- Email: [email protected]
Functionalized magnetic nanoparticle with poly(3-thiopheneacetic acid) and its application for electrogenerated chemiluminescence sensor
Synthetic Metals 159 (2009) 571–575
Contents lists available at ScienceDirect
Synthetic Metals journal homepage: www.elsevier.com/locate/synmet
Functionalized magnetic nanoparticle with poly(3-thiopheneacetic acid) and its application for electrogenerated chemiluminescence sensor Young-Ku Lyu 1 , Kyu Jin Kyung 1 , Won-Yong Lee ∗ Department of Chemistry and Center for Bioactive Molecular Hybrids, Yonsei University, 134 Shinchon-dong, Seodaemoon-ku, Seoul 120-749, Republic of Korea
a r t i c l e
i n f o
Article history: Received 6 August 2008 Received in revised form 6 November 2008 Accepted 27 November 2008 Available online 7 January 2009 Keywords: Electrogenerated chemiluminescence Ru(bpy)3 2+ Magnetic nanoparticle Chemically modified electrode
a b s t r a c t Polymer-coated magnetic nanoparticles (MNPs) have been prepared and used as an immobilization matrix for the fabrication of solid-state tris(2,2 -bipyridyl)ruthenium(II) (Ru(bpy)3 2+ ) electrogenerated chemiluminescence (ECL) sensor. The pre-synthesized maghemite (␥-Fe2 O3 ) MNPs were coated with poly(3-thiopheneacetic acid) based on an oxidative polymerization method using KMnO4 . The poly(3thiopheneacetic acid)-coated MNPs have formed the clusters with average diameter of 200–500 nm. The multilayer films of poly(3-thiopheneacetic acid)-coated MNPs were uniformly formed on the surface of a Pt electrode by an external magnet. The Ru(bpy)3 2+ was rapidly incorporated into the multilayer films within 5 min through the electrostatic interaction between the Ru(bpy)3 2+ and the negatively charged carboxylates of 3-thiopheneacetic acid at pH 7.0. Due to the fast mass transport in the multilayer films of the functionalized MNPs, the present Ru(bpy)3 2+ ECL sensor based on the functionalized MNPs showed improved ECL sensitivity for tripropylamine (TPA) compared to the Nafion-based ECL sensor. The present ECL sensor exhibited a quite low limit of detection for TPA (49 nM). The sensor-to-sensor reproducibility was very good (R.S.D. = 5%). © 2008 Elsevier B.V. All rights reserved.
1. Introduction Due to their compatibility to biological units, magnetic particles are of great interest for biomedical applications. In particular, magnetic particles of iron oxides functionalized with redox or biochemical components have been extensively used for the concentration and localization of chemical or biochemical components such as enzymes [1], DNA [2,3], and cells [4]. In addition, the magnetic localization of the functionalized particles to the electrode surface by an external magnetic field can be used to initiate the electrocatalytic and biocatalytic processes. Thus, magnetic-field switching of electrocatalytic and biocatalytic processes have been easily performed for selective dual biosensing, immunosensing, DNA sensing, and luminol ECL [5–8]. Although, the micron-sized magnetic particles have been previously used for such applications, recent studies are mainly focused on the use of smaller-sized nanoparticles because they provide high colloidal stability, minimal nonspecific binding, and enhanced magnetic behaviors [9]. However, a suitable coating of the nanopar-
∗ Corresponding author. Tel.: +82 2 2123 2649; fax: +82 2 364 7050. E-mail address: [email protected] (W.-Y. Lee). 1 These authors contributed equally to the work. 0379-6779/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.synthmet.2008.11.021
ticles is necessary because the magnetic nanoparticles are easily degraded when they are exposed to certain environments [10]. The preparation and application of the heterostructured magnetic nanoparticles (MNPs) are well summarized in recent papers [9,11]. Previously, we described the Ru(bpy)3 2+ ECL sensor based on the Nafion-stabilized MNPs (Nafion/Fe3 O4 ) [12]. Due to the fast mass transport of Ru(bpy)3 2+ in the Nafion/Fe3 O4 multilayer films, the resulting ECL sensor exhibited good sensitivity and fast response time. Dong and co-workers used bifunctional core–shell nanostructures of core MNPs and silica shell containing Ru(bpy)3 2+ for solid-state ECL sensor materials [13]. In the core–shell structure, silica outershell could protect the immobilized Ru(bpy)3 2+ from oxygen quenching and improve the ECL sensitivity. In the present work, monodisperse maghemite nanoparticles (␥-Fe2 O3 , ca. ∼10 nm in size) has been coated with poly(3thiopheneacetic acid) via an oxidative polymerization using KMnO4 as an oxidant (Scheme 1) and the magnetic-field driven multilayer films of the functionalized MNPs were formed as a matrix for the immobilization of Ru(bpy)3 2+ . The electrochemical and ECL behavior of the present Ru(bpy)3 2+ ECL sensor with typical coreactant tripropylmaine (TPA) [14–17] will be described in terms of sensitivity, selectivity, and long-term stability relative to the other ECL sensors.
572
Y.-K. Lyu et al. / Synthetic Metals 159 (2009) 571–575
Scheme 1. Oxidative polymerization of 3-thiopheneacetic acid.
2. Experimental 2.1. Reagents and apparatus Iron pentacarbonyl (99.999%), dodecylamine (98%), tripropylamine (TPA, 99%), 1,2-dichlorobenzene (99%), tris(2,2 -bipyridyl) dichlororuthenium(II) (Ru(bpy)3 2+ , 98%), 3-thiopheneacetic acid, potassium permanganate (KMnO4 , 99%) were purchased from Aldrich. Water for all solutions was purified using a Milli-Q water purification system (Millipore, Bedford, MA, USA). Cyclic voltammetric experiments were performed with an EG&G 273A potentiostat (Oak Ridge, TN, USA). A conventional threeelectrode system was employed with a platinum wire as counter electrode, a Pt plate (0.636 cm2 ) coated with PMNPs as a working electrode, and an Ag/AgCl (3 M NaCl) reference electrode. The ECL detection system described earlier has been used in the present study [18].
Fig. 1. Formation of poly(3-thiopheneacetic acid) based on the oxidative polymerization (a) and its use for the formation of PMNP-modified electrode (b).
initial potential of +850 mV to a high of +1300 mV and back to +850 mV at a scan rate of 100 mV/s.
2.2. Preparation of the functionalized magnetic nanoparticles According to the literature [19], monodisperse maghemite (␥-Fe2 O3 ) nanoparticles (10 nm diameter) were prepared from the thermal decomposition of Fe(CO)5 using a capping ligand of dodecylamine. In order to coat the maghemite MNPs with polymer, 1 mL of a MNP solution (1 mg/mL toluene) was mixed with 1 mL of 2.0 mM 3-thiopheneacetic acid dissolved in acetonitrile. The mixed solution was shaken for about 30 min and then blended with 1 mL solution of 2.0 mM KMnO4 dissolved in acetonitrile. Oxidative polymerization was immediately initiated. After shaking for additional 30 min, 2 mL ethanol was added. The poly(3-thiopheneacetic acid)-coated magnetic nanoparticles (PMNPs) were separated by a permanent magnet. PMNPs were washed by acetonitrile and then water. PMNP clusters were formed with average diameter of 200–500 nm. The zeta potential of PMNPs dispersed in 50 mM phosphate buffer at pH 7.0 was determined to be −21.5 mV (Zetasizer Nano ZS, Malvern Instruments Ltd., UK). 2.3. Fabrication of the ECL sensor For electrochemical experiments, the multilayer films of the PMNPs on a Pt electrode surface have been formed as shown in Fig. 1. An external magnet has been positioned behind the Pt electrode (surface area = 0.636 cm2 ) to attract the PMNPs dissolved in aqueous solution. The PMNP multilayer-modified Pt electrode was then placed in an electrochemical cell containing 10 mM Ru(bpy)3 2+ solution in 0.05 M phosphate buffer at pH 7. The incorporation of Ru(bpy)3 2+ into the PMNP multilayer films was electrochemically monitored by running consecutive cyclic potential scans from an
3. Results and discussion 3.1. Preparation of the functionalized magnetic nanoparticles and the nanoparticle-modified electrode Fig. 1 shows a schematic illustration of the preparation of the functionalized MNPs and the formation of the MNP-modified electrode. First, maghemite (␥-Fe2 O3 ) MNPs were prepared from the thermal decomposition of pentacarbonyl iron [Fe(CO)5 ] using a capping ligand of dodecylamine according to the literature [19]. As shown in the transmission electron micrograph (TEM) image of Fig. 2(a), maghemite MNPs were monodisperse and spherical with the average diameter of 10 nm. Then, as-prepared maghemite MNPs were coated with poly(3-thiopheneacetic acid) via an oxidative polymerization using KMnO4 as an oxidant. The polymerization was effective and was completed within 30 min. While the maghemite MNPs are well dispersed in toluene as shown in the TEM image of Fig. 2(a), the poly(3-thiopheneacetic acid)-coated magnetic nanoparticles (PMNP) has formed the clusters with average diameter of 200–500 nm as shown in the TEM image of Fig. 2(b). In order to characterize the charge state of the PMNPs, zeta potential of the PMNPs dispersed in 50 mM phosphate buffer at pH 7.0 has been measured. The zeta potential was determined to be −21.5 mV, which indicates the presence of the negatively charged carboxylates at the surface of the PMNPs. Since the PMNPs were well dispersed in deionized water, a small amount of PMNPs (1.0 mg) were introduced into an electrochemical cell containing 1 mL deionized water. Positioning of an external magnet behind a Pt electrode attracts the PMNPs to form the PMNP multilayer films. The formation of the PMNP-modified electrode
Y.-K. Lyu et al. / Synthetic Metals 159 (2009) 571–575
573
Fig. 3. FE-SEM images of the surface (a) and a cross-sectional view (b) of the PMNP multilayer films formed on a Pt plate electrode.
Fig. 2. TEM image of iron oxide nanoparticles before the polymerization (a) and FE-SEM image of iron oxide after the polymerization of 3-thiopheneacetic acid (b).
under the magnetic filed is so effective and can be completed in less than 20 min. As shown in Fig. 3(a), the PMNPs were evenly deposited on a Pt electrode surface. The micro-pore structures can be seen in the surface of the PMNP composite films, which can lead to fast diffusion of analytes within around 7 m-thick PMNP multilayer films as shown in Fig. 3(b). In fact, the thickness of the multilayer films can be easily controlled in the range of sub-m to several hundred m by the variation of the amount of the PMNPs initially introduced into the aqueous solution. 3.2. Electrochemical and ECL characteristics of PMNP-based Ru(bpy)3 2+ ECL sensor An electrochemiluminescent cation, Ru(bpy)3 2+ , can be easily incorporated into the PMNP multilayer films via electrostatic interaction between positively charged Ru(bpy)3 2+ and negatively charged carboxylates in the PMNP multilayer films. Therefore, the PMNP multilayer-modified Pt electrode was simply placed in an electrochemical cell containing 10 mM Ru(bpy)3 2+ solution in 0.05 M phosphate buffer at pH 7. The incorporation of Ru(bpy)3 2+
into the PMNP multilayer films was electrochemically monitored by running consecutive cyclic potential scans from an initial potential of +850 mV to a high of +1300 mV and back to +850 mV at a scan rate of 100 mV/s. The anodic and cathodic peak currents of Ru(bpy)3 2+ were rapidly increased and the steady-state currents were reached within 5 min. In contrast, the loading of Ru(bpy)3 2+ into the pure Nafion film was much slower under the identical experimental conditions with a steady-state current being achieved typically within 30 min. The fast uptake of the Ru(bpy)3 2+ into the PMNP multilayer films might be due to the micro-pore structures of the PMNP multilayer films as seen in Fig. 3. The ion-exchangeable carboxylate groups of the PMNP multilayer films seem to be easily accessible to Ru(bpy)3 2+ within the multilayer films, thus leading to fast diffusion of Ru(bpy)3 2+ in the films. The anodic peak currents of the Ru(bpy)3 2+ incorporated into the PMNP multilayer films were measured at different scan rates. The anodic peak currents were linearly proportional to the square root of the scan rate over the range of 50–500 mV. At those experimental time scales, the thickness of the diffusion layer is less than the thickness of the composite films (around 7 m) and therefore the semi-infinitive diffusion process prevails. Once a steady-state CV was obtained, the PMNP-modified electrode was removed from the Ru(bpy)3 2+ solution, rinsed with water, and then placed in 0.05 M phosphate buffer solution at pH 7. As shown in Fig. 4, the anodic peak current obtained with the Ru(bpy)3 2+ -immobilized at PMNP-modified electrode in the buffer solution has been gradually decreased and reached a state-state current within 1 h. The anodic peak current obtained with the Ru(bpy)3 2+ -immobilized PMNP-modified electrode in the buffer solution was about 25% that of the steady-state current obtained in the Ru(bpy)3 2+ solution, while Ru(bpy)3 2+ -immobilized in the
574
Y.-K. Lyu et al. / Synthetic Metals 159 (2009) 571–575
Fig. 4. Cyclic voltammograms of Ru(bpy)3 2+ immobilized in the PMNP multilayer films on a Pt electrode surface in 50 mM pH 7.0 phosphate buffer at a scan rate of 100 mV/s for 0 min (a), 15 min (b), 30 min (c), 45 min (d), and 60 min (e).
Nafion-stabilized MNPs film exhibited 30% loss in the above process [12]. The relatively fast initial leaching of Ru(bpy)3 2+ from the poly(3-thiopheneacetic acid)-coated MNP composite films are possibly due to the relatively weak interaction between Ru(bpy)3 2+ and carboxylates of PMNPs. Therefore, it was necessary to reimmobilize the Ru(bpy)3 2+ prior to each ECL experiment in order to get a high sensitivity of the ECL measurement. For reimmobilization process, the PMNP-modified electrode was placed in 10 mM Ru(bpy)3 2+ solution in 50 mM phosphate buffer at pH 7.0 for 5 min. Reproducible anodic peak current of the immobilized Ru(bpy)3 2+ in 50 mM phosphate buffer were observed with a good reproducibility (R.S.D. = 4.28%). The amount of PMNPs initially used in the coating process on a Pt electrode with an external magnet surface strongly affects the amount of Ru(bpy)3 2+ immobilized in the PMNP multilayermodified electrode. As shown in Fig. 5, the anodic peak current of Ru(bpy)3 2+ immobilized in the PMNP-modified electrode increased consistently as the amount of PMNPs coated on the modified electrode was increased because of the increased amount of the ion-exchange sites within the multilayer films. The ECL behavior of Ru(bpy)3 2+ incorporated into the PMNPmodified electrode has been studied with tripropylamine (TPA) as a representative analyte since Ru(bpy)3 2 –TPA system has been well characterized [20–23]. As shown in the inset of Fig. 6, the presence
Fig. 5. Anodic peaks currents in CVs of Ru(bpy)3 2+ immobilized in the PMNPmodified Pt electrode in 50 mM pH 7.0 phosphate buffer at a scan rate of 100 mV/s with different amount of PMNP used for the fabrication process.
Fig. 6. ECL potential profile recorded during the CV scan in the absence () and the presence (䊉) of 0.5 mM TPA. Inset: cyclic voltammograms of the Ru(bpy)3 2+ immobilized in the PSMNP multilayer films in the absence (a) and presence (b) of 0.5 mM TPA in 50 mM pH 8.0 phosphate buffer at a scan rate 100 mV/s.
of TPA causes the oxidation current to increase considerably while the reduction current decrease. This is due to the ECL reaction of Ru(bpy)3 2+ and TPA. The corresponding ECL-potential profile was recorded during the CV scan at a scan rate of 100 mV/s. As can be expected from the Ru(bpy)3 2 –TPA ECL mechanism, the onset of ECL seen at around 1.0–1.1 V, which is coincident with the oxidation of Ru(bpy)3 2+ immobilized in the PMNP-modified electrode. The amount of PMNP coated in the Pt electrode surface strongly affects the ECL intensity and the reproducibility of the ECL measurement. As shown in Fig. 7, the most sensitive and reproducible ECL signal was obtained when 1.0 mg of the PMNPs was used in the coating process. The smaller amount of PMNP less than 1 mg led to lower reproducibility of the ECL signals but greater amount of PMNP more than 1 mg led to lower intensity of the ECL signals although the films contain a greater amount of Ru(bpy)3 2+ as seen in Fig. 5. These results could be ascribed to the fact that increased amount of PMNP might absorb and scatter the ECL emission within the films as observed in the previous result with Nafion-stabilized Fe3 O4 nanoparticles [12]. The sensor-to-sensor reproducibility for ECL measurement of TPA was very good (R.S.D. = 5%) when 1.0 mg of the PMNPs was used in the coating process.
Fig. 7. Dependence of ECL intensity on the amount of the PMNPs used for the fabrication process for the PMNP-modified Pt electrode in 50 mM pH 7.0 phosphate buffer. The peak ECL intensity was obtained from the ECL-potential curves acquired at a scan rate of 100 mV/s with 0.5 mM TPA.
Y.-K. Lyu et al. / Synthetic Metals 159 (2009) 571–575
575
prepared by an oxidative polymerization with an oxidant of KMnO4 . The preparation method was quite simple and the charge state of the functionalized MNPs was negative. The multilayer films of the magnetic nanoparticles can be easily formed on a Pt electrode by an external magnet. The electrochemical and ECL behavior of the present ECL sensor was strongly dependant upon the amount of functionalized MNPs coated on the Pt electrode surface. Due to the large pore size of the present PMNP films, the ECL sensor based on the PMNP films exhibits one order of magnitude lower detection limit for TPA compared to those based on pure Nafion films. Because of good biocompatibility and fast mass transport in the PMNP-multilayer films, such PMNP-multilayer films also could be readily used to a bioanalysis combined with ECL detection as well as a preconcentration media for a variety of cations such as heart imaging agent, Re(DMPE)3 + (DMPE = 1,2bis(dimethylphosphino)ethane) [25].
Fig. 8. Calibration curve for TPA obtained with the ECL sensor of Ru(bpy)3 2+ ionexchanged in a PMNP-modified electrode (amount of the PMNP: 1.0 mg). The peak ECL intensity was obtained from the ECL–potential curves acquired at a scan rate of 100 mV/s.
Calibration curves for TPA have been constructed using the Ru(bpy)3 2+ ECL sensor based on the PMNP-modified electrode as shown in Fig. 8. Calibration curves are plotted on logarithmic axes to show the wide dynamic ranges. Each point is a mean of three or more ECL signals obtained by consecutive cyclic potential scans (100 mV/s) at a given concentration. The linear range extended from 0.5 M to 1.0 mM (R2 = 0.999) at the present ECL sensor compared to that from 1.0 M to 1.0 mM at the pure Nafion-modified electrode. The detection limit (S/N = 3) for TPA at the present ECL sensor was 49 nM, which is one order of magnitude lower than those obtained with other sol–gel ceramic–Nafion composite films [24,18]. The long-term storage stability of the present PMNP-modified electrode was studied by monitoring its ECL response to 0.5 mM TPA with the re-immobilized Ru(bpy)3 2+ on the PMNP-modified electrode in 50 mM phosphate buffer solution with intermittent usage and storage in the buffer solution at 4 ◦ C when not in use. The coating of the PMNP films did not come off during the test period, indicating that the PMNP films are well stick to the Pt electrode. It was found that the response of the ECL sensor gradually decreased to almost 62% of its initial value in 2 weeks. The stability of the PMNP-modified electrode is much better than that of the pure Nafion-modified electrode (stability less than a day). 4. Conclusions As an alternative new immobilization matrix, the poly(3thiopheneacetic acid)-coated magnetic nanoparticles has been
Acknowledgements This work was supported by Korea Research Foundation Grant (KRF-2007-C-00484) and Yonsei-CBMH. K.J.K., Y.K.L and J.H.H acknowledge the support of the BK 21 program of the Ministry of Education and Science. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
M.M.H. Liao, D.H. Chen, Biotechnol. Lett. 2 (2001) 1723. J. Wang, D. Xu, R. Polsky, J. Am. Chem. Soc. 124 (2002) 4208. J. Wang, Anal. Chim. Acta 500 (2003) 247. S.V. Sonti, A. Bose, J. Colloid Interf. Sci. 170 (1995) 575. I. Willner, E. Katz, Angew. Chem., Int. Ed. 42 (2003) 4576. R. Hirsch, E. Katz, I. Willner, J. Am. Chem. Soc. 122 (2000) 12053. E. Katz, L. Sheeney-Haj-Ichia, A.F. Bückmann, I. Willner, Angew. Chem., Int. Ed. 42 (2003) 4576. L. Sheeney-Haj-Ichia, E. Katz, J. Wasserman, I. Willner, Chem. Commun. (2002) 158. Y.W. Jun, J.S. Choi, J. Cheon, Chem. Commun. (2007) 1203. V.S. Maceira, M.A. Correa-Duarte, M. Spasova, L.M. Liz-Marzán, M. Farle, Adv. Funct. Mater. 16 (2006) 509. K. Landfester, L.P. Ramírez, J. Phys.: Condens. Matter 15 (2003) S1345. D.J. Kim, Y.K. Lyu, H.N. Choi, I.H. Min, W.Y. Lee, Chem. Commun. (2005) 2966. L. Zhang, B. Liu, S.J. Dong, J. Phys. Chem. B 111 (2007) 10448. W.Y. Lee, Microchim. Acta 127 (1997) 19. A.W. Knight, G.M. Greenway, Analyst 121 (1996) 101R. R.D. Gerardi, N.W. Barnett, S.W. Lewis, Anal. Chim. Acta 378 (1999) 1. M.M. Richter, Chem. Rev. 104 (2004) 3003. H.N. Choi, S.H. Cho, W.Y. Lee, Anal. Chem. 75 (2003) 4250. J. Cheon, N.-J. Kang, S.-M. Lee, J.-H. Lee, J.-H. Yoon, S.J. Oh, J. Am. Chem. Soc. 126 (2004) 1950. J.B. Noffsinger, N.D. Danielson, Anal. Chem. 59 (1987) 865. J.K. Leland, M.J. Powell, J. Electrochem. Soc. 137 (1990) 3127. T.M. Downey, T.A. Nieman, Anal. Chem. 64 (1992) 261. Y. Zu, A.J. Bard, Anal. Chem. 72 (2000) 3223. A.N. Khramov, M.M. Collinson, Anal. Chem. 72 (2000) 2943. B.H. Swaile, E.A. Blubaugh, C.J. Seliskar, W.R. Heineman, Anal. Chem. 70 (1998) 4326.
Contents lists available at ScienceDirect
Synthetic Metals journal homepage: www.elsevier.com/locate/synmet
Functionalized magnetic nanoparticle with poly(3-thiopheneacetic acid) and its application for electrogenerated chemiluminescence sensor Young-Ku Lyu 1 , Kyu Jin Kyung 1 , Won-Yong Lee ∗ Department of Chemistry and Center for Bioactive Molecular Hybrids, Yonsei University, 134 Shinchon-dong, Seodaemoon-ku, Seoul 120-749, Republic of Korea
a r t i c l e
i n f o
Article history: Received 6 August 2008 Received in revised form 6 November 2008 Accepted 27 November 2008 Available online 7 January 2009 Keywords: Electrogenerated chemiluminescence Ru(bpy)3 2+ Magnetic nanoparticle Chemically modified electrode
a b s t r a c t Polymer-coated magnetic nanoparticles (MNPs) have been prepared and used as an immobilization matrix for the fabrication of solid-state tris(2,2 -bipyridyl)ruthenium(II) (Ru(bpy)3 2+ ) electrogenerated chemiluminescence (ECL) sensor. The pre-synthesized maghemite (␥-Fe2 O3 ) MNPs were coated with poly(3-thiopheneacetic acid) based on an oxidative polymerization method using KMnO4 . The poly(3thiopheneacetic acid)-coated MNPs have formed the clusters with average diameter of 200–500 nm. The multilayer films of poly(3-thiopheneacetic acid)-coated MNPs were uniformly formed on the surface of a Pt electrode by an external magnet. The Ru(bpy)3 2+ was rapidly incorporated into the multilayer films within 5 min through the electrostatic interaction between the Ru(bpy)3 2+ and the negatively charged carboxylates of 3-thiopheneacetic acid at pH 7.0. Due to the fast mass transport in the multilayer films of the functionalized MNPs, the present Ru(bpy)3 2+ ECL sensor based on the functionalized MNPs showed improved ECL sensitivity for tripropylamine (TPA) compared to the Nafion-based ECL sensor. The present ECL sensor exhibited a quite low limit of detection for TPA (49 nM). The sensor-to-sensor reproducibility was very good (R.S.D. = 5%). © 2008 Elsevier B.V. All rights reserved.
1. Introduction Due to their compatibility to biological units, magnetic particles are of great interest for biomedical applications. In particular, magnetic particles of iron oxides functionalized with redox or biochemical components have been extensively used for the concentration and localization of chemical or biochemical components such as enzymes [1], DNA [2,3], and cells [4]. In addition, the magnetic localization of the functionalized particles to the electrode surface by an external magnetic field can be used to initiate the electrocatalytic and biocatalytic processes. Thus, magnetic-field switching of electrocatalytic and biocatalytic processes have been easily performed for selective dual biosensing, immunosensing, DNA sensing, and luminol ECL [5–8]. Although, the micron-sized magnetic particles have been previously used for such applications, recent studies are mainly focused on the use of smaller-sized nanoparticles because they provide high colloidal stability, minimal nonspecific binding, and enhanced magnetic behaviors [9]. However, a suitable coating of the nanopar-
∗ Corresponding author. Tel.: +82 2 2123 2649; fax: +82 2 364 7050. E-mail address: [email protected] (W.-Y. Lee). 1 These authors contributed equally to the work. 0379-6779/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.synthmet.2008.11.021
ticles is necessary because the magnetic nanoparticles are easily degraded when they are exposed to certain environments [10]. The preparation and application of the heterostructured magnetic nanoparticles (MNPs) are well summarized in recent papers [9,11]. Previously, we described the Ru(bpy)3 2+ ECL sensor based on the Nafion-stabilized MNPs (Nafion/Fe3 O4 ) [12]. Due to the fast mass transport of Ru(bpy)3 2+ in the Nafion/Fe3 O4 multilayer films, the resulting ECL sensor exhibited good sensitivity and fast response time. Dong and co-workers used bifunctional core–shell nanostructures of core MNPs and silica shell containing Ru(bpy)3 2+ for solid-state ECL sensor materials [13]. In the core–shell structure, silica outershell could protect the immobilized Ru(bpy)3 2+ from oxygen quenching and improve the ECL sensitivity. In the present work, monodisperse maghemite nanoparticles (␥-Fe2 O3 , ca. ∼10 nm in size) has been coated with poly(3thiopheneacetic acid) via an oxidative polymerization using KMnO4 as an oxidant (Scheme 1) and the magnetic-field driven multilayer films of the functionalized MNPs were formed as a matrix for the immobilization of Ru(bpy)3 2+ . The electrochemical and ECL behavior of the present Ru(bpy)3 2+ ECL sensor with typical coreactant tripropylmaine (TPA) [14–17] will be described in terms of sensitivity, selectivity, and long-term stability relative to the other ECL sensors.
572
Y.-K. Lyu et al. / Synthetic Metals 159 (2009) 571–575
Scheme 1. Oxidative polymerization of 3-thiopheneacetic acid.
2. Experimental 2.1. Reagents and apparatus Iron pentacarbonyl (99.999%), dodecylamine (98%), tripropylamine (TPA, 99%), 1,2-dichlorobenzene (99%), tris(2,2 -bipyridyl) dichlororuthenium(II) (Ru(bpy)3 2+ , 98%), 3-thiopheneacetic acid, potassium permanganate (KMnO4 , 99%) were purchased from Aldrich. Water for all solutions was purified using a Milli-Q water purification system (Millipore, Bedford, MA, USA). Cyclic voltammetric experiments were performed with an EG&G 273A potentiostat (Oak Ridge, TN, USA). A conventional threeelectrode system was employed with a platinum wire as counter electrode, a Pt plate (0.636 cm2 ) coated with PMNPs as a working electrode, and an Ag/AgCl (3 M NaCl) reference electrode. The ECL detection system described earlier has been used in the present study [18].
Fig. 1. Formation of poly(3-thiopheneacetic acid) based on the oxidative polymerization (a) and its use for the formation of PMNP-modified electrode (b).
initial potential of +850 mV to a high of +1300 mV and back to +850 mV at a scan rate of 100 mV/s.
2.2. Preparation of the functionalized magnetic nanoparticles According to the literature [19], monodisperse maghemite (␥-Fe2 O3 ) nanoparticles (10 nm diameter) were prepared from the thermal decomposition of Fe(CO)5 using a capping ligand of dodecylamine. In order to coat the maghemite MNPs with polymer, 1 mL of a MNP solution (1 mg/mL toluene) was mixed with 1 mL of 2.0 mM 3-thiopheneacetic acid dissolved in acetonitrile. The mixed solution was shaken for about 30 min and then blended with 1 mL solution of 2.0 mM KMnO4 dissolved in acetonitrile. Oxidative polymerization was immediately initiated. After shaking for additional 30 min, 2 mL ethanol was added. The poly(3-thiopheneacetic acid)-coated magnetic nanoparticles (PMNPs) were separated by a permanent magnet. PMNPs were washed by acetonitrile and then water. PMNP clusters were formed with average diameter of 200–500 nm. The zeta potential of PMNPs dispersed in 50 mM phosphate buffer at pH 7.0 was determined to be −21.5 mV (Zetasizer Nano ZS, Malvern Instruments Ltd., UK). 2.3. Fabrication of the ECL sensor For electrochemical experiments, the multilayer films of the PMNPs on a Pt electrode surface have been formed as shown in Fig. 1. An external magnet has been positioned behind the Pt electrode (surface area = 0.636 cm2 ) to attract the PMNPs dissolved in aqueous solution. The PMNP multilayer-modified Pt electrode was then placed in an electrochemical cell containing 10 mM Ru(bpy)3 2+ solution in 0.05 M phosphate buffer at pH 7. The incorporation of Ru(bpy)3 2+ into the PMNP multilayer films was electrochemically monitored by running consecutive cyclic potential scans from an
3. Results and discussion 3.1. Preparation of the functionalized magnetic nanoparticles and the nanoparticle-modified electrode Fig. 1 shows a schematic illustration of the preparation of the functionalized MNPs and the formation of the MNP-modified electrode. First, maghemite (␥-Fe2 O3 ) MNPs were prepared from the thermal decomposition of pentacarbonyl iron [Fe(CO)5 ] using a capping ligand of dodecylamine according to the literature [19]. As shown in the transmission electron micrograph (TEM) image of Fig. 2(a), maghemite MNPs were monodisperse and spherical with the average diameter of 10 nm. Then, as-prepared maghemite MNPs were coated with poly(3-thiopheneacetic acid) via an oxidative polymerization using KMnO4 as an oxidant. The polymerization was effective and was completed within 30 min. While the maghemite MNPs are well dispersed in toluene as shown in the TEM image of Fig. 2(a), the poly(3-thiopheneacetic acid)-coated magnetic nanoparticles (PMNP) has formed the clusters with average diameter of 200–500 nm as shown in the TEM image of Fig. 2(b). In order to characterize the charge state of the PMNPs, zeta potential of the PMNPs dispersed in 50 mM phosphate buffer at pH 7.0 has been measured. The zeta potential was determined to be −21.5 mV, which indicates the presence of the negatively charged carboxylates at the surface of the PMNPs. Since the PMNPs were well dispersed in deionized water, a small amount of PMNPs (1.0 mg) were introduced into an electrochemical cell containing 1 mL deionized water. Positioning of an external magnet behind a Pt electrode attracts the PMNPs to form the PMNP multilayer films. The formation of the PMNP-modified electrode
Y.-K. Lyu et al. / Synthetic Metals 159 (2009) 571–575
573
Fig. 3. FE-SEM images of the surface (a) and a cross-sectional view (b) of the PMNP multilayer films formed on a Pt plate electrode.
Fig. 2. TEM image of iron oxide nanoparticles before the polymerization (a) and FE-SEM image of iron oxide after the polymerization of 3-thiopheneacetic acid (b).
under the magnetic filed is so effective and can be completed in less than 20 min. As shown in Fig. 3(a), the PMNPs were evenly deposited on a Pt electrode surface. The micro-pore structures can be seen in the surface of the PMNP composite films, which can lead to fast diffusion of analytes within around 7 m-thick PMNP multilayer films as shown in Fig. 3(b). In fact, the thickness of the multilayer films can be easily controlled in the range of sub-m to several hundred m by the variation of the amount of the PMNPs initially introduced into the aqueous solution. 3.2. Electrochemical and ECL characteristics of PMNP-based Ru(bpy)3 2+ ECL sensor An electrochemiluminescent cation, Ru(bpy)3 2+ , can be easily incorporated into the PMNP multilayer films via electrostatic interaction between positively charged Ru(bpy)3 2+ and negatively charged carboxylates in the PMNP multilayer films. Therefore, the PMNP multilayer-modified Pt electrode was simply placed in an electrochemical cell containing 10 mM Ru(bpy)3 2+ solution in 0.05 M phosphate buffer at pH 7. The incorporation of Ru(bpy)3 2+
into the PMNP multilayer films was electrochemically monitored by running consecutive cyclic potential scans from an initial potential of +850 mV to a high of +1300 mV and back to +850 mV at a scan rate of 100 mV/s. The anodic and cathodic peak currents of Ru(bpy)3 2+ were rapidly increased and the steady-state currents were reached within 5 min. In contrast, the loading of Ru(bpy)3 2+ into the pure Nafion film was much slower under the identical experimental conditions with a steady-state current being achieved typically within 30 min. The fast uptake of the Ru(bpy)3 2+ into the PMNP multilayer films might be due to the micro-pore structures of the PMNP multilayer films as seen in Fig. 3. The ion-exchangeable carboxylate groups of the PMNP multilayer films seem to be easily accessible to Ru(bpy)3 2+ within the multilayer films, thus leading to fast diffusion of Ru(bpy)3 2+ in the films. The anodic peak currents of the Ru(bpy)3 2+ incorporated into the PMNP multilayer films were measured at different scan rates. The anodic peak currents were linearly proportional to the square root of the scan rate over the range of 50–500 mV. At those experimental time scales, the thickness of the diffusion layer is less than the thickness of the composite films (around 7 m) and therefore the semi-infinitive diffusion process prevails. Once a steady-state CV was obtained, the PMNP-modified electrode was removed from the Ru(bpy)3 2+ solution, rinsed with water, and then placed in 0.05 M phosphate buffer solution at pH 7. As shown in Fig. 4, the anodic peak current obtained with the Ru(bpy)3 2+ -immobilized at PMNP-modified electrode in the buffer solution has been gradually decreased and reached a state-state current within 1 h. The anodic peak current obtained with the Ru(bpy)3 2+ -immobilized PMNP-modified electrode in the buffer solution was about 25% that of the steady-state current obtained in the Ru(bpy)3 2+ solution, while Ru(bpy)3 2+ -immobilized in the
574
Y.-K. Lyu et al. / Synthetic Metals 159 (2009) 571–575
Fig. 4. Cyclic voltammograms of Ru(bpy)3 2+ immobilized in the PMNP multilayer films on a Pt electrode surface in 50 mM pH 7.0 phosphate buffer at a scan rate of 100 mV/s for 0 min (a), 15 min (b), 30 min (c), 45 min (d), and 60 min (e).
Nafion-stabilized MNPs film exhibited 30% loss in the above process [12]. The relatively fast initial leaching of Ru(bpy)3 2+ from the poly(3-thiopheneacetic acid)-coated MNP composite films are possibly due to the relatively weak interaction between Ru(bpy)3 2+ and carboxylates of PMNPs. Therefore, it was necessary to reimmobilize the Ru(bpy)3 2+ prior to each ECL experiment in order to get a high sensitivity of the ECL measurement. For reimmobilization process, the PMNP-modified electrode was placed in 10 mM Ru(bpy)3 2+ solution in 50 mM phosphate buffer at pH 7.0 for 5 min. Reproducible anodic peak current of the immobilized Ru(bpy)3 2+ in 50 mM phosphate buffer were observed with a good reproducibility (R.S.D. = 4.28%). The amount of PMNPs initially used in the coating process on a Pt electrode with an external magnet surface strongly affects the amount of Ru(bpy)3 2+ immobilized in the PMNP multilayermodified electrode. As shown in Fig. 5, the anodic peak current of Ru(bpy)3 2+ immobilized in the PMNP-modified electrode increased consistently as the amount of PMNPs coated on the modified electrode was increased because of the increased amount of the ion-exchange sites within the multilayer films. The ECL behavior of Ru(bpy)3 2+ incorporated into the PMNPmodified electrode has been studied with tripropylamine (TPA) as a representative analyte since Ru(bpy)3 2 –TPA system has been well characterized [20–23]. As shown in the inset of Fig. 6, the presence
Fig. 5. Anodic peaks currents in CVs of Ru(bpy)3 2+ immobilized in the PMNPmodified Pt electrode in 50 mM pH 7.0 phosphate buffer at a scan rate of 100 mV/s with different amount of PMNP used for the fabrication process.
Fig. 6. ECL potential profile recorded during the CV scan in the absence () and the presence (䊉) of 0.5 mM TPA. Inset: cyclic voltammograms of the Ru(bpy)3 2+ immobilized in the PSMNP multilayer films in the absence (a) and presence (b) of 0.5 mM TPA in 50 mM pH 8.0 phosphate buffer at a scan rate 100 mV/s.
of TPA causes the oxidation current to increase considerably while the reduction current decrease. This is due to the ECL reaction of Ru(bpy)3 2+ and TPA. The corresponding ECL-potential profile was recorded during the CV scan at a scan rate of 100 mV/s. As can be expected from the Ru(bpy)3 2 –TPA ECL mechanism, the onset of ECL seen at around 1.0–1.1 V, which is coincident with the oxidation of Ru(bpy)3 2+ immobilized in the PMNP-modified electrode. The amount of PMNP coated in the Pt electrode surface strongly affects the ECL intensity and the reproducibility of the ECL measurement. As shown in Fig. 7, the most sensitive and reproducible ECL signal was obtained when 1.0 mg of the PMNPs was used in the coating process. The smaller amount of PMNP less than 1 mg led to lower reproducibility of the ECL signals but greater amount of PMNP more than 1 mg led to lower intensity of the ECL signals although the films contain a greater amount of Ru(bpy)3 2+ as seen in Fig. 5. These results could be ascribed to the fact that increased amount of PMNP might absorb and scatter the ECL emission within the films as observed in the previous result with Nafion-stabilized Fe3 O4 nanoparticles [12]. The sensor-to-sensor reproducibility for ECL measurement of TPA was very good (R.S.D. = 5%) when 1.0 mg of the PMNPs was used in the coating process.
Fig. 7. Dependence of ECL intensity on the amount of the PMNPs used for the fabrication process for the PMNP-modified Pt electrode in 50 mM pH 7.0 phosphate buffer. The peak ECL intensity was obtained from the ECL-potential curves acquired at a scan rate of 100 mV/s with 0.5 mM TPA.
Y.-K. Lyu et al. / Synthetic Metals 159 (2009) 571–575
575
prepared by an oxidative polymerization with an oxidant of KMnO4 . The preparation method was quite simple and the charge state of the functionalized MNPs was negative. The multilayer films of the magnetic nanoparticles can be easily formed on a Pt electrode by an external magnet. The electrochemical and ECL behavior of the present ECL sensor was strongly dependant upon the amount of functionalized MNPs coated on the Pt electrode surface. Due to the large pore size of the present PMNP films, the ECL sensor based on the PMNP films exhibits one order of magnitude lower detection limit for TPA compared to those based on pure Nafion films. Because of good biocompatibility and fast mass transport in the PMNP-multilayer films, such PMNP-multilayer films also could be readily used to a bioanalysis combined with ECL detection as well as a preconcentration media for a variety of cations such as heart imaging agent, Re(DMPE)3 + (DMPE = 1,2bis(dimethylphosphino)ethane) [25].
Fig. 8. Calibration curve for TPA obtained with the ECL sensor of Ru(bpy)3 2+ ionexchanged in a PMNP-modified electrode (amount of the PMNP: 1.0 mg). The peak ECL intensity was obtained from the ECL–potential curves acquired at a scan rate of 100 mV/s.
Calibration curves for TPA have been constructed using the Ru(bpy)3 2+ ECL sensor based on the PMNP-modified electrode as shown in Fig. 8. Calibration curves are plotted on logarithmic axes to show the wide dynamic ranges. Each point is a mean of three or more ECL signals obtained by consecutive cyclic potential scans (100 mV/s) at a given concentration. The linear range extended from 0.5 M to 1.0 mM (R2 = 0.999) at the present ECL sensor compared to that from 1.0 M to 1.0 mM at the pure Nafion-modified electrode. The detection limit (S/N = 3) for TPA at the present ECL sensor was 49 nM, which is one order of magnitude lower than those obtained with other sol–gel ceramic–Nafion composite films [24,18]. The long-term storage stability of the present PMNP-modified electrode was studied by monitoring its ECL response to 0.5 mM TPA with the re-immobilized Ru(bpy)3 2+ on the PMNP-modified electrode in 50 mM phosphate buffer solution with intermittent usage and storage in the buffer solution at 4 ◦ C when not in use. The coating of the PMNP films did not come off during the test period, indicating that the PMNP films are well stick to the Pt electrode. It was found that the response of the ECL sensor gradually decreased to almost 62% of its initial value in 2 weeks. The stability of the PMNP-modified electrode is much better than that of the pure Nafion-modified electrode (stability less than a day). 4. Conclusions As an alternative new immobilization matrix, the poly(3thiopheneacetic acid)-coated magnetic nanoparticles has been
Acknowledgements This work was supported by Korea Research Foundation Grant (KRF-2007-C-00484) and Yonsei-CBMH. K.J.K., Y.K.L and J.H.H acknowledge the support of the BK 21 program of the Ministry of Education and Science. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
M.M.H. Liao, D.H. Chen, Biotechnol. Lett. 2 (2001) 1723. J. Wang, D. Xu, R. Polsky, J. Am. Chem. Soc. 124 (2002) 4208. J. Wang, Anal. Chim. Acta 500 (2003) 247. S.V. Sonti, A. Bose, J. Colloid Interf. Sci. 170 (1995) 575. I. Willner, E. Katz, Angew. Chem., Int. Ed. 42 (2003) 4576. R. Hirsch, E. Katz, I. Willner, J. Am. Chem. Soc. 122 (2000) 12053. E. Katz, L. Sheeney-Haj-Ichia, A.F. Bückmann, I. Willner, Angew. Chem., Int. Ed. 42 (2003) 4576. L. Sheeney-Haj-Ichia, E. Katz, J. Wasserman, I. Willner, Chem. Commun. (2002) 158. Y.W. Jun, J.S. Choi, J. Cheon, Chem. Commun. (2007) 1203. V.S. Maceira, M.A. Correa-Duarte, M. Spasova, L.M. Liz-Marzán, M. Farle, Adv. Funct. Mater. 16 (2006) 509. K. Landfester, L.P. Ramírez, J. Phys.: Condens. Matter 15 (2003) S1345. D.J. Kim, Y.K. Lyu, H.N. Choi, I.H. Min, W.Y. Lee, Chem. Commun. (2005) 2966. L. Zhang, B. Liu, S.J. Dong, J. Phys. Chem. B 111 (2007) 10448. W.Y. Lee, Microchim. Acta 127 (1997) 19. A.W. Knight, G.M. Greenway, Analyst 121 (1996) 101R. R.D. Gerardi, N.W. Barnett, S.W. Lewis, Anal. Chim. Acta 378 (1999) 1. M.M. Richter, Chem. Rev. 104 (2004) 3003. H.N. Choi, S.H. Cho, W.Y. Lee, Anal. Chem. 75 (2003) 4250. J. Cheon, N.-J. Kang, S.-M. Lee, J.-H. Lee, J.-H. Yoon, S.J. Oh, J. Am. Chem. Soc. 126 (2004) 1950. J.B. Noffsinger, N.D. Danielson, Anal. Chem. 59 (1987) 865. J.K. Leland, M.J. Powell, J. Electrochem. Soc. 137 (1990) 3127. T.M. Downey, T.A. Nieman, Anal. Chem. 64 (1992) 261. Y. Zu, A.J. Bard, Anal. Chem. 72 (2000) 3223. A.N. Khramov, M.M. Collinson, Anal. Chem. 72 (2000) 2943. B.H. Swaile, E.A. Blubaugh, C.J. Seliskar, W.R. Heineman, Anal. Chem. 70 (1998) 4326.