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Cytosensor Constructed with a Biomimetic Fibronectin-Functionalized Carbon Nanotubes on Glassy Carbon Heated Electrode Xue Zhong, Guang-Sheng Qian, Jing-Juan Xu,* and Hong-Yuan Chen The Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing UniVersity, Nanjing 210093, People’s Republic of China ReceiVed: July 10, 2010; ReVised Manuscript ReceiVed: September 4, 2010
In this paper, an interface constructed with fibronectin (Fn) functionalized multiple-walled carbon nanotubes (MWNTs) was prepared which can efficiently capture cells. The acid-treated MWNTs were covalently coupled with Fn in the presence of a coupling agent, thus realizing the efficient immobilization of Fn, and it was proven by infrared spectroscopy. Due to the specific binding of integrin receptor on cells and the arginineglycine-aspartic acid amino acids (RGD) peptide contained in Fn, this interface not only has a property for capturing SMMC 7721 cells, but also efficiently retains viability of immobilized cells. The presence of MWNTs not only increases the surface area of the electrode but also promotes the electrochemical behavior of SMMC 7721 cells. In addition, we applied homemade heated glassy carbon electrodes to cell detection, which were indirectly heated by direct current. Profiting from the heat convection generated by the joule heat from direct current, the sensitivity of the detection of cell concentration was clearly meliorated. The biosensor based on heated electrode modified with Fn-MWNTs, presents a promising platform for sensitive cell detection. 1. Introduction Over the past decade, significant progress has been made in detecting cells and monitoring their activity for life science research, toxicity monitoring, clinical diagnostics, and public health protection. Some techniques such as the spectrophotometric method, fluorescent microscopy, amperometric or potentiometric scanning probe technique, and flow cytometry have been developed for monitoring cell viability and proliferation.1-3 Since a living cell can be properly described as an electrochemical dynamic system, the use of cell-based biosensor platforms has attracted considerable attention,4 which has remarkable advantages in the electrochemical monitoring of cell viability and proliferation, such as low cost, convenient operation, rapid detection and good sensitivity. For cells growing on a substrate, the substrate properties such as roughness, hydrophobicity, topography, positive/negative charges, surface chemistry, and specific protein or cell-surface interactions play a key role in cellular adhesion and proliferation.5,6 Adsorption,7 sandwich,8 entrapment,9 covalent binding,10 and cross-linking technique11 have been utilized for cell immobilization. However, the stability of cell immobilized by passive trapping or adsorption during continuous use is not satisfactory, while covalent binding usually leads to the decrease of cell viability when the cells are exposed to reactive groups and harsh reaction conditions. Thus, the construction of a cytosensor with sensitivity of detection, and the interface of which can retain viability of immobilized cells, is very important. During the last two decades, a new tendency that consists in application of extra-electrochemical effects like illumination, heating, or mechanical attack to electrodes in situ has developed, leading to photoelectrochemistry, thermoelectrochemistry, triboelectrochemistry, and other fields. Among the fields mentioned, thermoelectrochemistry can be considered to be the most important technology.12 Heated electrodes have been proven to * To whom correspondence should be addressed. E-mail:
[email protected]. Tel/Fax: +86-25-83597294.
provide advantages and new opportunities for electroanalytical chemistry such as enhancing mass transport via establishing thermal convection in the vicinity of the electrode surface and increasing the diffusion coefficient, accelerating some reactions inert, thus improving the sensitivity.13-25 This work used fibronectin (Fn)-functionalized multiwalled carbon nanotubes (MWNTs) to construct a nontoxic biomimetic interface for immobilization of SMMC 7721 cells on an electrode surface through specific interaction between the integrin receptor on cells and the arginine-glycine-aspartic acid amino acids (RGD) peptide contained in Fn, providing an environment similar to a native system, thus efficiently retaining the activity of living tumor cells and preventing cell leakage from the electrode interface. The presence of MWNTs not only increases the surface area of the electrode but also promotes the electrochemical behavior of cells. In addition, we applied a unique indirect heating approach by copper wire to heat electrode to obtain a homemade heated glassy carbon electrode and introduced it to cell detection. Use of heated electrodes induced thermally efficient convection within a thin solution layer near the surface, meliorating sensitivity of the detection of cell concentration. 2. Experimental Section 2.1. Materials and Reagents. MWNTs were purchased from Shenzhen Nanotech Port Ltd. Co. (China). Fn was purchased from Sigma (U.S.A.). N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimehtylaminopropyl) carbodiimide (EDC) were purchased from Aldrich (U.S.A.). Acridine orange (AO) and ethidium bromide (1% w/v, EB) were products from Amresco (U.S.A.). All other reagents were of analytical grade. Phosphatebuffered saline (PBS) (pH 7.4) contained 137 mM NaCl, 2.7 mM KCl, 87.2 mM Na2HPO4 · 12H2O, and 14.1 mM KH2PO4. All aqueous solutions were prepared using ultrapure water (Milli-Q, Millipore). 2.2. Cell Line and Culture. The SMMC 7721 cell line was kindly provided by the Gulou Hospital, Nanjing, China. SMMC
10.1021/jp106383p 2010 American Chemical Society Published on Web 10/27/2010
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SCHEME 1: Schematic Diagram of (A) Fabrication of Heated Glassy Carbon Electrode, and (B) Heating and Sensing System
7721 cells were cultured in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO), penicillin (60 µg mL-1), and streptomycin (100 µg mL-1) at 37 °C in a humidified atmosphere containing 5% CO2. After 72 h, the cells were collected and separated from the medium by centrifugation at 1500 rpm for 5 min and then washed twice with sterile pH 7.4 PBS. The sediment was resuspended in PBS to obtain a homogeneous cell suspension at a certain concentration. The cell number was determined using a Petroff Hausser cell counter (U.S.A.). 2.3. Fabrication of Heated Glassy Carbon Electrode. A glassy carbon stick (diameter 3 mm) that connected to a copper stick was coiled by a lacquered wire (diameter 50 µm) twice in adverse direction avoiding induction voltage. The glassy carbon stick twisted by copper wire was spread by epoxy resin. Then the whole piece fabricated in the former step was covered by a glass tube. The room between the copper and the interface of the tube was filled with carbon powder for thermal insulation. The section of the glassy carbon stick was used as the working electrode and the lacquered wire was the heater (Scheme 1A). Before use, the working area of glassy carbon electrode (GCE) was carefully polished with a shammy and rinsed with pure water and corresponding buffer. The electrode can be used carefully for a long time avoiding heating to an extremely high temperature. A direct current (dc) power was connected to the lacquered wire of the heated glassy carbon electrode to provide steady current for heating (Scheme 1B). 2.4. Preparation of the Fn-MWNTs Film and Cell Adhesion. MWNTs were dispersed in 30% HNO3 and then refluxed for 24 h at 140 °C to shorten the nanotubes and to produce carboxylic acid groups mainly on the open ends as well as the sidewalls, which introduced negative charges to MWNTs and improved their dispersion in water. The resulting suspension was centrifuged, and the sediment was washed with ultrapure water until the pH reached 7.0. Then, the oxidized MWNTs were dispersed in ultrapure water to a concentration of 1 mg mL-1. A homemade heated glassy carbon electrode (3 mm diameter) was polished to a mirror using 0.3 and 0.05 µm alumina slurry (Buehler) followed by rinsing thoroughly with ultrapure water. After successive sonication in 1:1 nitric acid, acetone, and ultrapure water, the electrode was rinsed with ultrapure water and allowed to dry at room temperature. Five µL of 1 mg mL-1 carboxylic group functionalized MWNTs solution was dropped on the pretreated GCE and dried in a desiccator. The MWNTscoated GCE was then immersed in a solution containing 2 mM EDC and 5 mM NHS for 1 h. After the activated MWNTs/ GCE was thoroughly rinsed with deionized water, 5 µL of 1 mg mL-1 Fn was immediately dropped on its surface and then
incubated for 1 h to yield an Fn-MWNTs-modified GCE. Following a rinse with pH 7.4 PBS, 10 µL of SMMC 7721 cell suspension at a certain concentration was dropped on the FnMWNTs/GCE surface and incubated at 37 °C for 2 h. After carefully rinsing with pH 7.4 PBS to remove the noncaptured cells, the obtained SMMC 7721 cells/Fn-MWNTs/GCE was used for subsequent assay. The images of SMMC 7721 cells with different incubation temperature on the Fn-MWNTs film were recorded by fluorescent staining under an inverted microscope with a magnification of 100× and acquired with a CCD camera. 2.5. Apparatus. A DMIRE2 Inverted fluorescence microscope (Leica, Germany) equipped with a DP71 CCD (Olympus, Japan) was used for fluorescence microimaging. Scanning electron microscopic (SEM) images were obtained on a SSX550 SEM (Japan). Fourier-transform infrared spectra (FTIR) were carried out on a Nicolet 6700 FTIR spectrometer (Nicolet, U.S.A.). Resonance Raman spectra were measured on a LabRAM HR 800 Raman spectrophotometer (Jobin Yvon, France). A dc power supply RXN-303A (Shengzhen Zhao Xin Electronic Equipments and Instruments Producer, China) was used to heat the heated GCE. Electrochemical measurements were performed on a CHI 660B electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China). All experiments were carried out using a conventional three-electrode system comprising a platinum foil as the auxiliary electrode, a saturated calomel electrode as the reference electrode, and the modified homemade heated glassy carbon electrode as the working electrode. 3. Results and Discussion 3.1. Control and Measurement of the Temperature at the Surface of the Heated Glassy Carbon Electrode. Electrode temperature control and measurement is quite important for the investigation of the electrochemical signals acquired with heated GCE. Since direct measurement of the temperature was not practically feasible, indirect temperature calibration by open circuit potentiometry was reported.26 Here, a heated GCE and a Pt electrode were placed into a solution of 5 mM [Fe(CN)6]3-/4in 1.0 M KCl. The potential of the heated electrode changes due to the change in temperature, leading to change of open circuit potential of a reversible redox couple. The exact electrode temperature could be calculated with the temperature coefficient (1.56 mV/K for [Fe(CN)6]3-/4- of the electrode potential. The relationship between the electrode potential with the heating current was also determined, and then the relationship between the heating current and the temperature of electrode surface could be deduced (Figure 1). The voltammetric behavior of the heated GCE was tested in K3[Fe(CN)6] system. Increasing the temperature of electrode
Cytosensor Constructed with Biomimetic Fn-Functionalized CNTs
Figure 1. Relationship between the heating current and the temperature.
Figure 2. Cyclic voltammograms of [Fe3(CN)6] (5 mM) in KCl (1.0 M) on heated GCE at different temperature values: (a) 25, (b) 28, (c) 32, (d) 38, and (e) 48 °C. Scan rate: 50 mV s-1.
could greatly speed up diffusion and also lead to convection of redox couple at a thin layer near the surface of the electrode,22 resulting in the increase of the peak current (Figure 2). Comparing to the behavior shown at low temperature, the electrode showed the steady state behavior at higher temperature, which could be attributed to enhanced convection and diffusion at the electrode surface. Moreover, temperature gradient would exist resulting in enhanced convection. Thus, the diffusion and convection would be enhanced and the steady state behavior would be obtained. As a result, we could profit from that with the increase of the current, the analytical signal of the analytes would be enhanced. 3.2. Characterization of Fn-Functionalized MWNTs. Scanning electron microscopy images of an Fn-MWNTs-modified glassy carbon electrode showed a uniformly distributed MWNTs film with a large surface area (Figure 3A). As has been reported before, the roughness of the surface is a critical parameter for the immobilization of cells.27 By coating the GCE with FnMWNTs, the surface of the electrode became rough and porous, which not only served as a better medium for cell immobilization but also increased the surface area of the electrode. Moreover, the diameter of Fn-MWNTs was larger than the actual MWNTs, indicating that Fn has been combined on the surface of the MWNTs. The transmission electron microscope
J. Phys. Chem. C, Vol. 114, No. 45, 2010 19505 (TEM) images (Figure 3B) clearly showed that Fn has been modified to the surface of the MWNTs, which was in agreement with the results of SEM images. Before Fn functionalization, the walls of CNTs were smooth (data not shown), while after the functionalization, the walls became rough and some grains were attached to the walls, indicating that Fn had been bound to MWNTs. The oxidation and covalent modification of MWNTs with Fn was confirmed by both FTIR spectra and Raman spectra. The concentrated-acid oxidization treatment induced the formation of abundant oxygen-containing groups, such as ether or carboxyl groups, at the ends and on the surface of the MWNTs, which improved their dispersion in water and these groups could be surface functionalized. The FTIR spectrum of oxidized MWNTs displays two peaks at 1705 and 3425 cm-1 (Figure 4A, curve a), which could be assigned to the carbonyl stretch modes of COOH and OH, respectively. Successful immobilization of Fn to MWNTs was evidenced by the presence of a vibration mode at 1627 cm-1 belonging to CdO and C-N stretching induced by the formation of amide linkages between the carboxylic groups on the MWNTs and the amine groups of Fn (Figure 4A, curve b). The Raman spectrum of oxidized MWNTs (Figure 4B, curve a) showed the characteristic MWNT peaks at around 1355 cm-1, which were related to the disorder mode (D-band), and at around 1575 cm-1, which presented the tangential modes (G-band). Compared to the oxidized MWNTs, the relatively increased intensity of the disorder mode, which was diagnostic of disruptions in the hexagonal framework of MWNTs,28 for the Fn-modified MWNTs (Figure 4B, curve b) provided direct evidence of the covalent modification of the MWNTs. The bands in the Fn-MWNTs spectrum were only slightly red shifted, indicating that the functionalization did not damage the tube structure of the MWNTs. 3.3. Cell Adhesion. The cell adhesion abilities of MWNTsmodified and Fn-MWNTs-modified electrodes were tested. It could be seen that few cells attached themselves to the MWNTsmodified GCE, whereas Fn-MWNTs films captured numerous amounts of cells, as presented in Figure 5. It has been reported that surface charges play a particularly important role in governing nonspecific cellular adhesion to material substrates, and substrates with positive charges have a significantly higher level of cell adhesion.29 Thus, although the oxidized-MWNTs film showed a hydrophilic microenvironment that was also reported favorable for cell adhesion,30,31 the large amount of negative charges on its surface seems to inhibit cell adhesion. Because of the specific binding between the integrin receptor on the cell membrane and the RGD peptide belonging to Fn, cells could effectively attach to the Fn-MWNTs-modified GCE. The MWNTs here as a medium were used to immobilize Fn by covalent coupling between -NH2 groups in Fn and -COOH groups that not only exists on the ends but also on the walls of
Figure 3. (A) Scanning electron micrograph and (B) transmission electron micrograph of Fn-MWNTs.
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Figure 4. (A) FTIR and (B) Raman spectra of (a) oxidized MWNTs and (b) Fn-MWNTs.
Figure 5. Photos of 7721 cells cultivated on (A) MWNTs and (B) Fn-MWNTs for 2 h.
Figure 6. (A) Linear sweep voltammograms of Cells/Fn-MWNTs/GCE obtained with SMMC 7721 cell (concentration 5.0 × 105 cell mL-1) in 0.1 M pH 7.4 PBS at different temperature values: (a) 37, (b) 40, and (c) 42 °C. Inset: Linear sweep voltammograms of Cells/Fn-MWNTs/GCE obtained with SMMC 7721 cell (concentration 5.0 × 105 cell mL-1) in 0.1 M pH 7.4 PBS at different temperature values: (a) 44 and (b) 46 °C. Scan rate: 50 mV s-1. (B) Relationship (n ) 6) between current and temperature of heated electrodes.
the acid oxidized MWNT, because it is difficult to generate a stable Fn film directly onto the GCE surface via adsorbing. The presence of MWNTs not only acted as a good medium for Fn immobilization but also excessively increased the surface area and provided abundant RGD domains for cell capture. According to the Randles-Sevick formula Ip ) 2.69 × 105n3/2ADo1/2V1/2Co and peak current determined by cyclic voltammograms of 1 mM K4Fe(CN)6 in 0.1 M KCl at bare GCE and MWNTs/GCE, the effective area of MWNTs/GCE was 29.60 mm2, which was 4.2 times more than that of bare GCE. Many important processes in living cells have electrochemical characteristics. Redox reactions and changes in ionic composition derived from various cellular processes lead to electron generation and electron transfer at the interface of living cells.32-34 Linear sweep voltammograms of Cells/Fn-MWNTs/ GCE were performed in 0.1 M PBS (pH 7.4) with the cell concentration of 5.0 × 105 cell mL-1 (Figure 6A). An irreversible oxidation peak, which is attributed to the conversion of guanine in the cell cytoplasm to 8-oxo-guanine,35 appeared at +0.74 V for Cells/Fn-MWNTs/GCE. During the electrochemical process, the guanine molecules within the cytoplasm of the living cells were able to get across the cell membrane rapidly and to reach the electrode surface.35 MWNTs immobilized on
the electrode surface acted as a “molecular wire” to promote the transfer of electrons between guanine and the electrode, thus enhancing the electrochemical response of the cells. The oxidation peak did not show any corresponding reduction signal in the inverse scan, and they disappeared in the second scan (data not shown), which were characteristic of an irreversible electrode process. Therefore, Fn-MWNTs films have obvious advantages for both immobilizing and detecting SMMC 7721 cells by combining the good cell-adhesion ability of Fn and the excellent conductivity of MWNTs, which makes them suitable for biosensor applications for the detection of cells. 3.4. Evaluation of the Influence of Temperature in Cell Detection. Because the temperature plays an important role in cell detection, we constructed a unique indirect heating approach by copper wire to heat GCE for cell detection. The influence of temperature in cell detection was tested, as shown in Figure 6. With an increasing detecting temperature of electrode surface, the permeability of cellular membrane could be changed, leading to an easier effusion of the electroactive substances inside the cell, which enhanced the electron transfer rate between the electroactive substances and the electrode surface. Besides, a higher temperature strongly promoted the convection rate of the solution, and further increased the diffusion coefficient of
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Figure 7. Fluorescent images (856.8 µm × 644.8 µm) of SMMC 7721 cells cultivated on Fn-MWNTs modified glassy carbon slides at (A) 37, (B) 42, and (C) 46 °C for 2 h. Staining with AO and EB: live cells (green), dead cells (red).
4. Conclusion
Figure 8. Linear relationship (n ) 6) between current and logarithm of SMMC 7721 cell concentration. Temperature of electrode: (a) 37 and (b) 42 °C. Inset: Linear sweep voltammograms of Cells/FnMWNTs/GCE obtained with SMMC 7721 cell (concentration 5.0 × 103 cell mL-1) in 0.1 M pH 7.4 PBS at 42 °C.
the solution nearby the electrode.12 Hence, the heated electrode could improve the detection sensitivity. However, when the temperature was above 46 °C, the obtained electrode showed a decreasing peak current. The images of SMMC 7721 cells at different temperature values on the Fn-MWNTs film were recorded by fluorescent staining (Figure 7). As has been shown, cells cultivated at 37 and 42 °C both showed good viability, while at 46 °C, there were fewer cells on the electrode due to the cell killing ability at high temperature,36 and some cells lost their viability or even died presenting the red fluorescent. Thus, 42 °C was chosen for the following cell detection. 3.5. Cell Detection. With an increasing concentration of SMMC 7721 cells for their immobilization, the obtained Cells/ Fn-MWNTs/GCE showed an increasing peak current, indicating that a higher amount of cells were immobilized to the surface of the Fn-MWNTs/GCE. The peak current was proportional to the logarithmic value of the cell concentration ranging from 1.0 × 104 to 1.0 × 107 cell mL-1 with a correlation of 0.9997 at 37 °C, while it was proportional to the logarithmic value of the cell concentration ranging from 5.0 × 103 to 1.0 × 107 cell mL-1 with a correlation of 0.99588 at 42 °C as the optimized temperature (Figure 8). The detection limit at 42° was 5.0 × 103 cell mL-1 (inset, Figure 8), which was lower than those of 1.0 × 104 cell mL-1 at 37 °C, indicating that the detection sensitivity was obviously improved. Besides, the detection limit at 42 °C was lower than those of 7.1 × 103 cells mL-1 at an impedance sensor for K562A37 and 1.0 × 104 cells mL-1 at a piezoelectric immunosensor for Salmonella,38 suggesting a very efficient cell-capture ability of the Fn-MWNTs nanocomposites and a sensitive detecting ability of the heated GCE. Therefore, the Fn-MWNTs nanocomposite shows a good performance for the immobilization and detection of tumor cells and we can profit from the homemade heated glassy carbon electrode in the fabrication of cytosensor with the advantage of a simple fabrication process, broad detection range, and improved detection limit.
We constructed a unique indirect heating approach by copper wire to heat GCE to study the effect of temperature for cell detection. A novel nanocomposite consisting of Fn and MWNTs has been designed that showed excellent biocompatibility for living cells. The specific binding between the integrin receptor on the cell surface and the RGD peptide belonging to Fn improved attachment ability of the cells. Different temperatures had been tested, and the results indicated that the Fn-MWNTs/ GCE provides lower detection limit at 42 °C than the unheated one. This biosensor based on heat electrode modified with FnMWNTs presents a promising platform for sensitive cell detection. Acknowledgment. This work is supported by the National Natural Science Foundation (No. 20890021), the National Natural Science Funds for Creative Research Groups (20821063), and the 973 Program (2007CB936404) of China. We thank Professor Jian-Jun Sun from Fuzhou University for his help in fabrication of heated electrodes. References and Notes (1) Andreescu, S.; Sadik, O. A.; McGee, D. W. Anal. Chem. 2004, 76, 2321–2330. (2) Feng, W. J.; Rotenberg, S. A.; Mirkin, M. V. Anal. Chem. 2003, 75, 4148–4154. (3) Du, D.; Cai, J.; Ju, H. X.; Yan, F.; Chen, J.; Jiang, X. Q.; Chen, H. Y. Langmuir 2005, 21, 8394–8399. (4) Neufeld, T.; Biran, D.; Popovtzer, R.; Erez, T.; Ron, E. Z.; Rishpon, J. Anal. Chem. 2006, 78, 4952–4956. (5) Lee, S. J.; Choi, J. S.; Park, K. S.; Khang, G.; Lee, Y. M.; Lee, H. B. Biomaterials 2004, 25, 4699–4707. (6) Claase, M. B.; Riekerink, M. B. O.; de Bruijn, J. D.; Grijpma, D. W.; Engbers, G. H. M.; Feijen, J. Biomacromolecules 2003, 4, 57–63. (7) Mulchandani, A.; Mulchandani, P.; Janeva, I.; Chen, W. Anal. Chem. 1998, 70, 4140–4145. (8) Gonchar, M. V.; Maidan, M. M.; Moroz, O. M.; Woodward, J. R.; Sibirny, A. A. Biosens. Bioelectron. 1998, 13, 945–952. (9) NAssens, M.; Tran-Minh, C. Sens. Actuators, B. 1999, 59, 100– 102. (10) Erti, P.; Mikkelsen, S. R. Anal. Chem. 2001, 73, 4241–4248. (11) Skla´dal, P.; Morozova, N. O.; Reshetilov, A. N. Biosens. Bioelectron. 2002, 17, 867–873. (12) Gru¨ndler, P.; Flechsig, G.-U. Microchim Acta 2006, 154, 175–189. (13) Lau, C.; Borgmann, S.; Maciejewska, M.; Ngounou, B.; Gru¨ndler, P.; Schuhmann, W. Biosens. Bioelectron. 2007, 22, 3014–3020. (14) Boika, A.; Baranski, A. S. Anal. Chem. 2008, 80, 7392–7400. (15) Baranski, A. S. Anal. Chem. 2002, 74, 1294–1301. (16) Yang, H.; Choi, C. A.; Chung, K. H.; Jun, C.-H.; Kim, Y. T. Anal. Chem. 2004, 76, 1537–1543. (17) Wildgoose, G. G.; Giovanelli, D.; Lawrence, N. S.; Compton, R. G. Electroanalysis 2004, 16, 421–433. (18) Lee, D.-S.; Choi, H. G.; Chung, K. H.; Lee, B. Y.; Yoon, H. C. Sens. Actuators, B 2008, 130, 150–157. (19) Lin, Z. Y.; Sun, J. J.; Chen, J. H.; Guo, L.; Chen, Y. T.; Chen, G. N. Anal. Chem. 2008, 80, 2826–2831. (20) Lin, Z. Y.; Sun, J. J.; Chen, J. H.; Guo, L.; Chen, G. N. Anal. Chim. Acta 2006, 504, 226–230. (21) Beckmann, A.; Coles, B. A.; Compton, R. G.; Gru¨ndler, P.; Marken, F.; Neudeck, A. J. Phys. Chem. B 2000, 104, 764–769.
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