Novel Electrochemiluminescence-Sensing Platform for the Precise

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A Novel Electrochemiluminescence Sensing Platform for Precise Analysis of Multiple LTBI Markers Bin Zhou, Mingyao Zhu, Peihui Yang, and Peihui Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 12 May 2017 Downloaded from http://pubs.acs.org on May 14, 2017

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ACS Applied Materials & Interfaces

A Novel Electrochemiluminescence Sensing Platform for Precise Analysis of Multiple LTBI Markers

Bin Zhou, Mingyao Zhu, Youyi Qiu, 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: Latent tuberculosis infection (LTBI) is one of the major contributing factors for the high incidence of tuberculosis (TB), and the low contents of LTBI markers in human serum presents a great challenge for the diagnosis of LTBI. Here we reported a novel electrochemiluminescence (ECL) sensing platform for the precise analysis of multiple LTBI markers, interferon-gamma (IFN-γ) and interleukin (IL)-2. In this approach, self-prepared carbon quantum dots (CQDs) and luminol were integrated onto gold nanoparticles (AuNPs), which were further enriched on the surface of magnetic bead (MB) to create two solid-phase ECL nanoprobes (MB@Au@CQDs and MB@Au@luminol) for improving the detection sensitivity efficiently. Graphene oxide (GO) and AuNPs were electrodeposited onto a patterned indium tin oxide (ITO) electrode with two spatially-resolved areas in sequence to form two sensitive and stable sensing areas. INF-γ-antibody (Ab)1 and IL-2-Ab1 were separately immobilized on the two sensing areas to capture the corresponding LTBI markers, which were further recognized by INF-γ-Ab2 and IL-2-Ab2 labelled MB@Au@CQDs and MB@Au@luminol. The ECL intensity depended linearly on the content of INF-γ and IL-2 in the range from 0.01 to 1000 pg mL-1 with a low detection limit of 10 fg mL-1. The proposed ECL sensing platform is simple, sensitive, accurate, reliable, and specific to the detection of rare INF-γ and IL-2 in human serum, and provides a valuable protocol for facilitating fast and precise diagnosis of LTBI.

Keywords: electrochemiluminescence sensing platform; precise analysis; latent tuberculosis infection; interferon-gamma; interleukin-2


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1. INTRODUCTION Tuberculosis (TB), an infectious bacterial disease, kills almost two million people annually and is an international public health priority.1,2 According to the World Health Organization, an estimated third of the world’s population is infected with Mycobacterium tuberculosis (M. tb).3 The enormous reservoir of individuals with latent tuberculosis infection (LTBI) is one of the major contributing factors for the high incidence of TB patients,4 but the sensitivity and accuracy of the existing diagnosis methods in the diagnosis of LTBI are suboptimal.5-7 Multiple immunoregulatory cytokines are produced by immune cells upon stimulation with M. tb, and they have been found to play crucial roles in regulating immune response and can serve as biological markers for LTBI diagnosis.7-11 Interferon gamma (IFN-γ), a 15.5 kDa molecule that is secreted by peripheral blood mononuclear cells, can act as a diagnostic marker for LTBI.7-9 Moreover, interleukin (IL)-2, a cytokine produced by activated T lymphocytes, is known to regulate the activation and expansion of T cells, which can be used for testing individuals with LTBI.10,11 A considerable number of studies have confirmed that quantification of multiple disease markers could help to improve the effectiveness of disease diagnosis.12-14 Therefore, developing a sensitive and accurate analysis platform for the precise analysis of multiple LTBI markers in parallel will enhance the efficiency of LTBI diagnosis and significantly help to prevent and control the spread of TB worldwide. Traditionally, tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) serve as the gold standard for the detection of LTBI. However, TST has poor specificity and can easily result in false positives because of the influence of nontuberculous mycobacteria and vaccine strains of Bacille Calmette Gue'rin.2,15 In addition, for IGRAs, it is hard to measure IFN-γ when its concentration is below the cut-off value (15 pg mL-1), and it is laborious, time-consuming, and expensive for analysis due to the requirement of in vitro cell culture under defined conditions of stimulation.15,16 Currently, electrochemical approaches have been widely applied in bioassays because of their inherent simplicity, time-saving, and low-cost features.14 To date, some electrochemical immunoassays have been established for the individual determination of INF-γ and IL-2 associated with LTBI on the basis of electrochemical impedance spectroscopy,17,18 square wave voltammetry,9,19,20 and differential pulse voltammetry21,22. However, these electrochemical immunoassays reported previously were only performed on single cytokine assays, which is usually not sufficient to diagnose LTBI because LTBI is associated with



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abnormal secretion of multiple cytokines. Compared with single cytokine assays, quantification of multiple cytokines related to LTBI in parallel has remarkable advantages, including high sample throughput, shorter assay time, improved detection accuracy, and reduced cost per assay.23 Additionally, the sensitivity of most sensing systems were insufficient for the detection of rare INF-γ or IL-2 in human serum because the detection limits were in the magnitude order of ng/mL to pg/mL. Electrochemiluminescence (ECL), which combines the advantages of electrochemistry and chemiluminescence, has received much attention from many scholars as a result of its simplicity, high sensitivity, and low background signal.24 In our previous work, a sensitive ECL immunosensor for the evaluation of IFN-γ was successfully fabricated, and it has a low detection limit of 30 fg mL-1.8 Given these facts, the development of a simple, sensitive, accurate, time-saving, and low-cost ECL sensing platform has become an urgent need for the fast and precise diagnosis of multiple LTBI markers. In our work, the key issue for the successful development of the ECL sensing platform is the preparation of potential-resolved ECL probes. Herein, self-prepared carbon quantum dots (CQDs) were synthesized with sucrose and emit an ECL signal at the potential of –1.8 V (vsAg/AgCl), and luminol is a classic organic ECL species that can emit an ECL signal at +0.6 V.25 However, the two water-soluble probes had the problem of weak luminescence emission, which limited their application in bioassays. To solve this problem, a multiple amplification strategy was utilized for enhancing the sensitivity of the two ECL probes based on the high loading capacity of nanomaterials.26 Over the past decade, a wide variety of nanomaterials were designed as transducing materials to fabricate solid-state ECL nanoprobes for signal amplification, such as, noble metal nanoparticles,27-29 magnetic bead (MB),30-32 and metal oxides.33,34 In this research, gold nanoparticles (AuNPs) were used for the preparation of solid-phase ECL nanoprobes with signal amplification achieved by attaching many water-soluble probes onto their surface. However, further signal amplification was needed to overcome the limitation due to the restricted amplification efficiency of the AuNPs surface. For this reason, MB was introduced and combined with an abundance of AuNPs to attract a large number of water-soluble probes (MB@Au@CQDs and MB@Au@luminol) for further enhancing the ECL signal intensity. The integration of amplifying the effect of both AuNPs and MB on the ECL of the CQDs / luminol system endowed the ECL sensing platform with a high sensitivity.


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Graphene oxide (GO) is a two-dimensional derivative of grapheme that continues to be of interest for bioassays because of its excellent electronic properties, large specific surface area, and superior biocompatibility.35-37 In addition, GO has been proven to be a satisfactory biosensor substrate for enhancing the ECL signal intensity by promoting the electron transfer reaction on ITO electrode.38 AuNPs not only have the ability to promote the electron transfer rate, but can also improve the effective immobilization of antibodies, which greatly improves the detecting sensitivity.39,40 Previously, our lab has developed a patterned ITO electrode with two spatially-resolved areas spaced 3 mm apart for multiplex detection, which can exclude the potential cross reaction between different ECL probes.41 Given these facts, GO and AuNPs were electrodeposited onto the patterned ITO electrode in sequence to form two sensitive and stable sensing areas for the detection of multiple LTBI markers in parallel, which is helpful for increasing the accuracy of our proposed approach. In the present work, a novel ECL sensing platform with high sensitivity and accuracy was designed for the quantitative evaluation of multiple LTBI markers in human serum, IFN-γ and IL-2. GO and AuNPs were electrodeposited onto the patterned ITO electrode in sequence to form two sensitive and stable sensing areas, on which INF-γ-antibody (Ab)1 and IL-2-Ab1 were separately immobilized to capture corresponding LTBI markers by the specific recognition of antigen and antibody. INF-γ-Ab2 and IL-2-Ab2 labelled MB@Au@CQDs and MB@Au@luminol nanoprobes were employed to recognize the captured LTBI markers, respectively. The ECL sensing platform was capable of detecting rare IFN-γ and IL-2 in parallel, indicating that this approach could provide an effective tool for the precise analysis of multiple LTBI markers and has potential application in the clinical diagnosis of LTBI.

2. EXPERIMENTAL SECTION 2.1. Materials All antigens (Ag), antibodies (Ab) and ELISA kits were purchased from eBioscience (USA). (Carboxyl group)-functionalized magnetic bead (diameter ~300 nm) was achieved from BioCanal Scientific Inc. (Wuxi, China). HAuCl4 was obtained from Shanghai Chemical Reagent Co., Ltd. (China). Luminol, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and bovine serum albumin (BSA) were purchased from



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Sigma-Aldrich (USA). Polyethylenimine (PEI, Mw = 10,000, 99%) was achieved 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. For all dilutions, Milli-Q water (18 MΩ) was used from a Millipore Milli-Q purification system (Millipore GmbH, Schwalbach, Germany).

2.2. Preparation of CQDs Novel CQDs with excellent ECL were synthesized using an electrochemical method. Sucrose (6.0 g) was dissolved in NaOH aqueous solution in a glass beaker. Two Pt sheets employed as anode and cathode electrodes were immersed into this alkaline solution. A CHI 760D electrochemical workstation (Shanghai CH Instruments Inc., China) was used to apply a suitable potential between the two electrodes. A yellow solution formed and was dialyzed in a 1000 Da dialysis bag for 24 h against ultrapure water to remove large or agglomerated particles. The obtained yellow solution was freeze dried, and the finished product was a yellow powder of CQDs.

2.3. Preparation of Antibody-Functionalized ECL Nanoprobes AuNPs were prepared according to a classic method.42 HAuCl4 (0.6 mL, 0.04 M) was dispersed in ultrapure water (25 mL), and heated to boiling. Sodium citrate (2 mL, 1%) was added to the boiling solution, and heating was continued for 15 min. The obtained AuNPs were coated with negatively charged citrate ions, and hence were well suspended in ultrapure water. PEI (0.5 mL, 4 mg mL-1) was added to the as-prepared AuNPs solution (10 mL) with stirring for 1 h. Then the newly synthesized CQDs (2 mL, 10 mg mL-1) were added to the solution, and the solution was then stirred for 2 h to obtain Au@CQDs via ionic bond formation. Afterwards, (carboxyl group)-functionalized MB (50 µL, 5 mg mL-1) and IFN-γ-Ab2 (4 µL, 8 µg mL-1) were dispersed in the Au@CQDs solution to form MB@Au@CQDs-IFN-γ-Ab2 via amide bond conjugation with the aid of NHS (15 mM) and EDC (75 mM) (37 °C, 1 h). The pure MB@Au@CQDs-IFN-γ-Ab2 was obtained by magnetic separation and was resuspended in PBS (300 µL, 0.1 M, pH 7.4) for subsequent tests. In a similar way, luminol (1 mL, 0.2 M) was added to the as-prepared AuNPs solution (10 mL) with stirring for 1 h to prepare Au@luminol. Then MB (50 µL, 5 mg mL-1) and IL-2-Ab2 (4 µL, 8 µg mL-1) were added to the Au@luminol solution to


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prepare MB@Au@luminol-IL-2-Ab2 with the aid of NHS and EDC.

2.4. Fabrication of the Sensing Interface A patterned ITO electrode with two spatially-resolved areas spaced 3 mm apart was fabricated according to our previously published method.41 The finished ITO electrode was cleaned with ethanol and purified water in turn, and then was dried with nitrogen. GO was electrodeposited on the two spatially-resolved areas by cyclic voltammetry (CV) scan from 0 to –1.5 V for 20 circles in 0.1 M KCl solution containing GO (40 mg mL-1). Then AuNPs were electrodeposited onto the rGO/ITO surface from 5.0 mM HAuCl4 solution by CV scan from –0.2 to +0.8 V for 20 circles. INF-γ-Ab1 and IL-2-Ab1 (20 µL, 8 µg mL-1) were separately dropped onto the two sensing areas for 30 min under room temperature. Then the two Ab1-functionalized sensing areas were blocked with BSA (5 µL, 5%) for 30 min to prevent nonspecific adsorption. The as-prepared sensing interface was washed with PBS (0.1 M, pH 7.4) for further experiments.

2.5. Electrochemiluminescence Detection for INF-γ and IL-2 The proposed ECL detection was based on a sandwich immunoassay strategy. INF-γ-Ab1 and IL-2-Ab1-functionalized sensing areas were separately incubated with INF-γ-Ag and IL-2-Ag standard solutions for 30 min at 37 °C, followed by washing carefully with PBS buffer (0.1 M, pH 7.4) to exclude the effect of nonspecific adsorption. MB@Au@CQDs-IFN-γ-Ab2 and MB@Au@luminol-IL-2-Ab2 incubation solutions were separately dropped onto the two sensing areas for 20 min at 37 °C, as shown in Scheme 1. Then the fabricated ECL sensing platform was washed with PBS buffer and placed in the electrochemical cell. Photomultiplier tube voltage was set at 600 V, and potential scanned from –2.0 V to +1.0 V in PBS buffer (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) and scanning electron microscopy (SEM) were employed to characterize the morphologies and sizes of the nanomaterials. As shown in Figure 1A, the average diameter of the prepared CQDs with uniform morphology and size was ~6.5 nm.



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Figure 1B shows the prepared AuNPs had uniform spherical shape with an average particle size of ~18 nm. CQDs were coated on the surface of AuNPs to form Au@CQDs in the presence of PEI (Figure S1D), and luminol-capped gold nanoparticles is exhibited in Figure S1E. (Carboxyl group)-functionalized MB presented a smooth-surfaced spherical structure with an average diameter of ~300 nm (Figure S1F). After Au@CQDs and Au@luminol were separately loaded on the MB surface with INF-γ-Ab2 or IL-2-Ab2 via amide bond conjugation, the average diameter and the spherical particle number of MB@Au@CQDs-INF-γ-Ab2 (Figure 1C) were greater than that of MB@Au@luminol-IL-2-Ab2 (Figure 1D), because the size of CQDs was much larger than the size of luminol. UV–vis spectra of MB@Au@CQDs-INF-γ-Ab2 (Figure S2B) shows that CQDs, INF-γ-Ab2, and AuNPs were loaded onto the MB surface (Figures S2A and S2B). Moreover, MB@Au@luminol-IL-2-Ab2 (Figure S2C) had luminol, IL-2-Ab2, and AuNPs absorption bands, which was consistent with the separated luminol, IL-2-Ab2, and AuNPs (Figures S2A

and

S2C).

The

results

indicated

that

MB@Au@CQDs-INF-γ-Ab2

and

MB@Au@luminol-IL-2-Ab2 nanoprobes were prepared successfully.

3.2. Amplification Effect of ECL Nanoprobes The separated amplification effect of the MB@Au@CQDs or MB@Au@luminol nanoprobe was confirmed first. As shown in Figures 2A and 2B, the ECL emissions of CQDs and luminol were observed at –1.8 V and +0.6 V, respectively. Two remarkably enhanced ECL signals can be obtained by attaching many CQDs and luminol on the surface of AuNPs. MB was introduced and combined with an abundance of AuNPs to attract a large number of CQDs and luminol for further enhancing ECL signal intensity, which increased by about 8-fold for CQDs and 16-fold for luminol (Figures 2A and 2B). To confirm the amplification effect of the two ECL nanoprobes during one potential scanning, ECL emissions of the two nanoprobes (located on two spatially-resolved areas of a patterned ITO electrode) were detected simultaneously. Figure 2C shows two ECL signals at the potentials of –1.8 V and +0.6 V during one potential scanning, which were sensitive and well separated. The integration of amplifying effect of both AuNPs and MB on the ECL of the CQDs / luminol system endowed the ECL sensing platform with a high sensitivity.


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3.3. Characterization of the Sensing Interface The surface topography and structure of the sensing interface was characterized step by step using SEM. The bare ITO electrode shows a smooth surface, and the image is presented in Figure 3A. Figure 3B shows a crumpled and flaked structure on the ITO electrode surface, revealing that GO was electrodeposited on the patterned ITO electrode, which was beneficial to strengthen the conductivity and stability of the sensing interface. Figure 3C displays some spherical particles on the surface of the crumpled and flaked structure, indicating that AuNPs were assembled on the rGO/ITO surface for loading more biomolecules. INF-γ-Ab1 and IL-2-Ab1 were employed to separately functionalize the two working areas on a sensing interface, which displayed a rough surface (Figure 3D). Experimental results indicated that two Ab1-functionalized areas on the sensing interface were fabricated successfully.

3.4. Characterization of the ECL Sensing Platform The fabrication procedure of the ECL sensing platform was characterized by CV and ECL measurements. Figure 4A shows a pair of typical redox peaks of ferricyanide ions on the bare patterned ITO electrode (curve a). Peak currents increased in turn with the electrodeposition of GO (curve b) and AuNPs (curve c), attributed to the excellent electric conductivity of GO and AuNPs, which could greatly increase the sensitivity of the ECL sensing platform. After modification with Ab1 and BSA (curve d) and capture of IL-2-Ag and INF-γ-Ag (curve e), peak currents decreased rapidly; this was due to the electron inert feature of the biomolecules. Afterwards, peak current further decreased when incubated with MB@Au@CQDs-INF-γ-Ab2 and MB@Au@luminol-IL-2-Ab2 (curve f). Moreover, Figure 4B shows that no remarkable ECL response was found for the bare and assembled ITO electrode (curves a, b, c, d, and e) until the two ECL nanoprobes had been assembled; a pair of significant ECL signals were observed at the peak potentials of –1.8 V and +0.6 V (curve f) during one potential scanning. These results confirmed that the ECL sensing platform had been fabricated successfully.

3.5 Optimization of the ECL Sensing Platform To optimize the measurement capability of the ECL sensing platform, some key parameters were analyzed. Figures S3A, S3B, and S3C display the effect of CV scan number of GO and HAuCl4



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on ECL intensity of MB@Au@CQDs and MB@Au@luminol nanoprobes; the best number of scans was 20 as it could provide two well-separated ECL signals. The best concentration of INF-γ-Ab1 and IL-2-Ab1 was 8 µg mL-1 (Figure S3D). As shown in Figure S3F, the optimal dosage of INF-γ-Ab2 and IL-2-Ab2 was 4 µL. In addition, the capture time of IL-2-Ag and INF-γ-Ag was optimized as 30 min (Figure S3E), and the recognition time of MB@Au@CQDs-IFN-γ-Ab2 and MB@Au@luminol-IL-2-Ab2 nanoprobes was further optimized as 20 min (Figure S3G). The maximum ECL emissions were obtained when the volume of each of the two ECL nanoprobes was 30 µL (Figure S4). These optimal experimental parameters were used in subsequent measurements.

3.6. Analytical Performance of the ECL Sensing Platform The contents of IFN-γ-Ag and IL-2-Ag were quantitatively detected by the proposed ECL sensing platform to validate the sensitivity and specificity of this approach. As presented in Figures 5A and 5B, the ECL intensity of the two ECL nanoprobes gradually increased with an increase in the concentrations of INF-γ-Ag and IL-2-Ag. Both calibration plots displayed a good linear relationship between the ECL intensity and the logarithm of the analyte concentration in the range from 0.01 to 1000 pg mL-1 for both IFN-γ-Ag (Figure 5C) and IL-2-Ag (Figure 5D). The linear regression equations were IECL = 5154.7 + 2838 lg CIFN-γ-Ag with a correlation coefficient R = 0.9970 for IFN-γ-Ag, and IECL = 5118.2 + 2882 lg CIL-2-Ag with a correlation coefficient R = 0.9963 for IL-2-Ag. In addition, the detection limit for both INF-γ-Ag and IL-2-Ag was calculated to be 10 fg mL-1 (S/N = 3), which was more sensitive than that previously reported (Table S1). The results showed that the ECL sensing platform was capable of detecting INF-γ and IL-2 simultaneously and possessed low detection limits and wide linear ranges, indicating that this approach had high sensitivity and excellent accuracy and could provide an effective tool for the precise analysis of multiple LTBI markers.

3.7 Analysis of INF-γ and IL-2 in Human Serum To verify the reliability of the ECL sensing platform, the recovery and the relative standard deviation (RSD) of the ECL immunoassay at different concentrations of the two LTBI markers in three human serum are listed in Table 1. Table 1 shows that the detection results of INF-γ and IL-2


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were in good agreement with that from the commercial ELISA test. The recoveries of IFN-γ and IL-2 were calculated to be 97.6–103.9% and 97.2–100.3%, respectively. Moreover, the RSD was 0.8–6.1% for INF-γ and 2.5–5.1% for IL-2 (n = 5). These results clearly demonstrated that the reliability of the developed ECL sensing platform was good.

Table1. Result of the determination of IFN-γ and IL-2 in human serum.

Smples

Added

Found (pg mL-1)

Recovery

RSD(%)

(pg mL-1)

( x ±s, n=5)

(%)

(n=5)

0.0

5.6±1.0

–

–

6.4±1.3

25.0

30.0±0.3

97.6

0.8

31.8±2.8

50.0

57.4±1.3

103.6

6.1

57.6±1.1

100.0

109.5±4.0

103.9

3.7

107.6±3.7

0.0

10.2±1.1

–

–

11.3±1.4

25.0

34.5±1.7

97.2

5.1

34.8±1.4

50.0

59.0±1.4

97.6

2.5

63.2±4.5

100.0

110.5±2.7

100.3

2.7

106.6±1.8

ELISA ( x ±s, n=5)

Serum+IFN-γ

Serum+IL-2

3.8 Specificity, Reproducibility, and Stability of the ECL Sensing Platform The specificity of the proposed ECL sensing platform was investigated by analyzing the detection sample containing standard solution of 1.0 pg mL-1 INF-γ-Ag and 1.0 pg mL-1 IL-2-Ag and other interferences (100 pg mL-1) such as bovine serum albumin (BSA), transferrin (Tf), alpha-fetoprotein (AFP), and carcinoembryonic antigen (CEA). Figures S5A and S5B show that the outside interferences had no significant effect on the standard solution of INF-γ-Ag and IL-2-Ag, indicating a satisfactory specificity of the ECL sensing platform. In addition, ten sensing interfaces constructed in the same manner were applied to detect 10 pg mL-1 INF-γ-Ag and IL-2-Ag for evaluating the reproducibility of the ECL sensing platform. RSD for INF-γ-Ag and IL-2-Ag was 3.9% and 4.2%, respectively, declaring the acceptable reproducibility of the ECL sensing platform. The stability of the ECL sensing platform was examined by consecutive cyclic potential scans for 6 cycles; RSD = 1.9% for INF-γ-Ag and RSD = 2.2% for IL-2-Ag, showing the



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good stability of the ECL sensing platform (Figure S5C). Furthermore, the ECL intensity was 97.3% and 96.5% of the initial value for INF-γ-Ag and IL-2-Ag, respectively, after a storage period of one week at 4 °C. Consequently, the ECL sensing platform had satisfactory specificity, reproducibility, and stability.

4. CONCLUSION In conclusion, we have successfully fabricated a novel ECL sensing platform for the precise analysis of multiple LTBI markers in human serum. The proposed analysis platform can be used to detect INF-γ and IL-2 associated with LTBI in parallel, and it exhibited high sensitivity and accuracy, where the detection limit for both LTBI markers was 10 fg mL-1. In addition, the detection results of INF-γ and IL-2 were in good agreement with that from the commercial ELISA test, indicating that the ECL sensing platform possessed good reliability. The ECL sensing platform also showed excellent specificity for the detection of LTBI markers. Thus, the novel analysis platform is simple, sensitive, accurate, reliable, and specific; this provides an effective approach for the precise analysis of multiple LTBI markers in human serum in parallel and has potential application in the clinical diagnosis of LTBI.

Supporting Information Characterization of MB@Au@CQDs and MB@Au@luminol nanoprobes (Figure S1); UV–vis spectra of ECL nanoprobes (Figure S2); optimization of the proposed ECL sensing platform (Figure S3 and S4); specificity and stability of the ECL sensing platform (Figure S4); Comparison of the performance of the present immunosensors for detection of INF-γ or/and IL-2 (Table S1) ACKNOWLEDGMENTS This research was financed by grants from National Natural Science Foundation of China (No. 21375048), the Twelfth Five-Year National Science and Technology Major Project (No. 2014ZX10003002).

REFERENCES (1) Dheda, K.; Schwander, S. K.; Zhu, B.; Van Zyl-Smit, R. N.; Zhang, Y. The Immunology of Tuberculosis: From Bench to Bedside. Respirology 2010, 15, 433−450.


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Scheme 1. The preparation process of the electrochemiluminescence sensing platform for determination of INF-γ and IL-2. 224x227mm (300 x 300 DPI)


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Figure 1. TEM images of (A) CQDs (inset image showed size distributions) and (B) AuNPs. SEM images and TEM images (inset images) of (C) MB@Au@CQDs-INF-γ-Ab2 and (D) MB@Au@luminol-IL-2-Ab2. 115x83mm (300 x 300 DPI)



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Figure 2. ECL behaviors of (A) CQDs, Au@CQDs and MB@Au@CQDs; (B) luminol, Au@luminol and MB@Au@luminol; (C) MB@Au@CQDs and MB@Au@luminol. 179x51mm (300 x 300 DPI)


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Figure 3. SEM images of (A) bare ITO; (B) rGO/ITO; (C) AuNPs/rGO/ITO; (D) Ab1/AuNPs/rGO/ITO. 112x85mm (300 x 300 DPI)



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Figure 4. (A) CV responses of the different modified electrodes in PBS (0.1 M, pH 7.4) containing [Fe(CN)6]4-/3- (5.0 mM) and KCl (0.1 M); (B) ECL signal obtained on the different modified electrodes in PBS buffer (0.1 M, pH 7.4) containing K2S2O8 (0.1 M) and H2O2 (10 mM), (a) bare ITO, (b) rGO/ITO, (c) AuNPs/rGO/ITO, (d) BSA/IL-2-Ab1 and INF-γ-Ab1/c, (e) IL-2-Ag and INF-γ-Ag/d, (f) MB@Au@CQDs-IFN-γAb2 and MB@Au@luminol-IL-2-Ab2/e. 112x45mm (300 x 300 DPI)


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Figure 5. (A) ECL intensity-potential curves and (B) ECL intensity-time curves of the ECL sensing platform with different concentrations of INF-γ-Ag and IL-2-Ag; linear relationship between ECL intensity and (C) INF-γ-Ag or (D) IL-2-Ag concentrations of 0.01, 0.025, 0.05, 0.1, 1, 10, 50, 100, 500, 1000 pg mL-1. 156x109mm (300 x 300 DPI)



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Graphical Abstract 81x33mm (300 x 300 DPI)


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Novel Electrochemiluminescence-Sensing Platform for the Precise

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