Immuno-Electrochemiluminescent Imaging of a Single Cell Based on

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Immuno-Electrochemiluminescent Imaging of Single Cell Based on Functional Nanoprobes of Heterogeneous Ru(bpy)32+@SiO2/Au Nanoparticles Juntao Cao, Yu-Ling Wang, Jing-Jing Zhang, Yu-Xiang Dong, Fu-Rao Liu, Shu-Wei Ren, and Yan Ming Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02141 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 4, 2018

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Analytical Chemistry

1

Immuno-Electrochemiluminescent Imaging of Single Cell Based on

2

Functional Nanoprobes of Heterogeneous Ru(bpy)32+@SiO2/Au

3

Nanoparticles

4

Jun-Tao Cao†,∗, Yu-Ling Wang†, Jing-Jing Zhang†, Yu-Xiang Dong†, Fu-Rao Liu†,

5

Shu-Wei Ren‡, Yan-Ming Liu†,∗

6

†

7

Utilization of Agro-bioresources in Dabie Mountains, Xinyang Normal University,

8

Xinyang 464000, P.R.China

9

‡

College of Chemistry and Chemical Engineering, Institute for Conservation and

Xinyang Central Hospital, Xinyang 464000

10 11

ABSTRACT: It is valuable to develop a sensing platform for not only detecting a

12

tumor marker in body fluids but also measuring its expression at single cells. In the

13

present study, a simple closed bipolar electrodes-based electrochemiluminescence

14

(BPEs-ECL) imaging strategy was developed for visual immunoassay of prostate

15

specific antigen (PSA) at single cells using functional nanoprobes of heterogeneous

16

Ru(bpy)32+@SiO2/Au nanoparticles. Multiple-assisted ECL signal amplification

17

strategy was introduced into the detection system on the basis of the synergetic

18

amplifying effect of the anodic and cathodic amplification. Based on the synergetic

19

amplifying effect, the detection limits of PSA by using photomultiplier tube and CCD

20

imaging are 3.0 pg/mL and 31 pg/mL, respectively. The obtained immunosensor was

21

employed to evaluate PSA levels in serum samples with a satisfying result. Moreover,

22

the obtained functional nanoprobes were used to visually profile the PSA expression

23

on the surface of single LNCaP cells (a kind of prostate cancer cells) based on a bare

24

BPE. The results show that the functional nanoprobes-based ECL imaging

25

immunoassay provides a promising visual platform for detecting tumor markers

26

(proteins and cancer cells), and thus shows a high potential in cancer diagnosis.

27

KEYWORDS:

28

multiple-assisted ECL signal amplification strategy; closed bipolar electrodes;

29

functional nanoprobes; single LNCaP cells

Immuno-electrochemiluminescent

imaging

1

ACS Paragon Plus Environment

of

single

cell;

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

1

INTRODUCTION

2

Proteins as one of the most important classes of molecules in living cells and

3

tissues play essential role in all cellular processes such as providing structure support

4

to cells, catalyzing of biochemical reactions, transporting molecules across membrane,

5

controlling cell growth and adhension.1 The sensitive and accurate quantification of

6

proteins is crucial to understand their role in disease progression, cell differentiation

7

and fate, and for disease-related biomarker discovery and development of novel

8

therapeutics and diagnostics. Some methods such as gel electrophoresis,2 mass

9

spectrometry,3 and chromatography4 have been developed to analyze proteins.

10

However, the population-averaged data obtained by these methods from a large

11

number of cells can mask the information from individual cells owing to the

12

heterogeneity of cellular systems. Therefore, the acquirement of protein-associated

13

data at the level of single cells is desirable.

14

To date, various analysis techniques such as optical spectroscopy,5 electrochemical

15

assay,6 mass spectrometry,7 imaging techniques8 have been applied in single cell

16

analysis. These tools provide rich biochemical information of single cells. ECL

17

imaging assay featuring the merits of low background, high sensitivity, good temporal

18

and spatial resolution has attracted much attention for bioanalysis.9-11 Currently, it has

19

been proved to be a powerful tool for detection of proteins,12 polypeptides,13 small

20

molecules14 and cells.15 However, the reported ECL visual assay for targets was still

21

performed in the body fluids and/or a large number of cells. Specifically, the ECL

22

data only indirectly reflect the average outcome from a population of cells, the

23

individual information of single cells was hidden. Up to now, the straight forward

24

ECL imaging protein on the cell surfaces at single cell levels is still challenging. Very

25

recently, using L012 with higher luminescence as ECL emitter, Jiang’s group16,17 has

26

realized the direct ECL imaging analysis of small biomolecules including cholesterol

27

and intracellular glucose at single cell levels, providing ingenious strategy for single

28

cell imaging. Valenti et al.18 employed ECL as a confined microscopy to directly

29

image single cells and their membrane proteins on carbon nanotubes-based 2


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1

inkjet-printed disposable electrode. These works realized the direct ECL imaging of

2

single cells. Different from the above two examples which the redox reaction in the

3

ECL detection occurred in the same electrode interface, the closed bipolar

4

electrode-ECL (BPE-ECL) system could well avoid the chemical interference

5

between sensing and reporting reactions existed in conventional three-electrode ECL

6

system19 and improve the imaging sensitivity. It can be deduced that a novel ECL

7

imaging assay for single cells and their proteins would be achieved by integrating the

8

high efficiency ECL emitters with high specific immunoassay on the bipolar electrode

9

based-platform.

10

In recent years, Ru(bpy)32+-doped silica (Ru(bpy)32+@SiO2) has become an ideal

11

nanocomposite in ECL bioanalysis as SiO2 nanoparticles have been demonstrated to

12

be a good matrix for immobilizing a high concentration of Ru(bpy)32+ to promote

13

ECL intensity.20-23 For example, by introducing Ru(bpy)32+@SiO2 NPs into the ECL

14

detection system, Rusling’s group developed the ECL immunosensing platforms for

15

interleukin-621 and prostate specific antigen (PSA) detection.21,22 In our previous

16

report, Ru(bpy)32+@SiO2@Ru(bpy)32+ serving as signal tags was also used for the

17

PSA analysis.23 In the past few decades, gold nanoparticles (Au NPs) have been

18

widely used as carriers of proteins to construct biosensors,24,25 which is attributed to

19

their excellent conductivity, large active surface area, and good biocompatibility.

20

Therefore, the decoration of Ru(bpy)32+@SiO2 with Au NPs (Ru(bpy)32+@SiO2/Au

21

NPs) would be a promising ECL luminophore reagent for the construction of ECL

22

imaging biosensors.

23

Herein, a versatile BPE platform for the ECL immuno-imaging of PSA both in

24

serum samples and on the surface of single cancer cells (LNCaP cells) was achieved

25

for the first time using heterogeneous Ru(bpy)32+@SiO2/Au NPs as ECL emitters. To

26

improve the imaging sensitivity, multiple signal amplification technology was

27

introduced into the closed BPE-ECL system and depicted as follows. On the one hand,

28

the signal amplification on the anodic pole was acquired using heterogeneous

29

Ru(bpy)32+@SiO2/Au NPs, which is outlined as follows: (i) silica NPs could

30

immobilize a large amount of Ru(bpy)32+ molecules; (ii) Au NPs decorated on the 3



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1

surface of Ru(bpy)32+@SiO2 NPs could not only promote the ECL signal of

2

Ru(bpy)32+@SiO2, but also effectively increase the amount of loaded antibody. On the

3

other hand, dual amplifying effects from the cathodic pole were realized using

4

Au/indium tin oxide (ITO) hybrid BPEs dipped in K3Fe(CN)6: (i) electrodeposited Au

5

NPs on the ITO (Au/ITO) were taken as the cathodic pole which presented a higher

6

cathodic reduction current, and resulted in an increased ECL intensity of the

7

Ru(bpy)32+-tripropylamine (TPA) system on the anodic pole; (ii) the reduction

8

potential of [Fe(CN)6]3- (+0.36 V) is much lower than that of oxygen (+1.21 V), the

9

external voltage was effectively reduced when the reduction reaction of [Fe(CN)6]3-

10

occurred at the cathodic pole. Based on the synergetic amplifying effect of the

11

multiple amplifying strategies, the PSA was detected both using the traditional

12

photomultiplier tube (PMT)-based method and the ECL imaging protocol. To verify

13

the feasibility and adaptability of the proposed ECL platform for single cell imaging,

14

the PSA on LNCaP cells was evaluated at single cell levels. This BPE-ECL system

15

not only provides a sensitive approach for visually detecting tumor markers but

16

achieves visual immuno-imaging for single cancer cells, which has a good application

17

prospect in the field of life science.

18

EXPERIMENTAL SECTION

19

Preparation of Heterogeneous Ru(bpy)32+@SiO2/Au NPs and Ab2 Labeled

20

Ru(bpy)32+@SiO2/Au Bioprobes. The Ru(bpy)32+@SiO2 NPs and Ab2 labeled

21

Ru(bpy)32+@SiO2/Au

22

according to the previous report with a slight modifition.22 The detailed process was

23

described in the Supporting Information.

bioprobes

(Ru(bpy)32+@SiO2/Au-Ab2)

were

synthesized

24

Assembly of the ECL Assay Interface. First, ITO slices were treated by

25

photolithography and chemical etching and electrodeposition to fabricate Au/ITO

26

hybrid BPEs (the corresponding operation and characterization were included in the

27

Supporting Information). Then, a sandwich type ECL assay interface was fabricated

28

on the pretreated Au/ITO hybrid BPE (the pretreated process was shown in

29

Supporting Information) by stepwise modification. As shown in Scheme 1B, 5% 4


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1

glutaraldehyde (GLD) was placed onto the interface of the anodic pole with

2

pretreatment and left at room temperature for 30 min. Then, 20 µL of 50 µg/mL Ab1

3

was introduced onto the ITO/GLD activated surface by covalent coupling. After

4

washing with 0.01 M phosphate buffered saline (PBS), 20 µL of 1% BSA was

5

injected into the anodic reservoir and left for 1 h at 37 °C. The resulting anodic pole

6

was then incubated with 20 µL PSA solution at various concentrations at 37 °C for 40

7

min. Then, 20 µL of the Ru(bpy)32+@SiO2/Au-Ab2 was dropped onto the electrode

8

and incubated at 37 °C for 40 min.

9

Cell Culture and Treatment. LNCaP cells were cultured in RPMI 1640 medium

10

supplemented with fetal bovine serum (10%), penicillin, and streptomycin at 37 °C

11

under a humidified atmosphere containing 5% CO2. Hela cells were cultured similarly

12

except that RPMI 1640 was replaced by DMEM. The cells were harvested at the

13

logarithmic growth phase and the cell concentrations were determined using a

14

Petroff-Hausser cell counter.

15

To achieve ECL imaging analysis of PSA at single cells, living LNCaP cells and

16

Hela cells were seeded on the anodic pole of the BPEs at 37 °C, 5% CO2 for 6 h,

17

respectively. Then, the cells on the electrode were washed and fixed with 4%

18

paraformaldehyde. After blocking the nonspecific binding sites on the cell surface and

19

the electrode surface with 2% BSA for 30 min, the Ru(bpy)32+@SiO2/Au-Ab2 probes

20

were introduced and incubated with fixed cells for 1 h at 37 °C. The ECL single cell

21

imaging measurements were performed after washing the labeling cells with sterile

22

PBS.

23

ECL Imaging Measurements. ECL experiments were carried out using an MPI-B

24

electrochemical and ECL analyzer. The ECL-voltage curves were obtained by

25

applying a linearly increasing voltage (from 0.2 − 4.0 V) on the end of the two driving

26

electrodes with a scan rate of 0.1 V/s. ECL imaging experiments were performed by

27

using a set of equipment assembled with an air-immersion objective (4 ×) and a

28

water-immersion objective (20 ×, Olympus, Japan), a tube, electron multiplying CCD

29

(EM CCD) (Evolve, Photometrics, Tucson, AZ). A switching of the potential between

30

4.0 V (2 s) and 0.2 V (0.5 s) was continuously applied using a voltage generator (DG 5



Page 6 of 17

1

1021, Rigol, China) to induce the luminescence at room temperature. As shown in

2

Scheme 1, the PSA analysis in human serum sample was carried out on the pretreated

3

BPE (the process was shown in Supporting Information.) and the analysis on single

4

cell surface was executed on the bare BPE. The ECL detection solution was 0.1 M

5

PBS (pH 7.4) including 10 mM TPA. 20 µL of the above detection solution and the

6

same volume of 10 mM K3Fe(CN)6 were injected into the anodic and cathodic

7

reservoirs of the chip, respectively. The luminescence image recorded was analyzed

8

using Image J software.

9 10

Scheme 1. Construction of the ECL assay interface on the anodic pole of BPE and

11

schematic setup used for ECL imaging.

12

RESULTS AND DISCUSSION Ru(bpy)32+@SiO2

13

Characterization

of

14

Ru(bpy)32+@SiO2/Au

NPs.

15

heterogeneous Ru(bpy)32+@SiO2/Au NPs were characterized by scanning electron

16

microscopy (SEM) and transmission electron microscopy (TEM), and the

17

corresponding images were shown in Figure 1A−C. As demonstrated in Figure 1A,

18

the Ru(bpy)32+@SiO2 NPs are spheres with a mean diameter of 75 nm. The TEM

19

image of Ru(bpy)32+@SiO2 NPs in Figure 1B shows that the nanoparticles are highly

20

monodispersed. Modification of Au NPs on the surface of Ru(bpy)32+@SiO2 NPs

21

using a simple in-situ reduction method resulted in small Au NPs which were

22

decorated uniformly on the surface of Ru(bpy)32+@SiO2 (Figure 1C). The

The

morphologies

and of

6


Heterogeneous

Ru(bpy)32+@SiO2

and

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1

high-resolution TEM view shown in the inset of Figure 1C indicates that the diameter

2

of Au NPs immobilized on the surface of Ru(bpy)32+@SiO2 NPs is approximately 5.0

3

nm.

4

UV–visible absorption spectroscopy analysis was used to verify the formation of

5

Ru(bpy)32+@SiO2 and Ru(bpy)32+@SiO2/Au NPs. The characteristic absorption peaks

6

of Au NPs, Ru(bpy)32+, Ru(bpy)32+@SiO2 and Ru(bpy)32+@SiO2/Au NPs were

7

presented in Figure 1D. Peaks at 287 and 453 nm in Ru(bpy)32+ spectrum correspond

8

to π–π* electronic transition of bi-pyridine and metal to ligand charge transfer

9

adsorption, respectively. Compared to the absorption spectra of Ru(bpy)32+, the

10

characteristic absorption peak of Ru(bpy)32+@SiO2 NPs shows a red-shift from 453 to

11

458 nm, suggesting the effective encapsulation of Ru(bpy)32+ in silica. Similarly, the

12

red-shift also appeared in the absorption spectrum of Ru(bpy)32+@SiO2/Au NPs.

13

Furthermore, the peak at 525 nm from these nanoparticles was assigned to the

14

characteristic peak of Au NPs. All above results demonstrate the successful

15

preparation of Ru(bpy)32+@SiO2 and Ru(bpy)32+@SiO2/Au NPs.

16

The size and composition of Ru(bpy)32+@SiO2/Au could influence the transfer

17

efficiency of ECL signal. Thus, the size and composition of Ru(bpy)32+@SiO2/Au

18

were investigated. The experimental results indicate that the optimum size of

19

Ru(bpy)32+@SiO2/Au is 75 nm (the corresponding operation and data were included

20

in the Supporting Information). The composition of Ru(bpy)32+@SiO2/Au-Ab2 probe

21

was analyzed according to the method reported in a literature.22 The results show that

22

the ratio of Ab2 to Ru(bpy)32+@SiO2/Au is about 74:1 in all cases and the number of

23

Ru(bpy)32+ molecules in one Ru(bpy)32+@SiO2/Au particle is 136,000.

7



1

SEM image

(A) of

Ru(bpy)32+@SiO2 and TEM images of

2

Figure 1.

3

Ru(bpy)32+@SiO2 (B) and Ru(bpy)32+@SiO2/Au NPs (C). UV–visible spectra (D) of

4

Au NPs, Ru(bpy)32+, Ru(bpy)32+@SiO2 and Ru(bpy)32+@SiO2/Au NPs.

5

The Multiple Signal Amplification Strategy. A highly sensitive analysis strategy

6

is required, and many signal amplification methods have been introduced into the

7

BPE-ECL platform for the sensitive detection of biomarkers. However, the

8

amplification strategies mentioned are only anodic or cathodic amplification in many

9

existing reports. To date, there are few reports on the coupling of anodic and cathodic

10

amplification for the construction of a novel closed BPE sensing platform. It can be

11

deduced that the combination of these two amplification strategies may generate a

12

synergetic effect in the BPE system, which would result in better performance. In the

13

present study, to evaluate this synergetic effect, a series of experiments were designed

14

and the results were shown in Figure 2.

15

For amplification on the anodic pole, Ru(bpy)32+@SiO2/Au NPs labels were

16

prepared to achieve dual signal amplification as follows. (i) Ru(bpy)32+@SiO2/Au

17

NPs as a bioprobe could immobilize a large amount of Ru(bpy)32+ molecules. (ii) The

18

AuNPs with high conductivity and a highly active surface area were immobilized on

19

the surface of Ru(bpy)32+@SiO2 NPs, resulting in significantly enhanced ECL

20

intensity.

21

For amplification on the cathodic pole, the Au NPs with high conductivity and

22

large surface area were electrodeposited on the cathodic pole and K3Fe(CN)6 solution 8


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1

was used as supporting electrolyte. The Ru(bpy)32+@SiO2/Au NPs labeled probes

2

were firstly assembled on the anodic pole of BPE through layer by layer method.

3

Then, amplification effect on the cathodic pole was studied by changing the

4

conditions of the cathodic pole. In the absence of K3Fe(CN)6 and without Au NPs

5

modification in the cathodic pole (i.e. ITO-cathodic pole), the dissolve oxygen in the

6

water was reduced at the cathodic pole of the ITO-BPE, a basic ECL signal (curve a)

7

was observed. In the presence of K3Fe(CN)6, the cathodic pole of BPE without Au

8

NPs modification shows an enhanced ECL intensity (curve b), indicating the fine

9

ECL enhancement effect of K3Fe(CN)6 in the system. When the cathodic pole was

10

modified with Au NPs, the Au/ITO hybrid BPE gave a greatly increased ECL

11

intensity using K3Fe(CN)6 as the oxidant in the cathodic pole (curve c). Compared

12

with the ECL signal obtained without cathodic amplification (curve a), the intensity

13

was enhanced approximately 3.5-folds. It can be concluded that the synergistic effect

14

of multiple signal amplification strategies was successfully realized.

15 16

Figure 2. ECL behaviors of the BPE system under changed conditions in cathodic

17

pole. The cathodic pole without Au NPs modification containing only water (a), the

18

cathodic pole without Au NPs modification containing K3Fe(CN)6 solution (b) and

19

the cathodic pole with Au NPs modification (Au/ITO hybrid cathodic pole)

20

containing K3Fe(CN)6 solution (c). In the Ru(bpy)32+@SiO2/Au NPs modified anodic

21

pole, 0.1 M PBS (pH 7.4) containing 10 mM TPA were used as the detection solution.

22

PMT was set at -600 V.

23

The Analytical Performance of the Immunosensing Platform. In order to

24

investigate the potential application of the method, the model tumor marker PSA was 9



1

assayed. Under the optimized conditions (see the Supporting Information), the ECL

2

intensity of PSA at various concentrations was studied. As shown in Figure 3A, the

3

ECL intensity increased with increasing PSA concentration, due to increased signal

4

probe immobilized on the platform. A linear ECL relationship between the ECL

5

signal and logarithm of PSA concentration from 10 pg/mL to 50 ng/mL was obtained

6

(Figure 3B). The calibration curve is I = 1546.8 logCPSA + 5006.2 (R = 0.997). The

7

detection limit of PSA was experimentally found as 3.0 pg/mL. The developed

8

method was comparable with or even more sensitive than some the previously

9

literature reported (as shown in Table S1).

10 11

Figure 3. The ECL-voltage curves (A) of the sensing interface for PSA detection. The

12

linear relationship (B) between ECL intensity and the logarithm of PSA concentration.

13

The voltage of the PMT is -600 V.

14

Then the levels of PSA were visually analyzed using CCD imaging. As can be seen

15

from Figure 4(A1 – A6), clear visual ECL signals could be observed by our system

16

when the concentration of PSA is 0, 0.05, 0.50, 1.0, 10 and 50 ng/mL. The acquired

17

ECL signal enhanced with PSA concentration increasing, and a linear response to

18

concentration was observed between 0.05 ng/mL and 50 ng/mL (Figure 4B). The

19

regression equation can be described as G = 17.8 logCPSA + 27.5 (R = 0.997) with a

20

detection limit of 31 pg/mL (S/N = 3). The low detection limit could be ascribed to the

21

good performance of heterogeneous Ru(bpy)32+@SiO2/Au nanoparticles and the low

22

background of the developed system.

10


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1

In addition, the selectivity, reproducibility and stability of the ECL platform were

2

investigated as detail. The results were shown in Figure S5 of Supporting Information,

3

which indicated excellent performance of the proposal ECL platform for PSA assay.

4 5

Figure 4. ECL images captured at the anodic poles of the BPE system (A1–A6) for

6

detection of PSA (1–6: 0, 0.05, 0.50, 1.0, 10 and 50 ng/mL) and the linear relationship

7

(B) between gray value of ECL spots and the logarithm of PSA concentration. The

8

exposure time of the images taken by the CCD camera is 5 s.

9

Application in Complex Biological Samples. To assess the applicability of the

10

proposed strategy, the content of PSA in human serum samples was quantified. Seven

11

human serum samples (male) from Xinyang Central Hospital were measured using

12

both the fabricated chip and a reference method (the ROCHE ECL method) employed

13

in Xinyang Central Hospital. As shown in Figure 5, the results obtained using our

14

system were comparable to those obtained by the reference method in the seven

15

serum samples, demonstrating that the proposed BPE-ECL chip is satisfactory for

16

monitoring PSA in real samples. 11



Page 12 of 17

1

The recovery experiments were carried out using a standard addition method. PSA

2

with different concentrations (0.05, 0.50 and 5.0 ng/mL) were added to two human

3

serum samples, respectively. The results listed in Table S2 demonstrate that the

4

recovery varied from 80.0% to 108.0%, which indicated that the design shows good

5

accuracy for PSA detection in real biomedical samples.

6 7

Figure 5. Analytical results of the proposed methods and the reference ECL method

8

for PSA in complex biological samples.

9

ECL Imaging Analysis of PSA at Single LNCaP Cells. The LNCaP cell line is a

10

commonly applied model for prostate cancer and has been shown to overexpress PSA

11

antigen on the cell membrane.26-28 To investigate the performance of the present ECL

12

imaging platform for protein profiling at single cell levels, the PSA on the surface of

13

individual LNCaP cells were evaluated using Hela cells as control. Figure 6A1 and

14

B1 shows the bright field image of individual LNCaP cells and Hela cells. When the

15

potential was applied on the BPE electrode, imaging with the exposure time of 30 s

16

were performed. As shown in Figure 6A2-3 and B2-3, the high brightness ECL image

17

and the overlap of the ECL image and corresponding bright-field image associated

18

with PSA on individual LNCaP cells are observed while those images associated with

19

individual Hela cells do not present overtly detectable ECL signal under the same

20

imaging

21

Ru(bpy)32+@SiO2/Au-Ab2 nanoprobe by the PSA on LNCaP cells. ECL from 18

22

individual LNCaP cells and the same number of Hela cells were summarized and the

23

results were depicted in Figure 6C, respectively. The averaged ECL intensity from

procedure,

indicating

the

specific

recognition

12


of

functional

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1

LNCaP cells was calculated to be 73.7 ± 26.2 and that from Hela cells was 6.3 ± 2.9.

2

The data confirmed the high expression of PSA on LNCaP cells surface compared

3

with the negative control and the immunofluorescence staining analysis further

4

validated the results (Figure S6), in accordance with the previous reports.29-31 All

5

these results supported that our novel ECL imaging platform could be applied to

6

profile membrane protein at single cell levels, well avoiding the loss of individual

7

information of cells in the traditional population-averaged

8

Furthermore, the designed BPE-ECL system could be used to detect other molecules

9

on surface of cancer cells by just replacing the specific recognition element.

measurements.

10 11

Figure 6. Images of LNCaP cells (A) and Hela cells (B) on the BPE system:

12

bright-field images (A1, B1); ECL images of Ru(bpy)32+@SiO2/Au-Ab2 conjugated

13

LNCaP cells (A2) and Hela cells (B2); Overlay of the associated false color

14

elaborated ECL images and corresponding bright-field images (A3, B3). The

15

exposure time is 30 s. The error bar presents the RSD of ECL gray value from 18

16

cells.

17 18

CONCLUSIONS

19

In summary, a versatile BPE-ECL immunosensing platform for the visual

20

evaluation of PSA both in the serum samples and on the surface of individual cancer

21

cells was developed using heterogeneous Ru(bpy)32+@SiO2/Au-Ab2 as signal reporter

22

by virtue of a multiple amplification strategy. The proposed ECL platform

23

demonstrated three excellent properties: firstly, the closed BPEs could avoid the

24

interaction between electroactive compounds and the intermediate of the co-reactants, 13



1

which would quench the ECL of luminophores and limit the visual sensitivity;

2

secondly, combining anodic amplification using Ru(bpy)32+@SiO2/Au NPs as a signal

3

reporter with cathodic amplification using Au/ITO hybrid BPEs dipped in K3Fe(CN)6

4

produced a synergistic amplification effect on ECL detection; finally, the developed

5

strategy represents a sensitive and efficient system for visually assay of protein both

6

in body fluids and single cell surface. For all we know, the wireless and sensitive

7

visual immunoassay of tumor marker at single cells was achieved for the first time. It

8

is probably that a quantitative visual immunoassay for targets at single cells will be

9

attained by the proposed method in the future. Based on the properties described

10

above, we anticipate that this method has great applied potential in the development

11

of various BPE-ECL immuno-imaging for disease-related biomarkers detection.

12 13

ASSOCIATED CONTENT

14

Supporting Information

15

The contents include materials and reagents; apparatus; design and construction of

16

bipolar electrodes (BPE) chip; fabrication of the Au/ITO hybrid BPE and pretreatment

17

of its anodic pole; characterization and performance of the Au/ITO hybrid BPE;

18

Investigation for the Size of Ru(bpy)32+@SiO2/Au NPs; optimization of the signaling

19

scheme; selectivity, reproducibility and stability of the BPE chip; Scheme S1, Figure

20

S1-S6, Table S1 and Table S2. This material is available free of charge via the Internet

21

at http://pubs.acs.org.

22 23

AUTHOR INFORMATION

24

Corresponding Author

25

*Phone/Fax: +86-376-6392889. E-mail: [email protected] (Y.-M. Liu) &

26

[email protected] (J.-T. Cao)

27

Notes

28

The authors declare no competing financial interest.

29 14


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1

ACKNOWLEDGMENTS

2

We thank Dr. Lei Chen of College of Life Sciences in Xinyang Normal University for

3

his help in the immunofluorescence staining experiments. This work was supported

4

by the National Natural Science Foundation of China (Grant 21675136), Plan for

5

Scientific Innovation Talent of Henan Province (2017JR0016), Science & Technology

6

Innovation Talents in Universities of Henan Province (18HASTIT003), Funding

7

Scheme for the Young Backbone Teachers of Higher Education Institutions in Henan

8

Province (2016GGJS-097), and Nanhu Young Scholar Supporting Program of

9

XYNU.

10

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