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Phenol determination by an amperométrico biosensor based on lyophilized mushroom (Agaricus bisporus) tissue a
a
a
L.M.C. Silva , A.C.C. de Mello & A.M. Salgado a
Laboratory of Biological Sensors, Chemistry School, Technology Center, Federal University of Rio de Janeiro, Horácio Macedo Avenue, 2030 – University City, Ilha do Fundão, Rio de Janeiro, RJ 21949-909, Brazil Accepted author version posted online: 24 Oct 2013.Published online: 22 Nov 2013.
Click for updates To cite this article: L.M.C. Silva, A.C.C. de Mello & A.M. Salgado (2014) Phenol determination by an amperométrico biosensor based on lyophilized mushroom (Agaricus bisporus) tissue, Environmental Technology, 35:8, 1012-1017, DOI: 10.1080/09593330.2013.858755 To link to this article: http://dx.doi.org/10.1080/09593330.2013.858755
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Environmental Technology, 2014 Vol. 35, No. 8, 1012–1017, http://dx.doi.org/10.1080/09593330.2013.858755
Phenol determination by an amperométrico biosensor based on lyophilized mushroom (Agaricus bisporus) tissue L.M.C. Silva∗ , A.C.C. de Mello and A.M. Salgado Laboratory of Biological Sensors, Chemistry School, Technology Center, Federal University of Rio de Janeiro, Horácio Macedo Avenue, 2030 – University City, Ilha do Fundão, Rio de Janeiro, RJ 21949-909, Brazil
Downloaded by [Erciyes University] at 20:11 07 January 2015
(Received 1 April 2013; final version received 25 August 2013 ) A simple and inexpensive biosensor based on lyophilized mushroom tissue (Agaricus bisporus) was developed for amperometric determination of phenol. This fungi tissue contains tyrosinase (EC 1.14.18.1) enzyme that catalysis two sequential oxidation reactions with phenolic substrates. Both reactions involve molecular oxygen; therefore, the commercial Clark-type oxygen electrode was selected as a transducer. The lyophilized biocomponent was tested in two different forms: cubes (at two positions in the biosensor system) or powder. In characterization studies of the biosensor, some parameters such as time reaction, linear range and repeatability were investigated. For the best biosensor configuration, a linear response was observed from 0.1 to 10.0 mg L−1 phenol; variation coefficient and standard deviation were calculated as 0.02% and ±0.11 mg L−1 , respectively. Keywords: phenol; biosensor; fungi tissue; Clark-type oxygen electrode; tyrosinase
1. Introduction Over recent years, electrochemical biosensors are becoming an accepted part of analytical chemistry since they fulfil the expanding need for rapid and reliable measurements. Therefore, analytical technology based on these instruments is an extremely broad field that impacts on many major industrial sectors such as agriculture industries and environmental monitoring. A great number of agricultural and industrial activities discharge phenolic compounds in the environment. Phenols are also released into the environment by the degradation of pesticides with phenolic skeleton. The phenol levels control is very important for the environmental protection. Phenols and especially their chlorinated, nitro and alkyl derivatives have been defined as hazardous pollutants due to their high toxicity and persistence in the environment and are found in the hazardous substances and priority pollutants list of the European Commission and the U.S. Environmental Protection Agency.[1] The Brazilian Environmental Law (CONAMA no. 357/2005 and no. 397/2008) regulates that the phenol concentration limit is 0.5 mg L−1 in the wastewater discharged in the environment).[2] Traditionally, phenol analysis has been based on spectrophotometric or chromatographic methods, which do not always allow easy and continuous monitoring, are more expensive and often require sample pretreatment steps, resulting in increased time and low cost-effectiveness. Therefore, the need for disposable systems or tools for ∗ Corresponding
author. Email: [email protected]
© 2013 Taylor & Francis
environmental applications has encouraged the development of new technologies and more suitable methodologies. In this context, biosensors appear as a suitable alternative or as a complementary analytical tool.[3–5] A biosensor shown in Figure 1 is defined by the International Union of Pure and Applied Chemistry (IUPAC) as a self-contained integrated device that is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor), which is retained in direct spatial contact with a transduction element.[6] In this context, this study was aimed to develop an amperometric biosensor based on mushroom tissue (Agaricus bisporus) in a simple and inexpensive way to be applied in real environmental samples.[2,7] 2. Materials and methods 2.1. Reagents and solutions All chemicals were analytical reagent grade and were acquired from Vetec Química Fina (Rio de Janeiro, Brazil) and Sigma (St. Louis, USA). Phosphate buffer solutions were prepared using distilled water. 2.2.
Biocomponent: A. bisporus tissue
In a search for economic and efficient biological components to be used in phenolic compound biosensors,
Environmental Technology
Downloaded by [Erciyes University] at 20:11 07 January 2015
Figure 1.
A biosensor simplified scheme.
polyphenol oxidases, in particular, tyrosinase (EC 1.14.18.1) (a polyphenol oxidase with a relative selectivity for phenolic compounds)has been investigated in the last few years. The tyrosinase biosensors are applicable to the monitoring of phenol and ortho-benzenediols.[1] Many biosensors have been developed using tyrosinase from A. bisporus as the biocomponent.[8–15] However, few studies have been using this enzyme naturally immobilized in mushroom tissue. The purpose of this work was to continue the work of Silva et al. [16] in the development of an amperometric biosensor for phenol detection using lyophilized mushroom tissue (cube or powder) as the tyrosinase source. The mushrooms used in the biosensor preparation were purchased from a local market (Rio de Janeiro, Brazil) as fresh and culture vegetables. They were stored at 4◦ C until use.
2.3. Instrumentation: the biosensor system The schematic set-up for the biosensor system for phenol analysis is presented as Figure 2.[17] The set-up consists of a standard sample (1), peristaltic pump (Milan Equipamentos Ltda) (2), reaction chamber made from PVC pipe with a biocomponent (cube or powder) (3), transducer (oxygen electrode) (OD/O2/Saturação DM-4P Digimed) and data recorder (4) and discard sample (5). Silicone tubing was used for connections.
2.4. Measurement procedure For phenol analysis, calibration standards were prepared by dilution of phenol stock solution in phosphate buffer, pH 8.0. All measurements were carried out by injection of 50.00 mL standard sample (0.10–50.00 mg L−1 ) at a flow rate of 40.00 mL min−1 . After the sample had filled up the reaction chamber, the injection pump was shut down followed by the insertion of the calibrated oxygen electrode in the reaction chamber. Then, data were collected after the chosen reaction time. After each sample analysis, the system was thoroughly rinsed with distilled water for 2 min. The amperométrico measurements were made at room temperature (24 ± 1◦ C). Measurements were carried out by noting the decrease in dissolved oxygen concentration in relation to substrate concentration added into the biosensor system. This can be explained by the fact that dissolved oxygen electrode has a membrane that separates the internal electrolyte and the electrodes (anode and cathode) from the external medium. Oxygen can pass through this membrane by diffusion until platinum cathode. A redox reaction occurs with four electrons, and it generates a current, proportional to oxygen concentration. The enzymatic reaction involved is shown in Figure 3. Therefore, the higher substrate concentration in the sample will increase the dissolved oxygen variation detection. The biosensor response was defined by the decrease in dissolved oxygen concentration, which was linearly related to the ethanol concentration in the standard solutions. Thereby, the calibration curve of phenol concentration versus dissolved oxygen concentration change was obtained. 2.5.
Figure 2. analysis.
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Schematic set-up for the biosensor system for phenol
Optimization of the biosensor system configuration
The initial tests had the intent to choose the best configuration of the biosensor system (Figure 2). The mushroom tissue amount and form (cube or powder) and enzymatic reaction time were investigated. Assays were performed according to Section 2.4; however, data were collected throughout 30 min, in order to analyse the instrument
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Figure 3. Simplified scheme of the reaction catalysed by the tyrosinase, which represents the cresolase activity (1) and the catecholase activity (2).
room tissue amount and form in the linearity range of the instrument. For this, the range of 1.0–30.0 mg L−1 of phenol was chosen. The results of these assays are shown in Figures 5 and 6. Therefore, as can be seen in these figures, the curve related to the position A on the biosensor system had better results and the chosen reaction time was 6 min. Later, to detect the effect of the mushroom (powder) amount in position A in the biosensor system on instrument response, two quantities (0.5 and 1.0 g) were selected. The results of these assays (the average of triplicates) are shown in Figure 7, where the curve related to the amount of 0.5 g had better results. Therefore, Figure 8 shows the result of
Figure 4. The two positions (A and B) in the reaction chamber were chosen: directly coupled to the transducer system (A) or immediately preceding this (B).
response time. The biosensor response time is defined as the enzymatic reaction reaching the steady state. The lyophilized mushroom tissue was shaped as cubes with 1 cm (5.0 g) or was used as powder (0.5 and 1.0 g). Furthermore, to detect the effect of the mushroom (cubes) position into the biosensor system on instrument response, two sites were used as can be seen from Figure 4. 2.6.
Figure 5. Study of the biosensor response time when the biocomponent (cubes, 5 g) was placed in position A in contact with phenol solution (10 mg L−1 ) in the biosensor system.
Repeatability
The repeatability of the phenol biosensor response was also studied by measuring the response (n = 5) when it was used in phenol solution (10 mg L−1 ) under the chosen reaction time for each quantity of biocomponent form and the others at optimum working conditions. The assays were performed according to Section 2.4. 3. Results and discussion 3.1. Effect of the amount and form of lyophilized mushroom and time reaction The preliminary tests with the biosensor system were aimed to find the response time and the effect of lyophilized mush-
Figure 6. Study of the biosensor response time when the biocomponent (cubes, 5 g) was placed in position B in contact with phenol solution (10 mg L−1 ) in the biosensor system.
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Environmental Technology the test, in triplicate, using the amount of 0.5 g of the biocomponent. As shown, the biosensor response increased with time until few minutes reaction time. It can be noted that the time taken to achieve steady-state response was not longer. This could be due to the diffusion of analyte in the mushroom (powder) not affected by the biosensor response. Therefore, the chosen reaction time was 10 min. The reaction time value determined in this study is consistent with other studies in the literature that used plant material or fungal in nature in the biosensor development. Uchiyama et al. [18] used tissue from spinach leaves (Spinacea oleracea) stings supported by a dialysis membrane in the development of biosensor for catechol, with a response time of 5–10 min. Timur et al. [19] and Abdullah et al. [20] used the respective developing biosensors for the detection of phenolic compounds and found the response time of 5 min. Using the same transducer work, oxygen electrode, Campanella et al. [21] developed an amperometric biosensor for ascorbic acid that has the best response time of 2 min using 3 mg of tyrosinase placed under the transducer. 3.2. Calibration curve To determine the phenol concentration, oxygen consumption that occurred in the reaction catalysed by tyrosinase
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in the mushroom was detected. With the available studies on phenol biosensors using commercial tyrosinase immobilized on various matrices, the linearity in concentration range between 10−9 and 10−4 mol L−1 for several phenolic compounds was obtained.[8,22] The results of the construction of calibration curves are shown in Figures 9 and 10. For lyophilized mushroom (cubes), data analysis with STATISTICA Software Version Trial (StatSoft, Inc. 1984– 2011) showed significance (α = 95%) between the data with R2 above 0.93 in all three curves in the range of 5.0– 25.0 mg L−1 phenol, indicating that there is a direct linear correlation between the input (phenol concentration) and output (signal variation of the oxygen electrode). Although for lyophilized mushroom (powder), data analysis showed significance (α = 95%) between the data with R2 above 0.95 in all curves in the range of 0.1–10.0 mg L−1 phenol. 3.3. Repeatability Repeatability of the biosensor was also studied for phenol concentration of 10 mg L−1 (n = 5) under the optimum working conditions. According to the results obtained from the experiments for lyophilized mushroom (cubes), the standard deviation (SD) and coefficient of variation (CV %) were 0.54 mg L−1 and 47.83%, respectively.
Figure 7. Study of the biosensor response time when the biocomponent (powder; 0.5 and 1.0 g) was placed in position A in contact with phenol solution (10 mg L−1 ) in the biosensor system.
Figure 9. Calibration curves of the biosensor. Working conditions: phosphate buffer, pH 8.0. Five gram of the lyophilized mushroom tissue (cubes) was placed at position A on the system. Response time: 6 min.
Figure 8. Study of the biosensor response time with best working conditions (position A and 0.5 g of powder). Phenol concentration used as substrate in the experiments was 10 mg L−1 .
Figure 10. Calibration curves of the biosensor. Working conditions: phosphate buffer, pH 8.0. In total, 0.5 g of the lyophilized mushroom tissue (powder) was placed at position A on the system. Response time: 10 min.
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Possibly, this variation is due to the biocomponent and its maturation degree that has been observed by Silva et al. [2,7] Moreover, the problem of low reproducibility of analytical characteristics of biosensors that utilize plant and fungal tissue is reported in the literature by FatibeloFilho and Vieira,[23] worsening when treating polyphenol oxidases due to the formation of quinones that promote a decrease in the sensitivity of the system. Furthermore, Rosatto et al. [24] show that electrode biosensors that use oxygen Clark-type have the disadvantage of having your response influenced by fluctuations in the concentration of dissolved oxygen resulting from variations in pH, temperature, ionic strength or partial pressure. Therefore, it is clear that developing an instrument that uses fungal tissue and oxygen electrode as a transducer is a difficult task for being susceptible to large external influence and their own inherent biocomponent compounds. However, according to the results obtained from the experiments for lyophilized mushroom (powder), the SD and CV % were ± 0.11 mg L−1 and 0.02%, respectively. Therefore, a promising alternative is the use of powder, which helps to increase the catalytic activity and provide a larger surface area, imparting a higher sensitivity to the biosensor. In the literature, the repeatability of the biosensor developed by Topçu et al. [10] was tested by repeating the experiment eight times, finding a low value of 0.002 × 10−3 mol L−1 as SD. Akyilmaz and Dinçkaya [25] studied the repeatability of the instrument by performing 10 repetitions, finding a value of SD of 0.23 × 10−3 mol L−1 . Thereby, the results with the lyophilized powder in the phenol biosensor development show a promising future.
4. Conclusion A biosensor based on mushroom (A. bisporus) lyophilized tissue was developed for the amperometric determination of phenolic compounds. In biosensor design development, the best configuration was achieved when using the lyophilized powder placed at position A of the system – closer to the oxygen electrode position and a reaction time of 10 min is chosen. A linear response was observed for 0.1– 10.0 mg L−1 phenol. In repeatability studies, CV % and SD were calculated as 0.02% and ±0.11 mg L−1 , respectively. The results showed that biosensor developed offer an alternative to other biosensors based on isolated enzymes. The next step is the application of the biosensor in environmental samples and comparison with the colorimetric method described in Standard Methods of American Public Health Association.
Funding The authors thank the financial support of the National Council for Scientific and Technological Development
(CNPq) and National Institute of Metrology, Quality and Technology (INMETRO). References [1] Silva LMC, Melo AF, Salgado AM. Biosensor for environmental applications. In: Vernon Somerset, editor. Environmental biosensors. Croatia: InTech; 2011. ISBN: 978-953307-486-3. [2] Silva LMC, Salgado AM, Coelho MAZ. Development of an amperometric biosensor for phenol detection. Environ Technol. 2011;32(5):493–497. [3] Rogers KR, Gerlach CL. Environmental biosensors: a status report. Environ Sci Technol. 1996;30:486–491. [4] Rodriguez-Mozaz S, Marco M-P, Alda MJL, Barceló D. Biosensors for environmental applications: future development trends. Pure Appl Chem. 2004;76:723–752. [5] Rogers KR. Recent advances in biosensor techniques for environmental monitoring. Anal Chim Acta. 2006;568:222– 231. [6] Thévenot DR, Toth K, Durst RA, Wilson GS. Electrochemical biosensors: recommended definitions and classification. Pure Appl Chem. 1999;71:2333–2348. [7] Silva LMC, Salgado AM, Coelho MAZ. Agaricus bisporus as a source of tyrosinase for phenol detection for future biosensor development. Environ Technol. 2010;31(6):611– 616. [8] Kochana J, Nowak P, Jarosz-Wilkołazka A, Biero´n B. Tyrosinase/laccase bienzyme biosensor for amperometric determination of phenolic compounds. Microchem J. 2008;89:171–174. [9] Zejli H, Hidalgo-Hidalgo de Cisneros JL, NaranjoRodriguez I, Liu B, Temsamani KR, Marty JL. Phenol biosensor based on sonogel-carbon transducer with tyrosinase alumina sol–gel immobilization. Anal Chim Acta. 2008;612:198–203. [10] Topçu S, Sezginturk MK, Dinçkaya E. 2004, Evaluations of a new biosensor-based mushroom (Agaricus bisporus) tissue homogenate: investigation of certain phenolic compounds and some inhibitor effects. Biosens Bioelectron. 2004;20:592–597. [11] dos Santos VPS, Silva LMC, Salgado AM, Pereira KS. Application of Agaricus bisporus extract for benzoate sodium detection based on tyrosinase inhibition for biosensor development. Chem Eng Trans. 2013;32:1831–1836. [12] Sezgintûrk MK, Gôktug T, Dinçkaya E. Detection of benzoic acid by an amperometric inhibitor biosensor based on mushroom tissue homogenate. Food Bioprocess Technol. 2005;43(4):329–334. [13] Stoyanov NS, Neykov AN. Three parametric biosensor measurement system. Biotechnol Biotechnol Eq. 2005;19(2):211–214. [14] Abhijith KS, Sujith Kumar PV, Kumar MA, Thakur MS. Immobilised tyrosinase-based biosensor for the detection of tea polyphenols. Anal Bioanal Chem. 2007;389:2227–2234. [15] Sezginturk MK, Dinçkaya E. Sulfite determination by an inhibitor biosensor-based mushroom (Agaricus bisporus) tissue homogenate. Artif Cells Blood Substit Immobil Biotechnol. 2012;40(1–2):38–43. [16] Silva LMC, Salgado AM, Coelho MAZ. Amperometric biosensor for phenol determination. Chem Eng Trans. 2011;24:1249–1254. [17] Silva LMC. Eletrochemical biosensors development for phenol and urea for environmental application. Doctoral thesis. Rio de Janeiro, Brazil: Federal University of Rio de Janeiro; 2011.
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[18] Uchiyama S, Tamata M, Tofuku Y, Suzuki S. A catechol electrode based on spinach leaves. Anal Chim Acta. 1988;208:287–290. [19] Timur S, Pazarlioglu N, Pilloton R, Telefoncu A. Thick film sensors based on laccases from different sources immobilized in polyaniline matrix. Sensor Actuat B Chem. 2004;97: 132–136. [20] Abdullah J, Ahmad M, Karuppiah N, Heng LY, Sidek H. Immobilization of tyrosinase in chitosan film for an optical detection of phenol. Sensor Actuat B Chem. 2006;114: 604–609. [21] Campanella L, Beone T, Sammartino MP, Tomassetti M. Determination of phenol in wastes and water using an enzyme sensor. Analyst. 1993;118:979–986.
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[22] Adamski J, Nowak P, Kochana J. Simple sensor for the determination of phenol and its derivatives in water based on enzyme tyrosinase. Electrochim Acta. 2010;55:2363–2367. [23] Fatibelo-Filho O, Vieira IC. Uso analítico de tecidos e de extratos brutos vegetais como fonte enzimática. Quim Nova. 2002;25(3):455–464. [24] Rosatto SS, Freire RS, Durán N, Kubota LT. Amperometric biosensors for phenolic compounds determination in the environmental interess samples. Quim Nova. 2001;24(1):77–86. [25] Akyilmaz E, Dinçkaya E. 2000, A mushroom (Agaricus bisporus) tissue homogenate based alcohol oxidase electrode for alcohol determination in serum. Talanta. 2000;53: 505–509.
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Phenol determination by an amperométrico biosensor based on lyophilized mushroom (Agaricus bisporus) tissue a
a
a
L.M.C. Silva , A.C.C. de Mello & A.M. Salgado a
Laboratory of Biological Sensors, Chemistry School, Technology Center, Federal University of Rio de Janeiro, Horácio Macedo Avenue, 2030 – University City, Ilha do Fundão, Rio de Janeiro, RJ 21949-909, Brazil Accepted author version posted online: 24 Oct 2013.Published online: 22 Nov 2013.
Click for updates To cite this article: L.M.C. Silva, A.C.C. de Mello & A.M. Salgado (2014) Phenol determination by an amperométrico biosensor based on lyophilized mushroom (Agaricus bisporus) tissue, Environmental Technology, 35:8, 1012-1017, DOI: 10.1080/09593330.2013.858755 To link to this article: http://dx.doi.org/10.1080/09593330.2013.858755
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Environmental Technology, 2014 Vol. 35, No. 8, 1012–1017, http://dx.doi.org/10.1080/09593330.2013.858755
Phenol determination by an amperométrico biosensor based on lyophilized mushroom (Agaricus bisporus) tissue L.M.C. Silva∗ , A.C.C. de Mello and A.M. Salgado Laboratory of Biological Sensors, Chemistry School, Technology Center, Federal University of Rio de Janeiro, Horácio Macedo Avenue, 2030 – University City, Ilha do Fundão, Rio de Janeiro, RJ 21949-909, Brazil
Downloaded by [Erciyes University] at 20:11 07 January 2015
(Received 1 April 2013; final version received 25 August 2013 ) A simple and inexpensive biosensor based on lyophilized mushroom tissue (Agaricus bisporus) was developed for amperometric determination of phenol. This fungi tissue contains tyrosinase (EC 1.14.18.1) enzyme that catalysis two sequential oxidation reactions with phenolic substrates. Both reactions involve molecular oxygen; therefore, the commercial Clark-type oxygen electrode was selected as a transducer. The lyophilized biocomponent was tested in two different forms: cubes (at two positions in the biosensor system) or powder. In characterization studies of the biosensor, some parameters such as time reaction, linear range and repeatability were investigated. For the best biosensor configuration, a linear response was observed from 0.1 to 10.0 mg L−1 phenol; variation coefficient and standard deviation were calculated as 0.02% and ±0.11 mg L−1 , respectively. Keywords: phenol; biosensor; fungi tissue; Clark-type oxygen electrode; tyrosinase
1. Introduction Over recent years, electrochemical biosensors are becoming an accepted part of analytical chemistry since they fulfil the expanding need for rapid and reliable measurements. Therefore, analytical technology based on these instruments is an extremely broad field that impacts on many major industrial sectors such as agriculture industries and environmental monitoring. A great number of agricultural and industrial activities discharge phenolic compounds in the environment. Phenols are also released into the environment by the degradation of pesticides with phenolic skeleton. The phenol levels control is very important for the environmental protection. Phenols and especially their chlorinated, nitro and alkyl derivatives have been defined as hazardous pollutants due to their high toxicity and persistence in the environment and are found in the hazardous substances and priority pollutants list of the European Commission and the U.S. Environmental Protection Agency.[1] The Brazilian Environmental Law (CONAMA no. 357/2005 and no. 397/2008) regulates that the phenol concentration limit is 0.5 mg L−1 in the wastewater discharged in the environment).[2] Traditionally, phenol analysis has been based on spectrophotometric or chromatographic methods, which do not always allow easy and continuous monitoring, are more expensive and often require sample pretreatment steps, resulting in increased time and low cost-effectiveness. Therefore, the need for disposable systems or tools for ∗ Corresponding
author. Email: [email protected]
© 2013 Taylor & Francis
environmental applications has encouraged the development of new technologies and more suitable methodologies. In this context, biosensors appear as a suitable alternative or as a complementary analytical tool.[3–5] A biosensor shown in Figure 1 is defined by the International Union of Pure and Applied Chemistry (IUPAC) as a self-contained integrated device that is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor), which is retained in direct spatial contact with a transduction element.[6] In this context, this study was aimed to develop an amperometric biosensor based on mushroom tissue (Agaricus bisporus) in a simple and inexpensive way to be applied in real environmental samples.[2,7] 2. Materials and methods 2.1. Reagents and solutions All chemicals were analytical reagent grade and were acquired from Vetec Química Fina (Rio de Janeiro, Brazil) and Sigma (St. Louis, USA). Phosphate buffer solutions were prepared using distilled water. 2.2.
Biocomponent: A. bisporus tissue
In a search for economic and efficient biological components to be used in phenolic compound biosensors,
Environmental Technology
Downloaded by [Erciyes University] at 20:11 07 January 2015
Figure 1.
A biosensor simplified scheme.
polyphenol oxidases, in particular, tyrosinase (EC 1.14.18.1) (a polyphenol oxidase with a relative selectivity for phenolic compounds)has been investigated in the last few years. The tyrosinase biosensors are applicable to the monitoring of phenol and ortho-benzenediols.[1] Many biosensors have been developed using tyrosinase from A. bisporus as the biocomponent.[8–15] However, few studies have been using this enzyme naturally immobilized in mushroom tissue. The purpose of this work was to continue the work of Silva et al. [16] in the development of an amperometric biosensor for phenol detection using lyophilized mushroom tissue (cube or powder) as the tyrosinase source. The mushrooms used in the biosensor preparation were purchased from a local market (Rio de Janeiro, Brazil) as fresh and culture vegetables. They were stored at 4◦ C until use.
2.3. Instrumentation: the biosensor system The schematic set-up for the biosensor system for phenol analysis is presented as Figure 2.[17] The set-up consists of a standard sample (1), peristaltic pump (Milan Equipamentos Ltda) (2), reaction chamber made from PVC pipe with a biocomponent (cube or powder) (3), transducer (oxygen electrode) (OD/O2/Saturação DM-4P Digimed) and data recorder (4) and discard sample (5). Silicone tubing was used for connections.
2.4. Measurement procedure For phenol analysis, calibration standards were prepared by dilution of phenol stock solution in phosphate buffer, pH 8.0. All measurements were carried out by injection of 50.00 mL standard sample (0.10–50.00 mg L−1 ) at a flow rate of 40.00 mL min−1 . After the sample had filled up the reaction chamber, the injection pump was shut down followed by the insertion of the calibrated oxygen electrode in the reaction chamber. Then, data were collected after the chosen reaction time. After each sample analysis, the system was thoroughly rinsed with distilled water for 2 min. The amperométrico measurements were made at room temperature (24 ± 1◦ C). Measurements were carried out by noting the decrease in dissolved oxygen concentration in relation to substrate concentration added into the biosensor system. This can be explained by the fact that dissolved oxygen electrode has a membrane that separates the internal electrolyte and the electrodes (anode and cathode) from the external medium. Oxygen can pass through this membrane by diffusion until platinum cathode. A redox reaction occurs with four electrons, and it generates a current, proportional to oxygen concentration. The enzymatic reaction involved is shown in Figure 3. Therefore, the higher substrate concentration in the sample will increase the dissolved oxygen variation detection. The biosensor response was defined by the decrease in dissolved oxygen concentration, which was linearly related to the ethanol concentration in the standard solutions. Thereby, the calibration curve of phenol concentration versus dissolved oxygen concentration change was obtained. 2.5.
Figure 2. analysis.
1013
Schematic set-up for the biosensor system for phenol
Optimization of the biosensor system configuration
The initial tests had the intent to choose the best configuration of the biosensor system (Figure 2). The mushroom tissue amount and form (cube or powder) and enzymatic reaction time were investigated. Assays were performed according to Section 2.4; however, data were collected throughout 30 min, in order to analyse the instrument
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Downloaded by [Erciyes University] at 20:11 07 January 2015
Figure 3. Simplified scheme of the reaction catalysed by the tyrosinase, which represents the cresolase activity (1) and the catecholase activity (2).
room tissue amount and form in the linearity range of the instrument. For this, the range of 1.0–30.0 mg L−1 of phenol was chosen. The results of these assays are shown in Figures 5 and 6. Therefore, as can be seen in these figures, the curve related to the position A on the biosensor system had better results and the chosen reaction time was 6 min. Later, to detect the effect of the mushroom (powder) amount in position A in the biosensor system on instrument response, two quantities (0.5 and 1.0 g) were selected. The results of these assays (the average of triplicates) are shown in Figure 7, where the curve related to the amount of 0.5 g had better results. Therefore, Figure 8 shows the result of
Figure 4. The two positions (A and B) in the reaction chamber were chosen: directly coupled to the transducer system (A) or immediately preceding this (B).
response time. The biosensor response time is defined as the enzymatic reaction reaching the steady state. The lyophilized mushroom tissue was shaped as cubes with 1 cm (5.0 g) or was used as powder (0.5 and 1.0 g). Furthermore, to detect the effect of the mushroom (cubes) position into the biosensor system on instrument response, two sites were used as can be seen from Figure 4. 2.6.
Figure 5. Study of the biosensor response time when the biocomponent (cubes, 5 g) was placed in position A in contact with phenol solution (10 mg L−1 ) in the biosensor system.
Repeatability
The repeatability of the phenol biosensor response was also studied by measuring the response (n = 5) when it was used in phenol solution (10 mg L−1 ) under the chosen reaction time for each quantity of biocomponent form and the others at optimum working conditions. The assays were performed according to Section 2.4. 3. Results and discussion 3.1. Effect of the amount and form of lyophilized mushroom and time reaction The preliminary tests with the biosensor system were aimed to find the response time and the effect of lyophilized mush-
Figure 6. Study of the biosensor response time when the biocomponent (cubes, 5 g) was placed in position B in contact with phenol solution (10 mg L−1 ) in the biosensor system.
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Environmental Technology the test, in triplicate, using the amount of 0.5 g of the biocomponent. As shown, the biosensor response increased with time until few minutes reaction time. It can be noted that the time taken to achieve steady-state response was not longer. This could be due to the diffusion of analyte in the mushroom (powder) not affected by the biosensor response. Therefore, the chosen reaction time was 10 min. The reaction time value determined in this study is consistent with other studies in the literature that used plant material or fungal in nature in the biosensor development. Uchiyama et al. [18] used tissue from spinach leaves (Spinacea oleracea) stings supported by a dialysis membrane in the development of biosensor for catechol, with a response time of 5–10 min. Timur et al. [19] and Abdullah et al. [20] used the respective developing biosensors for the detection of phenolic compounds and found the response time of 5 min. Using the same transducer work, oxygen electrode, Campanella et al. [21] developed an amperometric biosensor for ascorbic acid that has the best response time of 2 min using 3 mg of tyrosinase placed under the transducer. 3.2. Calibration curve To determine the phenol concentration, oxygen consumption that occurred in the reaction catalysed by tyrosinase
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in the mushroom was detected. With the available studies on phenol biosensors using commercial tyrosinase immobilized on various matrices, the linearity in concentration range between 10−9 and 10−4 mol L−1 for several phenolic compounds was obtained.[8,22] The results of the construction of calibration curves are shown in Figures 9 and 10. For lyophilized mushroom (cubes), data analysis with STATISTICA Software Version Trial (StatSoft, Inc. 1984– 2011) showed significance (α = 95%) between the data with R2 above 0.93 in all three curves in the range of 5.0– 25.0 mg L−1 phenol, indicating that there is a direct linear correlation between the input (phenol concentration) and output (signal variation of the oxygen electrode). Although for lyophilized mushroom (powder), data analysis showed significance (α = 95%) between the data with R2 above 0.95 in all curves in the range of 0.1–10.0 mg L−1 phenol. 3.3. Repeatability Repeatability of the biosensor was also studied for phenol concentration of 10 mg L−1 (n = 5) under the optimum working conditions. According to the results obtained from the experiments for lyophilized mushroom (cubes), the standard deviation (SD) and coefficient of variation (CV %) were 0.54 mg L−1 and 47.83%, respectively.
Figure 7. Study of the biosensor response time when the biocomponent (powder; 0.5 and 1.0 g) was placed in position A in contact with phenol solution (10 mg L−1 ) in the biosensor system.
Figure 9. Calibration curves of the biosensor. Working conditions: phosphate buffer, pH 8.0. Five gram of the lyophilized mushroom tissue (cubes) was placed at position A on the system. Response time: 6 min.
Figure 8. Study of the biosensor response time with best working conditions (position A and 0.5 g of powder). Phenol concentration used as substrate in the experiments was 10 mg L−1 .
Figure 10. Calibration curves of the biosensor. Working conditions: phosphate buffer, pH 8.0. In total, 0.5 g of the lyophilized mushroom tissue (powder) was placed at position A on the system. Response time: 10 min.
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Possibly, this variation is due to the biocomponent and its maturation degree that has been observed by Silva et al. [2,7] Moreover, the problem of low reproducibility of analytical characteristics of biosensors that utilize plant and fungal tissue is reported in the literature by FatibeloFilho and Vieira,[23] worsening when treating polyphenol oxidases due to the formation of quinones that promote a decrease in the sensitivity of the system. Furthermore, Rosatto et al. [24] show that electrode biosensors that use oxygen Clark-type have the disadvantage of having your response influenced by fluctuations in the concentration of dissolved oxygen resulting from variations in pH, temperature, ionic strength or partial pressure. Therefore, it is clear that developing an instrument that uses fungal tissue and oxygen electrode as a transducer is a difficult task for being susceptible to large external influence and their own inherent biocomponent compounds. However, according to the results obtained from the experiments for lyophilized mushroom (powder), the SD and CV % were ± 0.11 mg L−1 and 0.02%, respectively. Therefore, a promising alternative is the use of powder, which helps to increase the catalytic activity and provide a larger surface area, imparting a higher sensitivity to the biosensor. In the literature, the repeatability of the biosensor developed by Topçu et al. [10] was tested by repeating the experiment eight times, finding a low value of 0.002 × 10−3 mol L−1 as SD. Akyilmaz and Dinçkaya [25] studied the repeatability of the instrument by performing 10 repetitions, finding a value of SD of 0.23 × 10−3 mol L−1 . Thereby, the results with the lyophilized powder in the phenol biosensor development show a promising future.
4. Conclusion A biosensor based on mushroom (A. bisporus) lyophilized tissue was developed for the amperometric determination of phenolic compounds. In biosensor design development, the best configuration was achieved when using the lyophilized powder placed at position A of the system – closer to the oxygen electrode position and a reaction time of 10 min is chosen. A linear response was observed for 0.1– 10.0 mg L−1 phenol. In repeatability studies, CV % and SD were calculated as 0.02% and ±0.11 mg L−1 , respectively. The results showed that biosensor developed offer an alternative to other biosensors based on isolated enzymes. The next step is the application of the biosensor in environmental samples and comparison with the colorimetric method described in Standard Methods of American Public Health Association.
Funding The authors thank the financial support of the National Council for Scientific and Technological Development
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