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Sensors and Actuators B 134 (2008) 273–280
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Quartz crystal microbalance and electrochemical cytosensing on a chitosan/multiwalled carbon nanotubes/Au electrode Xueen Jia, Liang Tan, Qingji Xie ∗ , Youyu Zhang, Shouzhuo Yao ∗ Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China
a r t i c l e
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Article history: Received 13 February 2008 Received in revised form 23 April 2008 Accepted 24 April 2008 Available online 2 May 2008 Keywords: Piezoelectric quartz crystal microbalance Chitosan/multiwalled carbon nanotubes composite Cyclic voltammetry Electrochemical impedance spectroscopy Human breast cancer cells (MCF-7)
a b s t r a c t The piezoelectric quartz crystal microbalance (QCM) was used to monitor the adhesion of mammalian cells on a chitosan (CS)/multiwalled carbon nanotubes (MWCNTs) composite modified gold electrode. The morphology and chemical properties of the CS/MWCNTs film were characterized with scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy. The human breast cancer cells (MCF-7) were adhered to and grown on the CS/MWCNTs film modified gold surface or a net CS film modified gold surface, and the process of which was continuously monitored and displayed by changes of the resonant frequency (f0 ) and the motional resistance (R1 ) of the QCM. The attachment/spreading process of the MCF-7 cells on the QCM Au electrode decreased the f0 and increased the R1 simultaneously, implying rather complicated effects (simultaneous mass, viscoelasticity and probable surface-stress load) on the sensor surface. The attachment rate and viability of the cells when proliferating on the two surfaces were detected by the MTT assay. The presence and state of cells on the electrode surface were confirmed by the fluorescent microscopy. Cyclic voltammetry and electrochemical impedance spectroscopy of the ferricyanide/ferrocyanide couple were examined before and after the cell adhesion. All data showed that the cell adhesion and proliferation processes were more efficient on the biocompatiable nanocomposite surfaces. The cell-based biosensor has potential for identification and screening of biologically active drugs and other biomolecules affecting cellular shape and attachment. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Cell adhesion is a complex progress contributing to the maintenance of tissue structure, the promotion of cell migration, and the transduction of information about the cell microenvironment across the plasma membrane [1]. Moreover, the cell adhesion on a biomaterial surfaces is an important aspect to evaluate its biocompatible character. In particular, a quantitative characterization of cell adhesion and its kinetics provides valuable information for the development of biomaterials. Therefore, a lot of efforts have been made to elucidate dynamic mechanisms of cell adhesion [2]. Current techniques for evaluating cell spreading and cell adhesion strength are labor intensive and destructive (e.g. flow detachment assay). Especially, they are not sufficient in providing the detailed kinetics of the adhesion process. Initial attachment and spreading patterns have been studied, and various techniques have been introduced to quantify the cell adhesion strength [3–7]. Morphology and topographical distribution of focal adhesions have been
∗ Corresponding authors. Tel.: +86 731 8865515; fax: +86 731 8865515. E-mail address: [email protected] (Q. Xie). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.04.043
investigated in real-time manner with confocal microscopy and immuno-staining techniques [8,9]. The piezoelectric quartz crystal microbalance (QCM) is a multiparameter sensor that mainly detects the film mass/viscoelasticity on and the solution viscodensity near an electrode [10], which has been widely used in various label-free bioanalyses, such as detecting enzymes [11], DNA and bacteria [12,13]. Recently, the QCM technique has been used to detect cells adhering to the sensor surface, and the cell adhesion processes were monitored by the resonance frequency shifts of the QCM [14,15]. The Willner group developed piezoelectric immunosensors for rapid and sensitive analyses of urine specimens of Chlamydia trachomatis cells [16]. The Wegener group showed that different cell types have their own characteristic frequency changes when they formed confluent monolayer, and tried to explain the frequency shifts with geometrical properties of cells [17]. Fohlerova et al. studied the adhesion of eukaryotic cell lines on the gold surface modified with extracellular matrix proteins monitored by the piezoelectric sensor [18]. Li et al. assessed the integrin-mediated cellular interactions with extracellular matrix (ECM) proteins by evaluating the bandwidth shift [19]. The Marx group had conducted a series of studies about cell adhesion with QCM, showing that morphological change of cells
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due to the depolymerization of cytoskeleton caused frequency and resistance shifts [20]. Sonnjin investigated the cell adhesion process and the molecular interactions that determine its kinetics using a thickness shear mode (TSM) sensor [21]. Chitosan (CS), a copolymer of 2-amino-2-deoxy--dglucopyranose and 2-acetamido-2-deoxy--d-glucopyranose, is the deacetylated derivative of chitin, the second most abundant natural polymer on earth [22]. Based on its biocompatibility, biodegradability, multiple functional groups, as well as its pH-dependent solubility in aqueous media, chitosan has been extensively investigated for molecular separation, food packaging film, artificial skin, bone substitutes, water treatment, and so on [23–25]. However, its mechanical properties are not good enough to meet this wide range of applications. Incorporation with inorganic fillers is an effective approach for improving the physico/chemical properties of chitosan and other biopolymers. Hydroxyapatite [26], calcium phosphate cements [27] and clay [28] are inorganic fillers that are frequently used to reinforce the chitosan matrix. Carbon nanotubes (CNTs) as the quintessential nanomaterial have already compiled an impressive list of superlatives since their discovery in 1991 [29]. Because of their nanometer-scale size, high surface area, and more importantly, their extraordinary mechanical strength and high electrical and thermal conductivity, CNTs have been widely used in electrochemical investigations, for instance, in direct electrochemistry of proteins [30], construction of electrochemical sensors and biosensors [31], electrocatalysis [32,33], and electrode materials in batteries [34]. Moreover, CNTs have been considered as ideal reinforcing fillers for polymer matrixes to achieve high performance and multifunctions especially in bioengineering field [35,36]. Electrochemical impedance spectroscopy (EIS) is a powerful electrochemical method for investigating electrode processes and determining surface-adsorption kinetics as well as mass-transport parameters, through adopting smaller electrochemical perturbations than some transient electrochemical techniques [37]. The electrochemical complex impedance (Z) can be represented as a sum of the √ real (Zre ) and imaginary (Zim ) components (Z = Zre + jZim where j = −1) that originate generally from the resistance and capacitance of an electrolytic cell, respectively. While EIS analyses with appropriate equivalent circuits allow one to obtain information of a film modified on an electrode surface, the modification of a chemical or biological substance on an electrode could also be monitored by measuring the electrochemical impedance (EI) at a fixed measurement frequency. Although lots of sensors for DNA, enzyme and cells based on electrochemical [38] and QCM technique have been developed [39–41], however, to our knowledge, the process of mammalian cells adhering to CNTs filled biocomposite film has not been studied with the QCM. In this work, we study the process of breast cancer cell line adhering to the CS/MWCNTs nanocomposite film modified QCM electrode in real time. It is suggested that the CS/MWCNTs nanocomposite film is more compatible for cell culturing, as also confirmed by MTT assay and optical microscopy. 2. Experimental 2.1. Cell culture and chemicals Chemicals for cell culture were obtained from Gibco (Grand Island, NY, USA). A human breast cancer cell line (MCF-7 cells) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 mg/ml), and 0.03% glutamate. The cultures were incubated at 37 ◦ C, in water sat-
Fig. 1. Schematic depiction of the QCM measurement setup (not to scale). The measurement chamber and the oscillator are placed inside the thermostated CO2 incubator.
urated with atmosphere containing 5% CO2 . When a cell monolayer was formed, the cells were harvested by trypsinization with 0.25% trypsin containing 0.02% EDTA. The viability of cells was determined by trypan blue staining and the cell number was counted using a hematocytometer. CS (from crab shells, >90% deacetylation) was obtained from Sinopharm Chemical Reagents Co., Ltd. They were used as received. Multiwalled CNTs (95%, 20–50 nm in diameter and 2–5 m length) purchased from Shenzhen Nanotech. Port. Co., Ltd. (Shenzhen, China) was purified by refluxing in concentrated nitric acid. All other reagents were of analytical or better grade. All solutions were prepared fresh prior to use, and double-distilled water was used throughout. 2.2. Apparatus A research QCM (RQCM, Maxtek Inc., USA) with simultaneous frequency (f0 ) and resistance (R1 ) recordings was used. A 9-MHz quartz wafer was fixed by two silica-gel O-rings to a Teflon holder, which was placed in a CO2 incubator with temperature control (37 ◦ C) and 5% (v/v) CO2 provision, as shown in Fig. 1. Electrochemical experiments were carried out with a CHI660A electrochemical workstation (CH Instruments, Shanghai Chenhua Instruments Inc., China) by using an one-compartment three-electrode electrolytic cell. The reference electrode was a saturated KCl calomel electrode (SCE). The counter electrode was a platinum electrode. 2.3. Preparation and characterization of the film The surface of the QCM gold electrode was treated by washing with 1.0 mol L−1 HNO3 for 10 s and then with double-distilled water, followed by drying via a stream of clean air. The treatment was repeated thrice. The treated electrode was then scanned between 0 and 1.5 V versus SCE in 0.20 mol L−1 HClO4 at 50 mV s−1 for sufficient cycles to obtain reproducible cyclic voltammograms. The electrode was then thoroughly washed with double-distilled water and dried with high-purity nitrogen stream. A 0.5% CS solution was prepared by dissolving CS in 1% acetic acid solution with magnetic stirring for about 2 h and then filtered by a 0.22 m filter. For preparation of a CS/MWCNTs electrode, an appropriate amount of MWCNTs was mixed first with 1 ml of 0.5% CS solution with ultrasonic agitation over 15 min. Then 10 L of the CS-MWCNTs mixture was spread evenly onto the QCM Au electrode surface with a syringe. Finally, the modified Au electrode was allowed to dry for 24 h at 37 ◦ C. Then the CS/MWCNTs membrane modified electrode was obtained.
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Scanning electron microscopy (SEM) images of CS and CS/MWCNTs film-covered electrodes were obtained with a Hitachi X-570 scanning electron microscope. The Fourier transform infrared (FT-IR) spectra (KBr pellets) were collected on a Nicolet Nexus 670 FT-IR spectrophotometer equipped with a DTGS KBr detector in the region 4000–400 cm−1 , and 25 scans with a 4 cm−1 resolution for each were averaged. The contact angles toward distilled water were measured with a contact angle meter (JJC-II, Changchun optical instrument) at room temperature by the sessile drop method. 2.4. EIS and cyclic voltammetry (CV) investigations of the modified surfaces The QCM electrodes before and after the film modification and in sequence cell incubation were characterized in 0.1 M aqueous KCl containing 1.0 mM K4 Fe(CN)6 and 1.0 mM K3 Fe(CN)6 via EIS and CV (all at 50 mV s−1 ). For EIS measurements, the working electrode potential was fixed at the formal potential of the ferricyanide/ferrocyanide couple. 2.5. Cell attachment rate and vitality study The MTT assay, reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrasodium bromide (MTT) to a purple formazan reaction product by living cells, was used to estimate cell attachment rates and viability/proliferation [42]. Both experiments were performed with tissue culture polystyrene (TCPS) plates modified with CS or CS/MWCNTs films, with a neat TCPS plate as control. For attachment rate test, control cultures and seeded specimens were incubated with 0.5 mg/ml MTT after the cell seeding on the prepared substrates for 3 h; the medium was then decanted, the water-insoluble
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formazan salts were dissolved with dimethylsulphoxide and the absorbance was measured at 570 nm. As for the attachment rate test, the growth vitality of cells cultured on the tow films were measured after cell seeding for 24, 48 and 72 h, respectively. 2.6. Fluorescence microscopy Adherent cells were stained with acridine orange for 24 h after seeding onto the QCM electrodes coated with the two different CS films. Pictures of stained cells were observed under an inverted fluorescence microscope (Leica DMI4000B, Germany). 3. Results and discussion 3.1. Characterization of the CS/MWCNTs composite The surface morphologies of CS and CS/MWCNTs films were studied by SEM, as shown in Fig. 2. The CS film surface (A and B) looks very smooth while a network-like structure of CNTs is observed on the (C and D). Obviously, on the CS/MWCNTs film surface there are many places looks like islands, due probably to the bunching of some CNTs. Such a surface was favorable for cells loading and adhesion. The FT-IR spectra of MWCNTs, CS/MWCNTs and CS are shown in Fig. 3. The CS (curve c) showed several peaks at 1152, 1068, and 1025 cm−1 characteristic of a saccharine structure and a strong acylamide characteristic peak at 1630 cm−1 , while the treated MWCNTs (curve a) showed the peaks of hydroxyl group at 3434 cm−1 , and of carboxyl group at 1621 cm−1 . These groups made the treated CNTs easy to be dispersed in water and CS. For the CS/MWCNTs film, the peaks of these groups could also be observed, but the positions for both hydroxyl and carboxylate groups blue shifted to 3425, 1563, and 1383 cm−1 , and the peaks at 1152 and
Fig. 2. SEM images of the CS film (A and B) and the CS/MWCNTs film (C and D) coated QCM electrodes.
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Fig. 3. FT-IR spectra of MWCNTs (a), CS/MWCNTs (b) and the CS (c).
1068 cm−1 for CS red shifted to 1141 and 1047 cm−1 , respectively, indicating their hydrogen-bond interactions. Contact angles of water on the films were measured (Fig. 4). The biocompatibility of a material for loading of biomolecules and preserving their bioactivity is positively related to its hydrophilicity, which could be characterized with the contact angle measurement of the substrate. Bare Au, CS and CS/MWCNTs modified Au electrodes gave the contact angles of 64, 52 and 47 ◦ C, respectively. The CS/MWCNTs gave a smaller contact angle than that of CS, demonstrating its excellent hydrophilicity. The hydrophilic CS/MWCNTs film provided a favorable microenvironment for living cells, and thus longer life of the adhered cells on the better biocompatible CS/MWCNTs film than CS film is expected, which is highly favorable for their adhesion, proliferation and retention of bioactivity.
Fig. 4. Contact angle toward the distilled water on the specimen surfaces (data presented are mean ± S.D., n = 3).
3.2. Real time QCM measurement of cell adhesion and growth on the electrode First the holder containing the prepared QCM electrodes were sterilized under UV light for almost 0.5 h, and then 800 L of the culture medium was added onto the electrode, which was then put into the incubator. After the QCM records became stable, 100 L of culture medium containing 50,000 cells was added evenly to the medium surface. The f0 and R1 responses are shown in Figs. 5A and 6A. There was a transient instrumental disturbance on the frequency when the cells were just injected into the incubation cell, which is reversible and thus should not spoil the following frequency response. After that, the f0 decreased and R1 increased quickly, then they changed moderately and finally reached their steady states. For the CS/MWCNTs modified electrode, in the whole
Fig. 5. The plot A shows real-time responses of frequency (curve a) and resistance (curve b) to the addition of 50,000 MCF-7 cells on the CS/MWCNTs film modified QCM electrode. The plot B shows the frequency–resistance plot. The plot C shows cyclic voltammograms for bare (a), CS/MWCNTs (b) and cell-CS/MWCNTs (c) Au electrodes. Scan rate: 50 mV s−1 . The plot D shows EIS for bare (a), CS/MWCNTs (b) and cell-CS/MWCNTs (c) Au electrodes. 100 kHz–10 mHz, 5 mVrms, 0.2 V vs. SCE. Solution: 0.1 M aqueous KCl containing 1.0 mM K4 Fe(CN)6 and 1.0 mM K3 Fe(CN)6 .
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Fig. 6. The plot A shows real-time responses of frequency (curve a) and resistance (curve b) to the addition of 50,000 MCF-7 cells on the CS film modified QCM electrode. The plot B shows the frequency–resistance plot. The plot C shows cyclic voltammograms for bare (a), CS (b) and cell-CS (c) Au electrodes. Scan rate: 50 mV s−1 . The plot D shows EIS for bare (a), CS (b) and cell-CS (c) Au electrodes. 100 kHz–10 mHz, 5 mV rms, 0.2 V vs. SCE. Solution: 0.1 M aqueous KCl containing 1.0 mM K4 Fe(CN)6 and 1.0 mM K3 Fe(CN)6 .
process f0 decreased by ∼−3405 Hz, correspondingly R1 increased by ∼150 , and for the CS modified electrode, f0 decreased by ∼−1510 Hz, correspondingly R1 increased by ∼130 . Obviously, based on the results shown above, cell adhesion and growth on the CS/MWCNTs surface induced a larger shift of f as well as R1 than those on the CS modified surface. Furthermore, both are larger than that on the bare Au electrode (about ∼−900, not shown). There may be several reasons leading to this findings, first, CS is one kind of best biocompatible material, cell can well adhere to it; second, as shown in Fig. 2, the CS/MWCNTs film has a rather rough surface, on one hand, this leads to a larger surface area of the electrode, on the other hand, there may be more anchoring sites between cell membrane and CS/MWCNTs film, so the cells can adhere to the CS/MWCNTs modified electrode more tightly. Also, we conducted the experiment with different cell numbers, the shift of f0 and R1 were enlarged as the number of detecting cell increased. When the whole electrode surface was covered by cell monolayer, the f0 and R1 will not change much even using increased amounts of cells. Figs. 5B and 6B show the f0 /R1 plots in the whole process of measurement. For the CS/MWCNTs modified QCM electrode (Fig. 5B), a linear regression of f0 vs. R1 responses gives f0 = −26.15R1 − 257.8 (r = 0.9838). The |f0 /R1 | ratio obtained here is larger than the characteristic value for a net viscous effect, i.e. 10 Hz −1 [10], indicating a notable mass effect. But for the CS modified surface, in the first stage, the |f0 /R1 | ratio obtained here is 5.8 Hz −1 , being smaller than the characteristic value for a net viscous effect, i.e. 10 Hz −1 , demonstrating that the CS film as a solid gel possessed specific mechanical properties, and to which the cells were attached, a complicated process that is not very clear at present occurred. The surface-stress effect induced by cells’ adhesion might be a responsible factor, since it has been proven that an increase in the surface stress on the QCM can simultaneously increase the f0 and the R1 [10]. In contrast, the CS/MWCNTs modified film showed an enhanced rigidity due to the reinforcement of implanted MWCNTs, the CS/MWCNTs nanocomposite is thus an appropriate and rather rigid material for cell culture.
3.3. Electrochemical measurement of modified surfaces In order to further examine the cell adhesion to the QCM electrode, we performed CV and EIS tests in 0.1 M aqueous KCl containing 1.0 mM K4 Fe(CN)6 and 1.0 mM K3 Fe(CN)6 before and after the film modification and in sequence cell incubation, as shown in Figs. 5C and D and 6C and D. As can be seen from Figs. 5C and 6C, for the CS/MWCNTs electrode, the peak currents (curve Fig. 5C-b) of the Fe(CN)6 3− /Fe(CN)6 4− redox couple increased after the CS/MWCNTs modification (versus those of the bare Au electrode, curve Fig. 5C-a). In contrast, the redox peak currents of the Fe(CN)6 3− /Fe(CN)6 4− couple (curve Fig. 6C-b) for the CS modification decreased (versus those of the bare one, curve Fig. 6C-a). It is reasonable to think that the insulating CS film retarded the electron transfer, but the CS/MWCNTs film enlarged the real electrode surface as well as enhanced the electron transfer rate because of the specific conducting character of MWCNTs. After the cell incubation, the peak currents (curves Fig. 5C-c and Fig. 6C-c) of the two electrodes notably decreased, but the peak-to-peak separation (Ep ) increased only slightly. The interesting results may be explained as follows. There were many cells on the electrode surface after the cell incubation, thus the electrochemistry of the redox couple might be largely inhibited at the cells-occupied locations on the electrode surface, but the redox activities of the couple might be almost invariable at the unoccupied locations, leading to the observations that the peak currents notably decreased but the peakto-peak separation did not change much. The EIS results at the two modified electrodes are shown in Figs. 5D and 6D. The diameter of the EIS semicircle did not change so much with the film modification, but evidently increased after the cell incubation, with increases in the corresponding interfacial resistance (Rct ) of 4.05 k for the CS/MWCNTs modified electrode (curve Fig. 5D-c) and of 3.20 k for the CS modified electrode (curve Fig. 6D-c). These findings should suggest the significant occupation of the cells on the electrode surface after the cell incubation, leading to larger Rct values for the redox probe due to the decreased active surface area, and the cells’ barrier effect against the electron com-
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Fig. 7. The plot A shows attachment rate of MCF-7 cells on different substrates after seeding on the two film modified substrates. The plot B shows viability of MCF-7 cells cultured on different matrices tested by MTT assay when the cells had been inoculated for 24, 48 and 72 h. The absorbance of formazan solution measured from TCPS after cell seeding as a control (data presented are mean S.D., n = 3).
munication between the electrochemical probe and the electrode surface. 3.4. Cell attachment rate and vitality study Fig. 7A shows cell attachment rate of MCF-7 cells cultured on the specimens and under control conditions (3 h after seeding), evaluated by the MTT assay. MTT is metabolized to an insoluble purple formazan salt by mitochondrial enzymes in living cells, and the absorbance is proportional to the number of viable cells. After cell
seeding for 3 h, the absorbance for the CS/MWCNTs substrate was a little lower than that at the neat TCPS plate (control), but obviously higher than that for the CS substrate (almost tripled). This is a reliable proof indicating that the CS/MWCNTs film is more compatible for cell adhesion. We also investigated the cell growth vitality on the two films. Fig. 7B shows the values for the MTT reduction of the MCF-7 cell cultures grown on the specimens and under control conditions (culture for 24, 48 and 72 h). Obviously, the pattern of the tendency was similar to that observed for the attachment rates test. The results show that the cell proliferation and growth
Fig. 8. Fluorescence microscopy images of stained MCF-7 cells cultured on the CS modified QCM electrode (A and B) and the CS/MWCNTs modified QCM electrode (C and D) for 24 h, recorded under ultraviolet irradiation.
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on the CS/MWCNTs substrate is more efficient than those on the CS substrate, though not better than the TCPS. Anyway, all the proofs indicate that the cells are suitable for culturing on the CS/MWCNTs nanocomposite specimens, because they adhered strongly to the surface and grew well. 3.5. Observation of MCF-7 cells on the two films by fluorescence microscopy Pictures of the stained cells cultured for 24 h on the different surfaces are presented in Fig. 8. These images show the differences in the number of adhered cells when cultured on two films, the CS modified QCM electrode (A and B), and the quartz coated with CS/MWCNTs nanocomposite (C and D). This is in agreement with the results obtained by the QCM and cell counting techniques that the cells prefer to reside on the CS/MWCNTs modified QCM electrode due to its biocompatibility higher than the CS surface. Moreover, this technique enabling the visualization of the cells adhered to the surfaces gives an overview of cell distribution on the different surfaces. The images with low magnification (A and C, ×100) show that cells cultured on the CS/MWCNTs film surface are numerous and looks like lots of green hills made by dozens of cells, and cells grown on the CS film surface are fewness and distribute dispersedly with a little wine color. The images with large magnification (B and D, ×200) show that the cells cultured on the CS/MWCNTs film surface are spread more completely than that on the CS film surface. In addition, acridine orange as vital staining allows confirmation that the cell adhesion inhibitory effect of CS film and CS/MWCNTs film is suitable for cell adhesion and proliferation. In conclusion, the observations by fluorescence microscopy technique confirm and validate the results obtained by the QCM technique. 4. Conclusions In summary, the QCM sensor was used to monitor the adhesion of breast cancer cell line (MCF-7) on the CS/MWCNTs composite and CS modified gold electrode in real time. The CS/MWCNTs composites have been successfully prepared by a simple solutionevaporation method. The process of cell attachment, spreading on the electrode surfaces was continuously monitored and displayed by changes of the resonant frequency and the motional resistance of the QCM. The cell attachment and spreading on the CS/MWCNTs surface induced a larger decrease of frequency and a larger increase of resistance than those on the CS surface. MTT assay and fluorescent microscopy confirmed that the CS/MWCNTs composite is suitable for cells culturing, it can be a reliable biomaterial for biosensing application and the development of biomedical devices. The proposed method presents a reliable way to investigate cell interaction with nanocomposite modified substrate in real time. The piezoelectric sensor is suitable for studies of the cell adhesion and proliferation processes on nanocomposite substrate. Also, it is interesting to further investigate electrochemical behavior of cells and other biomolecules. The cell-based biosensor has potential for identification and screening of biologically active drugs and other biomolecules affecting cellular shape and attachment. Acknowledgments This work was supported by the National Natural Science Foundation of China (20675029, 90713018, and 20335020), the Basic Research Special Program of the Ministry of Science and Technology of China (2003CCC00700), the Foundations of the Ministry of Education (MOE) of China and Hunan Provincial Education Department, and the State Key Laboratory of Electroanalytical Chemistry.
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Biographies Xueen Jia is pursuing his Ph.D. in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. His research interests include biocompatible interface constructing, biosensor fabricating, QCM and Electrochemistry study cells and biomolecules. Liang Tan is a teacher in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. He received his ME degree from College of Chemistry and Chemical Engineering, Hunan normal University in 2004. He research interests cover Electrochemistry analysis, biosensing and cell study. Qingji Xie is a professor in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. He received his Ph.D. degree in 1993 from Hunan University, China. His current research is focused on chemo/biosensing and electroanalytical chemistry, including the EQCM, SECM and spectroelectrochemistry characterizations of sensing events. Youyu Zhang is a professor of Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. Her research is focused on chemical and biological sensing. Her current research includes piezoelectric quartz crystal sensor and electrochemical sensor. Shouzhuo Yao is a professor in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. He has been appointed as an academician of Chinese Academy of Science in analytical chemistry.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Quartz crystal microbalance and electrochemical cytosensing on a chitosan/multiwalled carbon nanotubes/Au electrode Xueen Jia, Liang Tan, Qingji Xie ∗ , Youyu Zhang, Shouzhuo Yao ∗ Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China
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Article history: Received 13 February 2008 Received in revised form 23 April 2008 Accepted 24 April 2008 Available online 2 May 2008 Keywords: Piezoelectric quartz crystal microbalance Chitosan/multiwalled carbon nanotubes composite Cyclic voltammetry Electrochemical impedance spectroscopy Human breast cancer cells (MCF-7)
a b s t r a c t The piezoelectric quartz crystal microbalance (QCM) was used to monitor the adhesion of mammalian cells on a chitosan (CS)/multiwalled carbon nanotubes (MWCNTs) composite modified gold electrode. The morphology and chemical properties of the CS/MWCNTs film were characterized with scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy. The human breast cancer cells (MCF-7) were adhered to and grown on the CS/MWCNTs film modified gold surface or a net CS film modified gold surface, and the process of which was continuously monitored and displayed by changes of the resonant frequency (f0 ) and the motional resistance (R1 ) of the QCM. The attachment/spreading process of the MCF-7 cells on the QCM Au electrode decreased the f0 and increased the R1 simultaneously, implying rather complicated effects (simultaneous mass, viscoelasticity and probable surface-stress load) on the sensor surface. The attachment rate and viability of the cells when proliferating on the two surfaces were detected by the MTT assay. The presence and state of cells on the electrode surface were confirmed by the fluorescent microscopy. Cyclic voltammetry and electrochemical impedance spectroscopy of the ferricyanide/ferrocyanide couple were examined before and after the cell adhesion. All data showed that the cell adhesion and proliferation processes were more efficient on the biocompatiable nanocomposite surfaces. The cell-based biosensor has potential for identification and screening of biologically active drugs and other biomolecules affecting cellular shape and attachment. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Cell adhesion is a complex progress contributing to the maintenance of tissue structure, the promotion of cell migration, and the transduction of information about the cell microenvironment across the plasma membrane [1]. Moreover, the cell adhesion on a biomaterial surfaces is an important aspect to evaluate its biocompatible character. In particular, a quantitative characterization of cell adhesion and its kinetics provides valuable information for the development of biomaterials. Therefore, a lot of efforts have been made to elucidate dynamic mechanisms of cell adhesion [2]. Current techniques for evaluating cell spreading and cell adhesion strength are labor intensive and destructive (e.g. flow detachment assay). Especially, they are not sufficient in providing the detailed kinetics of the adhesion process. Initial attachment and spreading patterns have been studied, and various techniques have been introduced to quantify the cell adhesion strength [3–7]. Morphology and topographical distribution of focal adhesions have been
∗ Corresponding authors. Tel.: +86 731 8865515; fax: +86 731 8865515. E-mail address: [email protected] (Q. Xie). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.04.043
investigated in real-time manner with confocal microscopy and immuno-staining techniques [8,9]. The piezoelectric quartz crystal microbalance (QCM) is a multiparameter sensor that mainly detects the film mass/viscoelasticity on and the solution viscodensity near an electrode [10], which has been widely used in various label-free bioanalyses, such as detecting enzymes [11], DNA and bacteria [12,13]. Recently, the QCM technique has been used to detect cells adhering to the sensor surface, and the cell adhesion processes were monitored by the resonance frequency shifts of the QCM [14,15]. The Willner group developed piezoelectric immunosensors for rapid and sensitive analyses of urine specimens of Chlamydia trachomatis cells [16]. The Wegener group showed that different cell types have their own characteristic frequency changes when they formed confluent monolayer, and tried to explain the frequency shifts with geometrical properties of cells [17]. Fohlerova et al. studied the adhesion of eukaryotic cell lines on the gold surface modified with extracellular matrix proteins monitored by the piezoelectric sensor [18]. Li et al. assessed the integrin-mediated cellular interactions with extracellular matrix (ECM) proteins by evaluating the bandwidth shift [19]. The Marx group had conducted a series of studies about cell adhesion with QCM, showing that morphological change of cells
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due to the depolymerization of cytoskeleton caused frequency and resistance shifts [20]. Sonnjin investigated the cell adhesion process and the molecular interactions that determine its kinetics using a thickness shear mode (TSM) sensor [21]. Chitosan (CS), a copolymer of 2-amino-2-deoxy--dglucopyranose and 2-acetamido-2-deoxy--d-glucopyranose, is the deacetylated derivative of chitin, the second most abundant natural polymer on earth [22]. Based on its biocompatibility, biodegradability, multiple functional groups, as well as its pH-dependent solubility in aqueous media, chitosan has been extensively investigated for molecular separation, food packaging film, artificial skin, bone substitutes, water treatment, and so on [23–25]. However, its mechanical properties are not good enough to meet this wide range of applications. Incorporation with inorganic fillers is an effective approach for improving the physico/chemical properties of chitosan and other biopolymers. Hydroxyapatite [26], calcium phosphate cements [27] and clay [28] are inorganic fillers that are frequently used to reinforce the chitosan matrix. Carbon nanotubes (CNTs) as the quintessential nanomaterial have already compiled an impressive list of superlatives since their discovery in 1991 [29]. Because of their nanometer-scale size, high surface area, and more importantly, their extraordinary mechanical strength and high electrical and thermal conductivity, CNTs have been widely used in electrochemical investigations, for instance, in direct electrochemistry of proteins [30], construction of electrochemical sensors and biosensors [31], electrocatalysis [32,33], and electrode materials in batteries [34]. Moreover, CNTs have been considered as ideal reinforcing fillers for polymer matrixes to achieve high performance and multifunctions especially in bioengineering field [35,36]. Electrochemical impedance spectroscopy (EIS) is a powerful electrochemical method for investigating electrode processes and determining surface-adsorption kinetics as well as mass-transport parameters, through adopting smaller electrochemical perturbations than some transient electrochemical techniques [37]. The electrochemical complex impedance (Z) can be represented as a sum of the √ real (Zre ) and imaginary (Zim ) components (Z = Zre + jZim where j = −1) that originate generally from the resistance and capacitance of an electrolytic cell, respectively. While EIS analyses with appropriate equivalent circuits allow one to obtain information of a film modified on an electrode surface, the modification of a chemical or biological substance on an electrode could also be monitored by measuring the electrochemical impedance (EI) at a fixed measurement frequency. Although lots of sensors for DNA, enzyme and cells based on electrochemical [38] and QCM technique have been developed [39–41], however, to our knowledge, the process of mammalian cells adhering to CNTs filled biocomposite film has not been studied with the QCM. In this work, we study the process of breast cancer cell line adhering to the CS/MWCNTs nanocomposite film modified QCM electrode in real time. It is suggested that the CS/MWCNTs nanocomposite film is more compatible for cell culturing, as also confirmed by MTT assay and optical microscopy. 2. Experimental 2.1. Cell culture and chemicals Chemicals for cell culture were obtained from Gibco (Grand Island, NY, USA). A human breast cancer cell line (MCF-7 cells) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 mg/ml), and 0.03% glutamate. The cultures were incubated at 37 ◦ C, in water sat-
Fig. 1. Schematic depiction of the QCM measurement setup (not to scale). The measurement chamber and the oscillator are placed inside the thermostated CO2 incubator.
urated with atmosphere containing 5% CO2 . When a cell monolayer was formed, the cells were harvested by trypsinization with 0.25% trypsin containing 0.02% EDTA. The viability of cells was determined by trypan blue staining and the cell number was counted using a hematocytometer. CS (from crab shells, >90% deacetylation) was obtained from Sinopharm Chemical Reagents Co., Ltd. They were used as received. Multiwalled CNTs (95%, 20–50 nm in diameter and 2–5 m length) purchased from Shenzhen Nanotech. Port. Co., Ltd. (Shenzhen, China) was purified by refluxing in concentrated nitric acid. All other reagents were of analytical or better grade. All solutions were prepared fresh prior to use, and double-distilled water was used throughout. 2.2. Apparatus A research QCM (RQCM, Maxtek Inc., USA) with simultaneous frequency (f0 ) and resistance (R1 ) recordings was used. A 9-MHz quartz wafer was fixed by two silica-gel O-rings to a Teflon holder, which was placed in a CO2 incubator with temperature control (37 ◦ C) and 5% (v/v) CO2 provision, as shown in Fig. 1. Electrochemical experiments were carried out with a CHI660A electrochemical workstation (CH Instruments, Shanghai Chenhua Instruments Inc., China) by using an one-compartment three-electrode electrolytic cell. The reference electrode was a saturated KCl calomel electrode (SCE). The counter electrode was a platinum electrode. 2.3. Preparation and characterization of the film The surface of the QCM gold electrode was treated by washing with 1.0 mol L−1 HNO3 for 10 s and then with double-distilled water, followed by drying via a stream of clean air. The treatment was repeated thrice. The treated electrode was then scanned between 0 and 1.5 V versus SCE in 0.20 mol L−1 HClO4 at 50 mV s−1 for sufficient cycles to obtain reproducible cyclic voltammograms. The electrode was then thoroughly washed with double-distilled water and dried with high-purity nitrogen stream. A 0.5% CS solution was prepared by dissolving CS in 1% acetic acid solution with magnetic stirring for about 2 h and then filtered by a 0.22 m filter. For preparation of a CS/MWCNTs electrode, an appropriate amount of MWCNTs was mixed first with 1 ml of 0.5% CS solution with ultrasonic agitation over 15 min. Then 10 L of the CS-MWCNTs mixture was spread evenly onto the QCM Au electrode surface with a syringe. Finally, the modified Au electrode was allowed to dry for 24 h at 37 ◦ C. Then the CS/MWCNTs membrane modified electrode was obtained.
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Scanning electron microscopy (SEM) images of CS and CS/MWCNTs film-covered electrodes were obtained with a Hitachi X-570 scanning electron microscope. The Fourier transform infrared (FT-IR) spectra (KBr pellets) were collected on a Nicolet Nexus 670 FT-IR spectrophotometer equipped with a DTGS KBr detector in the region 4000–400 cm−1 , and 25 scans with a 4 cm−1 resolution for each were averaged. The contact angles toward distilled water were measured with a contact angle meter (JJC-II, Changchun optical instrument) at room temperature by the sessile drop method. 2.4. EIS and cyclic voltammetry (CV) investigations of the modified surfaces The QCM electrodes before and after the film modification and in sequence cell incubation were characterized in 0.1 M aqueous KCl containing 1.0 mM K4 Fe(CN)6 and 1.0 mM K3 Fe(CN)6 via EIS and CV (all at 50 mV s−1 ). For EIS measurements, the working electrode potential was fixed at the formal potential of the ferricyanide/ferrocyanide couple. 2.5. Cell attachment rate and vitality study The MTT assay, reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrasodium bromide (MTT) to a purple formazan reaction product by living cells, was used to estimate cell attachment rates and viability/proliferation [42]. Both experiments were performed with tissue culture polystyrene (TCPS) plates modified with CS or CS/MWCNTs films, with a neat TCPS plate as control. For attachment rate test, control cultures and seeded specimens were incubated with 0.5 mg/ml MTT after the cell seeding on the prepared substrates for 3 h; the medium was then decanted, the water-insoluble
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formazan salts were dissolved with dimethylsulphoxide and the absorbance was measured at 570 nm. As for the attachment rate test, the growth vitality of cells cultured on the tow films were measured after cell seeding for 24, 48 and 72 h, respectively. 2.6. Fluorescence microscopy Adherent cells were stained with acridine orange for 24 h after seeding onto the QCM electrodes coated with the two different CS films. Pictures of stained cells were observed under an inverted fluorescence microscope (Leica DMI4000B, Germany). 3. Results and discussion 3.1. Characterization of the CS/MWCNTs composite The surface morphologies of CS and CS/MWCNTs films were studied by SEM, as shown in Fig. 2. The CS film surface (A and B) looks very smooth while a network-like structure of CNTs is observed on the (C and D). Obviously, on the CS/MWCNTs film surface there are many places looks like islands, due probably to the bunching of some CNTs. Such a surface was favorable for cells loading and adhesion. The FT-IR spectra of MWCNTs, CS/MWCNTs and CS are shown in Fig. 3. The CS (curve c) showed several peaks at 1152, 1068, and 1025 cm−1 characteristic of a saccharine structure and a strong acylamide characteristic peak at 1630 cm−1 , while the treated MWCNTs (curve a) showed the peaks of hydroxyl group at 3434 cm−1 , and of carboxyl group at 1621 cm−1 . These groups made the treated CNTs easy to be dispersed in water and CS. For the CS/MWCNTs film, the peaks of these groups could also be observed, but the positions for both hydroxyl and carboxylate groups blue shifted to 3425, 1563, and 1383 cm−1 , and the peaks at 1152 and
Fig. 2. SEM images of the CS film (A and B) and the CS/MWCNTs film (C and D) coated QCM electrodes.
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Fig. 3. FT-IR spectra of MWCNTs (a), CS/MWCNTs (b) and the CS (c).
1068 cm−1 for CS red shifted to 1141 and 1047 cm−1 , respectively, indicating their hydrogen-bond interactions. Contact angles of water on the films were measured (Fig. 4). The biocompatibility of a material for loading of biomolecules and preserving their bioactivity is positively related to its hydrophilicity, which could be characterized with the contact angle measurement of the substrate. Bare Au, CS and CS/MWCNTs modified Au electrodes gave the contact angles of 64, 52 and 47 ◦ C, respectively. The CS/MWCNTs gave a smaller contact angle than that of CS, demonstrating its excellent hydrophilicity. The hydrophilic CS/MWCNTs film provided a favorable microenvironment for living cells, and thus longer life of the adhered cells on the better biocompatible CS/MWCNTs film than CS film is expected, which is highly favorable for their adhesion, proliferation and retention of bioactivity.
Fig. 4. Contact angle toward the distilled water on the specimen surfaces (data presented are mean ± S.D., n = 3).
3.2. Real time QCM measurement of cell adhesion and growth on the electrode First the holder containing the prepared QCM electrodes were sterilized under UV light for almost 0.5 h, and then 800 L of the culture medium was added onto the electrode, which was then put into the incubator. After the QCM records became stable, 100 L of culture medium containing 50,000 cells was added evenly to the medium surface. The f0 and R1 responses are shown in Figs. 5A and 6A. There was a transient instrumental disturbance on the frequency when the cells were just injected into the incubation cell, which is reversible and thus should not spoil the following frequency response. After that, the f0 decreased and R1 increased quickly, then they changed moderately and finally reached their steady states. For the CS/MWCNTs modified electrode, in the whole
Fig. 5. The plot A shows real-time responses of frequency (curve a) and resistance (curve b) to the addition of 50,000 MCF-7 cells on the CS/MWCNTs film modified QCM electrode. The plot B shows the frequency–resistance plot. The plot C shows cyclic voltammograms for bare (a), CS/MWCNTs (b) and cell-CS/MWCNTs (c) Au electrodes. Scan rate: 50 mV s−1 . The plot D shows EIS for bare (a), CS/MWCNTs (b) and cell-CS/MWCNTs (c) Au electrodes. 100 kHz–10 mHz, 5 mVrms, 0.2 V vs. SCE. Solution: 0.1 M aqueous KCl containing 1.0 mM K4 Fe(CN)6 and 1.0 mM K3 Fe(CN)6 .
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Fig. 6. The plot A shows real-time responses of frequency (curve a) and resistance (curve b) to the addition of 50,000 MCF-7 cells on the CS film modified QCM electrode. The plot B shows the frequency–resistance plot. The plot C shows cyclic voltammograms for bare (a), CS (b) and cell-CS (c) Au electrodes. Scan rate: 50 mV s−1 . The plot D shows EIS for bare (a), CS (b) and cell-CS (c) Au electrodes. 100 kHz–10 mHz, 5 mV rms, 0.2 V vs. SCE. Solution: 0.1 M aqueous KCl containing 1.0 mM K4 Fe(CN)6 and 1.0 mM K3 Fe(CN)6 .
process f0 decreased by ∼−3405 Hz, correspondingly R1 increased by ∼150 , and for the CS modified electrode, f0 decreased by ∼−1510 Hz, correspondingly R1 increased by ∼130 . Obviously, based on the results shown above, cell adhesion and growth on the CS/MWCNTs surface induced a larger shift of f as well as R1 than those on the CS modified surface. Furthermore, both are larger than that on the bare Au electrode (about ∼−900, not shown). There may be several reasons leading to this findings, first, CS is one kind of best biocompatible material, cell can well adhere to it; second, as shown in Fig. 2, the CS/MWCNTs film has a rather rough surface, on one hand, this leads to a larger surface area of the electrode, on the other hand, there may be more anchoring sites between cell membrane and CS/MWCNTs film, so the cells can adhere to the CS/MWCNTs modified electrode more tightly. Also, we conducted the experiment with different cell numbers, the shift of f0 and R1 were enlarged as the number of detecting cell increased. When the whole electrode surface was covered by cell monolayer, the f0 and R1 will not change much even using increased amounts of cells. Figs. 5B and 6B show the f0 /R1 plots in the whole process of measurement. For the CS/MWCNTs modified QCM electrode (Fig. 5B), a linear regression of f0 vs. R1 responses gives f0 = −26.15R1 − 257.8 (r = 0.9838). The |f0 /R1 | ratio obtained here is larger than the characteristic value for a net viscous effect, i.e. 10 Hz −1 [10], indicating a notable mass effect. But for the CS modified surface, in the first stage, the |f0 /R1 | ratio obtained here is 5.8 Hz −1 , being smaller than the characteristic value for a net viscous effect, i.e. 10 Hz −1 , demonstrating that the CS film as a solid gel possessed specific mechanical properties, and to which the cells were attached, a complicated process that is not very clear at present occurred. The surface-stress effect induced by cells’ adhesion might be a responsible factor, since it has been proven that an increase in the surface stress on the QCM can simultaneously increase the f0 and the R1 [10]. In contrast, the CS/MWCNTs modified film showed an enhanced rigidity due to the reinforcement of implanted MWCNTs, the CS/MWCNTs nanocomposite is thus an appropriate and rather rigid material for cell culture.
3.3. Electrochemical measurement of modified surfaces In order to further examine the cell adhesion to the QCM electrode, we performed CV and EIS tests in 0.1 M aqueous KCl containing 1.0 mM K4 Fe(CN)6 and 1.0 mM K3 Fe(CN)6 before and after the film modification and in sequence cell incubation, as shown in Figs. 5C and D and 6C and D. As can be seen from Figs. 5C and 6C, for the CS/MWCNTs electrode, the peak currents (curve Fig. 5C-b) of the Fe(CN)6 3− /Fe(CN)6 4− redox couple increased after the CS/MWCNTs modification (versus those of the bare Au electrode, curve Fig. 5C-a). In contrast, the redox peak currents of the Fe(CN)6 3− /Fe(CN)6 4− couple (curve Fig. 6C-b) for the CS modification decreased (versus those of the bare one, curve Fig. 6C-a). It is reasonable to think that the insulating CS film retarded the electron transfer, but the CS/MWCNTs film enlarged the real electrode surface as well as enhanced the electron transfer rate because of the specific conducting character of MWCNTs. After the cell incubation, the peak currents (curves Fig. 5C-c and Fig. 6C-c) of the two electrodes notably decreased, but the peak-to-peak separation (Ep ) increased only slightly. The interesting results may be explained as follows. There were many cells on the electrode surface after the cell incubation, thus the electrochemistry of the redox couple might be largely inhibited at the cells-occupied locations on the electrode surface, but the redox activities of the couple might be almost invariable at the unoccupied locations, leading to the observations that the peak currents notably decreased but the peakto-peak separation did not change much. The EIS results at the two modified electrodes are shown in Figs. 5D and 6D. The diameter of the EIS semicircle did not change so much with the film modification, but evidently increased after the cell incubation, with increases in the corresponding interfacial resistance (Rct ) of 4.05 k for the CS/MWCNTs modified electrode (curve Fig. 5D-c) and of 3.20 k for the CS modified electrode (curve Fig. 6D-c). These findings should suggest the significant occupation of the cells on the electrode surface after the cell incubation, leading to larger Rct values for the redox probe due to the decreased active surface area, and the cells’ barrier effect against the electron com-
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Fig. 7. The plot A shows attachment rate of MCF-7 cells on different substrates after seeding on the two film modified substrates. The plot B shows viability of MCF-7 cells cultured on different matrices tested by MTT assay when the cells had been inoculated for 24, 48 and 72 h. The absorbance of formazan solution measured from TCPS after cell seeding as a control (data presented are mean S.D., n = 3).
munication between the electrochemical probe and the electrode surface. 3.4. Cell attachment rate and vitality study Fig. 7A shows cell attachment rate of MCF-7 cells cultured on the specimens and under control conditions (3 h after seeding), evaluated by the MTT assay. MTT is metabolized to an insoluble purple formazan salt by mitochondrial enzymes in living cells, and the absorbance is proportional to the number of viable cells. After cell
seeding for 3 h, the absorbance for the CS/MWCNTs substrate was a little lower than that at the neat TCPS plate (control), but obviously higher than that for the CS substrate (almost tripled). This is a reliable proof indicating that the CS/MWCNTs film is more compatible for cell adhesion. We also investigated the cell growth vitality on the two films. Fig. 7B shows the values for the MTT reduction of the MCF-7 cell cultures grown on the specimens and under control conditions (culture for 24, 48 and 72 h). Obviously, the pattern of the tendency was similar to that observed for the attachment rates test. The results show that the cell proliferation and growth
Fig. 8. Fluorescence microscopy images of stained MCF-7 cells cultured on the CS modified QCM electrode (A and B) and the CS/MWCNTs modified QCM electrode (C and D) for 24 h, recorded under ultraviolet irradiation.
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on the CS/MWCNTs substrate is more efficient than those on the CS substrate, though not better than the TCPS. Anyway, all the proofs indicate that the cells are suitable for culturing on the CS/MWCNTs nanocomposite specimens, because they adhered strongly to the surface and grew well. 3.5. Observation of MCF-7 cells on the two films by fluorescence microscopy Pictures of the stained cells cultured for 24 h on the different surfaces are presented in Fig. 8. These images show the differences in the number of adhered cells when cultured on two films, the CS modified QCM electrode (A and B), and the quartz coated with CS/MWCNTs nanocomposite (C and D). This is in agreement with the results obtained by the QCM and cell counting techniques that the cells prefer to reside on the CS/MWCNTs modified QCM electrode due to its biocompatibility higher than the CS surface. Moreover, this technique enabling the visualization of the cells adhered to the surfaces gives an overview of cell distribution on the different surfaces. The images with low magnification (A and C, ×100) show that cells cultured on the CS/MWCNTs film surface are numerous and looks like lots of green hills made by dozens of cells, and cells grown on the CS film surface are fewness and distribute dispersedly with a little wine color. The images with large magnification (B and D, ×200) show that the cells cultured on the CS/MWCNTs film surface are spread more completely than that on the CS film surface. In addition, acridine orange as vital staining allows confirmation that the cell adhesion inhibitory effect of CS film and CS/MWCNTs film is suitable for cell adhesion and proliferation. In conclusion, the observations by fluorescence microscopy technique confirm and validate the results obtained by the QCM technique. 4. Conclusions In summary, the QCM sensor was used to monitor the adhesion of breast cancer cell line (MCF-7) on the CS/MWCNTs composite and CS modified gold electrode in real time. The CS/MWCNTs composites have been successfully prepared by a simple solutionevaporation method. The process of cell attachment, spreading on the electrode surfaces was continuously monitored and displayed by changes of the resonant frequency and the motional resistance of the QCM. The cell attachment and spreading on the CS/MWCNTs surface induced a larger decrease of frequency and a larger increase of resistance than those on the CS surface. MTT assay and fluorescent microscopy confirmed that the CS/MWCNTs composite is suitable for cells culturing, it can be a reliable biomaterial for biosensing application and the development of biomedical devices. The proposed method presents a reliable way to investigate cell interaction with nanocomposite modified substrate in real time. The piezoelectric sensor is suitable for studies of the cell adhesion and proliferation processes on nanocomposite substrate. Also, it is interesting to further investigate electrochemical behavior of cells and other biomolecules. The cell-based biosensor has potential for identification and screening of biologically active drugs and other biomolecules affecting cellular shape and attachment. Acknowledgments This work was supported by the National Natural Science Foundation of China (20675029, 90713018, and 20335020), the Basic Research Special Program of the Ministry of Science and Technology of China (2003CCC00700), the Foundations of the Ministry of Education (MOE) of China and Hunan Provincial Education Department, and the State Key Laboratory of Electroanalytical Chemistry.
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Biographies Xueen Jia is pursuing his Ph.D. in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. His research interests include biocompatible interface constructing, biosensor fabricating, QCM and Electrochemistry study cells and biomolecules. Liang Tan is a teacher in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. He received his ME degree from College of Chemistry and Chemical Engineering, Hunan normal University in 2004. He research interests cover Electrochemistry analysis, biosensing and cell study. Qingji Xie is a professor in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. He received his Ph.D. degree in 1993 from Hunan University, China. His current research is focused on chemo/biosensing and electroanalytical chemistry, including the EQCM, SECM and spectroelectrochemistry characterizations of sensing events. Youyu Zhang is a professor of Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. Her research is focused on chemical and biological sensing. Her current research includes piezoelectric quartz crystal sensor and electrochemical sensor. Shouzhuo Yao is a professor in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. He has been appointed as an academician of Chinese Academy of Science in analytical chemistry.