Potential-Resolved Electrochemiluminescence Nanoprobes for Visual

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Potential-Resolved Electrochemiluminescence Nanoprobes for Visual Apoptosis Evaluation at Single-Cell Level Gen Liu, Baokang Jin, Cheng Ma, Zixuan Chen, and Jun-Jie Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01401 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 13, 2019

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Analytical Chemistry

Potential-Resolved Electrochemiluminescence Nanoprobes for Visual Apoptosis Evaluation at Single-Cell Level Gen Liu,†,‡,§ Bao-Kang Jin,‡,§ Cheng Ma,†,§ Zixuan Chen,*,† and Jun-Jie Zhu† †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing

University, Nanjing, 210023, China ‡

College of Chemistry & Chemical Engineering, Anhui University, Hefei, Anhui 230601, China

§

Gen Liu, Bao-Kang Jin and Cheng Ma contributed equally to this work.

* Corresponding authors E-mail addresses: [email protected]

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Analytical Chemistry 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

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Abstract In this work, a potential-resolved electrochemiluminescence (ECL) method is developed and used for the apoptosis diagnosis at single-cell level. The apoptosis of cells usually induces the decreasing expression of epidermal growth factor receptor (EGFR), and promotes phosphatidylserine (PS) eversion in cell membrane. Here, Au@L012 and g-C3N4 as ECL probes are functionalized with epidermal growth factor (EGF) and peptide (PSBP) to recognize the EGFR and PS on cell surface, respectively, showing two well-separated ECL signals during a potential scanning. Experimental results reveal that the relative ECL change of g-C3N4 and Au@L012 correlates with the degree of apoptosis, which provides an accurate way to investigate apoptosis without interference that solely changes EGFR or PS. With a homemade ECL microscopy, we simultaneously evaluate the EGFR and PS expression of abundant individual cells, and therefore achieve the visualization analysis of the apoptosis rate for normal and cancer cell samples. This strategy contributes to visually studying tumor markers and pushing the application of ECL imaging for the disease diagnosis at single-cell level. Keywords: Electrochemiluminescence, Apoptosis, Microscopy, Cell imaging

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Apoptosis is a programmed cell death process to remove diseased cells. The diagnosis of apoptosis will contribute to evaluating the occurrence of diseases and the therapeutic effect of antitumor pharmaceuticals.1 Phosphatidylserine (PS) is generally confined to the inner leaflet of plasma membrane. In the early and intermediate stages of cell apoptosis, PS is externalized to the outer leaflet of cytomembrane.2-6 Epidermal growth factor receptor (EGFR), a member of tyrosine kinase receptors, is a cellular transmembrane glycoprotein and plays a significant role in some cell processes such as adhesion, proliferation, migration and apoptosis.7-9 Over-expressed EGFR is associated with multiple types of cancers and has been generally considered as a strong prognostic marker for examining likelihood of cancers.10 Besides, EGFR is also the vital mediator for anticancer drugs delivery.11 Some EGFR antibody conjugated immunoparticles can be ingested by cancer cells and effectively induce apoptosis.12 As a significant biological indicator for apoptotic cells, PS is interrelated with EGFR. Accordingly, it is necessary to figure out the relative variation of EGFR and PS in rather than solely measure each of them. However, as we know, there is no detailed report for in-situ study of the relative variation of the two receptors. Among various analytical methods, electrochemiluminescence (ECL) has presented outstanding advantages over common analytical methods, such as electrochemical, fluorescence, and colorimetric in recent years, owing to its low background interference, simplified optical setup and good spatiotemporal control.13,14 With the development of ECL microscopy, one can observe the local ECL emission from individual nanoprobes or cells.15,16 To achieve a sensitive analysis of micro targets, it is necessary to choose ECL probes with high emission efficiency. Graphitic-phase carbon nitride (g-C3N4), a 2D graphite-like material, mainly consists of carbon and nitrogen atoms and is easily synthesized by thermal polymerization of plentiful nitrogen-rich precursors.17 g-C3N4 has been proved to be an excellent ECL emitters by virtue of its large surface defect, proper band gap and lone electron pairs of nitrogen atoms.18 Besides, g-C3N4 exhibits high ECL activity in coreactants solutions of peroxydisulfate

(S2O82-)

and

hydrogen

peroxide

(H2O2).19,20

Another

candidate

is

L012

(8-Amino-5-chloro-7-phenylpyrido [3,4-d]pyridazine-1,4(2H,3H)-dione). This type of luminol analogue has become a popular ECL reagent in recent years because of it high properties in chemiluminescence and electrochemiluminescence.21,22 The two ECL agents share a same coreactant and generate ECL at different potentials,23,24 which offers an effective approach for simultaneous investigation of two biomarkers. In this study, two potential-resolved ECL probes Au@L012@EGF and g-C3N4-PSBP are successfully synthesized and used to specifically bind with EGFR and PS on cell surface. Thus, a new strategy for simultaneous evaluation of EGFR and PS on cell surface is designed, as shown in Figure 1. Firstly, polylysine 3



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(PLL), folic acid (FA) and bovine serum albumin (BSA) are modified on a carbon nanotube sponge (CNTSP) to construct a CNTSP/PLL/FA/BSA electrode that is used as a platform to immobilize and detect cells. Secondly, cells are labeled with Au@L012@EGF and g-C3N4-PSBP, denoted as cell@probes. Finally, the cells are captured on electrode through the specific binding of FA on electrode with FA receptors on cell surface. When the apoptosis of cells is induced by resveratrol (RVL), two sensitive ECL signals are provided, whose intensity correlates with the amount of EGFR and PS on cell surface. The degree of apoptosis can be reflected by the ECL ratio of g-C3N4-PSBP and Au@L012@EGF. Besides, we successfully detect the apoptotic cells at single-cell level using ECL imaging, which is difficult for previous ECL techniques or ECL probes to achieve it. The success in the establishment of dual ECL signal system provides a strategy to simultaneously evaluate two cell-surface receptors and realizes the visualized analysis of disease-related cells at single-cell level. This strategy may promote the application of ECL cytosensor for evaluation of drug-induced cell apoptosis and the accurate diagnosis of disease.

EXPERIMENTAL SECTION Materials, apparatus, cell culture and fabrication of ECL cytosensor are included in the Supporting Information.

Synthesis of g-C3N4 nanosheets. The bulk g-C3N4 material was prepared according to the previous report.25,26 Melamine was heated at 550 °C in a muffle furnace for 4 h with a ramp of 2.3 °C·min−1. The product was cooled to room temperature and then grounded to powder. 1.0 g of g-C3N4 powder and 100 mL of 4 M HNO3 were refluxed at 120 °C for 24 h. The refluxed product was centrifuged at 12,000 rpm and then washed with deionized water until pH reached 7.0. Finally, the product was dried in a vacuum oven for 12 h at 35°C to obtain the carboxylated g-C3N4. 200 mg of carboxylated g-C3N4 was dispersed into 100 mL of distilled water by sonicating for 12 h. Afterward, the obtained suspension was centrifuged at 8000 rpm for 15 min to remove the unexfoliated bulk g-C3N4 powder. Subsequently, the supernatant was concentrated by heating and then diluted to 5.0 mL with distilled water for future use.

Preparation of gold nanoparticles. In a typical experiment,27 500 μL of PDDA, 400 μL of 0.5 M NaOH, 80 of mL water, and 200 μL of HAuCl4 (1 %, w/w) were added into a flask. Then, the mixture was heated to 100 °C

4


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and maintains for a few minutes until the solution became red and no change in color. After cooling to room temperature, the PDDA-functionalized AuNPs were obtained.

Preparation of g-C3N4-PSBP and Au@L012@EGF. ECL probes g-C3N4-PSBP and Au@L012@EGF were prepared as following procedures. For g-C3N4-PSBP, first, the carboxylic acid groups on the surface of g-C3N4

nanosheets

(30

mg·mL−1,

1

mL)

were

activated

by

a

mixture

containing

20

mM

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 10 mM N-hydroxysuccinimide (NHS) for 1 h at room temperature. Then, 50 μL of 1.0 mg·mL−1 PSBP (FNFRLKAGAKIRFGRGC) was reacted with the activated g-C3N4 at 37 °C for 12 h. Next, the precipitum was centrifuged at 10000 rpm for 10 min and then washed with deionized water. Finally, the formed PSBP associated g-C3N4 nanosheets was diluted to 1.0 mL with PBS solution (10 mM, pH 7.4) and stored at 4 °C. To obtain Au@L012@EGF, 200 μL of 4 mM L012 was added into 20 mL of AuNPs solution (about 3.68 × 1011 AuNPs per milliliter) and stirred for 12 h. Then, the Au@L012 conjugates was obtained by centrifugation at 12000 rpm for 5 min and redispersed in water (1.0 mL) under mild ultrasonication. Afterward, 100 μL of 50 μg·mL−1 EGF was added into Au@L012 solution and kept gentle stirring at 37 °C for 12 h to get Au@L012@EGF. Finally, the precipitate was collected by centrifuging at 12000 rpm for 5 min and then washed for three times. The resulted Au@L012@EGF was dispersed in 1.0 mL of PBS solution (10 mM, pH 7.4) and stored at 4 °C.

Flow Cytometric Analysis. MCF-7 cells were seeded on six-well plates and incubated in the medium for 12 h at 37 °C. After the treatment of RVL (100 μg·mL−1, based on the MTT assay in Figure S1, Supporting Information) for 8 h, and 24 h, the cells were washed with sterile PBS and resuspended in 500 μL of binding buffer. Subsequently, the apoptotic cells were stained with annexin V-FITC/propidium iodide (PI) according to the manufacturer’s instructions and then used in flow cytometric assay.

RESULTS AND DISCUSSION The characterization of g-C3N4 nanosheets and Au@L012@EGF are described in the Supporting Information (Figure S2 and S3).

Fabrication of the carbon nanotube sponge-based electrochemical platform. L-lysine can be electro-polymerized on the CNTSP.28,29 In this work, CNTSP electrode was immersed into the electrolyte 5



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consisted of 0.1 M PBS (pH 9.0) and 50 mM L-lysine for 5 h to make the electrolyte molecules adequately enter CNTSP. Subsequently, the electropolymerization of L-lysine was performed by cyclic voltammetry from -1.0 to 2.2 V for 20 cycles with a scan rate of 0.1 V s−1. Figure 2A shows 20 cycles of the cyclic voltammograms (CV) of L-lysine during electro-polymerization on CNTSP. With the scanning cycles increasing, the cathodic and anodic current increases and finally tends to be stable, indicating that excessive scanning will slow down the electropolymerization procedure. Figure 2B displays the FT-IR spectra of CNTSP (curve a) and CNTSP/PLL (curve b). In the case of CNTSP, two obvious characteristic peaks appear at ~ 1630 cm-1 and ~ 3430 cm-1, which are assigned to the symmetric stretching vibration of COO- and the O-H stretching vibrations in carboxylic acid group, respectively.30 After electropolymerization of PLL, the absorption peaks at ~1679, ~1530, and ~1320 cm−1 are observed, which may result from the amide I, II, and III of polypeptides, respectively.31-33 The morphology of CNTSP and CNTSP/PLL are characterized by scanning electron microscopy (SEM), respectively. The CNTSP consists of CNTs self-assembled into a three-dimensional (3D), interconnected and porous framework (Figure 2C). After electropolymerization, PLL particles with approximately 30 nm diameter are produced on CNTs (Figure 2D).

Permeable CNTSP electrode avoiding the steric hindrance of cells. The steric hindrance of cells immobilized on electrode surface will block the diffusion of ECL reagents to the electrode surface where cells are located, and therefore induce shadows in ECL imaging. We have demonstrated in previous works that such steric hindrance can be eliminated by a permeable electrode,34,35 and we come to the same conclusion with CNTSP electrode (Figure S4, Supporting Information). In this work, ECL probes are labeled on the cell surface, but co-reactants still need to bypass cells and react with ECL probes between CNTSP and cells. To illustrate it, we detect the g-C3N4-labeled MCF-7 cells on a normal glass carbon electrode (GCE), and compare the ECL images of which with that detected on a permeable CNTSP electrode. As shown in Figure 3A, MCF-7 cells (e.g. 1, 2, 3, and 4) immobilized on GCE present a donut-like ECL profile with a shadow in center. In remarkable contrast, cells (e.g. 5, 6, 7, and 8) on CNTSP electrode present a solid profile (Figure 3B) and provide a Gaussian distribution of ECL emission, which gives a more accurate evaluation of the expression of EGFR and PS on cell surface (Figure S8, Supporting Information). That is to say, the proposed permeable CNTSP electrode avoids the steric hindrance of cells, and offers a more accurate description of in-situ ECL emission from single cells.

Optimization of detection conditions. To achieve a better performance of ECL cytosensor, the probe 6


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dosage and incubation time were optimized. In consideration of the low expression of EGFR in late apoptotic cells (24 h RVL-treated cells) and the scarce exposed PS in non-apoptotic cells (control group), the 8 h RVL-treated cells are used to optimize the signal of the two ECL probes on cell surface. The ECL intensity raises with the dosage of g-C3N4-PSBP and Au@L012@EGF increasing from 0 to 100 μL and 150 μL, respectively (Figure S5A and B, Supporting Information). The detailed steps are described in the Supporting Information. To make sure there’s no interplay of two probes in this dose range, the control tests are performed by comparing cells labeled with individual ECL probe and dual ECL probes according to the previous report.32 Figure S6 (Supporting Information) demonstrates that the ECL intensity of individual ECL probe with different amount is consistent with that of simultaneous determination, suggesting that the dual ECL signal system possesses two independent ECL signals and there is no cross reaction or energy transfer during ECL reaction. The incubation time is another important parameter. With incubation time progressively increases, as shown in Figure S5C and D (Supporting Information), the ECL intensity of g-C3N4-PSBP and Au@L012@EGF increase initially and then tend to stable after 50 min. indicating that the optimum capture time is 50 min. These optimal experiment parameters are used in the subsequent measurements.

Cell Detection The cytosensor was further employed to quantitatively detect HSMC cells in this work. As shown in Figure S7 (Supporting Information), we measured the potential-resolved ECL intensity of cell samples with and without RVL treatment, where the cell concentration ranges from 0 to 1.0 × 106 mL-1. In all samples, the ECL intensity scales up with the logarithm of cell concentration, and the corresponding linear ranges, detection limits, etc. are listed in Table S1.

Evaluation of cell apoptosis with EGFR and PS expression. To induce the cell apoptosis, we treat MCF-7 cells with RVL, which is a kind of antitumor agent naturally occurring in grapes, peanuts, berries, shows promising potential to treat cancer cells.36-38 RVL can induce tumor cell death by preventing the activation of tyrosine protein kinase, inhibiting the activity of EGFR and reducing the EGFR expression level.8,37,38 RVL and its analogues can also suppress tumorigenesis and cell proliferation.7,39 In brief, RVL plays a dual role in inhibiting EGFR expression and inducing apoptosis. We investigate the potential-resolved ECL response of cells labeled with g-C3N4-PSBP and Au@L012@EGF under the treatment of RVL (100 μg·mL−1) for 0, 8 h and 24 h, respectively. Without RVL treatment, cells only exhibit an ECL emission from L012 at 0.6 V (Figure 4A), 7



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demonstrating abundant EGFR on cell membrane. Flow cytometry analysis results reveal that almost all cells are non-apoptotic [annexin V (-)/PI (-)] at this moment (Figure 4D). After treated by RVL for 8 h, an ECL signal from g-C3N4 appears at -1.2 V, while the ECL intensity from L012 reduces, suggesting RVL causes the variation of PS and EGFR expression on cell surface (Figure 4B). The ECL ratio of g-C3N4 and L012 is about 3.4. As illustrated in Figure 4E, flow cytometry results show that 51.1 % of cells are in early apoptosis stage [annexin V (+)/PI (-)] and 4.1% in late stage [annexin V (+)/PI (+)]. After 24 h RVL treatment, the ECL intensity of g-C3N4 is further enhanced owing to the increasing amount of apoptotic cells (Figure 4C). Meanwhile, the ECL intensity of L012 keeps declining, indicating the weakened expression of EGFR on cell surface. About 10.3 of ECL ratio reveal that most of cells have entered the late apoptosis stage. As shown in Figure 4F, the percentage of cells in early and late apoptosis is 14.1 % and 82.5 %, previously. Both of ECL and flow cytometry results suggest that the more cells enter the apoptosis state, the more PS are exposed on the outer leaflet of cell membrane. According to above results, cell apoptosis could be well evaluated with EGFR and PS expression.

ECL imaging diagnosis of apoptosis at single-cell level. In-situ measurement of ECL at single-cell level is necessary for further understanding the apoptosis process. Thus, we use a home-made ECL microscopy to image the cell samples (Scheme S1). Because cells on the surface of CNTSP electrode are hard to be observed, we culture cells with DMEM medium containing 2-NBDG, a nontoxic and fluorescent D-glucose derivative widely used in cell research.40,41 As shown in Figure 5A4 and B4 and C4, MCF-7 cells with and without RVL treatment both present bright fluorescence of 2-NBDG. For ECL imaging experiments, cells are treated with the same procedures with PMT experiments. Without RVL treatment, most of cells present red spots (EGFR dominating, Figure 5A2) while only few cells present blue spots (PS dominating, Figure 5A1). The merged ECL image (Figure 5A3) demonstrates that the cell distribution is in accordance with that in fluorescence images. According to above results, we attribute the EGFR dominating cells to normal cells and the PS dominating one to apoptotic cells. Once treated with RVL for 8 h and 24 h, increasing amount of cells start to present blue spots (Figure 5B1 and C1). To quantitatively describe the apoptosis level, we measure the ECL ratio (PS to EGFR) of 100 cells in each state. Without any treatment, cells only show a concentrated distribution of ECL ratio near zero because of the lack of PS, and the percentage of cells in apoptosis stage is evaluated to be 3% (Figure 5D). In remarkable contrast, cells with RVL treatment have one additional broad distribution of ECL ratio at around 3.5, and the percentage of apoptotic cells rise to around 42% cells and 89%, respectively (Figure 5E and F). The general trend of apoptosis rate for control, 8 h and 24 h RVL-stimulated cells agrees with that in flow cytometry analysis, and 8


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also supports the elevated ECL responses of PS in Figure 4.

Apoptosis analysis for more types of cells. The ECL apoptosis analysis strategy can be further extended to other types of cells, including not only cancer cells (K562) but also normal cells (HSMC and H9C2). EGFR overexpression can be employed to distinguish cancer cells from normal cells, since the expression of EGFR on normal cells is about 50 times less than that on cancer cells.42 In our ECL imaging test (Figure 6), the EGFR-expressed cells (red spots) are observed in K562 cells. However, it fails to achieve the ECL images of normal cells under the same experimental conditions, no matter RVL induces apoptosis or not, because the amount of the specifically recognized Au@L012@EGF on normal cells is too small to support a detectable ECL imaging signal. More importantly, we are able to diagnose single apoptotic cells when try to detect the EGFR-expressed cells. For the cells without RVL stimulation, few PS-expressed cells (blue spots) appeare among K562, HSMC and H9C2 cells. However, after 8 h RVL stimulation, lots of apoptotic cells can be observed. Besides, it is easy to distinguish cancer cells from normal cells. K562 cells provide a dual ECL image of PS-expressed and EGFR-expressed cells. However, HSMC and H9C2 cells only present the ECL images of PS-expressed cells. The apoptosis rate of MCF-7, K562, HSMC and H9C2 cells are analyzed by ECL imaging technique. We performed three independent experiments for each kind of cells after the stimulation of RVL for 8 h. The apoptosis rate was calculated through counting the apoptotic cell number and the total cell number (more than 100 cells). As shown in Figure 7, 88% of HSMC cells and 94% of H9C2 cells are apoptotic, whereas only 55% of K562 cells and 42% of MCF-7 cells undergo apoptosis. The statistics suggest that the normal cells are particularly vulnerable to RVL and are easily apoptotic in comparison with cancer cells.

CONCLUSION In conclusion, the relative expression level of PS and EGFR on cell surface tends to make a difference when cells undergo apoptosis, which provides a new way to analyze the degree of apoptosis. In present work, a novel dual ECL signal system based on Au@L012@EGF and g-C3N4-PSBP is established to simultaneously investigate the expression of EGFR and PS on cell-surface. When cells undergo apoptosis, the expression of PS on cell surface increases, but meanwhile that of EGFR declines. Au@L012@EGF and g-C3N4-PSBP generate ECL signals under positive and negative potential, respectively and their ECL spectra have no overlap. Thus, the separable ECL profiles of the EGFR-expressed and PS-expressed cells could be observed. More importantly, the 9



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proposed method is successfully used to diagnose single apoptotic cells. This strategy provides a novel technique of single cell imaging assay for the disease-related biomarkers detection. Some drug-regulated receptors, such as thyroxine-regulated β-adrenergic, insulin-regulated GLUT4, tunicamycin-regulated N-glycan, etc. are also supposed to be expected to realize ECL imaging by similar method, which will contribute to guiding rational drug use at the single-cell level.

ASSOCIATED CONTENT Supporting Information. Experimental details including the MTT assay, characterizations of ECL probes, optimization of detection conditions, evaluation of interplay of dual probe on cell-surface, as well as additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Authors [email protected] (Z. Chen)

Author Contributions §

Gen Liu, Bao-Kang Jin and Cheng Ma contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS Financial supports from the National Natural Science Foundation of China (Grants Nos. 21427807, 21605081 and 21375001), the International Cooperation Foundation from Ministry of Science and Technology (2016YFE0130100), the Natural Science Foundation of Jiangsu Province (Grants No BK20160638), and the Fundamental Research Funds for the Central Universities.

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Figure Captions Figure 1. The fabrication process of the ECL cytosensor.

Figure 2. SEM images of CNTSP (A) and CNTSP/PLL (B); Cyclic voltammograms of the electropolymerization procedure of L-lysine (C); FT-IR spectra of CNTSP and CNTSP/PLL (D).

Figure 3. ECL images of g-C3N4-PSBP labeled cells for (A) GCE/PLL/FA/BSA/cell@probes, (B) CNTSP/PLL/FA/BSA/cell@probes in a coreactant of 0.1 M K2S2O8, respectively. The corresponding enlarged ECL images of cells and their plots of light intensity vs distance (pixel size 0.32 μm). Each diamond in the plot represented one pixel on the line across cell.

Figure 4. ECL responses of g-C3N4-PSBP and Au@L012@EGF on control cells (A), 8 h RVL-stimulated (B) and 24 h RVL-stimulated cells (C) in the coreactants of 0.1 M K2S2O8 and 0.1 M H2O2. Flow cytometric analysis based on annexin V-FITC and PI staining of (D) control cells, (E) 8 h RVL-stimulated and (F) 24 h RVL-stimulated cells.

Figure 5. Images of MCF-7 cells without RVL stimulation (A) and under the stimulation of RVL for 8 h (B) and 24 h (C). The first column (A1, B1, C1): ECL images of PS-exposed cells; the second column (A2, B2, C2): ECL images of EGFR-expressed cells; the third column (A3, B3, C3): overlay images of PS-exposed and EGFR-expressed cells; the forth column (A4, B4, C4): fluorescence images of 2-NBDG incubated cells. (D), (E) and (F) are the statistics of the each cell’s ECL ratio of PS to EGFR for control, 8 h and 24 h RVL-stimulated cells, respectively. The coreactants are 0.1 M K2S2O8 and 0.1 M H2O2. All images are pseudocolor.

Figure 6. ECL images images of PS-expressed and EGFR-expressed cells among K562, HSMC and H9C2 cells without RVL stimulation and under the stimulation of RVL for 8 h in the coreactants of 0.1 M K2S2O8 and 0.1 M H2O2. All images are pseudocolor.

Figure 7. Apoptosis rate HSMC, H9C2, K562 and MCF-7 cells after 8 h stimulation of RVL. The total cell number was more than 100 cells for each cell line.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Table of Contents (TOC)

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Potential-Resolved Electrochemiluminescence Nanoprobes for Visual

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