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Biosensor based on pequi polyphenol oxidase immobilized on chitosan crosslinked with cyanuric chloride for thiodicarb determination
Enzyme and Microbial Technology 47 (2010) 153–158
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Biosensor based on pequi polyphenol oxidase immobilized on chitosan crosslinked with cyanuric chloride for thiodicarb determination Fábio de Lima a , Bruno G. Lucca a , Antônio M.J. Barbosa a , Valdir S. Ferreira a , Sally K. Moccelini b , Ana C. Franzoi b,∗ , Iolanda C. Vieira b a b
Departamento de Química, Universidade Federal do Mato Grosso do Sul, 79070-900, Campo Grande, MS, Brazil Departamento de Química, Laboratório de Biossensores, Universidade Federal de Santa Catarina, 88040-970, Florianópolis, SC, Brazil
a r t i c l e
i n f o
Article history: Received 2 March 2010 Received in revised form 13 April 2010 Accepted 15 May 2010 Keywords: Biosensor Thiodicarb Polyphenol oxidase Pequi fruit
a b s t r a c t A novel pequi fruit (Caryocar brasiliense Camb.) homogenate source of polyphenol oxidase was obtained and immobilized in chitosan crosslinked with cyanuric chloride (CHcych-PPO). A biosensor was developed and used for the square-wave voltammetric determination of thiodicarb. Several parameters were investigated to evaluate the performance of this biosensor in the presence of hydroquinone and thiodicarb. The best response was obtained using 70:20:10% (w/w/w) of graphite powder:Nujol:CHcych-PPO, 250 U mL−1 of PPO and phosphate buffer solution (0.1 M; pH 7.0) with frequency, pulse amplitude and scan increment of 10 Hz, 150 mV, and 15 mV, respectively. Under optimized operational conditions the thiodicarb concentration was linear in the range of 3.75 × 10−7 to 2.23 × 10−6 M with a detection limit of 1.58 × 10−7 M. The biosensor was applied in the determination of thiodicarb in fresh fruit and vegetable samples and the results compared with those obtained using high-performance liquid chromatography. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Polyphenol oxidase (PPO) has received continuous attention from food chemists and processors since its discovery over 90 years ago. This is because it is involved in the enzymatic browning of many edible plant products, especially fruits and vegetables. The positive attributes of high catalytic activity and the ability to utilize different phenolic compounds as a substrate have led to a wide interest in its use for the construction of new biosensors. The level of PPO in plants is dependent on the species, cultivar, maturity and age. It is located in chloroplasts, mitochondria, microsomes, peroxisomes and cellular plasma [1–4]. PPO belongs to the group of oxidoreductases, and is a copper-containing enzyme capable of catalyzing the oxidation of monophenols and diphenols to the corresponding o-quinones [1–4]. The development of biosensors using plant homogenates as alternative biological materials to replace isolated enzymes has received considerable attention and has been successfully used by our group [5–9]. This class of materials maintains the enzyme of interest in its natural environment with higher stability and enzyme activity [1–4]. A biosensor can be defined as an analytical device that combines a biological material (e.g. enzymes, microorganisms, tissues)
∗ Corresponding author. Tel.: +55 48 3721 9852. E-mail address: [email protected] (A.C. Franzoi). 0141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2010.05.006
with an appropriate transducer (e.g. electrochemical, piezoelectric) capable of giving selective and/or quantitative analytical information. Electrochemical biosensors combine the analytical power of known techniques with the specificity of biological recognition processes. The aim is to biologically produce an electrical signal that relates to the concentration of the analyte. Enzymes may be immobilized by a variety of supports and methods, which may be classified as physical, where there are weak interactions between the support and the enzyme, and chemical, where covalent bonds are formed with the enzyme. A key factor in the construction of a biosensor is the need to achieve adequate and effective enzyme immobilization [10,11]. The PPO enzyme has been obtained from different plant tissues and immobilized in a variety of supports (e.g. chitosan, chitin) and these PPO biosensors employing alternative biological materials have been extensive used due to their high stability and catalytic activity [9,12–14]. Chitosan (CH) is a natural polysaccharide consisting of glucosamine and N-acetylglucosamine. It can be derived through the partial deacetylation of chitin, the major compound of the exoskeletons of crustaceans. The molecular unit of CH has one amino group and two hydroxyl groups that are potentially capable of being crosslinked with different substances. It is one of the most promising polymers and has considerable potential due to its biocompatibility, biodegradability, and low toxicity, along with a variety of biological activities including antimicrobial activity [15–17]. This biopolymer can be employed in the immobilization of the enzyme for the construction of biosensors for use in
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several fields of application, such as biomedical, industrial and chemical [5,7,15–18]. In a previous study, Franzoi et al. [18] successfully immobilized laccase onto CHcych. The Lac enzyme is entrapped within the interstitial space of the crosslinked waterinsoluble CH and this system mimics its natural environment which resulted in considerable stabilization of the enzyme structure and activity. Pesticides are a group of several organic compounds with different structures, which has been extensively used in agriculture. The determination of these substances is of great importance, because their abusive use poses a risk to human health [19–21]. Chromatographic methods [20–23] are commonly used for their determination. However, analysis of samples for pesticide content is generally time consuming, and requires expensive materials and preliminary steps to prepare the sample before injection. The electrochemical methods [24–26] described in the literature for the determination of these substances are performed using biosensors based on enzymes, such as organophosphorus hydrolase, organophosphorus acid hydrolase and parathion hydrolase for direct detection or biosensors based on the inhibition of the activity of cholinesterase immobilized using different techniques. These methods offer improved analytical characteristics such as high sensitivity, wider dynamic range, rapid response time, no need for elaborate sample preparation techniques and simple analysis in complex matrices. Reports published in the literature have demonstrated that inhibition of the PPO enzyme can be used for the determination of hydrazine, organophosphorus and carbamate pesticides [27–29]. Thiodicarb is a carbamate and its overuse poses a serious threat to non-target organisms in the environment, including fish. However, biosensors based on pequi (Caryocar brasiliense Camb.) fruit as a source of polyphenol oxidase enzyme for the determination this carbamate have not been reported in the literature. In this study, a novel pequi (C. brasiliense Camb.) tissue homogenate source of PPO was obtained and immobilized in CH crosslinked with cyanuric chloride (cych). A biosensor was developed and used for the square-wave voltammetric determination of thiodicarb. The characteristics and merits of the biosensor are described in the following sections. After optimization the proposed bioelectrode was used for the determination of thiodicarb in fresh fruit and vegetable samples and the results compared with those obtained using high-performance liquid chromatography (HPLC).
perature (25.0 ± 0.5 ◦ C) and all potentials were measured and reported vs. Ag/AgCl (3.0 M KCl). In a typical run, 10 mL of the supporting electrolyte was transferred to a clean, dry cell and the required volume of the thiodicarb and a fresh fruit or vegetable sample was added by micropipette. The SWV measurements were performed applying a sweep potential between +0.9 and −1.1 V, at a frequency of 10–100 Hz, pulse amplitude of 10–150 mV and scan increment of 5–50 mV, after successive additions of the analyte. After a stirring period of 60 s to homogenize the solution, square-wave voltammograms were recorded. High-performance liquid chromatography analysis of thiodicarb was carried out using a Varian Pro Star instrument fitted with a C18 column (250 mm × 4.6 mm × 5 m) with 65:35 (v/v) of water:acetonitrile as the mobile phase set at a flow rate of 1 mL min−1 and a UV detector set at 220 nm. The concentration was determined by recording the peak area and comparing the results with the calibration graph. 2.3. Crosslinked chitosan and measurement of polyphenol oxidase activity The solid support for polyphenol oxidase immobilization was obtained through the modification of the CH with cych, as previously described [18,30]. The PPO enzyme was obtained from the pequi (C. brasiliense Camb.) fruit. Twenty-five grams of the pulp of this material were homogenized in a mixer with 100 mL of phosphate buffer solution (0.1 M; pH 7.0) for 1 min. The homogenate was rapidly filtered and centrifuged at 18.000 rpm for 2 min at 4 ◦ C. The resulting supernatant was stored at 4 ◦ C in a refrigerator and utilized as a source of PPO after determination of the activity. One unit of activity was defined as the increase in the amount of enzyme that caused a change in absorbance of 0.001 min−1 at 420 nm for o-quinone produced by the reaction between 2.8 mL of catechol solution (0.05 M) in phosphate buffer (0.1 M; pH 7.0) and 0.2 mL of the enzymatic extract. 2.4. Biosensor construction The biosensor was prepared by hand-mixing 20 mg (10% w/w) of CHcych-PPO containing 250 U mL−1 and 140 mg (70%, w/w) of graphite powder with a mortar and pestle for 20 min to ensure a uniform mixture. To this mixture 40 mg (20%, w/w) of Nujol was added with subsequent mixing for least 20 min to produce the final paste The resulting modified carbon paste was tightly packed into a plastic syringe (1.0 mm internal diameter) and a copper wire was inserted to obtain the external electrical contact. The carbon paste electrode (bare CPE) was prepared in a similar way. 2.5. Preparation and analysis of samples Samples of two fresh fruits (peach and grape) and one of a leafy vegetable (lettuce) were acquired from local supermarkets (Campo Grande, Mato Grosso do Sul, Brazil) and analyzed using the proposed biosensor and HPLC. The samples were prepared by extracting 20 g of fresh fruit or vegetable with 50 mL of ethanol by hand-mixing in a mortar for 2 min and then filtering. Aliquots were transferred to an electrochemical cell and quantified by SWV, after successive additions of a thiodicarb standard solution. All measurements were performed in duplicate. An HPLC method for thiodicarb determination available in the Official Methods of Analysis [31] was used for the comparison of the analytical results with those obtained using the proposed biosensor.
2. Experimental 2.1. Chemicals and solutions Reagents were of analytical grade and employed without further purification. All solutions were prepared with ultrapure water (18.2 M cm) obtained from a MilliQ purification system. A phosphate buffer solution (0.1 M; pH 7.0) was used as the supporting electrolyte throughout the experiments. Chitosan with a deacetylation degree of 85% and thiodicarb were supplied by Sigma–Aldrich. Cyanuric chloride used to modify the CH was obtained from Fluka and toluene and ethanol from Sigma–Aldrich. Hydroquinone, catechol, ascorbic acid, uric acid, caffeic acid, tyrosine, fructose and glucose were purchased from Sigma–Aldrich. The carbon paste was prepared using graphite powder (Acheson 38, Fisher Scientific) and high purity Nujol purchased from Sigma–Aldrich. A thiodicarb standard solution (1.97 × 10−4 M) was prepared daily in phosphate buffer (0.1 M; pH 7.0) solution subjected to ultrasound for 2 min. 2.2. Instrumentation, electrochemical and HPLC measurements Electrochemical measurements, using square-wave voltammetry, 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 as the working electrode, a platinum wire as the counter electrode and Ag/AgCl (3.0 M KCl) as the reference electrode. Square-wave voltammetry (SWV) measurements were carried out in an unstirred, non de-aerated phosphate buffer solution (0.1 M; pH 7.0) at room tem-
3. Results and discussion 3.1. PPO immobilization and enzymatic reaction The good performance of biosensors is dependent on the success of the immobilization of the enzyme. The schematic representation of CHcych structure and the PPO immobilization are shown in Fig. 1. Thus, this water-insoluble support was found to be a very attractive matrix for PPO immobilization (the enzyme is entrapped within the interstitial space of the CHcych) and also an excellent material for the construction of biosensors, due to the interactions between the links, which can maintain the native enzyme behavior and activity. The PPO enzyme has a binding group with an affinity for aromatic compounds (the substrate site) and another, with an affinity for a metal binding agent (oxygen site). PPO activity is therefore affected by many inhibitors, e.g. triazine, phenyl-urea, herbicides, pesticides and a variety of environmental contaminants [27]. In this study, a biosensor containing CHcych-PPO was constructed and used for determination of thiodicarb based on the inhibition principle. Fig. 2 shows the possible inhibitory action of thiodicarb
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Fig. 1. Chemical modification of chitosan (CH) with cyanuric chloride (CHcych) and proposed immobilization of PPO in this support.
on the enzymatic activity of PPO on the biosensor surface. Initially, the hydroquinone (a) is oxidized by PPO in the presence of molecular oxygen to the corresponding p-benzoquinone (b), which is electrochemically reduced back to hydroquinone at +0.01 V vs. Ag/AgCl. Thus, when thiodicarb (c) is added, it is assumed that there will be a sulfoxidation reaction generating a product (d) [32]. 3.2. Optimization of experimental conditions of the biosensor Several experimental conditions were evaluated to optimize the biosensor performance: PPO concentration (150–2000 U mL−1 ),
supporting electrolyte and pH (3.5–8.0), along with the frequency (10–100 Hz), pulse amplitude (10–150 mV) and scan increment (5–50 mV) used in the SWV. The effect of different PPO unit values (150–2000 U mL−1 ) on the biosensor response was investigated and showed in Fig. 3(A). The analytical signals (cathodic peak currents) for 2.49 × 10−3 M hydroquinone in 0.1 M phosphate buffer solution at pH 7.0 increased with increases in the enzyme concentration up to 250 U mL−1 . Consequently, this enzyme concentration was selected for further studies. The effect of the different supporting electrolytes and pH, that is, 0.1 M acetate buffer solution (pH 3.0–5.0) and 0.1 M phosphate
Fig. 2. Possible inhibitory action of thiodicarb on the enzymatic activity of PPO on the biosensor surface: (a) hydroquinone, (b) p-benzoquinone, (c) thiodicarb and (d) sulfoxide product.
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Fig. 3. Effect of different (A) PPO unit values (150–2000 U mL−1 ) and (B) supporting electrolytes and pH (acetate buffer solution: pH 3.0–5.0 and phosphate buffer solution: pH 6.0–8.0) on the biosensor response to 2.49 × 10−3 M hydroquinone solution. These experiments were performed in triplicate.
buffer solution (pH 6.0–8.0), on the biosensor response to 2.49 × 10−3 M hydroquinone solution was investigated and illustrated in Fig. 3(B). The best voltammetric responses were obtained in phosphate buffer solution at pH 7.0. Finally, the instrumental parameters of the SWV were studied to obtain the best performance of the biosensor. Frequency, pulse amplitude and scan increment were then evaluated at between 10–100 Hz, 10–150 mV and 5–50 mV, respectively. The best analytical signal obtained for the biosensor was at frequency 10 Hz, pulse amplitude 120 mV and scan increment 15 mV. These experimental conditions were selected for subsequent experiments. Table 1 summarizes the range over which each variable was investigated and the optimal value found. 3.3. Analytical curve of hydroquinone and thiodicarb Fig. 4 shows the square-wave voltammograms (A) and analytical curve (A ) for hydroquinone, obtained employing the proposed biosensor. Under the optimal conditions established above, the analytical curve was linear from 5.06 × 10−6 to 3.03 × 10−4 M (−Ipc = 15.795 (±1.136) + 1.813 × 105 (±2.440 × 103 ) [hydroquinone]; r = 0.9989); where −Ipc is the cathodic peak current in A and [hydroquinone] is the concentration in M. Subsequently, the effect of hydroquinone concentration on the linearity of the thiodicarb analytical curve was investigated. The best agreement between the detection limit and correlation coefficient was observed at a hydroquinone concentration of 2.49 × 10−3 M. Therefore, this concentration was selected in this study. The thiodicarb response in the presence of the 2.49 × 10−3 M hydroquinone solution was obtained at a potential of +0.01 V vs. Ag/AgCl for the proposed biosensor. Fig. 4 shows the squarewave voltammograms (B) and analytical curve (B ), constructed Table 1 Optimization of the experimental conditions for the biosensor. Parameters investigated
Range studied
Optimal value
Polyphenol oxidase (U mL−1 ) Electrolyte support/pH
150–2000
250
Phosphate buffer/6.0–8.0 Acetate buffer/3.5–5.0 10–120 10–150 5–50
Phosphate buffer/7.0
Frequency (Hz) Pulse amplitude (mV) Scan increment (mV)
10 120 15
from cathodic current peak vs. thiodicarb concentration. As can be observed the cathodic current decreased with an increase in the thiodicarb concentration and this behavior is associated with inhibition of the enzyme activity by the pesticide added. The analytical curve was linear in the range of 3.75 × 10−7 to 2.23 × 10−6 M and the regression equation obtained for thiodicarb using this biosensor was: −Ipc = 295.103 (±0.896) − 5.247 × 107 (±1.006 × 106 ) with a 0.9990 correlation coefficient and detection limit (three times the standard deviation of the intercept/slope) of 1.58 × 10−7 M. 3.4. Repeatability, reproducibility and stability of the biosensor The repeatability of the current response for the proposed biosensor in a solution containing 7.49 × 10−7 M thiodicarb and 2.49 × 10−3 M of hydroquinone solution in phosphate buffer solution (0.1 M; pH 7.0) was investigated. The relative standard deviation was 1.3% for successive assays (n = 10). The reproducibility test using three biosensors prepared independently showed an acceptable reproducibility with a relative standard deviation of 0.4% under the optimized conditions described above. The good reproducibility may be due to the high sensitivity and efficiency of the immobilization of the polyphenol oxidase on the crosslinked CH. The long-term stability of the biosensor was investigated over a period of 30 days. It was dry-stored at room temperature and no noticeable change was found in the response using 7.49 × 10−7 M thiodicarb and 2.49 × 10−3 M of hydroquinone solution in phosphate buffer solution (0.1 M; pH 7.0). The CHcych-PPO biosensor was stable for at least 10 days (at least 250 determinations) without a change in the response. 3.5. Selectivity and interference studies Selectivity is important in terms of the practical use of the biosensors. In order to investigate this characteristic of the biosensor to different compounds, such as hydroquinone, catechol, ascorbic acid, uric acid, caffeic acid, tyrosine, fructose and glucose, a comparative study was carried out. The responses were obtained from solutions containing 2.49 × 10−3 M of the substrates in phosphate buffer solution, comparing the cathodic current values of each compound investigated. The sensitivity of the proposed biosensor followed the order: hydroquinone > catechol > caffeic acid. No response was observed for ascorbic acid, uric acid, tyrosine, fructose and glucose. Thus, in this study, hydroquinone was
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Fig. 4. (A) Square-wave voltammograms obtained using biosensor for (a) blank in phosphate buffer solution (0.1 M; pH 7.0) and hydroquinone solutions at the following concentrations: (b) 5.06 × 10−6 ; (c) 5.06 × 10−5 ; (d) 1.01 × 10−4 ; (e) 1.51 × 10−4 ; (f) 2.02 × 10−4 ; (g) 2.52 × 10−4 and (h) 3.03 × 10−4 M and (A ) calibration curve of hydroquinone. (B) Square-wave voltammograms obtained using the biosensor for (a) blank and (b) 2.49 × 10−3 M hydroquinone solution in phosphate buffer solution (0.1 M; pH 7.0) and thiodicarb solutions at the following concentrations: (c) 3.75 × 10−7 ; (d) 7.49 × 10−7 ; (e) 1.12 × 10−6 ; (f) 1.49 × 10−6 ; (g) 1.86 × 10−6 ; (h) 2.23 × 10−6 M and (B ) calibration curve of thiodicarb. The parameters used in these studies were frequency 10 Hz, pulse amplitude 120 mV and scan increment 15 mV.
selected for the optimization of the biosensor and application to real samples. To evaluate the interference in the analytical response of the biosensor, the influence of some possible interfering substance, i.e. catechol and caffeic acid, on the determination of hydroquinone in fresh fruit (peach and grape) and vegetable (lettuce) was investigated. The ratios of the concentration of hydroquinone to that of each substance were fixed at 1:1 and 1:5. The response of the biosensor in the presence of these excipients was compared with that obtained using only the hydroquinone standard solution. None of these substances interfered with the proposed procedure (the criterion for interference was a relative error of less than ±5%), that is, the biosensor was able to determine the amount of hydroquinone in the presence of these substances and can therefore be considered selective. The selectivity and interference studies were performed in duplicate.
3.6. Recovery tests and thiodicarb determination in fresh fruit and vegetable samples Analytical recovery measurements were obtained by adding different amounts of thiodicarb (0.198, 0.394 and 0.589 mg L−1 ) to fresh fruit (peach and grape) and vegetable (lettuce) samples. The percentage recovery values were calculated by comparing the concentrations obtained for the samples with and without the addition of known concentrations of thiodicarb standard solutions. Thiodicarb recoveries of 93.9–110.8% from these samples were obtained using the proposed biosensor as shown in Table 2. It can be clearly observed that the recovery results obtained suggest an absence of matrix effects in these determinations. To evaluate the applicability of the biosensor the thiodicarb concentrations of three fresh fruit and vegetable samples peach, grape and lettuce were determined applying the standard additions
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Table 2 Recovery of thiodicarb standard solutions (mg L−1 ) from fresh fruit and vegetable samples determined using the proposed biosensor. Thiodicarb (mg L−1 )
Sample
Added
Found
Recovered (%)
Lettuce
0.198 0.394 0.589
0.186 ± 0.001 0.400 ± 0.001 0.589 ± 0.002
93.9 101.6 101.4
Peach
0.198 0.394 0.589
0.212 ± 0.005 0.384 ± 0.004 0.607 ± 0.013
107.1 97.6 103.0
Grape
0.198 0.394 0.589
0.219 ± 0.043 0.418 ± 0.009 0.580 ± 0.005
110.8 106.1 98.5
Found: thiodicarb (mg L−1 ) detected by the biosensor; Recovered: [(found value/added value) × 100%]. Table 3 Determination of thiodicarb in fresh fruit and vegetable samples using the biosensor and the official method. Sample
Lettuce Peach Grape a b
Thiodicarb (mg kg−1 )
Relative error (%)a
Official method [31]
Biosensorb
3.17 ± 0.1 1.79 ± 0.1 1.67 ± 0.1
2.82 ± 0.2 1.83 ± 0.1 1.71 ± 0.2
−11.0 +2.2 +2.4
Biosensor vs. Official method. n = 3; confidence level of 95%.
method to overcome the matrix effects. The results obtained using the proposed biosensor were close to those obtained using the HPLC method (Table 3). As can be seen from the data, the results are in agreement at a 95% confidence level, within an acceptable range of error and it was therefore concluded that the biosensor is suitable for this application. 4. Conclusions Taking advantage of the excellent immobilization of PPO coupled with the inhibition effect of the thiodicarb on the PPOcatalyzed oxidation of hydroquinone to p-benzoquinone, a new effective biosensor for thiodicarb quantification was developed. It was optimized and characterized under different experimental conditions. The reported biosensor combines construction simplicity, linear calibration range, low detection limit, good repeatability and reproducibility and long-term stability with the sensitivity of the inhibition-based PPO. This device was applied to thiodicarb determination in fresh fruit and vegetable samples and the results were satisfactory when compared with those obtained using highperformance liquid chromatography. Acknowledgments Financial support from CNPq (Processes 470430/2009-5), MCT/CNPq/CT-Infra/CT-Petro/2008 and also a scholarship granted by CNPq to FL are gratefully acknowledged. References [1] Zamorano JP, Martínez-Álvarez O, Montero P, Gómez-Guillén MC. Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp (Parapenaeus longirostris). Food Chem 2009;112:104–11. [2] Mayer AM. Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry 2006;67:2318–31. [3] Fatibello-Filho O, Vieira IC. Uso analítico de tecidos e de extratos brutos vegetais como fonte enzimática. Quim Nova 2002;25:455–64.
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Biosensor based on pequi polyphenol oxidase immobilized on chitosan crosslinked with cyanuric chloride for thiodicarb determination Fábio de Lima a , Bruno G. Lucca a , Antônio M.J. Barbosa a , Valdir S. Ferreira a , Sally K. Moccelini b , Ana C. Franzoi b,∗ , Iolanda C. Vieira b a b
Departamento de Química, Universidade Federal do Mato Grosso do Sul, 79070-900, Campo Grande, MS, Brazil Departamento de Química, Laboratório de Biossensores, Universidade Federal de Santa Catarina, 88040-970, Florianópolis, SC, Brazil
a r t i c l e
i n f o
Article history: Received 2 March 2010 Received in revised form 13 April 2010 Accepted 15 May 2010 Keywords: Biosensor Thiodicarb Polyphenol oxidase Pequi fruit
a b s t r a c t A novel pequi fruit (Caryocar brasiliense Camb.) homogenate source of polyphenol oxidase was obtained and immobilized in chitosan crosslinked with cyanuric chloride (CHcych-PPO). A biosensor was developed and used for the square-wave voltammetric determination of thiodicarb. Several parameters were investigated to evaluate the performance of this biosensor in the presence of hydroquinone and thiodicarb. The best response was obtained using 70:20:10% (w/w/w) of graphite powder:Nujol:CHcych-PPO, 250 U mL−1 of PPO and phosphate buffer solution (0.1 M; pH 7.0) with frequency, pulse amplitude and scan increment of 10 Hz, 150 mV, and 15 mV, respectively. Under optimized operational conditions the thiodicarb concentration was linear in the range of 3.75 × 10−7 to 2.23 × 10−6 M with a detection limit of 1.58 × 10−7 M. The biosensor was applied in the determination of thiodicarb in fresh fruit and vegetable samples and the results compared with those obtained using high-performance liquid chromatography. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Polyphenol oxidase (PPO) has received continuous attention from food chemists and processors since its discovery over 90 years ago. This is because it is involved in the enzymatic browning of many edible plant products, especially fruits and vegetables. The positive attributes of high catalytic activity and the ability to utilize different phenolic compounds as a substrate have led to a wide interest in its use for the construction of new biosensors. The level of PPO in plants is dependent on the species, cultivar, maturity and age. It is located in chloroplasts, mitochondria, microsomes, peroxisomes and cellular plasma [1–4]. PPO belongs to the group of oxidoreductases, and is a copper-containing enzyme capable of catalyzing the oxidation of monophenols and diphenols to the corresponding o-quinones [1–4]. The development of biosensors using plant homogenates as alternative biological materials to replace isolated enzymes has received considerable attention and has been successfully used by our group [5–9]. This class of materials maintains the enzyme of interest in its natural environment with higher stability and enzyme activity [1–4]. A biosensor can be defined as an analytical device that combines a biological material (e.g. enzymes, microorganisms, tissues)
∗ Corresponding author. Tel.: +55 48 3721 9852. E-mail address: [email protected] (A.C. Franzoi). 0141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2010.05.006
with an appropriate transducer (e.g. electrochemical, piezoelectric) capable of giving selective and/or quantitative analytical information. Electrochemical biosensors combine the analytical power of known techniques with the specificity of biological recognition processes. The aim is to biologically produce an electrical signal that relates to the concentration of the analyte. Enzymes may be immobilized by a variety of supports and methods, which may be classified as physical, where there are weak interactions between the support and the enzyme, and chemical, where covalent bonds are formed with the enzyme. A key factor in the construction of a biosensor is the need to achieve adequate and effective enzyme immobilization [10,11]. The PPO enzyme has been obtained from different plant tissues and immobilized in a variety of supports (e.g. chitosan, chitin) and these PPO biosensors employing alternative biological materials have been extensive used due to their high stability and catalytic activity [9,12–14]. Chitosan (CH) is a natural polysaccharide consisting of glucosamine and N-acetylglucosamine. It can be derived through the partial deacetylation of chitin, the major compound of the exoskeletons of crustaceans. The molecular unit of CH has one amino group and two hydroxyl groups that are potentially capable of being crosslinked with different substances. It is one of the most promising polymers and has considerable potential due to its biocompatibility, biodegradability, and low toxicity, along with a variety of biological activities including antimicrobial activity [15–17]. This biopolymer can be employed in the immobilization of the enzyme for the construction of biosensors for use in
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several fields of application, such as biomedical, industrial and chemical [5,7,15–18]. In a previous study, Franzoi et al. [18] successfully immobilized laccase onto CHcych. The Lac enzyme is entrapped within the interstitial space of the crosslinked waterinsoluble CH and this system mimics its natural environment which resulted in considerable stabilization of the enzyme structure and activity. Pesticides are a group of several organic compounds with different structures, which has been extensively used in agriculture. The determination of these substances is of great importance, because their abusive use poses a risk to human health [19–21]. Chromatographic methods [20–23] are commonly used for their determination. However, analysis of samples for pesticide content is generally time consuming, and requires expensive materials and preliminary steps to prepare the sample before injection. The electrochemical methods [24–26] described in the literature for the determination of these substances are performed using biosensors based on enzymes, such as organophosphorus hydrolase, organophosphorus acid hydrolase and parathion hydrolase for direct detection or biosensors based on the inhibition of the activity of cholinesterase immobilized using different techniques. These methods offer improved analytical characteristics such as high sensitivity, wider dynamic range, rapid response time, no need for elaborate sample preparation techniques and simple analysis in complex matrices. Reports published in the literature have demonstrated that inhibition of the PPO enzyme can be used for the determination of hydrazine, organophosphorus and carbamate pesticides [27–29]. Thiodicarb is a carbamate and its overuse poses a serious threat to non-target organisms in the environment, including fish. However, biosensors based on pequi (Caryocar brasiliense Camb.) fruit as a source of polyphenol oxidase enzyme for the determination this carbamate have not been reported in the literature. In this study, a novel pequi (C. brasiliense Camb.) tissue homogenate source of PPO was obtained and immobilized in CH crosslinked with cyanuric chloride (cych). A biosensor was developed and used for the square-wave voltammetric determination of thiodicarb. The characteristics and merits of the biosensor are described in the following sections. After optimization the proposed bioelectrode was used for the determination of thiodicarb in fresh fruit and vegetable samples and the results compared with those obtained using high-performance liquid chromatography (HPLC).
perature (25.0 ± 0.5 ◦ C) and all potentials were measured and reported vs. Ag/AgCl (3.0 M KCl). In a typical run, 10 mL of the supporting electrolyte was transferred to a clean, dry cell and the required volume of the thiodicarb and a fresh fruit or vegetable sample was added by micropipette. The SWV measurements were performed applying a sweep potential between +0.9 and −1.1 V, at a frequency of 10–100 Hz, pulse amplitude of 10–150 mV and scan increment of 5–50 mV, after successive additions of the analyte. After a stirring period of 60 s to homogenize the solution, square-wave voltammograms were recorded. High-performance liquid chromatography analysis of thiodicarb was carried out using a Varian Pro Star instrument fitted with a C18 column (250 mm × 4.6 mm × 5 m) with 65:35 (v/v) of water:acetonitrile as the mobile phase set at a flow rate of 1 mL min−1 and a UV detector set at 220 nm. The concentration was determined by recording the peak area and comparing the results with the calibration graph. 2.3. Crosslinked chitosan and measurement of polyphenol oxidase activity The solid support for polyphenol oxidase immobilization was obtained through the modification of the CH with cych, as previously described [18,30]. The PPO enzyme was obtained from the pequi (C. brasiliense Camb.) fruit. Twenty-five grams of the pulp of this material were homogenized in a mixer with 100 mL of phosphate buffer solution (0.1 M; pH 7.0) for 1 min. The homogenate was rapidly filtered and centrifuged at 18.000 rpm for 2 min at 4 ◦ C. The resulting supernatant was stored at 4 ◦ C in a refrigerator and utilized as a source of PPO after determination of the activity. One unit of activity was defined as the increase in the amount of enzyme that caused a change in absorbance of 0.001 min−1 at 420 nm for o-quinone produced by the reaction between 2.8 mL of catechol solution (0.05 M) in phosphate buffer (0.1 M; pH 7.0) and 0.2 mL of the enzymatic extract. 2.4. Biosensor construction The biosensor was prepared by hand-mixing 20 mg (10% w/w) of CHcych-PPO containing 250 U mL−1 and 140 mg (70%, w/w) of graphite powder with a mortar and pestle for 20 min to ensure a uniform mixture. To this mixture 40 mg (20%, w/w) of Nujol was added with subsequent mixing for least 20 min to produce the final paste The resulting modified carbon paste was tightly packed into a plastic syringe (1.0 mm internal diameter) and a copper wire was inserted to obtain the external electrical contact. The carbon paste electrode (bare CPE) was prepared in a similar way. 2.5. Preparation and analysis of samples Samples of two fresh fruits (peach and grape) and one of a leafy vegetable (lettuce) were acquired from local supermarkets (Campo Grande, Mato Grosso do Sul, Brazil) and analyzed using the proposed biosensor and HPLC. The samples were prepared by extracting 20 g of fresh fruit or vegetable with 50 mL of ethanol by hand-mixing in a mortar for 2 min and then filtering. Aliquots were transferred to an electrochemical cell and quantified by SWV, after successive additions of a thiodicarb standard solution. All measurements were performed in duplicate. An HPLC method for thiodicarb determination available in the Official Methods of Analysis [31] was used for the comparison of the analytical results with those obtained using the proposed biosensor.
2. Experimental 2.1. Chemicals and solutions Reagents were of analytical grade and employed without further purification. All solutions were prepared with ultrapure water (18.2 M cm) obtained from a MilliQ purification system. A phosphate buffer solution (0.1 M; pH 7.0) was used as the supporting electrolyte throughout the experiments. Chitosan with a deacetylation degree of 85% and thiodicarb were supplied by Sigma–Aldrich. Cyanuric chloride used to modify the CH was obtained from Fluka and toluene and ethanol from Sigma–Aldrich. Hydroquinone, catechol, ascorbic acid, uric acid, caffeic acid, tyrosine, fructose and glucose were purchased from Sigma–Aldrich. The carbon paste was prepared using graphite powder (Acheson 38, Fisher Scientific) and high purity Nujol purchased from Sigma–Aldrich. A thiodicarb standard solution (1.97 × 10−4 M) was prepared daily in phosphate buffer (0.1 M; pH 7.0) solution subjected to ultrasound for 2 min. 2.2. Instrumentation, electrochemical and HPLC measurements Electrochemical measurements, using square-wave voltammetry, 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 as the working electrode, a platinum wire as the counter electrode and Ag/AgCl (3.0 M KCl) as the reference electrode. Square-wave voltammetry (SWV) measurements were carried out in an unstirred, non de-aerated phosphate buffer solution (0.1 M; pH 7.0) at room tem-
3. Results and discussion 3.1. PPO immobilization and enzymatic reaction The good performance of biosensors is dependent on the success of the immobilization of the enzyme. The schematic representation of CHcych structure and the PPO immobilization are shown in Fig. 1. Thus, this water-insoluble support was found to be a very attractive matrix for PPO immobilization (the enzyme is entrapped within the interstitial space of the CHcych) and also an excellent material for the construction of biosensors, due to the interactions between the links, which can maintain the native enzyme behavior and activity. The PPO enzyme has a binding group with an affinity for aromatic compounds (the substrate site) and another, with an affinity for a metal binding agent (oxygen site). PPO activity is therefore affected by many inhibitors, e.g. triazine, phenyl-urea, herbicides, pesticides and a variety of environmental contaminants [27]. In this study, a biosensor containing CHcych-PPO was constructed and used for determination of thiodicarb based on the inhibition principle. Fig. 2 shows the possible inhibitory action of thiodicarb
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Fig. 1. Chemical modification of chitosan (CH) with cyanuric chloride (CHcych) and proposed immobilization of PPO in this support.
on the enzymatic activity of PPO on the biosensor surface. Initially, the hydroquinone (a) is oxidized by PPO in the presence of molecular oxygen to the corresponding p-benzoquinone (b), which is electrochemically reduced back to hydroquinone at +0.01 V vs. Ag/AgCl. Thus, when thiodicarb (c) is added, it is assumed that there will be a sulfoxidation reaction generating a product (d) [32]. 3.2. Optimization of experimental conditions of the biosensor Several experimental conditions were evaluated to optimize the biosensor performance: PPO concentration (150–2000 U mL−1 ),
supporting electrolyte and pH (3.5–8.0), along with the frequency (10–100 Hz), pulse amplitude (10–150 mV) and scan increment (5–50 mV) used in the SWV. The effect of different PPO unit values (150–2000 U mL−1 ) on the biosensor response was investigated and showed in Fig. 3(A). The analytical signals (cathodic peak currents) for 2.49 × 10−3 M hydroquinone in 0.1 M phosphate buffer solution at pH 7.0 increased with increases in the enzyme concentration up to 250 U mL−1 . Consequently, this enzyme concentration was selected for further studies. The effect of the different supporting electrolytes and pH, that is, 0.1 M acetate buffer solution (pH 3.0–5.0) and 0.1 M phosphate
Fig. 2. Possible inhibitory action of thiodicarb on the enzymatic activity of PPO on the biosensor surface: (a) hydroquinone, (b) p-benzoquinone, (c) thiodicarb and (d) sulfoxide product.
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Fig. 3. Effect of different (A) PPO unit values (150–2000 U mL−1 ) and (B) supporting electrolytes and pH (acetate buffer solution: pH 3.0–5.0 and phosphate buffer solution: pH 6.0–8.0) on the biosensor response to 2.49 × 10−3 M hydroquinone solution. These experiments were performed in triplicate.
buffer solution (pH 6.0–8.0), on the biosensor response to 2.49 × 10−3 M hydroquinone solution was investigated and illustrated in Fig. 3(B). The best voltammetric responses were obtained in phosphate buffer solution at pH 7.0. Finally, the instrumental parameters of the SWV were studied to obtain the best performance of the biosensor. Frequency, pulse amplitude and scan increment were then evaluated at between 10–100 Hz, 10–150 mV and 5–50 mV, respectively. The best analytical signal obtained for the biosensor was at frequency 10 Hz, pulse amplitude 120 mV and scan increment 15 mV. These experimental conditions were selected for subsequent experiments. Table 1 summarizes the range over which each variable was investigated and the optimal value found. 3.3. Analytical curve of hydroquinone and thiodicarb Fig. 4 shows the square-wave voltammograms (A) and analytical curve (A ) for hydroquinone, obtained employing the proposed biosensor. Under the optimal conditions established above, the analytical curve was linear from 5.06 × 10−6 to 3.03 × 10−4 M (−Ipc = 15.795 (±1.136) + 1.813 × 105 (±2.440 × 103 ) [hydroquinone]; r = 0.9989); where −Ipc is the cathodic peak current in A and [hydroquinone] is the concentration in M. Subsequently, the effect of hydroquinone concentration on the linearity of the thiodicarb analytical curve was investigated. The best agreement between the detection limit and correlation coefficient was observed at a hydroquinone concentration of 2.49 × 10−3 M. Therefore, this concentration was selected in this study. The thiodicarb response in the presence of the 2.49 × 10−3 M hydroquinone solution was obtained at a potential of +0.01 V vs. Ag/AgCl for the proposed biosensor. Fig. 4 shows the squarewave voltammograms (B) and analytical curve (B ), constructed Table 1 Optimization of the experimental conditions for the biosensor. Parameters investigated
Range studied
Optimal value
Polyphenol oxidase (U mL−1 ) Electrolyte support/pH
150–2000
250
Phosphate buffer/6.0–8.0 Acetate buffer/3.5–5.0 10–120 10–150 5–50
Phosphate buffer/7.0
Frequency (Hz) Pulse amplitude (mV) Scan increment (mV)
10 120 15
from cathodic current peak vs. thiodicarb concentration. As can be observed the cathodic current decreased with an increase in the thiodicarb concentration and this behavior is associated with inhibition of the enzyme activity by the pesticide added. The analytical curve was linear in the range of 3.75 × 10−7 to 2.23 × 10−6 M and the regression equation obtained for thiodicarb using this biosensor was: −Ipc = 295.103 (±0.896) − 5.247 × 107 (±1.006 × 106 ) with a 0.9990 correlation coefficient and detection limit (three times the standard deviation of the intercept/slope) of 1.58 × 10−7 M. 3.4. Repeatability, reproducibility and stability of the biosensor The repeatability of the current response for the proposed biosensor in a solution containing 7.49 × 10−7 M thiodicarb and 2.49 × 10−3 M of hydroquinone solution in phosphate buffer solution (0.1 M; pH 7.0) was investigated. The relative standard deviation was 1.3% for successive assays (n = 10). The reproducibility test using three biosensors prepared independently showed an acceptable reproducibility with a relative standard deviation of 0.4% under the optimized conditions described above. The good reproducibility may be due to the high sensitivity and efficiency of the immobilization of the polyphenol oxidase on the crosslinked CH. The long-term stability of the biosensor was investigated over a period of 30 days. It was dry-stored at room temperature and no noticeable change was found in the response using 7.49 × 10−7 M thiodicarb and 2.49 × 10−3 M of hydroquinone solution in phosphate buffer solution (0.1 M; pH 7.0). The CHcych-PPO biosensor was stable for at least 10 days (at least 250 determinations) without a change in the response. 3.5. Selectivity and interference studies Selectivity is important in terms of the practical use of the biosensors. In order to investigate this characteristic of the biosensor to different compounds, such as hydroquinone, catechol, ascorbic acid, uric acid, caffeic acid, tyrosine, fructose and glucose, a comparative study was carried out. The responses were obtained from solutions containing 2.49 × 10−3 M of the substrates in phosphate buffer solution, comparing the cathodic current values of each compound investigated. The sensitivity of the proposed biosensor followed the order: hydroquinone > catechol > caffeic acid. No response was observed for ascorbic acid, uric acid, tyrosine, fructose and glucose. Thus, in this study, hydroquinone was
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Fig. 4. (A) Square-wave voltammograms obtained using biosensor for (a) blank in phosphate buffer solution (0.1 M; pH 7.0) and hydroquinone solutions at the following concentrations: (b) 5.06 × 10−6 ; (c) 5.06 × 10−5 ; (d) 1.01 × 10−4 ; (e) 1.51 × 10−4 ; (f) 2.02 × 10−4 ; (g) 2.52 × 10−4 and (h) 3.03 × 10−4 M and (A ) calibration curve of hydroquinone. (B) Square-wave voltammograms obtained using the biosensor for (a) blank and (b) 2.49 × 10−3 M hydroquinone solution in phosphate buffer solution (0.1 M; pH 7.0) and thiodicarb solutions at the following concentrations: (c) 3.75 × 10−7 ; (d) 7.49 × 10−7 ; (e) 1.12 × 10−6 ; (f) 1.49 × 10−6 ; (g) 1.86 × 10−6 ; (h) 2.23 × 10−6 M and (B ) calibration curve of thiodicarb. The parameters used in these studies were frequency 10 Hz, pulse amplitude 120 mV and scan increment 15 mV.
selected for the optimization of the biosensor and application to real samples. To evaluate the interference in the analytical response of the biosensor, the influence of some possible interfering substance, i.e. catechol and caffeic acid, on the determination of hydroquinone in fresh fruit (peach and grape) and vegetable (lettuce) was investigated. The ratios of the concentration of hydroquinone to that of each substance were fixed at 1:1 and 1:5. The response of the biosensor in the presence of these excipients was compared with that obtained using only the hydroquinone standard solution. None of these substances interfered with the proposed procedure (the criterion for interference was a relative error of less than ±5%), that is, the biosensor was able to determine the amount of hydroquinone in the presence of these substances and can therefore be considered selective. The selectivity and interference studies were performed in duplicate.
3.6. Recovery tests and thiodicarb determination in fresh fruit and vegetable samples Analytical recovery measurements were obtained by adding different amounts of thiodicarb (0.198, 0.394 and 0.589 mg L−1 ) to fresh fruit (peach and grape) and vegetable (lettuce) samples. The percentage recovery values were calculated by comparing the concentrations obtained for the samples with and without the addition of known concentrations of thiodicarb standard solutions. Thiodicarb recoveries of 93.9–110.8% from these samples were obtained using the proposed biosensor as shown in Table 2. It can be clearly observed that the recovery results obtained suggest an absence of matrix effects in these determinations. To evaluate the applicability of the biosensor the thiodicarb concentrations of three fresh fruit and vegetable samples peach, grape and lettuce were determined applying the standard additions
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Table 2 Recovery of thiodicarb standard solutions (mg L−1 ) from fresh fruit and vegetable samples determined using the proposed biosensor. Thiodicarb (mg L−1 )
Sample
Added
Found
Recovered (%)
Lettuce
0.198 0.394 0.589
0.186 ± 0.001 0.400 ± 0.001 0.589 ± 0.002
93.9 101.6 101.4
Peach
0.198 0.394 0.589
0.212 ± 0.005 0.384 ± 0.004 0.607 ± 0.013
107.1 97.6 103.0
Grape
0.198 0.394 0.589
0.219 ± 0.043 0.418 ± 0.009 0.580 ± 0.005
110.8 106.1 98.5
Found: thiodicarb (mg L−1 ) detected by the biosensor; Recovered: [(found value/added value) × 100%]. Table 3 Determination of thiodicarb in fresh fruit and vegetable samples using the biosensor and the official method. Sample
Lettuce Peach Grape a b
Thiodicarb (mg kg−1 )
Relative error (%)a
Official method [31]
Biosensorb
3.17 ± 0.1 1.79 ± 0.1 1.67 ± 0.1
2.82 ± 0.2 1.83 ± 0.1 1.71 ± 0.2
−11.0 +2.2 +2.4
Biosensor vs. Official method. n = 3; confidence level of 95%.
method to overcome the matrix effects. The results obtained using the proposed biosensor were close to those obtained using the HPLC method (Table 3). As can be seen from the data, the results are in agreement at a 95% confidence level, within an acceptable range of error and it was therefore concluded that the biosensor is suitable for this application. 4. Conclusions Taking advantage of the excellent immobilization of PPO coupled with the inhibition effect of the thiodicarb on the PPOcatalyzed oxidation of hydroquinone to p-benzoquinone, a new effective biosensor for thiodicarb quantification was developed. It was optimized and characterized under different experimental conditions. The reported biosensor combines construction simplicity, linear calibration range, low detection limit, good repeatability and reproducibility and long-term stability with the sensitivity of the inhibition-based PPO. This device was applied to thiodicarb determination in fresh fruit and vegetable samples and the results were satisfactory when compared with those obtained using highperformance liquid chromatography. Acknowledgments Financial support from CNPq (Processes 470430/2009-5), MCT/CNPq/CT-Infra/CT-Petro/2008 and also a scholarship granted by CNPq to FL are gratefully acknowledged. References [1] Zamorano JP, Martínez-Álvarez O, Montero P, Gómez-Guillén MC. Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp (Parapenaeus longirostris). Food Chem 2009;112:104–11. [2] Mayer AM. Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry 2006;67:2318–31. [3] Fatibello-Filho O, Vieira IC. Uso analítico de tecidos e de extratos brutos vegetais como fonte enzimática. Quim Nova 2002;25:455–64.
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