Direct Electrochemiluminescence Imaging of a Single Cell on a

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Direct electrochemiluminescence imaging of a single cell on a chitosan film modified electrode Gen Liu, Cheng Ma, Baokang Jin, Zixuan Chen, and Jun-Jie Zhu Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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Analytical Chemistry

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Direct electrochemiluminescence imaging of a single cell on a chitosan film

2

modified electrode Gen Liua,b, Cheng Mab, Bao-Kang Jina ,*, Zixuan Chen b ,*, and Jun-Jie Zhub ,*

3 4 5

a

6

b

7

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

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China

8 9

*To whom correspondence should be addressed:

10

[email protected] (B.-K. Jin),

11

[email protected] (Z.-X. Chen)

12

[email protected] (J.-J. Zhu).

13

1

ACS Paragon Plus Environment

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

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Single-cell imaging is essential for elucidating the biological mechanism of cell function

16

because it accurately reveals the heterogeneity among cells. The electrochemiluminescence (ECL)

17

microscopy technique has been considered a powerful tool to study cells because of its high

18

throughput and zero cellular background light. However, since cells are immobilized on the

19

electrode surface, the steric hindrance and the insulation from the cells make it difficult to obtain a

20

luminous cell ECL image. To solve this problem, direct ECL imaging of a single cell was

21

investigated and achieved on chitosan and nano-TiO2 modified fluoride-doped tin oxide conductive

22

glass (FTO/TiO2/CS). The permeable chitosan film is not only favorable for cell immobilization but

23

also increases the space between the bottom of cells and the electrode, thus, more ECL reagent can

24

exist below the cells compared with the cells on a bare electrode, which guarantees the high

25

sensitivity of quantitative analysis. The modification of nano-TiO2 strengthens the ECL visual

26

signal in luminol solution and effectively improves the signal-to-noise ratio. The light intensity is

27

correlated with the H2O2 concentration on FTO/TiO2/CS, which can be applied to analyze the H2O2

28

released from cells at the single-cell level. As far as we know, this is the first work to achieve cell

29

ECL imaging without the steric hindrance effect of the cell, and it expands the applications of a

30

modified electrode in visualization study.

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Keywords: Electrochemiluminescence, Chitosan film, Hydrogen peroxide, Cell imaging

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Electrochemiluminescence (ECL), also called electrogenerated chemiluminescence, involves

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the process where species generated at the electrode surface undergo high energy charge transfer

37

reactions to form excited states that emit light. 1 ECL was first reported by Dufford in 1927 2 and in

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recent years has become a widely used analytical technique in a diversity of fields including

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bioanalysis, nanomaterials and light-emitting devices.

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photomultiplier (PMT) detector have high sensitivity for detecting the optical signal from the whole

41

electrode, the fine details of the electrode are unclear because they have no spatial resolution.

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Fortunately, ECL imaging provides a high temporal-spatial resolution approach, and much work so

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far has focused on the visualized ECL analysis. For example, Su and co-workers successfully

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established a method for visualizing latent fingerprints on the electrode surface. 7 Willets et al.

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reported the generation of ECL at single gold nanowire electrodes. 8 A three-dimensional ECL

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imaging approach was developed by Sojic’s group and applied to study the ECL mechanistic route. 9

47

Single-cell analysis can offer significant information regarding the cellular heterogeneity and

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the expression protein on the cell surface. The imaging of a single cell is an effective means to

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obtain insight into the processes that occur between drugs and cells, and it can help us to intensively

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understand cell-to-cell communication. Because of the lack of an external light source, ECL

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microscopy for single-cell imaging possesses zero background and a high signal-to-noise ratio

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compared with photoluminescence imaging.

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electrochemical or ECL methods, 11-13 which rely on the information of cell groups, ECL imaging

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can offer visual details of a single cell. Jiang reported indirect ECL imaging of a single cell for the

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analysis of active cell membrane cholesterol. 14 Recently, Sojic realized the first spatially resolved

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ECL imaging of membrane proteins of a single cell. 15 However, the cells on the electrode surface

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unavoidably block the electron transfer and hinder the diffusion of the ECL reagent, 13 which leads

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to dark contrast in the cell regions or only ECL emission at the cell edges. It is a significant

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challenge to overcome this negative effect when we investigate cells using the ECL imaging

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technique.

10

3-6

Though ECL instruments based on a

Unlike the works of determining cells using

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H2O2 is one of the reactive oxygen species (ROS) that are associated with the regulation of

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cellular processes in vivo. H2O2 at an appropriate level is vital for cells to maintain normal

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functions and the excessive H2O2 usually induces biological damages, causing cancer, aging,

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diabetes and so on. 16,17 Therefore, the development of a sensitive and specific method for reliable 3



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detection of H2O2 concentration under physiological conditions is of great important for early

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cancer screening and diagnosis.

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Chitosan is one deacetylated form of a natural polymer derived from various sources (insects,

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crustaceans, the cell walls of fungi and yeast, etc.). 18,19 Due to its good biocompatibility, some cells

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can adhere and proliferate on chitosan or its composites.19-21 In addition, as the most popular binder,

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a chitosan-modified electrode has been widely applied in electrochemical analysis. 22-24 By virtue of

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these merits, we developed a direct ECL microscopy strategy of single cells on a chitosan

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film-modified electrode. The cervical cancer cells (HeLa) could be cultured and stably immobilized

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on the chitosan film. The cells were elevated by the film and did not touch the electrode surface, so

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the steric hindrance effect of cells was obviously reduced. The ECL reagent in the bulk solution

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could pass around the cell, enter into the chitosan film and finally realize ECL on the electrode

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surface. Upon being stimulated by N-formylmethionyl leucyl phenylalanine (fMLP), the cells

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secreted H2O2 immediately, and then a bright image was observed in the cell region because of the

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classic ECL reaction between luminol and H2O2. Furthermore, we found that the nano-TiO2

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modified FTO electrode could obviously amplify the ECL signal and offer high signal-to-noise ratio

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due to the electro-catalysis of TiO2 in the ECL reaction. FTO/TiO2/CS showed sensitive responses

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to H2O2 and demonstrated the heterogeneity of H2O2 released from individual cells. This work

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provides a new and simple ECL strategy to visually study cells at the single-cell level.

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84

EXPERIMENTAL SECTION

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Materials and Reagents. All reagents were used without further purification, and deionized

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distilled water was used throughout the experiments. Fluoride-doped tin oxide conductive glass

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(FTO, sheet resistance ≤ 10 Ω/sq) was purchased from Guangdong Huanan Xiangcheng Technology

88

Co., Ltd. (China). TiO2 powder (P25) was obtained from Degussa Co. (Germany).

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8-Amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione (L012, a luminol analogue)

90

was bought from Wako Chemical USA Inc. (Richmond, VA). Chitosan (CS, 80%~95%

91

deacetylation)

92

N-formylmethionyl-leucyl-phenylalanine (fMLP) and diphenyleneiodonium (DPI) were purchased

93

from

Aladdin

was

obtained

(USA),

and

from

they

Sinopharm

were

Chemical

dissolved

in

4


Reagent

dimethyl

Co.,

Ltd.

sulfoxide

(China).

(DMSO).

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3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from

95

Nanjing KeyGen Biotech. Co., Ltd. (China). H2O2 was purchased from Shanghai Ling Feng

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Chemical Reagent Co., Ltd. (China). All solutions were prepared using ultrapure water (18.2 MΩ

97

cm resistivity).

98 99

Apparatus. The ECL imaging setup, as demonstrated in Figure 1, was assembled with two major

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apparatuses. A CHI 660D electrochemical workstation (CH Instruments Co., China) was the signal

101

actuating device to generate and record electrochemical responses. An electron multiplying CCD

102

(EM-CCD) (Evolve, Photometrics, Scientifica, USA) was the signal collecting device to output

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images. The optical path system was built with a water immersion objective (60X, NA 1.1,

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Olympus, Japan), a convex lens, a reflector and tubes. The ECL imaging test must be operated in a

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shielding room.

106

A conventional three-electrode system was used for all electrochemical experiments, which

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consisted of a FTO glass or a modified electrode as the working electrode, an Ag/AgCl (saturated

108

KCl) as the reference electrode and a platinum wire as the auxiliary electrode.

109 110

Scanning electron microscopy (SEM) tests were performed on a Hitachi S-4800 scanning electron microscope (Hitachi Co., Japan) and a Shimadzu SSX-550 (Shimadzu Co., Japan).

111 112

Cell culture and immobilization on electrode. The reagents for the cells experiments were all

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obtained from Nanjing KeyGen Biotech. Co., Ltd. (China). The cervical cancer cells (HeLa) were

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cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf

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serum, 4.5 g/L of glucose, 0.584 g/L of L-glutamine, 3.7 g/L of sodium bicarbonate, 0.11 g/L of

116

sodium pyruvate, 80 U/mL of penicillin and 80 mg/mL of streptomycin. The cells were proliferated

117

and formed as a monolayer in an incubator of humidified air atmosphere (relative humidity of 95%)

118

with 5% CO2 at 37 °C.

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The cells in the exponential growth phase were digested for 10 min by trypsin (0.1%, m/v) and

120

then separated from the cell culture medium. Centrifuging at 1000 rpm for 5 min was essential to

121

gather the cells after washing thrice with a sterile phosphate buffer solution (PBS, 10 mM, pH 7.4).

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The cell precipitate was then resuspended in 2 mL of DMEM.

123

Prior to seeding cells, FTO slices were successively cleaned in an ultrasonic bath with NaOH 5



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solution, ethanol, and ultrapure water for 5 min and were then dried with nitrogen flow. TiO2

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aqueous dispersion was cast onto the FTO surface with an area of 1.5 × 1.5 cm2 and dried in air. The

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chitosan solution was prepared by dissolving into 1.0% (v/v) acetic acid aqueous solution. This

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obtained solution was cast on FTO/TiO2 and dried in air. After forming chitosan film, the electrode

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(FTO/TiO2/CS) was washed with 0.1 M of sodium hydroxide solution for a given time to neutralize

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the excess acetic acid. After that, FTO/TiO2/CS was gently washed in ultrapure water and dried in

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air again. Finally, HeLa cells were seeded on the FTO/TiO2/CS surface and cultured in an incubator

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for 8 h, which achieved the immobilization of cells. Before measurement, the electrode with the

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cells was washed with PBS two times to wash out the unattached cells. For comparison, the bare

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FTO and the chitosan-modified electrode (FTO/CS) were prepared and used to immobilize cells

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under the same conditions.

135 136

RESULTS AND DISCUSSION

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Reducing the steric hindrance effect of cells. HeLa cells were immobilized on a bare FTO and

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used for ECL imaging in a solution of 10 mM of PBS (pH 7.4) and 400 µM of L012. MTT

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experiments confirm that 400 µM of L012 has low cytotoxicity (Supporting Information Figure S1).

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To our knowledge, cells can release a greater deal of H2O2 within a short time when simulated by

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fMLP, 25 and H2O2 can enhance the ECL of luminol. Hence, fMLP was adopted to trigger the cell

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ECL signal in the L012 solution. The ECL images before the addition of fMLP (100 ng·mL-1) and

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after the addition of fMLP were collected and are shown in Figure 2A2 and 2A3, respectively. The

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adhered cells exert an undesirable effect on the diffusion of L012 and the charge transfer, which

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leads to the dark ECL imaging of cells, which is consistent with a previous report. 14

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To overcome the steric hindrance effect of cells on the electrode, a bigger space below the cells

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and the liquid exchange is needed. As a permeable and biocompatible polymer, chitosan is a

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suitable biomaterial candidate to modify the electrode. Chitosan film was applied to increase the

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space between the bottom of the cells and the electrode surface. Then, 100 µL of chitosan solution

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with concentrations of 0.5%, 1%, 2%, 3%, and 4% (w/v) was cast on the FTO (1.5 × 1.5 cm2). After

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air-drying, alkaline washing, water washing and air-drying again, the chitosan films were peeled off

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from the electrode carefully. The chitosan film has a homogeneous and dense structure, as shown in

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the SEM images (Figure 3). The thicknesses of the dry films prepared from 0.5%, 1%, 2%, 3%, and 6


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4% (w/v) chitosan solutions are ~1.8 µm, 3.9 µm, 8.3 µm, 11.9 µm, and 16.1 µm, respectively. Thus,

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the thickness of the chitosan film prepared from 0.1% (w/v) chitosan solution can be estimated to be

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~0.5 µm. In addition, the dark field imaging demonstrates that the thickness of the chitosan film

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changes little in the aqueous solution (Supporting Information Figure S2 and Table S1).

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Following the same imaging procedure, FTO/CS/cell modified by 0.1% (w/v) chitosan (~0.5

159

µm) also gives dark ECL imaging of cells (Figure 2B2 and 2B3) because the chitosan film is too thin

160

to effectively eliminate the steric hindrance effect of cells. Interestingly, for FTO/CS/cell modified

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by 0.5% (w/v) chitosan (~1.8 µm), the brightness of the immobilized cells is directly observed after

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fMLP stimulation (Figure 2C3), and no dark cell imaging appears before the addition of fMLP

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(Figure 2C2), suggesting the steric hindrance effect of cell has been eliminated. The ECL reagent

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L012 in the bulk solution passes around the cell, enters into the chitosan film and finally realizes

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ECL in the space between cells and electrode. Continuing to increase the thickness (the thickness of

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the chitosan film is the distance between the bottom of the cells and the FTO surface), FTO/CS/cell

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modified by 1.0% (w/v) chitosan (~3.9 µm) shows similar results (Figure 2D2 and 2D3), except for

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the low luminosity. Nevertheless, with further increase in the thickness of the chitosan film, for

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FTO/CS/cell modified by 2.0% (w/v) chitosan (~8.3 µm), it is difficult to see the cell ECL image

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after the addition of fMLP (Figure 2E3), indicating the brightness can be reduced when exceeding

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the optimal chitosan thickness.

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Without fMLP stimulation (Figure 4A), the background is brighter than the cell for chitosan

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concentration lower than 0.5%, and the light intensity of the background is approximately equal to

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the cell from 0.5% to 2%. However, after fMLP stimulation, the cell is brighter than the background

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for 0.5% and 1.0% (Figure 4B), particularly for 0.5%, indicating that a chitosan film with an

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appropriate thickness is beneficial to realize the direct ECL imaging of cells. For the cells adhered

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on the chitosan film, the film raises the cells and increases the space between the bottom of the cells

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and the electrode, which allows more L012 remaining in the space and guarantees the constant

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concentration of ECL reagent below the cells. It is crucial to accurately analyze the H2O2 released

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from the cells. In fact, chitosan film is also a poor diffusion barrier, and a thick film has a negative

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impact on mass transfer, which can weaken the luminescence. Therefore, a chitosan film with an

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appropriate thickness can reduce or offset the steric hindrance effect from cells in the process of the

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ECL reaction. The sufficient L012 and the fast substance exchange below the cells not only support 7



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the available brightness but also ensure a sensitive and quantitative analysis in our cell ECL

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imaging. Herein, the electrode modified by 0.5% (w/v) chitosan (~1.8 µm) was chosen in the

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following work, unless stated otherwise.

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Recently, some reports have indicated that TiO2 nanoparticles can greatly intensify the ECL of

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luminol, since the excitons of luminol and the reactive oxygen species are easily created on the TiO2

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surface.

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nano-TiO2 was used to enhance the ECL intensity of L012. The optimization of TiO2 modification

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and the electric parameters of chronoamperometry (CA) are clarified in the Supporting Information

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(Figures S3 and S4). Under the optimal conditions, FTO/TiO2/CS (Figure 5B2) exhibits more

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obvious cell ECL imaging compared with FTO/CS (Figure 5A2). The light intensities of the

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background and the cells on FTO/TiO2/CS are both higher than those on FTO/CS, as demonstrated

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in Figures 5A3 and 5B3. Moreover, the signal-to-noise ratio (S/N) is improved obviously on

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FTO/TiO2/CS (S/N=5.5:1) compared with FTO/CS (S/N=3.2:1), which is ascribed to the

197

electro-catalysis of nano-TiO2.

26,27

Moreover, nano-TiO2 has already proven to be of low toxicity.

28,29

Therefore,

198

Considering that the location of the cell is uncertain on FTO/TiO2/CS, we randomly chose six

199

areas with a size of 30 × 30 µm2 (the cell size) and recorded the luminescence. The light intensity

200

fluctuated within a narrow range with a relative standard deviation (RSD) of 5.3%. This low RSD

201

supported the uniform modification of chitosan and TiO2 and guaranteed the analysis accuracy of

202

cells. FTO/TiO2/CS was a practical ECL electrode in the visualization study, especially in cell

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imaging. More cells were stimulated by fMLP and tested on FTO/TiO2/CS, and the RSD of the light

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intensity of cells was 30.6% (from 15 cells), indicating the heterogeneity of H2O2 released from a

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single cell. Furthermore, the detachment and the morphological change of the cells were not

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observed after ECL imaging, suggesting that the ECL process has a negligible influence on the cells

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(Supporting Information Figure S5). In addition, the influence of DMSO (the solvent of fMLP) on

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ECL imaging was eliminated because it does not cause cells to produce H2O2 (Supporting

209

Information Figure S6).

210 211

Detection of H2O2 released from a single cell. According to the previous discussion, the steric

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hindrance of cells on the bare electrode results in the low light intensity in the cell region relative to

213

the background. Increasing the distance between the cell and the FTO via a chitosan film with a 8


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certain thickness can overcome this negative effect. Moreover, the transparent cells cannot block the

215

light emitted from the FTO surface. Herein, various concentrations of H2O2 were measured on

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FTO/TiO2/CS/cell in 400 µM of L012 solution containing PBS (10 mM, pH 7.4). With the increase

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in H2O2 concentration (Supporting Information Figure S7), the light intensity becomes higher and

218

has a linear correlation: I = 35.93 C + 3645, r = 0.9781, from 20 µM to 350 µM (n = 5); the

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detection limit is 10 µM. However, a non-linear relationship appears above 350 µM of H2O2, which

220

is probably caused by the deficiency of L012. Besides, FTO/TiO2/CS/cell has lower detection limit

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and higher sensitivity than FTO/CS/cell and FTO/cell (Supporting Information Table S2).

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To our knowledge, the H2O2 released from cells is at a micromole level under exogenous

223

stimulation. 14, 30 After fMLP stimulation, the average H2O2 concentration released for a single cell

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is ~45 µM (RSD=38.9%, n = 25), as shown in Figures 6A2 and 6C. In this study,

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diphenyleneiodonium (DPI, an NADPH oxidase inhibitor) was applied to inhibit the production of

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fMLP stimulated H2O2. DPI (30 ug·mL-1) was incubated with the cells for 30 min. Then, fMLP was

227

added and the ECL image was collected immediately. As shown in Figure 6B2, the luminescence of

228

HeLa cells with DPI pretreatment obviously decreases compared with those without DPI inhibition.

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A single cell secretes ~15 µM of H2O2 on average. The RSD of 67.1% (n = 25) illustrates that there

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are distinct individual differences in H2O2 secretion at the single-cell level, which reveals the

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superiority of single-cell ECL microscopy. The similar experiments were carried out on MCF-7

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cells (Supporting Information Figure S8). The average H2O2 concentration is ~ 17 µM (RSD=70.2%)

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for DPI pretreated cells and ~ 50 µM (RSD=40.6%) for non-DPI pretreated cells, respectively. The

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cells with DPI treatment secrete H2O2 less than those without DPI treatment, indicating that ECL

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imaging is a simple and effective technique to evaluate drug effects, especially promotion and

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inhibition effects from exogenous substrates.

237

238

CONCLUSION

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In summary, we successfully prepared a FTO/TiO2/CS electrode for direct ECL imaging of

240

single cells. This ECL imaging method overcame the steric hindrance effect caused by cells by the

241

virtue of a permeable chitosan film, and it was successfully used to evaluate the drug effect at the

242

single-cell level. As a binder with good biocompatibility, chitosan shows potential for applying the

243

classic and resourceful ECL detection strategy to ECL imaging, especially cell imaging. Expanding 9



244

the space between the cells and the electrode is favorable for the ECL visualization study of cells.

245

Hence, modifying electrodes with materials containing an open structure or good permeability, such

246

as gelatin and agarose, is worth considering. Apart from H2O2, other intracellular substances or

247

exocytosis including NO, H2S, glucose, lactic acid, etc. are also expected to realize ECL imaging by

248

a similar method. Therefore, based on this high throughput ECL imaging system, more visible

249

information regarding cells will be explored and presented in the future.

250

251

ASSOCIATED CONTENT

252

Supporting Information. Additional information as noted in the text. This material is available

253

free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

257

Corresponding Authors

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[email protected] (B.-K. Jin),

259

[email protected] (Z.-X. Chen)

260

[email protected] (J.-J. Zhu).

261

Notes

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The authors declare no competing financial interest.

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264 265 266

ACKNOWLEDGMENTS This research is supported by National Natural Science Foundation of China (Grants Nos. 21335004, 21427807, 21605081 and 21375001).

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REFERENCES 10


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Figure Captions

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Figure 1. The setup and strategy for the ECL imaging of cells.

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Figure 2. Images of HeLa cells on bare FTO (A), FTO/CS modified by 0.1% (B), 0.5% (C), 1.0%

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(D), and 2.0% (E) (w/v) chitosan and FTO/TiO2/CS modified by 0.5% chitosan (F). The first row

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(A1, B1, C1, D1, E1, and F1): bright-field images; the second row (A2, B2, C2, D2, E2, and F2): ECL

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images before the addition of fMLP; the third row (A3, B3, C3, D3, E3, and F3,): ECL images after

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the addition of fMLP. The L012 concentration was 400 µM, and the exposure time was 10 s. The

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pixel size of all images was 0.32 µm.

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Figure 3. SEM images of the cross-section of chitosan films prepared from 0.5% (A), 1% (B), 2% (C), 3% (D) and 4% (E) (w/v) chitosan solution.

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Figure 4. The light intensity of background and cells before (A) and after (B)the addition of fMLP.

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The data were obtained from three independent experiments (at least 10 cells).

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Figure 5. Bright field image of FTO/CS (A1) and FTO/TiO2/CS (B1); ECL image after the addition

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of fMLP in 400 µM of L012 on FTO/CS (A2) and FTO/TiO2/CS (B2); The light intensity across the

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yellow line including the background and the cells on FTO/CS (A3) and FTO/TiO2/CS (B3).

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Figure 6. ECL images of HeLa cells on FTO/TiO2/CS. (A1 and B1): Bright field image in PBS (pH

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7.4, 10 mM); (A2): ECL pseudocolor image without DPI pretreatment in 400 µM of L012; (B2):

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ECL pseudocolor image with DPI pretreatment in 400 µM of L012; (C): The H2O2 released from

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HeLa cells with and without DPI pretreatment and the data obtained from 25 HeLa cells.

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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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Table of Contents (TOC)

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Direct Electrochemiluminescence Imaging of a Single Cell on a

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