Biosensors and Bioelectronics 63 (2015) 407–413
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Electrochemical biosensor for carbofuran pesticide based on esterases from Eupenicillium shearii FREI-39 endophytic fungus Gregory Ferreira Grawe a, Tássia Regina de Oliveira a, Esther de Andrade Narciso b, Sally Katiuce Moccelini a, Ailton José Terezo a, Marcos Antonio Soares b, Marilza Castilho a,n a
Departamento de Química, Grupo de Eletroquímica e Novos Materiais, Universidade Federal de Mato Grosso, 78060-900, Cuiabá, MT, Brazil Departamento de Botânica e Ecologia, Laboratório de Biotecnologia e Ecologia Microbiana, Universidade Federal de Mato Grosso, 78060-900, Cuiabá, MT, Brazil b
art ic l e i nf o
a b s t r a c t
Article history: Received 5 April 2014 Received in revised form 24 July 2014 Accepted 25 July 2014 Available online 2 August 2014
In this work, a biosensor was constructed by physical adsorption of the isolated endophytic fungus Eupenicillium shearii FREI-39 esterase on halloysite, using graphite powder, multi-walled carbon nanotubes and mineral oil for the determination of carbofuran pesticide by inhibition of the esterase using square-wave voltammetry (SWV). Specific esterase activities were determined each 2 days over a period of 15 days of growth in four different inoculation media. The highest specific activity was found on 6th day, with 33.08 U on PDA broth. The best performance of the proposed biosensor was obtained using 0.5 U esterase activity. The carbofuran concentration response was linear in the range from 5.0 to 100.0 mg L 1 (r ¼0.9986) with detection and quantification limits of 1.69 mg L 1 and 5.13 mg L 1, respectively. A recovery study of carbofuran in spiked water samples showed values ranging from 103.8 7 6.7% to 106.77 9.7%. The biosensor showed good repeatability and reproducibility and remained stable for a period of 20 weeks. The determination of carbofuran in spiked water samples using the proposed biosensor was satisfactory when compared to the chromatographic reference method. The results showed no significant difference at the 95% confidence level with t-test statistics. The application of enzymes from endophytic fungi in constructing biosensors broadens the biotechnological importance of these microorganisms. & 2014 Elsevier B.V. All rights reserved.
Keywords: Endophytic fungi Biosensor Carbofuran E. shearii
1. Introduction The quality of life on earth is essentially linked to the quality of the entire environment. As the conservation of natural resources and environmental protection issues become increasingly important, the application of enzymes, in general, can help to maintain a cleaner environment by replacing chemical industry processes in waste treatment or as analytical tools to assist in environmental monitoring. Enzymes are natural catalysts found in all living organisms, which may contain more than a thousand different enzymes (Ahuja et al., 2004). Endophytic fungi, microorganisms that live in healthy plant tissues, can synthesize biologically active substances and secrete manifold extracellular enzymes (e.g., hydrolases) in order to maintain a stable symbiosis that contributes to colonization and growth. Although fungi are economically important enzymatic sources for environmental remediation and other fields, endophytes just recently have been prospected for n
Corresponding author. Tel.: þ 55 65 3615 8769. E-mail address:
[email protected] (M. Castilho).
http://dx.doi.org/10.1016/j.bios.2014.07.069 0956-5663/& 2014 Elsevier B.V. All rights reserved.
study. Therefore, they are an interesting niche to be explored (Wang and Dai, 2010). The utilization of extracts from endophytic fungi as a source of enzymes can be a better alternative than purified enzymes because it avoids the lengthy and expensive process of enzyme purification, preserving the enzyme in its natural environment while protecting it from inactivation by external toxicants (D'Souza, 2001). Esterase enzymes are widespread in nature and have been isolated from mammalian tissues as well as microorganisms and plants. They belong to the group of hydrolases and catalyze the formation or cleavage of ester bonds of water-soluble substrates (short-chain fatty acid esters). The mechanism involves four steps; initially, the substrate is bound to the active serine, yielding a tetrahedral intermediate stabilized by the catalytic His and Asp residues. Afterward, the alcohol is released and an acyl–enzyme complex is formed. Attack by a nucleophile (water in hydrolysis), again produces a tetrahedral intermediate that, after resolution, yields the product and free enzyme (Bornscheuer, 2002). These enzymes are widely used in biocatalysis, do not require cofactors, and are usually stable and active in organic solvents (Bornscheuer, 2002; Fahmy et al., 2008).
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Enzymes can also act as recognition elements in biosensors, that are analytical devices comprising a biological sensing element, where the protein is bound to a solid support and combined with physicochemical transducers to deliver bioanalytical measurements (Turner, 2013). When enzymes are used in sensors, there is a need for immobilization in order to maintain or improve the stability of the protein, since such devices may be stored for long periods and must be sufficiently robust. Various materials can be used as supports to immobilize enzymes. These include nanoparticles and mesoporous materials, which provide large surface large areas and increased stability in some cases (Wong et al., 2009). Halloysite nanotubes (HNTs) are a natural aluminosilicate of the kaolin group with the unit formula (Al2Si2O5(OH)4 nH2O). A nanotube made of this material is multi-walled and usually composed of more than 10 layers (Joussein and Petit, 2005; White et al., 2012). The outer surface of the tube has a sheet structure, tetrahedral, composed mainly of siloxane groups (Si–O– Si), while the inner surface has an octahedral structure with aluminol groups (Al–OH) (Lecouvet et al., 2011). This nanosilicate is an important starting material for the production of fine ceramics but, since it is characterized by a high specific surface area, it has also been used as a catalyst support, nanoreactor and adsorbent (Chen et al., 2012). Studies on the biocompatibility of HNTs suggest that this material is non-toxic for cells and could potentially be used with biological substances (Vergaro et al., 2010). Furthermore, this nanomineral has been demonstrated to be a good matrix for immobilization of molecules such as enzymes (Sun et al., 2010). The construction of electrochemical biosensors using carbon nanotubes (CNTs) together with nanoscale materials is an interesting research field. Devices with CNTs have fast response and low detection limit due to the increased analytical signal provided by the high surface area, low-voltage and fast electrode kinetics. The high thermal conductivity, mechanical strength and chemically stable nature of CNTs are very interesting for sensing applications (Vashist et al., 2011; Yang et al., 2010). One of the important applications of biosensors is the monitoring the concentration of pesticides in natural waters. Water is considered the most essential natural resource, but even so, freshwater systems are directly threatened by human activities. The intensive use of pesticides in agriculture has significantly increased agricultural productivity. However, the use of pesticides can contaminate and alter the functions of aquatic environments, air and soils, leading to detrimental effects on the nutrient cycle and toxicity to non-target organisms (Brethour and Weersink, 2001). Moreover, the export of pesticides from agricultural regions through atmospheric or fluvial transport to other ecosystems may also occur (Laabs et al., 2007). Carbofuran [2,3-dihydro-2,2-dimethyl-7-benzofuranyl-N-methylcarbamate] is a broad spectrum pesticide that kills insects, mites and nematodes on contact or after ingestion and is used against soil and foliar pests of field, fruit and vegetable crops (Sakunthala Tennakoon et al., 2013). Due to its extensive use in agriculture and relatively good solubility in water, carbofuran can contaminate surface and ground waters and, therefore, is a risk to consumers and the environment (Brkic et al., 2008). Commonly, the analytical methods for determining carbofuran are high performance liquid chromatography (HPLC) and spectrophotometry (Bertrand and Barceló, 1991; Ribeiro and Dores, 2013; Vera-Avila et al., 2012). These techniques are sensitive, reliable and precise, but they are also complex, time consuming analytical processes with a relatively high cost. It is known that carbofuran inhibits esterase activity, and the inhibition of these enzymes can be used to determine the concentration of this compound in environmental and food samples. Biosensors containing esterase
have been used to determine organophosphorus and carbamate pesticides levels in water samples based on this mechanism (Firdoz et al., 2010; Larsson et al., 1998; Sigolaeva et al., 2010; Tekaya et al., 2013; Zheng et al., 2006). Based on this context, the present work describes for the first time the use of extracellular esterase enzymes from Eupenicillium shearii FREI-39-endophytic fungi isolated from Echinodorus scaber Rataj-immobilized on HNTs to fabricate a biosensor, which was optimized for carbofuran pesticide detection in water samples. The results obtained with this biosensor by square wave voltammetry (SWV) were compared favorably with those obtained using the liquid chromatography method.
2. Material and methods 2.1. Chemicals and solution All reagents were of analytical grade, used without further purification, and all solutions were prepared with ultrapure water (18.2 MΩ cm) obtained from a Millipore (Bedford, USA) Milli-Q Gradient purification system. Halloysite nanotubes (Al2Si2 O5(OH)4 2H2O) were purchased from Sigma-Aldrich. This material has an average tube diameter of 50 nm and inner lumen diameter of 15 nm, a typical specific surface area of 65 m2 g 1 and pore volume of 1.25 mL g 1. Sodium carbonate (Na2CO3), potassium phosphate monobasic (KH2PO4), and potassium phosphate dibasic (K2HPO4) were purchased from synth. Sodium chloride (NaCl), multi-walled CNT, graphite powder, high purity mineral oil, bovine serum albumin (BSA), p-nitrophenyl acetate (pNPA) and p-nitrophenol (pNP) spectrophotometric grade were purchased from Sigma-Aldrich. Carbofuran was obtained from Dr. Ehrenstorfer's Laboratory (Augsburg, Germany) with 99.5% purity. A 0.1 mol L 1 phosphate buffered saline (PBS) (0.1 mol L 1 NaCl, pH 7.0) solution was used as the supporting electrolyte in the electrochemical measurements. 2.2. Apparatus Esterase activity and total protein in the enzymatic extracts from E. shearii FREI-39 were determined using a 50 Scan UV–vis spectrophotometer (Varian, Australia) in a quartz cell with an optical path of 1.0 cm. An ultrasonic bath, model Ultra Cleaner 1400 (Unique) and an analytical balance, model AX200 (Shimadzu), were used for sample preparations. The pH measurements were performed on a Metrohm 827 pH meter and the esterase determination was carried out in a hot bath with a circulation system, model Stern 6 (Sieger). The scanning electron microscopy (SEM) images of the halloysite and the endophytic extract immobilized on halloysite samples were obtained using a model Shimadzu SSX-550 Superscan microscope with an accelerating voltage of 15 kV and magnification of 4000 for all images. Electrochemical measurements using SWV were performed by an Autolab PGSTAT302 potentiostat/galvanostat (Eco Chemie, The Netherlands), connected to data processing software (GPES, software version 4.9.007, Eco Chemie). The square-wave voltammograms were recorded by applying a sweep potential between 0.6 and 1.1 V, performed under optimized conditions. A threeelectrode system was used in the measurements, with the biosensor as the working electrode, Ag/AgCl (3.0 mol L 1 KCl) as the reference electrode and a platinum plate as the counter-electrode. The chromatographic analysis of carbofuran was performed on a Varian ProStar 210 equipped with a Varian ProStar 325 UV–vis detector connected to a Galaxie Chromatography Data System
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(version 1.9.302.952), using a Luna C18 column (250 mm 4.6 mm 5 mm; Phenomenex, USA). 2.3. Enzymatic extracts from E. shearri The endophytic fungus E. shearii FREI-39 was isolated from the leaves of Echinodorus scaber Rataj, popularly known as hat-leather (chapéu-de-couro), collected in the Pantanal (Brazil) in previous work. It is deposited and preserved in the Department of Botany and Ecology, UFMT. The mycelium was grown in solid potato dextrose agar (PDA) for 3 days at a temperature of 28 °C. To obtain the enzymatic extracts, six fragments, with approximately 1 mm2 each, of the fungi were inoculated and cultivated at 28 °C for 15 days in 250 mL of broth under 150 rpm agitation. Four inoculation media were used: PDA broth, PDA with 10% Tween 80 broth, yeast broth (0.08 g NaCl, 0.08 g Mg2SO4 7H2O, 0.08 g K2HPO4, 0.05 g yeast extract and distilled water q.s.p. 250 mL) and yeast with 10% Tween 80 broth. 2.4. Esterase activity and total protein The total esterase (EC 3.1.1.X) activity was determined using a spectrophotometric method by measuring the absorbance at 400 nm of pNP produced from hydrolysis of pNPA substrate catalyzed by esterase in the endophytic extract (Esteves et al., 2009). For the reaction, 1600 mL of 75 mmol L 1 pNPA solution was added in a glass tube together with 800 mL of 50 mmol L 1 phosphate buffer (pH 7.0) and 100 mL of the endophytic extract. The solution was kept in a water bath at 37 °C and, after 1 h, 100 mL 0.1 mol L 1 Na2CO3 was added to stop the reaction. Control experiments were carried out using the same conditions described above but in the absence of the enzymatic extract. All measurements were performed in triplicate every 2 days for 2 weeks. The total esterase activity was the amount of pNP formed in the hydrolysis reaction. The total protein concentration of the endophytic extract was determined by the Bradford method (Bradford, 1976), using BSA as a standard. An analytical curve was constructed by adding the Bradford reagent in the albumin solution, which absorbs at 595 nm, at different concentrations (from 5.0 to 100.0 mg L 1). The absorbance of the endophytic extract was read to determine total protein. All measurements were performed in triplicate. The specific esterase activity was expressed as units of enzyme (U), determined by
U=
A1 1 6 f 10 εV t
(1)
where one unit (U) was defined as the amount of enzyme that liberates 1 nmol pNP min 1 mL 1 mg 1 protein; A is the absorbance; ε is the pNP molar absorption coefficient (L mol 1 cm 1); V is the volume of the endophytic extract (mL); f is the dilution factor and t is the time reaction (min) (Zeraik et al., 2008). 2.5. Immobilization of esterase and biosensor construction The endophytic extract was immobilized on halloysite nanotubes by physical adsorption. To immobilize the enzyme, 0.2 to 5.0 U was added to the support material (HNTs), homogenized and allowed to dry. The proposed biosensor was constructed by mixing 90.0 mg of graphite powder, 10.0 mg of multi-walled carbon nanotubes and 0.5 U of esterase immobilized in 20.0 mg of halloysite in a mortar. Then, 50.0 mg of mineral oil was added to the mixture and it was homogenized for 20 min to produce the final paste. The paste obtained was compacted in a 1 mL plastic syringe with 1.0 mm internal diameter surface and a polished copper wire was used to establish the external electrical contact.
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2.6. Sample preparation and carbofuran determination The proposed biosensor was tested for carbofuran determination in spiked water samples from the Cuiabazinho River (Nobres, MT), collected in December of 2013 in 4 L amber bottles and stored at 4 °C. The Cuiabazinho is closest to the source of the Cuiabá River, suffering minimal environmental impact. The samples were filtered through cellulose acetate membrane (0.2 mm) and fortified with carbofuran at three levels (5.0, 7.0 and 25.0 mg L 1) For the carbofuran determination, the biosensor was initially immersed into a 0.1 mol L 1 PBS (0.1 mol L 1 NaCl, pH 7.0) solution for 5 min (Viswanathan et al., 2009). In the sequence was registered the base signal (I0) in 0.1 mol L 1 PBS (0.1 mol L 1 NaCl, pH 7.0) containing 5.0 10 4 mol L 1 pNPA solution. The biosensor was incubated in standard carbofuran solutions with different concentrations for 5 min and then transferred to the electrochemical cell to record the reduction in base signal after inhibition (I). The percentage of inhibition was calculated for each carbofuran concentration as follows:
IR% =
I0 − I × 100 I0
(2)
where I0 is the reduction current value of pNP before the inhibition and I is the reduction current value of pNP after inhibition. All measurements were performed in triplicate at room temperature (725 °C). The chromatographic analyses were performed in the reversedphase mode, using an isocratic solvent condition. Manual injections were performed using a Rheodyne injector valve with a 20 mL sample loop. The mobile phase consisted of 60:40 (v/v) water/ acetonitrile set at a flow rate of 1.0 mL min 1. All the analyses were performed at ambient temperature ( 725 °C) at a length wave (λ) of 282 nm (Kumar and Naidu, 2013). The carbofuran concentration was determined by recording the peak area (tR ¼ 5.11 min).
3. Results and discussion 3.1. Esterase activity determination Specific esterase activity differed with inoculation media and inoculation time. In this study, four broths were used as inoculation media for the endophyte E. shearii FREI-39. Growth in PDA broth (without Tween 80) was shown to be the best inoculation medium for the specific esterase activity of the endophyte. The addition of Tween 80 increased both total esterase activity and total protein concentration. But, its addition increased the total protein concentration more than the total esterase activity, reducing its specific activity. The specific activity was investigated over a 15-day period, initiating the determination on day zero (inoculation day). The highest value was found on day 6, with 33.08 U in PDA broth. Thus, subsequent endophyte extracts were made in PDA broth with inoculation time of 6 days. 3.2. Esterase immobilization and enzymatic reaction The use of reversible methods for enzyme immobilization, such as physical adsorption, is highly attractive, mostly because it is an easy to perform process and preserves the catalytic activity of the enzyme, improving its stability against the denaturation process. This method is based on the interaction (hydrogen bonding, van der Waals forces, or hydrophobic interactions) between amino
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Fig. 1. SEM images of (A) HNTs and (B) endophytic extract immobilized on HNTs.
acid residues on the enzyme and groups present on the solid support surface (Ardao et al., 2011; Bayramoglu et al., 2010). In this study, the enzymatic extracts were physically adsorbed on the surface of the HNTs, which is a suitable material for immobilization due its fast adsorption rate, high adsorption capacity, large surface area and regeneration ability (Kamble et al., 2012; Rawtani and Agrawal, 2012). Fig. 1 shows SEM images of HNTs and the enzymatic extract immobilized on HNTs. The image of HNTs (Fig. 1A) exhibit clusters of cylindrical nanotubes, while the immobilized enzyme image (Fig. 1B) shows a more compacted and homogeneous surface. The use of HNTs as a support material provides a favorable environment for immobilized enzymes (Zhai et al., 2010). In this way, the stability of the esterases is improved, thus providing biosensors with higher operational and storage stabilities. To verify the efficiency of the enzymatic extract immobilization, one biosensor was constructed with the free enzyme and another with 0.5 U of immobilized esterase. The biosensors responses were evaluated in 5.0 10 4 mol L 1 pNPA, 0.1 mol L 1 PBS (0.1 mol L 1 NaCl, pH 7.0) (Fig. 2). The best response was obtained for the biosensor constructed with the endophytic extract immobilized on HNTs, reflected in the highest resulting current from pNP, demonstrating efficient immobilization on this support. The hydrolyzed product of the reaction from pNPA by esterase catalysis is pNP, which can be measured electrochemically. The advantages of this substrate are the simplicity of the measurement procedure and low applied potential (Miao et al., 2010). During its catalytic cycle, the esterase catalytic triad (histidine, serine and glutamate or aspartate) hydrolyzes the pNPA substrate, generating pNP (Esteves et al., 2009). Then, pNP is electrochemically reduced at the biosensor surface in 0.9 V vs. EAg/AgCl (3.0 mol L 1 KCl) (Fig. 3A), generating p-aminophenol (pAP) (Li et al., 2012) through the transfer of two electrons and three protons. The current obtained in the reduction is quantitatively proportional to the concentration of pNP. A schematic representation of the mechanism established on the surface of the biosensor in pNPA solution is exhibited in Fig. 3B. 3.3. Optimization of the experimental conditions Several experimental conditions were investigated, in triplicate, such as specific esterase activity units, concentration of the pNPA substrate, pH of the PBS (electrolyte support) and SWV parameters. The biosensor sensitivity was dependent on
Fig. 2. Square-wave voltammograms obtained using (–) enzyme free biosensor and ( ) enzyme immobilized on HNTs biosensor in 5.0 10 4 mol L 1 pNPA, 0.1 mol L 1 PBS (0.1 mol L 1 NaCl, pH 7.0). Frequency of 70 Hz, pulse amplitude of 100 mV and scan increment of 2 mV.
immobilized esterase concentration. The amount of esterase enzyme in the paste preparation was investigated in the range of 0.2–5.0 U. The high response for 5.0 10 4 mol L 1 pNPA and the best carbofuran inhibition analytical signal was obtained using 0.5 U of esterase. Based on this result, pastes containing 0.5 U of immobilized esterase on HNTs support were prepared to perform further experiments. The pH study was based in the fact that the endophytic fungi esterases have an activity for hydrolysis in the range of pH 6.0–8.5 and the optimum value can vary depending on its structure (Kuhnel et al., 2012; Lisboa et al., 2013). To determine the influence of pH on the biosensor response, 0.1 mol L 1 PBS (0.1 mol L 1 NaCl) solutions were used in a pH range of 6.0–8.5. The highest current response for pNP was obtained at pH 7.0. Therefore, a pH of 7.0 was used in further experiments. It has been reported that pesticide inhibition is a function of both pesticide and substrate concentrations. The optimal pNPA concentration value for inhibition measurements was 5.0 10 4 mol L 1. The SWV method offers the best sensitivity and detection limit of the electrochemical signal. The current response is dependent on various instrumental parameters such as frequency, pulse amplitude and scan increment. Thus, the effects of these parameters were optimized for the best performance of the biosensor under
G.F. Grawe et al. / Biosensors and Bioelectronics 63 (2015) 407–413
Fig. 3. (A) Square-wave voltammograms obtained using the proposed biosensor in (—) 0.1 mol L 1 PBS (0.1 mol L (B) Schematic representation of the mechanism established on the surface of the biosensor in pNPA solution.
1
NaCl, pH 7.0) and (─) 5.0 10
411
4
mol L
1
pNPA solution.
the conditions described above. Frequencies from 10 to 100 Hz, pulse amplitude between 10 and 100 mV and scan increment from 1 to 7 mV were evaluated. The best performance of the proposed biosensor was obtained at a frequency of 70 Hz, pulse amplitude of 100 mV and scan increment of 2 mV. These experimental conditions were selected for subsequent experiments. 3.4. Repeatability, reproducibility and stability of the biosensor The repeatability of the current response for the proposed biosensor was investigated in 0.1 mol L 1 PBS (0.1 mol L 1 NaCl, pH 7.0) with 5.0 10 4 mol L 1 pNPA. The relative standard deviation (RSD) was 4.5% in successive assays (n ¼10). A reproducibility test using five biosensors prepared and used independently showed satisfactory reproducibility with an RSD value of 3.8% under the optimized conditions described above. The stability was investigated in order to evaluate the biosensor performance and efficiency of the immobilized enzymatic extract over time, based on the inhibition percentage the esterase activity. The biosensor was dry-stored and kept at room temperature (7 25 °C), where the measures were made like described in the 2.6 session, over a period of 20 weeks, illustrated in Fig. 4. The stability measures were carried out each week, with the values remaining within the limits of statistical control with normal random fluctuations. This good result may be due to the efficiency of the endophytic extract immobilization on the HNTs. 3.5. Detection of carbofuran pesticide The detection principle of the pesticide carbofuran was based on inhibiting the esterase activity on the surface of the biosensor. When carbofuran was added to the substrate solution, a decrease in the cathodic peak current was observed, being proportional to
Fig. 4. Study of the stability of the biosensor stored at room temperature. IR% for a solution carbofuran 50.0 μg L 1. Confidence level 99%.
the increase in the carbofuran concentration, inhibiting the electrocatalytic activity of the enzyme and consequently decreasing the current response of the biosensor. Inhibition measurements were performed with 5.0 10 4 mol L 1 pNPA. With increasing concentrations of carbofuran in the solution the resulting current from pNP on the enzymatic biosensor decreased, as demonstrated in the square-wave voltammograms in Fig. 5A. The carbofuran inhibition was proportional to its concentration in the range from 5.0 to 100.0 mg L 1, according to the analytical curve of carbofuran presented in Fig. 5B. The inhibition was reversible and the response of the biosensor could be restored in 5 min in PBS solution, and could be used repeatedly with an acceptable reproducibility.
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Fig. 5. (A) Square-wave voltammograms obtained using the proposed biosensor in: (a) 0.1 mol L 1 PBS (0.1 mol L 1 NaCl, pH 7.0), (b) 5.0 10 4 mol L 1 pNPA, (c–h) with different concentrations of carbofuran (c) 5.0 mg L 1 (d) 10 mg L 1 (e) 25 mg L 1 (f) 50 mg L 1 (g) 75 mg L 1 (h) 100 mg L 1, frequency of 70 Hz, pulse amplitude of 100 mV and scan increment of 2 mV. (B) Analytical curve.
The analytical curve for carbofuran (Fig. 5B) showed a linear relationship in this range, with the relative percent inhibition IR % ¼28.064 (7 0.356)70.380 (7 0.009) [carbofuran], with r ¼0.9986, where [carbofuran] is the carbofuran concentration (mg L 1). The detection limit (LOD) and quantification limit (LOQ) were calculated using the standard deviation of the lower level concentration (s) and the slope of the analytical curve (S) given by: LOD ¼3.3 s/S and LOQ¼ 10 s/S. The LOD and LOQ were 1.69 mg L 1 and 5.13 mg L 1, respectively. According to the Brazilian National Health Surveillance Agency (ANVISA), through Ordinance No. 2914 of 12 December 2011, the maximum carbofuran concentration permitted in drinking water is 7.0 mg L 1, and the maximum contaminant level (MCL)¼ 40.0 mg L 1 according to the United States Environmental Protection Agency – US EPA. For application to water analysis, the proposed biosensor was sensitive because its LOD and LOQ values were below the limit allowed by ANVISA and EPA. 3.6. Recovery study and analytical application In order to demonstrate the applicability of the proposed biosensor, a recovery test was made on spiked water samples from Cuiabazinho River, with three levels of fortification: 5.0, 7.5 and 25.0 mg L 1. In this study, carbofuran was also quantified by a liquid chromatographic analysis method and the findings were compared with the biosensor results. The recovery percentages for the biosensor were found to vary from 103.8% to 105.3%, and 97.8% to 106.9% for the HPLC method, as presented in Table 1. According to the Student’s t-test, at the 95% confidence level, there were no significant differences between the mean
concentrations obtained using the biosensor and the HPLC method in the spiked water samples. These average recoveries demonstrate the accuracy of the biosensor.
4. Conclusions For the first time, an extracellular esterase from endophytic fungi – E. shearii FREI-39 –was applied to the construction of a biosensor. The best values of specific activity were obtained in PDA medium on the 6th incubation day. The proposed biosensor exhibits a satisfactory performance, which can be attributed to the efficiency of the endophytic extract immobilization on the HNTs. The excellent biocompatibility of HNTs provided a favorable microenvironment for the enzyme, which retained its biological activity to a large extent. The nanomaterials that make up the composition of the carbon paste also favored the sensitivity and response of the biosensor. The results of this study show that the proposed method offers good precision and accuracy for carbofuran pesticide determination in water. The LOD was found to be 1.69 mg L 1 for carbofuran, which is less than the maximum concentration permitted in drinking water by the ANVISA and EPA environmental protection agencies. Furthermore, the biosensor construction was simple and relatively inexpensive and the sensor displays long-term stability and good repeatability and reproducibility. Studies should be conducted to evaluate the selectivity of the biosensor proposed. Therefore, this design provides a promising base for the development of other biosensors using endophytic fungi extracts as enzyme source.
Acknowledgments Table 1 Recovery studies of carbofuran in water samples using the biosensor and HPLC methods. Carbofuran (mg L Fortified
5.0 7.0 25.0 a b
1
)
The authors are grateful for the financial support received from FAPEMAT (Processes no. 399612/2011) and the scholarships granted by CNPq and CAPES.
Recoverya (%)
Found Biosensorb
HPLCb
Biosensor
HPLC
References
5.28 7 0.63 7.54 7 0.73 25.95 7 1.73
5.34 7 0.40 7.78 7 0.27 24.427 0.39
105.3 711.9 106.7 79.7 103.8 76.7
106.97 7.5 103.8 7 3.5 97.8 7 1.6
Ahuja, S.K., Ferreira, G.M., Moreira, A.R., 2004. Crit. Rev. Biotechnol. 24 (2–3), 125– 154. Ardao, I., Alvaro, G., Benaiges, M.D., 2011. Biochem. Eng. J. 56 (3), 190–197. Bayramoglu, G., Metin, A.U., Altintas, B., Arica, M.Y., 2010. Bioresour. Technol. 101 (18), 6881–6887.
Recovery ¼ (mean found value/added value) 100% 7 RSD. Mean 7standard deviation; n¼ 3.
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