Attomole Antigen Detection Using Self-Electrochemiluminous

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Attomole Antigen Detection using Self-electrochemiluminous Graphene Oxide-Capped Au@L012 Nanocomposite Chen Cui, Ying Chen, Dechen Jiang, Junjie Zhu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 01 Feb 2017 Downloaded from http://pubs.acs.org on February 1, 2017

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Analytical Chemistry

Attomole Antigen Detection using Self-electrochemiluminous Graphene Oxide-Capped Au@L012 Nanocomposite

Chen Cui, Ying Chen, Dechen Jiang*, Jun-Jie Zhu*, Hong-Yuan Chen

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China

Corresponding author: Phone: 086-25-89684846 (D.J); 086-25-89687204 (J. Z) Fax: 086-25-89684846 (D.J); 086-25-89687204 (J. Z) Email: [email protected] (D.J); [email protected] (J. Z)

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ABSTRACT. In this work, a self-electrochemiluminous graphene oxide-capped Au@L012 nanocomposite was prepared as the label at carcinoembryonic (CEA) antibody to detect attomole CEA antigen. To maximize the luminescence intensity, L012 molecules (luminol analog) were linked with poly(diallyldimethyl ammonium chloride) (PDDA) to form positive charged PDDA&L012 pairs, which were modified on negative charged Au@nafion nanoparticles to construct Au@nafion@PDDA&L012 (Au@L012) complex.

Graphene oxide with carboxyl

groups was capped at Au@L012 complex through electrostatic interaction to serve as an effective matrix for the covalent attachment of CEA antibody. As compared with the traditional used Au nanoparticles modified with luminol, ~ 740 fold increase of self-luminescence was observed from this new complex so that CEA antigen as low as 0.5 attomole at electrode surface was measurable in absence of any co-reactant.

Moreover, the nanocomposite was attached with CEA antigen at

MCF-7 cells allowing the detection of CEA antigen from 72 cells. The success in the detection of surface antigen at small population of cells suggested the self-electrochemiluminescence nanocomposite as the new and bio-safe label for the ECL immunoassay, which might push the application of ECL for the cellular immunoanalysis.


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INTRODUCTION Highly sensitive detection of specific antigens at cells using immunoassay is important to identify the subtypes of disease and clinical diagnosis.1-5 Due to high cellular heterogeneity, the analysis at small population of cells or even single cells is critical to elucidate the pathology.6,7 Since the amount of an antigen at one cell was typically at attomole level or less, the sensitive detection of antigens in this range is needed. Currently, fluorescence based immunoassay that labels the fluorophore at antibody to bind surface antigen at single cells for the quantification is well developped.8-10

However, the excitation light in florescence analysis introduces different

background signals that does not facilitate the application in clinic test. Electrochemiluminescence based (ECL) is an alternative strategy that labels the luminescence probe at antibody or antigen and utilizes the electrochemical generated luminescence from the probe for the analysis.11,12 The absence of excitation light results in low background intensity and high detection sensitivity for antigens.13

Traditionally, Ru(bpy)32+ is chosen as the ECL

probe that links with biotinylated antibody to emit red luminescence under the potential for immune-assay.14

Previously, our group had labeled Ru(bpy)32+ groups at AFP and CEA

antibodies, and the detection of femtomole AFP and CEA antigens at 105 fixed MCF7 cells was achieved.15

To decrease the detection limit of ECL immunoassay, a serious of strategies

including the usage of Ru(bpy)32+-doped silica nanospheres or quantum dots were reported, and the detection of attomole antigens at electrode surface was obtained.16,17

Despite the fast

development of ECL probe, high concentration of co-reactants, such as millimolar S2O82- or tripropyl amine (TPA), are required to obtain the measurable luminescence. Considering these reagents are harmful to the cells, it is necessary to propose a self-electrochemiluminescence probe with high luminescence intensity as a bio-safe label to facilitate ECL immunoassay in cellular study. For this aim, the current strategy to form self-electrochemiluminescence probes focus on the covalent linkage of luminophores with the co-reactant groups through the organic syntheses, in which the electron transfer between luminophore and the co-reactive group occurs intramolecularly.18-20

As compared with classic intermolecular electrochemiluminescence

reaction, the electronic transmission distance in these probes is shortened providing higher luminescence.

As the result, these novel probes have been applied for the analysis of proteins



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and cells without the addition of concentrated co-reactants.19,20 To expand the field of self-electrochemiluminescence immunoassay, luminol, the other classic ECL probe emitting blue luminescence under the potenital21, is attempted for the preparation of self-electrochemiluminescence probe to detect of antigens.

Previously, gold

nanoparticles (Au NPs) with carboxylic group were chosen as the support that could covalently link or electrostatically adsorb antibody and luminol to form the complex.22,23

Femtomole AFP

and CEA antigens at 105 MCF7 cells were detectable in presence of co-reactant, hydrogen peroxide.15

Since hydrogen peroxide is unstable, it is technically challenging to synthesize a new

probe composed with luminol and hydrogen peroxide. Therefore, a new strategy to form luminol based self-electrochemiluminescence probe is still needed.

Considering that luminol could

generate weak self-electrochemiluminescence without any co-reactants, the key is to increase the loading of luminol in the new probe so that a remarkably high self- electrochemiluminescence could be produced. Herein, we designed graphene oxide - capped Au@nafion@PDDA&L012 nanocomposite (abbreviated as “GO-Au@L012” ) as a novel self-electrochemiluminous probe for the detection of attomole carcino-embryonic antigen (CEA) at MCF7 cells, as schemed in Figure 1. L012 as the luminol analog with higher self-luminescence was chosen in the nanocomposite to replace luminol. Considering that both of Au NPs and L012 had negative surface charges, the simple electrostatic adsorption between them should not load abundant L012 at Au NPs. Therefore, poly(diallyldim ethylammonium

chloride) (PDDA), a kind of cationic polyelectrolyte, was interacted with

negative charged L012 to form positive charged PDDA&L012 for the following assembling with negative charged Au NPs.24 To further increase the adsorption ability of Au NPs, nafion was decorated on Au NPs to form Au@nafion complex that was used as the support to assemble Au@nafion@PDDA&L012 nanocomposite via electrostatic adsorption.25,26

Finally, graphene

oxide owing excellent biocompatibility, good water solubility and plenty of carboxyl functional groups at its edge was modified at the nanocomposite to covalently link antibody for immunoassay.27-29

As compared with the traditional Au-luminol nanocomposite, this new

GO-Au@L012 nanocomposite was observed to generate much higher luminescence and could be applied as the label for the immunoassay at cells. The composition and the detection ability of this novel nanocomposite on CEA antigen was characterized in this paper.


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EXPERIMENTAL SECTION Reagents and Materials. MCF7 cells were from the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences of Chinese Academy of Science (Shanghai, China). CEA primary and secondary antibodies, and antigen were obtained from Zhengzhou Biocell Biotechnology Co., Ltd. (Zhengzhou, China) and Beijing Bioss Biotechnology Co., Ltd. (Beijing, China), respectively. Graphene oxide was obtained from JCNANO (Nanjing, China). Sylgard 184 (including PDMS monomer and curing agent) was from Dow Corning (Midland, MI, USA). The compound 8-amino-5-chloro-7-phenylpyrido [3,4-d]pyridazine-1,4 (2H,3H)-dione (L012) was obtained from Wako Chemical, Inc.(Richmond, VA). All the other chemicals were obtained from Sigma-Aldrich Chemicals Co. and used as received. Ultrapure water (Mill-Q, Millipore, 18.2 MΩ •cm resistivity) was used throughout the experiments. Cell Culture. MCF 7 cells were seeded in DMEM/high glucose medium with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin). Cultures were maintained at 37 °C under a humidified atmosphere containing 5% CO2. Preparation of GO capped Au@nafion@PDDA&L012 (GO-Au@L012) nanocomposite. Figure 1A showed the proposed formation process for GO - capped Au@nafion@PDDA&L012. Au NPs (13 nm) were prepared via the reduction of HAuCl4 by trisodium citrate according to the report.30

The density of Au NPs was estimated to be 2×1012/mL according to the reference.31

Nafion (20 wt.%) was added into 25 mL solution with Au NPs, stirred at room temperature for 6 h and centrifuged at 14000 rpm for 25 min to get Au@nafion conjugates. PDDA (0.2%) solution was mixed with 3 mM L012 solution and stirred for 4 h at 50 ºC to obtain PDDA&L012 complex. Then, the PDDA&L012 complex was added into the Au@nafion solution and stirred for 10 h at 25 ºC. The Au@nafion@PDDA&L012 nanocomposite was obtained by centrifugation, and re-dispersed in water upon mild ultrasonication.

GO (0.1 mg/ml) was added to the

Au@nafion@PDDA&L012 solution and kept with gentle stirring for 10 h.

Finally, the

precipitate was collected by centrifugation at 9000 rpm for 25 min and washed for three times. The as-prepared GO capped Au@nafion@PDDA&L012 (GO-Au@L012) nanocomposite was dispersed in 5 ml of water. Labeling the nanocomposite with CEA antibody. To activate the carboxylic acid groups on the surface of graphene oxide, the mixture of 1 mM ethyl(dimethylaminopropyl) carbodiimide (EDC)



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and N-hydroxysuccinimide (NHS) were reacted with GO-Au@L012 nanocomposite at 37 °C for 1 h. Then, the activated nanocompsite were reacted with 17.86 µg/mL CEA primary antibody at 37 °C for 12 h. The final product was re-suspended in 10 mM phosphate buffered saline (PBS, pH 7.4) and stored at 4 °C. Linkage of attomole antigen at the electrode surface. The cleaned indium tin oxide (ITO) electrodes was immersed in 1% NH4OH solution at 80 °C for 1 h generating an active hydroxyl surface layer, and then sealed in APTS steam flow for 40 min to introduce amine groups at the surface.32 5% (w/v) glutaraldehyde were added to activate the amine groups for 30 min. After the removal of redundant glutaraldehyde, 20 µL of 1 ng/ml CEA secondary antibody solution (10 mM PBS, pH 7.4) was reacted with ITO surface at 4 °C for over 12 h. The modified electrode was washed thoroughly with PBS to remove physically absorbed antibody. The active sites on the electrode were blocked using 20 µL 1% BSA solution for 1 h.

After washing with PBS, the

antibody modified electrode was incubated with 20 µL of CEA antigen for 1 h at 37 °C. The abundant antibody at the electrode could bind the entire aqueous CEA antigen from attomole to femtomole.

Finally, the electrode was incubated with 20 µL of GO-Au@L012 -labeled antibody

for 60 min at 37 °C and then washed thoroughly with PBS to remove nonspecifically bounded conjugates. Linkage of the complexes at cells. The cells were fixed by 2.5% glutaraldehyde, and then, exposed with GO-Au@L012 nanocomposite - labeled antibody at 37 °C for 2 h.

The

nanocomposites were pre-blocked by BSA. Finally, the cells were rinsed thoroughly with PBS buffer to remove nonspecifically bounded nanocomposite. ECL detection. The ITO electrode with a diameter of 5 mm was used as the working electrode for luminescence detection.

Ag/AgCl wire was connected as the reference electrode.

The

luminescence was collected using an MPI-A ECL analyzer (Ruimai, Xi-an) in 30 µL of 10 mM PBS (pH 7.4) at room temperature. The potential scanned from -1.0 to 1.2 V at a scan rate of 0.5 V s-1.

The luminescence spectra were measured using the band-pass filters with the bandwidth

of 25 nm from 375 to 625 nm between ITO electrode and PMT.


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RESULTS AND DISCUSSION. Characterization of GO-Au@L012 nanocomposite. The morphologies of GO-Au@L012 nanocomposite and the precursors were characterized using transmission electron microscopy (TEM). As compared with Au NPs with the average diameter of 13 nm, the modification of nafion and PDDA&L012 at Au NPs to form Au@nafion@PDDA&L012 nanocomposite did not increase the nanoparticle size (Figure S1, supporting information).

When positive charged

Au@nafion@PDDA&L012 complex was loaded on negative charge graphene oxide, the dark spots as large as 13 nm were observed at the surface of graphene oxide, as illustrated in Figure 2A. This observation confirmed the assembling of Au@L012 nanocomposite with graphene oxide through electrostatic interaction. UV-vis spectrum in Figure 2B was used to confirm the composition of GO-Au@L012 complex. The characteristic peaks of Au NPs, L012 and GO were measured to be 520 nm (trace a), 327 and 398 nm (trace b), and 221 nm (trace d), respectivily.33-36

For Au@L012

nanocomposite, the absorption peaks (trace c) in aqueous solution appeared at 400 and 525 nm corresponding to the absorption of L012 and Au NPs, respectively, indicating the successful assemble of Au NPs and L012.

After the decoration of graphene oxide at Au@L012 complex, a

new peak (trace e) was displayed at 223 nm that should be attributed to graphene oxide in the nanocomposite. To confirm electrostatic interaction among the components in the complex, the surface charges of the components were measured, also.

The mean zeta potential of Au NPs and

Au@nafion was measured to be -40.4 and -68.89 mV (Figure 2C). After the interaction with PDDA&L012, the zeta potential dramatically changed from negative to positive (+55.36 mV) owing to the successful functionalization of PDDA&L012 onto the surface of Au@nafion. All these results supported the electrostatic interaction between Au@nafion and PDDA&L012, and Au@nafion@PDDA&L012 and negative charged graphene oxide, as proposed. ECL of GO-Au@L012 nanocomposite. Traditionally, luminol was modified to Au NPs via covalent binding to form Au@luminol as the ECL labeling at the antibody for immunoassay. To enhance the luminescence, L012 was used to replace luminol and mixed with Au NPs to form simple Au-L012 complex initially. Figure S2 (supporting information) showed that the peak luminescence intensity was ~ 70-fold higher than that from Au-luminol complex. Although a



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potential from 1 to 1.2 V was applied on the electrodes, no obvious gas bubble was observed at the electrodes. This phenomenon indicated that the decomposition of water did not occur at ITO electrode due to the over-potential. And thus, the luminescence should be attributed to the self-luminescence from luminol or L012 in the complexes. Since both of Au NPs and L012 were negative charged, nafion was modified on the surface of Au NPs to enhance the adsorption of L012. As compared with the simple Au-L012 complex, Au@nafion@L012 generated ~8 more fold increase in luminescence, as shown in Figure 3A (trace a and b). To achieve electrostatic interaction between L012 and Au@nafion, positive charged PDDA&L012 complex was assembled with negative charged Au@nafion@L012. The luminescence in Figure 3A (trace c) Au@nafion@PDDA&L012 complex.

exhibited ~ 1.2

more

fold

increase

from

Finally, the Au@nafion@PDDA&L012 complex was

further attached at the surface of graphene oxide by electrostatic absorption. Figure 3A trace d exhibited the additional ~ 1.1 fold increase in luminescence from GO-Au@nafion@PDDA&L012 complex. Overall, the self-luminescence from GO-Au@nafion@PDDA&L012 complex was ~ 740 fold higher than that from the traditional Au-luminol nanoparticles. ECL spectrums of L012 and GO-Au@L012 nanocomposite in Figure S3 (supporting information) exhibited the same peak emission wavelengths at ~ 475 nm, which further demonstrated the incorporation of L012 in the nanocomposite. Based on the increases in the luminescence intensity during the assembling, L012 was the dominate molecule in the complex contributing self-luminescences. Similar to the ECL mechanism of luminol,

37

L012 was oxidized into the radical anion under the positive

potential in the neutral solution that emitted the luminescence. Additionally, a small part of GO in the complex was electrochemically reduced into graphene during the potential scanning from -1.0 to 1.2 V, which enhanced luminol ECL slightly due to the adsorption of oxygen at the reduced GO surface.38 After the establishment of the components in the nanocomposite, different concentration of L012 was used for the assembling of nanocomposite and a maximal luminescence intensity was observed when the concentration of L012 was at 50 µM, as shown in Figure S4 (supporting information). Eventually, 50 µM L012 was chosen for the preparation of the nanocomposite. To estimate ECL efficiency of GO-Au@L012 nanocomplex, the luminescence intensities from the nanocomplex was compared with that from 50 µM luminol in 10 mM following the literature.39


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As shown in Figure S5 (supporting information).

~ 40 fold increase in the luminescence

intensity was collected from the complex. Taking in account of ECL efficiency of luminol (0.0004),40 ECL efficiency of the nanocomplex was estimated to be 0.016. The stability of ECL intensity from GO-Au@L012 nanocomplex was investigated by the consecutive potential scanning from -1 to 1.2 V. As shown in Figure 3B, the near constant peak luminescence intensity was observed indicating stable luminescence from the nanocomplex, which should be suitable for the following analysis of cellular surface antigen. Detection of attomole CEA antigen. To demonstrate the analysis of attomole CEA antigen, ITO slide was modified with abundant CEA antibody and exposed to PBS solution with CEA antigen in the range of 0.5-100 attomole mimicking the cellular surface antigen. Then, Ab-GO-Au@ L012 nanocomposite was introduced to bind CEA antigen for the characterization of detection sensitivity of our immunoassay.

Figure 4A showed the ECL traces from the complex labeled

with different amounts of CEA antigen. The positive correlation between peak luminescence and antigen amount in Figure 4B confirmed that GO-Au@L012 nanocomposite could be a sensitive self-luminescence ECL probe for the analysis of attomole antigen at the surface. The relative standard deviation from three independent assays in the detection range was calculated to be less than 16%, which demonstrated the good reproducibility of this ECL immunoarray. The control experiment was performed on the electrode modified with CEA antibody without the introduction of antigen, no luminescence was observed in Figure S6 (supporting information) indicating that unspecific binding of the complex at the surface was minor. The further analysis of attomole antigen was performed at MCF 7 cells with 18-27 fg (0.1~0.15 attomole) CEA antigen per cell.41

The cells were cultured at ITO electrode, fixed and

exposed to Ab-GO-Au@L012 nanocomposite. After the linkage of the complex with surface antigen, extra nanocomposite was washed away from the solution. The obvious luminescence increases were observed from the antibody modified complex at 72 and 134 cells (Figure 4C, trace b and c), while, no luminescence increase was observed at 33 cells (Figure 4C trace a). The minimal cell number was 72 cells corresponding to 7~ 10 attomole CEA, which was slightly larger than the limit of the detection of antigen at the electrode surface. This discrepancy was attributed to partial detection of cellular antigen using our probe, which needed to be at the edge of the cell contacting with the electrode surface to generate the luminescence. Differently, the



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previous ECL detection utilized the diffusion of intermediates from co-reactants to analyze the whole cellular surface antigen. Therefore, more cells associated with more nanocomplex/antigen were needed in our assay. Overall, the self-electrochemiluminescence graphene oxide-capped Au@L012 nanocomposite could be successfully applied to detect attomole antigen at a small population of cells.

CONCLUSIONS Here, a self-electrochemiluminous graphene oxide-capped Au@L012 nanocomposite was prepared as the label at CEA antibody to detect attomole CEA antigen at the electrode and cell surfaces. The significant enhancement in the luminescence and the avoidance of co-reactants in the solution provided a new and biocompatible ECL label for the immunoassay that facilitated the future cellular study.

The continuous improvement of this nanocomposite to increase the

luminescence might achieve the ECL immunoassay at single cell level so that the cellular heterogeneity could be investigated. Meanwhile, the ECL imaging of this nanocomposite will be performed to visualize the cellular surface antigen for the high-throughput analysis.

ACKNOWLEDGEMENTS. This work was supported by the National Natural Science Foundation of China (nos. 21327902 and 21427807).

Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. More TEM images, luminescence trace of Au-luminol, Au-L012; the optimization of L012 in the assembling of nanocomposite; non-specific adsorption of nanocomposite on the electrode


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Matsudaira, P. Nat. Methods. 2007, 4, 567−569.



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Figures and Captions. Figure 1. The schematic preparation of GO-Au@L012 nanocomposite for the detection of CEA antigens at (A) cells and (B) antigen modified ITO slide. Figure 2. (A) TEM images of Au@L012 nanocomposite at the surface of GO; (B) UV-vis absorption spectra of (a) Au NPs, (b) L012, (c) Au@L012, (d) GO and (e) GO-Au@L012; (C) surface charges of Au NPs, Au@nafion and Au@nafion@PDDA&L012. Figure 3. (A) The luminescence traces of (a) Au-L012, (b) Au@nafion@L012; (c) Au@nafion@PDDA&L012; (d) GO-Au@nafion@PDDA&L012; (B) The consecutive ECL traces from GO-Au@nafion@PDDA&L012 nanocomposite.

The potential scanned from -1 V to 1 V

with the scan rate of 0.5 V/s. The buffer for ECL detection was 0.01M PBS (pH 7.4). Figure 4. (A) The luminescence curves for the detection of CEA antigen with the amount of (a) 0.5 amole, (b) 1 amole, (c) 5 amole, (d) 10 amole, (e) 50 amole, (f) 100 amole and (g) 500 amole at the electrode surface. (B) The correlation of luminescence intensity and the amount of CEA antigen at the electrode. The error bar presented the standard deviation from three independent measurements. (C) The luminescence curves for the detection of cellular CEA antigen from (a) 33, (b) 72 and (c) 134 cells.

.


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Figure 1.



Figure 2.

A

100 nm

1.2

2.5

B

2.0

Absorbance

Absorbance

1.0 0.8

1.0

e 0.5

c

0.6

d

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b

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500

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0.4

a

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60 Zeta potential/mv

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C

30 Au

Au@nafion

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Au@nafion@ PDDA&L012

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Figure 3

ECL Intensity / a.u.

12000

A

d c

8000

b

4000

a 0 -1.0

-0.5

0.0 0.5 Potential /V

1.0

B 12000

Figure 4.


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8000

4000

0 0

20

40 60 Time / s

80


100


Figure 4.


8000

A

g

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a

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10000 ECL Intensity / a.u.

TOC

-0.5

0.0 0.5 Potential / V

1.0

B

8000 6000 4000 2000 0 1

10 100 CEA / amole

1000

900

C ECL Intensity / a.u.

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c

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300 b a

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Attomole Antigen Detection Using Self-Electrochemiluminous

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