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Organic electrochemical transistor array for monitoring barrier integrity of epithelial cells invaded by nasopharyngeal carcinoma
Sensors & Actuators: B. Chemical 297 (2019) 126761
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Organic electrochemical transistor array for monitoring barrier integrity of epithelial cells invaded by nasopharyngeal carcinoma Sin Yu Yeunga, Xi Gua, Chi Man Tsangb, Sai Wah George Tsaoc, I-ming Hsinga,
T
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a
Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong Special Administrative Region Department of Anatomical and Cellular Pathology, The State Key Translational Laboratory, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Special Administrative Region c School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region b
A R T I C LE I N FO
A B S T R A C T
Keywords: Biosensors Semiconductors Organic electrochemical transistor Cancer Barrier integrity
Cancer metastasis, characterized by the migration of tumour cells to secondary, distant places, indicates a late stage of tumour progression and accounts for most cancer deaths. Barrier tissues, like epithelial cells, act as the frontline against circulating tumour cells and their disruption is often regarded as the hallmark of malignancy. Therefore, assessing barrier function is often involved in cancer-related studies and drug screening assays. Limitations of the current existing strategies have, however, led to a need for an accurate, intuitive option that carries the capability of simultaneous bioimaging. Taking advantages of the good biocompatibility and high transconductance, we made use of organic electrochemical transistor (OECT), an emerging bioelectronic platform, to study the effects of intruding cancer cells on normal epithelium monolayer. We performed multichannel recording of 16-channel OECT array to investigate the behaviour of a newly reported nasopharyngeal cancer cell line NPC43 on epithelial cells. Experimental results show that the platform can distinguish NPC43 from other epithelial or cancer cell types and retrieve spatial information during NPC43 invasion. The developed platform is thus envisioned to be a potential non-optical tool for monitoring cancer invasion.
1. Introduction Barrier tissues, a collective term describing tissues formed by epithelial and some endothelial cells, compartmentalize body cavities and control selective passage of substances across the barriers [1]. The dynamic feature of these semi-permeable tissues is attributed to multiple intercellular protein complexes, such as adherence junctions, desmosomes, gap junctions and tight junctions [2]. Degradation of these proteins can cause subsequent loss of barrier integrity or even irreversible breakage, which can lead to severe complications or progression of diseases, including cancers [3–5]. In particular, tight junction is well-studied for its critical role in cancer development [6]. Although the mechanisms of how tight junctions affect cancer invasion could vary case by case, nevertheless, the tight junctions seem to act as the frontline that being dissociated by cancer cells during metastasis [7]. Disruption of tight junctions could be caused by chemicals secreted by tumour cells (e.g. matrix metalloproteinases) and occur in all stages of metastatic cascade, including invasion, intravasation and extravasation [8]. For example, cancer cells need to destroy tight junctions to detach from the primary tumour [9].
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In addition, they may also invade the surrounding stroma elements and mesothelial cells [10]. Another example is that the tumour cells would have to disrupt endothelial tightness before penetrating into bloodstream [11]. In light of its importance in cancer metastasis, destruction of tight junctions is often regarded as the hallmark of malignancy, and assessment of cell-cell tightness is useful in in vitro pathophysiological and therapeutic studies. Optical-based permeability assay is one of the commonly employed methods to assess barrier function. The absorbance of the small, hydrophilic tag molecules (e.g. Lucifer Yellow) that can readily pass through tight junctions in paracellular spaces, are used to indirectly correlate the tightness of cell layer [2]. Nevertheless, due to its drawbacks of low-throughput and inaccuracy, researchers have switched to label-free electronic methods [12]. Transepithelial electrical resistance (TEER), defined as the ohmic resistance of epithelium, is often used in electronic platforms to quantitatively measure barrier integrity [13]. Epithelial voltohmmeter (EVOM) is one widely used benchtop equipment to measure TEER values. In EVOM measurement, alternating current (AC) square wave in low frequency is applied across a hanging porous filter cultured with cells and thereby TEER can be retrieved
Corresponding author. E-mail address: [email protected] (I.-m. Hsing).
https://doi.org/10.1016/j.snb.2019.126761 Received 11 March 2019; Received in revised form 4 June 2019; Accepted 29 June 2019 Available online 02 July 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Sensors & Actuators: B. Chemical 297 (2019) 126761
S.Y. Yeung, et al.
the MDCK cells were seeded in 50% MDCK medium and 50% NPC43 medium. Otherwise mentioned, the reagents and culture media were purchased from Thermofisher, MA.
[14]. Although EVOM measurement is straightforward and easy to handle, its accuracy could be affected by the size difference between electrodes and cell culture [15]. Moreover, the measurement is limited to filter-culturing environment, which gives rise to imaging difficulty. Electrical impedance spectroscopy (EIS) represents another choice to accurately characterize barrier status. By scanning across a range of frequencies, information not just limited to TEER, but cell capacitance that depends on high-frequency signals can also be obtained [16]. However, EIS is relatively expensive and requires highly trained personnel to operate and analyse data [17]. Organic electrochemical transistor (OECT) has been gaining significant attention in the field of organic bioelectronics recently [18]. Particularly, a number of applications have proven OECT’s excellent ability to monitor barrier integrity, under the impacts of epitheliumdestabilizing pathogen Samonella typhimurium and chemicals such as hydrogen peroxide (H2O2) and ethylene glycol tetraacetic acid (EGTA) [19–21]. OECT not only can characterize epithelial cells in a way that is similar to EIS (e.g. impedance spectrum), but also through its own responses (e.g. step function and transconductance), with results comparable to current standards [22,23]. Given its flexibility in fabrication and processing, OECT has been fabricated in versatile formats, including a planar configuration that allows electrical epithelial sensing combined optical imaging, offering advantages over current electronic tools [17,24–26]. OECT is also well-known to be biocompatible, electrochemically stable and has the highest transconductance among the electrolyte-gated transistor technologies thus making it a good choice for various biological applications [27,28]. Previously, we demonstrated the use of a 16-channel OECT to monitor action potential propagation in 2D and 3D primary cardiomyocyte culture [29,30]. Taking advantage of its multi-channel feature, we use the same device here to investigate the invasive behaviour of a newly established Epstein-Barr virus (EBV)-positive nasopharyngeal cancer cell line NPC43 [31]. Nasopharyngeal carcinoma, characterized by the cancer originating from nasopharynx, is a rare malignancy when considering worldwide population. However, it is endemic in Southeast Asia (e.g. Guangdong Province and Hong Kong), with a ranking of most common cancer at 23rd in the world comparing to 4th in Hong Kong [32,33]. With the developed platform, we were able to differentiate several epithelial and cancer cell types and more importantly, spatially map NPC43 invasion on the epithelial Madin–Darby Canine Kidney (MDCK) cells. As a proof-of-concept study, this work suggests that multi-channel OECT could be a promising electronic platform for studying cancer metastasis.
2.2. Immunofluorescence staining Cells were cultured directly on glass slices for immunofluorescence staining. After reaching confluency, they were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min at room temperature. Permeabilization was done by incubating the fixed cells in 0.25% Triton-X in PBS for 20 min. To block non-specific binding, samples were incubated in PBS with 1% bovine serum albumin (BSA) for 30 min. After that, samples were incubated in 5 μg/ml ZO-1 monoclonal antibody in 1% BSA-PBS solution for 1 h at room temperature. Fluorescence tags were conjugated by incubating cells in a mixture of 1 μg/ml goat anti-mouse IgG superclonal secondary antibody, Alexa Fluor 488 (1:1000) and Hoechst 33,342 (1:5000) in PBS for 1 h at room temperature. The cells on cover glass were mounted for fluorescence microscopy. All the involved reagents were procured from Thermofisher, MA. 2.3. OECT fabrication and assembly The OECT array was fabricated on quartz glass, which was precleaned by 10:1 sulfonic acid with hydrogen peroxide (H2O2). Gold tracks were deposited through evaporation and patterned by lift-off lithography. A layer of 2 μm SU-8 2002 (Microchem, MA) was used as passivation layer, with openings defining active channel area. Processing of SU-8 followed standard protocol suggested by manufacturer. PEDOT:PSS Clevios PH500 solution (Heraeus, Germany) consisting of 10% ethylene glycol (Sigma Aldrich, MO) and 1% 3-glycidoxypropyltri-methoxysilane (GOPS; Sigma Aldrich, MO) was spincoated at 3000 rpm and patterned again by lift-off process. A glass ring was assembled onto substrate using Slygard 184 polydimethylsiloxane (PDMS; Dow Corning, MI). 2.4. Frequency-dependent transconductance measurement Each OECT in the array was biased at VDS=−0.4 V, which was set based on the characterization results published previously [30]. Gate electrode utilized here was made of silver chloride-coated silver electrode (Ag/AgCl). Gate inputs were sinusoidal waves at 1–10 K Hz with amplitude of 0.01 V. The drain terminal was connected to a home-built negative feedback circuit for signal amplification, and the voltage signals were monitored by oscilloscope (MSO7034B, Agilent, CA) [34]. The recorded signals were fitted with the following formula:
2. Materials and methods 2.1. Epithelial and cancer cell culture
y = yo + Asin [π (X − Xc )/ ω]
Caco-2 and MDCK cells were kept in Dulbecco's Modified Eagle medium (DMEM) mixed with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) (100 U/ml and 100 μg/ml). NP460 cells were cultured in EpiLife + Defined Keratinocyte-SFM with 1% P/S added. NPC43 cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS, 4μM Rock inhibitor (Y-27632 dihydrochloride, Enzo Life Sciences Inc., Farmingdale, NY) and 1% P/S added. NPC43 cells were obtained from our collaborator Professor Sai Wah Tsao (HKU). For the study of invasion, MDCK was first cultured until confluency. Then the composition of medium was changed to 50% MDCK medium and 50% NPC43 medium. Subsequently, NPC43 cells were seeded to the confluent MDCK culture. For NPC-MDCK co-culture fluorescence imaging, NPC43-MDCK suspension was first prepared in 1:1 ratio. They were then seeded to the glass substrate in 50% MDCK medium and 50% NPC43 medium. Culture condition was maintained at 37 °C, 5% CO2 humidly. For NPC-MDCK co-culture in muti-channel OECT, NPC43 was firstly seeded to the chip and cultured for several days, until the NPC43 cells fully covered 3 to 8 OECT channels. Then
(1)
in which A is the amplitude of measured signal. The values of voltage amplitude at different frequencies were then extracted for calculating normalized transconductance and plotting diagram. The following equation describes transconductance gm:
gm = ΔIDS /ΔVGS
(2)
2.5. Impedance characterization The setup of impedance measurement was similar to that for transconductance measurement, except that signals were measured up to 3000 Hz because of the limitation of multi-meter’s sampling frequency. The gate current IGS was captured by digital multi-meter (34411A, Agilent, CA) and acquired through a built-in LabVIEW program. The recorded signals were again fitted with Eq. (1) and the amplitude value was plugged into ΔIGS of Eq. (3).
|Z| = ΔVGS /ΔIGS 2
(3)
Sensors & Actuators: B. Chemical 297 (2019) 126761
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3. Results and discussion 3.1. Configuration of 16-channel OECT array The configuration of our 16-channel OECT is presented in Fig. 1. The OECT array was fabricated on a glass substrate, with gold interconnects and conducting polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) as channel material. PEDOT:PSS is a commonly used channel material because of its high conductivity, facile processivity and electrochemical stability [35,36]. The devices were then passivated by a thin layer of SU-8, which was defined with openings, exposing the active PEDOT:PSS area. This sensing area and the SU-8 substratum were directly contacted with deposited cells and culture medium. Both of them were proven biocompatible and have been used in a number of in vitro and in vivo experiments [26,30,37–40]. As PEDOT:PSS is p-type doped, the current conducted is based on the mobile holes that are compensated by the negative sulfonate anions of PSS [41,42]. If a positive gate bias is applied, cations in electrolyte will drift into PEDOT:PSS film and compensate PSS−, resulting in lower hole density and thus lower drain-source current intensity IDS [43]. When cells are cultured on top of OECT, the epithelium can limit ion inflow and consequently, resulting in the reduction of the total number of cations in the channel with less electrochemical de-doping of the channel. Therefore, the relative leakiness of cultured cell layer can be reflected by the measured IDS intensity [44].
Fig. 1. A) Configuration of the assembled 16-chanbel OECT platform. B) A closer look of the OECT array. The array composes of 16 OECT channels with spacing of 200 μm, channel width as 6 μm and sensing area as 30 μm × 40 μm C) The OECT array composing of Au electrodes, SU-8 passivation and PEDOT:PSS channel was fabricated on a glass substrate. The gate electrode was immersed into the electrolyte to give bias.
where ΔVGS and ΔIGS corresponds to difference in applied gate voltage and gate current respectively. The impedance spectrum was plotted with impedance against corresponding frequency.
3.2. Determination of cellular epithelial properties by OECT measurement 2.6. Cell culture and on-chip measurement
Before studying cell-cell interactions with multi-channel OECT array, we first attempted to use single-channel OECT for cell-based measurement. The phase-contrast microscopic and immunofluorescence images of OECTs cultured separately with four types of cells (i.e., colorectal adenocarcinoma cells caco-2, MDCK, human telomerase immortalized nasopharyngeal epithelial cells NP460 [45] and NPC43) are shown in Fig. 2A and B respectively. In the fluorescence images of caco-2 and MDCK, the green fluorescence tags that labelled a domain of tight junction protein ZO-1, indicated that caco-2 and MDCK expressed intact epithelial junction networks. Although NP460 also
Before cell seeding, devices were disinfected by 70% ethanol and sterilized by UV light for 30 min. Measurements were taken when cell culture became confluent. During measurement, medium was replaced by PBS and an Ag/AgCl electrode was immersed into the electrolyte to give gate bias. For transconductance measurement, blank data was taken before seeding cells. For impedance sensing, cells were treated with 0.25% trypsin (Thermofisher, MA) before taking the blank data.
Fig. 2. A) Phase-contrast microscopic images of OECTs cultured with caco-2, MDCK, NP460 and NPC43 cells. Scale bar: 100 μm. B) Immunofluorescence images of cells stained with tight junction ZO-1 antibody-Alexa Fluor 488 (green) and nucleus stained with Hoechst 33342 (blue). Scale bar: 10 μm. C) Normalized transconductance plots of OECTs cultured with different cell types. D) Impedance spectrums of OECT with or without MDCK and NPC43 cell cultures. In our report, 16-channel OECT was not able to differentiate cells with different barrier tightness through impedance analysis.
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3.4. Multi-channel OECT array as an non-optical tool for NPC invasion study
showed a mesh-like structure in the phase-contrast image, the cells did not express ZO-1 as caco-2 or MDCK did. Interestingly, instead of forming a confluent monolayer, NPC43 cells aggregated and grew in a non-uniform, multi-layered and colonial manner. NPC43 also did not express ZO-1 as shown in the immunofluorescence image. In OECT, the frequency response is largely dominated by ion transport between electrolyte and channel [27]. Transconductance reflects the rate of ion flow into the channel at a given gate voltage bias. Therefore, if cells are tightly packed (i.e. exhibit high TEER value), the rate of ion inflow would be much lower and the characteristic transconductance value would drop at a much lower frequency. In Fig. 2C, the transconductance profiles of OECTs cultured with different types of cells are presented. Differences in cut-off frequencies, at which the maximal transconductance has dropped abruptly, were observed among different scenarios. Compared to the profile of OECTs cultured with NPC43, OECTs with MDCK or caco-2 cells had a much lower cutoff frequency, suggesting higher TEER values for these two cell lines. This observation is consistent with our EVOM data (data not shown) and other literature reports, where the TEER value of NPC43 was measured to have only 50 Ωcm2 and MDCK or caco-2 were reported to have more than 103 Ωcm2 [13]. As NPC43 cells tended to “huddle” together and did not completely cover OECT as shown in Fig. 2B, it might be easier for ions to leak through. Thus, it was easier for OECTs with NPC43 to be fully dedoped and have maximal transconductance value at a wider range of frequency. On the other hand, the normal epithelial cells NP460 also exhibited a low resistance value, suggesting NP460 was also leaky (Fig. 2C). Impedance spectrums of OECTs cultured with MDCK and NPC43 are shown in Fig. 2D. Similar to the other reported results, the plateau at mid frequencies (i.e. 50–5000 Hz) signifies the presence of cells, in which cell membranes act like dielectrics and paracellular junctions act like resistors [15,23]. Nevertheless, such impedance spectra were not sensitive enough to enable the differentiation between MDCK and NPC43. Therefore, in the following study, impedance measurement would not be used to assess the invasion of NPC43.
After exploring single-channel measurement, we applied our 16channel OECT to assess NPC43 invasion. NPC43 tagged with mCherry (marked in red) and MDCK (displayed in greyscale) were co-cultured on the device and their distribution was shown in Fig. 4A. Here the two representative OECT channel 9 and 12, which was covered by NPC43 and MDCK respectively, were selected for full spectrum transconductance analysis. Consistent with the results discussed in Section 3.2, the two OECTs displayed distinct frequency-dependent transconductance characteristics that represent the “fingerprints” of different cell lines (Fig. 4B). Because their difference in transconductance was more obvious at 100 Hz, a heat map showing the cell distribution was constructed based on the gm value at 100 Hz (Fig. 4C). Such spatial map successfully describes the approximate locations of MDCK and NPC 43 cells with the color intensity reflecting the corresponding transconductance magnitude. The redder the box, the larger the chance that the OECT would be covered by NPC43, and vice versa for MDCK. For example, the box color of channel 7 is white (Fig. 4C), meaning that the OECT had a relatively low transconductance and was most likely covered by the tightly packed MDCK cells, which could be observed in Fig. 4A. Again for channel 5, which has a higher transconductive value as depicted by its red box color (Fig. 4C), was completely covered by the leaky NPC43 cells shown in the microscopic image (Fig. 4A). To conclude, our 16-channel OECT array could be used as a complementary tool to microscopies for non-optical spatial mapping of cancer cell-cell interactions. 4. Conclusion Given the ion-sensitive feature of OECT, epithelial and cancer cell lines possessing different morphologies and TEER values could be distinguished based on frequency-dependent transconductance measurement. By arranging OECTs in a format of 4 × 4 matrix, spatial information on carcinoma invasion of normal epithelium could be retrieved. Moreover, this study partly reveals the invasion mechanism of a new nasopharyngeal cancer cell line NPC43, which was found to be leaky (i.e. has lower TEER value), and tendentious to intrude adjacent cells in a spheroid shape. With these results, the developed OECT platform shows a great potential in continuous monitoring of cancer invasion and metastasis. Integrating OECTs into in vitro barrier models as a complementary non-optical monitoring tool should benefit numerous pathological studies and drug screening assays.
3.3. Assessment of NPC metastatic potential by confocal microscopy Fig. 3 shows the three-dimensional confocal microscopic images illustrating how the suspended NPC43 cells interacted with MDCK cell monolayer. It showed that NPC43 cells proliferated to a spheroid structure and anchored onto MDCK monolayer. The bottom of the NPC43 spheroid was encapsulated by mesh-like ZO-1 network, which is believed to be part of the MDCK layer, while the top was not. It is likely that NPC43 cells might have broken down part of MDCK barrier, suggesting the invasiveness and metastatic potential of NPC43.
Acknowledgements This work was supported by Collaborative Research Funds (project number as [C1013-15G] & [C7027-16G]) and Innovative Technology Fig. 3. A) Confocal image of a NPC43 spheroid invaded into MDCK monolayer. The image was taken 7 days after adding NPC43 cells onto a confluent MDCK layer. B) Top and side views of the same image. Scale bar: 10 μm. Green: tight junctions, ZO-1 antibody-Alexa Fluor 488; blue: nucleus stained by Hoechst 33342; red: actin tagged with mCherry.
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Fig. 4. A) Microscopic image of NPC-43 and MDCK co-culture. NPC43 cells are tagged with red fluorescent marker mCherry. B) Normalized transconductance curves of channel 9 covered by NPC43 and channel 12 covered by MDCK only. C) The transconductance value at 100 Hz of each channel. OECT 1 was damaged so the signal was not avaliable and the respective box was illustrated as blue.
Fund Tier-3 (project number as [ITS/092/17]) of the HKSAR Government. S.Y.Y. would like to thank the Hong Kong Ph.D. Fellowship Scheme of HKSAR government for funding support and Nanosystem Fabrication Facility (NFF) of HKUST for technical support.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Organic electrochemical transistor array for monitoring barrier integrity of epithelial cells invaded by nasopharyngeal carcinoma Sin Yu Yeunga, Xi Gua, Chi Man Tsangb, Sai Wah George Tsaoc, I-ming Hsinga,
T
⁎
a
Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong Special Administrative Region Department of Anatomical and Cellular Pathology, The State Key Translational Laboratory, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Special Administrative Region c School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region b
A R T I C LE I N FO
A B S T R A C T
Keywords: Biosensors Semiconductors Organic electrochemical transistor Cancer Barrier integrity
Cancer metastasis, characterized by the migration of tumour cells to secondary, distant places, indicates a late stage of tumour progression and accounts for most cancer deaths. Barrier tissues, like epithelial cells, act as the frontline against circulating tumour cells and their disruption is often regarded as the hallmark of malignancy. Therefore, assessing barrier function is often involved in cancer-related studies and drug screening assays. Limitations of the current existing strategies have, however, led to a need for an accurate, intuitive option that carries the capability of simultaneous bioimaging. Taking advantages of the good biocompatibility and high transconductance, we made use of organic electrochemical transistor (OECT), an emerging bioelectronic platform, to study the effects of intruding cancer cells on normal epithelium monolayer. We performed multichannel recording of 16-channel OECT array to investigate the behaviour of a newly reported nasopharyngeal cancer cell line NPC43 on epithelial cells. Experimental results show that the platform can distinguish NPC43 from other epithelial or cancer cell types and retrieve spatial information during NPC43 invasion. The developed platform is thus envisioned to be a potential non-optical tool for monitoring cancer invasion.
1. Introduction Barrier tissues, a collective term describing tissues formed by epithelial and some endothelial cells, compartmentalize body cavities and control selective passage of substances across the barriers [1]. The dynamic feature of these semi-permeable tissues is attributed to multiple intercellular protein complexes, such as adherence junctions, desmosomes, gap junctions and tight junctions [2]. Degradation of these proteins can cause subsequent loss of barrier integrity or even irreversible breakage, which can lead to severe complications or progression of diseases, including cancers [3–5]. In particular, tight junction is well-studied for its critical role in cancer development [6]. Although the mechanisms of how tight junctions affect cancer invasion could vary case by case, nevertheless, the tight junctions seem to act as the frontline that being dissociated by cancer cells during metastasis [7]. Disruption of tight junctions could be caused by chemicals secreted by tumour cells (e.g. matrix metalloproteinases) and occur in all stages of metastatic cascade, including invasion, intravasation and extravasation [8]. For example, cancer cells need to destroy tight junctions to detach from the primary tumour [9].
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In addition, they may also invade the surrounding stroma elements and mesothelial cells [10]. Another example is that the tumour cells would have to disrupt endothelial tightness before penetrating into bloodstream [11]. In light of its importance in cancer metastasis, destruction of tight junctions is often regarded as the hallmark of malignancy, and assessment of cell-cell tightness is useful in in vitro pathophysiological and therapeutic studies. Optical-based permeability assay is one of the commonly employed methods to assess barrier function. The absorbance of the small, hydrophilic tag molecules (e.g. Lucifer Yellow) that can readily pass through tight junctions in paracellular spaces, are used to indirectly correlate the tightness of cell layer [2]. Nevertheless, due to its drawbacks of low-throughput and inaccuracy, researchers have switched to label-free electronic methods [12]. Transepithelial electrical resistance (TEER), defined as the ohmic resistance of epithelium, is often used in electronic platforms to quantitatively measure barrier integrity [13]. Epithelial voltohmmeter (EVOM) is one widely used benchtop equipment to measure TEER values. In EVOM measurement, alternating current (AC) square wave in low frequency is applied across a hanging porous filter cultured with cells and thereby TEER can be retrieved
Corresponding author. E-mail address: [email protected] (I.-m. Hsing).
https://doi.org/10.1016/j.snb.2019.126761 Received 11 March 2019; Received in revised form 4 June 2019; Accepted 29 June 2019 Available online 02 July 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Sensors & Actuators: B. Chemical 297 (2019) 126761
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the MDCK cells were seeded in 50% MDCK medium and 50% NPC43 medium. Otherwise mentioned, the reagents and culture media were purchased from Thermofisher, MA.
[14]. Although EVOM measurement is straightforward and easy to handle, its accuracy could be affected by the size difference between electrodes and cell culture [15]. Moreover, the measurement is limited to filter-culturing environment, which gives rise to imaging difficulty. Electrical impedance spectroscopy (EIS) represents another choice to accurately characterize barrier status. By scanning across a range of frequencies, information not just limited to TEER, but cell capacitance that depends on high-frequency signals can also be obtained [16]. However, EIS is relatively expensive and requires highly trained personnel to operate and analyse data [17]. Organic electrochemical transistor (OECT) has been gaining significant attention in the field of organic bioelectronics recently [18]. Particularly, a number of applications have proven OECT’s excellent ability to monitor barrier integrity, under the impacts of epitheliumdestabilizing pathogen Samonella typhimurium and chemicals such as hydrogen peroxide (H2O2) and ethylene glycol tetraacetic acid (EGTA) [19–21]. OECT not only can characterize epithelial cells in a way that is similar to EIS (e.g. impedance spectrum), but also through its own responses (e.g. step function and transconductance), with results comparable to current standards [22,23]. Given its flexibility in fabrication and processing, OECT has been fabricated in versatile formats, including a planar configuration that allows electrical epithelial sensing combined optical imaging, offering advantages over current electronic tools [17,24–26]. OECT is also well-known to be biocompatible, electrochemically stable and has the highest transconductance among the electrolyte-gated transistor technologies thus making it a good choice for various biological applications [27,28]. Previously, we demonstrated the use of a 16-channel OECT to monitor action potential propagation in 2D and 3D primary cardiomyocyte culture [29,30]. Taking advantage of its multi-channel feature, we use the same device here to investigate the invasive behaviour of a newly established Epstein-Barr virus (EBV)-positive nasopharyngeal cancer cell line NPC43 [31]. Nasopharyngeal carcinoma, characterized by the cancer originating from nasopharynx, is a rare malignancy when considering worldwide population. However, it is endemic in Southeast Asia (e.g. Guangdong Province and Hong Kong), with a ranking of most common cancer at 23rd in the world comparing to 4th in Hong Kong [32,33]. With the developed platform, we were able to differentiate several epithelial and cancer cell types and more importantly, spatially map NPC43 invasion on the epithelial Madin–Darby Canine Kidney (MDCK) cells. As a proof-of-concept study, this work suggests that multi-channel OECT could be a promising electronic platform for studying cancer metastasis.
2.2. Immunofluorescence staining Cells were cultured directly on glass slices for immunofluorescence staining. After reaching confluency, they were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min at room temperature. Permeabilization was done by incubating the fixed cells in 0.25% Triton-X in PBS for 20 min. To block non-specific binding, samples were incubated in PBS with 1% bovine serum albumin (BSA) for 30 min. After that, samples were incubated in 5 μg/ml ZO-1 monoclonal antibody in 1% BSA-PBS solution for 1 h at room temperature. Fluorescence tags were conjugated by incubating cells in a mixture of 1 μg/ml goat anti-mouse IgG superclonal secondary antibody, Alexa Fluor 488 (1:1000) and Hoechst 33,342 (1:5000) in PBS for 1 h at room temperature. The cells on cover glass were mounted for fluorescence microscopy. All the involved reagents were procured from Thermofisher, MA. 2.3. OECT fabrication and assembly The OECT array was fabricated on quartz glass, which was precleaned by 10:1 sulfonic acid with hydrogen peroxide (H2O2). Gold tracks were deposited through evaporation and patterned by lift-off lithography. A layer of 2 μm SU-8 2002 (Microchem, MA) was used as passivation layer, with openings defining active channel area. Processing of SU-8 followed standard protocol suggested by manufacturer. PEDOT:PSS Clevios PH500 solution (Heraeus, Germany) consisting of 10% ethylene glycol (Sigma Aldrich, MO) and 1% 3-glycidoxypropyltri-methoxysilane (GOPS; Sigma Aldrich, MO) was spincoated at 3000 rpm and patterned again by lift-off process. A glass ring was assembled onto substrate using Slygard 184 polydimethylsiloxane (PDMS; Dow Corning, MI). 2.4. Frequency-dependent transconductance measurement Each OECT in the array was biased at VDS=−0.4 V, which was set based on the characterization results published previously [30]. Gate electrode utilized here was made of silver chloride-coated silver electrode (Ag/AgCl). Gate inputs were sinusoidal waves at 1–10 K Hz with amplitude of 0.01 V. The drain terminal was connected to a home-built negative feedback circuit for signal amplification, and the voltage signals were monitored by oscilloscope (MSO7034B, Agilent, CA) [34]. The recorded signals were fitted with the following formula:
2. Materials and methods 2.1. Epithelial and cancer cell culture
y = yo + Asin [π (X − Xc )/ ω]
Caco-2 and MDCK cells were kept in Dulbecco's Modified Eagle medium (DMEM) mixed with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) (100 U/ml and 100 μg/ml). NP460 cells were cultured in EpiLife + Defined Keratinocyte-SFM with 1% P/S added. NPC43 cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS, 4μM Rock inhibitor (Y-27632 dihydrochloride, Enzo Life Sciences Inc., Farmingdale, NY) and 1% P/S added. NPC43 cells were obtained from our collaborator Professor Sai Wah Tsao (HKU). For the study of invasion, MDCK was first cultured until confluency. Then the composition of medium was changed to 50% MDCK medium and 50% NPC43 medium. Subsequently, NPC43 cells were seeded to the confluent MDCK culture. For NPC-MDCK co-culture fluorescence imaging, NPC43-MDCK suspension was first prepared in 1:1 ratio. They were then seeded to the glass substrate in 50% MDCK medium and 50% NPC43 medium. Culture condition was maintained at 37 °C, 5% CO2 humidly. For NPC-MDCK co-culture in muti-channel OECT, NPC43 was firstly seeded to the chip and cultured for several days, until the NPC43 cells fully covered 3 to 8 OECT channels. Then
(1)
in which A is the amplitude of measured signal. The values of voltage amplitude at different frequencies were then extracted for calculating normalized transconductance and plotting diagram. The following equation describes transconductance gm:
gm = ΔIDS /ΔVGS
(2)
2.5. Impedance characterization The setup of impedance measurement was similar to that for transconductance measurement, except that signals were measured up to 3000 Hz because of the limitation of multi-meter’s sampling frequency. The gate current IGS was captured by digital multi-meter (34411A, Agilent, CA) and acquired through a built-in LabVIEW program. The recorded signals were again fitted with Eq. (1) and the amplitude value was plugged into ΔIGS of Eq. (3).
|Z| = ΔVGS /ΔIGS 2
(3)
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3. Results and discussion 3.1. Configuration of 16-channel OECT array The configuration of our 16-channel OECT is presented in Fig. 1. The OECT array was fabricated on a glass substrate, with gold interconnects and conducting polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) as channel material. PEDOT:PSS is a commonly used channel material because of its high conductivity, facile processivity and electrochemical stability [35,36]. The devices were then passivated by a thin layer of SU-8, which was defined with openings, exposing the active PEDOT:PSS area. This sensing area and the SU-8 substratum were directly contacted with deposited cells and culture medium. Both of them were proven biocompatible and have been used in a number of in vitro and in vivo experiments [26,30,37–40]. As PEDOT:PSS is p-type doped, the current conducted is based on the mobile holes that are compensated by the negative sulfonate anions of PSS [41,42]. If a positive gate bias is applied, cations in electrolyte will drift into PEDOT:PSS film and compensate PSS−, resulting in lower hole density and thus lower drain-source current intensity IDS [43]. When cells are cultured on top of OECT, the epithelium can limit ion inflow and consequently, resulting in the reduction of the total number of cations in the channel with less electrochemical de-doping of the channel. Therefore, the relative leakiness of cultured cell layer can be reflected by the measured IDS intensity [44].
Fig. 1. A) Configuration of the assembled 16-chanbel OECT platform. B) A closer look of the OECT array. The array composes of 16 OECT channels with spacing of 200 μm, channel width as 6 μm and sensing area as 30 μm × 40 μm C) The OECT array composing of Au electrodes, SU-8 passivation and PEDOT:PSS channel was fabricated on a glass substrate. The gate electrode was immersed into the electrolyte to give bias.
where ΔVGS and ΔIGS corresponds to difference in applied gate voltage and gate current respectively. The impedance spectrum was plotted with impedance against corresponding frequency.
3.2. Determination of cellular epithelial properties by OECT measurement 2.6. Cell culture and on-chip measurement
Before studying cell-cell interactions with multi-channel OECT array, we first attempted to use single-channel OECT for cell-based measurement. The phase-contrast microscopic and immunofluorescence images of OECTs cultured separately with four types of cells (i.e., colorectal adenocarcinoma cells caco-2, MDCK, human telomerase immortalized nasopharyngeal epithelial cells NP460 [45] and NPC43) are shown in Fig. 2A and B respectively. In the fluorescence images of caco-2 and MDCK, the green fluorescence tags that labelled a domain of tight junction protein ZO-1, indicated that caco-2 and MDCK expressed intact epithelial junction networks. Although NP460 also
Before cell seeding, devices were disinfected by 70% ethanol and sterilized by UV light for 30 min. Measurements were taken when cell culture became confluent. During measurement, medium was replaced by PBS and an Ag/AgCl electrode was immersed into the electrolyte to give gate bias. For transconductance measurement, blank data was taken before seeding cells. For impedance sensing, cells were treated with 0.25% trypsin (Thermofisher, MA) before taking the blank data.
Fig. 2. A) Phase-contrast microscopic images of OECTs cultured with caco-2, MDCK, NP460 and NPC43 cells. Scale bar: 100 μm. B) Immunofluorescence images of cells stained with tight junction ZO-1 antibody-Alexa Fluor 488 (green) and nucleus stained with Hoechst 33342 (blue). Scale bar: 10 μm. C) Normalized transconductance plots of OECTs cultured with different cell types. D) Impedance spectrums of OECT with or without MDCK and NPC43 cell cultures. In our report, 16-channel OECT was not able to differentiate cells with different barrier tightness through impedance analysis.
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3.4. Multi-channel OECT array as an non-optical tool for NPC invasion study
showed a mesh-like structure in the phase-contrast image, the cells did not express ZO-1 as caco-2 or MDCK did. Interestingly, instead of forming a confluent monolayer, NPC43 cells aggregated and grew in a non-uniform, multi-layered and colonial manner. NPC43 also did not express ZO-1 as shown in the immunofluorescence image. In OECT, the frequency response is largely dominated by ion transport between electrolyte and channel [27]. Transconductance reflects the rate of ion flow into the channel at a given gate voltage bias. Therefore, if cells are tightly packed (i.e. exhibit high TEER value), the rate of ion inflow would be much lower and the characteristic transconductance value would drop at a much lower frequency. In Fig. 2C, the transconductance profiles of OECTs cultured with different types of cells are presented. Differences in cut-off frequencies, at which the maximal transconductance has dropped abruptly, were observed among different scenarios. Compared to the profile of OECTs cultured with NPC43, OECTs with MDCK or caco-2 cells had a much lower cutoff frequency, suggesting higher TEER values for these two cell lines. This observation is consistent with our EVOM data (data not shown) and other literature reports, where the TEER value of NPC43 was measured to have only 50 Ωcm2 and MDCK or caco-2 were reported to have more than 103 Ωcm2 [13]. As NPC43 cells tended to “huddle” together and did not completely cover OECT as shown in Fig. 2B, it might be easier for ions to leak through. Thus, it was easier for OECTs with NPC43 to be fully dedoped and have maximal transconductance value at a wider range of frequency. On the other hand, the normal epithelial cells NP460 also exhibited a low resistance value, suggesting NP460 was also leaky (Fig. 2C). Impedance spectrums of OECTs cultured with MDCK and NPC43 are shown in Fig. 2D. Similar to the other reported results, the plateau at mid frequencies (i.e. 50–5000 Hz) signifies the presence of cells, in which cell membranes act like dielectrics and paracellular junctions act like resistors [15,23]. Nevertheless, such impedance spectra were not sensitive enough to enable the differentiation between MDCK and NPC43. Therefore, in the following study, impedance measurement would not be used to assess the invasion of NPC43.
After exploring single-channel measurement, we applied our 16channel OECT to assess NPC43 invasion. NPC43 tagged with mCherry (marked in red) and MDCK (displayed in greyscale) were co-cultured on the device and their distribution was shown in Fig. 4A. Here the two representative OECT channel 9 and 12, which was covered by NPC43 and MDCK respectively, were selected for full spectrum transconductance analysis. Consistent with the results discussed in Section 3.2, the two OECTs displayed distinct frequency-dependent transconductance characteristics that represent the “fingerprints” of different cell lines (Fig. 4B). Because their difference in transconductance was more obvious at 100 Hz, a heat map showing the cell distribution was constructed based on the gm value at 100 Hz (Fig. 4C). Such spatial map successfully describes the approximate locations of MDCK and NPC 43 cells with the color intensity reflecting the corresponding transconductance magnitude. The redder the box, the larger the chance that the OECT would be covered by NPC43, and vice versa for MDCK. For example, the box color of channel 7 is white (Fig. 4C), meaning that the OECT had a relatively low transconductance and was most likely covered by the tightly packed MDCK cells, which could be observed in Fig. 4A. Again for channel 5, which has a higher transconductive value as depicted by its red box color (Fig. 4C), was completely covered by the leaky NPC43 cells shown in the microscopic image (Fig. 4A). To conclude, our 16-channel OECT array could be used as a complementary tool to microscopies for non-optical spatial mapping of cancer cell-cell interactions. 4. Conclusion Given the ion-sensitive feature of OECT, epithelial and cancer cell lines possessing different morphologies and TEER values could be distinguished based on frequency-dependent transconductance measurement. By arranging OECTs in a format of 4 × 4 matrix, spatial information on carcinoma invasion of normal epithelium could be retrieved. Moreover, this study partly reveals the invasion mechanism of a new nasopharyngeal cancer cell line NPC43, which was found to be leaky (i.e. has lower TEER value), and tendentious to intrude adjacent cells in a spheroid shape. With these results, the developed OECT platform shows a great potential in continuous monitoring of cancer invasion and metastasis. Integrating OECTs into in vitro barrier models as a complementary non-optical monitoring tool should benefit numerous pathological studies and drug screening assays.
3.3. Assessment of NPC metastatic potential by confocal microscopy Fig. 3 shows the three-dimensional confocal microscopic images illustrating how the suspended NPC43 cells interacted with MDCK cell monolayer. It showed that NPC43 cells proliferated to a spheroid structure and anchored onto MDCK monolayer. The bottom of the NPC43 spheroid was encapsulated by mesh-like ZO-1 network, which is believed to be part of the MDCK layer, while the top was not. It is likely that NPC43 cells might have broken down part of MDCK barrier, suggesting the invasiveness and metastatic potential of NPC43.
Acknowledgements This work was supported by Collaborative Research Funds (project number as [C1013-15G] & [C7027-16G]) and Innovative Technology Fig. 3. A) Confocal image of a NPC43 spheroid invaded into MDCK monolayer. The image was taken 7 days after adding NPC43 cells onto a confluent MDCK layer. B) Top and side views of the same image. Scale bar: 10 μm. Green: tight junctions, ZO-1 antibody-Alexa Fluor 488; blue: nucleus stained by Hoechst 33342; red: actin tagged with mCherry.
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Fig. 4. A) Microscopic image of NPC-43 and MDCK co-culture. NPC43 cells are tagged with red fluorescent marker mCherry. B) Normalized transconductance curves of channel 9 covered by NPC43 and channel 12 covered by MDCK only. C) The transconductance value at 100 Hz of each channel. OECT 1 was damaged so the signal was not avaliable and the respective box was illustrated as blue.
Fund Tier-3 (project number as [ITS/092/17]) of the HKSAR Government. S.Y.Y. would like to thank the Hong Kong Ph.D. Fellowship Scheme of HKSAR government for funding support and Nanosystem Fabrication Facility (NFF) of HKUST for technical support.
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