Potential-Resolved Electrochemiluminescence for Determination of

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Potential-Resolved Electrochemiluminescence for Determination of Two Antigens at the Cell Surface Fangfei Han,† Hui Jiang,§ Danjun Fang,*,‡ and Dechen Jiang*,† †

The State Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, China ‡ Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu 210029, China § State Key Laboratory of Bioelectronics-Chien-Shiung Wu Lab, School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu 210096, China S Supporting Information *

ABSTRACT: The potential-resolved electrochemiluminescence (ECL) was achieved for the determination of two antigens at the cell surface through a potential scanning on the electrode. Luminol and Ru(bpy)32+ groups as ECL probes were linked with the antibodies to recognize the corresponding antigens on the cell surface. A self-quenching of luminescence from the luminol group under negative potential was initialized by the introduction of concentrated aqueous luminol, which offered accurate measurements of the luminescence from luminol and Ru(bpy)32+ groups under positive and negative potentials, respectively. Using this strategy, carcinoembryonic (CEA) and alphafetoprotein (AFP) antigens on cells as the models were quantified serially through a potential scanning. Different patterns of luminescence were observed at MCF 7 and PC 3 cells, which exhibited that the assay can characterize the cells with a difference expression of antigens. Compared with fluorescence measurement, the potential resolved ECL for the detection of two analytes was not limited by the spectrum difference of probes. The strategy involving potential-induced signals required a simplified optical setup and eventually offered an alternative imaging method for multiply antigens in immunohistochemistry. probes, and thus, no beam splitter or filter is needed to distinguish the signals. Pioneering work on the potentialresolved ECL assay had been performed using Ir(ppy)3 and [Ru(bpy)2(L)]2+ complexes in solution, which emitted the light under different potentials.10,11 For the application of potentialresolved ECL assay in immunoassay, Ru(bpy)32+ and luminol are chosen as ECL probes, which have been reported to be linked with the antibody for the recognition of the antigen at the cell surface.12−14 In principle, applied with a positive potential, the luminol anion goes through electro-oxidation to diazaquinone, which is further oxidized into the excited 3aminophthalate species for the emission light.15−17 For the Ru(bpy)32+ probe, S2O82− as the coreactant is reduced into SO4•− under negative potential, which generates Ru(bpy)32+* to give out light.18−20 Given the fact that ECL reactions of luminol and Ru(bpy)32+ occur under different potentials, it is possible to detect two antigens using both of ECL probes through a potential scanning. Compared with the multiplexed ECL determination of surface antigens using an electrode array,21 the assay on one cell population can reveal the

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he assay of multiple biomarkers at cells is significant for the accurate diagnosis of cancers.1,2 In the past few years, the considerable efforts have been put in developing fluorescent immunoassays for the detection.3,4 Typically, the biomarkers at the cell surface are labeled with the fluorophores with different excitation/emission wavelengths. Under the irradiation of laser, the emissions from different fluorophores are distinguished through the beam splitters and filters before collected on a photomultiplier tube (PMT) or charge-coupled device (CCD). Although fluorescent immunoassay is advantagous for a wide spectrum of fluorophores, the ease of fluorescent labeling, and high throughput detection,5 the fluorescence assay relies on the spectrum difference of probes. Therefore, the components of the laser, the filter, or the beam splitter are required that complex the optical setup and increase instrumental cost. Electrochemiluminescence (ECL) is the other optical strategy for the immunoassay that replaces the fluorophore with the ECL probe.6−9 ECL does not need the light source resulting in low background, high detection sensitivity, and simple instrumentation. Since the emission of the luminescence from the ECL probe was controlled by the potential, it was feasible to develop potential-resolved ECL that detected multiply analytes through a potential scanning. This potential resolved strategy is not related with the spectrum difference of © 2014 American Chemical Society

Received: February 10, 2014 Accepted: June 25, 2014 Published: June 25, 2014 6896

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might introduce only one antibody on each particle. Au NPs without the addition of antibody was removed through the centrifugation after the following interaction with antigen modified silica particle or cells. Finally, 0.25 mM N-(4aminobutyl)-N-ethylisoluminol was applied on Au NPs-MPAantibody for 12 h in the dark to introduce luminol group on Au NPs. The amount of N-(4-aminobutyl)-N-ethylisoluminol was 1.5 × 1017 molecules/mL resulting in the averaged 1666 luminol groups on each Au NP. The final product (Au NPsMPA-antibody/luminol) was resuspended in 10 mM phosphate-buffered saline (PBS, pH 7.4) and stored at 4 °C. Preparation of Streptavidin Associated Ru(bpy)32+ Complex. One mg/mL streptavidin was mixed with 1 mg/ mL bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis (hexafluorophosphate) (Ru(bpy)32+) complex for 2 h at 4 °C. The complexes were purified by ultrafiltration using Amicon Ultra filters with a 10k molecular weight cut off membrane (Millipore). The streptavidin associated Ru(bpy)32+ complex was diluted to 20 μg/mL with PBS (pH 7.4) and stored at 4 °C. Synthesis of Antigen-Modified Silica Particles. Silica particles were reacted with 0.2 M NHS and 0.8 M EDC at 37 °C for 1 h to activate carboxylic acid groups. Then, the particles were linked with 12.88 μg/mL antibody at 37 °C for 24 h. The remaining active carboxylic acid groups were blocked with 0.1% (w/v) bovine serum albumin (BSA) at 37 °C for 1 h. After the removal of extra antibody in the solution, 2 μg/mL antigen was added and incubated at 37 °C for 2 h to form antigen-modified silica particles. Linkage of the Complexes on Antigen-Modified Silica Particles and Cells. To link Au NPs-MPA-luminol/antibody on the antigen modified silica particles, the particles were resuspended in PBS (pH 7.4)−0.02% (w/v) Tween-20 and incubated with luminol associated antibody at 37 °C for 2 h. For the linkage of streptavidin-modified Ru(bpy)32+ complex on antigen modified silica particles, the silica particles were incubated with 3 μg/mL biotinylated antibody at 37 °C for 30 min. After washing for 3 times, the product was mixed with 20 μg/mL streptavidin associated Ru(bpy)32+ complex for 2 min to form Ru(bpy)32+ labeled silica particles. The same procedure was applied for the linkage of the complexes on the cells. Before the linkage, the cells were fixed by 2.5% glutaraldehyde. Luminescence Detection. The indium tin oxide (ITO) electrode with a diameter of 2 cm was used as the working electrode for luminescence detection. Ag/AgCl and Pt electrodes were connected as a reference and counter electrode, respectively. PMT voltage was set at 600 V. For luminol associated analysis, the potential scanned from 0.6 to 0 V in 10 mM aerated PBS and the luminescence read at 0.6 V was taken as the signal. For Ru(bpy)32+ associated analysis, the potential scanned from 0 to −1.0 V in 10 mM PBS containing 20 mM luminol and 3 mM S2O82−, and the luminescence read at −1.0 V was taken as the signal. To decrease the measurement error induced by different ITO electrodes, the differences in the luminescence between luminescence probe-antibody/antigen modified particles/cells and antigen modified particles/cells were calculated and normalized by the background luminescence read at 0 V as the “luminescence ratio”. The luminescence spectra were measured using the band-pass filters with the bandwidth of 20 nm from 400 to 720 nm between ITO electrode and PMT.

correlation of surface antigens from the same cell state and, most importantly, be applied for cellular imaging of multiply antigens in immunohistochemistry. Although potential-resolved ECL based on the luminol/ Ru(bpy)32+ system is feasible in principle, we have previously incurred challenges in applying this system to achieve potentialresolved ECL because the cross-reactions between luminol and S2O82− gave the luminescence under the same potential with Ru(bpy)32+. The luminescence overlapping induced the difficulty to correlate the luminescence with each luminescence probe. To overcome this challenge, a self-quenching of luminescence from the luminol group in the presence of the Ru(bpy)32+ group and S2O82− under negative potential was initialized by the introduction of concentrated aqueous luminol, which achieved the restriction of the luminescence from luminol and Ru(bpy)32+ groups under positive and negative potentials, respectively. The luminescence related with specific potentials realized the potential-resolved ECL for the serial detection through a potential scanning. By labeling luminol and Ru(bpy)32+ groups on AFP and CEA antibodies, the potentialresolved method was validated on AFP and CEA antigen modified silica particles. Afterward, the assay was applied for the quantification of AFP and CEA antigen at cells. The success in the establishment of potential-resolved ECL offered a strategy for the analysis of multiply analytes at cells without any complex optical setup.

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EXPERIMENTAL SECTION Chemical. Au nanoparticles (Au NPs) with 13 nm in diameter were purchased from Beijing Deke Daojin Science and Technology Co., Ltd. (Beijing, China). CEA antibody and antigen were obtained from Zhengzhou Biocell Biotechnology Co., Ltd. (Zhengzhou, China) and Beijing Bioss Biotechnology Co., Ltd. (Beijing, China), respectively. AFP antibody and antigen were from Shuangliu Zhenglong Biochem Lab (Chengdu, China). Biotinylated CEA antibody was purchased from Beijing Key-bio Biotech Co., Ltd. (Beijing, China). MCF 7 cells and PC 3 cells were from the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences of Chinese Academy of Science (Shanghai, China). Silica particles (20 μm in diameter) with carboxylic acid groups were obtained from Micromod Partikeltechnologie GmbH (Germany). All other chemicals were from Sigma-Aldrich, unless indicated otherwise. Ultrapure water with a resistivity of 18.2 MΩ/cm was used throughout. Buffer solutions were sterilized. Cell Culture. MCF 7 cells and PC 3 cells were seeded in DMEM/high glucose medium and F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin), respectively. Cultures were maintained at 37 °C under a humidified atmosphere containing 5% CO2. Preparation of Antibody Associated Luminol Complex. 3-Mercaptopropionic acid (3-MPA) (1 mM) was added into the solution with 13 nm Au nanoparticles (Au NPs). The amount of Au NPs was 9 × 1013/mL. The mixture was stirred at 37 °C for 12 h and centrifuged at 14 000 rpm for 30 min to get the gold conjugates (Au NPs-MPA). To activate the carboxylic acid groups on the surface of gold conjugates, the mixture of 1 mM ethyl(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were reacted with Au NPs-MPA at 37 °C for 1 h. Then, the activated Au NPs were reacted with 5.4 × 1013 molecules/mL antibody (equal to 12.88 μg/mL) at 37 °C for 12 h. The little excessive Au NPs 6897

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RESULTS AND DISCUSSION Luminescence Distinction from Each Probe under Different Potentials. The aim of our work is the detection of two antigens at the cell surface using ECL probes through a potential scanning. Thus, the luminescence under different potential should be associated with each probe specifically. It was well-known that Ru(bpy)32+ in the presence of S2O82− emitted the luminescence under the negative potential. For the correlation of the luminescence under negative potential with Ru(bpy)32+ associated complex, the introduction of luminol group was not expected to generate luminescence in this potential window. To investigate the luminescence of the luminol group under negative potential, the luminescences of Ru(bpy)32+/ S2O82− in the absence and presence of luminol were compared. Although an intense luminescence was observed for Ru(bpy)32+ with the potential less than −1.0 V (Figure S1 in Supporting Information), the lowest potential applied for the analysis was chosen at −1.0 V to minimize the interruption of potential on the cells. As shown in Figure 1A

production of luminescence. The cogeneration of luminescence from luminol and Ru(bpy)32+ in the presence of S2O82− gave the difficulty to distinguish the luminescence from the Ru(bpy)32+ group under negative potential. To restrict the luminescence from luminol under negative potential, self-quenching of luminescence from concentrated luminol was initialized. As shown in Figure 1B and Figure S3 (Supporting Information), more aqueous luminol introduced in the presence of S2O82− led to a gradual decrease in the luminescence under negative potential. When the concentration of luminol was over 15 mM, a constant weak luminescence from luminol was obtained under negative potential that was independent of the amount of luminol. After obtaining the constant luminescence in the presence of 20 mM luminol and 3 mM S2O82− ion, more Ru(bpy)32+ was added in the solution and an increase in the luminescence was observed, as shown in Figure 1B. The luminescence increase was associated with the amount of Ru(bpy)32+ only in the presence of concentrated luminol and, thus, can be used to quantify the amount of Ru(bpy)32+ under negative potential. The same trend of luminescence with concentrated aqueous luminol was observed in the absence of oxygen, as shown in Figure S4 in the Supporting Information, which confirmed that oxygen was not related to the quenching process. As for positive potential, Figure 2 showed that the starting potentials for the emission of luminescence from luminol and

Figure 2. Luminescence curve of (a) 200 μM luminol and (b) 200 μM Ru(bpy)32+ under positive potential.

Ru(bpy) 3 2+ were 0.40 and 0.60 V, respectively. The luminescence spectra of Ru(bpy)32+ at the potential over 0.6 V, as shown in Figure S5 (Supporting Information), showed the maximum peak wavelength near 620 nm. The result indicated a small amount of Ru(bpy)32+* generated over this potential.18 To avoid the overlapping of luminescence from luminol and Ru(bpy)32+, the highest positive potential was set at 0.6 V. Hydrogen peroxide was a classic coreactant to enhance luminol ECL. However, Figure S6 (Supporting Information) exhibited that the coexistence of luminol, Ru(bpy)32+, hydrogen peroxide, and S2O82− generated chemiluminescence, which was independent of the potential and quenched ECL from Ru(bpy)32+ and S2 O82− under negative potential. The mechanism was most likely that S2O82− as a strong oxidant induced the production of oxygen radical from hydrogen peroxide for luminol chemiluminescence. The consumption of S2O82− in chemiluminescence inhibited the conversion of S2O82− into SO4•− under negative potential to generate ECL from Ru(bpy)32+. Since luminol itself generated luminescence under positive potential, hydrogen peroxide was excluded from the system to restore ECL from Ru(bpy)32+/S2O82−. Also, the contribution of oxygen in the luminescence of luminol and

Figure 1. (A) The luminescence curve of (a) 100 μM Ru(bpy)32+, (b) 100 μM luminol/Ru(bpy)32+ and (c) luminol in the presence of 3 mM S2O82− under negative potential; (B) the luminescence read at −1.0 V from 100 μM Ru(bpy)32+ and 3 mM S2O82− in the presence of 0.1, 1, 5, 10, 15, 20 mM luminol. The last point presents the luminescence from 200 μM Ru(bpy)32+ in 20 mM luminol and 3 mM S2O82−.

curves a and b, the mixture of luminol and Ru(bpy)32+ generated more luminescence than Ru(bpy)32+ itself in the presence of 3 mM S2O82− on the ITO electrode under negative potential. The additional luminescence was attributed to the cross-reaction between luminol and S2O82− under negative potential, as evidenced in Figure 1 curve c. Replacing the electrode material into gold did not change the phenomenon, which indicated that reaction between luminol and S2O82− was not related with electrode material. Also, since aerated buffer with oxygen was required for the following cell analysis, the contribution of oxygen in the generation of luminescence was investigated. As shown in Figure S2 (Supporting Information), the same luminescence was observed in the absence and presence of oxygen exhibiting no contribution of oxygen in the 6898

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Figure 3. Preparation procedure for antigen modified silica particles and luminol or Ru(bpy)32+-antibody/antigen modified silica particle.

Ru(bpy) 3 2+ was investigated. As shown in Figure S7 (Supporting Information), the same luminescence increase was observed in the presence and absence of oxygen. This phenomenon suggested that oxygen did not contribute to the generation of luminescence, which was similar to the literature result collected on a Pt or graphite electrode.22 Therefore, when the positive potential was scanned from 0.6 to 0 V, the luminescence in this potential range was only attributed to luminol that was independent of the amount of Ru(bpy)32+. Overall, the luminescences under the positive and negative potentials were determined to be associated with luminol and Ru(bpy)32+ groups, respectively. The potential-resolved ECL assay for the determination of two antigens at the cell surface through a potential scanning was proposed as (1) under the positive potential, only luminol associated antigen complex at the cell surface emitted the luminescence, which was used to the determination of the amount of luminol associated antigen; (2) under the negative potential, the addition of aqueous luminol in the solution quenched the luminescence from luminol and luminol associated complex in the presence of S2O82− resulting in a small constant luminescence; after the extracting of that constant luminescence from luminol, the additional luminescence under the negative potential was related with the amount of Ru(bpy)32+ associated antigen. Although the solution component was altered when the potential reached the negative region, the same cell population was analyzed through a potential scanning that could offer the information about two antigens at the cell surface. Potential-Resolved ECL Assay for the Surface Antigen at Silica Particles. The distinction of luminescence was further validated using antigen modified silica particles. The diameter of particles was 20 μm that was similar to the cell size. During the analysis, the particles sit on the electrode by gravity to mimic the behavior of adherent cells. To modify the antigen on silica particles, the particles with carboxylic groups were reacted with the amino group at the CEA antibody that bound CEA antigens. Then, the antigen modified particles interacted with luminol or Ru(bpy)32+ labeled CEA antibody for ECL detection. All the preparation procedures are shown in Figure 3. As for the labeling of luminol group on antibody, 13 nm gold nanoparticles (Au-NPs) with carboxylic acid groups were reacted with CEA antibody and NH2-coupling luminol in serial so that both the antibody and luminol groups were associated on Au-NP as a complex for the recognition of antigen.23 To associate CEA antibody with Ru(bpy)32+ group, the biotiny-

lated CEA antibody was used. The biotin on the antibody was linked with the streptavidin-Ru(bpy)32+ complex.12 Under positive potential from 0.6 to 0 V, Figure 4A curves a and b showed the “background luminescence” from CEA

Figure 4. (A) Luminescence curve under positive potential from (a) 105 CEA antigen modified silica particles, (b) 105 luminol-CEA antibody/antigen particles, (c) mixture of 105 luminol-CEA antibody/ antigen particles and 105 Ru(bpy)32+-CEA antibody/antigen particles, (d) 3 × 10 5 luminol-CEA antibody/antigen particles. (B) Luminescence curve in the presence of 20 mM luminol and 3 mM S2O82− under negative potential from (a) 105 CEA antigen modified particles, (b) 105 Ru(bpy)32+-CEA antibody/antigen particles, (c) mixture of 105 Ru(bpy)32+-CEA antibody/antigen particles and 105 luminol-CEA antibody/antigen particles, (d) 3 × 105 Ru(bpy)32+-CEA antibody/antigen particles.

antigen modified particles and more luminescence from luminol-CEA antibody/antigen particles on the ITO electrode. The maximum emission wavelength near 430 nm, as shown in Figure S8 (Supporting Information), supported the generation of luminescence from the luminol group on the particles, which was associated with CEA antigens. The weak luminescence was mainly caused by the limited number of luminol group on the electrode. On the basis of the fluorescence intensity (Ex/Em 280/335 nm) of antibody-Au NPs complex in the solution 6899

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before and after the linkage on the particle, the amount of Au NPs on each particle was estimated to be 1.60 × 10−19 mol. Taking into account that 1666 luminol groups were associated with one Au NP, ∼105 particles on the electrode had a maximum of 2.66 × 10−11 mol of luminol group. Since only luminol group at the interface of particles and the electrode generated the luminescence, the total amount of luminol was rare for weak luminescence. To investigate the interruption of the Ru(bpy)32+ group on the luminescence under positive potential, Ru(bpy)32+-CEA antibody/antigen particles were introduced on the electrode. No increase in the luminescence was observed in curve c confirming that the luminescence under positive potential was only attributed to luminol associated particles. The addition of more luminol-CEA antibody/antigen particles on the electrode increased the amount of antigens and an enhanced luminescence was observed in Figure 4 A curve d, which indicated that our assay can monitor the amount charge of antigen on particles. For ECL under negative potential, 20 mM aqueous luminol and 3 mM S2O82− were added into the solution after the potential scanned from 0.6 to 0 V. Figure 4B curve a exhibited the “background luminescence” from CEA antigen modified particles on the electrode with the potential from 0 to −1 V. The weak luminescence observed at −1.0 V came from aqueous concentrated luminol in the presence of S2O82−. When the particles were replaced by Ru(bpy)32+-CEA antibody/antigen particles, more luminescence observed in curve b was generated from the Ru(bpy)32+ group on the particles. The addition of luminol-antibody/antigen particles did not alter the luminescence, as shown in curve c. These results confirmed that the luminescence from Ru(bpy)32+ group was determined in the presence of luminol associated antigen. Similar to the result under positive potential, more Ru(bpy)32+-antibody/antigen modified particles were introduced on the electrode and an increase in the luminescence was observed, as expected in curve d. After the validation of our assay, the relation of luminescence with the amount of antigen was established for the following quantitative measurement in cells. To correlate the luminescence of luminol or Ru(bpy)32+ with different surface antigens, luminol and Ru(bpy)32+ groups were labeled with AFP and CEA antibody, respectively, for the recognition of the corresponding antigens at silica particles. For the regulation of antigen amount, the number of particles on the electrode was controlled. The estimation process of CEA and AFP antigen on each silica particle was discussed in the Supporting Information. Since concentrated luminol gave a weak and constant luminescence, the background luminescence collected on the unmodified cells needed to be excluded from the luminescence collected on luminescence probe-modified cells. This luminescence difference was further normalized by the background luminescence to minimize the luminescence deviation created by different ITO slides. As shown in Figure 5A,B, linear relationships of luminescence ratio on the amount of AFP and CEA antigens under positive and negative potentials were demonstrated. The correlation supported the quantitative measurement of surface antigens at cells using our assay. Potential-Resolved ECL Assay for the Detection of Two Antigens at Cell Surface. For the detection of two surface antigens at cells, the cells were fixed to ensure the interaction between the antigens and antibodies and minimize the interruption of concentrated aqueous luminol on cellular

Figure 5. Correlation of luminescence ratio with (A) AFP antigen and (B) CEA antigen on the electrode. The red lines were the linear fitting curves. The error bars represent the standard deviation from four independent experiments.

activity. MCF 7 cells with high expression of CEA and AFP antigens at the cell surface were used as a model.24 Using the same modification protocol, the cells were colabeled with AFP antibody-luminol and CEA antibody-Ru(bpy)32+ complexes. Figure 6A showed the luminescence with the potential scanning from 0.6 to −1.0 V. When the potential reached 0 V, 20 mM luminol and 3 mM S2O82− were added. Compared with the

Figure 6. (A) The luminescence curves from (a) 1.6 × 105 luminolAFP antibody and Ru(bpy)32+-CEA antibody labeled MCF 7 cells, and (b) 1.6 × 105 unlabeled MCF 7 cells; (B) the bar of luminescence ratio on the measurement of AFP and CEA antigen at 1.6× 105 MCF 7 and PC 3 cells. The bars labeled with “∗” were collected from the simultaneous detection of two antigens. The error bars were the standard deviation from three groups of cells. 6900

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electrochemiluminescence imaging system, the assay was promising for the coimaging of multiply analytes at cells. The success of this imaging method will provide an alternative technology for the cellular immunochemistry.

background luminescence on unmodified cells in curve a, the increases in luminescence under both of positive and negative potentials in curve b exhibited the analysis of two antigens at the cell surface. The luminescence ratios from three groups of cells were calculated to be 1.59 ± 0.09 for AFP antigen and 0.92 ± 0.13 for CEA antigen, as shown in Figure 6B. The control experiment was performed on bare ITO slide, which did not have the cells cultured. The luminescence curve collected from bare ITO slide, as shown in Figure S9 in the Supporting Information, was similar to the background luminescence curve with a slight increase in value. Since the luminescence probes were not located on the slide after the cell removal, and thus, the similarity of luminescence curve collected on bare ITO slide and ITO slide cultured with unmodified cells was reasonable. The slight increase in the luminescence value was ascribed to a more exposed electrode surface after the removal of cells. This result confirmed that the luminescence increase observed on curve b was attributed to the luminescence probe linked with the antigens on the cell surface. To confirm the accuracy of our detection, MCF 7 cells were labeled with either AFP antibody associated luminol or CEA antibody associated Ru(bpy)32+ to measure the luminescence. The luminescence ratios shown in Figure 6B were similar to those from the codetection in the presence of two luminescence probes, which suggested that the colabeling of two ECL probes and the detection did not give any measurement error. Meanwhile, PC 3 cells with no expression of CEA and AFP antigens on the surface were used as a negative control cell model.24 No luminescence change in the whole potential range was observed before and after the labeling of AFP antibody-luminol and CEA antibody-Ru(bpy)32+ on PC 3 cells, as shown in Figure 6B. All these results supported that our assay quantified surface antigens with a relative accuracy. Also, our assay needed 1.6 × 105 cells, which was similar to the cell number required in the clinic tests using fluorescence, HPLC, and mass spectroscopy. The success in the analysis of similar pool-sized cells exhibited a potential application of our assay for the real diagnosis. Referring to the luminescence ratio in Figure 5A,B, the average amount of AFP and CEA antigen at the cells were determined as 2.15 fg and 32.5 fg per cell. The results were close to the literature data, which was 0.21−1.75 fg for AFP and 18−27 fg for CEA per cell using cell lysate for analysis.24−26 The small difference in the antigen amount might be attributed to our analysis process. For ECL assay, only the antigen associated ECL probe at the boundary of cells/particles and electrode generated the luminescence, which was used to estimate the amount of antigen on the whole cell surface. As compared with the direct analysis of whole cell lysate, the addition of some measurement error might exist.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 086-25-86868477. Fax: 08625-86868477. *E-mail: [email protected]. Phone: 086-25-83594846. Fax: 086-25-83594846. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 Program (2013 Grant CB933800), the National Natural Science Foundation of China (Grant Nos. 21327902, 21135003, 21105045, and 21105049), and the open research fund from State Key Laboratory of Analytical Chemistry for Life Science (Grant SKLACLS 1212), Nanjing University.

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REFERENCES

(1) Wulfkuhle, J. D.; Liotta, L. A.; Petricoin, E. F. Nat. Rev. Cancer 2003, 3, 267−275. (2) Marquette, C. A.; Corgier, B. P.; Blum, L. J. Bioanalysis 2012, 4, 927−936. (3) Pepperkok, R.; Squire, A.; Geley, S.; Bastiaens, P. I. H. Curr. Biol. 1999, 9, 269−272. (4) Vignali, D. A. A. J. Immunol. Methods 2000, 243, 243−255. (5) Hemmila, I. Clin. Chem. 1985, 31, 359−370. (6) Bard, A. J., Ed. Electrogenerated Chemiluminescence; Marcel Dekker: New York, 2004. (7) Forster, R. J.; Bertoncello, P.; Keyes, T. E. Annu. Rev. Anal. Chem. 2009, 2, 359−385. (8) Miao, W. J. Chem. Rev. 2008, 108, 2506−2553. (9) Richter, M. M. Chem. Rev. 2004, 104, 3003−3036. (10) Doeven, E. H.; Zammit, E. M.; Barbante, G. J.; Francis, P. S.; Barnett, N. W.; Hogan, C. F. Chem. Sci. 2013, 4, 977−982. (11) Doeven, E. H.; Barbante, G. J.; Kerr, E.; Hogan, C. F.; Endler, J. A.; Francis, P. S. Anal. Chem. 2014, 86, 2727−2732. (12) Deiss, F.; LaFratta, C. N.; Symer, M.; Blicharz, T. M.; Sojic, N.; Walt, D. R. J. Am. Chem. Soc. 2009, 131, 6088−6089. (13) Tian, D. Y.; Duan, C. F.; Wang, W.; Cui, H. Biosens. Bioelectron. 2010, 25, 2290−2295. (14) Chen, Z. H.; Liu, Y.; Wang, Y. Z.; Zhao, X.; Li, J. H. Anal. Chem. 2013, 85, 4431−4438. (15) Sakura, S. Anal. Chim. Acta 1992, 262, 49−57. (16) Cui, H.; Xu, Y.; Zhang, Z. F. Anal. Chem. 2004, 76, 4002−4010. (17) Marquette, C. A.; Blum, L. J. Anal. Chim. Acta 1999, 381, 1−10. (18) White, H. S.; Bard, A. J. J. Am. Chem. Soc. 1982, 104, 6891− 6895. (19) Bolletta, F.; Ciano, M.; Balzani, V.; Serpone, N. Inorg. Chim. Acta 1982, 62, 207−213. (20) Bolletta, F.; Juris, A.; Maestri, M.; Sandrini, D. Inorg. Chim. Acta 1980, 44, L175−L176. (21) Wu, M. S.; Shi, H. W.; He, L. J.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2012, 84, 4207−4213. (22) Wróblewska, A.; Reshetnyak, O. V.; Koval’chuk, E. P.; Pasichnyuk, R. I.; Błażejowski, J. J. Electroanal. Chem. 2005, 580, 41−49.

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CONCLUSION In this paper, a potential resolved electrochemiluminescence assay was achieved for the detection of AFP and CEA antigens at the cell surface. The luminescence from each probe was restricted in the limited potential windows so that they were used for the quantification of surface antigens. Compared with the fluorescence assay, the analysis strategy using the potentialcontrolled signals avoids the limitation of spectrum difference for probes and simplifies the instrumental setup. The continuous introduction of more ECL probes with different potentials will permit the strategy to analyze more surface antigens during one potential scanning. Also, coupled with the 6901

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Analytical Chemistry

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(23) Yang, X. Y.; Guo, Y. S.; Wang, A. G. Anal. Chim. Acta 2010, 666, 91−96. (24) Grieve, R. J.; Woods, K. L.; Mann, P. R.; Smith, S. C. H.; Wilson, G. D.; Howell, A. Br. J. Cancer 1980, 42, 616−619. (25) Biddle, W.; Sarcione, E. J. Breast Cancer Res. Treat. 1987, 10, 279−286. (26) Zeskind, B. J.; Jordan, C. D.; Timp, W.; Trapani, L.; Waller, G.; Horodincu, V.; Ehrlich, D. J.; Matsudaira, P. Nat. Methods 2007, 4, 567−569.

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dx.doi.org/10.1021/ac501571a | Anal. Chem. 2014, 86, 6896−6902