Potential-Resolved Electrochemiluminescence for Simultaneous

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Potential-Resolved Electrochemiluminescence for Simultaneous Determination of Triple Latent Tuberculosis Infection Markers Bin Zhou, Mingyao Zhu, Yan Hao, and Peihui Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10343 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017

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ACS Applied Materials & Interfaces

Potential-Resolved Electrochemiluminescence for Simultaneous Determination of Triple Latent Tuberculosis Infection Markers Bin Zhou, Mingyao Zhu, Yan Hao, Peihui Yang* Department of Chemistry, Jinan University Guangzhou 510632, P. R. China

Corresponding author: Peihui Yang, Ph.D, Professor Department of Chemistry, Jinan University Guangzhou 510632, P. R. China E-mail: [email protected] Tel/Fax: +86-20-85223039

ACS Paragon Plus Environment


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Abstract:

A

novel

electrochemiluminescence

(ECL)

immunosensor

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based

on

the

potential-resolved strategy was firstly developed for simultaneous determination of triple latent tuberculosis infection (LTBI) markers with high sensitivity, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-2. In this work, luminol and self-prepared carbon quantum dots and CdS quantum dots were integrated onto gold nanoparticles and magnetic bead in sequence to fabricate potential-resolved ECL nanoprobes with signal amplification. IFN-γ-antibody (Ab)1, TNF-α-Ab1 and IL-2-Ab1 were separately immobilized on three spatially resolved areas of a patterned indium tin oxide electrode to capture the corresponding LTBI markers, which were further recognized by IFN-γ-Ab2, TNF-α-Ab2 and IL-2-Ab2-functionalized ECL nanoprobes. The binding reaction of antibody-functionalized ECL nanoprobes and the captured LTBI markers will generate three sensitive and potential-resolved ECL signals during one potential scanning, and the ECL intensities reflect the concentrations of IFN-γ, TNF-α and IL-2 in the range of 1.6 − 200 pg mL–1. Therefore, the multiplexd ECL immunosensor provided an effective approach for simultaneous detection of triple LTBI markers in human serum, so that it will benefit to facilitate more accurate and reliable clinical diagnosis for LTBI.

Keywords: multiplexed immunosensor, latent tuberculosis infection markers, interferon-gamma, tumor necrosis factor-alpha, interleukin-2


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1. Introduction Tuberculosis (TB) is a top infectious disease killer worldwide caused by intracellular pathogen Mycobacterium tuberculosis (M. tb) that most often affects the lungs.1-3 The World Health Organization 2016 global tuberculosis report estimates that in 2015 two million people died from TB.4 If individuals with latent tuberculosis infection (LTBI) are diagnosed early, TB control improves, as the infectivity of patients falls rapidly on appropriate treatment.5 Many studies have confirmed that various cytokines secreted by human peripheral blood immune cells in response to M. tb can be used as diagnostic biomarkers for LTBI.6-12 Interferon gamma (IFN-γ) is a crucial cytokines secreted by immune cell that can act as a diagnostic marker for LTBI.6,7 Tumor necrosis factor-alpha (TNF-α) plays an important part in the control of M. tb infection and it could be used to discriminate between TB and LTBI.8-10 Interleukin (IL)-2 has a central role in regulating T cell responses to M. tb, which can be used for testing individuals with LTBI.11,12 It was agreed that the determination of a panel of disease markers in parallel could enhance the effectiveness of disease diagnosis.13,14 Therefore, developing some new immunoassays for the simultaneous detection of multiple LTBI markers will improve the accuracy and reliability of LTBI diagnosis that is conducive for prevention and control of TB infection. Currently, tuberculin skin test and interferon-gamma release assay lack the ability to diagnose LTBI because of their poor specificity and high false positive rate.6,7,14,15 To date, some electrochemical and electrochemiluminescence (ECL) immunoassays with the advantages of simplicity, high sensitivity and low cost have been established for the detection of LTBI markers.16-23 However, most of the immunoassays in the previous studies were performed on single or double cytokine analyses that are not enough for a conclusive diagnosis of LTBI, since LTBI involves the abnormal secretion of multiple immunoregulatory cytokines.6-12 Therefore, the development of multiplexed immunoassays for identifying more LTBI markers has become an urgent need, for the advantages of improved sample throughput, shortened assay time, and enhanced analysis accuracy.14,24 But the multiplexed ECL immunoassays are always facing the challenges from the fabrication of ECL probes with different potential signals, particularly for the immunoassays identified more than two disease markers in one potential scanning. The ECL peaks of these probes need to be distinguishable and separate from each other.24,25 And in order to exclude the potential interference among ECL probes on one electrode, the distance of different



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ECL probes need to be larger than the nanometer scale.26,27 Therefore, it is a huge challenge to develop a multiplexed ECL immunosensor based on potential-resolved strategy for the simultaneous determination of triple LTBI marks during one potential scanning. The essential question for the successful fabrication of the multiplexed ECL immunosensor is the preparation of ECL probes with different potential signals. In our research, luminol and self-prepared CdS quantum dots (CdS QDs) and carbon quantum dots (CQDs) were chosen as potential-resolved ECL probes, which can emit ECL signals at the potential of +0.6 V, −1.2 V and −1.8 V (vs Ag/AgCl) respectively. Considerable works indicated that gold nanoparticles (AuNPs) and magnetic bead (MB) endowed ECL immunosensor with high sensitivity by enhancing the loading capacity of luminescence probes.14,25 Thus, AuNPs were introduced and combined with lots of ECL probes, which were further enriched on MB surface to fabricate ECL nanoprobes with signal amplification. In addition, a patterned indium tin oxide (ITO) electrode with three spatially resolved areas spaced 1.5 mm apart was prepared according to our published procedure,25 which can exclude the interference among ECL probes. Given these facts, IFN-γ-antibody (Ab)1, TNF-α-Ab1 and IL-2-Ab1 were separately immobilized on the three spatially resolved areas for capturing corresponding LTBI markers, which were further recognized by IFN-γ-Ab2, TNF-α-Ab2 and IL-2-Ab2-functionalized ECL nanoprobes. The proposed analysis approach was applicable for simultaneous detection of triple LTBI markers, which has significant value in improving the accuracy and reliability of the early diagnosis of LTBI.

2. Experimental Section 2.1. Materials All antigens, antibodies (Ab) and enzymelinked immunosorbent (immunoadsorbent) assay (ELISA) kits were obtained from eBioscience (USA). Alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) were purchased from Zhengzhou Biocell Biotechnology Co., Ltd (Zhengzhou, China).

Luminol,

transferrin

1-ethyl-3-(3-dimethylaminopropyl)

(Tf),

bovine

carbodiimide

serum hydrochloride

albumin (EDC),

(BSA), and

N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (USA). HAuCl4 was purchased from Shanghai Chemical Reagent Co., Ltd. (China). (Carboxyl group)-functionalized MB (300 nm diameter) was purchased from BioCanal Scientific Inc. (Wuxi, China).


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Polyethylenimine (PEI, Mw = 10 000, 99%) was obtained from Shanghai Macklin Biochemical Co., Ltd. (China). Phosphate buffered saline (PBS) solution included 2.7 mM KCl, 136.7 mM NaCl, 1.5 mM KH2PO4, and 9.7 mM Na2HPO4. All reagents were of analytical grade, and ultrapure water was used throughout the experiment (Millipore GmbH, Schwalbach, Germany). 2.2. Preparation of Antibody-Functionalized ECL Nanoprobes CQDs, L-cysteine-capped CdS QDs and AuNPs were prepared according to our published procedure (see the Supporting Information).14,28 Antibody-functionalized ECL nanoprobes were fabricated according to our recently published method.14 0.5 mL of PEI (4 mg mL−1) was added in 10 mL AuNPs solution with stirring at 37 °C for 1 h. Then add the as-prepared CQDs (2 mL, 10 mg mL−1) to the solution, which was stirred for 2 h at 37 °C to obtain Au@CQDs via ionic bond

formation. After that, 50 µL MB (5 mg mL−1) and 4 µL IFN-γ-Ab2 (8 µg mL−1) were added in the Au@CQDs solution to fabricate MB@Au@CQD-IFN-γ-Ab2 via amide bond conjugation with the presence of 15 mM NHS and 75 mM EDC (37 °C, 1 h). The pure MB@Au@CQD-IFN-γ-Ab2 was gained by magnetic separation and suspended in PBS for subsequent tests. In a similar way, L-cysteine-capped CdS QDs (2 mL 10 mg mL-1) or luminol (1 mL, 0.2 M) was added into AuNPs solution (10 mL) to prepare Au@CdS QDs or Au@luminol via amide bond conjugation. MB (50 µL, 5 mg mL−1) and TNF-α-Ab2 (or IL-2-Ab2) were dispersed into the as-prepared Au@CdS QDs (or

Au@luminol)

solution

to

form

MB@Au@CdS

QDs-TNF-α-Ab2

(or

MB@Au@luminol-IL-2-Ab2). Detailed preparation processes of the ECL nanoprobes were provided in Supporting Information. 2.3. Fabrication of the Sensing Interface A patterned ITO electrode (20 mm × 10 mm) with three spatially resolved areas spaced 1.5 mm apart was fabricated according to our previously published method.25 The as-prepared ITO electrode was cleaned up with ethanol and purified water, followed by drying with nitrogen. IFN-γ-Ab1, TNF-α-Ab1, and IL-2-Ab1 (20 µL, 8 µg mL-1) were separately dropped onto the three areas with coating buffer for 30 min under room temperature. Then, the three Ab1-functionalized areas were blocked with BSA (5 µL, 5%) for 30 min to prevent nonspecific adsorption. The obtained sensing interface was washed with PBS (0.1 M, pH 7.4) for the next stage of experiments. 2.4. ECL Detection for Triple LTBI Markers



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The measurement was based on a sandwich immunoassay method. The three Ab1-functionalized sensing areas were separately incubated with IFN-γ, TNF-α and IL-2 for 30 min at 37 °C and then were washed carefully with PBS (0.1 M, pH 7.4) to exclude the effect of nonspecific adsorption. MB@Au@CQD-IFN-γ-Ab2, MB@Au@CdS QDs-TNF-α-Ab2 and MB@Au@luminol-IL-2-Ab2 incubation solutions were separately dropped onto the three sensing areas for 20 min at 37 °C and then were washed carefully with PBS (Scheme 1). Afterwards, the fabricated multiplexed ECL immunosensor was placed in the electrochemical cell. The photomultiplier tube voltage was set at 600 V, and potential scanning was done from −2.0 to +1.0 V in PBS (0.1 M, pH 7.4) containing K2S2O8 (0.1 M) and H2O2 (10 mM).

3. Results and Discussion 3.1. Characterization of Antibody-Functionalized ECL Nanoprobes Transmission electron microscopy (TEM) was employed to characterize morphologies and sizes of the ECL nanoprobes. Figure S1A−C showed that the as-prepared CQDs, CdS QDs and AuNPs had uniform spherical structures with average particle sizes of ∼6.8 nm, ∼4.5 nm and ∼15 nm, respectively. The CQDs, CdS QDs and luminol were coated on the surface of AuNPs to form Au@CQDs, Au@CdS QDs, and Au@luminol, which were shown in Figure S1D−F in the Supporting Information. After Au@CQDs, Au@CdS QDs and Au@luminol were separately loaded on the surface of (carboxyl group)-functionalized MB with IFN-γ-Ab2, TNF-α-Ab2 and IL-2-Ab2,

lots

of

spherical

antibody-functionalized QDs-TNF-α-Ab2

and

ECL

particles

appeared

nanoprobes,

on

the

surface

of

MB@Au@CQDs-IFN-γ-Ab2,

MB@Au@luminol-IL-2-Ab2

(Figure

S1G−I).

MB

to

form

MB@Au@CdS In

addition,

ultraviolet-visible spectra of MB@Au@CQD-IFN-γ-Ab2 indicated that CQDs, IFN-γ-Ab2, and AuNPs were loaded on MB surface (Figure S2A,B). MB@Au@CdS QDs-TNF-α-Ab2 had CdS QDs, TNF-α-Ab2, and AuNPs absorption bands (Figure S2C). And MB@Au@luminol-IL-2-Ab2 possessed luminol, IL-2-Ab2, and AuNPs absorption bands (Figure S2D). These results demonstrated that the three antibody-functionalized ECL nanoprobes were prepared successfully. 3.2. Amplification Effect of the ECL Nanoprobes The introduction of AuNPs and MB could effectively enhance the ECL intensity of CQDs, CdS QDs, and luminol. Figure 1 showed the separated ECL behavior of CQDs, CdS QDs, and luminol


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at −1.8 V, −1.2 V and +0.6 V, respectively. Three enhanced ECL signals were observed by integrating CQDs, CdS QDs, and luminol onto AuNPs. With the introduction of MB, the ECL intensity could be further increased for improving the detection sensitivity, which increased by 5-fold compared to CQDs, 7-fold compared to CdS QDs and 11-fold compared to luminol (Figure 1A−C). The integration of amplifying the effect of both AuNPs and MB on the ECL of the CQDs/CdS QDs/luminol system endowed the ECL immunosensor with a high sensitivity. To characterize

the

ECL

behaviors

of


MB@Au@CdS

QDs-TNF-α-Ab2 and MB@Au@luminol-IL-2-Ab2 nanoprobes during one potential scanning, the ECL signals of them were detected simultaneously. As shown in Figure 1D, three potential-resolved ECL signals were observed at the potential of −1.8 V, −1.2 V and +0.6 V in one potential

scanning.

Thus,

the

prepared


MB@Au@CdS

QDs-TNF-α-Ab2 and MB@Au@luminol-IL-2-Ab2 nanoprobes exhibited three high sensitive and well-separated ECL signals, which could act as good indicators for simultaneous detection. 3.3. Characterization of the Multiplexed ECL Immunosensor The fabrication process of the multiplexed ECL immunosensor was characterized through scanning electron microscopy (SEM), cyclic voltammetry (CV), differential pulse voltammetry (DPV) and ECL. SEM images exhibited a rough structure on the surface of ITO electrode, which means that Ab1 was modified onto ITO electrode with the aid of coating buffer (Figure S3). Figure 2A showed a pair of typical redox peaks of ferricyanide ions on bare ITO (curve a), peak current decreased in sequence with the modification of Ab1, BSA, triple LTBI markers, and Ab2-functionalized ECL nanoprobes (curves b−e), which was due to the electron inert feature of biomolecules. DPV data (Figure 2B) was consistent with the result of CV analysis. Additionally, Ab2-functionalized ECL nanoprobes were employed for the recognition of triple LTBI markers captured on the sensing interface, respectively. Figure 2C displayed three redox peak signals coming from MB@Au@CQDs, MB@Au@CdS QDs and MB@Au@luminol (curve e), but there is no peak signal appeared in CV scan curves before the sensing interface incubated with ECL nanoprobes (curves a−d). ECL behaviors of the three ECL nanoprobes exhibited three well-separated and sensitive ECL signals at −1.8 V, −1.2 V and +0.6 V in Figure 2D (curve e). These results indicated that the multiplexed ECL immunosensor for simultaneous detection of triple LTBI marks during one potential scanning had been fabricated successfully.



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3.4. Optimization of the Proposed ECL Immunosensor Several important parameters were investigated for optimizing the detection performance of the multiplexed ECL immunosensor. Figure S4A showed the effect of incubation time of IFN-γ, TNF-α and IL-2 on the current intensity of sensing interface, the best incubation time of triple LTBI markers was 30 min. The dosages of IFN-γ-Ab2, TNF-α-Ab2 and IL-2-Ab2 in three ECL nanoprobes were optimized as 4 µL (Figure S4B). As shown in Figure S4C, the optimal recognition time of the ECL nanoprobes was 20 min. An increase in the dosage of ECL nanoprobes could enhance the ECL intensity of the multiplexed immunosensor but also reduce the distinguishability of the three ECL signals. The result displayed that the optimum dosages of ECL nanoprobes were all 5 µL (Figure S4D−F). 3.5. Analytical Performance of the Multiplexed ECL Immunosensor The multiplexed ECL immunosensor was employed for simultaneous detecting triple LTBI markers. Figure 3A,B indicated that the ECL intensity improved with the concentration increase of IFN-γ, TNF-α, and IL-2. The calibration plots exhibited the good linear relationship between the ECL intensity and the logarithm of IFN-γ, TNF-α, and IL-2 concentrations in the range of 1.6 − 200 pg mL–1 (Figure 3C−E). The linear regression equations were IECL = 713.28 lg CIFN-γ − 220.99 with a correlation coefficient R = 0.9760 for IFN-γ, IECL = 723.58 lg CTNF-α – 206.58 with a correlation coefficient R = 0.9817 for TNF-α, and IECL = 717.62 lg CIL-2 – 187.92 with a correlation coefficient R = 0.9944 for IL-2. Moreover, the detection limits for IFN-γ, TNF-α, and IL-2 were all 1.6 pg mL–1. The results showed that the multiplexed ECL immunosensor was able to quantitatively detect IFN-γ, TNF-α, and IL-2 during one potential scanning and possessed wide linear ranges and low detection limits, indicating that this proposed analysis approach had significant value in improving the accuracy and reliability of the early diagnosis of LTBI. 3.6. Analysis of IFN-γ, TNF-α, and IL-2 in Human Serum The reliability of the multiplexed ECL immunosensor was verified by using the commercial ELISA test. Table 1 listed that the detection results of ECL immunoassay for IFN-γ, TNF-α, and IL-2 in human serums were in good consistence with those from the ELISA test. Additionally, the recoveries of IFN-γ, TNF-α and IL-2 were 104.2–108.4%, 104.8–109.6% and 98.8–105.6%, respectively. RSD was 4.2–4.8% for IFN-γ, 3.0–4.7% for TNF-α, and 2.3–4.9% for IL-2 (n = 3). These results showed that the multiplexed ECL immunosensor had a capacity for determining


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IFN-γ, TNF-ɑ, and IL-2 in human serum and possessed good reliability. Table 1. Results of the Detection of IFN-γ, TNF-ɑ and IL-2 in Human Serum samples

serum + IFN-γ

serum + TNF-ɑ

serum + IL-2

added (pg/mL)

found (pg/mL)

recovery (%)

RSD (%) (n = 3)

ELISA

0.0

6.1 ± 1.0

–

–

7.6 ± 1.2

5.0 25.0

106.0 108.4 104.2

4.4 4.2

50.0

11.4 ± 0.5 33.2 ± 1.4 58.2 ± 2.8

4.8

12.8 ± 1.8 32.5 ± 2.7 57.4 ± 4.7

0.0

4.7 ± 1.3

–

–

–

5.0 25.0 50.0

10.1 ± 0.3 32.1 ± 1.5 57.1 ± 2.4

108.0 109.6 104.8

3.0 4.7 4.2

8.9 ± 1.1 30.3 ± 2.2 55.7 ± 3.5

0.0

12.3 ± 1.6

–

–

13.5 ± 1.1

5.0 25.0

17.4 ± 0.4 38.7 ± 1.9 61.7 ± 2.5

102.0 105.6 98.8

2.3

17.8 ± 1.6 40.2 ± 2.8 66.8 ± 5.3

50.0

(n = 3)

4.9 4.1

(n = 3)

3.7. Specificity, Reproducibility, and Stability of the Multiplexed ECL Immunosensor The as-prepared ECL immunosensor was investigated to evaluate its specificity, reproducibility, and stability. Figure S5A−C showed that the outside interferences, such as BSA, CEA, AFP and Tf, had no significant effect on the standard solution of IFN-γ, TNF-α, and IL-2, which means that the ECL immunosensor had a satisfactory specificity. Ten sensing interfaces fabricated at the same time were used for detecting 10 pg mL–1 IFN-γ, TNF-α and IL-2. RSD for IFN-γ, TNF-α and IL-2 were 4.2%, 4.6% and 3.9%, respectively, indicating an acceptable reproducibility of the ECL immunosensor. The stability of the ECL immunosensor was studied through consecutive cyclic potential scans for 5 cycles: RSD for IFN-γ, TNF-α and IL-2 were 3.2%, 2.9% and 2.6%, respectively (Figure S5D). And the ECL immunosensor stored at 4 °C for two weeks showed that the ECL intensity was 95.5%, 96.4% and 95.9% of the initial value for IFN-γ, TNF-α, and IL-2, respectively. These results demonstrated that the multiplexed ECL immunosensor possessed satisfactory specificity, reproducibility and stability.

4. Conclusions The multiplexed ECL immunosensor we have developed allowed us for the first time to



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simultaneously detect the concentrations of triple LTBI markers in human serum. The ECL immunosensor provided three potential-resolved and high sensitive ECL signals, and the ECL intensities were dependent on the concentrations of IFN-γ, TNF-α, and IL-2, so that it was capable of simultaneous detecting triple LTBI markers during one potential scanning. The proposed approach could exclude the potential interference among ECL nanoprobes on one electrode by spatial resolved strategy to gain more accurate detection results. Additionally, the commercial ELISA test results demonstrated that the ECL immunosensor possessed good reliability. Therefore, the multiplexed ECL immunosensor provided an effective approach for simultaneous detection of triple LTBI markers in human serum and has potential application in facilitating accurate and reliable clinical diagnosis of LTBI.

Supporting Information Characterization

of

antibody-functionalized

ECL

nanoprobes

(Figures

S1

and

S2);

characterization of the sensing interface (Figure S3); optimization of the multiplexed ECL immunosensor (Figure S4); specificity, reproducibility, and stability of the multiplexed ECL immunosensor (Figure S5)

Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 21375048).

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Scheme 1. Preparation and schematic illustration of the multiplexed electrochemiluminescence immunosensor for simultaneous detection of IFN-γ, TNF-α and IL-2. 208x196mm (300 x 300 DPI)



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Figure 1. ECL behaviors of (A) CQDs, Au@CQDs and MB@Au@CQDs; (B) CdS QDs, Au@CdS QDs and MB@Au@CdS QDs; (C) luminol, Au@luminol, and MB@Au@luminol; (D) MB@Au@CQDs, MB@Au@CdS QDs and MB@Au@luminol. 168x127mm (300 x 300 DPI)


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Figure 2. (A) CV responses and (B) DPV responses of the different modified electrodes in 0.1 M PBS (pH 7.4) containing 5.0 mM [Fe(CN)6]4-/3- and 0.1 M KCl; (C) CV responses and (D) ECL signals obtained on the different modified modified electrodes in 0.1 M PBS PBS (pH 7.4) containing 0.1 M K2S2O8 and 10 mM H2O2; (a) ITO, (b) IFN-γ-Ab1 + TNF-α-Ab1 + IL-2-Ab1/ITO, (c) BSA/b, (d) IFN-γ + TNF-α + IL-2/c, (e) MB@Au@CQDs-IFN-γ-Ab2 + MB@Au@CdS QDs-TNF-α-Ab2 + MB@Au@luminol-IL-2-Ab2/d. 179x136mm (300 x 300 DPI)



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Figure 3. (A) ECL intensity-potential curves and (B) ECL intensity-time curves of the multiplexed ECL immunosensor with different concentrations of IFN-γ, TNF-α and IL-2; linear relationship between the ECL intensity and (C) IFN-γ, (D) TNF-α or (E) IL-2 concentrations of 1.6，3.125， 6.25，12.5，25， 50，100， 200 pg mL−1. 175x107mm (300 x 300 DPI)


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Graphical Abstract 79x35mm (300 x 300 DPI)


Potential-Resolved Electrochemiluminescence for Simultaneous

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