RGD peptide doped polypyrrole film as a biomimetic electrode coating for impedimetric sensing of cell proliferation and cytotoxicity

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Journal of Applied Biomedicine journal homepage: www.elsevier.com/locate/jab

Original Research Article

RGD peptide doped polypyrrole film as a biomimetic electrode coating for impedimetric sensing of cell proliferation and cytotoxicity Yuan Lia,b , Chao Yub,* a b

Yongchuan Hospital, Chongqing Medical University, Chongqing, China Institute of Life Science, Chongqing Medical University, Chongqing, China

A R T I C L E I N F O

Article history: Received 13 August 2016 Received in revised form 24 March 2017 Accepted 19 June 2017 Available online xxx Keywords: RGD peptide Polypyrrole Cell-based sensor Proliferation Cytotoxicity Electrochemical impedance microscopy

A B S T R A C T

A novel and facile in vitro cell sensing system has been developed with one-step electropolymerization of the conducting polypyrrole(PPy) polymer using RGD peptide as the sole dopant on an indium tin oxide (ITO) surface. The resulted RGD peptide-doped polypyrrole (PPy/RGD) composite film had a robust surface, in which PPy provided a biocompatible matrix for cell growth and a conducting interface for electrical detection, while the RGD peptide entrapped in the PPy matrix conferred the desired biomimetic properties. Using the human lung cancer cell A549 as a model, this system can be used to monitor cell behaviors of proliferation and cytotoxicity. © 2017 Faculty of Health and Social Sciences, University of South Bohemia in Ceske Budejovice. Published by Elsevier Sp. z o.o. All rights reserved.

Introduction In vitro assay-based cultured cells have been widely used as an alternative to animal experiments to investigate cell biology behaviors and to assess the cytotoxicity of potential drug molecules. Some traditional and well-established cell-based assays, such as MTT, BrdU and LDH release need bench-top instruments and rely heavily on optical signal collection. The main disadvantages of these methods are complexity, high reagent cost and indirection. Simple and nondestructive electrochemical methods have been exhaustively explored for studying living cells (Tschirhart et al., 2016). As electrochemical impedance spectroscopy (EIS) is a noninvasive and powerful tool for monitoring the changes of interfacial properties at the electrode surface, an electrical impedance-based sensing system was intensively researched to track the morphological change of immobilized cells and monitor cell response to drugs (Ribaut et al., 2009; Yoon et al., 2014; Zhang et al., 2013). To fulfill the full potential offered by EIS, the cell/electrode interface should not only have high conductivity, but also possess good cell immobilization characteristics (Ding et al., 2007; Guo et al., 2008). Despite extensive efforts concerning this area, the construction of an interface with specific biophysical-chemical properties that fulfills both requirements remains challenging.

* Corresponding author at: Institute of Life Science, Chongqing Medical University, Chongqing, 40016, China. E-mail address: [email protected] (C. Yu).

Polypyrrole(PPy), one of the most important conducting polymers, has been considered the most promising electrode coating material for different kinds of electrochemical biosensor, such as DNA (Radhakrishnan et al., 2013), protein (Xiao et al., 2007) and enzyme (Gao et al., 2014), etc., due to its low cost, easy synthesis, inherent conductivity and high electrochemical activity. PPy has also been widely investigated as a biomaterial since it not only provides a biocompatible scaffold for cellular physical support (Vaitkuviene et al., 2013), but through its inherent conductivity can load electrical stimuli to cells (Gilmore et al., 2009) or detect electrical signal from cells (Ateh et al., 2009; Ding et al., 2009; Li et al., 2015a, 2015b). Therefore, PPy possesses the potential as a cell-electrode coupling interface for various biomedical applications, such as impedancebased cell biosensors. For example, Ding et al. (2009) reported a simple EIS cell sensor for monitoring ECA-109 cell adhesion and proliferation based on an electropolymerized canbon nanofiberdoped Polypyrrole(PPy/CNT) nanoscaffold. The highly conductive PPy/CNT provided a biocompatible substrate for the growth of ECA109 cells, which increase the electron transfer resistance and thus lead to cell growth detection. Ateh et al. (2009) synthesized a dermatan-doped PPy film on gold electrode and studied various skin-derived cells on them using EIS. The results showed that lower cell densities could be detected on PPy compared to the bare electrode. Very recently, we electropolymerized a dextran sulfatedoped PPy (PPy/DS) film on an ITO microelectrode in order to detect the proliferation and epithelial-mesenchymal transition of A549 cells using EIS. The results showed that the PPy/DS film modified ITO

http://dx.doi.org/10.1016/j.jab.2017.06.001 1214-021X/© 2017 Faculty of Health and Social Sciences, University of South Bohemia in Ceske Budejovice. Published by Elsevier Sp. z o.o. All rights reserved.

Please cite this article in press as: Y. Li, C. Yu, RGD peptide doped polypyrrole film as a biomimetic electrode coating for impedimetric sensing of cell proliferation and cytotoxicity, J. App. (2017), http://dx.doi.org/10.1016/j.jab.2017.06.001

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electrode had a lower electrical impedance and better biocompatibility than the bare ITO electrode (Li et al., 2015a). From a separate standpoint, as an attractive feature of PPy for cell growth, the choice of dopant for PPy electro-polymerization can confer the resulted PPy different biological-physical-chemical properties. On the one hand, the incorporation of dopants with different properties results in PPy scaffolds with different physical properties in terms of morphology, roughness, contact angle, etc., which in turn regulate the biological behavior of cells cultured on them (Thompson et al., 2011). On the other hand, by selecting bioactive molecules as the negatively charged dopants, the resulted PPy scaffold can directly confer biofunctionality due to the bioactive dopants in the PPy matrix that can be by accessed by cells. For example, studies by Collier JH et al. have reported that hyaluronic acid-doped PPy film(PPy/HA) can support cell growth for a few days in vitro and even promote the formation of blood vessels in vivo (Collier et al., 2000). Also, function fragment CDPGYGSR peptide of laminin protein was doped with PPy to study neural cell proliferation (Zhang et al., 2010). It was found that PPy films doped with different function fragment peptides, CDPGYGSR and RNIAEIIKDIA, showed different effects on supporting neuron cell adhesion, proliferation and migration (Stauffer and Cui, 2006). However, many of the bioactive molecules investigated as dopants in the literatures are large biological molecules, such as proteins and peptide fragments with molecular weights ranging from 1 to 2.3 KDa, which results in the degradation of the PPy’s mechanical and electrochemical stability. Fortunately, the relatively small Arg-Gly-Asp (RGD) peptide has been confirmed in many adhesive extracellular matrix proteins as a minimal cell-recognizable motif. The immobilization of the RGD motif on biomaterials is considered an effective strategy to provide a biomimetic surface for biomedical applications (Dettin et al., 2015). Until recently, immobilization of the RGD motif on PPy has been achieved by carboxyl-amino-mediated covalent conjugates (De Giglio et al., 2000; Lee et al., 2006), and by a T59 peptide-mediated linker (Nickels and Schmidt, 2013; Sanghvi et al., 2005). However, they were found to be too complex to complete (involving multiple steps) or needed a strict acidic environment (pH < 4). Thus, a PPy/ RGD composite prepared by a facile one-step electropolymerization using the RGD peptide as the dopant could be proposed. The PPy/RGD composite can not only be used as a biomimetic scaffold for cell growth, but also as a conductive interface for impedance detection of cell bio-behaviors. Until now, no such investigation has been reported. In this work, a biomimetic PPy/RGD interface combining the unique properties of PPy and RGD peptide was prepared using onestep coelectropolymerization of Py monomer and RGD peptide on ITO conductive glass. ITO glass was selected as the substrate due to its excellent conductivity and transparency, which allow electropolymerization, EIS detection and morphology observation during cell culture (Choi et al., 2015). The adhesion/spreading and proliferation of A549 cells on the PPy/RGD film were studied and the results were compared with other substrates, such as bare ITO and polystyrene sulfonate (PSS)-doped PPy film. PSS was selected as the control since it is the dopant commonly used in the preparation of PPy, and it has high conductivity, good stability and biocompatibility (George et al., 2005). Finally, the PPy/RGD film was coated on the surface of the ITO microelectrode, and the proliferation of A549 cells as well as the cytotoxicity of the natural anticancer molecule Polyphyllin I were assessed by EIS. Materials and methods Materials ITO conductive glass, which has a conductive layer thickness of 2200  300 nm and a surface resistance of 7 Vcm 2, was

purchased from Zhuhai Kaiva Optoelectronic Technology Co., Ltd. (Zhuhai, China). The ITO slides were cleaned by ultrasonication in acetone, alcohol and distilled deionized (DDI) water for 15 min successively and dried with nitrogen gas flow. Pyrrole monomer (Sigma Aldrich, China) was pre-treated by distillation in nitrogen atmosphere and then stored at 20  C. The RGD motif-containing peptide sequence GRGDSP was synthesized at SciLight Biotechnology Co., Ltd. (Beijing, China). Polystyrene sulfonate (PSS) was purchased from Sigma Aldrich. Unless otherwise stated, other reagents were of analytical grade and were used as received. All aqueous solutions were prepared with ultrapure water(>18 MV) form a Milli-Q Plus system(Millipore, USA). The electrochemical experiments were carried out on a CS350 electrochemical workstation (CorrTest Instruments Corp., LTD, Wuhan, China) using a home-made three-electrode cell, with an ITO electrode (diameter 6 mm) as the working electrode, an Ag/ AgCl wire as the reference electrode, and a platinum wire as the counter electrode. A Multimode VIII AFM (Vecco, USA) was used to determine the micromorphology and roughness of the prepared PPy film. The surface energy of PPy was measured at room temperature with the sessile drop method, using distilled water with a SDC200 optical contact angle meter (Shengding Precision Instruments, China). The three-dimensional micro-morphology of cells was measured with a VK-V150 laser microscopy system (Keyence, Japan). The phase-contrast observations of cells were performed on an inverted fluorescence microscopy (Olympus IX71, Japan). Electropolymerization of PPy/RGD composite film The PPy/RGD composite was electrodeposited on the ITO electrode using the constant potential method. In brief, the monomer solution containing 0.1 M pyrrole and 1.0 mg/ml GRGDSP peptide was de-oxygenated by nitrogen bubbling. Then, the polymerization was conducted by applying a constant potential of 0.7 V (vs. Ag/AgCl) on the ITO electrode. The thickness of the PPy/ RGD composite could be controlled by the polymerization charge. Our pre-experiment revealed that the most homogeneous and stable composite film could be formed on the surface of the ITO electrode when the polymerization charge was 0.8 mC cm 2 and the thickness could be calculated with a theoretical equation and was estimated to be 209 nm (Patois et al., 2011). As a control, PPy/ PSS film was synthesized in a solution containing 0.1 M Py and 1.0 mg/ml PSS. After polymerization, the PPy samples were cleaned with DDI water(18.0 MV) and dried at room temperature. Cell culture A549 cells were routinely cultured in RPMI-1640 medium (Gibco, China) supplemented with 10% fetal bovine serum (Gibco, China), 100 U/ml penicillin and 100 mg/ml streptomycin at 37  C with 5% CO2 in a humidified incubator. After the cells grew into a monolayer, they were harvested with 0.25% trypsin. The density and viability of the cells was determined with a hematocytometer, using the trypan blue staining. Before cell inoculation, the PPy/RGD composite film was sterilized with 70% ethanol and rinsed with sterilized DDI water. The A549 cell suspension (100 mL) was seeded on the surface of PPy/RGD film at a density of 5.0  104 cells/mL, and then placed in a humidified incubator(37  C, 5% CO2) for 12 h. Then, the A549 cells were fixed using 4.0% paraformaldehyde solution, and the threedimensional morphology observed using laser microscopy system to assess the adhesion/spreading of the A549 cells. To evaluate cell growth on the PPy/RGD film, time-dependent phase-contrast images of the A549 cells cultured on the PPy/RGD film were taken with a CCD camera equipped on the inverted fluorescence

Please cite this article in press as: Y. Li, C. Yu, RGD peptide doped polypyrrole film as a biomimetic electrode coating for impedimetric sensing of cell proliferation and cytotoxicity, J. App. (2017), http://dx.doi.org/10.1016/j.jab.2017.06.001

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microscopy from which the cell proliferation curves were plotted. For comparison, bare ITO and PPy/PSS were also used as control substrates for cell culture. Electrochemical impedance spectroscopy (EIS) measurements In order to minimize the negative effects of the redox probe on cell bioactivity and to accurately measure cell behaviors using EIS in the phosphate-buffered saline (PBS) electrolyte solution, first, the PPy/RGD film was electropolymerized on the surface of the ITO microelectrode with a diameter of 500 mm in a way that the measured total impedance of the system was exclusively dominated by the cell-covered working microelectrode (Arndt et al., 2014). The fabrication of the ITO microelectrode followed the protocol from our previously reported work (Li et al., 2015a, 2015b). In brief, the ITO microelectrode was fabricated by etching the insulating layer of photosensitive dry film using lithography technology. After sterilization of the PPy/RGD film covered ITO microelectrode, 100 mL A549 cells (2.0  104cells/ml) were loaded onto its surface, then the ITO microelectrode was placed in a humidified incubator (37  C, 5% CO2) to allow cell adhesion and

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proliferation. After inoculation for 2 h, 24 h and 48 h respectively, the weakly adherent cells were washed off with PBS. EIS measurements were carried out in 0.01 M pH 7.4 PBS to assess cells adhesion and proliferation. The impedance spectra were recorded within the 1 105 Hz frequency range with the applied sine wave potential of 10 mV amplitude and analyzed by curve fitting to an equivalent circuit model using the ZView 2.0 software (Scribner Associates, USA). Impedance spectra signals of PPy/RGD modified ITO microelectrodes without cell loading were measured as a control. To analyze of the cytotoxicity effect of the natural anticancer drug Polyphyllin I on A549 cells, 1 mL A549 cells (1.0  105 cells/ mL) were seeded on the PPy/RGD modified ITO microelectrode and incubated for 24 h at 37  C. Then different concentrations of Polyphyllin I were added into the cell and incubated for another 12 h, followed by an EIS measurement in PBS. The control group was made up of cells without drug treatment. The drug induced impedance change was calculated as (1-Zt/Zc)  100%, where Zt is the highest impedance change at a special frequency for the drug treated group and Zc is for the drug free group. In parallel, the effect of Polyphyllin I on A549 cells viability was analyzed using a WST-1

Fig. 1. Preparation and characterization of the PPy/RGD film. (A) Picture of the home-made electrolytic cell device with three-electrode system for preparation of the PPy/RGD film. (B) Current-time curves recording during the electropolymerization of PPy/RGD and PPy/PSS on ITO micro-electrode. Inset: microscope images of the bare ITO and ITO modified by PPy/RGD and PPy/PSS. Scale bar: 200 mm. (C, D) AFM images and height profiles of PPy/RGD (left) and PPy/PSS (right) electropolymerized on the surface of ITO.

Please cite this article in press as: Y. Li, C. Yu, RGD peptide doped polypyrrole film as a biomimetic electrode coating for impedimetric sensing of cell proliferation and cytotoxicity, J. App. (2017), http://dx.doi.org/10.1016/j.jab.2017.06.001

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assay. In brief, A549 cells were seeded into 96-well plates (1.0  105 cells/mL) for 24 h and treated with different concentrations of Polyphyllin I for 12 h. Then, 10 mL of WST-1 solution was added into each well and incubated for 1 h at 37  C. The absorbance value was recorded at 450 nm wavelength with a microplate reader (Varioskan, Thermo Scientific, USA). The drug induced cell cytotoxicity was characterized by (1-At/Ac)  100%, where At and Ac refer to the absorbance values of the drug treatment group and the untreated group, respectively. Results and discussion Preparation and characterization of the PPy/RGD film To facilitate the electropolymerization experiments, cell culture and EIS measurements, a home-made electrolytic cell device was constructed. The electrolytic cell is presented in Fig. 1(A). The ITO slide, serving as the working electrode, was fixed under the bottom of the electrolytic cell with four screws. The ITO working electrode had a diameter of 6 mm for cell culture and 0.5 mm for EIS detection. Each device had four independent cells, and the volume of each cell was 500 mL. The PPy/RGD composite film was formed on the surface of the ITO electrode by the electroploymerizaiton of 0.1 M pyrrole with 1.0 mg/ml GRGDSP peptide. The current-time (it) curves recorded during the polymerization process of PPy/RGD and PPy/PSS at a constant potential of +0.7 V (vs. Ag/AgCl) are shown in Fig.1(B). The increased current, along with the polymerization time, indicated that the formed PPy film can increase the active surface area and conductivity of the ITO electrode. As expected, the current obtained from the PPy/RGD

polymerization was smaller than the one from the PPy/PSS polymerization, which may be caused by the poor conductivity and inherently weak eletrolyte of the RGD peptide. Thus, in comparison to PPy/PSS, a longer time was required to finish the electropolymerization of the PPy/RGD under the same charge passed. In addition, it is worth noting that the Py monomer or RGD alone could not be electropolymerized on the ITO electrode under the same polymerization conditions, proving that the RGD peptide functions as the sole, integrant dopant for the polymerization of PPy/RGD composite film. This result indicated that the negative charge-bearing RGD peptide acted as the counterion and was incorporated into the PPy matix to balance the positive charge of the PPy backbone during the electropolymerization process. To date, as far as we know, this work is the most facile approach regarding the preparing PPy and RGD peptide composites. After electropolymerization, homogeneous grey films were formed on the surface of ITO electrodes, indicating successfully electrodeposited PPy/RGD and PPy/PSS films, as demonstrated in Fig.1(B). Fig.1(C, D) shows typical atomic force microscopy(AFM) images of the electropolymerized PPy/RGD and PPy/PSS films on the ITO surface. Uniformly distributed particles with average particle size in the range of 50–70 nm can be observed on both PPy films. This observation was in accordance with the results of other studies regarding electropolymerized PPy/DS film in the literature (Li et al., 2015a). A careful observation of the PPy/RGD film morphology has shown that the roughness value of the PPy/RGD film was significantly lower in magnitude (Ra = 5.57  0.08 nm) than that of the PPy/PSS film (Ra = 22.96  4.23 nm). However, the roughness values of both PPy films were relatively low (<100 nm). It has been

Fig. 2. 3D images of A549 cells cultured for 12 h on (A) bare ITO, (B) PPy/PSS and (C) PPy/RGD. (D) Proliferation curves of A549 cells cultured on different substrates: 1) bare ITO, 2) PPy/PSS, 3) PPy/RGD, 4) PPy/RGD + RGD inhibition.

Please cite this article in press as: Y. Li, C. Yu, RGD peptide doped polypyrrole film as a biomimetic electrode coating for impedimetric sensing of cell proliferation and cytotoxicity, J. App. (2017), http://dx.doi.org/10.1016/j.jab.2017.06.001

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reported that a material surface that has a low degree roughness is more preferable for cell adherence than flat or micro-scale roughness surfaces, which suggests that the prepared PPy films had a good potential for cells growth (Fahlgren et al., 2015; Vandrovcová and Ba9 cáková, 2011). In addition, the contact angles for the PPy/RGD and the PPy/PSS films were 48.6  4.6 and 28.8  1.2 respectively, which indicates that polymerized PPy films possess sufficient hydrophilicity for anchorage-dependent cells adhesion and proliferation (Chang et al., 2005; Fahlgren et al., 2015). Cell adhesion and proliferation behaviors on PPy/RGD film The surface characteristics of the sensing interface are vital for the impedimetric assessment of cellular behaviors and responses to external stimulations, because cells require a specific substrate for proliferation and regulation of cellular functions (Asakawa et al., 2008). In general, cell proliferation on an appropriate substrate takes place in three stages: attaching, spreading and dividing. Therefore, the adhesion/spreading behavior of cells allows evaluating the suitability of a substrate for cell culture. Fig. 2(A)–(C) show the typical three-dimensional (3D) micromorphology of A549 cells cultured for 12 h on the surfaces of bare ITO, PPy/PSS and PPy/RGD respectively. It can be observed clearly that on all surfaces the A549 cell cultures have a spread-out shape with pseudopodia, suggesting that these biocompatible substrates

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support the attachment and spreading of A549 cells. It was well-known that a cell’s attachment and spreading on a substrate depend not only on the surface properties of the substrate, but also on the proteins absorbed onto the surface from the culture medium or secreted from cells, which may lead to the observed cell attachment and spreading on bare ITO and PPy/PSS film. Most importantly, while on the bare ITO and PPy/PSS film, the A549 cells had a relatively round 3D micro-morphology, the stretched 3D micromorphology on PPy/RGD film suggests the improved adhesion capability of the PPy/RGD film. The proliferation activity of A549 cells on different substrates was further investigated through time-dependent phase-contrast images from 0 h to 72 h. The cell proliferation curves plotted form these successive images are presented in Fig. 2(D). It can be clearly observed that the PPy/PSS film(curve 2) and the PPy/RGD film (curve 3) provide better cell proliferation than bare ITO(curve 1), as indicated by a significant increase in cell density compare to bare ITO from 24 h to 72 h, suggesting that PPy can improved cell growth (Balint et al., 2014). In addition, it is clearly shown that the proliferation activity of A549 cells was further improved on the PPy/RGD film to achieve an approximately 72% increase in cell density compare to PPy/PSS film, showing that the incorporation of RGD into PPy was effective in promoting cell growth. It is well understood that the RGD motif-containing peptide mimics the cell-bonding sequence of most adhesive extracellular matrix proteins (Dettin et al., 2015; Guo et al., 2012; Susan 2011).

Fig. 3. EIS detection of the adhesion and proliferation of A549 cells on PPy/RGD modified ITO microelectrode. (A) Nyquist plots for PPy/RGD modified ITO microelectrode recorded at 0, 2, 24 and 48 h after inoculation of A549 cells. (B) Nyquist plots for PPy/RGD modified ITO microelectrode without cells inoculation. (C) Equivalent circuit model of PPy/RGD modified ITO microelectrode (left) in series with equivalent circuit model of biological cells (right). (D) Curve fit results of the A549 cells after inoculation on PPy/ RGD modified ITO microelectrode (red) and PPy/PSS modified ITO microelectrode (blue) for different time points (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Y. Li, C. Yu, RGD peptide doped polypyrrole film as a biomimetic electrode coating for impedimetric sensing of cell proliferation and cytotoxicity, J. App. (2017), http://dx.doi.org/10.1016/j.jab.2017.06.001

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Therefore, RGD peptide doping conferred biomimetic properties to the PPy film and greatly improved cell adhesion/spreading activity and proliferation as well. From another point of view, since it has been extensively reported that the type of dopant selected for PPy electropolymerization will affect the physical properties of the prepared PPy such as roughness, morphology and wettability, films with different dopants may possess different surface properties, which in turn influence cell growth and viability (Fahlgren et al., 2015). In this case, since PPy/RGD film has a distinct surface property from PPy/ PSS, as mentioned in Fig. 1, clarification of the factors that affect the proliferation activity is very crucial. For this purpose, in an inhibition study by carried out by adding RGD peptide into the culture media, the proliferation curve of A549 cells(curve 4) on PPy/RGD film showed a remarkable reduction and no significant difference compared to the PPy/PSS film, demonstrating that the improved proliferation of A549 cells was in fact due to the immobilized RGD peptide instead of secondary factors, such as roughness and wettability differences between the PPy/RGD film and the PPy/PSS film. EIS detection of cell behaviors In vitro assessment of cell behaviors using a cell-based biosensor is of great significance for studying cell biological mechanisms and the assessment of new anticancer drugs. EIS has recently gained popularity in cell-based biosensor studies due to its advantages such as high sensitivity, non-invasiveness and accessibility of quantitative data. Therefore, based on its improved biocompatibility and biomimetics, in this study, PPy/RGD film was electropolymerized on the ITO microelectrode as electrode coating for EIS assessment of cell proliferation and anticancer drug cytotoxicity. In comparison with most EIS cell biosensor studies that are based on the change of electron transfer resistance of redox probe, such as [Fe(CN)6]3 /4 , our proposed EIS cell biosensor was based on measuring the non-faradic impedance of the cell-covered microelectrode in PBS in the absence of exogenous redox probes. Using this method, we are able to avoid the negative effect of redox probes on immobilized cells on the electrode surface and to monitor cells behaviors in real-time. A similar measurement based on non-faradic EIS has been reported for a PPy-based electrochemical immunosensor (Ramanavicius et al., 2010). The basic mechanism of this EIS cell biosensor has been described in more detail before (Giaever and Keese, 1984), and only the primary principle is stated here. Modeled as insulating particles, when cells adhere and proliferate on the surface of a planar microelectrode, they can hinder unrestricted current flow from the electrode into the bulk electrolyte and thereby change the overall electrode impedance. Subsequently, morphological information about cell behaviors on the electrode surface will be extracted from the measured impedance signals by model fitting. EIS detection of the proliferation of A549 cells Fig. 3(A) showed the Nyquist plot responses of the PPy/RGD film modified ITO microelectrode upon the addition of 100 mL of culture media containing A549 cells at oh, followed by incubation for 2 h, 24 h and 48 h respectively. For comparison, a control experiment was also conducted by adding 100 mL of culture media without cells on the PPy/RGD film modified ITO microelectrode. The Nyquist plot responses of the control experiment are also shown in Fig. 3(B). Phase contrast microscopic observation of A549 cells was preformed before EIS measurement, and the results confirmed that the cells adhere and proliferate well on the surface of the PPy/RGD film modified ITO microelectrode since the number of cells was visibly increasing. From the inset of Fig. 3(A), it can be

clearly observed that the adhesion and proliferation of A549 cells caused changes in the high frequency region of the Nyquist plot. The main change can be characterized with the appearance of an increased diameter semicircle in the Nyquist plot along the process of cell incubation, which can be attributed to the electron transfer velocity slowdown and/or charge transfer resistance increase at the electrode interface, caused by the capacitance effect of the adherent cell’s plasma membrane (Ateh et al., 2007). In contrast, as shown in Fig. 3(B), the Nyquist plot of the control PPy/RGD film modified ITO microelectrode did not change significantly during the incubation process, which demonstrates the good stability of the PPy/RGD film modified ITO microelectrode, and further verifyies that the appearance of an increased diameter semicircle in Fig. 3(A) was only due to the adhesion and proliferation of A549 cells. The adhesion and proliferation of A549 cells can be further analyzed by fitting the measured impedance data to a proposed equivalent circuit model. By taking the difference between the electrode with no cell coverage (0 h) and the electrode after cell inoculation, we assumed that the impedance due to cells can be modeled as a series circuit consisting of an RC parallel circuit and an R component (Affar et al., 2013). For the bare PPy/RGD film modified ITO microelectrode, a series circuit consisting of an R element and a constant phase element (CPE) can yield a good fitting result. Thus, after cell inoculation, the equivalent circuit model consisted of the equivalent circuit of electrode and the equivalent circuit of cells, as shown in Fig. 3(C). From the equivalent circuit of cells, Rs‘ can be theoretically explained as the resistance of current in the small gap between the cell and the electrode, similarly to what Giaever and Keese have discussed (Giaever et al., 1984). Rcell is due to the resistance to current flow between the gaps of adjacent cells and Ccell can be attributed to the capacitive effect of cells adhering to the electrode surface. Using a similar approach, Qiu et al. (Qiu et al., 2008) have measured the cell-substrate distance for cardiomyocytes, where cells were simply modeled by a resistor in parallel to a capacity. The limitation of such model is that it is not valid when cells are only partially covering the electrode. Taking into consideration partial electrode coverage, our proposed model uses the theory of electrical elements similar to the one proposed by Qiu et al. (Qiu et al., 2008), but with a certain modification by adding the Rs‘ element. The impedance data measured during the process of adhesion and proliferation of A549 cells was further analyzed with a complex non-linear least squares curve fitting method, using the equivalent circuit model discussed above. The red curve in Fig. 3(D) shows the results of the curve fit after cells inoculation for different time points. It can be observed that with the increase of time after cell incubation, the values of Rs‘, Rcell and Ccell increase as well. After cell seeding, the value of Ccell rapidly reached a value around 5.2 nF at 2 h and slowly increased to about 9.1 nF at 24 h to 48 h. The changes of the Ccell value reflected the reducing of cell thickness, enlargement of cell area and the status of ion channels on the cell membrane(Hong et al., 2011). The value of Rcell kept increasing and reached a value of 1884 Vat 48 h, and Rs‘ had a constant increase just before 24 h and then a slow increase to 312 V. The increase in Rcell and Rs‘ can be described as a tighter cell–cell gap (increasing confluence) and a decrease in cell-substrate distance, respectively (Affar et al., 2013; Qiu et al., 2008). A similar analysis was performed on the PPy/PSS modified ITO microelectrode. The results of the curve fit for A549 cells incubated on the PPy/PSS modified ITO microelectrode are shown by the blue curve in Fig. 3(D). It is obvious that the Rs‘, Rcell and Ccell values for the PPy/PSS modified ITO microelectrode show a similar trend to those of PPy/RGD modified ITO microelectrode, due to the adhesion and proliferation of A549 cells on both electrode surfaces.

Please cite this article in press as: Y. Li, C. Yu, RGD peptide doped polypyrrole film as a biomimetic electrode coating for impedimetric sensing of cell proliferation and cytotoxicity, J. App. (2017), http://dx.doi.org/10.1016/j.jab.2017.06.001

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However, the curve fit results of the A549 cells measured by PPy/ PSS modified ITO microelectrode are lower than those measured by the PPy/RGD modified ITO microelectrode at 2 h, 24 h and 48 h, which was expectable due to the biomimetic properties of the PPy/ RGD film. In addition, based on the results of the value changes of Rs‘, Rcell and Ccell during the incubation of A549 cells, the PPy/RGD film also showed improved adhesion and proliferation compared to the electropolymerized dextran sulfate (DS) doped PPy film reported in our previous work (Li et al., 2015a). Therefore, the present work not only shows a facile method for the detection of cell proliferation, but also provides an elaborate approach for investigating the interaction between cells and the conducting PPy substrate. EIS detection of the cytotoxic effect of Polyphyllin I Polyphyllin I, a small molecular monomer extracted from Rhizoma of Paris polyphyllin, has been reported to have strong anticancer effects on many tumor cells and xenografts, including lung, breast and liver cancer in previous studies (Kong et al., 2010;

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Shi et al., 2015). In this work, Polyphyllin I was selected as a model to investigate the cytotoxic effect of drugs using the proposed EIS cell biosensor based on the PPy/RGD modified ITO microelectrode. A549 cells were allowed to grow for 24 h on the PPy/RGD modified ITO microelectrode before the addition of Polyphyllin I solution. The Nyquist plots responses and the impedance frequency responses of the microelectrode after the addition of Polyphyllin I with final different concentrations for 12 h are shown in Fig. 4(A) and (B) respectively. The concentration-dependent cytotoxic effect of Polyphyllin I can be observed by comparing the diameter of the well-defined semicircle at high frequency in the Nyquist plots and the impedance value at special frequency in the Bode plots. With the increase of Polyphyllin I concentration, both the diameter of the semicircle and the overall impedance value have decreased due to the constriction of cell plasma membrane and cell detachment from the electrode surface under the condition of cell apoptosis induced by Polyphyllin I. This assumption can be confirmed by observing the morphology, shown in Fig. 4(D), where it can be clearly seen that with the increase of Polyphyllin I concentration,

Fig. 4. EIS detection of cytotoxic effect of Polyphyllin I on A549 cells on PPy/RGD modified ITO microelectrode. (A) Nyquist plots responses recorded after addition of Polyphyllin I with different final concentrations for 12 h. (B) Impedance frequency responses recorded after addition of Polyphyllin I with different final concentrations for 12 h. The arrow shows the frequency where the maximum impedance change induced by Polyphyllin I. (C) Cytotoxic effect of Polyphyllin I on A549 cells determined by the proposed EIS cell biosensor and colorimetric WST-1 assay (n = 5). (D) Phase contrast microscopy images of A549 cells after addition of the Polyphyllin I with different final concentrations for 12 h. Scale bar: 100 mm.

Please cite this article in press as: Y. Li, C. Yu, RGD peptide doped polypyrrole film as a biomimetic electrode coating for impedimetric sensing of cell proliferation and cytotoxicity, J. App. (2017), http://dx.doi.org/10.1016/j.jab.2017.06.001

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more A549 cells were killed and detached from the culture substrate. Additionally, the cytotoxity effect of Polyphyllin I can be quantified from the maximum impedance changes at a frequency of 3.5 kHz. The relationship curves between the cytotoxic effect and Polyphyllin I concentration are shown in Fig. 4(C), where it can be seen that the cytotoxicity results obtained from the proposed EIS biosensor show a similar trend to those from the traditional colorimetric WST-1 assay. However, there are some slight differences between the two results, and while the underlying cause has yet to be fully elucidated, there are two main hypotheses. Firstly, the proposed EIS cell biosensor used PPy/RGD film as the cell growth substrate, while the WST-1 assay used polystyrene. The different biophysical-chemical properties of the substrates might regulate cell activity and affected cellular response to drugs. Secondly, the primary cause for the difference in results might be the essential difference between these two methods: one is based on the morphological changes of cells immobilized on the electrode surface, and the other is based on the enzymatic cleavage of the WST-1 to formazan by the mitochondrial dehydrogenases present in viable cells. Therefore, control experiments, parallel groups and strict quality control are needed in order to obtain more reliable results using the proposed EIS cell sensor. Furthermore, despite their diversity, the proposed EIS cell biosensor has several advantages over traditional optical methods, including low cost (no need for expensive instruments and reagents), noninvasiveness, laborsaving, and higher precision (Relative standard deviation (RSD) < 5% for the proposed EIS cell sensor vs. RSD < 10% for the WST-1 assays). Conclusions The present study demonstrated a facile approach to building an impedimetric cell biosensor based on one-step electropolymerization of PPy/RGD film for the detection of A549 cells proliferation and drug cytotoxity. The RGD-peptide entrapment in PPy matrix made the composite film biomimetic for enhanced human cells attachment and proliferation. Based on the biomimetics of the PPy/RGD film, this work successfully demonstrated that using the facile electropolymerization of RGD-peptide-doped conducting PPy is a promising way to design label-free and noninvasive EIS biosensors for detection of cells proliferation and anticancer drug cytotoxicity. Additionally, the proposed EIS approach allowed analyzing the interaction between various PPy-based interfaces and the cells cultured on them, which was considered beyond the capabilities of most optical methods. In brief, this study not only reports an approach to fabricated PPybased functional film with biomimetics, but also provides a powerful platform to build biomedical devices for live-cell assay and drug effect screening. It should be emphasized that the proposed EIS detection is performed in PBS in the absence of redox probes, possessing the potential for in-suit and real-time monitoring of live-cells. We are currently conducting these experiments. Conflict of interest The authors declare that there are no competing interests regarding the publication of this paper. Acknowledgments This study was supported financially by the Natural Science Foundation Project of Chongqing (cstc2012jjA10046), Innovation Ability Construction Platform Project of Yongchuan district (ycstc2014bf5001) and Key Research Project of Yongchuan

Hospital, Chongqing YJZQN201534).

Medical

University

(YJZD201302,

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