical sensors with rapid response and high sensitivity and selectivity [12,20,21]. The nanotechnology brings several possibilities for biosensor construction and for developing novel electrochemical bioassays [20]. The introduction of the metal nanoparticles into the sensing interface to facilitate the electron

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transfer can significantly improve the sensitivity [21]. Nanoparticles of noble metals have attracted special interest because they differ from their bulk metal counterparts. Their size controllability, chemical stability, high catalysis activity and surface tenability make them very advantageous for application in sensors [21,22]. Ionic liquids (ILs) also have attracted considerable attention in electroanalysis because of their unusual physical and chemical properties, mainly resulting from their peculiar structural organization [23]. Of particular interest are salts resulting from the combination of imidazolium cations with inorganic or organic anions that are liquid at room temperature. These materials display excellent properties such as good chemical and thermal stability, almost negligible vapor pressure, high ionic conductivity and wide electrochemical window. Among the various application ILs are considered promising fluids for the separation and electrochemical applications [22,24,25], in particular they are widely used to prepare modified electrodes and biosensors [10,25,26] and in preparation and stabilization of nanomaterials [27–30]. This paper describes the synthesis and characterization of platinum nanoparticles dispersed in ionic liquid 1-butyl-3methylimidazolium hexafluorophosphate (BMI.PF6 ) [30,31] for use in the construction of a novel biosensor for the determination of adrenaline in pharmaceutical formulations by square-wave voltammetry. This biosensor was constructed with laccase from Aspergillus oryzae and the results obtained with its use were compared to those obtained using the official method [32]. The influence of different experimental conditions including the percentage of IL-containing Pt nanoparticles (Pt-BMI.PF6 ) and laccase, pH and voltammetry parameters (frequency, pulse amplitude, scan increment and cathodic peak potential – Epc ) were investigated to optimize the proposed biosensor.

253

dichloromethane (3 × 15 mL), and dried under reduced pressure. The Pt-BMI.PF6 samples thus obtained were prepared for TEM and X-ray analysis (see below). 2.3. Sample preparation for TEM analysis The morphology and the electron diffraction (ED) of the obtained particles were carried out on a JEOL JEM-2010 equipped with an energy-dispersive X-ray spectroscopy (EDS) system and a JEOL JEM-1200 EXII electron microscope operating at accelerating voltages of 120 kV. The samples for TEM analysis were prepared by dispersion of the Pt-BMI.PF6 nanoparticles at room temperature and then collected on a carbon-coated copper grid. The histograms of the nanoparticle size distribution, assuming spherical shape, were obtained from the measurement of approximately 300 particles and were reproduced in different regions of the Cu grid, found in an arbitrarily chosen area of enlarged micrographs [33]. 2.4. Sample preparation and X-ray analysis (XRD) The phase structures of the Pt-BMI.PF6 were characterized by XRD. For the XRD analysis, the nanoparticles were isolated as a fine powder and placed in the sample holder. The XRD experiments were carried out on a SIEMENS D500 diffractometer equipped with a curved graphite crystal using Cu K␣ R radiation ( = 1.5406 Å). The diffraction data were collected at room temperature in a BraggBrentano  − 2 geometry. The equipment was operated at 40 kV and 20 mA with a scan range between 20◦ and 90◦ . The diffractograms were obtained with a constant step of 2 = 0.05. The indexation of Bragg reflections was obtained by a pseudo-Voigt profile fitting using the FULLPROF code [34].

2. Experimental 2.5. Construction of biosensors 2.1. Chemicals Laccase in microcapsules was purchased from Novozymes (Denmark), under the trade name Denilite® II BASE (E.C. 1.10.3.2; 800 U g−1 ), produced by genetically modified microorganisms (A. oryzae). The platinum nanoparticles dispersed in ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (Pt-BMI.PF6 ) were synthesized as previously described in the literature [30,31]. The carbon paste was prepared using graphite powder (Acheson 38, Fisher Scientific) and Nujol purchased from Aldrich. Adrenaline, carbidopa, catechol, dopamine, levodopa, and methyldopa were obtained from Sigma–Aldrich. Phosphate buffer solution (0.1 mol L−1 , pH 6.5) was used as the supporting electrolyte. The samples (A, B and C) of adrenaline for injection (1 mg mL−1 ) were supplied by the University Hospital of the Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil, and analyzed using both the proposed biosensors and a spectrophotometric method. All reagents were of analytical grade and used without further purification, and all solutions were prepared with deionized water. 2.2. Synthesis and characterization of platinum nanoparticles in ionic liquid BMI.PF6 In a typical experiment, a Fischer–Porter bottle containing BMI.PF6 (1 mL) and [Pt2 (dba)3 ] (dba = dibenzylidene acetone) (30 mg, 0.02 mmol) was stirred at room temperature for 15 min yielding a violet dispersion. The system was then heated to 75 ◦ C and hydrogen (4 bar) was admitted to the system. After stirring for 1.5 h, a black “solution” was obtained. The Pt nanoparticles were isolated by centrifugation (3500 rpm) with the addition of acetone (5 mL) for 3 min, washed with acetone (3 × 15 mL) and

The biosensors were constructed by hand-mixing laccase (40.0 mg; 20%, w/w) and graphite powder (100.0 mg; 50%, w/w) with a mortar and pestle for 20 min to ensure the uniform dispersion of the enzyme. Subsequently, the Nujol (30.0 mg; 15%, w/w) and platinum nanoparticles dispersed in ionic liquid BMI.PF6 (30.0 mg; 15%, w/w) were added and mixed for at least 20 min to produce the final paste. The resulting modified carbon paste was placed in a 1 mL syringe (1.0 mm internal diameter) and a copper wire (0.4 × 10.0 cm) was inserted to obtain the external electric contact. The biosensor without platinum nanoparticles was prepared following the same steps. The carbon paste electrode (CPE) was prepared in a similar way by mixing graphite powder and Nujol in a mortar. The constructed electrodes were stored at room temperature in a dry place when not in use [19]. 2.6. Instrumentation and electrochemical measurements A Hewlett-Packard (Boise, ID, USA) UV–vis spectrophotometer, model 8452A, with a quartz cell (optical path of 1.00 cm) was used for the determination of adrenaline by comparative method. A Unique 1400A ultrasonic bath was used in preparation of the solutions. Electrochemical measurements, using square-wave and cyclic voltametry, were performed on an Autolab PGSTAT12 potentiostat/galvanostat (Eco Chemie, The Netherlands) connected to data processing software (GPES, software version 4.9.006, Eco Chemie). A conventional three-electrode system was used with a biosensor based on platinum nanoparticles dispersed in ionic liquid (PtBMI.PF6 ) and laccase as the working electrode, a platinum wire as the counter electrode and an Ag/AgCl (3.0 mol L−1 KCl) electrode as the reference electrode.

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Square-wave voltammetry and cyclic measurements were carried out in an unstirred, non de-aerated phosphate buffer solution (0.1 mol L−1 ; pH 6.5) at room temperature (25.0 ± 0.5 ◦ C) and all potentials were measured and reported vs. Ag/AgCl (3.0 mol L−1 KCl). In a typical run, 10 mL of the supporting electrolyte was transferred to a clean, dry cell and the required volume of the adrenaline or pharmaceutical sample was added by micropipette. The cyclic voltammograms were recorded by cycling the potential between −0.6 and +0.4 V at a scan rate of 100 mV s−1 . The square-wave voltammetry measurements were performed applying a sweep potential between −0.5 and 0.0 V, at a frequency of 10–100 Hz, pulse amplitude of 10–100 mV and scan increment of 1.0–7.0 mV, after successive additions of the analyte. After a stirring period of 60 s to homogenize the solution, a square-wave or cyclic voltammogram was recorded. 2.7. Preparation of pharmaceutical samples and determination of the adrenaline Samples of adrenaline for intramuscular injection (A, B and C) containing sodium bisulphite (NaHSO3 ), as a preservative, were used after pre-treatment. The samples were appropriately diluted with acetate buffer solution (0.1 mol L−1 ; pH 4.0) and then bubbled with nitrogen for 5 min. This period was sufficient to quantitatively remove the interfering species in the form of H2 SO3 [3,7,10]. These adrenaline samples were quantified using the proposed biosensor and a spectrophotometric method. The method of standard addition was used for determination of adrenaline in the pharmaceutical samples. Aliquots of the previously prepared sample were transferred to the cell and quantified using square-wave voltammetry, after successive additions of standard solutions of adrenaline. All measurements were performed in triplicate. A spectrophotometric method for adrenaline determination recommended in the United States Pharmacopoeia [32] was used to compare the analytical results obtained with the proposed biosensor.

Fig. 1. X-ray diffraction pattern of the Pt-BMI.PF6 .

teristic diameter results in a monomodal particle size distribution. A mean diameter dm ≈ 2.3 ± 0.4 nm of the Pt-BMI.PF6 nanoparticles was estimated from ensembles of 300 particles found in an arbitrarily chosen area of the enlarged micrographs. Fig. 2B shows the particle size distributions obtained, which can be reasonably well fitted by a Gaussian curve.

3. Results and discussion 3.1. Preparation and characterization of platinum nanoparticles dispersed in BMI.PF6 ionic liquid The Pt-BMI.PF6 nanoparticles of 2.3 ± 0.4 mm were prepared by simple decomposition of Pt2 (dba)3 dispersed in the BMI.PF6 under hydrogen atmosphere using the classical procedure described earlier [30,31]. These nanoparticles were isolated and characterized by XRD and TEM analysis. The XRD pattern (Fig. 1) of the isolated material confirmed the obtainment of crystalline Pt-BMI.PF6 and the mean diameter (2.6 ± 0.4 nm) was estimated from the XRD pattern by means of the Debye–Scherrer equation calculated from the full width at halfmaximum (fwhm) of the (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) planes. TEM analysis was performed on samples prepared using the Pt-BMI.PF6 suspension ultrasonically shaken and then deposited on holey carbon film supported on a copper grid. The grids were placed on filter paper to remove the excess material and allowed to dry for 2 h under high vacuum. The TEM samples were then examined using a JEM-2010 microscope operating at an accelerating voltage of 120 kV. Bright field TEM observations were performed under slightly under-focused conditions (f ≈ −300 nm), and particle size distributions were determined once the original negative had been digitalized and enlarged to 470 pixels/cm (Fig. 2A). The particles show an irregular shape, but evaluation of their charac-

Fig. 2. TEM micrographs (A) and histograms (B) showing the particle size distribution of Pt-BMI.PF6 .

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(89.0%), dopamine (31.2%), carbidopa (7.4%), methyldopa (6.1%) and levodopa (3.7%). In this study, adrenaline was selected for the optimization of the proposed biosensor and determination in pharmaceutical formulations. 3.4. Principle of the enzymatic process

Fig. 3. Square-wave voltammograms obtained using (a) CPE; (b) BMI.PF6 -biosensor; (c) Pt-BMI.PF6 -biosensor in 1.0 × 10−4 mol L−1 adrenaline in 0.1 mol L−1 phosphate buffer solution (pH 6.5); pulse amplitude 80 mV, frequency 20 Hz and scan increment 5.0 mV.

3.2. Voltammetric behavior of adrenaline in the biosensors and bare CPE The electrochemical behavior of adrenaline using the biosensor based on laccase and platinum nanoparticles dispersed in ionic liquid (Pt-BMI.PF6 -biosensor), the biosensor without platinum nanoparticles (BMI.PF6 -biosensor) and the bare carbon paste electrode was investigated by square-wave voltammetry in the potential range of −0.5 and 0.0 V vs. Ag/AgCl. Fig. 3 shows the voltammograms obtained using the (a) CPE; (b) BMI.PF6 -biosensor; and (c) Pt-BMI.PF6 -biosensor in 1.0 × 10−4 mol L−1 adrenaline in 0.1 mol L−1 phosphate buffer solution (pH 6.5); pulse amplitude 80 mV, frequency 20 Hz and scan increment 5.0 mV. As can be observed, for the Pt-BMI.PF6 -biosensor a higher response of the reduction wave of o-quinone to adrenaline at a potential of −0.21 V was observed and compared with the biosensor without platinum nanoparticles and the bare carbon paste electrode. The cathodic peak current of the BMI.PF6 -biosensor corresponded to 64.2% of the response of the biosensor based on platinum nanoparticles dispersed in ionic liquid, while the CPE had a response corresponding to only 13.8% of the response of the reduction of the selected electrode (Pt-BMI.PF6 -biosensor).

Extensive research efforts have been dedicated to evaluating the possibilities offered by enzymes in biotechnological and environmental applications [16]. Commercially, laccases have been used mostly to delignify woody tissues and produce ethanol, and in wastewater treatment and bioremediation. Research in recent years has been intense, much of it elicited by the wide diversity of laccases, their utility and their very interesting enzymology [15,16]. Reactions catalyzed by enzymes have been extensively used in the determination of different analytes, particularly of pharmaceutical interest. Numerous biosensors based on laccases have been described in the literature [13,14,18,19], as discussed previously. Fig. 4 shows a schematic representation of the enzymatic reaction involving adrenaline and laccase on the Pt-BMI.PF6 -biosensor surface. Initially, (A) adrenaline is oxidized by laccase in presence of molecular oxygen to (B) adrenalinequinone [3,10]. This o-quinone can be reduced electrochemically at a potential of −0.21 V vs. Ag/AgCl on the biosensor surface. The resulting current reduction is used as the analytical response for adrenaline determination. 3.5. Optimization of the biosensor construction and experimental conditions The effect of the Nujol:Pt-BMI.PF6 ratios (a) 100:0; (b) 75:25; (c) 50:50; (d) 25:75; and (e) 0:100% (w/w) were investigated and the biosensor responses in the adrenaline determination by (A) cyclic voltammetry and (B) square-wave voltammetry are compared in Fig. 5. As can been seen in the cyclic voltammograms (Fig. 5A), the background current increased with an increase in the ionic liquid content. As previously reported [35], the addition of ILs to a CPE modifies the microstructure of the paste and this charge transfer resistance decreases and the charge transfer rate increases, because of the higher conductivity of the electrode containing the IL in comparison with the use of other binders. Nevertheless, when the contribution of the capacitive current was

3.3. Response of the biosensor to catecholamines Catecholamines are compounds containing an o-catechol nucleus and amine groups on a chain of two-carbon m or p atoms bound to the phenolic hydroxyl groups [7]. These compounds play a regulatory function in physiology and are therefore of interest both as disease markers and pharmaceuticals [26]. Several observations on the electrochemical behavior of catecholamines, such as adrenaline [1-(3,4-dihydroxyphenyl)-2-methylamino-ethanol] – one of the most significant compounds of this group – have been made in many papers [1–3,10,11,26]. Thus, in order to investigate the response of the biosensor to different catecholamines (adrenaline, catechol, dopamine, carbidopa, levodopa and methyldopa) a comparative study was carried out. The biosensor responses to the investigated substrates were obtained from solutions containing 2.0 × 10−4 mol L−1 of catecholamine in phosphate buffer solutions (0.1 mol L−1 ; pH 6.5). Table 1 shows the potential (cathodic peak current) and relative response (%). As can be seen, the proposed biosensor showed decreasing sensitivity in the order: catechol (100.0%), adrenaline

Fig. 4. Schematic representation of the enzymatic process between (A) adrenaline, oxygen and laccase and electrochemical reduction of the (B) adrenalinequinone at the biosensor surface. LACred : laccase reduced form and LACoxid : laccase oxidized form.

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Table 1 Relative response of the biosensor for the catecholamines investigated. Catecholamines

Structure

Epc (V)

Relative response (%)

Catechol

+0.18

100.0

Adrenaline

−0.21

89.0

Dopamine

+0.15

31.2

Carbidopa

+0.07

7.4

Methyldopa

+0.06

6.1

Levodopa

+0.06

3.7

suppressed (square-wave voltammograms – Fig. 5B), an increase in the cathodic peak current was observed up to a Nujol:Pt-BMI.PF6 ratio of 50%. After this, the response decreased considerably. This same behavior has been reported for biosensors containing ILs in previous studies carried out by our group [10,19]. Hence, the Nujol: Pt-BMI.PF6 ratio of 50:50% (w/w) was used for subsequent biosensor construction and square-wave voltammetry analysis for the determination of adrenaline in pharmaceutical samples. In addition, the effect of laccase in percentages of 5–40% (w/w) on the modified carbon paste electrode containing platinum nanoparticles dispersed in IL was also investigated. The cathodic peak current increased with increases in the enzyme composition up to 20.0% (w/w). Therefore, this enzyme composition was selected for further studies and a composition of 50:20:15:15% (w/w/w/w) graphite powder:laccase:Nujol:Pt-BMI.PF6 , respectively, was used in the construction of the Pt-BMI.PF6 -biosensor. Recently, similar parameters were investigated for construction of a biosensor for rosmarinic acid by Franzoi et al. [19], who obtained similar results. After the optimization of the biosensor composition, the experimental conditions were evaluated, in triplicate, including solution pH (4.0–7.5) and the square-wave voltammetry parameters (frequency of 10–100 Hz, pulse amplitude of 10–100 mV, and scan increment of 1.0–7.0 mV) in order to obtain the best response for the proposed Pt-BMI.PF6 -biosensor. The response was ana-

lyzed in terms of the cathodic peak current for a 1.3 × 10−4 mol L−1 adrenaline standard solution. Table 2 summarizes the range over which each variable was investigated and the optimal value found. The best experimental conditions were selected for subsequent experiments. 3.6. Repeatability, reproducibility and stability of the biosensor The repeatability of the resulting reduction current (analytical response) obtained using the same biosensor was examined in phosphate solutions (pH 6.5) containing 1.3 × 10−4 mol L−1 adrenaline. The relative standard deviation (R.S.D.) was 3.4% for eight successive measures. The biosensor-to-biosensor reproducibility was also investigated considering four biosensors prepared independently. A good reproducibility was obtained, with Table 2 Optimization of biosensor parameters. Parameter

Range investigated

Optimal value

Nujol:Pt.BMIPF6 (%, w/w) Laccase (%, w/w) pH Frequency (Hz) Pulse amplitude (mV) Scan increment (mV)

100:0–0:100 5–40 4.0–7.5 10–100 10–100 1.0–7.0

50:50 20 6.5 20 80 5.0

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Fig. 6. Study on stability of the proposed biosensor using 1.3 × 10−4 mol L−1 adrenaline in 0.1 mol L−1 phosphate buffer solution (pH 6.5).

Analytical features observed for adrenaline determination and those reported previously in the literature using modified carbon paste electrodes are shown in Table 3. The linear range and limit of detection of the developed biosensor are comparable, or better than, those described in the literature [2,3,10,11]. The satisfactory analytical performance for the proposed biosensor can be attributed to the good stability of laccase, high conductivity of the ionic liquid and the fact that the metal nanoparticles facilitate the electron transfer, significantly improving the sensitivity. 3.8. Interference, recovery study and analytical application

Fig. 5. (A) Cyclic and (B) square-wave voltammograms obtained using the Nujol:PtBMI.PF6 ratios (a) 100:0; (b) 75:25; (c) 50:50 (d) 25:75 and (e) 0:100% (w/w) in the biosensor containing laccase submerged in phosphate buffer pH 6.5 (A) containing 4.0 × 10−4 mol L−1 adrenaline at 100 mV s−1 and (B) 1.3 × 10−4 mol L−1 adrenaline at frequency 20 Hz, pulse amplitude 80 mV, scan increment 5.0 mV. Inset: current values for each biosensor composition.

In agreement with previous studies carried out by our group [10] and reported by other researchers [3,7] a strong interference of the bisulphite (or metabisulphite) anion was observed in the measurements employing biosensors for determination of adrenaline pharmaceutical formulations. Thus, to remove efficiently the bisulphite interference the adrenaline samples were bubbled with nitrogen for 5 min in the presence of acetate buffer solution (0.1 mol L−1 ; pH 4.0).

relative standard deviations of approximately 4.8% for the proposed biosensor. The stability and lifetime of the biosensor are important parameters in analytical determinations. Thus, these parameters were investigated for the proposed biosensor stored at room temperature with measurements taken every 1–10 days, over a 90-day period (over 300 determinations for each quantity of carbon paste used in the syringe). As can be seen in Fig. 6, the biosensor showed an excellent operational stability, obtaining a response of over 80% for 90 days. 3.7. Square-wave voltammograms and analytical curve Under the optimized experimental conditions, the square-wave voltammograms and analytical curve for adrenaline were obtained employing the proposed biosensor (shown in Fig. 7). The analytical curve obtained for biosensor was linear in the range 9.99 × 10−7 to 2.13 × 10−4 mol L−1 of adrenaline (−I = 0.21 (±0.03) + 2.10 (±0.02) × 105 [adrenaline]; r = 0.9998), where I is the resultant peak current in ␮A and [adrenaline] is the adrenaline concentration in mol L−1 . The detection limit (three times the signal blank/slope) and quantification limit (10 times the signal blank/slope) were found to be 2.93 × 10−7 and 9.76 × 10−7 mol L−1 , respectively.

Fig. 7. Square-wave voltammograms obtained using the proposed biosensor for (a) blank in phosphate buffer solution (0.1 mol L−1 ; pH 6.5) and adrenaline solutions at the following concentrations: (b) 9.99 × 10−7 mol L−1 ; (c) 4.97 × 10−6 mol L−1 ; (d) 9.90 × 10−6 mol L−1 ; (e) 1.96 × 10−5 mol L−1 ; (f) 2.91 × 10−5 mol L−1 ; (g) 3.85 × 10−5 mol L−1 ; (h) 5.66 × 10−5 mol L−1 ; (i) 7.41 × 10−5 mol L−1 ; (j) 9.09 × 10−5 mol L−1 ; (k) 1.23 × 10−4 mol L−1 ; (l) 1.52 × 10−4 mol L−1 ; (m) 1.74 × 10−4 mol L−1 and (n) 2.13 × 10−4 mol L−1 at frequency 20 Hz, pulse amplitude 80 mV, and scan increment 5.0 mV. Inset: the analytical curve of adrenaline.

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Table 3 Analytical response of modified carbon paste electrodes for determination of adrenaline. Linear range (mol L−1 )

Composition of modified CPE

−7

7.0 × 10 to 1.2 × 10 5.0 × 10−5 to 3.5 × 10−4 1.98 × 10−6 to 1.06 × 10−4 9.89 × 10−7 to 1.22 × 10−4 5.0 × 10−6 to 1.0 × 10−4 9.99 × 10−7 to 2.13 × 10−4

Graphite powder–carbon nanotube–EBNBH complex–paraffin Graphite powder–mineral oil–polyphenol oxidase Graphite powder–Nujol–peroxidase immobilized on chitin Graphite powder–Nujol–BMIPF6 –peroxidase immobilized on chitin Graphite powder–binder–poly(isonicotinic acid) Graphite powder–Nujol–Pt.BMIPF6 –laccase

Table 4 Recovery of adrenaline in pharmaceuticals using the proposed biosensor. Adrenaline (mg L−1 )

Sample

Recovery (%)

Added

Founda

A

3.30 6.54 9.73

3.24 ± 0.06 6.74 ± 0.07 9.62 ± 0.04

98.2 103.1 98.9

B

3.30 6.54 9.73

3.44 ± 0.07 6.63 ± 0.05 9.66 ± 0.06

104.2 101.4 99.3

C

3.30 6.54 9.73

3.15 ± 0.04 6.43 ± 0.06 9.88 ± 0.05

95.5 98.3 101.5

Table 5 Determination of adrenaline in pharmaceutical formulation using a standard method [32] and the proposed biosensor.

A B C

Detection limit (mol L−1 ) −7

2.16 × 10 1.5 × 10−5 3.96 × 10−7 2.27 × 10−7 1.0 × 10−6 2.93 × 10−7

Reference [2] [3] [10] [10] [11] This work

ceutical formulations by square-wave voltammetry. The addition of Pt-BMI.PF6 to the biosensor led to an increase in sensitivity, probably because of the high conductivity of the IL combined with the electron transfer facilitated by the metal nanoparticles. The proposed biosensor exhibited high sensitivity, rapid response, suitable selectivity, good reproducibility and stability. In addition, the low-cost, simplicity and fast construction of the biosensor makes it superior to other techniques used for catecholamines determination. Satisfactory results were obtained using this biosensor when compared with the official method and, thus, it represents an alternative method to determine adrenaline in pharmaceutical formulations. Acknowledgments

A, B and C = adrenaline injection. a n = 3.

Sample

−3

Adrenaline (mg mL−1 )

Relative error (%)

Financial support from CNPq (Processes 472169/2004-1 and 472541/2006-4), MCT/CNPq/PADCT, NOVOZYMES and also the scholarship granted by CAPES to DB are gratefully acknowledged. References

Label

Standard methoda

Biosensora

RE1

RE2

RE3

1.00 1.00 1.00

1.02 ± 0.08 0.97 ± 0.07 0.98 ± 0.05

1.04 ± 0.03 0.96 ± 0.04 0.95 ± 0.04

+2.0 −3.0 −2.0

+2.0 −1.0 −3.0

+4.0 −4.0 −5.0

a n = 3; confidence level 95%; A, B and C = adrenaline injection; RE1 : standard method vs. label value; RE2 : Biosensor vs. standard method; RE3 : Biosensor vs. label value.

A recovery study was performed, in triplicate, using three pharmaceutical formulation samples (A, B and C: adrenaline injection) with three different standard concentrations (3.30, 6.54 and 9.73 mg L−1 ) of adrenaline and the concentrations measured were compared with the concentrations added. The results are summarized in Table 4 and show that average recoveries varied from 95.5% to 104.2%, demonstrating the satisfactory accuracy of the developed biosensor and indicating the absence of matrix effects on these determinations. The proposed method was validated by applying it to the determination of adrenaline in pharmaceuticals. The adrenaline concentrations in three samples of injectable formulations were determined, in triplicate, using the proposed biosensor and the standard spectrophotometric method [32]. The results of the quantifications using the biosensor, the standard method and label values were compared (shown in Table 5). According to the Student’s t-test, at a 95% confidence level, there are no significant differences between the standard method, the results obtained with the biosensor, and the label value. It can thus be concluded that the method is suitable for the determination of adrenaline in pharmaceutical formulations. 4. Conclusions In this paper, platinum nanoparticles dispersed in ionic liquid (Pt-BMI.PF6 ) and laccase were successfully used in the construction of a novel biosensor for the determination of adrenaline in pharma-

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Biographies Daniela Brondani received her BSc in industrial chemistry in 2007 from the Universidade Regional Integrada do Alto Uruguai e das Missões, Frederico Westphalen, Brazil. Currently, she is studying for an MSc at the Universidade Federal de Santa Catarina, Florianópolis, Brazil. Her research work is concentrated on biosensor determination of catecholamines in pharmaceuticals. Carla Weber Scheeren received her Doctorate in 2006 from Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil and postdoctorate in 2007 from Université Louis Pasteur de Strasbourg, France. Her research interests include the synthesis and characterization of ionic liquids and metal nanoparticles. Jairton Dupont received his Doctorate in 1988 from Univerité Louis Paster de Strasbourg, France and postdoctorate in 1990 from University of Oxford, England. Currently, he is a full professor in the chemistry department at the Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. His research work is concentrated on organometallic chemistry and ionic liquids. Iolanda Cruz Vieira received her doctorate and postdoctorate in 1997 and 1999, respectively, from the Federal University of São Carlos, São Carlos, Brazil. Currently she is professor of analytical chemistry of the Federal University of Santa Catarina, Florianópolis, Brazil. Her research interests include the development of novel chemical sensors and biosensors and their application in the determination of phenolic compounds in samples of interest pharmaceutical and food.