Analytical Biochemistry 465 (2014) 121–126

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Electrochemical immunosensor for a-fetoprotein detection using ferroferric oxide and horseradish peroxidase as signal amplification labels Huan Wang a, Xiaojian Li b, Kexia Mao b, Yan Li a,b, Bin Du b, Yihe Zhang a,⇑, Qin Wei b,⇑ a

School of Materials Science and Technology, China University of Geosciences, Beijing 100083, People’s Republic of China Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 4 July 2014 Received in revised form 13 August 2014 Accepted 17 August 2014 Available online 26 August 2014 Keywords: Immunosensor Mesoporous silica nanoparticles Graphene Horseradish peroxidase a-Fetoprotein

a b s t r a c t An electrochemical immunosensor for quantitative detection of a-fetoprotein (AFP) in human serum was developed using graphene sheets (GS) and thionine (TH) as electrode materials and mesoporous silica nanoparticles (MSNs) loaded with ferroferric oxide (Fe3O4) nanoparticles and horseradish peroxidase (HRP) as labels for signal amplification. In this study, the compound of GS and TH (GS–TH) was used as a substrate for promoting electron transfer and immobilization of primary antibody of AFP (Ab1). MSNs were used as a carrier for immobilization of secondary antibody of AFP (Ab2), Fe3O4, and HRP. The synergistic effect occurred between Fe3O4 and HRP and greatly improved the sensitivity of the immunosensor. This method could detect AFP over a wide concentration range from 0.01 to 25 ng ml1 with a detection limit of 4 pg ml1. This strategy may find wide potential application in clinical analysis or detection of other tumor markers. Ó 2014 Elsevier Inc. All rights reserved.

Cancer is one of the greatest threats to human health. Early detection and early treatment are the keys to curing cancer, and early determination of cancer biomarkers is of great importance in clinical diagnosis [1]. To meet requirements for monitoring trace biomarkers, many researchers have focused on detection of cancer biomarkers over the years [2,3]. a-Fetoprotein (AFP)1 is the most common and important liver cancer tumor marker. Many research works choose it as a model to discuss the feasibility of the analysis methods [4–8]. Many research works have been focused on immunoassay because of high specificity, and a number of analytical methods

⇑ Corresponding authors. Fax: +86 531 82765969 (Y. Zhang). E-mail addresses: [email protected] (Y. Zhang), [email protected] (Q. Wei). Abbreviations used: AFP, a-fetoprotein; GS, graphene sheets; TH, thionine; MSN, mesoporous silica nanoparticle; Fe3O4, ferroferric oxide; HRP, horseradish peroxidase; Ab1, primary antibody of AFP; Ab2, secondary antibody of AFP; BSA, bovine serum albumin; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; PBS, phosphate-buffered saline; SEM, scanning electron microscopy; TEM, transmission electron microscopy; GCE, glassy carbon electrode; SCE, saturated calomel electrode; CV, cyclic voltammetry; CEA, carcinoembryonic antigen; PSA, prostate-specific antigen; RSD, relative standard deviation; ELISA, enzyme-linked immunosorbent assay. 1

http://dx.doi.org/10.1016/j.ab.2014.08.016 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.

are developed on the basis of immunoreaction [9–12]. Among the numerous immunoassay methods, electrochemical immunosensors have aroused extensive interest during the past few years because of high sensitivity, low detection limit, low cost, fast response, and ease of handling and miniaturization. Up to now, electrochemical immunosensors have been applied to clinical diagnosis, biotechnology, pharmaceutical chemistry, environmental pollutant detection, and food analysis [13–15]. To improve sensitivity of the electrochemical immunosensors, all kinds of nanomaterials were developed for immobilization of antibody or antigen as labels. Among the numerous nanomaterials, noble metal nanoparticles were often used for signal amplification, including Au, Ag, Pt, and their composite [16–19], whereas widespread use of noble metals will increase the cost of testing, so it is necessary to look for cheaper materials. In this work, a sandwich-type electrochemical immunosensor for quantitative detection of AFP was developed by using graphene sheets (GS) and thionine (TH) as a sensing platform and mesoporous silica nanoparticles (MSNs) loaded with ferroferric oxide (Fe3O4) nanoparticles and horseradish peroxidase (HRP) as labels for a signal amplification strategy. GS has received widespread attention in electrochemical analysis for large surface area, rich functional groups, and good conductivity [20–22]. TH has been

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widely used as a kind of electron transfer mediator for analytical applications in the development of immunosensors [23,24]. TH can be directly adsorbed onto GS through p–p stacking [25]; moreover, the compound of GS and TH (GS–TH) has been confirmed as an excellent sensing material in our previous reports [26,27]. Due to large surface area and uniform structure, MSNs are a good biological carrier and can be considered as a promising sensing material for electrochemical immunosensors. Fe3O4 nanoparticles have some intrinsic good catalytic activity toward H2O2, according to our previous reports [28,29]; hence, MSNs loaded with Fe3O4 nanoparticles and HRP as labels was taken into account for synergistic signal amplification. Moreover, HRP not only can enhance electrochemical signal as a biological enzyme but also can reduce nonspecific adsorption between the electrode surface and the label. This strategy may find potential application in clinical analysis or detection of other tumor markers. Materials and methods Apparatus and reagents The primary antibody of AFP (Ab1), secondary antibody of AFP (Ab2), and AFP were purchased from Beijing Kwinbon Biotechnology (Beijing, China). Bovine serum albumin (BSA, 96–99%) was purchased from Sigma (USA) and used as received. Glutaraldehyde was obtained from Sinopharm Chemical Reagent (Shanghai, China). N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were obtained from Sinopharm Chemical Reagent. All other chemicals were of analytical reagent grade and used without further purification. Phosphate-buffered saline (PBS, 0.067 mol L1) was used as an electrolyte for all electrochemistry measurements. Ultrapure water was used throughout the experiments. Electrochemical measurements were performed on a CHI 760D electrochemical workstation (Shanghai CH Instruments, China). Scanning electron microscopy (SEM) images were obtained from a Quanta FEG-250 microscope (USA). Transmission electron microscopy (TEM) images were obtained from a JEM-2100 microscope (Japan). A conventional three-electrode system was used for all electrochemical measurements: a glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire electrode as the counter electrode.

Preparation of amino-functionalized MSNs MSNs were synthesized according to Zhao and coworkers [30]. The obtained MSNs were dissolved in 15 ml of absolute ethanol and then transferred to a 100-ml three-necked flask. It was heated to 70 °C under magnetic stirring, followed by the addition of 50 ll (3-aminopropyl)triethoxysilane (APTES), and then maintained at 70 °C for 3 h. The resulting amino-functionalized MSNs were obtained by centrifuging, washing with absolute ethanol, and then drying under high vacuum at room temperature for 12 h. Preparation of bromine-functionalized Fe3O4 Fe3O4 nanoparticles were prepared according to the method reported previously [31]. Next, 0.1668 g of citric acid and 1.6633 g of 2-bromo-2-methyl-propionic acid were dissolved in a solution of chloroform and N,N-dimethylformamide (DMF) (1:1 by volume) and diluted to 50 ml. Subsequently, 30 ml of the solution described above was taken out and added into Fe3O4. It was maintained at 30 °C and stirred overnight. The brominefunctionalized Fe3O4 nanoparticles were obtained by centrifuging, washing, and drying. Preparation of Fe3O4–MSN–HRP–Ab2 conjugation First, equal weights of MSNs and Fe3O4 nanoparticles were dispersed in ethanol, shocked overnight, and then centrifuged and washed with ethanol and ultrapure water, respectively. They were then redispersed in ultrapure water and incubated with glutaraldehyde solution (2.5% by volume) for 2 h, followed by centrifuging and washing. The synthesized Fe3O4–MSNs were redispersed in PBS (1 ml, 2 mg ml1) and then successively incubated with Ab2 solution (1 ml, 10 lg ml1) and HRP solution (1 ml, 100 lg ml1) for 1 h. The mixture was finally centrifuged and washed with PBS. The Fe3O4–MSN–HRP–Ab2 conjugation was redispersed in 1 ml of PBS and stored at 4 °C until use. Preparation of AFP immunosensor Fig. 1 shows the fabrication procedure of the immunosensor. GCE was polished carefully with Al2O3 powder of 1.0, 0.3, and 0.05 lm, respectively, to a mirror-like surface, and then it was cleaned and dried in air. Onto the electrode surface, 5 ll of GS–TH solution was added and dried. Primary AFP antibody (Ab1)

Fig.1. Schematic representation of the electrochemical immunosensor for detection of AFP.

Immunosensor for a-fetoprotein detection / H. Wang et al. / Anal. Biochem. 465 (2014) 121–126

was immobilized onto the surface of the GS–TH modified electrode through an amidation reaction between the carboxylic acid groups on GS and the available amine groups of Ab1. Then, 3 ll of EDC/ NHS (0.1 mol L1) was added to promote the amidation reaction. After washing, the electrode was incubated with 1% BSA solution for 30 min to block nonspecific binding sites. Following that, the electrode was incubated with different concentrations of AFP solution for 1 h. Finally, the prepared Fe3O4–MSN–HRP–Ab2 solution was dropped onto the electrode surface and incubated for another 1 h. After washing, the electrode was ready for measurement. As a comparison, MSN–HRP–Ab2 and Fe3O4–MSN–Ab2 were used as labels to fabricate analogous immunosensors. Experimental measurements Unless stated otherwise, all of the cyclic voltammetry (CV) experiments were performed in a conventional electrochemical cell, and the potential was swept from 0.6 to 0.6 V (vs. SCE) with a sweeping rate of 100 mV s1 in PBS (pH 7.4) in the presence or absence of 5 mmol L1 H2O2. Results and discussion Characterization of prepared MSNs, Fe3O4 nanoparticles, and Fe3O4– MSNs Fig. 2A and B show typical TEM and SEM images of the synthesized MSNs, respectively. Mesoporous structure can be seen from Fig. 2A, and the synthesized MSNs have a spherical shape with particle size of approximately 100 nm. Fig. 2C shows a TEM image of the synthesized Fe3O4 nanoparticles. The particle size of synthesized Fe3O4 is approximately 8 nm. Fig. 2D shows a TEM image of the synthesized Fe3O4–MSNs. As seen from Fig. 2D, mesoporous structure still can be seen and Fe3O4 nanoparticles are attached on the surface of MSNs, suggesting that the synthesis of Fe3O4– MSNs was a success.

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Comparison of immunosensors with three kinds of labels The choice of labels is very important for sandwich-type electrochemical immunosensors because it is the source of the generated electrochemical signal. MSN–HRP–Ab2, MSN–Fe3O4– Ab2, and MSN–Fe3O4–HRP–Ab2 were used as labels for fabrication of electrochemical immunosensors. Fig. 3 shows a comparison of the three kinds of immunosensors by CV. H2O2 was used as catalytic substrate for the test. Curves a and b represent the presence and absence of H2O2, respectively. For three immunosensors, the best catalytic response toward H2O2 appeared at the potential of 0.3 V; hence, the potential at 0.3 V was selected for the comparison. The amount of the three kinds of labels is the same, and the concentrations are both at 2 mg ml1. For the test, 1 ng ml1 AFP was used. As shown in Fig. 3, for the immunosensor with MSN–HRP–Ab2 (panel A) there was only a small current change of 0.5 lA, whereas the current change for the immunosensor with MSN–Fe3O4–Ab2 (panel B) was 1.6 lA. For the immunosensor with MSN–Fe3O4–HRP–Ab2 (panel C), the current change was 5.0 lA and much higher than the sum of the current changes obtained with the other two immunosensors, suggesting that a synergistic effect occurred between Fe3O4 and HRP. That may be because Fe3O4 could enhance the enzyme activity and generate a synergistic effect [32], leading to a great increase of the catalytic current. Optimization of experimental condition The influence of the concentration ratio of GS to TH in the GS– TH film on the response of the immunosensor toward H2O2 was also investigated. As shown in Fig. 4, the biggest current change appeared at the concentration ratio of 5:2. TH is easy to leak as a kind of small molecule, whereas more GS are helpful for the immobilization of TH, thereby keeping the stability of the immunosensor system; hence, 5:2 was selected as the optimal concentration ratio.

Fig.2. (A) TEM image of the synthesized MSNs. (B) SEM image of the synthesized MSNs. (C,D) TEM images of the Fe3O4 nanoparticles (C) and Fe3O4–MSNs (D).

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Fig.3. Electrochemical responses toward H2O2 using different kinds of labels: (A) MSN–HRP–Ab2; (B) Fe3O4–MSN–Ab2; (C) Fe3O4–MSN–HRP–Ab2.

Table 1 Comparison with previous AFP detection methods.

Fig.4. Optimization of concentration ratio of GS to TH.

Fig.5. Calibration curve of the AFP immunosensor at concentrations of 0.01, 0.1, 0.5, 1, 2, 5, 8, 10, and 25 ng ml1 in PBS (pH 7.4).

Calibration curve of immunosensor Under the optimal conditions, the current changes to different concentrations of AFP were recorded and are shown in Fig. 5. The calibration curve shows a good linear relationship between current change and concentrations of AFP (0.01–25 ng ml1) with a low detection limit of 4 pg ml1 (signal/noise [S/N] = 3). Thus, the method was a suitable means to quantify AFP concentration. Table 1 shows a comparison with previous reports about AFP detection. As seen in the table, this work obtained good results and was superior to some of these reports.

Selectivity, reproducibility, and stability To investigate the selectivity of the immunosensor, it was incubated with 1 ng ml1 AFP and some interfering agents that potentially coexisted with AFP in serum samples. Carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), glucose, and BSA

Method

Linear range (ng ml1)

Detection limit

Reference

DNA hybridization assay Electrochemiluminescence Amperometric immunosensor Electrochemical immunosensor Electrochemical immunosensor Electrochemical immunosensor Electrochemical immunosensor Optical microfiber biosensor Magnetic immunoassay

0.002–20 0.005–14 0.01–40 0.01–25 0.01–12 0.05–30 0.6–80 0.2–1000 1–10

1.0 pg ml1 2.0 pg ml1 2.3 pg ml1 4.0 pg ml1 5.0 pg ml1 5.0 pg ml1 80 pg ml1 0.2 ng ml1 –

[33] [34] [35] This work [36] [7] [37] [38] [39]

Fig.6. Selectivity study of the AFP immunosensor.

(all of which were at a concentration of 10 ng ml1) were used as the interfering agents. As shown in Fig. 6, only the immunosensor incubated with AFP showed an obvious current change; other interfering substances resulted in little changes in current, indicating that the selectivity of the immunosensor was good. Reproducibility needed to be checked to confirm the reliability of the developed immunosensor. The reproducibility of the current response of the immunosensor was investigated by analysis of the same concentration of AFP (1 ng ml1) using five equally prepared electrodes. The relative standard deviation (RSD) was less than 5%. Thus, the precision and reproducibility of the immunoassay were acceptable. The stability of the immunosensor was also examined by checking current responses periodically. An immunosensor was measured every day, and each reading represents the average value of three assays. When the immunosensor was not in use, it was stored in PBS (pH 7.4) at 4 °C. After 15 days, there is only a small change of 4.6% compared with its initial current change. After

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Table 2 Real sample analysis and comparison with ELISA method. Sample number

ELISA (ng/ml)

RSD (%, n = 5)

This method (ng/ml)

RSD (%, n = 5)

Relative deviation (%)

1 2 3

6.93 ± 0.24 10.89 ± 0.42 8.95 ± 0.33

2.9 3.5 3.1

7.02 ± 0.23 10.45 ± 0.27 8.40 ± 0.46

2.5 1.8 3.5

1.3 4.0 6.1

1 month, the current change retained 91.2% of its initial current, suggesting that the stability of the immunosensors was also good. Real sample analysis To evaluate the reliability and application potential of the designed biosensor, the immunosensor was used for the detection of serum sample and the assay results were compared with the reference values obtained by enzyme-linked immunosorbent assay (ELISA). As shown in Table 2, the relative deviations were less than 6.1% and the RSDs of this method were less than 5%, indicating that the proposed method has good accuracy and is a suitable method for real sample detection. Conclusions In this work, a novel electrochemical immunosensor for detection of AFP was developed using GS–TH as substrate material and MSNs loaded with Fe3O4 nanoparticles and HRP as labels. MSNs with large specific surface area and good biocompatibility were very suitable to be used as a carrier for the immobilization of secondary antibody, Fe3O4, and HRP. Both Fe3O4 and HRP have catalytic activity toward H2O2, and a synergistic effect that occurred between Fe3O4 and HRP greatly enhanced the signal response. This strategy provided a more powerful signal than single Fe3O4 or HRP and improved the sensitivity of the proposed immunosensor. Acknowledgment This study was supported by the National Natural Science Foundation of China (21375047, 21377046, and 21175057). References [1] M. Yang, S. Gong, Immunosensor for the detection of cancer biomarker based on percolated graphene thin film, Chem. Commun. 46 (2010) 5796–5798. [2] M. Grunnet, J.B. Sorensen, Carcinoembryonic antigen (CEA) as tumor marker in lung cancer, Lung Cancer 76 (2012) 138–143. [3] P. Aggarwal, S. Kehoe, Serum tumour markers in gynaecological cancers, Maturitas 67 (2010) 46–53. [4] D. Liu, F. Wu, C. Zhou, H. Shen, H. Yuan, Z. Du, L. Ma, L.S. Li, Multiplexed immunoassay biosensor for the detection of serum biomarkers—b-HCG and AFP of down syndrome based on photoluminescent water-soluble CdSe/ZnS quantum dots, Sens. Actuators B 186 (2013) 235–243. [5] S. Yuan, R. Yuan, Y. Chai, L. Mao, X. Yang, Y. Yuan, H. Niu, Sandwich-type electrochemiluminescence immunosensor based on Ru-silica@Au composite nanoparticles labeled anti-AFP, Talanta 82 (2010) 1468–1471. [6] X. Wang, Q. Zhang, Z. Li, X. Ying, J. Lin, Development of high-performance magnetic chemiluminescence enzyme immunoassay for a-fetoprotein (AFP) in human serum, Clin. Chim. Acta 393 (2008) 90–94. [7] L. Zhao, S. Li, J. He, G. Tian, Q. Wei, H. Li, Enzyme-free electrochemical immunosensor configured with Au–Pd nanocrystals and N-doped graphene sheets for sensitive detection of AFP, Biosens. Bioelectron. 49 (2013) 222–225. [8] H. Zhou, N. Gan, T. Li, Y. Cao, S. Zeng, L. Zheng, Z. Guo, Enzyme-free electrochemical immunosensor configured with Au–Pd nanocrystals and Ndoped graphene sheets for sensitive detection of AFP, Anal. Chim. Acta 746 (2012) 107–113. [9] L. Shu, J. Zhou, X. Yuan, L. Petti, J. Chen, Z. Jia, P. Mormile, The sandwich-type electrochemiluminescence immunosensor for a-fetoprotein based on enrichment by Fe3O4–Au magnetic nano probes and signal amplification by CdS–Au composite nanoparticles labeled anti-AFP, Talanta 123 (2014) 161– 168.

[10] J. Luo, X. Cui, W. Liu, B. Li, Highly sensitive homogenous chemiluminescence immunoassay using gold nanoparticles as label, Spectrochim. Acta A 131 (2014) 243–248. [11] W. Hu, H. Chen, Z. Shi, L. Yu, Dual signal amplification of surface plasmon resonance imaging for sensitive immunoassay of tumor marker, Anal. Biochem. 453 (2014) 16–21. [12] Q. Zhang, X. Wang, Z. Li, J. Lin, Evaluation of a-fetoprotein (AFP) in human serum by chemiluminescence enzyme immunoassay with magnetic particles and coated tubes as solid phases, Anal. Chim. Acta 631 (2009) 212–217. [13] F. Ricci, G. Adornetto, G. Palleschi, A review of experimental aspects of electrochemical immunosensors, Electrochim. Acta 84 (2012) 74–83. [14] G. Liu, Y. Lin, Nanomaterial labels in electrochemical immunosensors and immunoassays, Talanta 74 (2007) 308–317. [15] W. Lai, D. Tang, X. Que, J. Zhuang, L. Fu, G. Chen, Enzyme-catalyzed silver deposition on irregular-shaped gold nanoparticles for electrochemical immunoassay of a-fetoprotein, Anal. Chim. Acta 755 (2012) 62–68. [16] B. Kavosi, R. Hallaj, H. Teymourian, A. Salimi, Au nanoparticles/PAMAM dendrimer functionalized wired ethyleneamine–viologen as highly efficient interface for ultrasensitive a-fetoprotein electrochemical immunosensor, Biosens. Bioelectron. 59 (2014) 389–396. [17] T. Xu, X. Jia, X. Chen, Z. Ma, Simultaneous electrochemical detection of multiple tumor markers using metal ions tagged immunocolloidal gold, Biosens. Bioelectron. 56 (2014) 174–179. [18] Y. Li, R. Yuan, Y. Chai, Z. Song, Electrodeposition of gold–platinum alloy nanoparticles on carbon nanotubes as electrochemical sensing interface for sensitive detection of tumor marker, Electrochim. Acta 56 (2011) 6715–6721. [19] Y. Wu, W. Xu, Y. Wang, Y. Yuan, R. Yuan, Silver–graphene oxide nanocomposites as redox probes for electrochemical determination of a-1fetoprotein, Electrochim. Acta 88 (2013) 135–140. [20] N. Ruecha, R. Rangkupan, N. Rodthongkum, O. Chailapakul, Novel paper-based cholesterol biosensor using graphene/polyvinylpyrrolidone/polyaniline nanocomposite, Biosens. Bioelectron. 52 (2014) 13–19. [21] Y. Zhang, Y. Fan, L. Cheng, L. Fan, Z. Wang, J. Zhong, L. Wu, X. Shen, Z. Shi, A novel glucose biosensor based on the immobilization of glucose oxidase on layer-by-layer assembly film of copper phthalocyanine functionalized graphene, Electrochim. Acta 104 (2013) 178–184. [22] W. Gao, Y. Chen, J. Xi, S. Lin, Y. Chen, Y. Lin, Z. Chen, A novel electrochemiluminescence ethanol biosensor based on tris(2,20 -bipyridine) ruthenium(II) and alcohol dehydrogenase immobilized in graphene/bovine serum albumin composite film, Biosens. Bioelectron. 41 (2013) 776–782. [23] W. Wang, L. Song, Q. Gao, H. Qi, C. Zhang, Highly sensitive detection of DNA using an electrochemical DNA sensor with thionine-capped DNA/gold nanoparticle conjugates as signal tags, Electrochem. Commun. 34 (2013) 18– 21. [24] X. Sun, Z. Ma, Electrochemical immunosensor based on nanoporous gold loading thionine for carcinoembryonic antigen, Anal. Chim. Acta 780 (2013) 95–100. [25] C. Chen, W. Zhai, D. Lu, H. Zhang, W. Zheng, A facile method to prepare stable noncovalent functionalized graphene solution by using thionine, Mater. Res. Bull. 46 (2011) 583–587. [26] S. Yu, Q. Wei, B. Du, D. Wu, H. Li, L. Yan, H. Ma, Y. Zhang, Label-free immunosensor for the detection of kanamycin using Ag@Fe3O4 nanoparticles and thionine mixed graphene, Biosens. Bioelectron. 48 (2013) 224–229. [27] Q. Wei, K. Mao, D. Wu, Y. Dai, J. Yang, B. Du, M. Yang, H. Li, A novel label-free electrochemical immunosensor based on graphene and thionine nanocomposite, Sens. Actuators B 149 (2010) 314–318. [28] Q. Wei, T. Li, G. Wang, H. Li, Z. Qian, M. Yang, Fe3O4 nanoparticles-loaded PEG– PLA polymeric vesicles as labels for ultrasensitive immunosensors, Biomaterials 31 (2010) 7332–7339. [29] Q. Wei, Z. Xiang, J. He, G. Wang, H. Li, Z. Qian, M. Yang, Dumbbell-like Au– Fe3O4 nanoparticles as label for the preparation of electrochemical immunosensors, Biosens. Bioelectron. 26 (2010) 627–631. [30] Y. Zhao, B.G. Trewyn, I.I. Slowing, V.S.Y. Lin, Mesoporous silica nanoparticlebased double drug delivery system for glucose-responsive controlled release of insulin and cyclic AMP, J. Am. Chem. Soc. 131 (2009) 8398–8400. [31] S. Sun, H. Zeng, D.B. Robinson, S. Raoux, P.M. Rice, S.X. Wang, G. Li, Monodisperse MFe2O4 (M = Fe Co, Mn) nanoparticles, J. Am. Chem. Soc. 126 (2004) 273–279. [32] X. Dong, X. Mi, B. Wang, J. Xu, H. Chen, Signal amplification for DNA detection based on the HRP-functionalized Fe3O4 nanoparticles, Talanta 84 (2011) 531– 537. [33] H. Li, W. Zhang, H. Zhou, Electrochemical biosensor based on base-stackingdependent DNA hybridization assay for protein detection, Anal. Biochem. 449 (2014) 26–31.

126

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[34] X. Li, Q. Guo, W. Cao, Y. Li, B. Du, Q. Wei, Enhanced electrochemiluminescence from luminol at carboxyl graphene for detection of a-fetoprotein, Anal. Biochem. 457 (2014) 59–64. [35] G.K. Parshetti, F. Lin, R. Doong, Sensitive amperometric immunosensor for afetoprotein detection based on multifunctional dumbbell-like Au–Fe3O4 heterostructures, Sens. Actuators B 186 (2013) 34–43. [36] T. Qi, J. Liao, Y. Li, J. Peng, W. Li, B. Chu, H. Li, Y. Wei, Z. Qian, Label-free afetoprotein immunosensor established by the facile synthesis of a palladium– graphene nanocomposite, Biosens. Bioelectron. 61 (2014) 245–250.

[37] D. Feng, L. Li, X. Han, X. Fang, X. Li, Y. Zhang, Simultaneous electrochemical detection of multiple tumor markers using functionalized graphene nanocomposites as non-enzymatic labels, Sens. Actuators B 201 (2014) 360– 368. [38] K. Li, G. Liu, Y. Wu, P. Hao, W. Zhou, Z. Zhang, Gold nanoparticle amplified optical microfiber evanescent wave absorption biosensor for cancer biomarker detection in serum, Talanta 120 (2014) 419–424. [39] T. Wang, Z. Yang, C. Lei, J. Lei, Y. Zhou, An integrated giant magnetoimpedance biosensor for detection of biomarker, Biosens. Bioelectron. 58 (2014) 338–344.

Electrochemical immunosensor for α-fetoprotein detection using ferroferric oxide and horseradish peroxidase as signal amplification labels.

An electrochemical immunosensor for quantitative detection of α-fetoprotein (AFP) in human serum was developed using graphene sheets (GS) and thionine...
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