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Live monitoring of microenvironmental pH based on extracellular acidosis around cancer cells with cell-coupled gate ion-sensitive field-effect transistor Toshiya Sakata, Haruyo Sugimoto, and Akiko Saito Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03070 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 8, 2018

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Analytical Chemistry

Live monitoring of microenvironmental pH based on extracellular acidosis around cancer cells with cellcoupled gate ion-sensitive field-effect transistor Toshiya Sakata*, Haruyo Sugimoto and Akiko Saito Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan 113-8656 *Corresponding author. E-mail: [email protected] CORRESPONDING AUTHOR FOOTNOTE: Affiliation: Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan 1138656, TEL: +81-3-5841-1842, FAX: +81-3-5841-1842

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ABSTRACT: We demonstrated the live monitoring of cellular respiration using an ion-sensitive field-effect transistor (ISFET), focusing on different types of living cells, namely, cancer and normal cells. In particular, we realized the label-free, real-time, and noninvasive monitoring of microenvironmental pH behavior based on extracellular acidosis around cancer cells in the long term and in situ. The change in interfacial pH (∆pHint), which was analyzed based on the change in interfacial potential (∆Vout), at the cell/gate nanogap interface gradually decreased for every cell-based ISFET. Moreover, the ∆pHint for cancer cells shifted by a factor of five to six, which was larger than that for normal cells. This is because cancer cells cause dysbolism and are activated, thereby suppressing oxidative phosphorylation in mitochondria so as not to induce their apoptosis. Therefore, cancer cellular respiration proceeds via the glycolysis pathway, through which lactic acid is eventually released. Additionally, the pH sensitivity of the ISFET device was maintained even when the device was immersed into a cell culture medium for 24 h and 1 w; thus, the effect of nonspecific adsorption of proteins contained in the medium on the pH sensitivity of the ISFET device was negligible in the live monitoring of cellular respiration. KEYWORDS: cancer cell; cellular respiration; microenvironmental pH; field-effect transistor; live monitoring BRIEFS: In this study, we demonstrated the live monitoring of microenvironmental pH behavior based on extracellular acidosis around cancer cells in the long term and in situ.


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INTRODUCTION Cancer cells produce adenosine triphosphate (ATP) through glycolysis rather than oxidative phosphorylation, which is well known as the Warburg effect, although glycolysis yields less ATP and occurs in hypoxic tissues that cannot obtain ATP through aerobic respiration,1-4 resulting in the generation of lactic acid. In particular, cancer cells are activated via the suppression of oxidative phosphorylation in mitochondria so as not to induce their apoptosis,5 resulting in their proliferation and metastasis. Cellular respiration, which proceed aerobically or anaerobically, involves a series of metabolic reactions to produce ATP by the uptake of nutrients such as glucose, then living cells release waste products such as carbon dioxide or lactic acid. On the basis of the amount of such waste products dissolved in a medium, cellular metabolism is noninvasively monitored as a change in pH for living cells in situ, resulting in the elucidation of the mechanisms of disease development and the investigation of drug effects. Additionally, extracellular acidosis is a feature of cancer cells; thus, the microenvironmental pH of cancer cells is different from that of normal cells. Therefore, various detection methods are proposed to evaluate the acid-producing activity of cancer cells and the effects of microenvironmental pH on their metabolism.6-11 Fluorescent probes have often been developed to image the pH around tumors,9-11 but optical setups are mostly expensive and the preparation of reagents is timeconsuming. Even pH meters have not contributed to the long-term monitoring of pH in the immediate vicinity of cancer cells in situ, because living cells are not directly cultured on the sensors.6-8 As one of the sensing technologies to monitor cellular respiration, a cell-coupled gate ionsensitive field-effect transistor (ISFET) (Fig. 1) is used to monitor it noninvasively as a change in pH at the cell/gate nanogap interface (∆pHint) in real time.12-18 This device enables the label-


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free, real-time, and noninvasive monitoring of pH behavior around cancer cells at the cell/gate interface, that is, pH behavior in the immediate vicinity of cancer cells. Because the gate insulator used as an electrode usually consists of an oxide with hydroxyl groups at the surface in a solution, the ISFET sensors are sensitive to changes in the concentration of hydrogen ions with positive charges based on the equilibrium reaction (-OH2+ ↔ -OH ↔ -O-); thus, they can be utilized as pH sensors (Fig. S1(a) in Supporting Information).19,20 Thus, ∆pHint due to cellular respiration can be monitored at the cell/gate interface on the basis of the amount of respiration products released from cells. In fact, various respiratory activities of cells, such as rat pancreatic β cells,12 a single mouse embryo,13 and bovine chondrocytes,14 or respiratory activities based on the allergic responses of mast cells15 and autophagy under nutrient starvation18 on a gate were monitored noninvasively, quantitatively, and continuously as ∆pHint using the cell-coupled gate ISFET sensors in our previous works. Moreover, ∆pHint at the cell/gate nanogap interface was clearly observed using a pH fluorescent indicator fixed at the cell membrane (the outside layer of cells).16 However, it remains unclear whether such electrical signals are valid with different types of cells. In particular, cancer cells would show a different respiration activity from that of normal cells; therefore, in this study, we investigated the live monitoring of cellular respiration as a change in pH at the cell/gate nanogap interface using the cell-coupled gate ISFET sensors, focusing on different cell types, namely, cancer and normal cells. That is, the microenvironmental pH of cancer cells is continuously monitored in situ. In this study, cellular respiration can be simply induced by changing a cell culture medium with a fresh one containing various nutrients such as serum and glucose. Moreover, the detection limit of cellular respiration using the cell-coupled gate ISFET sensors should be investigated, because a cell culture medium used as a measurement solution


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includes various ions and cell-related compounds resulting in shielding ionic or molecular charges at the gate surface. That is, the Debye length at a solution/gate interface based on the electrical double layer (EDL) structure should become very small in a culture medium with high ionic strengths for the potentiometric biosensors. In general, FET biosensors have the limit of detection depending on the Debye length,21-23 as expressed by λ=ට

ఌ బ ఌ ೝ ௞ಳ ் ଶேಲ ௘ మ ூ

, (1)

where I is the ionic strength of an electrolyte, ε0 is the permittivity of free space, εr is the dielectric constant, kB is the Boltzmann constant, T is the absolute temperature, NA is the Avogadro number, and e is the elementary charge.24 For example, the Debye length is calculated to be 0.75 nm even in a 150 mM NaCl solution. Therefore, a cell-coupled gate ISFET sensor is assumed to be insensitive to ionic charges of macromolecules such as proteins nonspecifically adsorbed on the gate, whereas small ions such as hydrogen are expected to be specifically detected at the oxide gate of the ISFET sensors, the surface of which has hydroxyl groups in a solution showing the above equilibrium reaction, regardless of the nonspecific adsorption of proteins. However, such an effect of a measurement environment in a cell culture medium on the electrical signal of the cell-coupled gate ISFET sensor are still not clearly demonstrated. Hence, the pH sensitivity of the ISFET sensors used in a culture medium is also analyzed in this study.

EXPERIMENTAL SECTION Electrical measurement using ISFET device. We used a silicon-based n-channel depletionmode FET with a Ta2O5/SiO2 (100 nm/50 nm) layer as a gate insulator with a width (W) and a length (L) of 340 and 10 µm, respectively (ISFETCOM Co., Ltd.). A glass ring with a diameter of 18 mm (1 ml) was fixed on a substrate excluding a gate sensing area using


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polydimethylsiloxane (PDMS) (Fig. S2 in Supporting information). The Ta2O5 thin film was used as a passivation layer to prevent the leakage currents as well as a pH-responsive layer in a buffer solution. Gate voltage (VG)–drain current (ID) electrical characteristics were measured using a semiconductor parameter analyzer (B1500A, Agilent). A change in VG in VG–ID electrical characteristics was estimated as a threshold voltage (VT) shift, which was evaluated at a constant ID of 700 µA and at a constant of drain voltage (VD) of 2 V. A Ag/AgCl reference electrode with a KCl solution was connected to the measurement solution through a salt bridge [Fig. 1(a)]. As shown in Fig. S3 (Supporting information), the time course of the surface potential at the gate surface (Vout) was monitored using a source follower circuit25 with which the potential change at the interface between an aqueous solution and a gate insulator can be read out directly at a constant ID (RadianceWare Inc.). In this study, VD and ID were set to 2.5 V and 700 µA, respectively. The gate size was enough to detect signals from cells with a diameter of about 20– 30 µm. In particular, W/L at the channel was designed to be adequate for the sensitivity of ID for VG. Additionally, the thickness of gate insulating layers was enough to prevent leakage currents through the insulating layer caused by bias application and invasion of ions in solutions. As the pH measurement solution, phosphate buffer solutions were prepared with pHs from 5.8 to 8.0 at intervals of 0.2 by controlling the mixing ratio of Na2HPO4 to NaH2PO4. The standard buffer solutions with pHs of 4.01, 6.86, 7.41, and 9.18 (Wako Pure Chemical Industries, Ltd.) were also prepared. For the live monitoring of cells, cellular respiration was induced by changing the cell culture medium for preculture with a fresh one, which contained glucose and serum. To examine the effects of background noises, such as the changes in temperature and ion concentration, on


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electrical signals, an ISFET without cells was utilized as a control sensor. We set a standard signal drift of less than a few mV per hour as the steady potential baseline. Cell culture. HeLa and HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) with 1 g/L glucose and 10% fetal bovine serum (FBS) including 50 U/mL penicillin and 50 µg/mL streptomycin on a conventional cell culture dish (Falcon® cell culture dishes) in an incubator (37 °C, 5% CO2) for 3 days. They were then transferred to the gate insulator of ISFET for electrical monitoring, where the same culture medium was added and kept at 37 °C and 5% CO2 for 24 h as the preculture [Figs. 1(b) and (c)]. The number of cells seeded on the gate insulator of ISFET was controlled to 1×105 cells/mL for both cells. HUVECs were cultured in endothelial cell growth medium (EGM-2, Lonza) containing 1 g/L glucose and 2% FBS and vascular endothelial growth factor (VEGF, Lonza) on a collagencoated dish (Falcon® cell culture dishes) in an incubator (37 °C, 5% CO2), and harvested using trypsin after cell culture for 1 week. Then, they were transferred to the gate insulator of ISFET, where the same culture medium was added and kept at 37 °C and 5% CO2 for 1 week as the preculture (Fig. 1(d)). The gate surface was coated with 0.1% gelatin in advance. The number of cells seeded on the dish was controlled to 1×105 cells/mL for HUVECs. There was no significant difference in the buffering effect of different culture media in this study because an appropriate medium was utilized to culture each cell.

RESULTS AND DISCUSSION Real-time monitoring of interfacial pH at cancer or normal cell/gate nanogap. For respiration monitoring, human cervical carcinoma (HeLa) and human hepatocellular carcinoma (HepG2) cells were utilized as the cancer cell models, and human umbilical vein endothelial


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cells (HUVECs) were utilized as the normal cell model in this study. Each cell was confluently cultured on the gate (Figs. 1(b), (c), and (d)). That is, the whole gate sensing area was substantially covered by living cells during the electrical measurements. Figure 2 shows the change in surface potential (∆Vout) at the gate of ISFET during incubation for cell culture. As shown in Fig. 2, ∆Vout for every cell-coupled gate ISFET (Figs. 2(a), (b), and (c)) gradually increased after changing the culture medium, which was intended to resupply fresh nutrients such as glucose in the culture medium, although the control sensor without cells hardly showed electrical responses even after changing the medium (Fig. 2(d)). The positive shift of ∆Vout for the cell-coupled gate ISFETs indicated the increase in the numbers of positive charges at the gate surface, that is, the increase in H+ concentration at the cell/gate interface. This is because the interfacial pH variation due to cellular respiration was monitored at the cell/gate interface on the basis of the amount of carbon dioxide or lactic acid generated by aerobic or anaerobic cellular respiration and dissolved in a medium. Indeed, the interfacial pH behavior at a cell/substrate nanogap was clarified by laser scanning confocal fluorescence microscopy.16 However, the amount of ∆Vout differed among the cells used in this study; ∆Vout for cancer cells (Figs. 2(a) and (b)) shifted more largely than that for normal cells (Fig 2(c)). In the case of normal cells, respiration predominantly occurs via the oxidative phosphorylation pathway in an aerobic environment. On the other hand, cancer cells cause dysbolism in which cellular respiration proceeds via the glycolysis pathway, through which ATP is more simply yielded than through the oxidative phosphorylation pathway and lactic acid is eventually released even under aerobic condition.1-4 Moreover, cancer cells release CO2 through the pentose phosphate pathway, resulting in the decrease in pH around tumors.26-28 Extracellular acidosis is a feature of cancer cells, and so the microenvironmental pH of cancer cells is different from that of normal cells, as


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reported previously.6-11 Thus, the change in pH based on a metabolic disorder of cancer cells should be larger than that for normal cells. The saturation of ∆Vout was observed with the lapse of time, which may have resulted from the consumption of nutrients based on the cellular metabolism in the culture medium. Moreover, in the early stage after exchanging the cell culture medium with a fresh one, hydrogen ions generated by cellular respiration were localized at the cell/gate nanogap, the concentration of which was monitored as ∆pHint at the nonequilibrium state using the ISFET device, then they diffused to the bulk solution, resulting in the equilibrium state of the pH at the cell/gate nanogap. The ISFET sensors are sensitive to changes in the concentration of hydrogen ions with positive charges based on the equilibrium reaction (-OH2+ ↔ -OH ↔ -O-), as shown in Fig. S1(a) (Supporting Information). The gate insulator of ISFET used in this study was composed of Ta2O5/SiO2, as shown in Fig. 1. The oxide surface is mostly covered by hydroxyl groups in solutions, which interact with hydrogen ions in an equilibrium reaction. In Fig. S1(b) (Supporting Information), pH was changed from 1.68 to 9.18 at designated time points, as shown in the left graph; that is, the measurement solution was changed with next buffer solution at the time point indicated by an arrow. As a result, the correlation between the gate voltage and the pH corresponding to electrical responses is shown in the right graph (Fig. S1(b) in Supporting Information). The averaged gate voltages for four ISFETs were calculated and plotted for the last 1 minute for each pH response. The gate voltage as a function of pH variation showed about 58 mV/pH on average near the Nernstian response at 25 °C, which was the ideal response of interfacial potential at the solution/gate interface.19,20 Considering the average pH sensitivity (58 mV/pH) of the ISFETs used in this study, ∆Vout shown in Fig. 2 was converted to ∆pHint for each cell-coupled gate ISFET, as shown in Fig. 3. Thus, cellular respiration can be directly monitored


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as ∆pHint at the cell/gate nanogap interface using the ISFET. In fact, ∆pHint for cancer cells (HeLa and HepG2 cells) was found to be shifted by a factor of five to six, which was larger than that for normal cells (HUVECs). As shown in the above results, we found that ∆pHint for cancer cells was larger than that for normal cells. This means that ionic behaviors at the cell/gate nanogap interface should be focused on for monitoring cellular respiration in real time using the ISFET sensor. That is, the nanogap interface is regarded as the closed nanospace between the cell and the gate (Fig. 4), where released ions and biomolecules are concentrated, resulting in the increase in H+ concentration in cellular respiration. In fact, previous works showed a gap of approximately 50150 nm at the cell/substrate interface,29-31 and the pH behavior at the cell/substrate nanogap has been observed by laser scanning confocal microscopy using phospholipid fluorescein inserted at the cell membrane.16 The results showed that the pH at the cell/substrate interface gradually decreased (shifted to acidity) and was lower than that at the cell/medium interface, which means that hydrogen ions were concentrated in the nanogap space. A platform based on the cell-coupled gate ISFET sensor is very useful for the in situ monitoring of ∆pHint based on cellular functions. This study shows that for the respiratory monitoring of living cells, it is required that the ISFET sensor should show a high sensitivity to pH variation which is induced at the cell/gate nanogap interface. If various types of membrane sensitive to other ions such as sodium and potassium ions are coated on all gates of an arrayed ISFET chip, various ionic behaviors of living cells may be simultaneously monitored in real time. In this case, we believe that it is very important for living cells to be directly adhered onto the gate sensing surface because the change in ion concentration based on cellular functions can be significantly monitored at the cell/gate nanogap interface.


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Effect of cell culture medium on pH sensitivity of ISFET. Figure 5 shows ∆Vout for pH using the ISFET immersed into a cell culture medium. An ISFET devices was immersed into a cell culture medium for 0 h, 24 h, and 1 w at 25 °C to investigate the effect of nonspecific adsorption of ions and biomolecules such as proteins contained in the medium on pH sensitivity. In this case, cells were not cultured on the gates. DMEM with 10% FBS was utilized as the cell culture medium. At 0 h, the ISFET device, which was not immersed in the medium, showed a pH sensitivity of 59.4 mV/pH near the Nernstian response at 25 °C (Fig. 5(a)). Moreover, the pH sensitivity was almost maintained at approximately 58 mV/pH even when the ISFET devices were immersed into the cell culture medium for 24 h and 1 w (Figs. 5(b) and (c)), although the nonspecific adsorption of proteins was observed using fluorescein-isothiocyanate (FITC) (Fig. S4 in Supporting Information). Therefore, the effect of nonspecific adsorption of proteins contained in the medium on the pH sensitivity can almost be neglected. Also, on the basis of the dimensions of nonspecifically adsorbed proteins such as fibronectin (MW ≈ 480,000), these proteins can be considered as cylindrical proteins, namely, length a = 60 (nm) and base diameter b = 6 (nm). Various proteins are included in 10% FBS, which corresponds to about 5 mg/mL of proteins. Also, a glass ring of 18 mm diameter was placed around the gate sensing area, to which 1 mL of a cell culture medium was poured. Here, we assume fibronectin to be the only protein that nonspecifically adsorbed on the gate. Considering these parameters, we can roughly calculate the density of adsorbed fibronectin at the gate sensing area to be approximately 300 molecules/µm2. Therefore, the distance between nonspecifically adsorbed proteins at the gate was assumed to be about 60 nm. Moreover, living cells produce extracellular matrix (ECM) proteins around themselves at the cell/gate interface during cell culture. That is, we need to consider the ECM as well as the FBS for nonspecifically adsorbed proteins on the gate surface.


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Therefore, the distance between nonspecifically adsorbed proteins at the gate is expected to be several tens of nanometers at most. In general, the FET biosensors have detection limit depending on the Debye length expressed by equation (1) shown in Introduction. In this case, the changes in the numbers of charges based on biomolecular recognition events can be detected within the Debye length, which depends on the ionic strength in a solution; therefore, the electrical charges based on macromolecules such as proteins can be easily shielded by counter ions in a cell culture medium with high ionic strengths. This means that the cell-based ISFET sensors are insensitive to proteins adsorbed to the gate in a cell culture medium because proteins are also nonspecifically adsorbed on the gate during preculture (Fig. 4). Also, the adhesive proteins with cells included in the cell culture medium should have been adsorbed on the gate surface, because the cancer cells used in this study attached and proliferated on the gate surface. However, the ISFET sensor was sensitive to pH even when it was immersed in a cell culture medium (Fig. 5), because hydrogen ion is the smallest among ions and its equilibrium reaction with the hydroxyl group at the oxide gate is expected without being affected by the adsorbed proteins (Fig. 4). Thus, the cell-based ISFET sensors can specifically monitor the cellular respiration activity as ∆pHint at the cell/oxide gate nanogap interface in real time, preventing nonspecific electrical signals derived from proteins because the Debye length is smaller in a cell culture medium with a higher ionic strength (< 1 nm even in 150 mM NaCl solution).

CONCLUSIONS In this study, we demonstrated the live monitoring of cellular respiration with a cell-coupled gate ISFET sensor. The change in output voltage of the cell-coupled gate ISFET was regarded as


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that in pH at the cell/gate nanogap interface. In particular, the change in the interfacial pH for cancer cells (HeLa and HepG2 cells) was found to be shifted by a factor of five to six, which was larger than that for normal cells (HUVEC), regardless of the nonspecific adsorption of proteins at the gate surface, although it was difficult to distinguish the difference in the change in the interfacial pH between different kinds of cancer cells (HeLa and HepG2 cells). Thus, a platform based on a cell-coupled gate ISFET sensor is suitable for the live and label-free monitoring of cellular functions such as metabolism with different types of cells. In particular, our electrical method is very attractive for the evaluation of microenvironmental pH based on extracellular acidosis around cancer cells. Moreover, the development of an ISFET-based flexible microelectrode will lead to the continuous monitoring of tumor tissues in vivo in the future.

ACKNOWLEDGMENT: This work was partly performed at the Center for NanoBio Integration (CNBI), the University of Tokyo, Japan.

ASSOCIATED CONTENT: Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:***. Fundamental characteristics of ISFET sensor (Figure S1); Photograph of FET biosensor for electrical measurement (Figure S2); Electrical circuit (Figure S3); Fluorescence image on the gate of ISFET immersed into cell culture medium for 1 w (Figure S4).

CONFLICT OF INTEREST: The authors declare no conflict of interest.


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29. Burmeister, J. S.; Olivier, L. A.; Reichert, W. M.; Truskey, G. A. Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials. Biomaterials 1998, 19, 307–325. 30. Stock, K.; Sailer, R.; Strauss, W. S. L.; Lyttek, M.; Steiner, R.; Schneckenburger, H. Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device. J. Microsc. 2003, 211, 19–29. 31. Santos, M. C. D.; Deturche, R.; Vezy, C.; Jaffiol, R. Topography of Cells Revealed by Variable-Angle Total Internal Reflection Fluorescence Microscopy. Biophys. J. 2016, 111, 1316–1327.

FIGURE CAPTIONS: Figure 1 (a) Schematic illustration of cell-coupled gate ISFET. Cells are cultured on the Ta2O5 gate surface. The charge density changes at the gate surface induce the electrostatic interaction with electrons at the channel in the silicon crystal, resulting in the change in drain current. The Ta2O5 gate membrane with the hydroxyl group in a solution is sensitive to pH based on the concentration of hydrogen ions (Fig. S1 in Supporting Information). HeLa cells (b), HepG2 cells (c), and HUVECs (d) were cultured on the gate, where the channel width (W) and length (L) show 340 and 10 µm, respectively. A few tens cells were cultured on each gate. Scale bar, 100 µm. Figure 2 Change in surface potential (∆Vout) for each cell detected using cell-coupled gate ISFET sensor. (a) HeLa cells, (b) HepG2 cells, (c) HUVECs, and (d) No cells (medium only). Figure 3 Change in interfacial pH (∆pHint) at cell/gate nanogap for each cell detected using cellcoupled gate ISFET sensor. ∆pHint was calculated from ∆Vout in Fig. 2, on the basis of the


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average pH sensitivity of 58 mV/pH for the ISFETs used in this study. (a) HeLa cells, (b) HepG2 cells, (c) HUVEC, (d) No cells (medium only). Figure 4 Schematic illustration of cell/gate nanogap interface. Some proteins in a cell culture medium were adsorbed at the oxide gate surface during preculture, resulting in the adhesion of cells at the substrate. These macromolecules prevent targeted ionic charges from coming in contact with the gate, but hydrogen ions easily approach to the oxide gate surface, where the equilibrium reaction between hydroxyl groups and hydrogen ions contributes to the change in the charge density at the gate. Also, hydrogen ions are concentrated in the closed nanogap space between the cell membrane and the gate. Figure 5 Change in surface potential with pH variation detected using ISFET sensor immersed into cell culture medium for 0 h (a), 24 h (b), and 1 w (c). Note that cells were not cultured on the gate surfaces.


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Figure 4


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Figure 5


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for TOC only


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Live Monitoring of Microenvironmental pH Based ... - ACS Publications

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