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Dual screen-printed electrodes with elliptic working electrodes arranged in parallel or perpendicular to the strip
Sensors and Actuators B 198 (2014) 302–308
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Dual screen-printed electrodes with elliptic working electrodes arranged in parallel or perpendicular to the strip Raquel García-González, Agustín Costa-García, M. Teresa Fernández-Abedul ∗ Departamento de Química Física y Analítica, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain
a r t i c l e
i n f o
Article history: Received 18 October 2013 Received in revised form 29 January 2014 Accepted 8 March 2014 Available online 19 March 2014 Keywords: Dual screen-printed electrodes Electrochemical bisensor Multiplexed determination Nanostructured electrodes Methylene blue
a b s t r a c t The number of works based on electrochemical mono-, bi- and multi-sensors is increasing, especially in which multiplexing is concerning. Determination of a panel of analytes in clinical analysis or of different related molecules in environmental or food analysis has increased the efforts for designing appropriate sensors, becoming the design, a very important step in their development. In this paper, two different dual designs for screen-printed electrodes, one of them symmetrical, were electrochemically evaluated with methylene blue as electroactive species. Two elliptic working electrodes substituted the traditional circled and centered working electrodes in the monosensing mode. Ellipses perpendicular to the strip and with the same configuration than conventional for reference and auxiliary electrodes constituted the asymmetrical design. Placing the ellipses in parallel with the strip, surrounding them with a counter electrode and locating the reference at the basis, produces a symmetrical design. Although both could be employed, more precise peak intensities are obtained for the more symmetrical design as well as with the employ of an external reference and auxiliary electrode system. Since a more sensitive detection is increasingly required, comparison is made also after nanostructuration with carbon nanotubes. The symmetrical design appears also to be more appropriate for all the electrochemical techniques tested: cyclic voltammetry, differential pulse voltammetry and square wave voltammetry. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Screen-printing methodology was introduced worldwide over a thousand years ago and exists today in fields such as art, textiles, and advertising. This technique was applied to biosensor manufacture in the middle of the 1980s [1] and is nowadays well established for the production of thick-film electrochemical transducers [2]. In this technology, the paste is deposited and distributed on the screen mesh using a squeegee or a roller and is forced to pass through the open areas of the screen mesh to the substrate. Finally, a protective ink coating is used to insulate the conductive track from the electrodes [3]. It allows the mass production of reproducible and inexpensive, mechanically robust and miniaturized strip solid electrodes. They are also of easy handling and manipulation in a disposable manner, which avoids inconveniences of conventional electrodes such as memory-effects due to difficult, time-consuming and sometimes inefficient cleaning steps, allowing the performance of parallel assays. Their precision makes possible the development of sensitive and quantitative methodologies, which converts
∗ Corresponding author. Tel.: +34 985102968; fax: +34 985103125. E-mail address: [email protected] (M.T. Fernández-Abedul). http://dx.doi.org/10.1016/j.snb.2014.03.036 0925-4005/© 2014 Elsevier B.V. All rights reserved.
them into versatile tools for biosensor development [4]. In addition, the extensive range of modifications to screen-printed electrodes (SPEs) opens numerous fields of application. On the other hand, since the discovery of carbon nanotubes (CNTs) Ref. [5], they have acquired a great relevance because of their advantageous properties. Modification of electrodes (nanostructuration) has been employed for the improvement of the electroanalytical behavior of analytes of interest. Numerous advantages of CNTs as electrode materials have been attested for analysis of diversified chemicals of food quality, clinical and environmental interest. CNT-electrochemical sensors exhibit low limit of detection and fast response due to the signal enhancement provided by high surface area, low overvoltage, and rapid electrode kinetics. A highly thermal conductive, mechanically strong and chemically stable nature of CNTs is very appealing for lot of applications [6–11]. Their high surface-to-volume ratio is also a definite asset toward the development of biosensing platforms for molecule detection [12]. The use of gold SPEs is very appropriate due to their adsorptive and electrochemical properties and the combination with CNTs is very promising [13]. Dual and multi-analysis of a variety of electroactive species based on SPEs have been reported, e.g. the simultaneous determination of maltose and glucose using a SPE systemed Ref. [14]
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simultaneous detection of free and total prostate specific antigen, where the measurement depends one on each other, on a screen-printed electrochemical dual sensor [15]. In this way, in one working electrode (WE) total prostate-specific antigen (PSA) is detected meanwhile free PSA is determined in the second one. The simultaneous detection of four pneumoniae bacteria using gold nanostructured carbon SPEs [16] or a multiplexed electrochemical immunosensor for detection of celiac disease serological markers [17] were developed in our research group too. The configuration of the electrode has influence on the measurements and has been optimized with respect to size and layout in the determination of highly electronegative metals with bismuth WE, Ag/AgCl RE and gold CE. It was especially important since generation of gas bubbles at the counter electrode (CE) due to electrolysis of water resulted in poor stability and reproducibility [18]. This was solved by increasing the area of the auxiliary electrode as well as the distance to the WE. Similarly, the WE and RE relative size is of relevance on the design of potentiometric CO2 sensors [19]. A narrow spacing between electrodes slightly improves the quality of the responses and the longer the facing lengths between electrodes, the better the shape of the responses. Moreover, when the surface of the RE (this is a two-electrode system) is smaller than that of the WE, the sensor does not operate properly (baseline instability and smaller response). Geometry is therefore very relevant for achieving an adequate performance of sensors. Electrode geometry plays a critical role in electrochemical sensors, and therefore multiple sensor designs incorporating the three electrode cell with different electrode geometries were explored, e.g. in the development of a lab-on-a-chip sensor for detection of highly electronegative heavy metals by anodic stripping voltammetry [18]. Commercially, there are two possible configuration designs of dual screen-printed electrodes: asymmetrical (with two WE perpendicular to the strip) and symmetrical (with two WE parallel to the strip) designs. The symmetrical design proposed maintains distance between electrodes and length of connections identical for each WE. Many dual sensors were developed using the symmetrical and asymmetrical designs proposed in this work as transducer surfaces. But this is the first time that a comparison study between these different dual electrode configurations was made. In this paper, a comparative study of two configurations of dual SPEs is made. The electrochemical cell of the bisensor, or dual sensor (a screen-printed strip that contains two working electrodes) consists of a four-electrode system: two elliptic gold WE, a silver pseudo-RE and a gold CE. This is the first time that the electrochemical characterizations of these designs were carried out, in this case, with Methylene Blue, 3,7-bis(dimethylamino)phenothiazine5-ium-chloride, (MB) as model electroactive species to characterize the effect of the electrode geometry. MB is a commonly employed biolabel that had been used as electroactive marker in biosensors to detect the hybridization event [20,21].
2. Experimental 2.1. Reagents and solutions Nafion® (perfluorinated ion-exchange resin, 5 wt% solution in a mixture of lower aliphatic alcohols and water) and methylene blue (certified by the BSC, Biological Stain Commission) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sulphuric (95–97% of purity) acid and potassium chloride were purchased from Merck (Darmstadt, Germany). Finally, amine functionalized multi-wall carbon nanotubes (MWCNT-NH2 ) were purchased from Belgium
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Nanocyl (Auvelais, Belgium). Water was purified employing a Milli-Q directQS system from Millipore. The resistivity value of the water used was 18.2 M cm. 2.2. Apparatus and instruments Voltammetric measurements were performed with an Autolab PGSTAT 10 (ECO Chemie) potentiostat interfaced to a Pentium 120 computer system and controlled by Autolab GPES software version 4.8. An Elma ultrasonic bath, a Nahita centrifuge with interchangeable car, a Mettler Toledo (AB54) balance, a Crison Micro-pH 2001 pH-meter, a magnetic stirrer Asincro (J.P.Selecta), a Sanyo refrigerator and a Sanyo (MIR-162) incubator were also used. 2.3. Dual SPEs Electrode configuration plays a critical role in electrochemical sensors, and therefore two sensor designs incorporating the four- electrode cell with different electrode configurations were explored. Bisensors based on gold SPEs purchased from DropSens (Asturias, Spain) include a four-electrode system configuration printed on the same strip. The format of these SPEs includes two elliptic WE (6.3 mm2 each one, semi-major axis of 4 mm and semiminor axis of 2 mm), a silver pseudo-RE and a gold CE that uses the same ink of the WE. All of them are screen-printed on a ceramic substrate (3.4 × 1.0 × 0.05 cm) and subjected to high-temperature curing. An insulating layer serves to delimit the working area and electric contacts. The production characteristics of commercial SPEs are regarded by the manufacturers as proprietary information. A specific connector supplied by DropSens allows their connection to the potentiostat. The two designs of dual SPEs studied are presented in Fig. 1. Two WE are included in the same strip, and they share RE and CE. The asymmetrical design (AS design), in Fig. 1B, derives from the traditional design of a monosensor (Fig. 1A), where the WE is a circle (12.6 mm2 ) surrounded by the CE, and a small silver arc situated as a continuation of the counter acts as a pseudo-reference electrode. In this case the central WE has been substituted by two ellipses (6.3 mm2 each one) with the major axis perpendicular to the strip. The electrochemical arrangement as a whole is not symmetrical. The counter (12.6 mm2 and 1 mm wide) and pseudo-reference electrodes are similar to those in the monosensor. A stable RE is an important component of an integrated electrochemical system and is a prerequisite for achieving reliable performance [18]. For the sake of clarity, the electrode closest to the CE top-WE to this that is nearer the connections. In some studies, an external homemade RE and CE system was used coupled to AS design. A 250 L micropipette tip included a RE that consisted of an anodized silver wire (Goodfellow, Huntingdon, UK) introduced through a rubber syringe piston. Then the tip was filled with saturated KCl solution containing a low-resistance liquid junction. The platinum wire (250 m diameter) that acted as CE was fixed with insulating tape to the micropipette tip. For recording the voltammograms, the tip was held on a support allowing horizontal and vertical movement. The symmetric design (S design), in Fig. 1C, has also two ellipses but the major axis of the ellipses are parallel to the ceramic strip. In this case, electrodes are called right and left-WE (6.3 mm2 each one). The CE (19.8 mm2 and 1 mm wide) surrounds equally both electrodes, and the pseudo-reference electrode is equidistant from both WEs, in a much more symmetrical design. The connections are at the same length of both WEs. In both biosensors the space between electrodes is covered by the insulating layer meanwhile in the monosensor this layer surrounded all the three electrodes.
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A
C
B
Ref erence electrod e (RE)
RE
CE
RE
CE
Working electrod e (WE) Counter electrod e (CE) Top-WE
Bottom-WE
(Design AS)
Lef t-WE
Right-WE
(Design S)
Fig. 1. Schematic design of SPEs with (A) one WE and two WEs with the main axis of the ellipses: (B) perpendicular (AS design) or (C) parallel (S design) to the strip.
2.4. Nanostructuration of AuSPEs The nanostructuration of WE with CNTs was carried out by evaporation at room temperature of a drop of 3 L of MWCNTs-NH2 (dispersed in a 0.5% Nafion® /ethanol solution [13]) deposited on the WE. In this way, CNTs-modified AuSPEs were obtained. Before the measurements the CNTs-AuSPEs were washed with the supporting electrolyte (0.1 M Tris–H2 SO4 pH 8.0) and dried at room temperature (24 ± 1 ◦ C). 2.5. Electrochemical measurements The reduction signal of MB was measured ten times (in ten different bare or nanostructured AuSPEs) by scanning the potential between 0.0 and −0.7 V using cyclic voltammetry (CV, with a scan rate of 250 mV s−1 ), differential pulse voltammetry (DPV, with a step potential of 0.008 V, modulation amplitude of 0.05 V and standby potential of 0 V) and square wave voltammetry (SWV, with a step potential of 0.008 V, amplitude of 0.05 V and standby potential of 0 V). Drops of 40 L of 20 and 100 M MB were used. All experiments were performed at room temperature (25 ◦ C). 3. Results and discussion Multianalyte determinations as well as recording repetitive measurements require the employ of appropriate analytical devices that maintains the miniaturization and simplicity of monosensors. The design has to be carefully checked in order to attain the best performance, and specially required before a massproduction is made. Two different designs for dual detection (those described in Section 2), with two elliptic WE (perpendicular and parallel to the ceramic strip) were evaluated. A pseudo-reference electrode consisting of a screen-printed silver ink is employed. Even when a real reference electrode (e.g. Ag/AgCl) is recommended, this is not very adequate for this type of electrodes and good precision of potentials is usually obtained [13]. A model molecule that has been employed as biolabel for genosensing [22], methylene blue, has been chosen for the electrochemical characterization. MB presents a well-defined two-electron redox process [23] with cathodic (conversion to leucomethylene blue (LB)) and anodic (oxidation to MB) peaks. MB studies were initially performed on bare electrodes but since
high sensitivity improvements are obtained on nanostructured electrodes, a comparative study of configuration designs was also carried out with nanostructured WEs. In a previous work performed in our research group, it has been demonstrated that nanostructuration of AuSPEs with MWCNTs-NH2 produces the enhancement of the analytical signal recorded for MB [13]. These nanostructures were efficiently dispersed in a 1:1 H2 O/0.5% Nafion® in ethanol. This dispersion of CNTs was used throughout this work for the nanostructuration of electrodes. Stability of the nanostructures was studied in a previous work of our research group [24]. However, it is a very simple and fast procedure that would not be difficult to reproduce at any time. 3.1. Electrochemical behavior of dual elliptic WEs perpendicular to the strip In order to know the influence of the design of the dual SPEs on the analytical signal of methylene blue, different electrochemical techniques: CV, DPV and SWV were employed. Voltammograms were recorded in a solution of MB in the supporting electrolyte (0.1 M Tris–H2 SO4 pH 8.0). Due to the sensitivity enhancement produced by electrode nanostructuration, a 100 M solution is employed for bare electrodes meanwhile voltammograms are recorded in 20 M solutions on nanostructured electrodes. First, bare WEs were tested, and further, they were nanostructured one by one, recording the analytical signal. In the case of bare electrodes (see Fig. 2A), a clear difference between signals for top and bottom electrodes can be seen. The peak intensities recorded for top-WE are higher than those for bottom-WE with ratios of 1.3, 1.4 and 1.2 for CV, DPV and SWV, respectively. It has been reported that when the distance between RE and WE increases, the electrical current decreases [25]. In this case, the top WE (closer to the RE) will always record higher intensities. In all cases, a notorious increase in the signal is observed after the nanostructuration of the electrodes, which was already reported [13]. When CV was used, intensities obtained were almost triple for a concentration five times lower. In the case of nanostructured electrodes both WEs present similar behavior, i.e. slightly higher for the top-WE too (see Fig. 2B). Here the ratio is 1.1 for all the techniques, CV, DPV and SWV. The nanostructuration with CNTs of only one of the WEs (the data are presented in Table 1) leads to results like those already commented, signals are similar to those obtained
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Fig. 2. Bar diagrams obtained with CV (250 mV s−1 ), DPV (s = 0.008 V, A = 0.05 V) and SWV (s = 0.008 V, A = 0.05 V) for the AS design on bare (A), and nanostructured (B) WEs. Colorless (top-WE) and colored (bottom-WE).
Table 1 Methylene blue cathodic peak currents on bare and nanostructured WEs perpendicular to the strip (AS design). AS design
ip (A)
CMB (M)
CNTs on top-WE
20
CNTs on bottom-WE
20
Top-WE Bottom-WE Top-WE Bottom-WE
for either bare or nanostructured electrodes. Following the same trend, higher intensities were obtained when top-WE were modified. The highest intensity difference was observed with SWV and when the top-WE was nanostructured: 58 ± 7 A were recorded, 1.4 times the intensity recorded with the modified bottom-WE. This could be explained by the asymmetry in the design. The CE does not extend equally to both WEs: the facing length is lower for the top-WE and RE is much closer to the top-WE. However, one of the most probable causes is the difference in the length of WEs connections. They are not the same length and they have an important role in the final current. Conduction of electrons is made through a screen-printed conductive ink and more resistance is obtained for longer connector pad (bottom-WE) with subsequent lower analytical signal. However, there are many parameters to take into account when designing a sensor: the solution IR drop (distance between WE and RE, concentration of supporting electrolyte), the dimensions and the specific surface area of the working electrodes [26], and edge effects on plain electrode [27] and diffusion on a porous electrode [28]. In a classic electrochemical cell with a system of three electrodes, the generated current flows through the WE and CE and the potential is controlled between the WE and RE. The threeelectrode system is advantageous because it prevents the RE from driving the current which could change its potential [18]. The effect of the surface area of the WE on the value of the peak
CV
DPV
SWV
−11 ± 1 −1.2 ± 0.1 −0.9 ± 0.1 −9.2 ± 0.8
−34 ± 4 −2.8 ± 0.2 −3.8 ± 0.3 −25 ± 2
−58 ± 7 −5.9 ± 0.4 −7.9 ± 0.6 −42 ± 3
current was discussed in several papers [29,30] too. Following the Randles–Sevcik equation, the higher the WE area, the higher the current intensity. A linear dependence of the reduction current on the WE surface area was reported for a NO2 sensor [31]. However, the area of the WE had a strong impact on the stability and current noise of the nitrogen dioxide sensor: the smaller the electrode area, the better the sensor stability and the lower the current noise. In this work, the surface area of each WE in the dual-SPE are half of the area of the WE in a monosensor-SPE (6.3 vs. 12.6 mm2 ). In this way, the current density generated is similar for both WEs and CE. In order to know if this design is the responsible of the differences observed, an external RE and CE were coupled to AS design. The results obtained are presented in Fig. 3A and B. When bare electrodes were tested, both WE (top and bottom) generated the same intensities in all cases and no differences between both were observed. When nanostructured electrodes were evaluated, slight differences in the recorded intensities were observed but in all cases these were lower than in the case before commented. The highest intensity corresponds in this case to the bottom-WE, and since it is only observed with nanostructured electrodes might be caused by the modification step. Since the design directly influences the currents generated and the use of external RE and CE is not of easy use, a new design was proposed and evaluated.
Table 2 Methylene blue cathodic peak currents on bare and nanostructured WEs parallel to the strip (S design). S design
CMB (M)
CNTs on right-WE
20
CNTs on left-WE
20
ip (A)
Right-WE Left-WE Right-WE Left-WE
CV
DPV
SWV
−9.1 ± 0.6 −0.36 ± 0.04 −0.32 ± 0.03 −9.2 ± 0.5
−30 ± 3 −1.4 ± 0.1 −1.5 ± 0.1 −28 ± 2
−47 ± 5 −2.1 ± 0.2 −1.9 ± 0.1 −43 ± 3
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A
B
25
50
(CNTs electrodes)
20
40
15
30
|ip| / A
|ip| / A
(Bare electrodes)
10
5
20
10
0
0
CV
DPV
SWV
CV
DPV
SWV
(AS design – external RE and CE) Fig. 3. Bar diagrams obtained with CV, DPV and SWV for the AS design coupled with external RE and CE from bare (A) and nanostructured (B) WEs. Colorless (top-WE) and colored (bottom-WE). (Conditions as in Fig. 2).
B
A
50
12
(Bare electrodes)
(CNTs electrodes) 40
|ip| / A
|ip| / A
9
6
30
20
3 10
0
0
CV
DPV
SWV
CV
DPV
SWV
(S design) Fig. 4. Bar diagrams obtained with CV, DPV and SWV for the S design from bare (A) and nanostructured (B) WEs. Colorless (left-WE) and colored (right-WE). (Conditions as in Fig. 2).
3.2. Electrochemical behavior of dual elliptic WEs parallel to the strip The S design of the electrochemical cell was proposed as the most symmetrical possible. In this case the CE and RE are equally shared, and connections for all the electrodes are at the same length. In this way, the same resistance in the internal circuit is produced. On bare electrodes (see Fig. 4A), the intensities recorded by any of the voltammetric techniques, CV, DPV or SWV on each WE were
very similar for right and left WEs. The intensity ratio in this case was 1.0 for all the techniques. When the nanostructuration is performed on both WEs (see Fig. 4B), the two intensities recorded on each WE were very similar too. Ratio of 1.0 is also found for all the techniques. When only one of the electrodes is nanostructured (data shown in Table 2), comparison between right and left WE gave ratios of 1.0 for CV and 1.1 for both DPV and SWV. RSD values were 6, 9 and 10% for CV, DPV and SWV, respectively.
Table 3 Analytical parameters of calibration plots from CV (100 mV s−1 ). Design
Electrode
Signal
Slope (A M−1 )
Intercept (A)
R
Linear range (M)
n
LD (M)
AS design
Bare E
Top Bottom Top Bottom Left Right Left Right
−0.0230 −0.0259 −0.7906 −0.5747 −0.0126 −0.0123 −0.2100 −0.2038
0.0558 0.0776 1.6931 0.5165 0.0117 0.0006 0.8279 0.6503
0.9979 0.9983 0.9957 0.9961 0.9985 0.9993 0.9962 0.9993
8–100 8–100 2–10 2–25 5–100 5–100 5–25 5–25
5 6 7 6 8 9 6 6
6.5 4.2 1.9 1.7 3.2 2.0 4.0 3.9
CNTs E S design
Bare E CNTs E
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Fig. 5. Differential pulse voltammograms obtained using the AS design (A), AS design coupled to external RE and CE (B) and S design (C). (Conditions as in Fig. 2).
For the sake of comparison, cyclic, differential pulse and square wave voltammograms for both, the AS design (with and without external RE and CE) and S design are shown in Fig. 5. Differences between signals decrease when external electrodes are used, and disappear when a symmetrical design is employed. Although in any case differences are not very notorious, errors are additive and total imprecision could be decreased by improving the measurement step. The best definition of the peaks is also obtained for this design, indicating that adequate electrode geometry was achieved.
3.3. Calibration plots Calibration plots were performed in order to calculate analytical parameters such as sensitivity or limit of detection. With this aim, cyclic voltammograms (100 mV s−1 ) were recorded for both designs, asymmetric and symmetric. Bare and CNTs modified electrodes were compared. In Table 3 the parameters obtained from the linear relationship between ip (A) and C (M), performing three repeated observations at each concentration value, are reported. The lower detection limit (calculated as the concentration corresponding to three times the standard deviation of the intercept) and higher sensitivity were obtained with nanostructured dual sensors, specifically, with AS Design (LOD lower than 2 M and sensitivity higher than 0.7 A M−1 ). In both cases, the nanostructuration allows obtaining higher sensitivities than with bare electrodes.
4. Conclusions A basic comparative study of two different designs of screenprinted bisensors was made by recording the analytical signal of a molecule that has been employed as electroactive label, methylene blue. This study demonstrates the relevance of the design of the screen-printed strips. Two elliptic WEs substituted the circled WE in traditional SPEs. Bare and nanostructured electrodes with carbon nanotubes were evaluated. In both cases, an asymmetrical design (with the major axis of the ellipses perpendicular to the strip) implies higher differences in the recorded intensities on each WE. These differences can be avoided with the use of an external RE and CE system or with the use of a more symmetrical design (major axis of the ellipses parallel to the strip with equidistant CE and RE to both WEs). Acknowledgment This work has been supported by the Ministry of Science and Innovation (MICINN) under project CTQ2011-25814. References [1] D.R. Matthews, E. Bown, A. Watson, R.R. Holman, J. Steemson, S. Hughes, D. Scott, Pen-sized digital 30-second blood glucose meter, Lancet 329 (1987) 778–779.
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[2] M. Tudorache, C. Bala, Biosensors based on screen-printing technology, and their applications in environmental and food analysis, Anal. Bioanal. Chem. 388 (2007) 565–578. [3] M. Li, Y.-T. Li, D.-W. Li, Y.-T. Long, Recent developments and applications of screen-printed electrodes in environmental assays—a review, Anal. Chim. Acta 734 (2012) 31–44. [4] J.P. Metters, R.O. Kadara, C.E. Banks, New directions in screen printed electroanalytical sensors: an overview of recent developments, Analyst 136 (2011) 1067–1076. [5] S. Iijima, Helical microtubules of graphitic carbon, Nature 354 (1991) 56–58. [6] A. Speltini, D. Merli, A. Profumo, Analytical application of carbon nanotubes, fullerenes and nanodiamonds in nanomaterials-based chromatographic stationary phases: a review, Anal. Chim. Acta 783 (2013) 1–16. [7] C. Herrero Latorre, J. Álvarez Méndez, J. Barciela García, S. García Martín, R.M. ˜ Crecente, Carbon nanotubes as solid-phase extraction sorbents prior to Pena atomic spectrometric determination of metal species: a review, Anal. Chim. Acta 749 (2012) 16–35. [8] A.V. Herrera-Herrera, M.Á. González-Curbelo, J. Hernández-Borges, M.Á. Rodríguez-Delgado, Carbon nanotubes applications in separation science: a review, Anal. Chim. Acta 734 (2012) 1–30. [9] D. Vairavapandian, P. Vichchulada, M.D. Lay, Preparation and modification of carbon nanotubes: review of recent advances and applications in catalysis and sensing, Anal. Chim. Acta 626 (2008) 119–129. ˜ ˜ J.M. Pingarrón, Role of carbon nanotubes in electro[10] L. Agüí, P. Yánez-Sede no, analytical chemistry: a review, Anal. Chim. Acta 622 (2008) 11–47. [11] J. Gooding, Nanostructuring electrodes with carbon nanotubes: a review on electrochemistry and applications for sensing, Electrochim. Acta 50 (2005) 3049–3060. [12] S.K. Vashist, D. Zheng, K. Al-Rubeaan, J.H.T. Luong, F.-S. Sheu, Advances in carbon nanotube based electrochemical sensors for bioanalytical applications, Biotechnol. Adv. 29 (2011) 169–188. [13] R. García-González, A. Costa-García, M.T. Fernández-Abedul, Enhanced detection of the potential electroactive label Methylene blue by optimized electrode nanostructuration with carbon nanotubes, Sens. Actuators, B (2014) (under revision). [14] F. Ge, X.-E. Zhang, Z.-P. Zhang, X.-M. Zhang, Simultaneous determination of maltose and glucose using a screen-printed electrode system, Biosens. Bioelectron. 13 (1998) 333–339. [15] V. Escamilla-Gómez, D. Hernández-Santos, M.B. González-García, J.M. Pingarrón-Carrazón, A. Costa-García, Simultaneous detection of free and total prostate specific antigen on a screen-printed electrochemical dual sensor, Biosens. Bioelectron. 24 (2009) 2678–2683. [16] G. Martínez-Paredes, M.B. González-García, A. Costa-García, Genosensor for detection of four pneumoniae bacteria using gold nanostructured screenprinted carbon electrodes as transducers, Sens. Actuators, B 149 (2010) 329–335. [17] M.M.P.S. Neves, M.B. González-García, C. Delerue-Matos, A. Costa-García, Multiplexed electrochemical immunosensor for detection of celiac disease serological markers, Sens. Actuators, B 187 (2013) 33–39. [18] P. Jothimuthu, R.A. Wilson, J. Herren, E.N. Haynes, W.R. Heineman, I. Papautsky, Lab-on-a-chip sensor for detection of highly electronegative heavy metals by anodic stripping voltammetry, Biomed. Microdevices 13 (2011) 695–703. [19] F. Ménil, B.O. Daddah, P. Tardy, H. Debéda, C. Lucat, Planar LiSICON-based potentiometric CO2 sensors: influence of the working and reference electrodes relative size on the sensing properties, Sens. Actuators, B 107 (2005) 695–707. [20] R. Singh, G. Sumana, R. Verma, S. Sood, M.K. Pandey, R.K. Gupta, B.D. Malhotra, DNA biosensor for detection of Neisseria gonorrhoeae causing sexually transmitted disease, J. Biotechnol. 150 (2010) 357–365.
[21] N. Zhu, A. Zhang, Q. Wang, P. He, Y. Fang, Electrochemical detection of DNA hybridization using methylene blue and electro-deposited zirconia thin films on gold electrodes, Anal. Chim. Acta 510 (2004) 163–168. [22] R. García-González, A. Costa-García, M.T. Fernández-Abedul, Methylene blue covalently attached to single-stranded DNA as electroactive label for geneosensors development, Sens. Actuators B 191 (2014) 784–790. [23] E.M. Boon, D.M. Ceres, T.G. Drummond, M.G. Hill, J.K. Barton, Mutation detection by electrocatalysis at DNA-modified electrodes, Nat. Biotechnol. 18 (2000) 1096–1100. [24] R. García-González, A. Costa-García, M.T. Fernández-Abedul, Dispersion studies of carboxyl, amine and thiol-functionalized carbon nanotubes for improving the electrochemical behavior of screen printed electrodes, Sens. Actuators, B 181 (2013) 353–360. [25] N. Modirshahla, M.A. Behnajady, S. Kooshaiian, Investigation of the effect of different electrode connections on the removal efficiency of Tartrazine from aqueous solutions by electrocoagulation, Dyes Pigm. 74 (2007) 249–257. [26] F.G. Cottrell, Z. Phys. Chem. 42 (1903) 385. [27] M.J. Sims, N.V. Rees, E.J.F. Dickinson, R.G. Compton, Effects of thin-layer diffusion in the electrochemical detection of nicotine on basal plane pyrolytic graphite (BPPG) electrodes modified with layers of multi-walled carbon nanotubes (MWCNT-BPPG), Sens. Actuators, B 144 (2010) 153–158. [28] I.K. Kookos, On the diffusion in porous electrodes of SOFCs, Chem. Eng. Sci. 69 (2012) 571–577. [29] M. Nádherná, F. Opekar, J. Reiter, K. Stulík, A planar, solid-state amperometric sensor for nitrogen dioxide, employing an ionic liquid electrolyte contained in a polymeric matrix, Sens. Actuators, B 161 (2012) 811–817. [30] G.J. Maclay, W.J. Buttner, J.E. Stetter, Microfabricated amperometric gas sensors, IEEE Trans. Electron Devices 35 (1988) 793–799. ´ A. Hamáˇcek, S. Neˇspurek, ˚ [31] P. Kubersky, R. Soukup, R. Vik, Effect of the geometry of a working electrode on the behavior of a planar amperometric NO2 sensor based on solid polymer electrolyte, Sens. Actuators, B 187 (2013) 546–552.
Biographies Raquel García-González obtained her Ph.D. in 2013 (University of Oviedo) with the work “Nanostructuration of gold screen printed electrodes for transduction of genosensors with direct labels”. Her research is focused on electrochemistry and development of new electrodic surfaces as sensor platforms. Agustín Costa-García obtained his B.Sc. degree in chemistry, focus in analytical chemistry, in 1974 (University of Oviedo) and the Ph.D. in chemistry in 1977 (University of Oviedo). Since February 2000 he is professor in analytical chemistry (University of Oviedo). He leads the Immunoelectroanalytical Research Group of the University of Oviedo and has been supervisor of several research projects developed at the electrochemistry laboratories of the Department of Physical and Analytical Chemistry of the University of Oviedo. Nowadays his research is focused on the development of nanostructured electrodic surfaces and its use as transducers for electrochemical immunosensors and genosensors employing electrochemical labels. M. Teresa Fernández-Abedul received her Ph.D. in Chemistry in 1995 at University of Oviedo, Spain. Since 2002 is working as Senior Lecturer in Analytical Chemistry at the University of Oviedo. Her current research interests are the development of immunosensors and genosensors employing nanostructured transducers as well as the development of miniaturized analytical devices (microchip electrophoresis) for the sensitive electrochemical detection of analytes of interest, even those nonelectroactive through adequate electroactive labeling systems.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Dual screen-printed electrodes with elliptic working electrodes arranged in parallel or perpendicular to the strip Raquel García-González, Agustín Costa-García, M. Teresa Fernández-Abedul ∗ Departamento de Química Física y Analítica, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain
a r t i c l e
i n f o
Article history: Received 18 October 2013 Received in revised form 29 January 2014 Accepted 8 March 2014 Available online 19 March 2014 Keywords: Dual screen-printed electrodes Electrochemical bisensor Multiplexed determination Nanostructured electrodes Methylene blue
a b s t r a c t The number of works based on electrochemical mono-, bi- and multi-sensors is increasing, especially in which multiplexing is concerning. Determination of a panel of analytes in clinical analysis or of different related molecules in environmental or food analysis has increased the efforts for designing appropriate sensors, becoming the design, a very important step in their development. In this paper, two different dual designs for screen-printed electrodes, one of them symmetrical, were electrochemically evaluated with methylene blue as electroactive species. Two elliptic working electrodes substituted the traditional circled and centered working electrodes in the monosensing mode. Ellipses perpendicular to the strip and with the same configuration than conventional for reference and auxiliary electrodes constituted the asymmetrical design. Placing the ellipses in parallel with the strip, surrounding them with a counter electrode and locating the reference at the basis, produces a symmetrical design. Although both could be employed, more precise peak intensities are obtained for the more symmetrical design as well as with the employ of an external reference and auxiliary electrode system. Since a more sensitive detection is increasingly required, comparison is made also after nanostructuration with carbon nanotubes. The symmetrical design appears also to be more appropriate for all the electrochemical techniques tested: cyclic voltammetry, differential pulse voltammetry and square wave voltammetry. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Screen-printing methodology was introduced worldwide over a thousand years ago and exists today in fields such as art, textiles, and advertising. This technique was applied to biosensor manufacture in the middle of the 1980s [1] and is nowadays well established for the production of thick-film electrochemical transducers [2]. In this technology, the paste is deposited and distributed on the screen mesh using a squeegee or a roller and is forced to pass through the open areas of the screen mesh to the substrate. Finally, a protective ink coating is used to insulate the conductive track from the electrodes [3]. It allows the mass production of reproducible and inexpensive, mechanically robust and miniaturized strip solid electrodes. They are also of easy handling and manipulation in a disposable manner, which avoids inconveniences of conventional electrodes such as memory-effects due to difficult, time-consuming and sometimes inefficient cleaning steps, allowing the performance of parallel assays. Their precision makes possible the development of sensitive and quantitative methodologies, which converts
∗ Corresponding author. Tel.: +34 985102968; fax: +34 985103125. E-mail address: [email protected] (M.T. Fernández-Abedul). http://dx.doi.org/10.1016/j.snb.2014.03.036 0925-4005/© 2014 Elsevier B.V. All rights reserved.
them into versatile tools for biosensor development [4]. In addition, the extensive range of modifications to screen-printed electrodes (SPEs) opens numerous fields of application. On the other hand, since the discovery of carbon nanotubes (CNTs) Ref. [5], they have acquired a great relevance because of their advantageous properties. Modification of electrodes (nanostructuration) has been employed for the improvement of the electroanalytical behavior of analytes of interest. Numerous advantages of CNTs as electrode materials have been attested for analysis of diversified chemicals of food quality, clinical and environmental interest. CNT-electrochemical sensors exhibit low limit of detection and fast response due to the signal enhancement provided by high surface area, low overvoltage, and rapid electrode kinetics. A highly thermal conductive, mechanically strong and chemically stable nature of CNTs is very appealing for lot of applications [6–11]. Their high surface-to-volume ratio is also a definite asset toward the development of biosensing platforms for molecule detection [12]. The use of gold SPEs is very appropriate due to their adsorptive and electrochemical properties and the combination with CNTs is very promising [13]. Dual and multi-analysis of a variety of electroactive species based on SPEs have been reported, e.g. the simultaneous determination of maltose and glucose using a SPE systemed Ref. [14]
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simultaneous detection of free and total prostate specific antigen, where the measurement depends one on each other, on a screen-printed electrochemical dual sensor [15]. In this way, in one working electrode (WE) total prostate-specific antigen (PSA) is detected meanwhile free PSA is determined in the second one. The simultaneous detection of four pneumoniae bacteria using gold nanostructured carbon SPEs [16] or a multiplexed electrochemical immunosensor for detection of celiac disease serological markers [17] were developed in our research group too. The configuration of the electrode has influence on the measurements and has been optimized with respect to size and layout in the determination of highly electronegative metals with bismuth WE, Ag/AgCl RE and gold CE. It was especially important since generation of gas bubbles at the counter electrode (CE) due to electrolysis of water resulted in poor stability and reproducibility [18]. This was solved by increasing the area of the auxiliary electrode as well as the distance to the WE. Similarly, the WE and RE relative size is of relevance on the design of potentiometric CO2 sensors [19]. A narrow spacing between electrodes slightly improves the quality of the responses and the longer the facing lengths between electrodes, the better the shape of the responses. Moreover, when the surface of the RE (this is a two-electrode system) is smaller than that of the WE, the sensor does not operate properly (baseline instability and smaller response). Geometry is therefore very relevant for achieving an adequate performance of sensors. Electrode geometry plays a critical role in electrochemical sensors, and therefore multiple sensor designs incorporating the three electrode cell with different electrode geometries were explored, e.g. in the development of a lab-on-a-chip sensor for detection of highly electronegative heavy metals by anodic stripping voltammetry [18]. Commercially, there are two possible configuration designs of dual screen-printed electrodes: asymmetrical (with two WE perpendicular to the strip) and symmetrical (with two WE parallel to the strip) designs. The symmetrical design proposed maintains distance between electrodes and length of connections identical for each WE. Many dual sensors were developed using the symmetrical and asymmetrical designs proposed in this work as transducer surfaces. But this is the first time that a comparison study between these different dual electrode configurations was made. In this paper, a comparative study of two configurations of dual SPEs is made. The electrochemical cell of the bisensor, or dual sensor (a screen-printed strip that contains two working electrodes) consists of a four-electrode system: two elliptic gold WE, a silver pseudo-RE and a gold CE. This is the first time that the electrochemical characterizations of these designs were carried out, in this case, with Methylene Blue, 3,7-bis(dimethylamino)phenothiazine5-ium-chloride, (MB) as model electroactive species to characterize the effect of the electrode geometry. MB is a commonly employed biolabel that had been used as electroactive marker in biosensors to detect the hybridization event [20,21].
2. Experimental 2.1. Reagents and solutions Nafion® (perfluorinated ion-exchange resin, 5 wt% solution in a mixture of lower aliphatic alcohols and water) and methylene blue (certified by the BSC, Biological Stain Commission) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sulphuric (95–97% of purity) acid and potassium chloride were purchased from Merck (Darmstadt, Germany). Finally, amine functionalized multi-wall carbon nanotubes (MWCNT-NH2 ) were purchased from Belgium
303
Nanocyl (Auvelais, Belgium). Water was purified employing a Milli-Q directQS system from Millipore. The resistivity value of the water used was 18.2 M cm. 2.2. Apparatus and instruments Voltammetric measurements were performed with an Autolab PGSTAT 10 (ECO Chemie) potentiostat interfaced to a Pentium 120 computer system and controlled by Autolab GPES software version 4.8. An Elma ultrasonic bath, a Nahita centrifuge with interchangeable car, a Mettler Toledo (AB54) balance, a Crison Micro-pH 2001 pH-meter, a magnetic stirrer Asincro (J.P.Selecta), a Sanyo refrigerator and a Sanyo (MIR-162) incubator were also used. 2.3. Dual SPEs Electrode configuration plays a critical role in electrochemical sensors, and therefore two sensor designs incorporating the four- electrode cell with different electrode configurations were explored. Bisensors based on gold SPEs purchased from DropSens (Asturias, Spain) include a four-electrode system configuration printed on the same strip. The format of these SPEs includes two elliptic WE (6.3 mm2 each one, semi-major axis of 4 mm and semiminor axis of 2 mm), a silver pseudo-RE and a gold CE that uses the same ink of the WE. All of them are screen-printed on a ceramic substrate (3.4 × 1.0 × 0.05 cm) and subjected to high-temperature curing. An insulating layer serves to delimit the working area and electric contacts. The production characteristics of commercial SPEs are regarded by the manufacturers as proprietary information. A specific connector supplied by DropSens allows their connection to the potentiostat. The two designs of dual SPEs studied are presented in Fig. 1. Two WE are included in the same strip, and they share RE and CE. The asymmetrical design (AS design), in Fig. 1B, derives from the traditional design of a monosensor (Fig. 1A), where the WE is a circle (12.6 mm2 ) surrounded by the CE, and a small silver arc situated as a continuation of the counter acts as a pseudo-reference electrode. In this case the central WE has been substituted by two ellipses (6.3 mm2 each one) with the major axis perpendicular to the strip. The electrochemical arrangement as a whole is not symmetrical. The counter (12.6 mm2 and 1 mm wide) and pseudo-reference electrodes are similar to those in the monosensor. A stable RE is an important component of an integrated electrochemical system and is a prerequisite for achieving reliable performance [18]. For the sake of clarity, the electrode closest to the CE top-WE to this that is nearer the connections. In some studies, an external homemade RE and CE system was used coupled to AS design. A 250 L micropipette tip included a RE that consisted of an anodized silver wire (Goodfellow, Huntingdon, UK) introduced through a rubber syringe piston. Then the tip was filled with saturated KCl solution containing a low-resistance liquid junction. The platinum wire (250 m diameter) that acted as CE was fixed with insulating tape to the micropipette tip. For recording the voltammograms, the tip was held on a support allowing horizontal and vertical movement. The symmetric design (S design), in Fig. 1C, has also two ellipses but the major axis of the ellipses are parallel to the ceramic strip. In this case, electrodes are called right and left-WE (6.3 mm2 each one). The CE (19.8 mm2 and 1 mm wide) surrounds equally both electrodes, and the pseudo-reference electrode is equidistant from both WEs, in a much more symmetrical design. The connections are at the same length of both WEs. In both biosensors the space between electrodes is covered by the insulating layer meanwhile in the monosensor this layer surrounded all the three electrodes.
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A
C
B
Ref erence electrod e (RE)
RE
CE
RE
CE
Working electrod e (WE) Counter electrod e (CE) Top-WE
Bottom-WE
(Design AS)
Lef t-WE
Right-WE
(Design S)
Fig. 1. Schematic design of SPEs with (A) one WE and two WEs with the main axis of the ellipses: (B) perpendicular (AS design) or (C) parallel (S design) to the strip.
2.4. Nanostructuration of AuSPEs The nanostructuration of WE with CNTs was carried out by evaporation at room temperature of a drop of 3 L of MWCNTs-NH2 (dispersed in a 0.5% Nafion® /ethanol solution [13]) deposited on the WE. In this way, CNTs-modified AuSPEs were obtained. Before the measurements the CNTs-AuSPEs were washed with the supporting electrolyte (0.1 M Tris–H2 SO4 pH 8.0) and dried at room temperature (24 ± 1 ◦ C). 2.5. Electrochemical measurements The reduction signal of MB was measured ten times (in ten different bare or nanostructured AuSPEs) by scanning the potential between 0.0 and −0.7 V using cyclic voltammetry (CV, with a scan rate of 250 mV s−1 ), differential pulse voltammetry (DPV, with a step potential of 0.008 V, modulation amplitude of 0.05 V and standby potential of 0 V) and square wave voltammetry (SWV, with a step potential of 0.008 V, amplitude of 0.05 V and standby potential of 0 V). Drops of 40 L of 20 and 100 M MB were used. All experiments were performed at room temperature (25 ◦ C). 3. Results and discussion Multianalyte determinations as well as recording repetitive measurements require the employ of appropriate analytical devices that maintains the miniaturization and simplicity of monosensors. The design has to be carefully checked in order to attain the best performance, and specially required before a massproduction is made. Two different designs for dual detection (those described in Section 2), with two elliptic WE (perpendicular and parallel to the ceramic strip) were evaluated. A pseudo-reference electrode consisting of a screen-printed silver ink is employed. Even when a real reference electrode (e.g. Ag/AgCl) is recommended, this is not very adequate for this type of electrodes and good precision of potentials is usually obtained [13]. A model molecule that has been employed as biolabel for genosensing [22], methylene blue, has been chosen for the electrochemical characterization. MB presents a well-defined two-electron redox process [23] with cathodic (conversion to leucomethylene blue (LB)) and anodic (oxidation to MB) peaks. MB studies were initially performed on bare electrodes but since
high sensitivity improvements are obtained on nanostructured electrodes, a comparative study of configuration designs was also carried out with nanostructured WEs. In a previous work performed in our research group, it has been demonstrated that nanostructuration of AuSPEs with MWCNTs-NH2 produces the enhancement of the analytical signal recorded for MB [13]. These nanostructures were efficiently dispersed in a 1:1 H2 O/0.5% Nafion® in ethanol. This dispersion of CNTs was used throughout this work for the nanostructuration of electrodes. Stability of the nanostructures was studied in a previous work of our research group [24]. However, it is a very simple and fast procedure that would not be difficult to reproduce at any time. 3.1. Electrochemical behavior of dual elliptic WEs perpendicular to the strip In order to know the influence of the design of the dual SPEs on the analytical signal of methylene blue, different electrochemical techniques: CV, DPV and SWV were employed. Voltammograms were recorded in a solution of MB in the supporting electrolyte (0.1 M Tris–H2 SO4 pH 8.0). Due to the sensitivity enhancement produced by electrode nanostructuration, a 100 M solution is employed for bare electrodes meanwhile voltammograms are recorded in 20 M solutions on nanostructured electrodes. First, bare WEs were tested, and further, they were nanostructured one by one, recording the analytical signal. In the case of bare electrodes (see Fig. 2A), a clear difference between signals for top and bottom electrodes can be seen. The peak intensities recorded for top-WE are higher than those for bottom-WE with ratios of 1.3, 1.4 and 1.2 for CV, DPV and SWV, respectively. It has been reported that when the distance between RE and WE increases, the electrical current decreases [25]. In this case, the top WE (closer to the RE) will always record higher intensities. In all cases, a notorious increase in the signal is observed after the nanostructuration of the electrodes, which was already reported [13]. When CV was used, intensities obtained were almost triple for a concentration five times lower. In the case of nanostructured electrodes both WEs present similar behavior, i.e. slightly higher for the top-WE too (see Fig. 2B). Here the ratio is 1.1 for all the techniques, CV, DPV and SWV. The nanostructuration with CNTs of only one of the WEs (the data are presented in Table 1) leads to results like those already commented, signals are similar to those obtained
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Fig. 2. Bar diagrams obtained with CV (250 mV s−1 ), DPV (s = 0.008 V, A = 0.05 V) and SWV (s = 0.008 V, A = 0.05 V) for the AS design on bare (A), and nanostructured (B) WEs. Colorless (top-WE) and colored (bottom-WE).
Table 1 Methylene blue cathodic peak currents on bare and nanostructured WEs perpendicular to the strip (AS design). AS design
ip (A)
CMB (M)
CNTs on top-WE
20
CNTs on bottom-WE
20
Top-WE Bottom-WE Top-WE Bottom-WE
for either bare or nanostructured electrodes. Following the same trend, higher intensities were obtained when top-WE were modified. The highest intensity difference was observed with SWV and when the top-WE was nanostructured: 58 ± 7 A were recorded, 1.4 times the intensity recorded with the modified bottom-WE. This could be explained by the asymmetry in the design. The CE does not extend equally to both WEs: the facing length is lower for the top-WE and RE is much closer to the top-WE. However, one of the most probable causes is the difference in the length of WEs connections. They are not the same length and they have an important role in the final current. Conduction of electrons is made through a screen-printed conductive ink and more resistance is obtained for longer connector pad (bottom-WE) with subsequent lower analytical signal. However, there are many parameters to take into account when designing a sensor: the solution IR drop (distance between WE and RE, concentration of supporting electrolyte), the dimensions and the specific surface area of the working electrodes [26], and edge effects on plain electrode [27] and diffusion on a porous electrode [28]. In a classic electrochemical cell with a system of three electrodes, the generated current flows through the WE and CE and the potential is controlled between the WE and RE. The threeelectrode system is advantageous because it prevents the RE from driving the current which could change its potential [18]. The effect of the surface area of the WE on the value of the peak
CV
DPV
SWV
−11 ± 1 −1.2 ± 0.1 −0.9 ± 0.1 −9.2 ± 0.8
−34 ± 4 −2.8 ± 0.2 −3.8 ± 0.3 −25 ± 2
−58 ± 7 −5.9 ± 0.4 −7.9 ± 0.6 −42 ± 3
current was discussed in several papers [29,30] too. Following the Randles–Sevcik equation, the higher the WE area, the higher the current intensity. A linear dependence of the reduction current on the WE surface area was reported for a NO2 sensor [31]. However, the area of the WE had a strong impact on the stability and current noise of the nitrogen dioxide sensor: the smaller the electrode area, the better the sensor stability and the lower the current noise. In this work, the surface area of each WE in the dual-SPE are half of the area of the WE in a monosensor-SPE (6.3 vs. 12.6 mm2 ). In this way, the current density generated is similar for both WEs and CE. In order to know if this design is the responsible of the differences observed, an external RE and CE were coupled to AS design. The results obtained are presented in Fig. 3A and B. When bare electrodes were tested, both WE (top and bottom) generated the same intensities in all cases and no differences between both were observed. When nanostructured electrodes were evaluated, slight differences in the recorded intensities were observed but in all cases these were lower than in the case before commented. The highest intensity corresponds in this case to the bottom-WE, and since it is only observed with nanostructured electrodes might be caused by the modification step. Since the design directly influences the currents generated and the use of external RE and CE is not of easy use, a new design was proposed and evaluated.
Table 2 Methylene blue cathodic peak currents on bare and nanostructured WEs parallel to the strip (S design). S design
CMB (M)
CNTs on right-WE
20
CNTs on left-WE
20
ip (A)
Right-WE Left-WE Right-WE Left-WE
CV
DPV
SWV
−9.1 ± 0.6 −0.36 ± 0.04 −0.32 ± 0.03 −9.2 ± 0.5
−30 ± 3 −1.4 ± 0.1 −1.5 ± 0.1 −28 ± 2
−47 ± 5 −2.1 ± 0.2 −1.9 ± 0.1 −43 ± 3
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A
B
25
50
(CNTs electrodes)
20
40
15
30
|ip| / A
|ip| / A
(Bare electrodes)
10
5
20
10
0
0
CV
DPV
SWV
CV
DPV
SWV
(AS design – external RE and CE) Fig. 3. Bar diagrams obtained with CV, DPV and SWV for the AS design coupled with external RE and CE from bare (A) and nanostructured (B) WEs. Colorless (top-WE) and colored (bottom-WE). (Conditions as in Fig. 2).
B
A
50
12
(Bare electrodes)
(CNTs electrodes) 40
|ip| / A
|ip| / A
9
6
30
20
3 10
0
0
CV
DPV
SWV
CV
DPV
SWV
(S design) Fig. 4. Bar diagrams obtained with CV, DPV and SWV for the S design from bare (A) and nanostructured (B) WEs. Colorless (left-WE) and colored (right-WE). (Conditions as in Fig. 2).
3.2. Electrochemical behavior of dual elliptic WEs parallel to the strip The S design of the electrochemical cell was proposed as the most symmetrical possible. In this case the CE and RE are equally shared, and connections for all the electrodes are at the same length. In this way, the same resistance in the internal circuit is produced. On bare electrodes (see Fig. 4A), the intensities recorded by any of the voltammetric techniques, CV, DPV or SWV on each WE were
very similar for right and left WEs. The intensity ratio in this case was 1.0 for all the techniques. When the nanostructuration is performed on both WEs (see Fig. 4B), the two intensities recorded on each WE were very similar too. Ratio of 1.0 is also found for all the techniques. When only one of the electrodes is nanostructured (data shown in Table 2), comparison between right and left WE gave ratios of 1.0 for CV and 1.1 for both DPV and SWV. RSD values were 6, 9 and 10% for CV, DPV and SWV, respectively.
Table 3 Analytical parameters of calibration plots from CV (100 mV s−1 ). Design
Electrode
Signal
Slope (A M−1 )
Intercept (A)
R
Linear range (M)
n
LD (M)
AS design
Bare E
Top Bottom Top Bottom Left Right Left Right
−0.0230 −0.0259 −0.7906 −0.5747 −0.0126 −0.0123 −0.2100 −0.2038
0.0558 0.0776 1.6931 0.5165 0.0117 0.0006 0.8279 0.6503
0.9979 0.9983 0.9957 0.9961 0.9985 0.9993 0.9962 0.9993
8–100 8–100 2–10 2–25 5–100 5–100 5–25 5–25
5 6 7 6 8 9 6 6
6.5 4.2 1.9 1.7 3.2 2.0 4.0 3.9
CNTs E S design
Bare E CNTs E
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Fig. 5. Differential pulse voltammograms obtained using the AS design (A), AS design coupled to external RE and CE (B) and S design (C). (Conditions as in Fig. 2).
For the sake of comparison, cyclic, differential pulse and square wave voltammograms for both, the AS design (with and without external RE and CE) and S design are shown in Fig. 5. Differences between signals decrease when external electrodes are used, and disappear when a symmetrical design is employed. Although in any case differences are not very notorious, errors are additive and total imprecision could be decreased by improving the measurement step. The best definition of the peaks is also obtained for this design, indicating that adequate electrode geometry was achieved.
3.3. Calibration plots Calibration plots were performed in order to calculate analytical parameters such as sensitivity or limit of detection. With this aim, cyclic voltammograms (100 mV s−1 ) were recorded for both designs, asymmetric and symmetric. Bare and CNTs modified electrodes were compared. In Table 3 the parameters obtained from the linear relationship between ip (A) and C (M), performing three repeated observations at each concentration value, are reported. The lower detection limit (calculated as the concentration corresponding to three times the standard deviation of the intercept) and higher sensitivity were obtained with nanostructured dual sensors, specifically, with AS Design (LOD lower than 2 M and sensitivity higher than 0.7 A M−1 ). In both cases, the nanostructuration allows obtaining higher sensitivities than with bare electrodes.
4. Conclusions A basic comparative study of two different designs of screenprinted bisensors was made by recording the analytical signal of a molecule that has been employed as electroactive label, methylene blue. This study demonstrates the relevance of the design of the screen-printed strips. Two elliptic WEs substituted the circled WE in traditional SPEs. Bare and nanostructured electrodes with carbon nanotubes were evaluated. In both cases, an asymmetrical design (with the major axis of the ellipses perpendicular to the strip) implies higher differences in the recorded intensities on each WE. These differences can be avoided with the use of an external RE and CE system or with the use of a more symmetrical design (major axis of the ellipses parallel to the strip with equidistant CE and RE to both WEs). Acknowledgment This work has been supported by the Ministry of Science and Innovation (MICINN) under project CTQ2011-25814. References [1] D.R. Matthews, E. Bown, A. Watson, R.R. Holman, J. Steemson, S. Hughes, D. Scott, Pen-sized digital 30-second blood glucose meter, Lancet 329 (1987) 778–779.
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[2] M. Tudorache, C. Bala, Biosensors based on screen-printing technology, and their applications in environmental and food analysis, Anal. Bioanal. Chem. 388 (2007) 565–578. [3] M. Li, Y.-T. Li, D.-W. Li, Y.-T. Long, Recent developments and applications of screen-printed electrodes in environmental assays—a review, Anal. Chim. Acta 734 (2012) 31–44. [4] J.P. Metters, R.O. Kadara, C.E. Banks, New directions in screen printed electroanalytical sensors: an overview of recent developments, Analyst 136 (2011) 1067–1076. [5] S. Iijima, Helical microtubules of graphitic carbon, Nature 354 (1991) 56–58. [6] A. Speltini, D. Merli, A. Profumo, Analytical application of carbon nanotubes, fullerenes and nanodiamonds in nanomaterials-based chromatographic stationary phases: a review, Anal. Chim. Acta 783 (2013) 1–16. [7] C. Herrero Latorre, J. Álvarez Méndez, J. Barciela García, S. García Martín, R.M. ˜ Crecente, Carbon nanotubes as solid-phase extraction sorbents prior to Pena atomic spectrometric determination of metal species: a review, Anal. Chim. Acta 749 (2012) 16–35. [8] A.V. Herrera-Herrera, M.Á. González-Curbelo, J. Hernández-Borges, M.Á. Rodríguez-Delgado, Carbon nanotubes applications in separation science: a review, Anal. Chim. Acta 734 (2012) 1–30. [9] D. Vairavapandian, P. Vichchulada, M.D. Lay, Preparation and modification of carbon nanotubes: review of recent advances and applications in catalysis and sensing, Anal. Chim. Acta 626 (2008) 119–129. ˜ ˜ J.M. Pingarrón, Role of carbon nanotubes in electro[10] L. Agüí, P. Yánez-Sede no, analytical chemistry: a review, Anal. Chim. Acta 622 (2008) 11–47. [11] J. Gooding, Nanostructuring electrodes with carbon nanotubes: a review on electrochemistry and applications for sensing, Electrochim. Acta 50 (2005) 3049–3060. [12] S.K. Vashist, D. Zheng, K. Al-Rubeaan, J.H.T. Luong, F.-S. Sheu, Advances in carbon nanotube based electrochemical sensors for bioanalytical applications, Biotechnol. Adv. 29 (2011) 169–188. [13] R. García-González, A. Costa-García, M.T. Fernández-Abedul, Enhanced detection of the potential electroactive label Methylene blue by optimized electrode nanostructuration with carbon nanotubes, Sens. Actuators, B (2014) (under revision). [14] F. Ge, X.-E. Zhang, Z.-P. Zhang, X.-M. Zhang, Simultaneous determination of maltose and glucose using a screen-printed electrode system, Biosens. Bioelectron. 13 (1998) 333–339. [15] V. Escamilla-Gómez, D. Hernández-Santos, M.B. González-García, J.M. Pingarrón-Carrazón, A. Costa-García, Simultaneous detection of free and total prostate specific antigen on a screen-printed electrochemical dual sensor, Biosens. Bioelectron. 24 (2009) 2678–2683. [16] G. Martínez-Paredes, M.B. González-García, A. Costa-García, Genosensor for detection of four pneumoniae bacteria using gold nanostructured screenprinted carbon electrodes as transducers, Sens. Actuators, B 149 (2010) 329–335. [17] M.M.P.S. Neves, M.B. González-García, C. Delerue-Matos, A. Costa-García, Multiplexed electrochemical immunosensor for detection of celiac disease serological markers, Sens. Actuators, B 187 (2013) 33–39. [18] P. Jothimuthu, R.A. Wilson, J. Herren, E.N. Haynes, W.R. Heineman, I. Papautsky, Lab-on-a-chip sensor for detection of highly electronegative heavy metals by anodic stripping voltammetry, Biomed. Microdevices 13 (2011) 695–703. [19] F. Ménil, B.O. Daddah, P. Tardy, H. Debéda, C. Lucat, Planar LiSICON-based potentiometric CO2 sensors: influence of the working and reference electrodes relative size on the sensing properties, Sens. Actuators, B 107 (2005) 695–707. [20] R. Singh, G. Sumana, R. Verma, S. Sood, M.K. Pandey, R.K. Gupta, B.D. Malhotra, DNA biosensor for detection of Neisseria gonorrhoeae causing sexually transmitted disease, J. Biotechnol. 150 (2010) 357–365.
[21] N. Zhu, A. Zhang, Q. Wang, P. He, Y. Fang, Electrochemical detection of DNA hybridization using methylene blue and electro-deposited zirconia thin films on gold electrodes, Anal. Chim. Acta 510 (2004) 163–168. [22] R. García-González, A. Costa-García, M.T. Fernández-Abedul, Methylene blue covalently attached to single-stranded DNA as electroactive label for geneosensors development, Sens. Actuators B 191 (2014) 784–790. [23] E.M. Boon, D.M. Ceres, T.G. Drummond, M.G. Hill, J.K. Barton, Mutation detection by electrocatalysis at DNA-modified electrodes, Nat. Biotechnol. 18 (2000) 1096–1100. [24] R. García-González, A. Costa-García, M.T. Fernández-Abedul, Dispersion studies of carboxyl, amine and thiol-functionalized carbon nanotubes for improving the electrochemical behavior of screen printed electrodes, Sens. Actuators, B 181 (2013) 353–360. [25] N. Modirshahla, M.A. Behnajady, S. Kooshaiian, Investigation of the effect of different electrode connections on the removal efficiency of Tartrazine from aqueous solutions by electrocoagulation, Dyes Pigm. 74 (2007) 249–257. [26] F.G. Cottrell, Z. Phys. Chem. 42 (1903) 385. [27] M.J. Sims, N.V. Rees, E.J.F. Dickinson, R.G. Compton, Effects of thin-layer diffusion in the electrochemical detection of nicotine on basal plane pyrolytic graphite (BPPG) electrodes modified with layers of multi-walled carbon nanotubes (MWCNT-BPPG), Sens. Actuators, B 144 (2010) 153–158. [28] I.K. Kookos, On the diffusion in porous electrodes of SOFCs, Chem. Eng. Sci. 69 (2012) 571–577. [29] M. Nádherná, F. Opekar, J. Reiter, K. Stulík, A planar, solid-state amperometric sensor for nitrogen dioxide, employing an ionic liquid electrolyte contained in a polymeric matrix, Sens. Actuators, B 161 (2012) 811–817. [30] G.J. Maclay, W.J. Buttner, J.E. Stetter, Microfabricated amperometric gas sensors, IEEE Trans. Electron Devices 35 (1988) 793–799. ´ A. Hamáˇcek, S. Neˇspurek, ˚ [31] P. Kubersky, R. Soukup, R. Vik, Effect of the geometry of a working electrode on the behavior of a planar amperometric NO2 sensor based on solid polymer electrolyte, Sens. Actuators, B 187 (2013) 546–552.
Biographies Raquel García-González obtained her Ph.D. in 2013 (University of Oviedo) with the work “Nanostructuration of gold screen printed electrodes for transduction of genosensors with direct labels”. Her research is focused on electrochemistry and development of new electrodic surfaces as sensor platforms. Agustín Costa-García obtained his B.Sc. degree in chemistry, focus in analytical chemistry, in 1974 (University of Oviedo) and the Ph.D. in chemistry in 1977 (University of Oviedo). Since February 2000 he is professor in analytical chemistry (University of Oviedo). He leads the Immunoelectroanalytical Research Group of the University of Oviedo and has been supervisor of several research projects developed at the electrochemistry laboratories of the Department of Physical and Analytical Chemistry of the University of Oviedo. Nowadays his research is focused on the development of nanostructured electrodic surfaces and its use as transducers for electrochemical immunosensors and genosensors employing electrochemical labels. M. Teresa Fernández-Abedul received her Ph.D. in Chemistry in 1995 at University of Oviedo, Spain. Since 2002 is working as Senior Lecturer in Analytical Chemistry at the University of Oviedo. Her current research interests are the development of immunosensors and genosensors employing nanostructured transducers as well as the development of miniaturized analytical devices (microchip electrophoresis) for the sensitive electrochemical detection of analytes of interest, even those nonelectroactive through adequate electroactive labeling systems.