Electrochemiluminescence Imaging for Parallel Single-Cell Analysis of

Page 1 of 19

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

Analytical Chemistry

Electrochemiluminescence Imaging for Parallel Single-cell Analysis of Active Membrane Cholesterol

Junyu Zhou,1 Guangzhong Ma,2 Yun Chen,1 Danjun Fang1*, Dechen Jiang2*, Hong-yuan Chen

1

School of Pharmacy, Nanjing Medical University, Jiangsu, 210000, China;

2

Key State Laboratory of Analytical Chemistry for Life Science and School of Chemistry and

Chemical Engineering, Nanjing University, Jiangsu, 210093, China

Corresponding Authors: Phone: 086-25-86868477 (D.F.); 086-25-83594846 (D.J.) Fax: 086-25-86868477 (D.F.); 086-25-83594846 (D.J.) E-mail: [email protected] (D.F.); [email protected] (D.J.)

1

ACS Paragon Plus Environment


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

Page 2 of 19

ABSTRACT. Luminol electrochemiluminescence (ECL) imaging was developed for the parallel measurement of active membrane cholesterol at single living cells, thus establishing a novel electrochemical detection technique for single cells with high analysis throughput and low detection limit. In our strategy, the luminescence generated from luminol and hydrogen peroxide upon the potential was recorded in one image so that hydrogen peroxide at the surface of multiple cells could be simultaneously analyzed. Compared with the classic microelectrode array for the parallel single-cell analysis, the plat electrode only was needed in our ECL imaging avoiding the complexity of electrode fabrication. The optimized ECL imaging system exhibited that hydrogen peroxide as low as 10 µM was visible and the efflux of hydrogen peroxide from cells could be determined. Coupled with the reaction between active membrane cholesterol and cholesterol oxidase to generate hydrogen peroxide, active membrane cholesterol at cells on the electrode was analyzed at single cell level. The luminescence intensity was correlated with the amount of active membrane cholesterol validating our system for single cell cholesterol analysis.

The

relative high standard deviation on the luminescence suggested high cellular heterogeneities on hydrogen peroxide efflux and active membrane cholesterol, which exhibited the significance of single cell analysis.

This success in ECL imaging for single-cell analysis opens a new field in

the parallel measurement of surface molecules at single cells.

2


Page 3 of 19

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


INTRODUCTION.

Electrochemical detection of single living cells provides valuable

information such as surface chemical composition and local activities of cells.1 The typical strategy includes the position of microelectrode near or at the surface of single living cells and the electrochemical detection of chemical species.2

For higher analysis throughput, microelectrode

arrays are fabricated and the cells are cultured on the cell-sized microelectrodes individually to achieve the parallel single cell measurement.3,4

Although the fast development of

microfabrication advances the preparation of microelectrode array, the complexity in the fabrication process and the need of multi-channel electrochemical station limit the application of microelectrode array for single cell analysis.

As an alternative to microelectrode array, recently

developed electrochemical imaging techniques based on surface- plasmon-resonance (SPR) utilized the relationship between local electrochemical and SPR signals for single cell imaging.5,6 The parallel recording of the optical signal from multiple cells on one electrode avoids the electrode fabrication, however, the inherent high detection limit associated with SPR complicates observation of low levels of chemical efflux or membrane molecules at singe cells. Electrochemiluminescence (ECL) is a highly sensitive electrochemical method utilizing the emission light during the relaxation of electronically excited products to the ground state after an electrochemical reaction.7-10

Since the light from the entire electrode surface is collected by a

charge coupled device (CCD), ECL-based imaging of single cells cultured on the electrode should allow for high analysis throughput and low detection limit, and thus, offset the limitations on the existing electrochemical techniques. The pioneer work on ECL imaging was performed on latent fingerprint and protein layers at electrode surfaces.11-13 Typically, the ECL probe and co-reactant were introduced and different luminescence intensity was observed on fingerprint- or protein-covered electrode region. The co-reactant concentrations in the millimolar range are required to generate detectable luminescence. Further developments in ECL imaging achieved the high throughput analysis of antigens on microbeads, in which the antigen was linked with Ru(bpy)32+ (ECL probe) through the antibody to generate luminescence in the presence of co-reactant.14-16 In spite that ECL imaging demonstrates potential for the analysis of antigens on the surface of cells, this strategy is more significant to image small molecules at cells. As compared with fluorescence microscopy that needs specific fluorescence probe for each small molecule, ECL 3



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

Page 4 of 19

imaging can utilize the corresponding oxidase only for the reaction with the target molecule to produce hydrogen peroxide.

In the presence of luminol, the luminol anion and hydrogen

peroxide go through electro-oxidation to diazaquinone and oxygen radicals, respectively, which are further reacted together to generate the excited 3-aminophthalate species for the emission light.17-20

The universal imaging strategy avoids the individual design of the fluorescence probe

and facilitates the single cell analysis. Moreover, recent researches revealed that some molecules at plasma membrane, such as cholesterol, had different chemical activity due to the interaction with the surrounding molecules in cellular microenviourment, which could not be distinguished by fluorescence observation.21

However, under certain conditions, these molecules with high

chemical activity was reactive with the oxidase generating hydrogen peroxide and the luminescence, while, the molecules with low chemical activity could not react with the oxidase.22 Therefore, our ECL imaging has the ability to analysis the active molecule activity at the cells with high throughput, which is significant for the biological study. Although the ECL imaging for the analysis of small molecules at single cells is important, the technical achievement faces the fact of low concentration of molecules generated from the cells, typically in the micromolar range. As the result, the luminescence intensity generated from micromolar hydrogen peroxide is too weak to be detected from single cells. To realize the ECL imaging for single cell analysis, an upright optical configuration coupled with double potential mode was optimized so that hydrogen peroxide at concentrations as low as 10 µM was visible. Our successful ECL imaging of hydrogen peroxide at micromolar concentrations enabled the parallel analysis of cellular hydrogen peroxide from single living cells.

Coupled with the

generation of hydrogen peroxide from the reaction between active membrane cholesterol and cholesterol oxidase, active membrane cholesterol at multiple cells was analyzed simultaneously.

EXPERIMENTAL SECTION. Chemical. Hela cells were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences of Chinese Academy of Science (Shanghai, China). 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L012, molecular formula: C13H9ClN4NaO2) was obtained from Wako Chemical USA Inc (Richmond, VA). chemicals were from Sigma−Aldrich, unless indicated otherwise. 4


All other

Buffer solutions were

Page 5 of 19

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


sterilized. Cell Culture. Hela cells were seeded in DMEM/high glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) at 37°C under a humidified atmosphere containing 5% CO2.

To inhibit intracellular acyl-coA/cholesterol acyltransferase

(ACAT) activity, the cells on ITO slide were cultured with 2 µg/mL sandoz 58035 at 37°C for 24 h. For the inhibition of hydroxy methylglutaryl coenzyme A reductase (HMGCoA) activity inside the cells, the ACAT inhibited cells were further treated with 100 µM mevastatin for 24h. Luminescence imaging. The luminescence imaging system shown in Figure 1 was assembled with a water-immersion objective (20 X, Olympus, Japan), a tube and EM CCD (Evolve, Photometrics, Tucson, AZ). The distance between the imaging system and the sample was adjusted using a one-dimensional translation stage. As compared with the classic inverted setup, this direction connection between the objective and CCD in the upright design offered the shortest optical pathway to minimize the luminescence loss during the transmission.

Two-electrode

system, including an indium tin oxide (ITO) slide cultured with cells as the working electrode and Ag/AgCl wire as the reference electrode, was used. 10 mM phosphate buffered saline (PBS, pH 7.4) with 200 µM L012 was used as the solution for cell imaging. The potential was applied through the electrodes using a voltage generator (DG 1021, Rigol, China). The luminescence image recorded was analyzed using Image J software.

RESULTS AND DISCUSSIONS. Double potential mode for the imaging of hydrogen peroxide. The feasibility of our system for the visualization of hydrogen peroxide was demonstrated using an ITO slide partially covered with a layer of tape, so that the bare ITO surface yielded luminescence under applied potential while the tape-covered ITO surface did not generate luminescence. L012, a luminol analog with higher luminescence than luminol, was chosen for the generation of luminescence under positive potential.23

We have previously shown that the peak luminescence from L012/hydrogen

peroxide was generated at 1.0 V on an ITO electrode, and no luminescence was initiated at potentials lower than 0.40 V.23

Therefore, a constant potential of 1.0 V was needed for the

maximum intensity from L012/hydrogen peroxide on the CCD during the exposure time. To minimize the fouling of the electrode after the positive potential, a switching of the potential 5



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

Page 6 of 19

between 1.0 and -1.0 V was employed, as shown in Figure 1 (inset).24, 25 The optimization process of double potential mode was clarified in Figure S1 (supplementary materials).. Under the optimized condition, the electrochemiluminescence image of aqueous hydrogen peroxide at the boundary between the bare and tape-covered regions was exhibited in Figure 2. Figure 2A and B showed the bright field and background luminescence imaging of the boundary before the addition of 10 µM of hydrogen peroxide, respectively. Since L012 could generate luminescence in absence of hydrogen peroxide under the positive potential,23 weak luminescence was observed in the bare ITO region and darkness was observed in the tape-covered region. When 10 µM of hydrogen peroxide was introduced, the image was acquired under dark conditions again (Figure 2C). To exhibit the intensity of luminescence generated from hydrogen peroxide, the luminescence intensity in bare ITO region was measured using Image J software and shown in Figure 2 B and C (inset). More intense luminescence was observed on the bare ITO region, which was ascribed to the ECL process of L012 and hydrogen peroxide. The large noise in Figure 2C (inset, pixel size 0.8 µm) was attributed to the low concentration of hydrogen peroxide (10 µM), which reached the detection limit.

A better S/N ratio was observed from higher

concentration of hydrogen peroxide (Figure S2A in supplementary information). Since the cell size was ~ 30 µm, we calculated the total luminescence in the total region of 30 × 30 µm. The relative standard deviation of luminescence observed at five random regions was 3.23%. Considering that the whole single cell was analyzed as one unit in our work, this uniform generation of luminescence from this region size (30 × 30 µm) guaranteed the analysis accuracy of cells cultured on different regions of the ITO slide. To examine the relationship between luminescence intensity and the concentrations of hydrogen peroxide, more hydrogen peroxide was added into the system and the imaging process was repeated.

The luminescence difference before and after the introduction of hydrogen

peroxide had a near-linear relationship with the concentration of hydrogen peroxide in the range of 10 and 200 µM, as shown in Figure 2D.

When hydrogen peroxide was over 200 µM, a

non-linear relationship observed might be caused by the insufficient amount of L012 (200 µM) in the solution, as supported in Figure S2B (supplementary information).

Since the local

concentration of hydrogen peroxide from cells was typically lower than 100 µM, 200 µM L012 with less cytotoxicity was chosen for the imaging of single cells. 6


In this situation, the

Page 7 of 19

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


luminescence intensity was dependent on the concentration of L012 and hydrogen peroxide, and thus, the near-linear relationship supported that the intensity collected from the solution could be used to estimate the concentration of hydrogen peroxide from single cells. Imaging of hydrogen peroxide efflux from single cells. To validate the system for the imaging of hydrogen peroxide from single cells, Hela cells cultured on an ITO slide were stimulated with 20 ng/ml phorbol myryslate acetate (PMA).

The introduction of PMA has been reported to

stimulate intracellular NADPH oxidase and accumulated hydrogen peroxide inside, thus increasing the hydrogen peroxide efflux.26

Following the same imaging procedure, the

bright-field and background luminescence images of cells in PBS with L012 were collected (Figures 3A and B). Within the exposure time of 5 s, the cells appeared dark in the luminescence image, owing to their adherence on the electrode slowing down the diffusion of L012 to the electrode surface. After the cells were stimulated by PMA to release hydrogen peroxide, a luminescence image was recorded immediately (Figure 3C).

By extracting the background

luminescence (Figure 3B) from the luminescence in Figure 3C, the brightness of the cells was observed (Figure 3D). More typical luminescence image of hydrogen peroxide release from single cells was shown in Figure S3A in supplementary information.

A relative standard

deviation of 37.4% from 20 cells exhibited the cellular heterogeneity on hydrogen peroxide efflux from single cells.

For the control experiment, the un-stimulated cells were imaged by replacing

PMA with PBS. The series of images was shown in Figure S3B (supplementary information). No luminescence increase was observed at the cells indicating no hydrogen peroxide released from the control cells, as expected. The correlation of luminescence with hydrogen peroxide efflux suggested that our strategy can visualize the release of hydrogen peroxide from cells, which achieved the parallel single-cell analysis of hydrogen peroxide efflux. Referring the luminescence intensity generated from aqueous hydrogen peroxide in Figure S2 (supplementary information), the luminescence intensity at PMA stimulated cells (Figure 3 D) was converted in the concentration of hydrogen peroxide and labelled with colors. The different colors in Figure S4A exhibited the local heterogeneity of hydrogen peroxide efflux, which concentration was ranged from 10 to 30 µM. However, it was noted that the intermediate, oxygen radicals, with the lifetime as long as millisecond from the electro-oxidation of hydrogen 7



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

Page 8 of 19

peroxide could induce the delocalization between the bright-field and ECL images affecting the quality of the image (Figure S5 and S6, more discussion at “Quality of ECL image” in the supplementary information).

Therefore, our imaging method could be applied only for the

whole-cell analysis with high analysis rate, and had the difficulty to offer the accurate sub-cellular information. The more luminescence observed at the stimulated cells was believed to be generated from hydrogen peroxide from the bottom surface of cells, which was reacted with L012 under the potential to give the luminescence. Although the cells suffered some electric charges under the potential, this charge on the upper surface of cells was not likely to induce the luminescence from L012 and hydrogen peroxide, as discussed in supplementary information (Figure S7).

Also,

different from the generation mechanism of luminescence on Ru(bpy)32+-antibody/antigen modified microbeads (3 µm)14,16, the luminescence at center of cells was not likely caused by the diffusion of oxygen radicals from cell-electrode interface to the upper surface of cell because no apparent luminescence was observed surrounding the cells, as evidenced in the overlaying image (Figure S6, supplementary information). Moreover, the morphology change, if happened after the stimulation, will give more space between the cells and the electrode so that L012 could diffuse into this space more easily for higher luminescence intensity. To exclude the possibility, SPR microscopy was applied to monitor the morphology change of the cells during the treatments.27,28 The more space between the cells and the electrode should give a decreased intensity in SPR signals, however, no any change in SPR signal were observed in Figure S7 (supplementary information). The result supported that the morphology might not change during the addition of PMA. After excluding the source of luminescence from the upper surface of the cells and the morphology change, the existence of the reaction between L012 and hydrogen peroxide at the bottom surface of the cells was the only possible explanation for more luminescence at cells.

For

the adherent cells, membrane ruffling had been confirmed to create a small space between the bottom surface of the cell and the support surface29,30, which resulted in the accumulation of L012 between the bottom surface of the cells and the electrode. To confirm the presence of L012 between the cells and the electrodes, one part of ITO electrode was covered with the tape and the cells were cultured near the edge of the tape on the electrode. Since no L012 was existed under 8


Page 9 of 19

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


the tape, the accumulation of L012 under the cells should generate an increase in background luminescence.

As shown in Figure S9 (supplementary information), more luminescence

observed under the cells than the tape suggested that L012 might diffuse into the small space between the cells and the electrode. After the stimulation, the release of hydrogen peroxide from the bottom surface of the cells generated oxygen radicals under applied potential for luminol luminescence. Imaging active cholesterol at cellular plasma membrane. After the validation of our strategy for the parallel single-cell analysis, active membrane cholesterol at cells was measured. Active membrane cholesterol, which possesses high chemical activity (escape tendency), plays a key function in cellular cholesterol trafficking.31,32

Many groups used fluorescence or radio-labeling

method to confirm the presence of active membrane cholesterol.22,33-35

Recently, microelectrodes

modified with cholesterol oxidase had been applied successfully for the detection of membrane cholesterol at single cells, however, the position of the microelectrode at each cell surface limited the analysis rate.36,37

Since active cholesterol reacts with cholesterol oxidase to generate

hydrogen peroxide,38 our previous results exhibited the addition of luminol induced the luminescence under the positive potential for the quantitative detection of active membrane cholesterol.23,39

In current work, our ECL imaging strategy could visualize the active cholesterol

in parallel so that high analysis rate was achieved. To activate the membrane cholesterol, cells were pretreated with sandoz 58035 overnight to inhibit intracellular acyl-coA/cholesterol acyltransferase (ACAT), so that more cholesterol was accumulated in the membrane to increase cholesterol acitivity.40

When the cells were exposed to cholesterol oxidase and L012, both

species diffused into the small spaces under the cells.

After the oxidase reacted with active

membrane cholesterol, the applied potential induced luminescence from hydrogen peroxide and L012 for imaging. The bright-field image and the luminescence difference image before and after the addition of cholesterol oxidase were shown in Figure 4 A and B, respectively. exposure time for luminescence images was 5 s.

The

Similar to the result on PMA stimulated cells,

SPR microscopy did not show any morphology change at the cells after the introduction of cholesterol oxidase. Therefore, more luminescence observed at cells exhibited the visualization of active membrane cholesterol. Referring the luminescence intensity generated from aqueous hydrogen peroxide in Figure S2, the luminescence intensity at cells (Figure 4 B) was converted in 9



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

the concentration of hydrogen peroxide and labelled with colors. The different colors in Figure S4 B suggested that the concentration of active membrane cholesterol might be ranged from 10 to 20 µM. The negative control experiment was performed by the inhibition of 3-hydroxy -3-methyl glutaryl-CoA (HMGCoA) activity in ACAT inhibited cells using the drug of statin, which decreased the intracellular cholesterol synthesis resulting in less active membrane cholesterol.23 The luminescence difference image was shown in Figure 4C and the other images, including the bright field and the overlapping images, were shown in Figure S7 (supplementary information). The luminescence observed at HMGCoA/ACAT cells exhibited that active membrane cholesterol was still existed, even though the total amount of intracellular and membrane cholesterol decreased significantly after the inhibition. Compared with ACAT inhibited cells, the weak luminescence at HMGCoA/ACAT cells (Figure 4D) confirmed that our ECL imaging strategy can analyze active membrane cholesterol for the drug study. Overall, this first image of active membrane cholesterol at cells will offer more biological information for the study of cellular cholesterol trafficking.

CONCLUSIONS.

In conclusion, ECL imaging was successfully applied for the parallel

single-cell electrochemical analysis. The simultaneous recording of luminescence from multiple cells offered the information about the efflux of hydrogen peroxide efflux and the amount of active membrane cholesterol. This strategy needed the plat electrodes only, and therefore, the complexity for the fabrication of microelectrode array was avoided facilitating single cell analysis. Ongoing studies are focused on the design of highly luminescent ECL probes to increase luminescence intensity.

The achievement of a larger signal-to-noise ratio in ECL image will

permit a shorter exposure time and smaller pixel size for higher temporal and spatial resolutions. Also, our ECL imaging platform is being applied for the visualization of more molecular efflux from cells, such as oxalate and pyruvate, which are the co-reactants of Ru(bpy)32+.

ACKNOWLEDGEMENTS. This work was supported by the 973 Program (2013 CB933800), the National Natural Science Foundation of China (nos. 21327902, 21135003).

10


Page 10 of 19

Page 11 of 19

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


SUPPORTING INFORMATION. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

11



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

REFERENCES. 1. Wightman, R. M. Science 2006, 311, 1570-1574. 2. Amatore, C.; Arbault, S.; Guille, M.; Lemaitre, F. Chem. Rev. 2008, 108, 2585−2621. 3.

Liu, X.; Barizuddin, S.; Shin, W.; Mathai, C.J.; Gangopadhyay, S.; Gillis, K.D. Anal Chem. 2011, 83, 2445–2451.

4. Wang, J.; Trouillon, R.; Dunevall, J.; Ewing, A. G. Anal. Chem. 2014, 86, 4515−4520. 5. Shan, X. N.; Patel, U.; Wang, S. P.; Iglesias, R.; Tao, N. J. Science 2010, 327, 1363-1366. 6. Wang, W.; Foley, K.; Shan, X.; Wang, S. P.; Eaton, S.; Nagaraj, V. J.; Wiktor, P.; Patel, U.; Tao, N. J. Nat. Chem. 2011, 3, 249-255. 7. Bard, A. J. Electrogenerated Chemiluminescence, Marcel Dekker, New York, 2004. 8. Richter, M. M. Chem. Rev. 2004, 104, 3003-3036. 9. Miao, W. Chem. Rev. 2008, 108, 2506-2553. 10. Forster, R. J.; Bertoncello P.; Keyes, T. E. Annu. Rev. Anal. Chem. 2009, 2, 359-385. 11. Xu, L. R.; Li, Y.; Wu, S. Z.; Liu, X. H.; Su, B. Angew. Chem.-Int. Edit. 2012, 51, 8068-8072. 12. Wu, M. S.; Yuan, D. J.; Xu, J. J.; Chen, H. Y. Chem. Sci. 2013, 4, 1182-1188. 13. Xu, L.R.; Zhou, Z.Y.; Zhang, C.Z.; He, Y.Y.; Su B. Chem Commun. 2014, 50, 9097-9100. 14. Deiss, F.; LaFratta, C. N.; Symer, M.; Blicharz, T. M.; Sojic, N.; Walt, D. R. J. Am. Chem. Soc. 2009, 131, 6088-6089. 15. Dolci, L. S.; Zanarini, S.; Della Ciana, L.; Paolucci, F.; Roda. A. Anal. Chem. 2009, 81, 6234-6241. 16. Sentic, M.; Milutinovic, M.; Kanoufi, F.; Manojlovic, D.; Arbaulta, S.; Sojic, N. Chem. Sci. 2014, 5, 2568-2572. 17. Knight, A.W. TrAC. 1999, 18, 47-62. 18. Haapakka, K.E.; Kankare, J.J. Anal. Chim. Acta. 1982, 138, 263-275. 19. Marquette, C. A.; Blum, L. J. Anal. Chim. Acta. 1999, 381, 1−10. 20. Cui, H.; Xu, Y.; Zhang, Z. F. Anal. Chem. 2004, 76, 4002−4010. 21. Ikonen, E. Nat. Rev. Mol. Cell Biol. 2008, 9, 125-138. 22. Lange, Y. J. Lipid Res. 1992, 33, 315-319. 23. Tian, C. X.; Zhou, J. Y.; Wu, Z. Q.; Fang, D. J.; Jiang, D. C. Anal Chem. 2014, 86, 678-684. 24. Jackson, B.P.; Dietz, S.M.; Wightman, R.M. Anal Chem. 1995, 67, 1115–20. 12


Page 12 of 19

Page 13 of 19

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


25. Robinson, D.L.; Venton, J.B.;. Heien, M.; Wightman, R. M. Clin Chem 2003, 49, 1763–1773. 26. Lundqvist, H.; Follin, P.; Khalfan, L.; Dahlgren, C. J. Leukoc. Biol. 1996, 59, 270-279. 27. Wang, W.; Yang, Y.; Wang, S.; Nagaraj, V.J.; Liu, Q.; Wu, J.; Tao, N. Nat. Chem. 2012, 4, 846-853. 28. Wang, W.; Foley, K.; Shan, X.; Wang, S.; Eaton, S.; Nagaraj, V.J.; Wiktor, P.; Patel, U.; Tao, N. Nat. Chem. 2011, 3, 249-255. 29. Wang, W.; Wang, S. P.; Liu, Q.; Wu, J.; Tao, N. J. Langmuir, 2012, 28, 13373-13379. 30. Iwanaga, Y.; Braun, D.; Fromherz, P. Eur. Biophys. J. Biophys. Lett. 2001, 30, 17-26. 31. Steck, T.L.; Lange, Y. Trends Cell Biol. 2010, 20, 680-687 32. Maxfield, F. R.; Wustner, D. J. Clin. Invest. 2002, 110, 891-898. 33. Lange, Y.; Ye, J.; Steck, T. L. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11664-11667 34. Wüstner, D. Mol Biol Cell. 2007, 18, 211-28. 35. Lange, Y. J. Lipid Res. 1992, 33, 315-319. 36. Devadoss, A.; Burgess, J.D. J Am Chem Soc. 2004, 126, 10214-5. 37. Jiang, D.C.; Devadoss, A.; Palencsár, M.S.; Fang, D.J.; White, N.M.; Kelley, T.J.; Smith, J.D.; Burgess, J.D. J Am Chem Soc. 2007, 129, 11352-3. 38. Lange, Y.; Cutler, H.B.; Steck, T.L. J. Biol. Chem. 1980, 255, 9331– 9337. 39. Ma, G.Z.; Zhou, J.Y.; Tian, C.X.; Jiang, D.C.; Fang, D.J.; Chen, H.Y. Anal Chem. 2013, 85, 3912-7. 40. Warner, G. J.; Stoudt, G.; Bamberger, M.; Johnson, W. J.; Rothblat, G. H. J. Biol. Chem. 1995, 270, 5772-5778.

13



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

Page 14 of 19

Figures and Legends. Figure 1. Schematic setup used for ECL imaging and double potential mode (inset). Figure 2. The boundary of the tape covered ITO electrode and bare electrode. (A) bright-field image; (B) the luminescence image with 200 µM L012; (C) the luminescence image with 200 µM L012 and 10 µM hydrogen peroxide; (D) the relationship of luminescence intensity with the concentration of hydrogen peroxide in the presence of 200 µM L012. The insets in image B and C were the luminescence intensity across the red line in the images.

The exposure time was 5 s.

The error bar presented the standard deviation of the luminescence intensity from three independent experiments. Figure 3. Images of Hela cells on an ITO electrode. (A) bright-field image; (B) luminescence image with 200 µM L012; (C) luminescence image with 200 µM L012 after stimulation by PMA; (D) image of luminescence difference between images B and C. The exposure time was 5 s. Figure 4. Images of ACAT inhibited Hela cells on an ITO electrode with 200 µM L012. (A) bright-field image; (B) image of luminescence difference before and after the addition of cholesterol oxidase; (C) image of luminescence difference selected on ACAT/HMGCoA inhibited cells before and after the addition of cholesterol oxidase; (D) bar of the luminescence intensity collected from twenty ACAT inhibited and ACAT/HMGCoA inhibited cells. The exposure time was 5 s. The error bar presented the standard deviation of luminescence intensity from 20 cells.

14


Page 15 of 19

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.

15



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 2.

16


Page 16 of 19

Page 17 of 19

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.

17



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 4.

18


Page 18 of 19

Page 19 of 19

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


For TOC only

19


Electrochemiluminescence Imaging for Parallel Single-Cell Analysis of

Recommend Documents