Microchim Acta 152, 261–265 (2006) DOI 10.1007/s00604-005-0438-0
Original Paper Microchip Capillary Electrophoresis with a Single-Wall Carbon Nanotube==Gold Electrochemical Detector for Determination of Aminophenols and Neurotransmitters Martin Pumera, Xavier Llopis, Arben Merkoc° i , and Salvador Alegret Grup de Sensors i Biosensors, Departament de Quı´mica, Universitat Aut onoma de Barcelona, E-08193 Bellaterra, Barcelona, Catalonia, Spain Published online November 30, 2005 # Springer-Verlag 2005
Abstract. A new SWCNT modified gold detector for microchip capillary electrophoresis–electrochemistry is described. SWCNT modified gold electrode displays greatly improved sensitivity and separation resolution compared to bare gold electrode, reflecting the electrocatalytic activity of SWCNT. The SWCNT=Au electrode exhibits low background noise levels. Parameters such as separation voltage and detection potential of the microchip electrophoresis–electrochemistry with SWCNT modified gold electrode were optimized. Key words: Single-wall carbon nanotubes; microchip capillary electrophoresis; electrochemistry; phenols.
Distinctive properties of CNT, such as a high surface area, ability to accumulate analyte, minimization of surface fouling and electrocatalytic activity are very attractive for electrochemical sensing [1, 2]. Recent studies demonstrated that CNT exhibits strong electrocatalytic activity for a wide range of compounds, such as neurotransmitters [3–6], NADH [7–11], hydrogen peroxide [3, 8, 9, 11, 12], ascorbic [3–5] and uric acid [3], cytochrome c [13], hydrazines [14], hydrogen sulfide [15], amino acids [16] and DNA [17]. The above described favorable characteristics of CNT Author for correspondence. E-mail:
[email protected]
were studied in batch systems; however, the attractive properties of CNT suggest the possibility of developing powerful electrochemical detector for microfluidic systems. Microfluidic systems (Lab-on-a-chip) can dramatically change the speed and scale at which chemical and biological analyses are performed [18, 19]. Such miniaturized systems represent the ability to shrink conventional ‘‘benchtop’’ analytical systems with major advantages of speed, integration, cost, portability, and sample=reagent consumption. Electrochemical detection [20–22], in particular amperometric detection [23], offers great promise for microfluidic systems, with features that include high sensitivity, inherent miniaturization, low-power requirements, compatibility with advanced micromachining and microfabrication technologies, and low cost. Various electrode materials have been used for fabrication of detection electrodes for microfluidic devices. The carbon electrodes proved to be very suitable due to their minimal fouling, lower noise and larger potential range for organic compound than metal electrodes [21]. Variety of types of carbon electrodes were used, i.e. thick-film screen-printed electrodes [24–26], carbon fibers [27], carbon paste [28] or graphite-epoxy composite electrodes [29].
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The coupling of the attractive properties of the carbon nanotube electrochemical detectors with microfluidic systems was demonstrated very recently. Wang et al. described microfluidic electrophoretic system-electrochemistry with carbon nanotube modified carbon thick-film screen-printed electrodes for analysis of purines, chlorophenols, hydrazines and amino acids [30]. The aim of this work is to investigate the behavior of single-wall carbon nanotubes cast on gold electrode for determination of aminophenols and neurotransmitters.
Experimental Reagents Single wall carbon nanotubes (SWCNT) were received from Aldrich (product no. 519308; dimensions, diameter length: 1.2–1.5 2–5 mm). Further purification of nanotubes was accomplished by stirring the carbon nanotubes in 2 M nitric acid (PanReac, Spain) at 25 C for 24 hours. 1 mg of SWCNT was dissolved in 1 mL of dimethylformamide (DMF, from Sigma-Aldrich). p-Aminophenol (p-AP), o-aminophenol (o-AP), dopamine, catechol, 2-(4-morpholino)ethanesulfonic acid (MES), sodium hydroxide and sodium acetate were received from Sigma-Aldrich (Spain), while acetic acid was received from PanReac, Spain. Stock solutions of catecholamines (10 mM) were prepared daily in deionized water while stock solutions (1 mM) of aminophenols were prepared directly in running acetate buffer (20 mM, pH 5.0). The working electrode was modified by casting of 5 mL of a DMF containing 1 mg mL1 SWCNT and allowing drying at room temperature for 13 hours.
Apparatus The glass chip was fabricated by Micralyne (Model MC-BF4-001, Edmonton, Canada) by means of wet chemical etching and thermal bonding techniques. The 88 mm 16 mm chip consisted of four-
Fig. 1. Top view of the microchip electrophoresis device: (a) the glass chip, (b) separation channel, (c) run buffer reservoir, (d) sample reservoir, (e) unused reservoir, (f ) platinum cathode for separation, (g) SWCNT modified gold disk electrode detector, (h) Ag=AgCl wire reference electrode, (i) platinum counter electrode, (j) detection reservoir
M. Pumera et al. way injection cross, with 74 mm long separation channel and side arms of 5 mm long each. The original waste reservoir was cut off, leaving the channel outlet at the end side of the chip, thus facilitating the end-column amperometric detection [31]. The channels were 50 mm wide and 20 mm deep. A plexiglas holder, with minor modifications to the previously described in Ref. [31], was fabricated for holding the separation chip and housing the detector and reservoirs. The amperometric detector was placed in the waste reservoir (at the channel outlet side), and consisted of a platinum wire counter (Pt wire of 0.1 mm diameter), an Ag wire=AgCl reference (0.1 mm diameter, home made) and gold (or SWCNT modified gold) disk working electrode (CH Instruments, TX, USA). The working electrode, housed in the plastic screw, was placed opposite to the channel outlet, at 50 mm distance controlled under microscope. For scheme of the setup, see Fig. 1.
Electrophoretic Procedure The channels of the glass chip were treated before use by rinsing with 0.1 M NaOH and deionized water for 10 and 10 min, respectively. The electrophoresis buffer for analysis of catecholamines consisted of a MES (25 mM, pH 6.5) [24] while running buffer for analysis of aminophenols consisted of acetate buffer (20 mM, pH 5.0) [32, 33]. The electrophoretic procedure (injection, separation) was carried out in the same manner as described previously [24]. Amperometric detection was performed with an electrochemical analyzer CHI 630B (CH Instruments, Austin, TX, USA) connected to a personal computer. The electropherograms were recorded with a time resolution of 0.1 s while applying the detection potential. Sample injections were performed after stabilization of the baseline. All experiments were performed at room temperature.
Results and Discussion To study the electrocatalytic activity of SWCNTmodified gold detector in comparison to the bare unmodified gold detector, the hydrodynamic voltammograms (HDV) were constructed for aminophenols and catecholamine and they were compared. Figure 2 shows typical HDV for the oxidation of p-aminophenol (A), o-aminophenol (B) and dopamine (C) at the bare gold disk electrode (a) and SWCNT modified gold disk electrode (b). These voltammograms shows the SWCNT electrode provide higher sensitivity (around 5 times) for the detection of aminophenols (A, B) and lowering operation potential for detection of dopamine (C) of 200 mV compared to bare gold electrode. The lowering of the overvoltages is coupled to sharper voltammograms and higher sensitivities which allow for better amperometric detection in the case of SWCNT modified gold electrodes. Representative electrophoregrams for aminophenols recorded with bare gold disk electrode (A) and SWCNT modified gold electrode (B) are shown in the
Determination of Aminophenols and Neurotransmitters with a SWCNT=Au Electrode
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Fig. 2. Hydrodynamic voltammograms for 1 mM p-aminophenol (A), 1 mM o-aminophenol (B) and 200 mM dopamine (C) at bare gold electrode (a) and SWCNT modified gold electrode (b). Conditions: running buffer, 20 mM acetate (pH 5.0) (A, B), 25 mM MES (pH 6.5) (C); separation voltage, þ1500 V; injection voltage, þ1500 V; injection time, 5 s. SWCNT loading, 5 mg
Fig. 3. Electrophoregrams for mixture of p-aminophenol (a) and o-aminophenol (b) at the bare gold (A) and SWCNT modified gold (B) electrodes. Detection potential, þ0.5 V. Other conditions, as in Fig. 2
Fig. 3. The analytes are well separated in well-defined and resolved peaks with SWCNT modified gold electrode within 150 seconds. The sharp peaks of aminophenols on SWCNT modified detector strongly contrast with tailing peaks on bare gold detector. The sharp and well resolved responses of SWCNT detector reflects its faster electron transfer resulting in smaller values of the half-peak widths for p-aminophenol and o-aminophenol at the SWCNT modified electrode when compared to unmodified electrode, 5.8 vs. 6.6 s (for p-AP) and 8.7 vs. 9.9 s (for o-AP), respectively. This results in improved separation resolution (Rs)
Fig. 4. Influence of detection potential upon electrochemical response of background noise of bare gold (a) and SWCNT modified gold (b) detectors. Conditions: Separation Voltage, þ1500 V; running buffer, 25 mM MES (pH 6.5). SWCNT loading, 5 mg
of the aminophenols, Rs ¼ 6.46 vs. Rs ¼ 5.72 for SWCNT modified detector and bare gold detector, respectively. The elevated background noise is often problem when using carbon nanotube electrodes for amperometric measurements. Figure 4 depicts the dependence of the background noise upon the detection voltage of SWCNT modified gold detector compared to bare gold detector. It is clearly shown that the background
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noise of bare gold electrode has similar values as noise of SWCNT modified electrode with the values around 0.17 nA in the range of 0 to þ0.6 for both electrodes while the background noise of SWCNT increases more rapidly thereafter. Such a low background noise of SWCNT modified gold electrode is coupled with higher sensitivity. The effect of the separation voltage upon electrochemical output of microchip electrophoresis–electrochemistry with SWCNT modified gold detector was examined (see Fig. 5). As expected, increasing the separation voltage from þ1000 V to þ4000 V (A–G)
dramatically decreases migration time for both dopamine (a) and catechol (b) from 150.2 to 41.1 s and from 335.4 to 94.6 s, respectively. The separation voltage had negligible effect upon the background noise level of SWCNT modified gold electrode for voltages ranging from þ1000 to þ2500 V with sharper increase afterwards. The SWCNT modified gold detector for microchip electrophoresis displays well-defined concentration dependence. Electrophoregrams for sample mixtures containing increasing levels of dopamine and catechol (in 50 mM steps) provided defined peaks, proportional to analyte concentration for both compounds. The resulting calibration plots are linear with sensitivities of 117.5 and 35.6 pA mM1 for dopamine and catechol, respectively (correlation coefficients, 0.999 and 0.998, respectively). Sensitivity of microchip electrophoresis-SWCNT detector system for p-aminophenol is stated in Table 1, together with sensitivities of comparable CE–EC methods.
Conclusion
Fig. 5. Influence of separation voltage on the migration times of 200 mM dopamine (a) and 200 mM catechol (b). Also shown (as inset) is the effect of the separation voltage upon the amperometric response at þ1500 V (A) and þ2500 V (B). Injection voltage, þ1500 V; injection time, 5 s; detection potential, þ0.5 V; Other conditions as in Fig. 4
The SWCNT modified gold electrode can be used as an attractive electrochemical detector for microchip electrophoresis. SWCNT detector displays greatly improved sensitivity and resolution compared to bare gold electrode, reflecting the electrocatalytic activity of SWCNT. The SWCNT=Au electrode exhibits low noise levels and shows a great promise for future bioanalytical and environmental applications.
Table 1. Sensitivities of comparable CE–EC methods for determination of p-aminophenol Detector
Analyte
Interferences
Buffer
Sensitivity (nA=mM)
Reference
Single-wall carbon nanotube film on gold electrode Boron doped diamond electrode
p-aminophenol
o-aminophenol, m-aminophenol
20 mM acetate, pH 5.0
106
[this work]
p-aminophenol
1,2-phenylenediamine, 2-aminonaphthalene, 2-chloroaniline, o-aminobenzoic acid dopamine, isoproterenol, chlorogenic acid None
30 mM acetate, pH 4.5
226
[34]
25 mM MES buffer, pH 5.65
N=A
[35]
N=A
[36]
o-aminophenol, m-aminophenol o-aminophenol, m-aminophenol
20 mM acetate, pH 5.0 20 mM acetate, pH 5.0
50
[32]
40
[33]
Array of gold microband electrodes Screen-printed thick-film carbon electrode Gold coated screen-printed thick-film carbon electrode Gold coated screen-printed thick-film carbon electrode N=A Non-available.
p-aminophenol p-aminophenol p-aminophenol p-aminophenol
Determination of Aminophenols and Neurotransmitters with a SWCNT=Au Electrode Acknowledgements. This work was financially supported by MEC (Madrid) (Projects BIO2004-02776, MAT2004-05164), by the Spanish ‘‘Ramon Areces’’ foundation (project ‘Bionanosensores’) and the ‘‘Ramon y Cajal’’ program of the Ministry of Science and Technology (Madrid) that supports A. Merkoc° i. M. Pumera is grateful for the support from the Marie Curie Intra-European Fellowship from European Community under 6th FP (MEIF-CT-2004-005738). The authors would like to thank to J. L opez Santos and M. Garcia Rigol (Dept. of Physics, UAB) for the construction of an adjustable high-voltage power supply and machine shop staff (UAB) for chip holder fabrication.
References [1] Merkoc° i A, Pumera M, Llopis X, Perez B, del Valle M, Alegret S (2005) Trends Anal Chem 24: 826 [2] Wang J (2005) Electroanalysis 17: 7 [3] Luo H, Shi Z, Li N, Gu Z, Zhuang Q (2001) Anal Chem 73: 915 [4] Wang J, Li M, Shi Z, Li N (2002) Electroanalysis 14: 225 [5] Wang Z H, Liu J, Liang Q L, Wang T M, Luo G (2002) Analyst 127: 653 [6] Rubianes M D, Rivas G A (2003) Electrochem Commun 5: 689 [7] Musameh M, Wang J, Merkoc° i A, Lin Y (2002) Electrochem Commun 4: 743 [8] Wang J, Musameh M (2003) Anal Chem 75: 2075 [9] Valentini F, Amine A, Orlanducci S, Terranova M L, Palleschi G (2003) Anal Chem 75: 5413 [10] Moore R R, Banks C E, Compton R G (2004) Anal Chem 76: 2677 [11] Pumera M, Merkoci A, Alegret S (2005) Sens Actuators B (in press) doi:10.1016=j.snb.2005.07.010 [12] Hrapovic S, Liu Y, Male K B, Luong J H T (2004) Anal Chem 76: 1083 [13] Wang J, Li M, Shi Z, Li N (2002) Anal Chem 74: 1993 [14] Zhao Y, Zhang W D, Chen H, Luo Q M (2002) Talanta 58: 529
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[15] Lawrence N, Deo R P, Wang J (2004) Anal Chim Acta 517: 131 [16] Wang J X, Li M X, Shi Z J, Li N Q, Gu Z N (2004) Electroanalysis 16: 140 [17] Pedano M L, Rivas G A (2004) Electrochem Commun 6: 10 [18] Vilkner T, Janasek D, Manz A (2004) Anal Chem 76: 3373 [19] Pumera M (2005) Talanta 66: 1048 [20] Pumera M, Merkoci A, Alegret S (2005) Trends Anal Chem (in press) doi: 10.1016=j.trac.2005.08.005 [21] Vandaveer IV W R, Pasas-Farmer S A, Fischer D J, Frankenfeld C N, Lunte S M (2004) Electrophoresis 25: 3528 [22] Pumera M, Wang J, Opekar F, Jelı´nek I, Feldman J, Lowe H, Hardt S (2002) Anal Chem 74: 1968 [23] Wang J (2005) Electroanalysis 17: 1133 [24] Wang J, Tian B, Sahlin E (1999) Anal Chem 71: 5436 [25] Wang J, Pumera M, Chatrathi M P, Rodriquez A, Spillman S, Martin R S, Lunte S M (2002) Electroanalysis 14: 1251 [26] Wang J, Chen G, Pumera M (2003) Electroanalysis 15: 862 [27] Liu Y, Vickers J A, Henry C S (2004) Anal Chem 76: 1513 [28] Martin R S, Gawron A J, Fogarty B A, Regan F B, Dempsey E, Lunte S M (2001) Analyst 126: 277 [29] Pumera M, Merkoc° i A, Alegret S (2005) Electroanalysis (in press) [30] Wang J, Chen G, Chatrathi M P, Musameh M (2004) Anal Chem 76: 298 [31] Wang J, Escarpa A, Pumera M, Feldman J (2002) J Chromatogr A 952: 249 [32] Pumera M, Wang J, Grushka E, Polsky R (2001) Anal Chem 73: 5625 [33] Wang J, Pumera M, Chatrathi M P, Escarpa A, Konrad R, Griebel A, Dorner W, Lowe H (2002) Electrophoresis 23: 596 [34] Shin D C, Tryk D A, Fujishima A, Muck A, Chen G, Wang J (2004) Electrophoresis 25: 3017 [35] Kiett O, Nischang L, Nyholm L (2002) Electrophoresis 23: 3678 [36] Wang J, Ibanez A, Chatrathi M P, Escarpa A (2001) Anal Chem 75: 23
