Disposable electrodes modified with multi-wall carbon nanotubes for biosensor applications

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ITBM-RBM 29 (2008) 202–207

Original article

Disposable electrodes modified with multi-wall carbon nanotubes for biosensor applications Électrodes jetables modifiées avec nanotubes de carbone pour le développement de biocapteurs S. Laschi a,∗ , E. Bulukin a , I. Palchetti a , C. Cristea b , M. Mascini a a

Dipartimento di Chimica, Polo Scientifico e Tecnologico, Università degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy b Facultatea de Farmacie, Chimie Analiticã s¸ i Analizã Instrumentalã, Universitatea de Medicinã s¸ i Farmacie Iuliu Hat¸ieganu, str. Pasteura nr. 4, Cluj Napoca, Romania Received 24 October 2007; accepted 11 November 2007 Available online 26 December 2007

Abstract This paper deals with the development of a disposable electrochemical sensor for the detection of hydrogen peroxide, using screen-printed carbonbased electrodes (SPCEs) modified with multi-wall carbon nanotubes (MWCNs) dispersed in a polyethylenimine (PEI) mixture. The modified sensors showed an excellent electrocatalytic activity towards the analyte, respect to the high overvoltage characterising unmodified screen-printed sensors. The composition of the PEI/MWCNT dispersion was optimised in order to improve the sensitivity and reproducibility. The optimised sensor showed good reproducibility (10% RSD calculated on three experiments repeated on the same electrode), whereas a reproducibility of 15% as RSD was calculated on electrodes from different preparations. Preliminary experiments carried out using glucose oxidase (GOD) as biorecognition element gave rise to promising results indicating that these new devices may represent interesting components for biosensor construction. © 2007 Elsevier Masson SAS. All rights reserved. Résumé Cet article traite le développement d’une sonde électrochimique jetable pour la détection du peroxyde d’hydrogène, en usant des électrodes sérigraphiées de carbone (SPCEs) modifiées avec nanotubes (MWCNs) dispersés dans une solution de polyéthylenimine (PEI). Les sondes modifiées ont montré une excellente activité électrocatalytique vers l’analyte, respect à la surtension élevée caractérisant les sondes sérigraphiées pas modifiées. La composition de la dispersion de PEI/MWCNT a été optimisée afin d’améliorer la sensibilité et la reproductibilité. La sonde optimisée a montré une bonne reproductibilité (10 % RSD, calculé sur trois expériences répétées sur la même électrode), tandis qu’une reproductibilité de 15 % comme RSD a été calculé sur des électrodes produits pendant différentes préparations. Expériences préliminaires effectuées en utilisant l’enzyme glucose oxidase (GOD) a donné des résultats prometteurs, indiquant que ces nouveaux dispositifs peuvent représenter des instruments intéressants pour la construction de biocapteurs. © 2007 Elsevier Masson SAS. All rights reserved. Keywords: Screen-printed electrode; Multi-wall carbon nanotubes; PEI; Hydrogen peroxide; Biosensor Mots clés : Électrode sérigraphiée ; Nanotubes ; PEI ; Peroxyde d’hydrogène ; Biocapteur

1. Introduction

∗

Corresponding author. E-mail address: [email protected] (S. Laschi).

1297-9562/$ – see front matter © 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.rbmret.2007.11.002

Carbon nanotubes (CNTs) are new and interesting members of the carbon family offering unique mechanical and electronic properties combined with chemical stability [1]. Carbon nanotubes are constructed from sp2 carbon units, presenting a

S. Laschi et al. / ITBM-RBM 29 (2008) 202–207

seamless structure with hexagonal honeycomb lattices, being several nanometers in diameter and many microns in length [2,3]. There are two groups of carbon nanotubes, multi-wall carbon nanotubes (MWNTs) and single-wall carbon nanotubes (SWNTs) [2]. MWNTs can be visualised as concentric and closed graphite tubules with multiple layers of graphite sheet, defining a hole typically from 2 to 25 nm, separated by a distance of approximately 0.34 nm. A SWNT consists of a single graphite sheet rolled seamlessly, defining a cylinder of 1–2 nm diameter. Carbon nanotubes can behave as metals or semiconductors depending on the structure, mainly on the diameter and helicity [4]. Their electronic properties suggest that carbon nanotubes have the ability to mediate electron-transfer reactions with electroactive species when used as an electrode [5]. It has been suggested that the electrocatalytic properties of the CNTs originate from the open ends [6]. The properties of carbon nanotubes also make them extremely attractive for chemical and biochemical sensor assembly [7,8]. The electrical conductivity and flexibility of the CNTs have suggested the very interesting possibility of using such materials to promote electron transfer reactions with enzymes in biosensor development [9,10]. It is very difficult for the enzymes to exchange electrons directly with the electrode surface; however, CNTs can form a network and project outward from the electrode, acting like bundled ultramicroelectrodes that permit access to the active sites of enzymes, facilitating direct electron transfer [9,11]. The ability of CNTs to promote the electron-transfer reaction of hydrogen peroxide suggests interesting applications of the CNTs for oxidase-based amperometric biosensors [13,12]. Amperometric biosensors, which combine the bioselectivity of redox enzymes with the inherent sensitivity of amperometric transductions have proven to be very useful for the detection of glucose [14–16]. A major barrier for developing CNT-based devices is the insolubility of CNT in all solvents [13]. Nevertheless, the challenge in CNT solubilisation has been addressed through their covalent modification or noncovalent functionalisation [18,19]. The “wrapping” of CNTs in polymeric chains has been found useful for improving their solubility without impairing their physical properties; this strategy, based on wrapping watersoluble linear polymers around the tubes, is robust and simple; another advantage of noncovalent attachment is that the structure of the nanotube is not altered, thus its mechanical properties remains unvaried [20]. Among different families of polymers, Nafion films due to their unique ion-exchange and biocompatibility properties have been used extensively for the modification of electrode surfaces and for the construction of amperometric biosensors [21,13]. Similar to other polymers used for wrapping and solubilisation of CNT, Nafion bears a polar side chain. For this reason, CNT can be easily suspended in solutions of Nafion in phosphate buffer or alcohol [17]. Another approach for improving the dispersion of CNT involves polyethylenimine (PEI), which is indicated as a very suitable reagent to immobilise carbon nanotubes onto the working surface of an electrode without loss of sensitivity. It has been reported that PEI is irreversibly adsorbed onto the sidewalls of SWCNTs. Because PEI contains one of the highest densities of

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amine groups among all polymers, in these conditions n-doping of SWCNTs takes place [24]. This could be the reason for the highly efficient dispersion of CNTs in PEI as well as for the electroactivity of the PEI/CNT layer immobilized on the electrode surface, [4] developing glassy carbon electrodes modified with a dispersion of multi-wall carbon nanotubes in polyethylenimine (GCE/PEI/CNT). The resulting electrodes show an excellent electrocatalytic activity toward different bioanalytes such as ascorbic acid, dopamine, 3,4-dihydroxyphenylacetic acid and hydrogen peroxide in amperometric measurements. This strategy has also been successfully applied to the development of electrochemical sensors for use as detectors in flow injection and capillary electrophoresis [22]. In this paper, we report the advantages of dispersing multiwall carbon nanotubes (MWCNT) in PEI for the development of electrochemical sensors for the detection of hydrogen peroxide, using screen-printed graphite-based electrodes as transducers. For this purpose, a suspension of PEI/MWCNT was deposited onto the working electrode of a screen-printed sensor. Different compositions of a PEI/MWCNT mixture were tested, and a specific ratio was chosen considering the best performances in terms of sensitivity and reproducibility. The developed sensors used for the determination of hydrogen peroxide demonstrated to be highly reproducible, with 3% RSD for 10 different electrodes. In order to verify the possible use of these modified electrodes as disposable electrochemical biosensors, preliminary evaluations were carried out using glucose oxidase (GOD) as biological recognition element for the detection of glucose. 2. Experimental 2.1. Materials The planar, screen-printed electrochemical cell consisted of a graphite-working electrode, a graphite-counter electrode, and a silver pseudoreference electrode as reported in [23]. SPCEs were printed with a Model 248 screen-printer, obtained from DEK (Weimouth, UK). A graphite-based ink (Electrodag 423 SS, Acheson Italiana, Milan, Italy), a silver ink (Electrodag 410 PF, Acheson Italiana, Milan, Italy) and an insulating ink (Vinyl fast 36-100, Argon, Lodi, Milan, Italy) were used. The substrate was a polyester flexible film (Autostat HTS) obtained from Autotype Italia (Milan, Italy). The graphite working electrode surface was 3 mm in diameter. Each electrode was disposable. 2.2. Reagents Hydrogen peroxide (30% v/v aqueous solution), KCl, sodium dihydrogenophosphate, disodium hydrogenophosphate, and glucose were purchased from Merck (Milan, Italy). PEI (P-3143) and glucose oxidase were purchased from Sigma Aldrich (Milan, Italy). Multi-wall carbon nanotubes powder (diameter 30 ± 10 nm, length 1–5 ␮m) were obtained from Nano-Lab, (Newton, MA, USA). Other chemicals were reagent grade and used without further purification. Ultrapure water

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from a Millipore–MilliQ system was used for preparing all the solutions. A 0.050 M phosphate buffer solution pH 7.40 containing 0.1 M KCl was employed as measuring buffer. 2.3. Screen-printed graphite-based electrodes modification Screen-printed graphite-based electrodes (SPE) were modified with carbon nanotubes dispersed in PEI following a procedure similar to that reported in [4]. The dispersion of nanotubes was obtained by suspending MWCNTs in 1.0 mL of PEI solution (prepared in 50:50 v/v ethanol/water) followed by 15 min sonication. To find the best composition of the dispersion mixture, variable amount of MWCNTs and PEI were tested, then different volumes of the obtained dispersion were casted onto the surface of the working electrode, dried in the air at room temperature. 2.4. Instrumentation and experimental conditions Electrochemical measurements on the screen-printed electrodes were performed with a ␮Autolab type II electrochemical analysis system with a GPES 4.9 software package (Metrohm, The Netherlands). The amperometric experiments were carried out in a phosphate buffer solution (0.050 M, pH 7.40) by applying the desired potential and allowing the transient current to decay to a steadystate value prior to the addition of the analyte and the subsequent current monitoring. All the experiments were conducted at room temperature. All the potentials are referred vs. the Ag pseudoreference SPE. 3. Results and discussion 3.1. Overall characterisation Hydrodynamic voltammetry experiments using 3.0 × 10−3 M of hydrogen peroxide were performed with modified and unmodified SPEs in order to verify the sensitivity of the two electrochemical devices against the analyte. For this purpose, a PEI/MWCNT mixture composition was chosen accordingly to values reported in the literature [25]: 1.0 mg of MWCNTs in 1.0 mL of 1.0 mg mL–1 PEI solution. To perform the experiment, 5 mL of the measuring buffer was placed in a beaker and the electrode was equalised at a fixed potential in stirring conditions; then, hydrogen peroxide was added and the current was monitored. Fig. 1 shows the current values obtained versus the applied potential. The investigated potential values ranged from 0 to +750 mV; as seen, in the case of PEI/MWCNT modified electrodes the current increased up to a value of +700 mV, whereas after this value the current levelled off. Thus, +700 mV was considered to be the best working potential. On the contrary, a poor response was obtained on the bare electrodes, compared to very large current signals obtained on the electrodes modified with CNTs. The same experiment was also carried out with SPEs modified only with PEI; also in this case a weak response to hydrogen perox-

Fig. 1. Hydrodynamic voltammetry experiments for 10 mM of hydrogen peroxide performed using SPCEs (䊉) unmodified, () modified with PEI/MWCNT mixture and () modified with only PEI SPEs. Potential range investigated: 0 to +700 mV. Electrodes were modified with the following PEI/MWCNT mixture composition: 1.0 mg of MWCNTs within 1.0 mL of 1.0 mg mL–1 PEI solution.

ide was observed, demonstrating that the noticeable increase in sensitivity for hydrogen peroxide oxidation with graphite-based electrodes could be attributed to the presence of MWCNT. A calibration curve for hydrogen peroxide using PEI/MWCNT modified electrodes was performed and the resulting amperogram is shown in Fig. 2. The corresponding calibration plot, carried in the range of 0 to 100 mM hydrogen peroxide, showed a good linear trend (r2 = 0.998) and a sensitivity of 3.33 ± 0.63 ␮A mM−1 , calculated on three repetitions performed using the same screen-printed modified electrode. A high relative standard deviation (RSD%: 19%) could be attributed to the loss of MWCNTs from the electrode surface, observed during measurements under stirring conditions. This could have been due to the fact that the ratio between PEI and CNT was not well balanced, giving rise to a loss of material. Nevertheless, the standard deviation calculated by repeating the calibration curve using three different electrodes was larger than in the previous case [(3.53 ± 1.38) ␮A mM−1 ], probably

Fig. 2. Calibration curve for successive addition of 1 mM of hydrogen peroxide obtained using PEI/CNT modified electrodes. Electrodes were modified with the following PEI/MWCNT mixture composition: 1.0 mg of MWCNTs within 1.0 mL of 1.0 mg mL–1 PEI solution. Applied potential: +700 mV (vs. Ag pseudoreference SPE).

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Table 1 PEI/MWCNT mixture composition of the different dispersion tested during the optimisation step. The quantities are referred to a total volume of 1 mL Type of electrode

PEI (mg mL–1 )

CNT (mg)

Deposited volume (␮l)

Sensitivity to H2 O2 (␮A mM−1 )

1 2 3 4 5

1 1 5 5 5

1 1 1 5 5

5 10 10 5 10

3.33 30.32 26.32 98.19 101.23

± ± ± ± ±

0.63 12.08 5.18 10.24 13.05

r2 0.998 0.998 0.984 0.992 0.993

caused by the low reproducibility of intraelectrode deposition procedures. For this reason, further experimental optimisation of the coating suspension was needed. 3.2. PEI/MWCNT mixture composition optimisation Experiments were carried out to optimise the sensor coating composition. Different compositions of PEI/MWCNT suspension were tested. The sensitivity of the sensor (estimated on the basis of the slope of the calibration curve obtained for hydrogen peroxide, and reported as ␮A mM−1 ) was used to compare the performance of sensors obtained with different coatings. Each composition was analysed in triplicate with the same sensor and the average sensitivity values are reported in Table 1. As reported in the table, not only the amounts of PEI and MWCNT were varied, but also the volume of the dispersion deposited onto the working electrode. As it can be seen the linear trend was always maintained, but an increase in sensitivity from 3.33 to 30.32 ␮A mM−1 towards hydrogen peroxide was obtained by doubling the deposited volume from 5 to 10 ␮l. Nevertheless in the second case (electrode type 2) a very high relative standard deviation was observed (RSD% = 40%); this was attributed, as a result of visual inspection of the electrode, to loss of material from the electrode surface. This effect was the main influencing factor affecting the reproducibility of the measurements. To overcome this problem, the quantity of PEI in the dispersion was increased to 5 mg mL–1 (electrode type 3), as illustrated in Table 1, also in this case the obtained sensitivity towards hydrogen peroxide was higher than that obtained for electrode type 1; nevertheless, the RSD% was close to 20%. Experiments were thereafter carried out by increasing the amount of CNTs added to the dispersion from 1 to 5 mg mL–1 . Hence, 5 (electrode type 4) and 10 ␮l (electrode type 5) of dispersion volume were deposited on the working electrodes. Also in this case an enhancement of the sensitivity towards hydrogen peroxide was achieved (98.19 and 101.23 ␮A mM−1 ), whereas the relative standard deviation was dramatically reduced compared to the previous cases. Fig. 3 shows a comparison between all the tested sensors, together with unmodified sensors and sensors modified only with PEI. The graph shows that the best analytical performances were obtained by increasing the CNT concentration together with an increased amount of PEI (Fig. 3). The attention was finally focused on electrode type 4. To better analyse the problem related to the loss of material during measurements, the electrode type 4 was used to carry out 10 consecutive calibrations with H2 O2 . The resulting sensitiv-

Fig. 3. Optimisation of the PEI/MWCNT mixture composition by the comparison of the slope of the calibration curves obtained for hydrogen peroxide. PEI/MWCNT mixture compositions tested were reported in Table 1. Standard deviations were calculated on three measurement repetitions on each tested electrode.

ity, compared to that obtained for an electrode type 2, was hence plotted, and the results are reported in Fig. 4. The trends (reported as adimensional residual percentage sensitivities) show that in the case of the electrode type 4, the analytical response was maintained at 80% of the original value after 10 consecutive measurements, whereas for the electrode type 2 the analytical response decreased to 40%. This demonstrated that the dispersion composition containing a higher quantity of PEI allowed the formation of a more stable adhesion of MWCNTs to the working electrode surface. The analytical performances of the optimised sensor composition were also tested by considering the reproducibility of the preparation procedure. For this purpose, calibration

Fig. 4. Performance of PEI/MWCNTs modified sensors during repetitive measurements. Ten consecutive calibration curves of hydrogen peroxide were performed with () a SPE type 4 electrode and () a type 2. Signals are reported as residual percentage of the original sensitivity.

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Fig. 5. Evaluation of the reproducibility of the preparation procedure. Comparison of five sensitivities toward hydrogen peroxide obtained by carrying out five calibration curves with different modified sensors.

experiments with five different screen-printed PEI/MWCNT modified sensors were performed; the H2 O2 calibration curve for each electrode was repeated three times. The results are reported in Fig. 5. Among the tested electrodes only one resulted in an average sensitivity not within the standard deviation interval (represented in the graph by the dashed lines). Nevertheless, the overall average sensitivity obtained was 88.1 ± 13.1 ␮A mM−1 , associated with a quite high relative standard deviation (RSD% = 15%). The lack in reproducibility was attributed to the deposition procedure, even if carried out just after sonication, the settling of CNTs due to the gravity greatly affected the homogeneity of CNTs dispersion. 3.3. Amperometric detection of glucose at PEI/MWCNT modified electrode The developed sensor was tested in order to verify its possible suitability for building up disposable electrochemical biosensors based on H2 O2 measurements. This preliminary evaluation was carried out by using glucose oxidase (GOD) as the biological recognition element, with glucose as analyte. For this purpose the biosensor was prepared by casting screen-printed

Fig. 6. Amperometric recording for successive additions of 0.5 mM of glucose. The biosensor was prepared by casting screen-printed electrodes with the optimised PEI/MWCNT mixture containing 1 mg mL–1 of GOD solution. Working potential: +700 mV (vs. Ag pseudoreference SPE).

electrodes with the optimised PEI/MWCNT mixture, containing 1.0 mg mL–1 of GOD solution. After drying the modified electrodes were washed with measuring buffer and kept at +4 ◦ C until use. Fig. 6 shows the amperometric response of a PEI/MWCNT/GOD modified screen-printed electrode where the potential was kept at +700 mV in 0.05 M phosphate buffer solution with pH 7.4. As shown in Fig. 6, during successive additions of 0.5 mM glucose, a well-defined current response was observed. For each addition of glucose, a sharp rise in the current was observed, while no response was recorded in analogous measurements at the bare PEI/MWCNT electrode (data not shown). The calibration plot was linear over a wide concentration range, 0.5–3.0 mM. The data interpolation in this concentration range had a slope of 22.18 ␮A mM−1 , with a correlation coefficient of 0.998. 4. Conclusions In this work a new disposable electrochemical sensor for the rapid detection of hydrogen peroxide based on the dispersion of CNTs in a PEI solution deposited onto the surface of graphite-based screen-printed electrodes was purposed. The performances of the PEI/CNT layer as a new tool for the modification of carbon-based electrodes have been experimentally demonstrated; the addition of the enzyme GOD also allowed the demonstration of the suitability of the developed sensors for biosensors assembly. Taking into account the simplicity of the proposed method, the used strategy offers interesting opportunities for building up reliable and inexpensive biosensors based on oxidise enzymes. References [1] Luo H, Shi Z, Li N, Gu Z, Zhuang Q. Investigation of the electrochemical and electrocatalytic behavior of single-wall carbon nanotube film on a glassy carbon electrode. Anal Chem 2001;73:915–20. [2] Zhao Q, Gan Z, Zhuang Q. Electrochemical sensors based on carbon nanotubes. Electroanalysis 2002;14:1609–13. [3] Cohen ML. Nanotubes, nanoscience, and nanotechnology. Mater Sci Eng C 2001;15:1–11. [4] Rubianes MD, Rivas GA. Carbon nanotubes paste electrode. Electrochem Commun 2003;5:689–94. [5] Britto PJ, Santhanam KSV, Ajayan PM. Carbon nanotube electrode for oxidation of dopamine. Bioelectrochem Bioenerg 1996;41:121–5. [6] Moore RR, Banks CE, Compton RG. Basal plane pyrolytic graphite modified electrodes: comparison of carbon nanotubes and graphite powder as electrocatalysts. Anal Chem 2004;76:2677. [7] Liu G, Lin Y, Tub Y, Renb Z. Ultrasensitive voltammetric detection of trace heavy metal ions using carbon nanotube nanoelectrode array. Analyst 2005;130:1098–101. [8] Baughman RH, Zakhidov A, De Heer WA. Carbon nanotubes–the Route Toward Applications. Science 2002;297:787–92. [9] Rochette J-F, Sacher E, Meunier M, Luong JHT. A mediatorless biosensor for putrescine using multi-walled carbon nanotubes. Analytical Biochem 2005;336:305–11. [10] Marcus RA, Sutin N. Electron transfers in chemistry and biology. Biochim Biophys Acta 1985;811:265–318. [11] Anthony G-E, Lei C, Baughman RH. Direct electron transfer of glucose oxidase on carbon nanotubes. Nanotechnology 2002;13:559–64.

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