Induced Proton Perturbation for Sensitive and Selective Detection of

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Induced Proton Perturbation for Sensitive and Selective Detection of Tight Junction Breakdown Hiroaki Hatano, Tatsuro Goda, Akira Matsumoto, and Yuji Miyahara Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05237 • Publication Date (Web): 31 Dec 2018 Downloaded from http://pubs.acs.org on January 4, 2019

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Analytical Chemistry

Induced Proton Perturbation for Sensitive and Selective Detection of Tight Junction Breakdown Hiroaki Hatano†, Tatsuro Goda†*, Akira Matsumoto†,‡, Yuji Miyahara†* †Institute

of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 KandaSurugadai, Chiyoda, Tokyo 101-0062, Japan ‡Kanagawa

Japan

Institute of Industrial Science and Technology (KISTEC), 705-1 Shimoimaizumi, Ebina-shi, 243-0435,

Keywords: Tight junction, Ion-sensitive field-effect transistor, pH perturbation, Trans-epithelial electrical resistance, Clostridium perfringens enterotoxin ABSTRACT: Tight junctions (TJs) in the epithelial cell gap play primary roles in body defense and nutrient absorption in multicellular organisms. Standard in vitro assays lack sensitivity, selectivity, temporal resolution, and throughput for bioengineering applications. Prompted by the rigorous barrier functions of TJ, we developed a TJ assay that senses proton leaks in the cell gap using ion-sensitive field-effect transistors. Upon exposure of Madin-Darby canine kidney (MDCK) cells cultured on a gate dielectric to a calcium-chelator EGTA, ammonia-assisted pH perturbation was enhanced solely in TJforming cells. This was supported by simulations with increased ion permeability in the paracellular pathway. Following administration of Clostridium perfringens enterotoxin as a specific TJ inhibitor to the MDCK cells, our method detected TJ breakdown at a 13-times lower concentration than a conventional trans-epithelial electrical resistance assay. Thus, the semiconductor-based active pH sensing could offer an alternative to current in vitro assays for TJs in pathological, toxicological, and pharmaceutical studies.

INTRODUCTION Tight junctions (TJs) are a cell-cell adhesion modality found specifically in the apical side of polar epithelial and endothelial cells. Claudin,1-4 occluding,5 and the tricellulin family6,7 are the main constituents of TJs. The multiprotein junctional complex acts as a molecular gate by blocking the free permeation of small molecules such as ions and water in the cell gap.8 In addition, TJs are essential for the maintenance of homeostasis in multicellular organisms because they perform a biological defense mechanism by sealing cell gaps. Such epithelial sheets enable nutritional reabsorption in the small intestine9 and form ion gradients in sensory organs.10 As such, TJ failure causes various diseases such as infection,11 ulcers,12 irritable bowel syndrome,13 inflammatory bowel disease,14 and atopic dermatitis.15 In addition, epithelial-mesenchymal transition, known as the primary cause of cancer invasion and metastasis,16 leads to TJ disruption by decreasing expression of claudin and occludin. Therefore, TJ biosensing is an important subject from the viewpoint of cell physiology, pathology, and cancer research. Moreover, TJs are recognized as a new target for drug discovery.17 In the field of drug delivery systems (DDS), intercellular gaps are considered a route for the delivery of hydrophilic compounds into tissues without restrictions of drug size. Thus, researchers are developing drug carriers that lead to temporary TJ breakdown to achieve efficient DDS.18-21 For

example, compounds incorporating Clostridium perfringens enterotoxin (CPE) have been developed for enhancing drug absorption across epithelial barriers in recent years.21,22 The development of a new simple, sensitive, and selective TJ evaluation system may facilitate the understanding of molecular mechanisms involved in physiology and pathology, as well as support the development of novel drugs and carriers. Conventional in vitro TJ evaluation methods include trans-epithelial electrical resistance (TEER) and Transwell®-based transmittance tests. TEER examines cellular state, migration, and proliferation by measuring impedance spectra when alternating current is applied to epithelial sheets.23 Ionic currents flow through the cell sheets, which have resistance and capacitance components.24 TJs can be evaluated as an inherent resistance component because they function as a physical barrier for electrolytes. However, data modeling by an appropriate equivalent circuit is necessary to identify the TJ element because TEER signals reflect whole changes in epithelial sheets.23,25 Whereas, evaluating transmittance is a technique for evaluating TJ formation by optically measuring the amount of low molecular weight marker permeated through epithelial sheets cultured on a Transwell.26-28 Although this simple assay has become a standard research method, it is difficult to evaluate minor TJ breakdown that causes

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leakage of molecules smaller than the marker.25 In addition, the spatiotemporal resolution is low because this assay monitors the macroscopic process of marker transmission.28 Therefore, it is unsuitable for sensing of acute TJ breakdown and high-throughput screening for drug discovery. Ion-sensitive-field effect transistor (ISFET)-based sensors have provided several integrated bioanalytical devices with features in label-free and real-time modalities.29,30 Semiconductor microfabrication technologies allow low-cost production of high-density arrays suitable for multi-parallel assays.30,31 We have succeeded in the development of a cell function analysis system through passive sensing of physiological ions using ISFETs.32,33 Moreover, in recent years, we developed a new technique for detecting cell death and biomembrane injury through pH perturbation induced by instant exposure of cells cultured on ISFETs to weak acid such as the NH4+/NH3 system.34-37 Temporary pH alterations during the ammonia stimulus in a buffered solution originate from ion barrier properties inherent to intact plasma membranes. Therefore, pH perturbations realize noninvasive and reproducible analysis of cell membrane barriers. Indeed, monitoring the leakage of proton and ammonium ions as the smallest indicator (hydrodynamic radius: RH < 0.33 nm) allows us to detect the formation of molecularly sized pores on cell membranes.36,37 Prompted by the ion blocking features of epithelial cell gaps in the presence of TJs, we aimed to extend our ammonia-induced pH perturbation technique to the development of a noninvasive TJ evaluation assay. Changes in pH perturbation upon challenges with a calcium-chelator or cytotoxin were measured. The results showed advanced sensitivity and specificity of our system for TJ breakdown compared with impedance changes obtained by conventional TEER methods. This technique is noteworthy in that it can evaluate TJ barrier functions by simply measuring local pH alterations in a small volume of cell assembly. EXPERIMENTAL SECTION Materials. A poly-L-lysine solution (Mw 150-300 kDa), bis(tris(hydroxymethyl)methylamino)propane (BTP) and Ethylene glycol tetraacetic acid (EGTA) were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Madin-Darby canine kidney (MDCK) cells, NIH/3T3 mouse embryo fibroblast cells, and HeLa cells were purchased from RIKEN BRC under the support by the National BioResource Project (MEXT, Japan). Human Caucasian hepatocyte carcinoma (HepG2) were purchased from DS Pharma Biomedical Japan (Osaka, Japan). Fetal bovine serum (FBS) was purchased by MP Biomedicals Japan (Tokyo, Japan) and used after heat treatment for 1 h at 65°C. CPE was purchased from Bioacademia (Osaka, Japan). An open gate n-channel depletion type ISFET with a 40 nm-thick Ta2O5/140 nm-thick Si3N4/125 nm-thick SiO2 layer as a gate insulator was purchased from Isfetcom Co. Ltd. (Saitama, Japan).

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Cell culture. MDCK and HeLa cells were cultured on a 75-cm2 tissue culture polystyrene dish in Minimum Essential Media containing 1% Penicillin-Streptomycin (PS), 10% FBS and 1% non-essential amino acids with 5% CO2 at 37°C. NIH/3T3 and HepG2 cells were cultured in Dulbecco's Modified Eagle Medium containing 10% FBS and 1% PS. Subconfluent cell cultures were harvested by treating with 0.25% trypsin/EDTA for 5 min at 37°C for passaging at 4.38 × 105 cells cm-2. Scanning Electron Microscope (SEM). Cells were grown on a poly-L-lysine-coated glass in 5% CO2 at 37°C. After washing by DPBS, the cells were fixed by 2.5% glutaraldehyde for 1 h at room temperature. Then, cells were dehydrated step-by-step by treating with 30, 50, 70, 90, 95, and 99% ethanol in Ca2＋BTP buffer (pH 7.4). After drying the ethanol, the samples were coated by a thin layer of gold, for taking images using S-3400N microscope (Hitachi, Tokyo, Japan) under 5 kV accelerating voltage. Immunofluorescence microscopy. After wash by DPBS, the cells were immersed in 4% paraformaldehyde for 15 min for immobilization. Then, the cells immersed with 0.5% Triton X-100 (TX-100) in DPBS for 10 min to permeabilize. After rinse, the cells were immersed by blocking solution (1% bovine serum albumin, 10% normal goat serum, and 0.3 mol L-1 glycine in DPBS) at room temperature for an hour. After wash, anti-zonula occludens-1 (ZO-1) (ab59720; 1:25 dilution in DPBS; Abcam) was used to apply the cells overnight at 4°C. Then, Alexa Fluor 488 goat anti-rabbit IgG H&L (ab150077; 1:500 dilution in DPBS; Abcam) was used to apply the cells at room temperature for 1 h. After wash, cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (1:100 dilution in DPBS; FUJIFILM Wako Pure Chemical Corporation) for 10 min at room temperature. After rinse, the cells were observed by Nikon eclipse Ti-E confocal microscope with LU-N series laser unit (Nikon, Tokyo, Japan). The ammonia-induced pH perturbation assay. A cylindrical glass tube (6 mm in inner diameter) was fixed on an ISFET chip by a thermosetting water-proof epoxy resin. The gate insulator surface was coated by poly-Llysine solution prior to use. Cells were seeded on the chip at 4.38 × 105 cells mm-2 for cultures in 5% CO2 at 37°C. Before measurement, cells were conditioned in a flow of the Ca2+-containing BTP buffer (1 mmol L-1 [BTP], 137 mmol L-1 [NaCl], 1 mmol L-1 [KCl], 1 mmol L-1 [MgCl2], 1mmol L-1 [CaCl2], and 20 mmol L-1 [sucrose]; pH 7.4) using an automated fluidic system at 37°C. In measurement, Ca2+ BTP buffer containing 10 mmol L-1 [NH4Cl] instead of 20 mmol L-1 [sucrose] (pH 7.2) was used. The cellssurrounding solutions with/without NH4Cl were alternately switched by the perfusion system (30 μL min-1) for 1 min interval. The ISFET was set with a voltage of 0.5-1.0 V between the drain and source and 0.5 mV current. Ag/AgCl pellet (Warner Instruments, Hamden, CT, USA) as a reference electrode was used. A LabJack U6-Pro (LabJack Corp., Lakewood, CO) was connected to record between the reference electrode and drain.


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TEER measurement. Cells were grown on a pattern of sputtered gold as working, counter, and reference electrodes on planar glass substrate with poly-L-lysine precoating. Electrochemical impedance spectroscopy was performed at no DC bias (vs. R.E.), the AC voltage of 10 mV, and the frequency of 10-100 000 Hz using VersaSTAT 3 Potentiostat Galvanostat (Princeton Applied Research, Oak Ridge, TN). For the measurements under EGTA treatment, a solution of EGTA (500 mmol L-1 [EGTA], 1 mmol L−1 [BTP], 140 mmol L−1 [NaCl], 4 mmol L−1 [KCl], and 1 mmol L−1 [MgCl2]; pH 7.4) was used to give a final concentration of 5 mmol L-1. CPE was diluted in the Ca2+containing BTP buffer as a stock solution and used to give a desired concentration for measurements. Complex impedance was analyzed by ZView and ZPlot software (Scribner Associates Inc., Southern Pines, NC). Permeability test. To secure the cell attachment, the insert of transwell (Corning Inc., Corning, NY) having a micro-pore of 0.4 µm was coated by poly-L-lysine. MDCK cells were seeded on the insert at 4.38 × 105 cells mm-2, and cultured at 37°C with 5% CO2. For transmittance measurement, cell-culture medium in the insert was replaced with a medium containing the 30 μg mL-1 Lucifer yellow CH (LY). The LY permeability was determined by microplate reader INFINITE 200 (Tecan, Männedorf, Switzerland). RESULTS AND DISCUSSION ISFET system. We developed and constructed a device that combines an ISFET analyzer and electronically controlled perfusion system for in vitro cell function assays (Figure 1A).34-37 Prior to the cell assay, epithelial MDCK cell monolayers were formed on a poly-L-lysine-coated ISFET chip under stationary conditions (Figure 1B). Individual cells became interconnected through cell-cell junctions including TJs during the extended period of incubation.3840 The pH-sensing fluidic system enabled instant ammonium ion exposure to cultured cells. This stimulation was used for real-time noninvasive recording of pH perturbations in the cell microenvironment in a buffered solution as a result of epithelial ion-barrier functions. Namely, we expected to evaluate the integrity of TJs by the degree of pH perturbation during NH4Cl stimulations because TJs block the permeation of protons and ammonium ions in the paracellular pathway of epithelial tissues (Figure.1C). The pH response after 72-h incubation of MDCK cells was −22.4 ± 5.0 mV pH-1, which was significantly lower than values observed after 24-h incubation (−56.5 ± 0.8 mV pH-1) or in cell-free conditions (−55.4 ± 1.8 mV pH-1) (Figure 1D). One possible reason for the decreased Nernstian response is that epithelial monolayers physically separated the cell-ISFET interspace from the bulk solution.38-40 Confirmation of TJ formation. A time course of MDCK cell density was measured (Figure 2A). The cell density peaked at 24 h and gradually decreased thereafter, reaching a plateau at 72 h. This implies the growth of individual cells, as occupation area increased over time. In SEM observations after fixation, intercellular gaps were

observed after 24-h incubation, but had disappeared by 72 h (Figure 2B). Immunostaining revealed expression of ZO1 as a constituent protein for TJs in the periphery of cell gaps after 72-h incubation (Figure 2C). Absolute impedance and phase angle measured by TEER changed dramatically during the initial 48 h of incubation (Figure 2D). Bode plots were successfully modeled by an equivalent circuit comprising serial connections of the resistance of bulk solution (Rsol), resistance and capacitance of the epithelial cell sheet (Rcell and Ccell), and capacitance at the electrode/cell interface (Cint), i.e., [Rsol(RcellCcell)Cint].23 Among these elements, a change in Rcell is responsible for TJ formation/breakdown because the ionic current passes through transcellular/paracellular pathways.24 TJ formation was also evaluated by a Transwell-based transmittance assay using LY as a cellimpermeable low molecular weight fluorescent marker. LY permeation was dramatically reduced with 48-h incubation and completely blocked at 72 h (Figure 2E). Therefore, we regarded successful TJ formation to have occurred at 72 h and applied this condition for subsequent assays. TJ evaluation by pH perturbation. We previously showed that pH perturbation is induced by an instant NH4Cl exposure to cells cultured on a gate dielectric even in a buffered condition because of the ion barrier properties of healthy cell membranes.34-37 Prompted by the ion blockade of TJ in intercellular gaps,1,41,42 we aimed to evaluate TJ breakdown by pH perturbation in a nondestructive way. EGTA, a calcium-chelating agent, was used as a model reagent for inducing TJ breakdown. By chelating extracellular Ca2+, a series of cell-cell junctions including TJs and adherence junctions are dissociated.43,44 We measured NH4Cl-induced pH perturbations directly from the gate potential when MDCK cell sheets were subjected to either 1 mmol L-1 EGTA or 1 mg mL-1 TX-100 (Figure 3A). We defined the recovery from pH overshoots of upward and downward as ΔVup and ΔVdown, respectively. Both ΔVup and ΔVdown increased significantly 2 min after EGTA treatment of MDCK cells cultured for 72 h, but they remained unchanged for cells at 24 h (Figure 3B). However, pH perturbations decreased when cell lysis occurred as a result of TX-100 exposure, which was confirmed by optical microscope.36,37 Increases in ΔV were exclusively observed with EGTA treatment of TJ-forming MDCK cells. ΔVdown, which showed a larger signal than ΔVup, was used in subsequent evaluations of TJ breakdown. To understand the molecular dynamics for decreases of ΔV, pH perturbations were numerically determined by a mass transfer model in the cell microenvironment (Figure S1, S2).36 TJ breakdown was evaluated as an increase in the permeability of NH4+ at the paracellular pathway. Changes in ΔV were successfully reproduced by PNH4+ values (Figure S1). The model denotes that ΔV increased significantly by increasing PNH4+ in paracellular pathways. Accordingly, numerical calculations support our experimental results that ΔV increases as a result of epithelial TJ breakdown. Moreover, trends were similar between ΔVup and ΔVdown in



these calculations. The model also denotes that ΔV decreases significantly when cell membranes lyse.36,37 Selectivity for TJ breakdown. To investigate whether an increase in ΔVdown by EGTA is a specific signal for TJ breakdown, we performed our ISFET assay using MDCK, HepG2, HeLa, and NIH/3T3 mouse embryonic fibroblast cells (Figure 4A). In observations of cell morphology with SEM, cell gaps appeared in response to EGTA only in epithelial MDCK cells cultured for 72 h. For other cells and MDCK at 24 h, gaps inherently existed before treatments. In accordance with SEM observations, a significant increase in ΔVdown was exclusively observed for 72-h cultured MDCK cells exposed to EGTA (Figure 4B). The degree of pH perturbation did not change significantly for other cell types examined, or even decreased for HepG2 at 24 h and 72 h. Therefore, increased ΔVdown is a specific phenomenon caused by TJ breakdown. We also verified whether TJ breakdown can be specifically detected by conventional TEER measurements or not (Figure S3). We extracted |Z| and θ at 10 kHz as characteristics of TJ (Figure 4C).45-47 |Z| was decreased significantly for non-TJ forming MDCK at 24 h and HepG2, as well as for TJforming MDCK at 72 h. θ changed significantly for HepG2 as well as MDCK at 72 h. As shown, |Z| and θ were not specific to TJ breakdown as compared with ΔV for the following two reasons.23,25 First, the impedance consists of many resistive and capacitive components at the cell/electrode interface (Figure 2D). Second, EGTA takes up Ca2+ from cells, leading to alterations of adherence and desmosome junctions in addition to TJ disruption. Therefore, most likely, TEER contains information about the whole cell sheet including individual cells and cell-cell junctions. Sensitivity for TJ breakdown. pH perturbation is altered by the permeability of the smallest ions, i.e. H+ and NH4+ (RH < 0.33 nm),34-37 in intercellular gaps. Therefore, we expected to measure TJ breakdown with high sensitivity using our ISFET assay. CPE, which specifically binds to claudin to induce TJ breakdown, was used as a model cytotoxin.48-50 First, we measured pH perturbations in MDCK cells cultured for 72 h in the presence of 0.01, 0.05, or 0.1 μg mL-1 CPE (Figure 5A). The normalized potential signal (ΔVi/ΔV0−1) indicated that the proportion of pH perturbation increased over time with ≥ 0.05 μg mL-1 CPE (Figure 5B). For comparison, TEER measurements were also performed using MDCK cells cultured for 72 h at varying concentrations of CPE (0.1, 0.5, or 1 μg mL-1) (Figure 5C). |Z| and θ apparently changed over time in the frequency window with ≥ 0.5 μg mL-1 CPE. Bode plots were modeled by the equivalent circuit (Figure 2D) to analyze CPE-induced TJ breakdown using normalized epithelial cell sheet resistance (1−Ri/R0) and capacitance (Ci/C0−1) (Figure 5D). Again, time-dependent changes in normalized resistance and capacitance were observed with ≥ 0.5 μg mL-1 CPE. Changes in resistance were attributed to TJ breakdown. The cell capacitance originates from the cell membrane. Therefore, we speculate that the capacitive changes are caused by an increase in the effective surface area of cell membranes as a result of TJ breakdown. We

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determined the sensitivity for TJ breakdown by CPE from pH perturbation and sheet resistance results at 30 min (Figure 5E). The limit of detection (LOD) of our assay was 0.03 μg mL-1 based on three times the difference of standard deviation (SD) from values at the lowest CPE concentration. The value was approximately 13-times lower than the LOD of TEER (0.4 μg mL-1). Notably, the CPEtriggered TJ breakdown identified by ISFET (≥ 0.03 µg mL1) and TEER (≥ 0.4 µg mL-1) cannot be judged by cell morphology in SEM observations (Figure S4). The advanced sensitivity of the pH perturbation assay may derive from the high signal-to-background ratio in TJ sensing. In contrast, TEER measurements include whole resistive and capacitive elements in the epithelial cell sheet.45-47 Therefore, changes in the resistance and capacitance by TJ breakdown is buried in the relatively large background noise. Discussion. We succeeded in applying active pH sensing for evaluating TJ barriers. The advantages of our assay are the ability to detect TJ breakdown with high sensitivity and selectivity in real-time using a noninvasive approach. Specific TJ sensing is not possible for conventional TEER or transmittance assays (Figure 4). In addition, distinction of TJ breakdown from cell membrane injuries is achieved by focusing on changes in the direction of pH perturbation (Figure 3). Compared with the passive mode as the majority of cell-based ISFET sensing, our active pH sensing which measures the pH transient by the NH4+ stimulus is less prone to signal drift and biofouling. 36,37 This is advantageous in many applications that require an extended period of culture to mature the cells as exemplified by the formation of microvilli for intestinal tissue models and by studies on endothelial cell adaptation in models of stem cells-based organoid and organ-onchips. Moreover, our assay is tolerant to geometrical effects that are the major hurdle in TEER. With TEER, the placement of the electrodes and the geometry of channels cause that the impedance signals are not equally selective to each area of the cell membrane.51 The ISFET transducer is advantageous in developing a high-density sensor array in a microfluidic chip for multi-parallel analysis with the virtue of a CMOS-compatible fabrication process.52-55 Therefore, our semiconductor-based device could be effective for in vitro evaluation of TJ breakdown by biological toxins and chemical compounds. In recent years, TJs have garnered increasing attention because endothelial cells have manifested plastic features in organ repairs and maladaptive fibrosis/tumorigenesis.11-14,56 In addition, TJs have been recognized as a new drug target17 since the discovery of TJ-constituting protein structures.8 In the DDS field, crossing epithelial barriers is a primary concern for realizing efficient DDS to target organs and tissues.18-21 Our achievements in measuring TJ barrier functions will be useful for elucidating adaptive mechanisms of endothelial cells and developing drugs/carriers. CONCLUSIONS We developed a new assay for TJ function in live epithelial sheets using a pH-sensing transistor. Ion


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impermeability of TJs was monitored as pH perturbations at the ISFET/cell interface by NH4Cl stimulation using a perfusion system in a buffered condition. Increased pH perturbation upon EGTA stimulation was found to be specific to TJ breakdown, in sharp contrast to impedance changes in TEER, which are commonly observed for nonTJ forming cells. The LOD for CPE was about 13-times greater in the pH perturbation assay than TEER. Thus, our semiconductor-based active pH-sensing system may have applications in real-time noninvasive monitoring of TJ barrier functions with high sensitivity and selectivity.

ASSOCIATED CONTENT Supporting Information. Simulation results on the time course of gate potential during the NH4Cl stimulation (Figure S1, S2); Time course of Bode plots following EGTA treatments to various cell types (Figure S3); SEM observation of MDCK cells with 72 h cultures before and after CPE treatment for 30 min (Figure S4).

AUTHOR INFORMATION *Corresponding Author Phone: +81 3 5280 8095. Fax: +81 3 5280 8135. Email: [email protected]; [email protected]

ORCID Tatsuro Goda: 0000-0003-2688-8186 Yuji Miyahara: 0000-0003-2703-0958

Author Contributions H.H. conducted the experiments. H.H., T.G., and A.M. wrote the paper. T.G. conceived the research. T.G. and Y.M. supervised the project.

Funding Sources We are grateful for financial support in part from the Nakatani Foundation of Electronic Measuring Technology Advancement, the Tateisi Science and Technology Foundation, and KISTEC.

Notes The authors declare that they have no competing interests.

ACKNOWLEDGMENT We acknowledge Dr. Y. Imaizumi at TMDU for discussion and support. Electrodes for TEER were fabricated under the support by NIMS Nanofabrication Platform in Nanotechnology Platform Project sponsored by MEXT, Japan

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Page 7 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. An in vitro evaluation method for intercellular TJs. (A) A schematic diagram showing a combined system of ISFET and automated fluidics. MDCK cells with TJs were cultured on the poly-L-lysine-coated gate insulator of the ISFET in an incubator. The perfusion system enabled instant switching of cell-surrounding solutions with/without NH4Cl. (B) An optical micrograph of the ISFET chip covered by MDCK cell sheets after 72-h incubation. Scale bar = 150 μm. (C) Ions permeate through cell gaps as a result of TJ breakdown. TJ is mainly composed of claudin, occludin, JAMs, and ZO-1. (D) Gate potential as a function of solution pH with/without MDCK cells at 24- or 72-h incubation. Data are shown as the mean±SD (N = 3).

Figure 2. Confirmation of TJ formation in MDCK cell sheets. (A) Time course of MDCK cell density on an ISFET chip during incubation. Data are shown as the mean±SD (N = 3). (B) SEM images of MDCK cells after incubation for 24, 48, or 72 h. Scale bar = 30 μm. (C) Immunostaining of ZO-1 (green) in MDCK cells at 24 and 72 h cultures. Blue: nuclei. Scale bar = 10 μm. (D) Bode plots for MDCK cells on poly-L-lysine-coated Au electrodes at different time points. The illustration shows the equivalent circuit for an epithelial cell sheet. (E) Transmittance of LY through Transwell cultures of MDCK cells at 24, 48, and 72 h incubations. Mean±SD (N = 3).



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Figure 3. Changes in the degree of pH perturbation by TJ breakdown. (A) Time course of gate potential in MDCK cells cultures at 72 h during the NH4Cl-induced pH perturbation assay with/without exposures to 1 mmol L-1 EGTA or 1 mg mL-1 TX-100. (B) ΔVup and ΔVdown of MDCK cells cultured for 24 or 72 h following treatment with EGTA or TX-100. Mean±SD (N = 3). *p < 0.05, **p < 0.01.

Figure 4. Changes in pH perturbation and TEER by EGTA exposure. (A) Time course of gate potential during NH4Cl exchanges with 1 mmol L-1 EGTA exposure to various cell types. SEM images showing cell morphologies before and after 4-min exposures to EGTA. Scale bar = 30 μm. (B) ΔVdown before and after EGTA treatments for various cell types. Data are shown as the mean±SD (N = 3). *p < 0.05, **p < 0.01. (C) Changes in absolute impedance and phase angles at 10 kHz following EGTA treatment at different cell types. Mean±SD (N = 3). *p < 0.05, **p < 0.01.


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Figure 5. Detection of TJ breakdown by CPE. (A) Time course of pH perturbations with exposure of MDCK cells at 72 h to 0.01, 0.05, or 0.1 μg mL-1 CPE. (B) Changes in the normalized degree of pH perturbation (ΔVi/ΔV0−1) as a function of CPE concentration and time. Data are shown in the mean±SD (N = 3). (C) Bode plots at different time points following exposure of MDCK cells at 72 h to 0.1, 0.5, or 1.0 μg mL-1 CPE. (D) Changes in normalized cell resistance (1−Ri/R0) and cell capacitance (Ci/C0−1) in the equivalent circuit as a function of CPE concentration and time. Mean±SD (N = 3). (E) Signal comparison between ISFET and TEER assays after CPE treatment for 30 min in the semi-logarithmic plot. Dotted lines indicate three times the difference of SD from the baselines. Solid lines show the fitting of the plots by linear regression. Mean±SD (N = 3).


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Figure 1. An in vitro evaluation method for intercellular TJs. (A) A schematic diagram showing a combined system of ISFET and automated fluidics. MDCK cells with TJs were cultured on the poly-L-lysine-coated gate insulator of the ISFET in an incubator. The perfusion system enabled instant switching of cell-surrounding solutions with/without NH4Cl. (B) An optical micrograph of the ISFET chip covered by MDCK cell sheets after 72-h incubation. Scale bar = 150 μm. (C) Ions permeate through cell gaps as a result of TJ breakdown. TJ is mainly composed of claudin, occludin, JAMs, and ZO-1. (D) Gate potential as a function of solution pH with/without MDCK cells at 24- or 72-h incubation. Data are shown as the mean±SD (N = 3). 174x51mm (300 x 300 DPI)



Figure 2. Confirmation of TJ formation in MDCK cell sheets. (A) Time course of MDCK cell density on an ISFET chip during incubation. Data are shown as the mean±SD (N = 3). (B) SEM images of MDCK cells after incubation for 24, 48, or 72 h. Scale bar = 30 μm. (C) Immunostaining of ZO-1 (green) in MDCK cells at 24 and 72 h cultures. Blue: nuclei. Scale bar = 10 μm. (D) Bode plots for MDCK cells on poly-L-lysine-coated Au electrodes at different time points. The illustration shows the equivalent circuit for an epithelial cell sheet. (E) Transmittance of LY through Transwell cultures of MDCK cells at 24, 48, and 72 h incubations. Mean±SD (N = 3). 168x91mm (300 x 300 DPI)


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Page 13 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Changes in the degree of pH perturbation by TJ breakdown. (A) Time course of gate potential in MDCK cells cultures at 72 h during the NH4Cl-induced pH perturbation assay with/without exposures to 1 mmol L-1 EGTA or 1 mg mL-1 TX-100. (B) ΔVup and ΔVdown of MDCK cells cultured for 24 or 72 h following treatment with EGTA or TX-100. Mean±SD (N = 3). *p < 0.05, **p < 0.01. 82x64mm (300 x 300 DPI)



Figure 4. Changes in pH perturbation and TEER by EGTA exposure. (A) Time course of gate potential during NH4Cl exchanges with 1 mmol L-1 EGTA exposure to various cell types. SEM images showing cell morphologies before and after 4-min exposures to EGTA. Scale bar = 30 μm. (B) ΔVdown before and after EGTA treatments for various cell types. Data are shown as the mean±SD (N = 3). *p < 0.05, **p < 0.01. (C) Changes in absolute impedance and phase angles at 10 kHz following EGTA treatment at different cell types. Mean±SD (N = 3). *p < 0.05, **p < 0.01. 170x102mm (300 x 300 DPI)


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Figure 5. Detection of TJ breakdown by CPE. (A) Time course of pH perturbations with exposure of MDCK cells at 72 h to 0.01, 0.05, or 0.1 μg mL-1 CPE. (B) Changes in the normalized degree of pH perturbation (ΔVi/ΔV0−1) as a function of CPE concentration and time. Data are shown in the mean±SD (N = 3). (C) Bode plots at different time points following exposure of MDCK cells at 72 h to 0.1, 0.5, or 1.0 μg mL-1 CPE. (D) Changes in normalized cell resistance (1−Ri/R0) and cell capacitance (Ci/C0−1) in the equivalent circuit as a function of CPE concentration and time. Mean±SD (N = 3). (E) Signal comparison between ISFET and TEER assays after CPE treatment for 30 min in the semi-logarithmic plot. Dotted lines indicate three times the difference of SD from the baselines. Solid lines show the fitting of the plots by linear regression. Mean±SD (N = 3). 170x129mm (300 x 300 DPI)



TOC graphic 81x42mm (300 x 300 DPI)


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Induced Proton Perturbation for Sensitive and Selective Detection of

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