Microsoft Word - 12 HUTANU Florentina.doc 78 journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XII, Issue 1 – 2013, pag. 78 - 83 M ODIFIED PRUS SIAN BLU E SCR EEN PR INTED ELECTROD ES FO R H2 O2 DETEC TION Florentina HUTANU1, Gheorghe GUTT1* 1Stefan cel Mare University of Suceava, Faculty of Food Engineering, Universității Street, 720229, Suceava, Romania, g.gutt@usv.ro *Corespending author Received 10 January 2013, accepted 21 February 2013 Abstract In this work, presents recent developments in the electrochemical application of disposable screen-printed sensors, to the type of materials used to modify the working electrode.The sensor was based on the electrocatalytic reduction of H2O2 on Prussian Blue modified screen-printed electrode. A comparative study regarding different procedures for modifying the carbon screen-printed electrodes (SPE) with Prussian Blue (PB) was carried out in this work. Two procedures for PB deposition on the SPE electrodes were tested: electrochemical deposition (galvanostatic, cyclic voltammetry) and chemical deposition by the reaction of K3[Fe(CN)6] with FeCl3.Also, the influence of the pretreatment of SPE (+1.7V for 3 min in PBS, pH 7.4) and of the stabilization of PB deposited on SPE (by heating at 100°C) were evaluated. The developed sensors were optimized with respect to the lowest limit of detection achieved for amperometric detection of H2O2. Analytical parameters, such as detection limit, linearity range and sensitivity have been evaluated, together with operational and storage stability. The improved electro-deposition methods, pH stability and permeability of the optimized PB film provide a further boost in its sensitivity for H2O2 detection, which is a critical parameter in biosensor design and application. Keywords: Screen printed electrodes, H2O2 determination, Prussian Blue. 1. Introduction In 1978, Neff [1] reported a thin layer of Prussian Blue deposition on platinum foil by chemical method. Prussian Blue, is a prototype of mixed-valence transition metal hexacyanoferrates, has been widely used as an electron transfer mediator in amperometric biosensor due to its for electrocatalysis.[2-5] Only 4 years later, Itaya et al. [6] showed that the reduced from of Prussian blue (also called Prussian white, PW) had a catalytic effect on the reduction of both O2 and H2O2, many publications have appeared exploring its electrocatalytic, electromagnetic and electrochemic properties [7]. Itaya et al. [8] demonstrated the most important feature of Prussian Blue (in terms of analytical application). It was in fact shown that the reduced from of Prussian Blue (also called Prussian White) had a catalytic effect for the reduction of O2 and hydrogen peroxide. Many efforts have been made to improve the selectivity of carbon based electrochemical sensors, mainly through their modification with redox mediators [9]. However, direct hydrogen peroxide Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XII, Issue 1 – 2013 FLORENTINA HUTANU, GHEORGHE GUTT, MODIFIED PRUSSIAN BLUE SCREEN PRINTED ELECTRODES FOR H2O2 DETECTION, Food and Environment Safety, Volume XII, Issue 1 – 2013, pag. 78 - 83 79 amperometric detection at conventional electrodes is only possible 0.6 V vs. Ag/AgCl. At this potential, the presence of easlily oxidisable compounds such as ascorbate, bilirubin, urate etc. can easily interfere in the measurements, being oxidised at the electrode together with hydrogen peroxide [10]. Prussian Blue modified electrodes represent very attractive detectors for hydrogen peroxide because they work at an applied potential around 0 V vs. Ag/AgCl with no other electrochemical interferences [11]. Objectives The aim of this work was to compare three procedures for PB film formation on the working electrode of screen printed carbon electrodes in order to prepare sensitive and robust PB sensors for H2O2 determination. The tested procedure were based on: chemical, galvanostatic and cyclic voltammetry based deposition. Recently it was reported that the surface modification of electrodes with PB in presence of added surfactant offers enhanced film growth, improved stability and excellent electrochemical reversibility [13]. This approach also increases the PB sensor stability in neutral and basic media. Although, studies to date have focused mainly on the use of cationic surfactants like cetyltrimethyl ammonium bromide (CTAB), in this work we have studied the influence of the anionic surfactant dioctyl sulfo-succinate sodium salt (AOT) on the electrochemical properties of the PB film deposed on screen printed carbon electrode. 2. Materials and methods 2.1. Apparatus Electrochemical measurements were carried out using a µ Autolab type III potentiostat/galvanostat computer controlled by the GPES software, as well as a portable PalmSens potentiostat/ galvanostat controlled via the PalmSensPC software. 2.2. Reagents All chemicals from commercial sources were of analytical grade. Iron chloride (FeCl3), potassium ferricyanide K3[Fe(CN)6], HCl 37%, sodium chloride, hydrogen peroxide (30%), were purchased from Sigma-Aldrich. AOT (Dioctyl sulfo- succinate sodium salt) was from Carlo Erba. Double-distilled water was used throughout. 2.3. Electrodes Screen-printed carbon electrodes (SPCEs) model DRP-110 purchased from DropSens (Spain) were used for electrochemical measurements. In this case the electrochemical cell is composed by a graphite working electrode (d = 4mm), a graphite auxiliary electrode and a silver pseudoreference electrode, with silver electric contacts deposed on a ceramic substrate. In this case the electrochemical cell is composed by a graphite working electrode (d = 4 mm), a graphite auxiliary electrode and a silver pseudoreference electrode, with silver electric contacts and ceramic substrate. These electrodes are for working with microvolumes, for decentralized assays or to develop specific sensors.The electrodes were produced in 75 units packs. Each sensor consists of three printed electrodes, a carbon working and two silver electrodes, acting as counter and pseudoreference, respectively. Screen-printed electrodes [12] are frequently used in analytical applications because of their unique properties such as small size, low detection limit, fast response, high reproducibility, etc. [13]. Advantages of screen-printed electrodes is Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XII, Issue 1 – 2013 FLORENTINA HUTANU, GHEORGHE GUTT, MODIFIED PRUSSIAN BLUE SCREEN PRINTED ELECTRODES FOR H2O2 DETECTION, Food and Environment Safety, Volume XII, Issue 1 – 2013, pag. 78 - 83 80 that they are inexpensive, simple to prepare, versatile and suitable for the mass-production of disposable electrodes [14] 2.4 Chemical deposition of Prussian Blue Prior to Prussian Blue modification, screen-printed electrodes were pretreated in presence of 50 mM phosphate buffer in 0.1 M KCl, pH 7.4, by applying the potential of + 1.7 V versus Ag/Ag Cl for 3 minute [15]. For the chemical deposition of PB films two solutions were prepared. Solution 1: 100 mM K3[Fe(CN)] in solution 10 mM HCl. Solution 2: 100 mM FeCl3 in solution 10 mM HCl Prussian Blue modification of screen printed was then accomplished by placing 10 µl onto the working electrode area. The drop was carefully placed exclusively on the working electrode area, in order to avoid the formation of PB on the reference and counter electrodes which may increase the internal resistance of the system. The solution was left onto the electrode for 10 min and then rinsed with a few militers of 10 mM HCl. The electrodes were then left 90 min in the oven at 100° C to obtained more stable and active layer of Prussian Blue. [14] 2.5. Electrochemical deposition of Prussian Blue Prior to Prussian Blue modification, screen-printed electrodes were pretreated as in previous section. PB modification of SPE eas a accomplished by placing a drop (40 ml of total volume) of precursor solution onto the working electrode area. The deposition was made in a mixture KCl 100 mM of 0.1 M K3[Fe(CN)6,], 0.1M FeCl3 prepared in 100 mM KCl and 100 mM HCl solution by applying the potential of 0.4V for 40 sec [15]. After a gentle rinsing with water, the sensor was placed in a solution of 100 mM KCl in 100 mM HCl and a number of 20 cycles, between - 0.2 and 0.4 V, at a scan rate of 50 mV/s. After some deposition cycles, the PB modified electrode was dried at 100° for 1 h to obtain a more stable and active PB layer [16] 3. Results and discussion 3.1. Prussian Blue deposition The influence of the pre-treatment and thermal stabilization steps in the chemical and electrochemical procedures for PB deposition on the SPE electrodes were tested. -0.1 0.0 0.1 0.2 0.3 -1.5x10-4 -1.0x10-4 -5.0x10-5 0.0 5.0x10-5 1.0x10-4 I( A ) E(V) VC in KCl 0.1M in hydrochloric acid 0.1M SPE-II-pretreated SPE-II A (procedure I) -0.2 0.0 0.2 0.4 0.6 0.8 1.0 -2.0x10-4 -1.5x10-4 -1.0x10-4 -5.0x10-5 0.0 5.0x10-5 1.0x10-4 1.5x10-4 I( A ) E(V) P II - with pretreatment P II - without pretreatment B (procedure II) Fig. 1. Cyclic voltammograms obtained for the SPCE modified PB using the chemical andelectrochemical procedure 0.1 M KCl in 0.1M HCl; 50mV/s. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XII, Issue 1 – 2013 FLORENTINA HUTANU, GHEORGHE GUTT, MODIFIED PRUSSIAN BLUE SCREEN PRINTED ELECTRODES FOR H2O2 DETECTION, Food and Environment Safety, Volume XII, Issue 1 – 2013, pag. 78 - 83 81 In Fig. 2 are presented the cyclic voltammograms obtained for the SPCE modified PB using the chemical procedure in presence and absence of the anionic surfactant AOT at concentartion H2O2 3mM. The PB sensor prepared in presence of AOT showed for the first redox couple higher Iox and Ired. -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 -2,0x10-4 -1,5x10-4 -1,0x10-4 -5,0x10-5 0,0 5,0x10-5 1,0x10-4 1,5x10-4 2,0x10-4 I( A ) E(V) SPE/PB deposition chemical without AOT - VC H 2 O 2 3mM SPE/PB deposition chemical with AOT - VC H 2 O 2 3mM Figure 2. Cyclic voltamograms, in KCl 0.1 M in HCl 0.1 M; the scane rate 50 mV/s, modified PB/AOT using the chemical procedure. -0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 -2,5x10-4 -2,0x10-4 -1,5x10-4 -1,0x10-4 -5,0x10-5 0,0 5,0x10-5 1,0x10-4 1,5x10-4 2,0x10-4 2,5x10-4 I( A ) E(V) P-I VC in H2O2 1mM in phosphate buffer pH6,5 P-II VC in H2O2 1mM in phosphate buffer pH6,5 P-III VC in H2O2 1mM in phosphate buffer pH6,5 P-IV VC in H2O2 1mM in phosphate buffer pH6,5 Figure 3. Cyclic voltammograms of PB sensor 0.1 M KCl in HCl; E= -50mV/s, phophate buffer pH 6,5, H2O2 1mM. The influence of the thermal stabilization by keeping the electrodes at 100° for 90 min was also studied [17] No evident differences between the treated and nontreated PB electrodes were observed regarding the response of the electrodes in KCl, phosphate buffer or for H2O2, but the operational stability was greater improved for the electrodes stabilized via the thermal treatment. 0 5 10 15 20 25 -600 -400 -200 0 200 400 I( A ) v1/2 P II without pretreatment I ox =-41.1321+18.2348x r=0.9991 Ired=32.3735-22.9104x r=0.9998 Figure 4. Variation of oxidation and reduction vs. square root of scan rate in electrolyte solution 0.1M KCl, 0.1M HCl Plotting the oxidation and reduction vs. square root of scan rate showed a linear relationship (Fig. 4), the result indicating that diffusion of electrolyte across the PB layer controls the electrode process. This behaviour was observed for all the two tested procedures used for PB deposition. 3.2. Electrocatalytic reduction of H2O2 The pH influence on the electrochemical determination of H2O2 using the PB modified SPCE was studied at pH ranging from 6.5 (Fig. 3). For all the tested electrodes the highest reduction peaks were obtained for the pH 6.5. In order to select the working potential to be applied when measuring H2O2 it was used the amperometry technique. Therefore, we selected the -50 mV as working potential, considering both the response intensity and operational stability. The applied potential used -50 mV was selected in our previous paper [18] Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XII, Issue 1 – 2013 FLORENTINA HUTANU, GHEORGHE GUTT, MODIFIED PRUSSIAN BLUE SCREEN PRINTED ELECTRODES FOR H2O2 DETECTION, Food and Environment Safety, Volume XII, Issue 1 – 2013, pag. 78 - 83 82 0 20 40 60 80 100 0 200 400 600 800 1000 I( nA ) H 2 O 2 (M) Amperometry PII-A (pretreated) E=-50mV Figure 5. Amperometry response procedure II, from these sensors was evaluated by amperometric measurement in turbulent solution. 0 20 40 60 80 100 0 200 400 600 800 1000 1200 1400 1600 I( nA ) H 2 O 2 (M) Deposition electrochemical E=-50mV; 120s 1-100 M H 2 O 2 y=6.8071+15.1112x R=0.9986 Figure 6. Calibration curves obtained by successive additions of solution H2O2 phosphate buffer pH 6.5, E= -50 mV/s. 0 20 40 60 80 100 0 100 200 300 400 500 600 I( nA ) H 2 O 2 (M) Depositon galvanostatic Cronoamperometrie E=-50mV 1 - 100 M H 2 O 2 y=-1.7741+5.5562x R=0.9996 Figure 7. Calibration curves obtained for H2O2detection performed at electrodes with PB layers in the 1-100 µM concentration range. Applied potential -50mV/s . Equation obtained for the linearity is y= 1.77+5.55, with a correlation coeficient r2 = 0.9996 deposition galvanostatic for the deposition electrochemical y=-6.8+15 r2=0.998 The time required to achieve 90% of corresponding steady state current was 10 s. 3.3. Performances of the PB sensor The developed PB sensors was used for H2O2 determination using as electrochemical techniques: amperometry and chronoamperometry. According to the measurement results, the linear range of the PB sensors was from 1-100 µM, H2O2 with the liniar corelation of 0.9996 for the deposition galvanostatic and chemical deposition r2=0.998. For both type of sensors the detection limit was 0.5 µM H2O2. The relative standard deviation (RSD) was 3.6 %, for PB sensors prepared according to the procedures mentioned. 4. Conclusions Two methods for preparation of PB modified SPCEs were compared in order to design a sensitive and robust sensor for hydrogen peroxide as a platform for oxidase based biosensors, resulting in the selection of the galvanostatic procedure (SPE II-A) as the most efficient. 5. Acknowledgments The authors acknowledge to the project POSDRU/CPP107/DMI1.5/S/78534 for financial support. 6. References [1].V. D. NEFF, Electrochem. Soc. 125 (1978) 886. 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