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A copper based enzyme-free fluorescence ELISA for HER2 detection
Journal of Immunological Methods xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
A copper based enzyme-free fluorescence ELISA for HER2 detection Shiyu Tiana, Ke Zenga, Aijun Yangb, Qin Wangb, Minghui Yanga,⁎ a b
College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China Center for Reproductive medicine, Affiliated Hospital of Jining Medical University, Jining, Shandong 272029, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Fluorescence Breast cancer ELISA CuO nanoparticle Serum
We reported an enzyme-free ELISA to detect breast cancer biomarker human epidermal growth factor receptor 2 (HER2) in human serum samples. Instead of enzymes (such as horseradish peroxidase) used in traditional ELISA, CuO nanoparticles were utilized as the signal probe. Compared to traditional enzymes, CuO nanoparticles have the advantages of low cost and good stability. After dissolving CuO nanoparticles with acid, the Cu (II) ions generated catalyzed the reaction of o-phenylenediamine with ascorbic acid to produce fluorescent quinoxaline derivative molecules. The immunoassay displays high sensitivity and good selectivity towards HER2 with detection limit as low as 9.65 pg·mL− 1. The assay was successfully applied to the analysis of HER2 in serum of breast cancer patients. The analysis results demonstrated the HER2 level in the serum samples determined by our assay were in good agreement with those determined by commercial HER2 ELISA kit. This enzyme-free ELISA assay can be easily adapted to the detection of other analytes. With these merits, the simple, sensitive and cost effective fluorescence immunoassay shows great potential for clinical applications.
1. Introduction The enzyme-linked immunosorbent assay (ELISA), a well developed testing method for detecting antibodies/antigens or different other indicators, is considered to be the most popular analysis tool in hospital diagnosis, medicinal and food industry (Zeng et al., 2017; Satija et al., 2016; Li et al., 2013; Yang et al., 2014). ELISA has advantages of good specificity, low cost and high-throughput compared to other methods, such as electrochemistry, surface-enhanced Raman spectroscopy (SERS) and surface plasmon resonance (SPR) (Kiyota et al., 2017; Pappert et al., 2010). In traditional ELISA, enzymes, such as horseradish peroxidase and alkaline phosphatase are often employed to generate amplified detection signals (He et al., 2017; Shen et al., 2016; Guo et al., 2017). However, the enzymes are usually expensive and the catalytic activity of enzymes is sensitive to the environmental changes, such as temperature and acidity. Enzyme denaturation caused by these reasons will make ELISA results inaccurate and lack of reproducibility. Enzyme-free ELISA is then developed to solve the above issues. In enzyme-free ELISA, inorganic or small organic substances are used instead of enzymes to be linked onto binding molecules to catalyze specific reaction and generate amplified detection signals. These kinds of substance are usually much more stable and cost effective than enzymes. Such substances include DNA and different nanomaterials (Hu et al., 2015; Li et al., 2009; Si et al., 2014). Breast cancer is the highest incidence of women malignant
⁎
carcinoma and the second leading cause of cancer death in women in United States. Human epidermal growth factor receptor 2 (HER2), encoded by the ErbB2 gene, is one of the human epidermal growth factor receptor family members (Hu et al., 2017; Ngamcherdtrakul et al., 2016; Elster et al., 2015). It is found that the fluctuation of HER2 content is closely related to the occurrence of breast cancer. According to the statistical data, up to 30% of breast cancer patients are accompanied with the over-expression of the HER2 (Yang et al., 2017; Ross and Fletcher, 1998). Therefore, it is important to develop methods for precise detection of HER2 protein for early breast cancer diagnosis. Two FDA-approved detective methods are applied to clinical testing of HER2, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These two methods focus on the cellular HER2 analysis that require medical infrastructure, pathology, and cytology expertise which is not accessible to some region with few medical equipment especially in developing countries. Recent studies have shown that extracellular HER2 value also reflects the status of breast cancer. The serum HER2 concentration was elevated in 20%–50% of patients with primary breast cancer and 50%–62% for metastatic cancer (Mitri et al., 2012). Normal individuals have a HER2 concentration between 2 and 15 ng·mL− 1 in the blood, and breast cancer patients have blood HER2 levels from 15 to 75 ng·mL− 1. Our group recently developed an approach for detecting extracellular HER2 by using electrochemical aptasensor based on DNA generated electrochemical current (Hu et al., 2017). However, efforts are still needed to
Corresponding author. E-mail address: [email protected] (M. Yang).
http://dx.doi.org/10.1016/j.jim.2017.09.002 Received 30 July 2017; Received in revised form 22 August 2017; Accepted 11 September 2017 0022-1759/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Tian, S., Journal of Immunological Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.09.002
Journal of Immunological Methods xxx (xxxx) xxx–xxx
S. Tian et al.
Fig. 1. Schematic illustration of the copper ion based ELISA.
the CuO NPs solution and then vortex-shaken for about 3 h at 37 °C to conjugate peptide onto the CuO. Finally, the solution was centrifuged for 10 min at 12,000 rpm to remove the excess peptide and the precipitate was re-dispersed in 7.5 mL phosphate buffer. Then, 200 μL of 25 mM 6-mercapto-1-hexanol (MCH) solution was added into 800 μL peptide modified CuO NPs solution, and after 4.5 h stirring at room temperature, unbounded MCH were removed by centrifuge. Finally, the obtained nanoparticles were re-dispersed in 800 μL of pH 7.4 buffer and stored at 4 °C when not in use.
develop simple and easy operating methods for HER2 analysis in serum samples. In this work, we reported an enzyme-free ELISA for HER2 detection utilizing copper oxide nanoparticles (CuO NPs) as signal amplification probe. After dissolving CuO NPs with acid, the generated Cu (II) ions catalyzed the reaction of o-phenylenediamine (OPDA) with ascorbic acid (AA) to produce fluorescent quinoxaline derivative molecules. The new ELISA is easy to operate, highly sensitive and selective to HER2 contents in serum samples of breast cancer patients, which means the assay has wide potential clinical applications.
2.3. ELISA measurements 2. Experimental The 5 μg·mL− 1 anti-HER2 antibody (100 μL) was added into the wells of the 96-well plate and incubated overnight. The plate was washed with phosphate buffer containing 0.1% Tween 20 three times and the wells were then blocked with 1 mg·mL− 1 BSA for 1.5 h at 37 °C. After washing again, 100 μL of various concentrations of HER2 standard solutions were added into the plate and incubated for 3 h at 37 °C. Similarly, the plate was washed and then, 100 μL peptide modified CuO NPs were added and incubated for 2 h at 37 °C. After another round of washing, 50 μL HCl solution (pH = 2) was added into each well and reacted for 30 min at room temperature under shaking to dissolve the CuO nanoparticles. The resulting acid solution containing the dissolved Cu2 + ions was then reacted with 200 μL solution containing AA (0.5 mM) and OPDA (6.5 mM). After another 30 min shaking at room temperature, the fluorescence emission spectra were collected at the excitation wavelength of 347 nm.
2.1. Regents and equipment The polyclonal rabbit anti-HER2 antibody was purchased from Sangon Biotech Company (Shanghai, China). Peptide with sequence of CKLRLEWNR was synthesized from GL Biochem Co. Ltd. (Shanghai, China). HER2 proteins and L-ascorbic acid (AA) were obtained from Abcam (Cambridge, UK). CuO nanoparticles were purchased from Sigma–Aldrich. All other reagents used were of analytical grade. Double-deionized water was used throughout the experiments. A 0.01 M phosphate buffer (pH 7.4) containing 0.1% Tween 20 was used as the washing buffer. Transmission electron microscope (TEM) images of CuO were obtained from Tecnai G2 60-300 (FEI. Co. Ltd). UV–Vis experiments were performed on a Shimadzu UV2450 spectrophotometer (Shimadzu, Japan). Size distribution data were obtained using Zetasizer ZEN 3600 (Malvern Instruments Ltd). The fluorescence spectra were recorded on a fluorescence spectrophotometer (Hitachi, F-7000). The electrospray ionization mass spectrometry (ESI-MS) data were measured by Bruker compact mass spectrometry. The 1H NMR was tested by Bruker ascend 400 NMR spectrometer.
3. Results and discussion 3.1. Feasibility study The schematic representation of the fluorescence enzyme-free ELISA immunoassay is shown in Fig. 1. We occasionally found acid solution containing OPDA and AA can produce strong fluorescence emission at around 430 nm (excited at 347 nm) in the presence of either Cu (II) or CuO nanoparticles. However, the fluorescence intensity generated due to the same amount of Cu (II) was about two times higher than that due to CuO nanoparticles. So the catalytic behavior of Cu (II) towards OPDA and AA was investigated. To study this phenomenon, UV–Vis spectra
2.2. Preparation of CuO–peptide conjugates The CuO–peptide conjugates were prepared according to a previous report (Qu et al., 2011). Briefly, about 0.75 mg CuO NPs were added into 7.5 mL phosphate buffer followed by ultrasonication for about 30 min. Then, 750 μg·mL− 1 peptide solution (0.5 mL) was added into 2
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0.6
at a characteristic ion peak of m/z 247.07. For NMR characterization, the characteristic peaks of quinoxaline protons were observed as a doublet peak at 6.09 ppm of protons 1 and a multiple peak at 8.09 ppm of protons 2–5(Supporting information, Fig. S2). Combining the 1H NMR with ESI-MS results, we confirm the formed fluorescence molecule is quinoxaline-one. In order to optimize the performance of the assay, different parameters that affect the fluorescence of the system were optimized, such as the molar ratio of OPDA versus AA and the pH of solution for Cu (II) catalyzed reaction. More details are shown in supporting information (Supporting information, Figs. S3–S4). The optimal pH value for the reaction was found to be 2 and the ratio of OPDA/AA was 13:1. Based on the above results, CuO nanoparticles were utilized to carry out the ELISA assy. The dissolving of CuO nanoparticles by acid will produce Cu (II) ions. The CuO NPs utilized were characterized by SEM and TEM, which indicated the spherical morphology of the CuO NPs (Fig. 4). The average size of CuO NPs was 104 nm calculated by dynamic light scattering (DLS) (Supporting information, Fig. S5). To perform the ELISA assay, peptides with the sequence of CKLRLEWNR that can bind specifically with HER2 molecules were conjugated onto CuO to turn the CuO NPs into signal probe (Hu et al., 2017). The thiol moiety on the peptide can form SeCu bond with Cu atom on the surface of CuO and the MCH immobilized onto the CuO surface can prevent non-specific adsorption between the nanoparticles and molecules in detection solution. With the immobilization of anti-HER2 antibodies into the wells of the 96 plate, the following specific capture of HER2 molecules and then CuO probe, the Cu (II) generated by dissolving CuO probe will then catalyze the reaction of OPDA and AA to generate fluorescence. The resulted fluorescence intensity is proportional to the HER2 concentration in a specific range.
AA OPDA OPDA+AA OPDA+Cu (II) AA+Cu (II) OPDA+AA+ Cu (II)
A Absorbance (a.u.)
0.4
0.2
0.0 300
Fluorecence intensity (a.u.)
8000
350 Wavelength (nm)
400
AA OPDA OPDA+AA OPDA+Cu (II) AA+Cu (II) OPDA+AA+Cu (II)
B
6000 4000 2000 0
3.2. The performance of the assay
400
440
480
520
Under the optimized experimental conditions, different concentrations of HER2 standard solutions were analyzed to test the linear range of the assay towards HER2. As can be seen from Fig. 5, the fluorescence intensity increased with the increase of HER2 concentrations and a good linear relationship for HER2 was obtained range from 25 pg·mL− 1 to 5 ng·mL− 1 with R square value of 0.996 (inset of Fig. 5). The limit of detection of the assay for HER2 was calculated to be about 9.65 pg·mL− 1 based on S/N of 3. The American Medical Association (AMA) has approved a serum HER-2 oncoprotein test intended to quantitatively measure HER-2 protein in serum with reference range (s) < 12.8 ng·mL− 1. The sensitivity of this ELISA means it can be applied for clinical testing of HER2 in diluted samples. The analytical performances of our assay were compared with literature reported assays for HER2 detection. From Table 1, it can be seen the performance of our assay were comparable or even better than literature reports. In addition, this enzyme-free ELISA has advantages of low cost and high-throughput. In addition of sensitivity, good selectivity is of great importance for precise detection of target analytes in complex samples. Different proteins and molecules that present in the serum samples, such as the β-site amyloid precursor protein cleaving enzyme 1 (BACE1), human IgG, mucin 1 (MUC1), carcinoembryonic antigen (CEA) and P53 were utilized as potential interferents to study the selectivity of the assay. The concentration of HER2 tested was 100 pg·mL− 1 while the concentration of the above potential interferents studied was 10 times higher than HER2. The fluorescence intensity occurred by the above potential interferents were negligible when compared to HER2 (Fig. 6), indicating good selectivity of the assay.
Wavelength (nm) Fig. 2. UV–Vis spectra (A) and fluorescence spectra (B) of acid solution (pH = 2) containing different compounds.
and fluorescence spectra of the mixing solutions were recorded. As shown in Fig. 2A, for UV–Vis spectrum, a broad peak is appeared at 350 nm for acid solution (pH = 2) containing 100 μM Cu (II), 6.5 mM OPDA and 0.5 mM AA. No absorbance were observed in the absent of Cu (II), which means the reaction between OPDA and AA did not occur without Cu (II). The fluorescence data was also in consistent with the above results (Fig. 2B). The fluorescence intensity of the mixture solution was increased either with the increase of reaction time or with the increase of Cu (II) concentrations (Supporting information, Fig. S1). These data indicated that the three components, Cu (II), OPDA as well as AA are all indispensable for the generation of fluorescence, and Cu (II) was acted as catalyst to catalyze the reaction between OPDA and AA. The generated fluorescence compound was believed to be quinoxaline derivative. To prove the formed fluorescence molecules were quinoxaline derivative, the formed molecules were characterized by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy (1H NMR). OPDA was commonly employed for the detection of AA in the presence of oxidants such as ascorbate oxidase, tempol and hydroxide (Vislisel et al., 2007; Sánchez-Mata et al., 2000; Wu et al., 2003). In this assay, the oxidant was substituted by Cu (II). We suppose in this system, Cu (II) was first reacted with AA to form dehydro-ascorbic acid (DHAA), and then the condensation reaction of OPDA with DHAA occurred to produce 3-(dihydroxyethyl)furo[3,4-b] quinoxaline-1-one (abbreviated as quinoxaline-one, Fig. 3). The ESI-MS data was obtained using the mixed solution containing OPDA/AA/ Cu2 + without further separation. [Quinoxaline-one + H]+ was found
3.3. Real sample analysis To further prove the potential of the assay in clinical applications, eight serum samples from four different breast cancer patients were 3
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Fig. 3. The possible mechanism of the reaction process use Cu2 + as catalyst.
obtained. For each patient, two samples were collected, one collected before tumor removal surgery, and one collected 5 days after tumor removal surgery. The HER2 contents in the 8 samples were determined by commercial HER2 ELISA kit and our assay. To test the samples by our assay, the serum samples were diluted 500 times, which can lower sample consumption (< 1 μL of serum samples is needed, lower than that needed for commercial assay) and minimize sample matrix effect. The HER2 concentrations acquired by our assay were in good consistent with the value obtained from the commercial kits (as shown in Table 2). In addition, the testing results demonstrated the HER2 levels of the four patients are all dropped after surgery. These results demonstrated the assay has wide potential clinical application in diagnosis and in breast cancer therapy.
Fig. 4. SEM and TEM (inset) image of the CuO NPs.
5 ng/mL
Fluorescence intensity
Fluorencence intensity (a.u.)
2500 2000
0 pg/mL 1500
4. Conclusion In summary, we reported an enzyme-free ELISA strategy for HER2 detection. The enzyme used in conventional ELISA is replaced with inorganic CuO nanoparticles. The assay shows low detection of limits of 9.65 pg·mL− 1 and a wide linear relationship for detecting HER2 from 25 pg·mL− 1 to 5 ng·mL− 1. Compared with traditional ELISA immunoassay, our strategy of using CuO nanoparticle as signal probe lowered the cost and improved the stability of the assay. By replace the binding ligands, this assay can be extended to detect other kinds of biomarkers and find wide clinical applications. However, further efforts are still needed to simplify the detection procedures, for example, using nanoparticles itself to trigger the generation of fluorescence.
2000 1600 1200 800 400 10
1000
100 1000 10000 Concentration of HER2 (pg/mL)
500 0 350
400
450 Wavelength (nm)
500
Acknowledgments
550
The authors thank the support of this work by the National Key Basic Research Program of China (2014CB744502), the National Natural Science Foundation of China (No. 21575165), the Natural Science Foundation of Shandong Province (NO: ZR2012HL002 and NO: ZR2015HL022) and the Open Foundation of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (2016008).
Fig. 5. The fluorescence response of the enzyme-free ELISA towards different concentrations of HER2. The inset is the calibration curve of the assay towards HER2. Error bar = RSD (n = 3).
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Table 1 Compare the performance of the assay towards HER2 with literature reports. Detection method
Signal tag
Linear range (ng·mL− 1)
Detection limit (ng·mL− 1)
Reference
Fluorescence PEMS PEMS Fluorescence Fluorescence Electrochemical Optical Resonator Fluorescence
CdSe/ZnS nanocrystal None None Silica nanospheres Photonic crystals Magnetic beads None CuO nano
\ 0.05–2 6–60 1–10,000 3–200 0–15 13–100 0.025–5
8 0.0253 5 1 0.3 6 11 0.00965
(Qiu et al., 2016) (Loo et al., 2011) (Capobianco et al., 2011) (Rossi et al., 2006) (Sinibaldi et al., 2017) (Mitri et al., 2012) (Gohring et al., 2010) This work
PEMS = piezoelectric microcantilever. the OptoFluidic ring resonator biosensor. Sensors Actuators B Chem. 146, 226–230. Guo, L., Chen, D., Yang, M., 2017. DNA-templated silver nanoclusters for fluorometric determination of the activity and inhibition of alkaline phosphatase. Microchim. Acta 184, 2165–2170. He, S., Li, X., Gao, J., Tong, P., Chen, H., 2017. Development of sandwich ELISA for testing bovine beta-lactoglobulin allergenic residues by specific polyclonal antibody against human IgE binding epitopes. Food Chem. 227, 33–40. Hu, R., Liu, T., Zhang, X.-B., Yang, Y., Chen, T., Wu, C., et al., 2015. DLISA: a DNAzymebased ELISA for protein enzyme-free immunoassay of multiple analytes. Anal. Chem. 87, 7746–7753. Hu, L., Hu, S., Guo, L., Shen, C., Yang, M., Rasooly, A., 2017. DNA generated electric current biosensor. Anal. Chem. 89, 2547–2552. Kiyota, K., Kawatsu, K., Sakata, J., Yoshimitsu, M., Akutsu, K., Satsuki-Murakami, T., et al., 2017. Development of monoclonal antibody-based ELISA for the quantification of orange allergen Cit s 2 in fresh and processed oranges. Food Chem. 232, 43–48. Li, J., Zhang, T., Ge, J., Yin, Y., Zhong, W., 2009. Fluorescence signal amplification by cation exchange in ionic nanocrystals. Angew. Chem. Int. Ed. 48, 1588–1591. Li, D., Ying, Y., Wu, J., Niessner, R., Knopp, D., 2013. Comparison of monomeric and polymeric horseradish peroxidase as labels in competitive ELISA for small molecule detection. Microchim. Acta 180, 711–717. Loo, L.N., Capobianco, J.A., Wei, W., Gao, X., Wan, Y.S., Shih, W.H., et al., 2011. Highly sensitive detection of HER2 extracellular domain in the serum of breast cancer patients by piezoelectric microcantilevers. Anal. Chem. 83, 3392. Mitri, Z., Constantine, T., O'Regan, R., 2012. The HER2 receptor in breast cancer: pathophysiology, clinical use, and new advances in therapy. Chemother. Res. Pract. 2012, 743193. Ngamcherdtrakul, W., Castro, D.J., Gu, S., Morry, J., Reda, M., Gray, J.W., et al., 2016. Current development of targeted oligonucleotide-based cancer therapies: perspective on HER2-positive breast cancer treatment. Cancer Treat. Rev. 45, 19–29. Pappert, G., Rieger, M., Niessner, R., Seidel, M., 2010. Immunomagnetic nanoparticlebased sandwich chemiluminescence-ELISA for the enrichment and quantification of E. coli. Microchim. Acta 168, 1–8. Qiu, X., Wegner, K.D., Wu, Y., Henegouwen, P.M.P.V.B.E., Jennings, T.L., Hildebrandt, N., 2016. Nanobodies and antibodies for duplexed EGFR/HER2 immunoassays using Terbium-to-Quantum Dot FRET. Chem. Mater. 28, 8256–8267. Qu, W., Liu, Y., Liu, D., Wang, Z., Jiang, X., 2011. Copper-mediated amplification allows readout of immunoassays by the naked eye. Angew. Chem. Int. Ed. 50, 3442–3445. Ross, J.S., Fletcher, J.A., 1998. The HER-2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy. Stem Cells 16, 413–428. Rossi, L.M., Shi, L., Rosenzweig, N., Rosenzweig, Z., 2006. Fluorescent silica nanospheres for digital counting bioassay of the breast cancer marker HER2/nue. Biosens. Bioelectron. 21, 1900–1906. Sánchez-Mata, M.C., Cámara-Hurtado, M., Díez-Marqués, C., Torija-Isasa, M.E., 2000. Comparison of high-performance liquid chromatography and spectrofluorimetry for vitamin C analysis of green beans (Phaseolus vulgaris L.). Eur. Food Res. Technol. 210, 220–225. Satija, J., Punjabi, N., Mishra, D., Mukherji, S., 2016. Plasmonic-ELISA: expanding horizons. RSC Adv. 6, 85440–85456. Shen, C., Li, X., Rasooly, A., Guo, L., Zhang, K., Yang, M., 2016. A single electrochemical biosensor for detecting the activity and inhibition of both protein kinase and alkaline phosphatase based on phosphate ions induced deposition of redox precipitates. Biosens. Bioelectron. 85, 220–225. Si, Z., Li, Y., Quan, H., Qi, H., Li, T., Yang, M., 2014. ELISA type fluorescence turn-on immunoassay based on Fe3 + induced fluorescence enhancement. Sensors Actuators B Chem. 202, 663–666. Sinibaldi, A., Sampaoli, C., Danz, N., Munzert, P., Sibilio, L., Sonntag, F., et al., 2017. Detection of soluble ERBB2 in breast cancer cell lysates using a combined label-free/ fluorescence platform based on Bloch surface waves. Biosens. Bioelectron. 92, 125–130. Vislisel, J.M., Schafer, F.Q., Buettner, G.R., 2007. A simple and sensitive assay for ascorbate using a plate reader. Anal. Biochem. 365, 31–39. Wu, X., Diao, Y., Sun, C., Yang, J., Wang, Y., Sun, S., 2003. Fluorimetric determination of ascorbic acid with o-phenylenediamine. Talanta 59, 95–99. Yang, M., Yi, X., Wang, J., Zhou, F., 2014. Electroanalytical and surface plasmon resonance sensors for detection of breast cancer and Alzheimer's disease biomarkers in cells and body fluids. Analyst 139, 1814. Yang, H.-Y., Ma, D., Liu, Y.-R., Hu, X., Zhang, J., Wang, Z.-H., et al., 2017. Impact of hormone receptor status and distant recurrence-free interval on survival benefits from trastuzumab in HER2-positive metastatic breast cancer. Sci. Report. 7. Zeng, K., Tian, S., Wang, Z., Shen, C., Luo, J., Yang, M., et al., 2017. An ELISA for the determination of human IgG based on the formation of a colored iron(II) complex and photometric or visual read-out. Microchim. Acta 184, 2791–2796.
FLuorescence intensity
1000
750
500
250
0
BACE1
IgG
MUC1
CEA
P53
HER2
Different samples Fig. 6. Selectivity of the enzyme-free ELISA for HER2 over other potential interferents. Error bar = RSD (n = 3).
Table 2 Determination of HER2 in breast cancer patients before and 5 days after surgery by the enzyme-free assay and commercial HER2 kit. Sample
Status
Commercial ELISA assay (ng·mL− 1)
Our assay (ng·mL− 1)
1
Before surgery 5 days after surgery Before surgery 5 days after surgery Before surgery 5 days after surgery Before surgery 5 days after surgery
42.00 ± 4.04 38.45 ± 3.38
40.70 ± 3.14 38.17 ± 4.25
39.53 ± 4.70 31.13 ± 2.47
36.2 ± 3.74 27.04 ± 2.75
70.67 ± 9.06 54.52 ± 8.40
59.78 ± 3.06 43.83 ± 4.05
34.83 ± 1.73 33.52 ± 2.55
37.22 ± 2.88 32.00 ± 3.23
2
3
4
Results are mean ± S.D. (n = 5).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jim.2017.09.002. References Capobianco, J., Shih, W.Y., Adams, G.P., Shih, W., 2011. Label-free Her2 detection and dissociation constant assessment in diluted human serum using a longitudinal extension mode of a piezoelectric microcantilever sensor. Sensors Actuators B Chem. 160, 349–356. Elster, N., Collins, D.M., Toomey, S., Crown, J., Eustace, A.J., Hennessy, B.T., 2015. HER2-family signalling mechanisms, clinical implications and targeting in breast cancer. Breast Cancer Res. Treat. 149, 5–15. Gohring, J.T., Dale, P.S., Fan, X., 2010. Detection of HER2 breast cancer biomarker using
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Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
A copper based enzyme-free fluorescence ELISA for HER2 detection Shiyu Tiana, Ke Zenga, Aijun Yangb, Qin Wangb, Minghui Yanga,⁎ a b
College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China Center for Reproductive medicine, Affiliated Hospital of Jining Medical University, Jining, Shandong 272029, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Fluorescence Breast cancer ELISA CuO nanoparticle Serum
We reported an enzyme-free ELISA to detect breast cancer biomarker human epidermal growth factor receptor 2 (HER2) in human serum samples. Instead of enzymes (such as horseradish peroxidase) used in traditional ELISA, CuO nanoparticles were utilized as the signal probe. Compared to traditional enzymes, CuO nanoparticles have the advantages of low cost and good stability. After dissolving CuO nanoparticles with acid, the Cu (II) ions generated catalyzed the reaction of o-phenylenediamine with ascorbic acid to produce fluorescent quinoxaline derivative molecules. The immunoassay displays high sensitivity and good selectivity towards HER2 with detection limit as low as 9.65 pg·mL− 1. The assay was successfully applied to the analysis of HER2 in serum of breast cancer patients. The analysis results demonstrated the HER2 level in the serum samples determined by our assay were in good agreement with those determined by commercial HER2 ELISA kit. This enzyme-free ELISA assay can be easily adapted to the detection of other analytes. With these merits, the simple, sensitive and cost effective fluorescence immunoassay shows great potential for clinical applications.
1. Introduction The enzyme-linked immunosorbent assay (ELISA), a well developed testing method for detecting antibodies/antigens or different other indicators, is considered to be the most popular analysis tool in hospital diagnosis, medicinal and food industry (Zeng et al., 2017; Satija et al., 2016; Li et al., 2013; Yang et al., 2014). ELISA has advantages of good specificity, low cost and high-throughput compared to other methods, such as electrochemistry, surface-enhanced Raman spectroscopy (SERS) and surface plasmon resonance (SPR) (Kiyota et al., 2017; Pappert et al., 2010). In traditional ELISA, enzymes, such as horseradish peroxidase and alkaline phosphatase are often employed to generate amplified detection signals (He et al., 2017; Shen et al., 2016; Guo et al., 2017). However, the enzymes are usually expensive and the catalytic activity of enzymes is sensitive to the environmental changes, such as temperature and acidity. Enzyme denaturation caused by these reasons will make ELISA results inaccurate and lack of reproducibility. Enzyme-free ELISA is then developed to solve the above issues. In enzyme-free ELISA, inorganic or small organic substances are used instead of enzymes to be linked onto binding molecules to catalyze specific reaction and generate amplified detection signals. These kinds of substance are usually much more stable and cost effective than enzymes. Such substances include DNA and different nanomaterials (Hu et al., 2015; Li et al., 2009; Si et al., 2014). Breast cancer is the highest incidence of women malignant
⁎
carcinoma and the second leading cause of cancer death in women in United States. Human epidermal growth factor receptor 2 (HER2), encoded by the ErbB2 gene, is one of the human epidermal growth factor receptor family members (Hu et al., 2017; Ngamcherdtrakul et al., 2016; Elster et al., 2015). It is found that the fluctuation of HER2 content is closely related to the occurrence of breast cancer. According to the statistical data, up to 30% of breast cancer patients are accompanied with the over-expression of the HER2 (Yang et al., 2017; Ross and Fletcher, 1998). Therefore, it is important to develop methods for precise detection of HER2 protein for early breast cancer diagnosis. Two FDA-approved detective methods are applied to clinical testing of HER2, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These two methods focus on the cellular HER2 analysis that require medical infrastructure, pathology, and cytology expertise which is not accessible to some region with few medical equipment especially in developing countries. Recent studies have shown that extracellular HER2 value also reflects the status of breast cancer. The serum HER2 concentration was elevated in 20%–50% of patients with primary breast cancer and 50%–62% for metastatic cancer (Mitri et al., 2012). Normal individuals have a HER2 concentration between 2 and 15 ng·mL− 1 in the blood, and breast cancer patients have blood HER2 levels from 15 to 75 ng·mL− 1. Our group recently developed an approach for detecting extracellular HER2 by using electrochemical aptasensor based on DNA generated electrochemical current (Hu et al., 2017). However, efforts are still needed to
Corresponding author. E-mail address: [email protected] (M. Yang).
http://dx.doi.org/10.1016/j.jim.2017.09.002 Received 30 July 2017; Received in revised form 22 August 2017; Accepted 11 September 2017 0022-1759/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Tian, S., Journal of Immunological Methods (2017), http://dx.doi.org/10.1016/j.jim.2017.09.002
Journal of Immunological Methods xxx (xxxx) xxx–xxx
S. Tian et al.
Fig. 1. Schematic illustration of the copper ion based ELISA.
the CuO NPs solution and then vortex-shaken for about 3 h at 37 °C to conjugate peptide onto the CuO. Finally, the solution was centrifuged for 10 min at 12,000 rpm to remove the excess peptide and the precipitate was re-dispersed in 7.5 mL phosphate buffer. Then, 200 μL of 25 mM 6-mercapto-1-hexanol (MCH) solution was added into 800 μL peptide modified CuO NPs solution, and after 4.5 h stirring at room temperature, unbounded MCH were removed by centrifuge. Finally, the obtained nanoparticles were re-dispersed in 800 μL of pH 7.4 buffer and stored at 4 °C when not in use.
develop simple and easy operating methods for HER2 analysis in serum samples. In this work, we reported an enzyme-free ELISA for HER2 detection utilizing copper oxide nanoparticles (CuO NPs) as signal amplification probe. After dissolving CuO NPs with acid, the generated Cu (II) ions catalyzed the reaction of o-phenylenediamine (OPDA) with ascorbic acid (AA) to produce fluorescent quinoxaline derivative molecules. The new ELISA is easy to operate, highly sensitive and selective to HER2 contents in serum samples of breast cancer patients, which means the assay has wide potential clinical applications.
2.3. ELISA measurements 2. Experimental The 5 μg·mL− 1 anti-HER2 antibody (100 μL) was added into the wells of the 96-well plate and incubated overnight. The plate was washed with phosphate buffer containing 0.1% Tween 20 three times and the wells were then blocked with 1 mg·mL− 1 BSA for 1.5 h at 37 °C. After washing again, 100 μL of various concentrations of HER2 standard solutions were added into the plate and incubated for 3 h at 37 °C. Similarly, the plate was washed and then, 100 μL peptide modified CuO NPs were added and incubated for 2 h at 37 °C. After another round of washing, 50 μL HCl solution (pH = 2) was added into each well and reacted for 30 min at room temperature under shaking to dissolve the CuO nanoparticles. The resulting acid solution containing the dissolved Cu2 + ions was then reacted with 200 μL solution containing AA (0.5 mM) and OPDA (6.5 mM). After another 30 min shaking at room temperature, the fluorescence emission spectra were collected at the excitation wavelength of 347 nm.
2.1. Regents and equipment The polyclonal rabbit anti-HER2 antibody was purchased from Sangon Biotech Company (Shanghai, China). Peptide with sequence of CKLRLEWNR was synthesized from GL Biochem Co. Ltd. (Shanghai, China). HER2 proteins and L-ascorbic acid (AA) were obtained from Abcam (Cambridge, UK). CuO nanoparticles were purchased from Sigma–Aldrich. All other reagents used were of analytical grade. Double-deionized water was used throughout the experiments. A 0.01 M phosphate buffer (pH 7.4) containing 0.1% Tween 20 was used as the washing buffer. Transmission electron microscope (TEM) images of CuO were obtained from Tecnai G2 60-300 (FEI. Co. Ltd). UV–Vis experiments were performed on a Shimadzu UV2450 spectrophotometer (Shimadzu, Japan). Size distribution data were obtained using Zetasizer ZEN 3600 (Malvern Instruments Ltd). The fluorescence spectra were recorded on a fluorescence spectrophotometer (Hitachi, F-7000). The electrospray ionization mass spectrometry (ESI-MS) data were measured by Bruker compact mass spectrometry. The 1H NMR was tested by Bruker ascend 400 NMR spectrometer.
3. Results and discussion 3.1. Feasibility study The schematic representation of the fluorescence enzyme-free ELISA immunoassay is shown in Fig. 1. We occasionally found acid solution containing OPDA and AA can produce strong fluorescence emission at around 430 nm (excited at 347 nm) in the presence of either Cu (II) or CuO nanoparticles. However, the fluorescence intensity generated due to the same amount of Cu (II) was about two times higher than that due to CuO nanoparticles. So the catalytic behavior of Cu (II) towards OPDA and AA was investigated. To study this phenomenon, UV–Vis spectra
2.2. Preparation of CuO–peptide conjugates The CuO–peptide conjugates were prepared according to a previous report (Qu et al., 2011). Briefly, about 0.75 mg CuO NPs were added into 7.5 mL phosphate buffer followed by ultrasonication for about 30 min. Then, 750 μg·mL− 1 peptide solution (0.5 mL) was added into 2
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0.6
at a characteristic ion peak of m/z 247.07. For NMR characterization, the characteristic peaks of quinoxaline protons were observed as a doublet peak at 6.09 ppm of protons 1 and a multiple peak at 8.09 ppm of protons 2–5(Supporting information, Fig. S2). Combining the 1H NMR with ESI-MS results, we confirm the formed fluorescence molecule is quinoxaline-one. In order to optimize the performance of the assay, different parameters that affect the fluorescence of the system were optimized, such as the molar ratio of OPDA versus AA and the pH of solution for Cu (II) catalyzed reaction. More details are shown in supporting information (Supporting information, Figs. S3–S4). The optimal pH value for the reaction was found to be 2 and the ratio of OPDA/AA was 13:1. Based on the above results, CuO nanoparticles were utilized to carry out the ELISA assy. The dissolving of CuO nanoparticles by acid will produce Cu (II) ions. The CuO NPs utilized were characterized by SEM and TEM, which indicated the spherical morphology of the CuO NPs (Fig. 4). The average size of CuO NPs was 104 nm calculated by dynamic light scattering (DLS) (Supporting information, Fig. S5). To perform the ELISA assay, peptides with the sequence of CKLRLEWNR that can bind specifically with HER2 molecules were conjugated onto CuO to turn the CuO NPs into signal probe (Hu et al., 2017). The thiol moiety on the peptide can form SeCu bond with Cu atom on the surface of CuO and the MCH immobilized onto the CuO surface can prevent non-specific adsorption between the nanoparticles and molecules in detection solution. With the immobilization of anti-HER2 antibodies into the wells of the 96 plate, the following specific capture of HER2 molecules and then CuO probe, the Cu (II) generated by dissolving CuO probe will then catalyze the reaction of OPDA and AA to generate fluorescence. The resulted fluorescence intensity is proportional to the HER2 concentration in a specific range.
AA OPDA OPDA+AA OPDA+Cu (II) AA+Cu (II) OPDA+AA+ Cu (II)
A Absorbance (a.u.)
0.4
0.2
0.0 300
Fluorecence intensity (a.u.)
8000
350 Wavelength (nm)
400
AA OPDA OPDA+AA OPDA+Cu (II) AA+Cu (II) OPDA+AA+Cu (II)
B
6000 4000 2000 0
3.2. The performance of the assay
400
440
480
520
Under the optimized experimental conditions, different concentrations of HER2 standard solutions were analyzed to test the linear range of the assay towards HER2. As can be seen from Fig. 5, the fluorescence intensity increased with the increase of HER2 concentrations and a good linear relationship for HER2 was obtained range from 25 pg·mL− 1 to 5 ng·mL− 1 with R square value of 0.996 (inset of Fig. 5). The limit of detection of the assay for HER2 was calculated to be about 9.65 pg·mL− 1 based on S/N of 3. The American Medical Association (AMA) has approved a serum HER-2 oncoprotein test intended to quantitatively measure HER-2 protein in serum with reference range (s) < 12.8 ng·mL− 1. The sensitivity of this ELISA means it can be applied for clinical testing of HER2 in diluted samples. The analytical performances of our assay were compared with literature reported assays for HER2 detection. From Table 1, it can be seen the performance of our assay were comparable or even better than literature reports. In addition, this enzyme-free ELISA has advantages of low cost and high-throughput. In addition of sensitivity, good selectivity is of great importance for precise detection of target analytes in complex samples. Different proteins and molecules that present in the serum samples, such as the β-site amyloid precursor protein cleaving enzyme 1 (BACE1), human IgG, mucin 1 (MUC1), carcinoembryonic antigen (CEA) and P53 were utilized as potential interferents to study the selectivity of the assay. The concentration of HER2 tested was 100 pg·mL− 1 while the concentration of the above potential interferents studied was 10 times higher than HER2. The fluorescence intensity occurred by the above potential interferents were negligible when compared to HER2 (Fig. 6), indicating good selectivity of the assay.
Wavelength (nm) Fig. 2. UV–Vis spectra (A) and fluorescence spectra (B) of acid solution (pH = 2) containing different compounds.
and fluorescence spectra of the mixing solutions were recorded. As shown in Fig. 2A, for UV–Vis spectrum, a broad peak is appeared at 350 nm for acid solution (pH = 2) containing 100 μM Cu (II), 6.5 mM OPDA and 0.5 mM AA. No absorbance were observed in the absent of Cu (II), which means the reaction between OPDA and AA did not occur without Cu (II). The fluorescence data was also in consistent with the above results (Fig. 2B). The fluorescence intensity of the mixture solution was increased either with the increase of reaction time or with the increase of Cu (II) concentrations (Supporting information, Fig. S1). These data indicated that the three components, Cu (II), OPDA as well as AA are all indispensable for the generation of fluorescence, and Cu (II) was acted as catalyst to catalyze the reaction between OPDA and AA. The generated fluorescence compound was believed to be quinoxaline derivative. To prove the formed fluorescence molecules were quinoxaline derivative, the formed molecules were characterized by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy (1H NMR). OPDA was commonly employed for the detection of AA in the presence of oxidants such as ascorbate oxidase, tempol and hydroxide (Vislisel et al., 2007; Sánchez-Mata et al., 2000; Wu et al., 2003). In this assay, the oxidant was substituted by Cu (II). We suppose in this system, Cu (II) was first reacted with AA to form dehydro-ascorbic acid (DHAA), and then the condensation reaction of OPDA with DHAA occurred to produce 3-(dihydroxyethyl)furo[3,4-b] quinoxaline-1-one (abbreviated as quinoxaline-one, Fig. 3). The ESI-MS data was obtained using the mixed solution containing OPDA/AA/ Cu2 + without further separation. [Quinoxaline-one + H]+ was found
3.3. Real sample analysis To further prove the potential of the assay in clinical applications, eight serum samples from four different breast cancer patients were 3
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Fig. 3. The possible mechanism of the reaction process use Cu2 + as catalyst.
obtained. For each patient, two samples were collected, one collected before tumor removal surgery, and one collected 5 days after tumor removal surgery. The HER2 contents in the 8 samples were determined by commercial HER2 ELISA kit and our assay. To test the samples by our assay, the serum samples were diluted 500 times, which can lower sample consumption (< 1 μL of serum samples is needed, lower than that needed for commercial assay) and minimize sample matrix effect. The HER2 concentrations acquired by our assay were in good consistent with the value obtained from the commercial kits (as shown in Table 2). In addition, the testing results demonstrated the HER2 levels of the four patients are all dropped after surgery. These results demonstrated the assay has wide potential clinical application in diagnosis and in breast cancer therapy.
Fig. 4. SEM and TEM (inset) image of the CuO NPs.
5 ng/mL
Fluorescence intensity
Fluorencence intensity (a.u.)
2500 2000
0 pg/mL 1500
4. Conclusion In summary, we reported an enzyme-free ELISA strategy for HER2 detection. The enzyme used in conventional ELISA is replaced with inorganic CuO nanoparticles. The assay shows low detection of limits of 9.65 pg·mL− 1 and a wide linear relationship for detecting HER2 from 25 pg·mL− 1 to 5 ng·mL− 1. Compared with traditional ELISA immunoassay, our strategy of using CuO nanoparticle as signal probe lowered the cost and improved the stability of the assay. By replace the binding ligands, this assay can be extended to detect other kinds of biomarkers and find wide clinical applications. However, further efforts are still needed to simplify the detection procedures, for example, using nanoparticles itself to trigger the generation of fluorescence.
2000 1600 1200 800 400 10
1000
100 1000 10000 Concentration of HER2 (pg/mL)
500 0 350
400
450 Wavelength (nm)
500
Acknowledgments
550
The authors thank the support of this work by the National Key Basic Research Program of China (2014CB744502), the National Natural Science Foundation of China (No. 21575165), the Natural Science Foundation of Shandong Province (NO: ZR2012HL002 and NO: ZR2015HL022) and the Open Foundation of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (2016008).
Fig. 5. The fluorescence response of the enzyme-free ELISA towards different concentrations of HER2. The inset is the calibration curve of the assay towards HER2. Error bar = RSD (n = 3).
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Table 1 Compare the performance of the assay towards HER2 with literature reports. Detection method
Signal tag
Linear range (ng·mL− 1)
Detection limit (ng·mL− 1)
Reference
Fluorescence PEMS PEMS Fluorescence Fluorescence Electrochemical Optical Resonator Fluorescence
CdSe/ZnS nanocrystal None None Silica nanospheres Photonic crystals Magnetic beads None CuO nano
\ 0.05–2 6–60 1–10,000 3–200 0–15 13–100 0.025–5
8 0.0253 5 1 0.3 6 11 0.00965
(Qiu et al., 2016) (Loo et al., 2011) (Capobianco et al., 2011) (Rossi et al., 2006) (Sinibaldi et al., 2017) (Mitri et al., 2012) (Gohring et al., 2010) This work
PEMS = piezoelectric microcantilever. the OptoFluidic ring resonator biosensor. Sensors Actuators B Chem. 146, 226–230. Guo, L., Chen, D., Yang, M., 2017. DNA-templated silver nanoclusters for fluorometric determination of the activity and inhibition of alkaline phosphatase. Microchim. Acta 184, 2165–2170. He, S., Li, X., Gao, J., Tong, P., Chen, H., 2017. Development of sandwich ELISA for testing bovine beta-lactoglobulin allergenic residues by specific polyclonal antibody against human IgE binding epitopes. Food Chem. 227, 33–40. Hu, R., Liu, T., Zhang, X.-B., Yang, Y., Chen, T., Wu, C., et al., 2015. DLISA: a DNAzymebased ELISA for protein enzyme-free immunoassay of multiple analytes. Anal. Chem. 87, 7746–7753. Hu, L., Hu, S., Guo, L., Shen, C., Yang, M., Rasooly, A., 2017. DNA generated electric current biosensor. Anal. Chem. 89, 2547–2552. Kiyota, K., Kawatsu, K., Sakata, J., Yoshimitsu, M., Akutsu, K., Satsuki-Murakami, T., et al., 2017. 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ELISA type fluorescence turn-on immunoassay based on Fe3 + induced fluorescence enhancement. Sensors Actuators B Chem. 202, 663–666. Sinibaldi, A., Sampaoli, C., Danz, N., Munzert, P., Sibilio, L., Sonntag, F., et al., 2017. Detection of soluble ERBB2 in breast cancer cell lysates using a combined label-free/ fluorescence platform based on Bloch surface waves. Biosens. Bioelectron. 92, 125–130. Vislisel, J.M., Schafer, F.Q., Buettner, G.R., 2007. A simple and sensitive assay for ascorbate using a plate reader. Anal. Biochem. 365, 31–39. Wu, X., Diao, Y., Sun, C., Yang, J., Wang, Y., Sun, S., 2003. Fluorimetric determination of ascorbic acid with o-phenylenediamine. Talanta 59, 95–99. Yang, M., Yi, X., Wang, J., Zhou, F., 2014. Electroanalytical and surface plasmon resonance sensors for detection of breast cancer and Alzheimer's disease biomarkers in cells and body fluids. Analyst 139, 1814. Yang, H.-Y., Ma, D., Liu, Y.-R., Hu, X., Zhang, J., Wang, Z.-H., et al., 2017. 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FLuorescence intensity
1000
750
500
250
0
BACE1
IgG
MUC1
CEA
P53
HER2
Different samples Fig. 6. Selectivity of the enzyme-free ELISA for HER2 over other potential interferents. Error bar = RSD (n = 3).
Table 2 Determination of HER2 in breast cancer patients before and 5 days after surgery by the enzyme-free assay and commercial HER2 kit. Sample
Status
Commercial ELISA assay (ng·mL− 1)
Our assay (ng·mL− 1)
1
Before surgery 5 days after surgery Before surgery 5 days after surgery Before surgery 5 days after surgery Before surgery 5 days after surgery
42.00 ± 4.04 38.45 ± 3.38
40.70 ± 3.14 38.17 ± 4.25
39.53 ± 4.70 31.13 ± 2.47
36.2 ± 3.74 27.04 ± 2.75
70.67 ± 9.06 54.52 ± 8.40
59.78 ± 3.06 43.83 ± 4.05
34.83 ± 1.73 33.52 ± 2.55
37.22 ± 2.88 32.00 ± 3.23
2
3
4
Results are mean ± S.D. (n = 5).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jim.2017.09.002. References Capobianco, J., Shih, W.Y., Adams, G.P., Shih, W., 2011. Label-free Her2 detection and dissociation constant assessment in diluted human serum using a longitudinal extension mode of a piezoelectric microcantilever sensor. Sensors Actuators B Chem. 160, 349–356. Elster, N., Collins, D.M., Toomey, S., Crown, J., Eustace, A.J., Hennessy, B.T., 2015. HER2-family signalling mechanisms, clinical implications and targeting in breast cancer. Breast Cancer Res. Treat. 149, 5–15. Gohring, J.T., Dale, P.S., Fan, X., 2010. Detection of HER2 breast cancer biomarker using
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