Immunochromatographic assay for quantitative and sensitive detection of hepatitis B virus surface antigen using highly luminescent quantum dot-beads

Talanta 142 (2015) 145–149

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Immunochromatographic assay for quantitative and sensitive detection of hepatitis B virus surface antigen using highly luminescent quantum dot-beads Jun Shen a,b, Yaofeng Zhou a, Fen Fu c, Hengyi Xu a,n, Jiaofeng Lv c, Yonghua Xiong a,b,nn, Andrew Wang d a

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, PR China Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, PR China The Second Affiliated Hospital, Nanchang University, Nanchang 330000, PR China d Ocean NanoTech, LLC., San Diego, CA 92126, USA b c

art ic l e i nf o

a b s t r a c t

Article history: Received 16 February 2015 Received in revised form 18 April 2015 Accepted 20 April 2015 Available online 27 April 2015

Hepatitis B virus infection is one of the major causes of hepatitis, liver cirrhosis and liver cancer. In this study, we used highly luminescent quantum dot-beads (QBs) as signal amplification probes in the sandwich immunochromatographic assay (ICA) for ultrasensitive and quantitative detection of hepatitis B virus surface antigen (HBsAg) in human serum. Various parameters that influenced the sensitivity and stability of the QB-based ICA (QB-ICA) sensor were investigated. Two linear independent regression equations for detection of serum HBsAg were expressed with Y ¼0.3361X 0.0059 (R2 ¼ 0.9983) for low HBsAg concentrations between 75 pg mL  1 and 4.8 ng mL  1, and Y¼ 0.8404 X  2.9364 (R2 ¼0.9939) for high HBsAg concentrations in the range from 4.8 ng mL  1 to 75 ng mL  1. The detection limit of the proposed ICA sensor achieved was 75 pg mL  1, which is much higher than that of the routinely-used gold nanoparticle based ICA. The intra- and inter-assays recovery rates for spiked serum samples at HBsAg concentrations of 75 pg mL  1, 3.75 ng mL  1 and 18.75 ng mL  1 ranged from 90.14% to 97.6%, and coefficients of variation were all below 7%, indicating that the QB-ICA sensor has an acceptable accuracy for HBsAg detection. Additionally, the quantitative method developed showed no false positive results in an analysis of 49 real HBsAg-negative serum samples, and exhibited excellent agreement (R2 ¼0.9209) with a commercial chemiluminescence immunoassay kit in identifying 47 HBsAg-positive serum samples. In summary, due to its high fluorescence intensity, the sandwich QB-ICA sensor is a very promising point-of-care test for rapid, simple and ultrasensitive detection of HBsAg, as well as other disease-related protein biomarkers. & 2015 Elsevier B.V. All rights reserved.

Keywords: Quantum dot-beads Immunochromatographic assay Hepatitis B virus surface antigen (HBsAg) Quantitative determination

1. Introduction Hepatitis B virus (HBV) is the leading cause of liver cancer and the most common reason for liver transplantation, and is mainly spread by transfusing blood or blood products [1,2]. Therefore, there is an

Abbreviations: QBs, quantum dot-beads; ICA, immunochromatographic assay; FCS, fetal calf serum; NC, nitrocellulose; D-pAbs, donkey anti-mouse polyclonal antibodies; G-pAbs, goat anti-HBsAg polyclonal antibodies; POC, point-of-care; LED, light emitting diode n Corresponding author. Tel.: þ 86 791 8830 4447x9512; fax: þ86 791 8830 4400. nn Corresponding author at: Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, PR China. Tel.: þ 86 791 8833 4578; fax: þ 86 791 8833 3708. E-mail addresses: [email protected] (H. Xu), [email protected] (Y. Xiong). http://dx.doi.org/10.1016/j.talanta.2015.04.058 0039-9140/& 2015 Elsevier B.V. All rights reserved.

urgent need for the development of a point-of-care (POC) test to control the rapid spread of the disease. The detection of hepatitis B virus surface antigen (HBsAg) in serum has been confirmed as one of the pivotal steps in the discovery and/or quantitation of HBV, because the HBsAg antigen overexpressed and secreted into the blood by virus-infected liver cells [3]. Traditional methods for HBsAg detection usually involve solid-phase enzyme-linked immunosorbent assay (ELISA) [4], time-resolved fluoroimmunoassay (TRFIA) [5], chemiluminescence immunoassay (CLIA) [6,7], or capillary electrophoresis (CE)-electrochemical immunoassay [8]. These methods are highly sensitive, reliable, and widely used, but require sophisticated instruments, well-trained personnel and time-consuming procedures. Such disadvantages restrict their value in clinical practice, especially for POC diagnostic tests at the physician's office or patient's bedside [9–11].

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The immunochromatographic assay (ICA) is a popular POC device in clinical diagnostics because of its rapidity, simplicity, practicality and user-friendliness [12,13]. However, use of the traditional gold nanoparticle based ICA for HBsAg detection is always limited by its relatively low sensitivity. To date, the lowest detection limit of the conventional strip test for HBsAg using gold nanoparticles as the reporter remains at 0.7 ng mL  1 [14]. To improve the sensitivity, many novel signal-amplified reagents including fluorescent and magnetic nanomaterials have been introduced into the ICA platform. Quantum dots (QDs) are an ideal fluorescent reporter and have been widely used to improve the detection sensitivity of ICA due to their narrow emission spectra, broad excitation range, and highly fluorescent quantum yields [15,16]. In our previous study, highly luminescent quantum dot beads (QBs) were synthesized and successfully applied in a competitive ICA method for ultrasensitive detection of aflatoxin B1 in maize with a detection limit (LOD) of 0.42 pg mL  1 [17]. Herein, we further report a sandwich ICA method for ultrasensitive and quantitative detection of HBsAg in sera. Owing to the signal amplification using highly luminescent QBs, the ICA sensor (QB-ICA) exhibits a low LOD of 75 pg mL  1, which corresponds to 5.3 pg HBsAg for each assay. The sensitivity of QB-ICA is significantly higher than that of the conventional commercial ELISA method (0.2 ng mL  1) [18], and of the previously reported QB-based dot-blot immunoassay (78 pg HBsAg) [19]. Significantly, as a newly POC test for HBsAg, the QB-based ICA will be a versatile strategy for applying in the field of medicine and even business.

2.2. Preparation of carboxyl-modified QBs and anti-HBsAg mAblabeled QBs Carboxyl-modified QBs (247 713 nm, 6 mg mL  1) were prepared according to our previously reported method [17]. Briefly, 20 mg of CdSe/ZnS QDs with a maximum emission wavelength at 623 nm was dissolved with 2 mL of CHCl3 containing 60 mg mL  1 of poly (methyl methacrylate) and 40 mg mL  1 of poly (maleic anhydride-alt-1-octadecene) and then mixed with 5 mL of sodium dodecyl sulfonate aqueous solution (3 mg mL  1). After ultrasonic homogenizing for 2 min, the emulsion was treated using a rotary evaporator to evaporate the non-polar solvent (CHCl3). Finally, the carboxyl-modified QBs were isolated by centrifugation (11,336 g, 10 min), and purified by washing with pure water for three times. The anti-HBsAg mAb-labeled QBs (QB probes) were prepared as previously reported with some modifications [17]. Briefly, 0.15 mg of the QBs and 30 mg of anti-HBsAg mAbs were mixed with 2.5 mg of EDC  HCl in 5 mL of pH 6.0 phosphate buffer (PB, 0.01 M), and incubated at room temperature for 1 h under continuous magnetic stirring. Then excess cross-linkers or antibodies in the reaction mixture were removed by centrifugation at 11,336 g for 10 min at 4 °C. In order to block the surplus carboxyl groups on the surface of the QB, the QB probes were re-suspended in 5 mL of pH 6.0 PB solution containing 1% BSA and 2.5 mg EDC  HCl, and reacted at room temperature for a further 1 h. The resultant QB probes were washed with pure water at least twice. The purified QB probes were then dissolved in 500 mL of PB containing 2% fructose, 5% sucrose, 1% polyethylene glycol 20,000, 1% BSA, 0.4% Tween-20, and then stored at 4 °C for further use.

2. Materials and methods 2.3. Preparation of QB-ICA sensor 2.1. Chemicals, materials and apparatus N-(3-dimethylaminopropyl)-Nʹ-ethylcarbodiimide hydrochloride (EDC  HCl) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Fetal calf serum (FCS) was bought from Tianhang Biological Technology Co., Ltd. (Zhejiang, China). Donkey anti-mouse polyclonal antibodies (D-pAbs, 5 mg mL  1) were purchased from Beijing Zhongshan Biotechnology, Inc. (Beijing, China). Nitrocellulose (NC) membrane, sample pad, backing card and absorbent pad were obtained from Schleicher and Schuell GmbH (Dassel, Germany). Deactivated hepatitis B virus surface antigen (HBsAg, 14 μg mL  1), mouse anti-HBsAg monoclonal antibodies (anti-HBsAg mAbs, 7.2 mg mL  1) and goat anti-HBsAg polyclonal antibodies (G-pAbs, 6.8 mg mL  1) were kindly supplied by Jinyuan Jiahe Biotechnology Co., Ltd. (Beijing, China). The conventional ELISA kit for HBsAg semi-quantitative analysis was purchased from Zhongshan Biotech. Co., Ltd. (Guangdong, China). The commercial CLIA kit for HBsAg quantitative analysis was received from Xiamen Xinchuang, Inc. (Xiamen, China). The composition of the phosphate buffer (PB, 0.01 M) was 1.37 g of NaH2PO4  2H2O, 0.44 g of Na2HPO4  12H2O in 1 L Milli-Q water, and the pH value of PB buffer was adjusted to 6.0 by using 0.01 moL L  1 HCl. All others chemical reagents were of analytical grade. The strip fabrication system consists of the BioDot XYZ platform, automatic programmable cutter and fluorescence strip reader. The BioDot XYZ platform equipped with a motion controller, BioJet Quanti3000k dispenser and AirJet Quanti3000k dispenser for solution dispensing were supplied by BioDot (Irvine, CA). An automatic programmable cutter was purchased from Shanghai Jinbiao Biotechnology Co., Ltd. (Shanghai, China). The portable fluorescent strip reader was obtained from Huguo Science Instrument Co., Ltd. (Shanghai, China). All the solutions were prepared with ultra-pure water, which is produced by Elix-3 and Milli-QA (Molsheim, France).

The QB-ICA sensor consists of three sections: sample pad, NC membrane, and absorbent pad. To reduce nonspecific adsorption on the QB probes, the sample pads were pretreated with 20 mM sodium borate buffer (pH 8.0) containing 1.0% (w/v) BSA, 0.1% (w/v) NaN3, and 0.25% Tween-20 as described in our previous report, and then oven dried at 60 °C for 2 h [20]. G-pAbs (1.0 mg mL  1) and D-pAbs (1.0 mg mL  1) were dispensed onto the NC membrane as test (T) and control (C) lines with a density of 0.75 μL cm  1. The distance between the T line and C line was 6 cm. After drying at 37 °C for 12 h, the sample pad, NC membrane and absorbent pad were laminated and adhered onto the backing card. All prepared strips were cut into 4.0 cm width using automatic programmable cutter and packaged at ambient temperature for subsequent usage. 2.4. Detection strategy of the QB-ICA sensor As illustrated in Fig. 1, the detection strategy of the QB-ICA sensor is based on the sandwich immunoassay. Briefly, 1.0 μL aliquots of QB probe (0.3 mg mL  1) were premixed with a serum sample (70 μL) in the tube for 1 min and then added into the well of the sample pad. When the target HBsAg was included in the serum sample, the QB probe would bind with HBsAg to form a QB-mAb-HBsAg complex, and then the immunocomplex would be captured by G-pAbs and D-pAbs on the NC membrane to form fluorescent signals on both the test and control lines (Fig. 1A). In contrast, when no analyte was present in the serum sample, the QB probes would not be captured by the G-pAbs to form a sandwich formation, which resulted in no fluorescent signal on the test line (Fig. 1B). For quantitative determination of HBsAg concentration in serum, fluorescence intensity signals (FIs) on both lines were recorded by a commercial fluorescent strip reader (Fig. 1C). Firstly, the strip was transported into the reader by a step motor controller, and then the QBs on both lines were excited by a high power 450 nm light emitting diode (LED). The emission fluorescence of QBs was focused with a cylindrical lens and

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Fig. 1. Schematic representation of the sandwich procedure for the detection of HBsAg using QB based ICA platform. (A) The five components assembly of conventional ICA sensor, the positive tests consequence consisting of a test line (T) and a control line (C). (B) Immunochromatographic assay shows a negative result with the presence of a control line. (C) The fluorescence strip reader.

Fig. 2. (A) Typical results of the QBs loaded with different amounts of target anti-HBsAg. Conditions: the concentration of positive serum, 0.3 ng mL  1; incubation time, 20 min; (B) immunoreaction dynamics of FIT, FIC and FIT/FIC at serum of 0.3 ng mL  1 HBsAg concentrations from 1 to 45 min.

received by a light sensitive cell after passing through an optical slit, whereas the background light produced by the 450 nm LED was eliminated with a 540 nm cut off filter.

2.5. Validation analysis of QB-ICA sensor To evaluate the accuracy and precision of QB-ICA sensor, three different concentrations of HBsAg serum samples were used to analyze the recoveries and variation coefficients of intra- and inter-assays. The intra-assays were determined by one batch of test strips for five replicates, while the inter-assays were analyzed once every day for three sequential days. The practicality and reliability of the QB-ICA sensor were performed by analyzing 96 real serum samples, which were collected from the second affiliated hospital of Nanchang University (Nanchang, China). Prior to measurement by using the ICA test, these samples were evaluated by a commercial semi-quantitative ELISA kit. The HBsAg negative serum samples were used for false positive analysis of QB-ICA sensor. The HBsAg positive serum samples were further determined by QB-ICA and a commercial CLIA kit. The CLIA method detected serum HBsAg linearly and dynamically over the range from 25 pg mL  1 to 125 ng mL  1 with a LOD of 25 pg mL  1. The CLIA test was performed according to the operating instructions.

3. Results and discussion 3.1. Optimization of QB probes The QBs were synthesized by encapsulating CdSe/ZnS using a microemulsion method as previously reported [17]. Transmission electron microscope (TEM) images (Fig. S1) show that the QBs have relatively uniform size distribution with an average diameter of 247713 nm. Meanwhile, the resultant QBs exhibited luminescence intensity more than 2800 times brighter than that of the corresponding QDs (Fig. S2). The QB probes were mainly prepared by covalently coupling the amino group of anti-HBsAg monoclonal antibodies with the carboxyl group of QBs in the presence of EDC  HCl [21]. Moreover, to ensure the captured efficiency of QB probes on HBsAg in serum samples, the amount of antibodies on the surface of the QB was optimized by coupling 50, 100, 150, 175, 200, 250 and 300 μg of anti-HBsAg antibodies to 1 mg of QBs and comparing the results. The mixtures of QB probes and HBsAg spiked serum samples (0.3 ng mL  1) were analyzed using a QB-ICA sensor, and the fluorescence signal on the test zone (FIT) was recorded using a portable strip reader. The results shown in Fig. 2A indicate that the FIT increased with the increasing amount of antibodies on the surface of the QBs. When the concentration of labeled antibodies reached 200 μg mg  1 QBs, the FIT of QB-ICA achieved a maximum value of 474.5674.39. However, the higher titer labeled antibodies (over

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Table 1 “checkerboard titration” experiment with 2 factors at 3 levels to optimize the concentration of anti-HBsAg antibody on T lines, the volume of QB probe pre-mixed with the sample solution. No. The concentration of anti-HBsAg mAbs (mg mL  1)

The volume of QB probe (μL)

The FI of test lines

1 2 3 4 5 6a 7 8 9

0.5 0.75 1.0 0.5 0.75 1.0 0.5 0.75 1.0

322 7 20 362 7 8.0 426 7 11 408 7 26 4767 11 5617 7.0 4337 25 5317 17 563 7 20

a

0.75 0.75 0.75 1.0 1.0 1.0 1.25 1.25 1.25

The optimal condition under the concentration of 0.3 ng mL  1 serum HBsAg.

200 μg mg  1) could result in a lower FIT, which could be attributed to the changeable configuration of antibodies degrading the active sites [22]. Therefore, 200 μg of anti-HBsAg antibodies per mg QBs was selected as the optimal saturated labeled concentration for the preparation of QB probes. 3.2. Optimization of QB-based ICA sensor To acquire the best sensitivity and a higher FI signal on test line, the reaction conditions should be optimized. Since the designed ICA sensor mainly consisted of QB probe concentrations and a series of G-pAbs concentrations, we initially investigated the effect of them by performing a “checkerboard titration” analysis. The results are presented in Table 1 and indicate that the optimal combination was 1.0 μL of QB probe (0.3 mg mL  1) and 1.0 mg mL  1 of G-pAbs on the test line. The fluorescent signal of the QB-ICA on the test line reached 561.7176.61 for detection of 0.3 ng mL  1 of HBsAg in a serum sample (n¼ 3). Moreover, the immunological kinetics of the QB-ICA was analyzed to elaborate the effect of the interpretation time on the stability of HBsAg quantitative analysis. The dynamic curves were indicated by plotting the values of FIT, FIC and the ratio of FIT/FIC against immunoreaction time according to our previously reported method [23]. Briefly, a positive serum sample containing 0.3 ng mL  1 of HBsAg was mixed with QB probe and then added into the sample well, and the strip was scanned with a commercial fluorescent reader to record the values of FIT, FIC and the ratio of FIT/FIC every 30 s for a total of 45 min immunoreaction time. As shown in Fig. 2B, the values of FIT and FIC continued to increase over the 45 min observation time, but the ratio of FIT/FIC reached a stable value after 10 min. 3.3. Analytical performance and validation of ICA sensor To demonstrate the accuracy of HBsAg quantitative analysis, we concluded that the FIT/FIC value should be recorded at 15 min after sample addition in all the succeeding studies. Under the optimal conditions, the calibration curve for the quantitative QB-ICA sensor was obtained by determining the FIT/FIC values of sixteen (16) HBsAg standard solutions against HBsAg concentration. The HBsAg standard solutions were prepared by diluting the HBsAg stock solution (14 μg mL  1) in FCS to a final concentration of 0, 0.015, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.4, 18.75, 37.5, 750, 150, 750, and 1500 ng mL  1. Error bars were obtained based on three duplicate measurements at different HBsAg concentrations. Fig. 3 shows that the FIT/FIC values augment along with the increase of target HBsAg concentration from 0 to 1500 ng mL  1. In order to better explain the quantitative relationship between the ratio of FIT/FIC and HBsAg concentration, two linear independent regression equations were expressed with Y¼0.3361X  0.0059 (R2 ¼0.9983) for low HBsAg concentrations between 75 pg mL  1 and 4.8 ng mL  1,

Fig. 3. Optimized standard curve for HBsAg was obtained by diluting stock serum samples and control with FCS; the insets of two independent show an enlarged view for HBsAg concentrations. Data were obtained by averaging three independent experiments. All above experiments are under the condition of 70 μL of samples containing different antigens with enhanced factor at 3.

and Y ¼0.8404 X 2.9364 (R2 ¼0.9939) in high HBsAg concentrations in the range from 4.8 ng mL  1 to 75 ng mL  1. Moreover, when HBsAg concentration was beyond 150 ng mL  1, a significant “hook effect” was observed, which exhibited that the FIT/FIC values declined with further increases in HBsAg concentration from 150 to 1500 ng mL  1. Thus, a real serum sample with HBsAg concentration over 150 ng mL  1 should be diluted with HBsAg-free FCS to avoid false negative or falsely lower results [24]. In addition, when the HBsAg concentration was as low as 15 pg mL  1, no fluorescent signal was observed on the test line, whereas for a 75 pg mL  1 HBsAg serum sample, the average FIT and ratio of FIT/FIC achieved were 687 6.28 and 0.03370.005, respectively (n ¼6). Thus, the detection limit for HBsAg was established as 75 pg mL  1, which is equivalent to 5.3 pg HBsAg antigen for each strip test. The sensitivity of the proposed QB-ICA method is approximately ten-fold and three-fold higher, respectively, than those of the previously reported gold nanoparticle-based ICA method (0.7 ng mL  1) [15] and the conventional commercial ELISA method (0.2 ng mL  1). The precision of the QB-ICA method was evaluated by calculating the intra- and inter-assay recoveries and coefficients of variation (CV) of three HBsAg spiked serum samples. The HBsAg concentrations were 0.75, 3.75, and 18.75 ng mL  1, which represented low, medium and high HBsAg spiked amounts respectively. The intra-assay evaluation was completed within a day for five replicates at each HBsAg spiked concentration. These series of analyses were repeated over 3 consecutive days to assess inter-assay precision. The outcomes, summarized in Table 2, indicated that the CV of the assays using the same-batch QB probes were 4.23% and 1.58% at 0.75 ng mL  1, 5.46% and 6.17% at 3.75 ng mL  1, 6.38% and 0.23% at 18.75 ng mL  1 of HBsAg, respectively, whereas the average recoveries varied from 90.8% to 97.6%, and 90.14% to 94.83% for intra- and inter-assay respectively. These results revealed that the precision of the QB-ICA method for HBsAg quantification was acceptable. 3.4. Application in the real serum samples The analytical reliability and practicability of the QB-ICA sensor were evaluated by analyzing 96 real serum specimens. Prior to measurement by using the ICA test, these samples were determined by a commercial semi-quantitative ELISA kit. Then the negative serum samples were determined with the QB-ICA sensor, and the results showed no fluorescent signal on the test line for any of the 49 HBsAg negative samples indicating that the QB-ICA sensor produced

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Table 2 The precision of the ICA platform in HBsAg serum. Diluted concentration (ng mL  1)

0.75 3.75 18.75 a b

Inter-assay precisiona

Intra-assay precision The meanb of T/C

SD

CV (%)

Recovery (%)

The meanb of T/C

SD

CV (%)

Recovery (%)

0.167 1.134 9.367

0.007 0.062 0.597

4.23 5.46 6.38

90.8 97.6 94.9

0.165 1.068 9.391

0.003 0.066 0.021

1.58 6.17 0.23

90.14 94.67 94.83

Assay was completed every 3 d for 15 d continuously; Mean value of five replicates at each diluted concentration.

National Institutes of Health of United State (1R43AI092962), Training Plan for the Main Subject of Academic Leaders of Jiangxi Province (20142BCB22004), and Training Plan for the Young Scientist (Jinggang Star) of Jiangxi Province (20142BCB23004).

Appendix A. Supplementary material Supplementary material associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta. 2015.04.058.

References Fig. 4. Correlation between results from CLIA (y-axis) and QB-ICA platform (x-axis) analyses of HBsAg in 47 positive sera samples.

no false positive results. The 47 HBsAg-positive serum samples were quantified using both QB-ICA and a commercially available human HBsAg CLIA kit. The results were performed using a linear regression analysis between two methods shown in Fig. 4. The regression line was fitted to Y¼ 1.3922Xþ42.962 (R2 ¼ 0.9209, n¼ 47) where X stands for the HBsAg concentrations estimated with the developed ICA test and Y stands for the results of the reference procedure, revealing that the consequences obtained from these two methods matched within the experimental error.

4. Conclusions Within this study, we describe the successful development of a sandwich ICA platform for highly sensitive detection of low-abundance proteins (HBsAg used as a model) in serum by using highly luminescent QBs as the signal amplification probe. As described above, the proposed QB-ICA sensor shows excellent performance for rapid detection of serum HBsAg within 15 min, and the superior sensitivity of 75 pg mL  1 is better than that of the conventional ELISA method, and is even comparable with the CLIA method. In general, the novel highly luminescent QBs were demonstrated to act as an efficient signal amplification probe in the POC test platform for the ultrasensitive detection of serum HBsAg, and even could also be applicable to other clinical biomarkers.

Acknowledgments This work was supported in part by the National Key Technology Research and Development of the Ministry of Science and Technology of the People's Republic of China (2013BAD19B02),

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