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Vanadium determination in water using alkaline phosphatase based screen-printed carbon electrodes modified with gold nanoparticles
Journal of Electroanalytical Chemistry 693 (2013) 51–55
Contents lists available at SciVerse ScienceDirect
Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Vanadium determination in water using alkaline phosphatase based screen-printed carbon electrodes modified with gold nanoparticles Ana L. Alvarado-Gámez a,b, M.A. Alonso-Lomillo c,⇑, O. Domínguez-Renedo c, M.J. Arcos-Martínez c a
Centro de Electroquímica y Energía Química Universidad de Costa Rica, San Pedro de Montes de Oca, San José, Costa Rica, Apartado 11500-2060, Spain Escuela de Química, Universidad de Costa Rica, San Pedro de Montes de Oca, San José, Costa Rica, Apartado 11500-2060, Spain c Department of Chemistry, Faculty of Sciences, University of Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, Spain b
a r t i c l e
i n f o
Article history: Received 7 November 2012 Received in revised form 9 January 2013 Accepted 17 January 2013 Available online 4 February 2013 Keywords: Biosensor Vanadium Alkaline phosphatase Riboflavin 5-monophosphate Screen printed carbon electrode
a b s t r a c t A chronoamperometric assay for vanadium ions, based on the inhibition of the enzyme alkaline phosphatase, is reported. Screen-printed carbon electrodes modified with gold nanoparticles were used as transducers for the immobilization of the enzyme. The enzymatic activity over riboflavin-5-monophosphate sodium salt is affected by vanadium ions, which results in a decrease in the chronoamperometric current registered. The assay has a detection limit of 3.9 ± 0.4 lM, a repeatability of 7.6% (n = 4) and a reproducibility of 11.6% (n = 4). A study of the possible interferences shows that the presence of Mo (VI) may affect vanadium determination. The method was successfully applied to the determination of vanadium in tap water. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Vanadium is a trace element that can be found in natural environments, such as waters, soils, rocks and fossil fuels, since it is the 22nd most abundant element in the earth’s crust [1]. This element is frequently used in many industrial processes, including steel industry and electrical engineering [2–4]. Food is the main source of vanadium exposure for general population, with an estimated daily intake of a few tens of lg. Drinking water contributes to a lesser extent, even though it has been found relatively high concentrations of vanadium in some water supply sources [3,4]. Although vanadium has been considered essential for different organisms [5,6], its function is not clear; however, it is well known the adverse effects of vanadium compounds in mammals, observed in vitro, which can induce DNA breakdown and chromosomal aberrations among others [7–9]. On the contrary, vanadium supplements provide various insulin-like effects in vitro and in vivo systems, and have shown its potential to improve insulin resistance in diabetes [10–13]. Therefore, a narrow threshold between essential and toxic levels in living organisms has been established, which points out the need of sensitive and field portable methods of analysis of vanadium [10]. ⇑ Corresponding author. Tel.: +34 947258818; fax: +34 947258831. E-mail addresses: [email protected] (A.L. Alvarado-Gámez), malomillo@ ubu.es (M.A. Alonso-Lomillo), [email protected] (O. Domínguez-Renedo), jarcos@ ubu.es (M.J. Arcos-Martínez). 1572-6657/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jelechem.2013.01.019
Spectrometric and chromatographic methods have been used to analyze vanadium in several matrices [14–16]. Electrochemical techniques applied to the determination of vanadium provide an interesting alternative to them. Together with the recognized advantage of the relatively low cost of electrochemical instrumentation, it should be taken into account the high sensitivity of methods such as adsorptive stripping voltammetry in combination with complexing ligands such as chloroanilic acid [17–20], catechol [21] or cupferron [22]. A very interesting alternative to conventional electrodes has been introduced in the determination of different analytes with the use of screen printed carbon electrodes (SPCEs) [23–25]. Moreover, the fabrication of biosensors based on the modification of SPCEs by enzymes has given rise to a wide range of applications in environmental, food, pharmaceutical and biomedical analysis [26–34]. Electrochemical enzymatic biosensors have been also applied to the determination of numerous metal ions [35–39], taking into account their inhibitory effect over the activity of enzymes. Among the different enzymes used with this aim [38], alkaline phosphatase (ALP) is a metalloenzyme sensitive simultaneously to pesticides and metals, such as vanadium [40–43]. Though, to the best of author’s knowledge, the determination of vanadium using biosensors based on the inhibitory effect of this metal over ALP have not been described up to now. Thus, chronoamperometric biosensors, based on ALP linked to SPCEs, have been developed in this work for the quantification of vanadium, using riboflavin-5-monophosphate sodium salt as substrate [44]. Immobilization and operational conditions that affect
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the chronoamperometric response of vanadium have been optimized. The performance of the developed biosensors has been checked in terms of precision, limit of detection (LOD) and application to water samples. 2. Materials and methods 2.1. Reagents Several inks were used in the fabrication of SPEs, namely Electrodag PF-407 A (carbon ink), Electrodag 6037 SS (silver/silver chloride ink) and Electrodag 452 SS (dielectric ink) supplied by Acheson Colloiden (Scheemda, The Netherlands). Analytical grade chemicals with no further purification were used. All solutions were prepared in ultrapure water, conductivity of 0.05 lS/cm (Gen-Pure TKA, Niederelbert, Germany). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4) from Sigma Chemical Co. (St. Louis, MO, USA) was used for the synthesis of gold nanoparticles (AuNPs). Polyvinyl alcohol (PVA) obtained from Mallinckrodt Baker (New Jersey, USA) and maleic acid, bovine serum albumine (BSA) and glutaraldehyde (GA) obtained from Sigma Chemical Co. (St. Louis, MO, USA) were used in the immobilization of ALP (Sigma Chemical Co., St. Louis, MO, USA). Riboflavin 5-monophosphate sodium salt was obtained from Fluka Analytical (Buchs, Switzerland). Ammonium metavanadate (Merck, Darmstadt, Germany) was used as stock solution of vanadium. 28 mM Tris(hydroxymethyl)aminomethane buffer (Aldrich Chemical Co., Buchs, Switzerland) was used together with 19 mM of MgCl2 (Merck, Darmstadt, Germany) and 0.1 M of KCl (Merck, Darmstadt, Germany) as supporting electrolyte. HCl (Merck, Darmstadt, Germany) was used to adjust the pH value.
Fig. 1. Scheme of a SPCE used for the construction of ALP based biosensors for vanadium determination.
a potential of 0.18 V (vs screen-printed Ag/AgCl electrode) during 15 s under stirring conditions [33,46]. 2.5. Functionalization of AuNPs–SCPEs by ALP The enzyme was immobilized by entrapment with PVA on the surface of AuNPs–SPCEs. The optimum immobilization process was adapted from the earlier work reported by Szydlowska et al. [47]: 30 lL of a 3% of enzyme solution were carefully mixed with 10 lL of a 0.25% (w/v) BSA, 10 lL of a pH 8.5 tris buffer and 10 lL of a 5% (w/v) PVA solution to complete homogenization. Then, 10 lL of this mixture was dropped onto the surface of a screen-printed working electrode. Next, the electrode was exposed to a fluorescent light for 2 h to allow photopolymerization and enzyme entrapment [47,48]. Finally, the electrode was stored for 24 h at 4 °C before first use. Under these storage conditions the developed biosensor showed a good stability for at least 3 weeks. 2.6. Amperometric procedure for the determination of vanadium
SPCEs were produced on a DEK 248 printing machine (DEK, Weymouth, UK) using polyester screens with appropriate stencil designs mounted at 45° to the printer stroke. Electrochemical measurements were made with an Autotolab 128N electrochemical system with GPES software (Echo Chemie, Utrech, Netherlands). pH measurements were performed using a Daigger (Vernon Hills, Illinois, USA) pHmeter 5000.
An ALP based biosensor was placed in the electrochemical cell containing 5 mL of supporting electrolyte solution. An adequate potential was applied and, once a steady-state current was set, a defined amount of riboflavin stock solution was added to the measuring cell. A large oxidation current was observed due to the addition of this substrate. Then, once a plateau corresponding to the steady-state response was reached again, fixed portions of the vanadium stock solution were added consecutively. Enzyme electrodes were conditioned in a buffer solution for 5 min between each calibration setting.
2.3. Manufacturing of SCPEs
3. Results and discussion
A DEK 248 screen-printer (DEK, Weymouth, UK), screen polyester mesh and polyurethane squeegees were used to fabricate the electrodes. Sequential layer deposition has been performed on a polyester film (0.5 mm thickness) obtaining 44 screen-printed configurations of three electrodes each one (working, reference and counter electrode) per film. 4 mm2 carbon working electrodes were designed (Fig. 1) [30,31,33,39]. Before modifying the SPCEs an electrochemical cleaning was carried out. The working electrode surface is activated by recording 30 cycle voltammograms between 1.8 V and 1.8 V vs screenprinted Ag/AgCl reference electrode, scan rate, 100 mV s1, in a 0.1 M KCl solution [33,45].
As it has been above-mentioned, riboflavin-5-monophosphate has been used as substrate for determination of the activity of ALP by using amperometric biosensors. In this way, in the presence of the immobilized ALP, riboflavin 5-monophosphate is hydrolyzed to riboflavin [44], which originates a well-defined oxidation current. Since vanadium inhibits the activity of ALP, the addition of a solution of this metal to the electrochemical cell results in a current decrease proportional to the amount of metal added (Fig. 2). Thus, vanadium concentration can be quantitatively related to the enzymatic inhibition current, that is to say, to the difference between the steady-state current in the absence of vanadium (I0) and the steady-state current in the presence of vanadium (I) (DI (I0 I)). A key step in the fabrication of these biosensors is the enzyme immobilization. Different immobilization procedures were attempted to link the enzyme to the working electrode surface. In order to obtain a biosensor with improved conductivity and performance for vanadium detection, AuNPs were deposited on
2.2. Apparatus
2.4. Modification of SCPEs with AuNPs (AuNPs–SPCEs) AuNPs were obtained by direct electrochemical deposition on the carbon working electrode surface, using a 0.1 mM solution of HAuCl4 in 0.5 M H2SO4. The deposition was performed by applying
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4000000
1/I = 802366 + 80.971 (1/[Substrate]) R2 = 0.9938
(1/I)/A-1
3000000
2000000
1000000 1/I = 545654 + 39.956 (1/[Substrate]) R2 = 0.9932
Fig. 2. Amperogram registered using an ALP based biosensor under the optimum conditions (applied potential, 1.45 V vs screen-printed Ag/AgCl electrode; supporting electrolyte pH 8.5 and riboflavin concentration, 0.25 mM) in the vanadium concentration range from 1.8 lM to 15.0 lM.
0 -20000
-10000
0
10000
20000
30000
40000
(1/[SUBSTRATE])/M -1
pH ¼ 8:5;
Fig. 3. Inhibitory effect of vanadium in the ALP/riboflavin-5-monophosphate reaction: Lineweaver–Burk plot in absence () and presence of (d) vanadium.
120 100
INHIBITION (%)
the working electrode previous to the enzyme immobilization [33,46]. First, a crosslinking immobilization procedure for ALP, using GA and BSA, was followed according to method developed by Blankert et al. [49]. Preliminary results were not satisfactory, since the biosensor showed a poor stability and a brief lifetime. Then, a new immobilization procedure based on the use of PVA and maleic acid as crosslinker was attempted [50]. Maleic acid is able to react with the hydroxyl groups of PVA, binding also the enzyme. Therefore, a 3% of enzyme solution was mixed with a solution containing 5% (w/v) PVA and different concentrations of maleic acid (0%, 20%, 40% or 60%). Then, the mixture was dropped onto the surface of a screen-printed working electrode and finally, the electrode was exposed to a fluorescent light for 2 h. In the case of electrodes in which the enzyme was also crosslinked by maleic acid, no amperometric response was obtained. This fact can be attributed to the well-known disadvantages of the crosslinking method, which can cause damage to the enzyme and limit diffusion of the substrate [51]. Hence, ALP entrapment using exclusively PVA was used as procedure for the enzyme immobilization (Section 2.5). The response of a measuring system in most analytical techniques depends on many experimental factors. In this case, three parameters were studied in order to maximize the current registered, DI (I0 I), for a 1.8 lM vanadium solution: applied potential (from 1.1 V to 1.5 V, vs screen-printed Ag/AgCl electrode), pH of supporting electrolyte (from 7 to 9) and substrate concentration (from 0.1 mM to 0.5 mM). The different experiments carried out in different experimental conditions led to establish the following optimum values:
80 60 40 20 0 V (V)
Mo (IV) W (VI)
Fe (III)
Al (III)
Cr (III)
Sn (II)
Fig. 4. Inhibition current of ALP based AuNPs/SPCEs in presence of 3.5 lM of different metals.
that vanadium can be determined by its inhibitory effect on the response of ALP to riboflavin-5-monophosphate. In addition, the inhibitory effect of vanadium in the ALP/riboflavin-5-monophosphate reaction was studied by the kinetic parameters (vmax and Km) of the Lineweaver–Burk plot (Fig. 4). It was observed a vmax decrease and a Km increase, which suggest a mixed inhibition [52]. Thus, it could be said that the enzyme has higher affinity for its substrate in the absence of vanadium, which confirms the inhibitory effect of this metal and as a consequence, the possibility of vanadium quantification using these electrochemical biosensors.
½Substrate ¼ 0:25 mM; Eap ¼ 1:45 V
Fig. 2 shows the calibration plot recorded under these conditions for vanadium, and under which the amperometric current was improved giving easily quantifiable signals. 3.1. Characterization of the developed biosensor for vanadium determination In order to guarantee that the inhibition response registered after the addition of the substrate was related to vanadium concentration, control experiments were carried out. Amperograms were recorded under the optimum conditions using SPCEs, AuNPs– SPCEs and AuNPs–SPCEs modified as has been reported in Section 2.5., but without enzyme. According to the amperometric procedure described for vanadium determination in Section 2.6., volumes of 100 lL of a 1.96 104 M of a vanadium solution were added. No analytical signal was obtained, thus, it can be concluded
3.2. Limit of detection Once experimental conditions were established for the analysis of vanadium using riboflavin-5-monophosphate as substrate, a series of calibration curves were performed using ordinary least squares regressions to determine the dynamic range of the biosensor (from 1.8 lM to 15.0 lM). LOD was calculated by the method based on standard deviation (Sy/x) of the responses for the blank injection in triplicate and the slope of the calibration curve. This figure of merit, calculated under the optimum conditions, was 3.9 ± 0.4 lM. 3.3. Precision Precision was studied to find out intra-biosensor (repeatability) and inter-biosensors (reproducibility) variations (Table 1).
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Table 1 Summary of the analytical characteristics of the developed biosensors for vanadium detection. Linear range (lM)
Limit of detection (lM)
Reproducibility (% RSD)
Repeatability (% RSD)
Recovery
1.8–15.0
3.9 ± 0.4 lM
11.6% (n = 4)
7.6% (n = 4)
98% (% RSD, 5.8)
Concentration range (lM)
Cadded (lM)
Cfound (lM)
% RSD
Recovery
1.8–15.0
3.64
3.45 ± 0.23 (n = 4, a = 0.05)
6.73
95%
Analysis of spiked tap water samples
Repeatability was determined as the relative standard deviation (RSD) of the slopes of four calibration curves using the same electrode under the optimum conditions of the experimental variables. A value of 7.6% (n = 4) was yielded. Likewise, reproducibility was evaluated as the RSD of the slope of different calibration curves using different biosensors, with a value 11.6% (n = 4). 3.4. Accuracy and trueness determination Accuracy and trueness were calculated in order to check the performance of the developed biosensor. The accuracy of the proposed method was evaluated by means of the analysis of a vanadium certified sample (High Purity StandardsÒ Vanadium Standard solution with a Certificate of Analysis confirmed against SRM 3165 (lot 992706), certified value 1000 ± 4 lg/mL) The vanadium mean concentration quantified by the developed procedure, 955 ± 62 lg/mL (n = 8; a = 0.05), matches the certified value of the sample considering the associated uncertainty. Trueness was evaluated by recovery studies. The method showed a satisfactory value for this parameter since the added vanadium concentration values (20.6 nM) were in good agreement with the found concentration value 20.2 ± 1.2 nM (n = 4, a = 0.05). The average recovery for this analysis was 98.0% with a RSD of 5.8%. Therefore, the proposed method is both accurate and suitable for the analysis of vanadium. 3.5. Study of possible interferences in the vanadium determination The presence of metals, such as Mo (VI), Al (III), Fe (III), Cr (VI), Sn (II), and W (VI), may interfere on the vanadium amperometric response. Their effect was then analyzed by measuring the current after consecutive additions of 3.5 lM solutions of each metal. It can be seen in Fig. 3 that only Mo (VI) presents a significant signal, so this metal must be taken into account in the analysis of vanadium. 3.6. Analysis of vanadium in tap water samples Finally, the developed procedure was applied to the determination of vanadium in spiked tap water samples (3.64 lM), by standard addition methodology in quadruplicate. The concentration of vanadium found was [3.45 ± 0.23] lM (n = 4, a = 0.05, RSD = 6.73), with an average recovery of 95%. 4. Conclusions The use of ALP based biosensors using AuNPs/SPCEs allows the selective chronoamperometric determination of vanadium. The method developed in this work presents for the first time the use of this enzyme and riboflavin-5-monophosphate sodium salt for the analysis of this metal. The effect of vanadium in the ALP/riboflavin-5-monophosphate reaction results a mixed inhibition, which allows the quantification of vanadium in tap water. The developed procedure has showed a limit of detection of 3.9 ± 0.4 lM, as well
as reproducibility and repeatability values of RSD for the slopes of several calibrations lower than 12%.
Acknowledgments Authors would like to acknowledge funding via Research Vicerrectory of Costa Rica University (Project 804-B0-058) and Spanish Ministry of Science and Innovation (TEC-2009/12029).
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Contents lists available at SciVerse ScienceDirect
Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Vanadium determination in water using alkaline phosphatase based screen-printed carbon electrodes modified with gold nanoparticles Ana L. Alvarado-Gámez a,b, M.A. Alonso-Lomillo c,⇑, O. Domínguez-Renedo c, M.J. Arcos-Martínez c a
Centro de Electroquímica y Energía Química Universidad de Costa Rica, San Pedro de Montes de Oca, San José, Costa Rica, Apartado 11500-2060, Spain Escuela de Química, Universidad de Costa Rica, San Pedro de Montes de Oca, San José, Costa Rica, Apartado 11500-2060, Spain c Department of Chemistry, Faculty of Sciences, University of Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, Spain b
a r t i c l e
i n f o
Article history: Received 7 November 2012 Received in revised form 9 January 2013 Accepted 17 January 2013 Available online 4 February 2013 Keywords: Biosensor Vanadium Alkaline phosphatase Riboflavin 5-monophosphate Screen printed carbon electrode
a b s t r a c t A chronoamperometric assay for vanadium ions, based on the inhibition of the enzyme alkaline phosphatase, is reported. Screen-printed carbon electrodes modified with gold nanoparticles were used as transducers for the immobilization of the enzyme. The enzymatic activity over riboflavin-5-monophosphate sodium salt is affected by vanadium ions, which results in a decrease in the chronoamperometric current registered. The assay has a detection limit of 3.9 ± 0.4 lM, a repeatability of 7.6% (n = 4) and a reproducibility of 11.6% (n = 4). A study of the possible interferences shows that the presence of Mo (VI) may affect vanadium determination. The method was successfully applied to the determination of vanadium in tap water. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Vanadium is a trace element that can be found in natural environments, such as waters, soils, rocks and fossil fuels, since it is the 22nd most abundant element in the earth’s crust [1]. This element is frequently used in many industrial processes, including steel industry and electrical engineering [2–4]. Food is the main source of vanadium exposure for general population, with an estimated daily intake of a few tens of lg. Drinking water contributes to a lesser extent, even though it has been found relatively high concentrations of vanadium in some water supply sources [3,4]. Although vanadium has been considered essential for different organisms [5,6], its function is not clear; however, it is well known the adverse effects of vanadium compounds in mammals, observed in vitro, which can induce DNA breakdown and chromosomal aberrations among others [7–9]. On the contrary, vanadium supplements provide various insulin-like effects in vitro and in vivo systems, and have shown its potential to improve insulin resistance in diabetes [10–13]. Therefore, a narrow threshold between essential and toxic levels in living organisms has been established, which points out the need of sensitive and field portable methods of analysis of vanadium [10]. ⇑ Corresponding author. Tel.: +34 947258818; fax: +34 947258831. E-mail addresses: [email protected] (A.L. Alvarado-Gámez), malomillo@ ubu.es (M.A. Alonso-Lomillo), [email protected] (O. Domínguez-Renedo), jarcos@ ubu.es (M.J. Arcos-Martínez). 1572-6657/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jelechem.2013.01.019
Spectrometric and chromatographic methods have been used to analyze vanadium in several matrices [14–16]. Electrochemical techniques applied to the determination of vanadium provide an interesting alternative to them. Together with the recognized advantage of the relatively low cost of electrochemical instrumentation, it should be taken into account the high sensitivity of methods such as adsorptive stripping voltammetry in combination with complexing ligands such as chloroanilic acid [17–20], catechol [21] or cupferron [22]. A very interesting alternative to conventional electrodes has been introduced in the determination of different analytes with the use of screen printed carbon electrodes (SPCEs) [23–25]. Moreover, the fabrication of biosensors based on the modification of SPCEs by enzymes has given rise to a wide range of applications in environmental, food, pharmaceutical and biomedical analysis [26–34]. Electrochemical enzymatic biosensors have been also applied to the determination of numerous metal ions [35–39], taking into account their inhibitory effect over the activity of enzymes. Among the different enzymes used with this aim [38], alkaline phosphatase (ALP) is a metalloenzyme sensitive simultaneously to pesticides and metals, such as vanadium [40–43]. Though, to the best of author’s knowledge, the determination of vanadium using biosensors based on the inhibitory effect of this metal over ALP have not been described up to now. Thus, chronoamperometric biosensors, based on ALP linked to SPCEs, have been developed in this work for the quantification of vanadium, using riboflavin-5-monophosphate sodium salt as substrate [44]. Immobilization and operational conditions that affect
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the chronoamperometric response of vanadium have been optimized. The performance of the developed biosensors has been checked in terms of precision, limit of detection (LOD) and application to water samples. 2. Materials and methods 2.1. Reagents Several inks were used in the fabrication of SPEs, namely Electrodag PF-407 A (carbon ink), Electrodag 6037 SS (silver/silver chloride ink) and Electrodag 452 SS (dielectric ink) supplied by Acheson Colloiden (Scheemda, The Netherlands). Analytical grade chemicals with no further purification were used. All solutions were prepared in ultrapure water, conductivity of 0.05 lS/cm (Gen-Pure TKA, Niederelbert, Germany). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4) from Sigma Chemical Co. (St. Louis, MO, USA) was used for the synthesis of gold nanoparticles (AuNPs). Polyvinyl alcohol (PVA) obtained from Mallinckrodt Baker (New Jersey, USA) and maleic acid, bovine serum albumine (BSA) and glutaraldehyde (GA) obtained from Sigma Chemical Co. (St. Louis, MO, USA) were used in the immobilization of ALP (Sigma Chemical Co., St. Louis, MO, USA). Riboflavin 5-monophosphate sodium salt was obtained from Fluka Analytical (Buchs, Switzerland). Ammonium metavanadate (Merck, Darmstadt, Germany) was used as stock solution of vanadium. 28 mM Tris(hydroxymethyl)aminomethane buffer (Aldrich Chemical Co., Buchs, Switzerland) was used together with 19 mM of MgCl2 (Merck, Darmstadt, Germany) and 0.1 M of KCl (Merck, Darmstadt, Germany) as supporting electrolyte. HCl (Merck, Darmstadt, Germany) was used to adjust the pH value.
Fig. 1. Scheme of a SPCE used for the construction of ALP based biosensors for vanadium determination.
a potential of 0.18 V (vs screen-printed Ag/AgCl electrode) during 15 s under stirring conditions [33,46]. 2.5. Functionalization of AuNPs–SCPEs by ALP The enzyme was immobilized by entrapment with PVA on the surface of AuNPs–SPCEs. The optimum immobilization process was adapted from the earlier work reported by Szydlowska et al. [47]: 30 lL of a 3% of enzyme solution were carefully mixed with 10 lL of a 0.25% (w/v) BSA, 10 lL of a pH 8.5 tris buffer and 10 lL of a 5% (w/v) PVA solution to complete homogenization. Then, 10 lL of this mixture was dropped onto the surface of a screen-printed working electrode. Next, the electrode was exposed to a fluorescent light for 2 h to allow photopolymerization and enzyme entrapment [47,48]. Finally, the electrode was stored for 24 h at 4 °C before first use. Under these storage conditions the developed biosensor showed a good stability for at least 3 weeks. 2.6. Amperometric procedure for the determination of vanadium
SPCEs were produced on a DEK 248 printing machine (DEK, Weymouth, UK) using polyester screens with appropriate stencil designs mounted at 45° to the printer stroke. Electrochemical measurements were made with an Autotolab 128N electrochemical system with GPES software (Echo Chemie, Utrech, Netherlands). pH measurements were performed using a Daigger (Vernon Hills, Illinois, USA) pHmeter 5000.
An ALP based biosensor was placed in the electrochemical cell containing 5 mL of supporting electrolyte solution. An adequate potential was applied and, once a steady-state current was set, a defined amount of riboflavin stock solution was added to the measuring cell. A large oxidation current was observed due to the addition of this substrate. Then, once a plateau corresponding to the steady-state response was reached again, fixed portions of the vanadium stock solution were added consecutively. Enzyme electrodes were conditioned in a buffer solution for 5 min between each calibration setting.
2.3. Manufacturing of SCPEs
3. Results and discussion
A DEK 248 screen-printer (DEK, Weymouth, UK), screen polyester mesh and polyurethane squeegees were used to fabricate the electrodes. Sequential layer deposition has been performed on a polyester film (0.5 mm thickness) obtaining 44 screen-printed configurations of three electrodes each one (working, reference and counter electrode) per film. 4 mm2 carbon working electrodes were designed (Fig. 1) [30,31,33,39]. Before modifying the SPCEs an electrochemical cleaning was carried out. The working electrode surface is activated by recording 30 cycle voltammograms between 1.8 V and 1.8 V vs screenprinted Ag/AgCl reference electrode, scan rate, 100 mV s1, in a 0.1 M KCl solution [33,45].
As it has been above-mentioned, riboflavin-5-monophosphate has been used as substrate for determination of the activity of ALP by using amperometric biosensors. In this way, in the presence of the immobilized ALP, riboflavin 5-monophosphate is hydrolyzed to riboflavin [44], which originates a well-defined oxidation current. Since vanadium inhibits the activity of ALP, the addition of a solution of this metal to the electrochemical cell results in a current decrease proportional to the amount of metal added (Fig. 2). Thus, vanadium concentration can be quantitatively related to the enzymatic inhibition current, that is to say, to the difference between the steady-state current in the absence of vanadium (I0) and the steady-state current in the presence of vanadium (I) (DI (I0 I)). A key step in the fabrication of these biosensors is the enzyme immobilization. Different immobilization procedures were attempted to link the enzyme to the working electrode surface. In order to obtain a biosensor with improved conductivity and performance for vanadium detection, AuNPs were deposited on
2.2. Apparatus
2.4. Modification of SCPEs with AuNPs (AuNPs–SPCEs) AuNPs were obtained by direct electrochemical deposition on the carbon working electrode surface, using a 0.1 mM solution of HAuCl4 in 0.5 M H2SO4. The deposition was performed by applying
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4000000
1/I = 802366 + 80.971 (1/[Substrate]) R2 = 0.9938
(1/I)/A-1
3000000
2000000
1000000 1/I = 545654 + 39.956 (1/[Substrate]) R2 = 0.9932
Fig. 2. Amperogram registered using an ALP based biosensor under the optimum conditions (applied potential, 1.45 V vs screen-printed Ag/AgCl electrode; supporting electrolyte pH 8.5 and riboflavin concentration, 0.25 mM) in the vanadium concentration range from 1.8 lM to 15.0 lM.
0 -20000
-10000
0
10000
20000
30000
40000
(1/[SUBSTRATE])/M -1
pH ¼ 8:5;
Fig. 3. Inhibitory effect of vanadium in the ALP/riboflavin-5-monophosphate reaction: Lineweaver–Burk plot in absence () and presence of (d) vanadium.
120 100
INHIBITION (%)
the working electrode previous to the enzyme immobilization [33,46]. First, a crosslinking immobilization procedure for ALP, using GA and BSA, was followed according to method developed by Blankert et al. [49]. Preliminary results were not satisfactory, since the biosensor showed a poor stability and a brief lifetime. Then, a new immobilization procedure based on the use of PVA and maleic acid as crosslinker was attempted [50]. Maleic acid is able to react with the hydroxyl groups of PVA, binding also the enzyme. Therefore, a 3% of enzyme solution was mixed with a solution containing 5% (w/v) PVA and different concentrations of maleic acid (0%, 20%, 40% or 60%). Then, the mixture was dropped onto the surface of a screen-printed working electrode and finally, the electrode was exposed to a fluorescent light for 2 h. In the case of electrodes in which the enzyme was also crosslinked by maleic acid, no amperometric response was obtained. This fact can be attributed to the well-known disadvantages of the crosslinking method, which can cause damage to the enzyme and limit diffusion of the substrate [51]. Hence, ALP entrapment using exclusively PVA was used as procedure for the enzyme immobilization (Section 2.5). The response of a measuring system in most analytical techniques depends on many experimental factors. In this case, three parameters were studied in order to maximize the current registered, DI (I0 I), for a 1.8 lM vanadium solution: applied potential (from 1.1 V to 1.5 V, vs screen-printed Ag/AgCl electrode), pH of supporting electrolyte (from 7 to 9) and substrate concentration (from 0.1 mM to 0.5 mM). The different experiments carried out in different experimental conditions led to establish the following optimum values:
80 60 40 20 0 V (V)
Mo (IV) W (VI)
Fe (III)
Al (III)
Cr (III)
Sn (II)
Fig. 4. Inhibition current of ALP based AuNPs/SPCEs in presence of 3.5 lM of different metals.
that vanadium can be determined by its inhibitory effect on the response of ALP to riboflavin-5-monophosphate. In addition, the inhibitory effect of vanadium in the ALP/riboflavin-5-monophosphate reaction was studied by the kinetic parameters (vmax and Km) of the Lineweaver–Burk plot (Fig. 4). It was observed a vmax decrease and a Km increase, which suggest a mixed inhibition [52]. Thus, it could be said that the enzyme has higher affinity for its substrate in the absence of vanadium, which confirms the inhibitory effect of this metal and as a consequence, the possibility of vanadium quantification using these electrochemical biosensors.
½Substrate ¼ 0:25 mM; Eap ¼ 1:45 V
Fig. 2 shows the calibration plot recorded under these conditions for vanadium, and under which the amperometric current was improved giving easily quantifiable signals. 3.1. Characterization of the developed biosensor for vanadium determination In order to guarantee that the inhibition response registered after the addition of the substrate was related to vanadium concentration, control experiments were carried out. Amperograms were recorded under the optimum conditions using SPCEs, AuNPs– SPCEs and AuNPs–SPCEs modified as has been reported in Section 2.5., but without enzyme. According to the amperometric procedure described for vanadium determination in Section 2.6., volumes of 100 lL of a 1.96 104 M of a vanadium solution were added. No analytical signal was obtained, thus, it can be concluded
3.2. Limit of detection Once experimental conditions were established for the analysis of vanadium using riboflavin-5-monophosphate as substrate, a series of calibration curves were performed using ordinary least squares regressions to determine the dynamic range of the biosensor (from 1.8 lM to 15.0 lM). LOD was calculated by the method based on standard deviation (Sy/x) of the responses for the blank injection in triplicate and the slope of the calibration curve. This figure of merit, calculated under the optimum conditions, was 3.9 ± 0.4 lM. 3.3. Precision Precision was studied to find out intra-biosensor (repeatability) and inter-biosensors (reproducibility) variations (Table 1).
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Table 1 Summary of the analytical characteristics of the developed biosensors for vanadium detection. Linear range (lM)
Limit of detection (lM)
Reproducibility (% RSD)
Repeatability (% RSD)
Recovery
1.8–15.0
3.9 ± 0.4 lM
11.6% (n = 4)
7.6% (n = 4)
98% (% RSD, 5.8)
Concentration range (lM)
Cadded (lM)
Cfound (lM)
% RSD
Recovery
1.8–15.0
3.64
3.45 ± 0.23 (n = 4, a = 0.05)
6.73
95%
Analysis of spiked tap water samples
Repeatability was determined as the relative standard deviation (RSD) of the slopes of four calibration curves using the same electrode under the optimum conditions of the experimental variables. A value of 7.6% (n = 4) was yielded. Likewise, reproducibility was evaluated as the RSD of the slope of different calibration curves using different biosensors, with a value 11.6% (n = 4). 3.4. Accuracy and trueness determination Accuracy and trueness were calculated in order to check the performance of the developed biosensor. The accuracy of the proposed method was evaluated by means of the analysis of a vanadium certified sample (High Purity StandardsÒ Vanadium Standard solution with a Certificate of Analysis confirmed against SRM 3165 (lot 992706), certified value 1000 ± 4 lg/mL) The vanadium mean concentration quantified by the developed procedure, 955 ± 62 lg/mL (n = 8; a = 0.05), matches the certified value of the sample considering the associated uncertainty. Trueness was evaluated by recovery studies. The method showed a satisfactory value for this parameter since the added vanadium concentration values (20.6 nM) were in good agreement with the found concentration value 20.2 ± 1.2 nM (n = 4, a = 0.05). The average recovery for this analysis was 98.0% with a RSD of 5.8%. Therefore, the proposed method is both accurate and suitable for the analysis of vanadium. 3.5. Study of possible interferences in the vanadium determination The presence of metals, such as Mo (VI), Al (III), Fe (III), Cr (VI), Sn (II), and W (VI), may interfere on the vanadium amperometric response. Their effect was then analyzed by measuring the current after consecutive additions of 3.5 lM solutions of each metal. It can be seen in Fig. 3 that only Mo (VI) presents a significant signal, so this metal must be taken into account in the analysis of vanadium. 3.6. Analysis of vanadium in tap water samples Finally, the developed procedure was applied to the determination of vanadium in spiked tap water samples (3.64 lM), by standard addition methodology in quadruplicate. The concentration of vanadium found was [3.45 ± 0.23] lM (n = 4, a = 0.05, RSD = 6.73), with an average recovery of 95%. 4. Conclusions The use of ALP based biosensors using AuNPs/SPCEs allows the selective chronoamperometric determination of vanadium. The method developed in this work presents for the first time the use of this enzyme and riboflavin-5-monophosphate sodium salt for the analysis of this metal. The effect of vanadium in the ALP/riboflavin-5-monophosphate reaction results a mixed inhibition, which allows the quantification of vanadium in tap water. The developed procedure has showed a limit of detection of 3.9 ± 0.4 lM, as well
as reproducibility and repeatability values of RSD for the slopes of several calibrations lower than 12%.
Acknowledgments Authors would like to acknowledge funding via Research Vicerrectory of Costa Rica University (Project 804-B0-058) and Spanish Ministry of Science and Innovation (TEC-2009/12029).
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