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Simultaneous Quantitation of Na+ and K+ in Single Normal and Cancer Cells Using a New Near-infrared Fluorescent Probe Lu Li, Ping Li, Juan Fang, Qingling Li, Haibin Xiao, Hui Zhou, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00571 • Publication Date (Web): 14 May 2015 Downloaded from http://pubs.acs.org on May 18, 2015

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Analytical Chemistry

Simultaneous Quantitation of Na+ and K+ in Single Normal and Cancer Cells Using a New Near-infrared Fluorescent Probe Lu Li,‡ Ping Li,‡ Juan Fang, Qingling Li, Haibin Xiao, Hui Zhou and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan, 250014, P.R. China. ABSTRACT: Considering the important functions of cellular Na+ and K+ together with their cooperative efforts on various biological processes, it is significant to simultaneous detection of Na+ and K+ at a single-cell level. Here, we present a novel method to discriminate and quantify simultaneously Na+ and K+ in single cells using a new near-infrared fluorescent probe associated with microchip electrophoresis. The fluorescent probe selectively responds to both Na+ and K+. The microchip electrophoresis allows accurate single-cell manipulation and effective distinction of Na+ and K+. Based on the method, the concentration of Na+ and K+ in single normal and cancer cells was compared and the variation of Na+ and K+ in single cancer cells during the early stage of apoptotic volume decrease was monitored, which would help us to better understand the critical roles of Na+ and K+ in malignant cells and apoptosis. This method has paved a new way for the research of the synergistical function of Na+ and K+ in the regulation of various biological processes at a single-cell level.

As two most important cations in living organisms, intracellular sodium (Na+) and potassium (K+) synergistically contribute to large numbers of biological processes.1-4 For example, an increase in the resting-state intracellular Na+/K+ ratio leads to sustained depolarization of cell membrane, which is involved in the regulation of cell divisions during both normal and cancerous growth of tissues.5-7 Besides, the movement of intracellular Na+ and K+ plays a pivotal role in both the activation and execution of apoptosis.8-11 Redistribution of intracellular Na+ and K+ has been shown to occur at the early stage of apoptotic volume decrease (AVD).12,13 Considering the important functions of Na+ and K+ in the cells together with their cooperative efforts on various biological events, it is significant to simultaneous detection of cellular Na+ and K+. To deeply understand the relationship between the concentration of Na+ and K+ and the regulated biologic processes, quantitative analysis at a single-cell level is very valuable, which address the issue of heterogeneity within cell populations.14-16 To date, various analytical techniques have been used for the detection of Na+ and K+, such as atomic absorption spectrophotometry, flame photometry, radioactive ion analogs and nuclear magnetic resonance.17-21 These methods only provide information concerning the average concentration of Na+ and K+ in a solution. The ion-selective microelectrode allows measurement of Na+ and K+ in single cells, but complicated operation and large cells are still required.22,23 Fluorescence detection method based on specific fluorescent probes is a powerful tool to analysis the bioactive molecules due to its high sensitivity, good reproducibility and convenient operation.24-33 Although flow cytometry has been developed to simultaneously detect the Na+ and K+ with a semi-quantitative manner in cell populations using two fluorescent probes.12 Absolute quantitative analysis of the two ions at a single-cell

level has not be realized. To solve the problem, a fluorescent probe specifically responding to both Na+ and K+ and a technology integrated with single-cell manipulation, fluorescent signal separation and absolute quantitation are in urgent need. Thus, we have devoloped a novel method to simultaneously quantify Na+ and K+ in single cells using a new near-infrared fluorescent probe associated with the technology of microchip electrophoresis. A novel BODIPY-based fluorescent probe (cBDP) was designed and synthesized for recognition of Na+ or K+ (Figure 1A). On the basis of lone electron pairs of four nitrogen atoms and the cavity size of the ring structure, cBDP can selectively bind Na+ or K+.34 After the cells are derived by the probe, they are controlled and analyzed by the technology of microchip electrophoresis with laser-induced fluorescence (MCE−LIF) detection. The microchip electrophoresis is a powerful tool to perform single-cell component analysis.35-39 The dimensions of microchannel are ideally comparable to the size of single cells and the networks permit accurate manipulation of cells.40-44 The electrophoresis can effectively separate multiple components in complex biological system according to the difference in charge-to-mass ratio. Taking the advantages of the unique properties of microchip electrophoresis, the fluorescent products of Na+ and K+ in single cells can be effectively distinguished by the migration time and directly detected without concerning spectral overlap of the fluorescence signal. Based on the proposed method, we quantified and compared the concentration of Na+ and K+ in normal and cancer cells and further monitored the variaton of Na+ and K+ in cancer cells during the early stage of apoptotic volume decrease. EXPERIMENTAL SECTION

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Materials and reagents. Mannitol, cetyltrimethyl ammonium bromide (CTAB), resveratrol, 4',6-diamidino-2- phenylindole (DAPI), 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), pinacidil (K+ channel opener) and veratridine (Na+ channel opener) were obtained from SigmaAldrich Chemicals (St. Louis, MO, USA). Sodium chloride (NaCl), potassium chloride (KCl), acetophenone, 2,6- Pyridinedicarboxaldehyde and boron trifluoride diethyl etherate were purchased from Aladdin Chemical Company (Shanghai, P.R. China). 5% glucose solution was obtained by dissolving appropriate glucose solid. Tris-HCl, HEPES and MES/His electrophoresis buffer was prepared by dissolving appropriate amount of Tris, HEPES and MES/His in ultrapure water respectively and adding moderate HCl to obtain the required pH. All solutions were stored at 4 ℃ in darkness and filtered through a 0.22 µm membrane filter before introduction into the microchip. MCF-7 (human breast cancer cell line) was purchased from KeyGEN biotechnology Company (Nanjing, China), MCF10A (normal immortalized human mammary epithelial cell line), BEAS-2B (normal human lung epithelial cell line) was purchased from Shanghai Bioleaf Biotechnology Company (Shanghai, China), HepG2 (human hepatocellular liver carcinoma cell line), HL-7702 (human hepatocyte cell line), A549 (human lung cancer cell line) and Hepa 1-6 (mouse hepatocellular liver carcinoma cell line) were obtained from the Committee on Type Culture Collection of the Chinese Academy of Sciences. Mouse hepatocytes were primary cells isolated from mouse liver. For cell treatment, 5.0 mM stock solution of resveratrol, pinacidil and veratridine were prepared in DMSO respectively and the working solutions through dilution in culture medium were used further experiment. All cell culture products were obtained from Gibco (Invitrogen). All chemicals and solvents used were of analytical grade and used without further purification unless otherwise noted. Sartorius ultrapure water (18.2 MΩ·cm) which was purified with a Sartorius Arium 611 VF system (Sartorius AG, Germany) was used throughout all the experiments. Instrumentation. 1H NMR spectra were taken on a Bruker Advance 300-MHz spectrometer (Bruker, Germany). The mass spectra were obtained by Bruker maXis ultra-high resolution-TOF MS system (Bruker Co., Ltd., Germany). Fluorescence measurements were performed on using Cary Eclipse fluorescence spectrophotometer with a xenon lamp and 1.0 cm quartz cells (Varian, Austrialia). Confocal fluorescence imaging studies were performed with a TCS SP5 confocal laser scanning microscopy using a 405nm and 633 nm excitation source (Leica Company, Ltd., Germany). Centrifugation was performed with a Sigma 3K15 refrigerated centrifuge. Cells were disrupted using a BILON92-IIL ultrasonic disintegrator (Shanghai Bilon Materials Inc.). The total electrophoresis processes were completed by a homemade MCE-LIF system which is comprised of a microchip with a cross design, a laser-induced fluorescence detector (LIFD), a versatile programmable eight-path-electrode power supply (PEPS), a data acquisition and a computer (Figure S1). All components above except LIFD was described previously.45 The chip channel was 70 µm width × 25 µm depth and the laser detection point lied 33 mm downstream from the cross C. LIFD was builted newly with a confocal optics mode. A lownoise semiconductor double-pumped solid state laser (MLLIII-633 nm/15 mW, Changchun Xinchanye Guangdianjishu

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Co.Ltd, China) was used as a light source. The emitted fluorescence was collected by a narrow band filter with a wavelength in the range of 675 ±10 nm (Omega Optical, Brattleboro, VT) and finally detected by a R928 photomultiplier (PMT, Hamamatsu, Japan). Synthesis of cBDP probe. The synthetic route of cBDP probe was shown in Figure S2. First, acetophenone and 2,6pyridinedicarboxaldehyde were mixed in the presence of sodium hydroxide to obtain intermediate a routinely. Intermediate a was dissolved in methanol following by adding nitromethane and diethylamine, then the mixed solution was acidified to obtain b. Next, b and ammonium acetate were mixed in ethanol and heated under reflux for 24 h. The mixture was cooled down, concentrated, washed with water, and product c as a blackish green solid was obtained without further purification. Solid c was dissolved in dry dichloromethane, and then the solution was stirred for 30 min after triethylamine was added under an argon atmosphere. Later boron trifluoride diethyl etherate was added dropwise and the mixture was stirred for 48 h at room temperature. After the reaction was stopped, the product was washed with water repeatedly and treated with liquid separation. The organic layer was dried, concentrated and isolated by column chromatography on silica with ethyl acetate and methanol (4:1) as eluent to give a light green solid d (overall yield 23%). Cell Culture. MCF-7, MCF-10A, HepG2, BEAS-2B, A549, Hepa 1-6 cells and mouse primary hepatocytes were cultured in Dulbecco’s modified Eagles medium (DMEM) with 10% fetal bovine serum. HL-7702 cells were cultured in RPMI 1640 with 20% fetal bovine serum medium. All cell culture medium was supplemented with 100 U/ml 1% antibiotic penicillin/streptomycin and all cells were maintained at 37 ℃ in a 5% CO2 / 95% air humidified incubator (MCO-15AC, SANYO). Cells were passaged every 2-3 days. Sample Pretreatment. For cell-free assays, sample was prepared through mixing cBDP (60 µM) solution with a series of different concentration of NaCl or KCl solution and diluting to a certain concentration in 5 mM Tris-HCl (pH 7.4) at room temperature. The reaction was fast enough so that the sample could be used for fluorescence and electrophoresis measurements immediately. For cell assays, adherent cells in culture medium were washed with 5% glucose solution three times to remove the culture media and incubated at 37 ℃ for 15 min in 5% glucose solution with the presence of 60 µM cBDP, then washed with 5% glucose solution to wash any excess probe. Adherent cells after incubation were used for fluorescence imaging. Besides, 0.25% trypsin-EDTA would be used to digest cBDP-incubated cells. After removel of trypsin-EDTA, the cells were resuspended in 5 mM Tris-HCl (pH 7.4). The density of cells was 5 × 105 cells·mL-1 counted by a hemocytometer for electrophoresis analysis of Na+ and K+ in single cells. To get cell extract, cells in culture medium were collected and resuspended as described above. The cells were disrupted with a BILON92-IIL ultrasonic disintegrator (Shanghai BiLon Materials Inc.). Then the broken cell suspension is centrifugated at 15000 rpm for 30 min by speed refrigerated centrifuge (Sigma 3K 15, Germany), and the pellet was discarded to obtain the cleared cell extract. All steps were performed below 4 ℃ and the extract was immediately analyzed .

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Fluorescence Measurements. The fluorescent spectra experiments were carried out in Cary Eclipse fluorescence spectrophotometer with a xenon lamp and 1.0 cm quartz cells (Varian, Austrialia). The excitation wavelength was 630 nm, and the emission spectra were recorded between 650 and 730 nm. The fluorescence emission maximum was at 675 nm. The fluorescent imaging studies were performed with a TCS SP5 confocal laser scanning microscopy using a 405 nm and 633 nm excitation source (Leica Co., Ltd. Germany). The fluorescence images of Na+ and K+ in HepG2 were acquired after cells were incubated as the sample pretreatment procedure. HepG2 cells were plated on chamber slides for 24 h. When cells were in a logarithmic growth phase, cells were incubated with cell medium containing 100 µM resveratrol for different time periods. After the incubation, the cells were treated with DAPI staining according to the instructions. Finally, the fluorescence images of DAPI staining of HepG2 were obtained. Microchip operation. To achieve single-cell electrophoresis analysis, electrokinetic gated injection was adopted as the sampling method.37 The same experiment process was applied in cell-free assay. During the pretreatment of microchip, the microchannels was rinsed sequentially with 0.1 mM NaOH and ultrapure water for 10min and then steeped with electrophoresis buffer containing 20 µM CTAB for 5min followed by injecting electrophoresis buffer finally. Reservoirs B, SW and BW were filled with 13 uL electrophoresis buffers, and reservoir S was filled with 13 uL sample solution. Fluid and cells manipulation were conducted by PEPS for each reservoir. In the single cell injection step, 280 V was applied to the reservoir S for 19 s, and 300 V was applied to the reservoir B, while reservoir SW and BW were grounded. Next, at the single-cell loading step, a set of electric potentials of four reservoirs were switched, with S at 500 V, B at 450 V, SW floating, BW at 0 V for 1s. Finally, cytolysis and electrophoresis separation step was immediately carried out by applying 2000 V at S, 2200 V at B, and 0 V at SW and BW for 100 s.

living cells (Figure 1D). However, it is difficult to distinguish Na+ and K+ due to the similar fluorescence spectra of the Na+cBDP and K+-cBDP.

Figure 1. Derivatization of Na+/K+ by fluorescent probe (cBDP). (A) The reaction of cBDP with Na+/K+. (B, C) Excitation and emission spectra of 60 uM cBDP in the absence (1, 1’) and presence of 100 uM NaCl (2, 2’) or KCl (3, 3’) in 20 mM Tris-HCl (pH 7.4), λex/λem=630/675nm, respectively. (D) Fluorescence imaging of Na+ and K+ in HepG2 cells without treatment (a), incubated with 100 µM veratridine for 12 h (b), incubated with 100 µM pinacidil for 12 h (c).

RESULTS AND DISCUSSION In this study, cBDP probe was synthesized and the structure of the probe was confirmed by 1H NMR, 13C NMR and HRMS (see supporting information). The fluorescent properties of cBDP were also investigated detailly. cBDP shows a very weak fluorescence at 675 nm (λex = 630 nm); Na+/K+ addition can create a remarkable enhancement (8/6.8 fold) and fluorescence quantum yield increased from 0.012 to 0.11 and 0.10. Near-infrared fluorescence is shown as in Figure 1B and 1C. Determination of complexing ratio through the mole ratio method revealed that the inflection points of fluorescence intensity of Na+-cBDP (fluorescent product of Na+) and K+cBDP (fluorescent product of K+) were at 1.15 and 1.25 respectively, indicating that cBDP forms a 1:1 species with Na+ or K+ in solution (Figure S4). Binding affinity (Kd) of cBDP to Na+ and K+ is determined to be 18.62 mM and 19.50 mM respectively, which could match the physiological concentration of Na+ and K+ well. Interference test demonstrates that relative biological substance do not mainly influence fluorescence of cBDP (Figure S5), which proves its selectivity for the Na+ and K+. MTT asssay indicates that the probe shows almost no cytotoxicity or side effects in living cells (Figure S6). Confocal imaging of HepG2 cells further verifies the specific fluorescent response of the probe to Na+ and K+ in

Figure 2. Electropherograms of fluorescent derivatives in standard solution. From top to bottom: a, Na+-cBDP; b, K+-cBDP; c, Na+-cBDP (1) and K+-cBDP(2); d, cBDP. The concentration of cBDP is 60 uM, and the concentration of Na+ and K+ are both 100 uM. Electrophoresis conditions: running buffer, 20 mM mannitol, 20 mM Tris-HCl, pH 9.0; separation electric field, 440 V/cm; effective separation distance, 33 mm. RFU (mV), relative fluorescence intensity units.

The microchip electrophoresis is believed to be an ideal tool to perform component analysis in single cells because of its unique properties including reduced reagent consumption and analysis time, accurate manipulation of cells and effective separation of multiple components in complex biological

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Table 1. Linearity, reproducibilities of migration time and peak area, and LODs for Na+/K+-cBDP derivatives. Analyte

Linearity range

Regression equation[a]

0.008-2(mM)

Y=0.8450+10.21X (concentration linear curve)

Na+ 3-750(fmol)

Q=0.8450+0.02723N (mass linear curve)

0.008-2(mM)

Y=0.7537+9.752X (concentration linear curve)

3–750(fmol)

Q=0.7537+0.02601N (mass linear curve)

+

K

R2

RSD(%,n=11) (migration time)

RSD(%,n=11) (peak area)

0.9992

0.59

3.16

LODs 2.5 µM 0.9375 fmol 2.7µM

0.9991

0.62

3.30 1.013 fmol

[a] Y, Q: peak area in mV·s; X: concentration of the analyte in mM; N: amount of the analyte in fmol.

system.45-50 When coupled with laser induced fluorescence (LIF) detection, the MCE-LIF system will offer ultrasensitive analysis for various biological molecules. To realize the simultaneous detection of cellular Na+ and K+, a MCE-LIF system reported in our previous study was used to carry out the single cell injection, lysis, electrophoresis separation and fluorescence detection.37 Due to the tiny difference between the Na+-cBDP and K+-cBDP in charge-to-mass ratio, high performance on-chip electrophoresis separation is distinctly advantageous. To obtain a good resolution, some factors affecting the separation such as the varieties, pH and concentrations of the electrophoresis buffers, separation electric field and buffer additive were systematically optimized (Figure S7). Finally, to decrease electroosmosis, cetyltrimethyl ammonium bromide (CTAB) was chose as a buffer addition to be introduced into the microchannel, that could lead to a obvious increase from 1.24 to 2.4 in the resolution, then 20 mM Tris-HCl buffer (pH 9.0) containing 20 mM mannitol and a separation electric field of 440V/cm were selected for the on-chip electrophoresis separation to obtain a compromise among a short analysis time, a higher column efficiency, and a better resolution. Under the optimized conditions, electrophoresis separation of positively charged Na+-cBDP and K+-cBDP was investigated to achieve simultaneous determination of Na+ and K+. The typical electrophoregrams of Na+-cBDP and K+-cBDP are shown in Figure 2. Peaks 1 and 2 at curve c were identified as Na+cBDP and K+-cBDP with migration times of 42 s and 45 s respectively based on their individual electrophoretic mobility (curve a presents Na+-cBDP and curve b presents K+-cBDP). The resolution was calculated to be 2.4, indicating complete separation. Good reproducibility was affirmed by the RSD with 0.59% and 0.62% obtained from 11 measurements of migration time for Na+ and K+, respectively. The Na+ and K+ with different concentrations were then detected to evaluate the linear range, limit of detection (LOD) and reproducibility of the proposed method. The results are shown in Table 1 and the data were obtained from the statistics of peak areas. Calibration curves for Na+ and K+ were tested over the concentration range both from 0.008–2.0 mM (Figure S8). The LODs were calculated to be 2.5 and 2.7 µM for Na+ and K+, respectively. Considering an electrokinetic pinched injection volume of 375 pL, the mass LODs for Na+ and K+ were calculated to be 0.938 and 1.01 fmol, respectively. Reproducibility was studied by 11 injections of a standard

solution consecutively and the RSD were calculated to be 3.2% and 3.3% for Na+ and K+, respectively. To verify the biological applicability of the proposed method, simultaneous quantify of Na+ and K+ in cell extract and single cells was performed. After a solution and a cell suspension was injected into the microchip individually, electrophoregram was recorded with a PMT. Two peaks corresponding to Na+ and K+ in the samples could be identified according to the standard. As shown in the Figure 3, there are good fluorescence response and electrophoresis separation in both the cell extract and the single cell. The concentration of the Na+ and K+ in single HepG2 cells were calculated to be 35.2 and 132.3 mM on average, which were in good agreement with those obtained from the cell extract (33.2/126.5 mM) and the previous reports.51

Figure 3. Electropherograms obtained from one individual HepG2 cell and cell extract. From top to bottom: a,standard Na+ and K+ derivatives with cBDP; b, single cell incubated with cBDP; c, cellular extract of HepG2 cells incubated with cBDP; d, single cell without treatment. Electrophoresis conditions were the same as figure 2.

To further demonstrate the applicability of the method, veratridine and pinacidil were selected as Na+ channel opener and K+ channel opener to regulate the flow of intracellular Na+ and K+ respectively. Intracellular levels of Na+ and K+ in single MCF-7 and MCF-10A cells were examined before and after induction of the openers. As shown in Figure 4, MCF-7 cells treated with 100 µM veratridine for 12 h showed significantly increased Na+ concentration and MCF-7 cells treated with 100 µM pinacidil for 12 h shows significantly decreased

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K+ concentration. The similar change was also observed in MCF-10A cells and the result was presented in Figure S9.

Figure 4. Intracellular Na+ and K+ concentration in MCF-7 cells before and after Na+ or K+ channel activator induction for 12 h. One point represents one cell. The number of measured cells are all 100. +

+

It has been reported that, increase of the intracellular Na :K ratio as an indication of a sustained depolarization of cell membrane, is implicated in the regulation of cell divisions during both normal and cancerous growth of tissues.5-7 While the intracellular levels of Na+ and K+ are directly involved in mitotic control.6,52 A higher intracellular Na+ concentration is associated with the high mitotic activity of the cancer cells and there is a positive correlation between the high intracellular Na+ concent and Na+:K+ ratio and malignantcell proliferation as well.7,51,53 In the view of the important biological function of the Na+ and K+ in mitogenesis and oncogenesis, it is very important to detect and compare the concentration of Na+ and K+ in normal and cancer cells. In this study, the free Na+ and K+ in single normal cells (human mammary epithelial cell MCF-10A, human hepatocyte cell HL7702) and cancer cells (human breast cancer cell MCF-7, human hepatocellular liver carcinoma cell HepG2) were simultaneously quantified by the proposed method. Typical electropherograms obtained from individual MCF-10A cells and MCF-7 cells are shown in Figure S10a and quantitative data of more cell lines is shown in Table S1. The distributions of Na+:K+ ratio in MCF-10A cells and MCF-7 cells is represented in Figure 5. These results revealed that there was not obvious difference in the concentration of K+ between the normal and cancer cells while elevated Na+ concentration appeared in cancer cells. Increase in the Na+/K+ ratio in the tumor cells (average: 0.6), as compared with normal cells (average: 0.16), was also verified, which support the notion that a high Na+/K+ ratio is closely associated with the oncogenesis. The result from single-cell analysis further displays the heterogeneity of Na+/K+ ratio within cell populations. The broader range of Na+/K+ ratios in cancer cells illustrates inhomogeneity. Although most MCF-7 cells possessed high Na+/K+ ratio, there were ~17% of MCF-7 cell showing lower Na+/K+ ratio as shown in the histograms. Such a feature would not have been seen if the cells were not examined at the single-cell level. The movementof Na+ and K+ also has been shown to play a pivotal role in both the activation and execution of apoptosis.811 The apoptotic volume decrease (AVD) is a classical feature of apoptosis that has been thought to reflect a redistribution of intracellular ions.54-57 Direct analysis of intracellular ion

Figure 5. Distribution of the concentration ratios of Na+ and K+ in the normal cells (MCF-10A) and cancer cells (MCF-7). Each histogram includes data from ∼102 cells. The curves were added based on the assumption of normal distribution for each population.

concentrations during the primary stage of AVD revealed anionic imbalance with a decrease in intracellular K+ coupled to an increase in intracellular Na+.12,13 Here, a simultaneous change of intracellular Na+ and K+ was investigated at singlecell level using the proposed method during the the primary stage of AVD. The resveratrol-induced apoptosis of HepG2 cell was chose as the model. After 6 hours, a distinct loss of cell volume (20–40%) indicating the primary stage of AVD was observed through the DIC image, as well as the chromatin condensation as an early signal of apoptosis was evidenced by the DAPI staining (Figure S11). Meanwhile, the concentration of Na+ and K+ in single resveratrol-stimulating HepG2 cells were simultaneously quantified. Typical electropherograms obtained from individual HepG2 cells and resveratrolstimulated HepG2 cells are shown in Figure S10b. The quantified result is shown in Figure 6. The single-cell analysis can exactly present simultaneous exchange of intracellular K+ and Na+. Comparing with the control cells that were not treated with resveratrol, cells at the primary stage of AVD displayed obvious ionic reversal with a decrease in intracellular K+ coupled to an increase in intracellular Na+. These suggest that the normal tight control of cationic ion fluxes is loss, resulting in decreasein intracellular ionic strength, decrease in cell volume, and eventual cell apoptosis.

Figure 6. Intracellular Na+ and K+ concentration in HepG2 cells before and after apoptosis induction. One point represents one cell. The number of measured cells both are 100.

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Besides, the variation of the levels of Na+ and K+ in HepG2 cells and MCF-7 cells and the kinetic loss of the cell volume after simulation by different concentration of resveratrol for different time were detected. As shown in Figure S12, with the increase in stimulation time and concentration of resveratrol, cell volume declined with a time- and dose- dependent manner, meanwhile, the concentration of K+ showed a consistent decrease and the concentration of Na+ increased first as the compensation of loss of K+ to maintain cell viability12 and then decreased continuously with the process of apoptosis. The samilar trends were observed in both HepG2 cells and MCF-7 cells. CONCLUSIONS In summary, we have developed a novel method to simultaneously quantify Na+ and K+ in single cells using a new fluorescent probe associated with the technology of microchip electrophoresis. The fluorescent probe cBDP can simultaneously respond to both Na+ and K+ with near-infrared fluorescence enhancement. Accurate single-cell manipulation and high performance on-chip electrophoresis separation of Na+-cBDP and K+-cBDP can be realized through the microchip electrophoresis. The Na+ and K+ in single cells were directly quantified without concerning spectral overlap of the fluorescence signal. Based on the proposed method, the Na+/K+ ratio was compared between normal and cancer cells, and the variation of Na+ and K+ at primary stage of AVD was accurately monitored. This method has paved a new way for us to better understand the cooperative efforts of Na+ and K+ on the regulation of various biological processes at a singlecell level. More importantly, it provides a novel strategy for the simultaneous discrimination and quantitative detection of multiple components in single cells when fluorescence probes suffer from serious spectral overlap problem.

ASSOCIATED CONTENT Supporting Information Supporting Information including extensive Figures S1−S12 and Table S1 as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: tangb@ sdnu.edu.cn.

Author Contributions ‡These authors contributed equally.

Notes The authors declare no competing financial interests..

ACKNOWLEDGMENT This work was supported by 973 Program (2013CB933800), National Natural Science Foundation of China (21227005, 21390411, 91313302,21205074).

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