Biosensors and Bioelectronics 71 (2015) 82–87
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Sensitive electrochemical immunosensor for α-fetoprotein based on graphene/SnO2/Au nanocomposite Junfeng Liu a, Guanhua Lin b, Can Xiao a, Ying Xue a, Ankang Yang a, Hongxuan Ren c,n, Wensheng Lu b,n, Hong Zhao a,n, Xiangjun Li a, Zhuobin Yuan a a
School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, 19A YuQuan Road, Beijing 100049, China Beijing National Laboratory for Molecular Sciences, Key Laboratory of Colloids and Surfaces, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China c National Center for Nanoscience and Technology of China, Beijing 100080, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 7 January 2015 Received in revised form 19 March 2015 Accepted 5 April 2015 Available online 8 April 2015
A label-free electrochemical immunosensor for sensitive detection of α-fetoprotein (AFP) was developed based on graphene/SnO2/Au nanocomposite. The graphene/SnO2/Au nanocomposite modified glassy carbon electrode was used to immobilize α-fetoprotein antibody (anti-AFP) and to construct the immunosensor. Results demonstrated that the peak currents of [Ru(NH3)6]3 þ decreased due to the interaction between antibody and antigen on the modified electrode. Thus, a label-free immunosensor for the detection of AFP was realized by monitoring the peak current change of [Ru(NH3)6]3 þ . The factors influencing the performance of the immunosensor were investigated in details. Under optimal conditions, the peak currents obtained by DPV decreased linearly with the increasing AFP concentrations in the range from 0.02 to 50 ng mL 1 with a linear coefficient of 0.9959. This electrochemical immunoassay has a low detection limit of 0.01 ng mL 1 (S/N¼ 3) and was successfully applied to the determination of AFP in serum samples. & 2015 Elsevier B.V. All rights reserved.
Keywords: Electrochemical immunosensor α-Fetoprotein Graphene Nanocomposite
1. Introduction
α-Fetoprotein (AFP), a plasma protein produced by the yolk sac and the liver during fetal life (Du et al., 2010), is one of the most extensively used clinical cancer biomarkers. An elevated AFP concentration in adult plasma is widely accepted as an early indicator for diagnosis and prognostics of some cancerous diseases such as hepatocellular cancer, yolk sac cancer, liver metastasis from gastric cancer, testicular cancer, and nasopharyngeal cancer (Alpert et al., 1971; Stefanova et al., 1988; Tomasi, 1977; Yamagata et al., 1998). Hence, it is very important to detect trace amount of AFP for early discovery, early diagnosis and early treatment. Several conventional methods have been reported for the detection of AFP, such as atomic absorption spectrometry (Wang et al., 2001), quartz crystal microbalance (Chou et al., 2002), surface plasmon resonance (Chang et al., 2009), inductively coupled plasma mass spectrometry (Zhang et al., 2004), chemiluminescence assay (Bi et al., 2009; Yang et al., 2009), electrochemiluminescence (Cao et al., 2012; Yuan et al., 2010) and n
Corresponding authors. E-mail addresses: [email protected] (H. Ren), [email protected] (W. Lu), [email protected] (H. Zhao). http://dx.doi.org/10.1016/j.bios.2015.04.012 0956-5663/& 2015 Elsevier B.V. All rights reserved.
electrochemical immunosensors (Gao et al., 2013; Liang et al., 2012; Liu and Ma, 2014; Liu et al., 2011; Peng et al., 2014; Shen et al., 2014; Tang et al., 2011; Wang et al., 2009; Wang and Xue, 2013; Xu et al., 2014). Among these methods, electrochemical immunosensors possessed of significant advantages, such as simple pretreatment procedure, instrument simplicity, small analytical volumes, high sensitivity and selectivity. Compared with the labeled immunosensors, the electrochemical label-free immunosensors have been attracted increasing attention due to its simple, low cost, ease of preparation and no need of secondary antibody. Graphene, a two-dimensional sheet of sp2 conjugated atomic carbon with one-atom thickness (Meyer et al., 2007), has captured worldwide interest in the fields of material science, physics, chemistry and nanotechnology during recent years due to its unique electronic (Neto et al., 2009), thermal (Balandin, 2011), mechanical (Ovidko, 2013) and optical (Falkovsky, 2008a, 2008b) properties. Owing to its high electrical conductivity, high surfaceto-volume ratio, high electron transfer rate and exceptional thermal stability, it has been widely applied for the fabrication of electrochemical sensors as modified material on the surface of glassy carbon and graphite electrodes (Hu et al., 2011; Kang et al., 2010; Shao et al., 2010). One of the most common methods to obtain graphene was chemical reduction of exfoliated graphite
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oxide in the presence of stabilizers such as sodium dodecyl sulfate (SDS) and sodium dodecyl benzene sulfonate (SDBS), which could prevent the aggregation of graphene sheet via coulomb repulsion between the charged hydrophilic parts of the ion surfactants and graphene nanosheets (Yang et al., 2013). Although the reduced graphene oxide (RGO) obtained by this method has high electric current intensity in electrochemical analysis, many of its unique properties and more extensive practical application might be realized after it is integrated into more complex assemblies (Fu et al., 2009). For example, more sensitive current response, good biocompatibility and electrocatalytic activity were obtained when assembled with noble metal or metal oxide nanoparticles (C. Xu et al., 2008; Zhou et al., 2010). Noble metal nanoparticles have been commonly utilized as intermediator to immobilize antibody with efficiently retaining its activity and to enhance current response in the sensitive amperometric immunosensor (Wang et al., 2014; Yang et al., 2014; Zhu et al., 2013). The metal-oxide nanoparticles have excellent stability in acidic and oxidative environments, and also have a remarkable promotional effect on electrochemical properties of materials. Thus, many kinds of graphene/metal nanocomposite and graphene/metal-oxide nanocomposite such as graphene/Au, graphene/ZnO, graphene/Fe3O4 and graphene/SnO2 have been widely used for the fabrication of electrochemical sensors (Kavitha et al., 2012; Wang et al., 2013; Yang et al., 2013; Zhang et al., 2013). However, the synthesis process of these materials was usually time-consuming, complicated and strict conditions need to be controlled. In this work, a simple synthesis method of graphene/SnO2/Au nanocomposite was developed for the detection of AFP. In room temperature, SnO2/Au nanocomposite was synthesized by reduction of HAuCl4 using SnCl2 as a reductant and a single-chain surfactant with multi-amine headgroups, bis(amidoethyl-carbamoylethyl) octadecylamine (C18N3) (Fig. S1) as a stabilizer. The good water solubility and enhanced bonding ability with metal nanoparticles made C18N3 wide application for synthesis of various size of nanoparticles (Jia et al., 2013, 2011). Besides, the excellent characteristics of amine-terminated surfactants such as biocompatible, nonimmunogenic and low mammalian toxicity (Kukowska-Latallo et al., 2005) made C18N3 attractive for biological applications. Based on the unique characteristics of RGO and SnO2/Au nanocomposite synthesized by this method, a novel electrochemical immunosensor was designed through layer-bylayer self-assembly. After immobilization of anti-AFP on the modified electrode, the detection of AFP was realized by monitoring the peak current change of [Ru(NH3)6]3 þ resulting from the antigen–antibody reaction. Compared to other reported methods, there were some great advantages of this proposed method: (1) the synthesis method of SnO2/Au nanocomposite was simple. (2) The good biocompatible of graphene/SnO2/Au (Cao et al., 2012; Kuila et al., 2011; Wu et al., 2012; Yuan et al., 2010; Zhu et al., 2011) provide a suitable condition for the interaction between antibody and antigen. (3) The synergistic effect among graphene, SnO2 and Au (Yang et al., 2015) made the sensitive detection of AFP be easily realized. Particularly, the proposed method could be applied to the detection of AFP in real human serum samples.
2. Experimental 2.1. Materials and reagents AFP and anti-AFP were purchased from Biocell Co. (ZhengZhou, China). Graphite powder, chloroauric acid (HAuCl4) and hydrazine hydrate (40 wt%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). KMnO4, H2O2 (30%), H2SO4, HCl, KCl, NaH2PO4 and Na2HPO4 were bought from Beijing Chemical
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Reagent Company (Beijing, China). Sodium dodecyl sulfate (SDS) was purchased from Jing Wei Chemical Co., Ltd (Shanghai, China). L-Arginine (L-Arg) and SnCl2 were obtained from J&K scientific Co., Ltd (Shanghai, China). L-Cysteine (L-Cys) was from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). Glutathione (GSH) and bovine serum albumin (BSA) were brought from Beijing CellChip Biotechnology Co., Ltd (Beijing, China). Casein was purchased from Acros Organics (New Jersey, USA). Hexaammineruthenium chloride was bought from Johnson Matthey (Ward Hill, MA). C18N3 was synthesized according to the method reported by Wang et al., (2008). All chemicals and solvent used were of analytical grade and were used as received without further purification. Aqueous solutions were prepared with doubly distilled water. Phosphate buffer saline (PBS) was prepared by mixing the stock solutions of 0.01 M NaH2PO4 and 0.01 M Na2HPO4. 2.2. Apparatus Electrochemical measurements were conducted on a CHI 760 C Electrochemical Workstation (Shanghai, China). The UV–vis spectra were measured with a UV-2550 spectrophotometer (Shimadzu, Japan) at room temperature. Fourier Transform-Infrared spectra (FT-IR) were measured on a FT-IR Bruker Vertex 70 spectrophotometer in transmission mode (Karlsruhe, Germany). X-ray diffraction (XRD) data were collected on MSALXD2 using Cu Kα (1.5406 Å ) radiation with a 2θ range from 20° to 80° at a step width of 0.01°. The Raman measurements were performed at room temperature using a Raman spectrometer (DXR, USA) with a 514 nm laser excitation source. For a morphological characterization of the modified electrode, a field-emission scanning electron microscope (SEM) Hitachi S-4800 (Tokyo, Japan) was used. TEM images were obtained using a Philips EM-400 transmission electron microscopy at 80 kV (Eindhoven, Netherlands). TEM samples were prepared by placing a drop of solution on carbon-coated copper grid and dried at room temperature. The pH measurements were made with a PB-10 precision pH meter (Sartorius, Germany), and sonication was carried out with a KuDos Ultrasonic Cleaner (Shanghai, China). All electrochemical experiments were carried out using a three-electrode system consisting of a working electrode (3 mm in diameter, Tianjin Aidahengsheng Technology Co., Ltd., China), a counter electrode (platinum wire electrode), and a reference electrode (saturated calomel electrode). 2.3. Synthesis of SnO2/Au nanocomposite The SnO2/Au nanocomposite was synthetized as follows: 50 μL of HAuCl4 (0.02 M) was added to 1875 μL of C18N3 aqueous solution (0.1 mM). The mixture was shaken gently about 20 min, then 75 μL of SnCl2 (0.02 M) was added to the mixture and shaken slightly for 30 min. The reaction was performed at room temperature. The prepared sample was storage at 5 °C for further use. The process of the synthesis was shown in Scheme 1A. 2.4. Preparation of GO and RGO Graphene oxide (GO) was prepared from purified natural graphite according to a modified Hummers’ method (Hummers and Offeman, 1958). First, about 1 g of graphite powder and 0.5 g of sodium nitrate were added to 70 mL of concentrated H2SO4 (in an ice bath). Then 3 g of KMnO4 was gradually added in the mixture. After continually stirred for 2 h, the mixture was diluted with de-ionized (DI) water. Then 5% H2O2 was added into the solution until the color of the mixture changed to brilliant yellow, indicating the fully oxidization of graphite. The mixture was filtered and washed with dilute HCl solution and DI water respectively to remove metal ions and the acid. Finally, the obtained
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Scheme 1. The schematic diagrams of (A) the synthesis of SnO2/Au nanocomposite and (B) the fabrication of the proposed immunosensor.
graphene oxide slurry was re-dispersed in DI water and then the yellow brown GO were obtained after exfoliated by ultra-sonication. The RGO were synthesized by reducing graphene oxide based on our previous reports (Yang et al., 2013), and the black product was storage at 5 °C for electrode modification. 2.5. Fabrication of proposed immunosensor The glassy carbon electrode was polished with a piece of diamond paper and 0.05 mm alumina slurry sequentially and then cleaned by ultrasonic in ethanol and water for 2 min, respectively. The cleaned GCE was dried with high-purity nitrogen steam for further modification. After the cleaning procedure, 6 μL of the RGO was first dropped on the pretreated GCE surface and dried under an infrared lamp. Then 4 μL of the prepared SnO2/Au nanocomposite was dropped on RGO/GCE surface and dried slowly in the air. Next, 6 μL of anti-AFP (100 μg mL 1) was evenly distributed on the modified electrode surface and kept in 37 °C for 2 h. Following that, 4 μL of 0.25% BSA was added on the surface for 30 min at 37 °C to block the possible remaining active sites and avoid the non-specific adsorption. After every modificatory step, the modified electrode was cleaned with PBS (pH 7.2) to remove the physically absorbed species and dried with high-purity nitrogen steam. The proposed immunosensor was stored at 4 °C when not in use. The stepwise assembly of the proposed immunosensor was shown in Scheme 1B. 2.6. Experimental measurements Electrochemical measurements were conducted by coupling the CHI 760C electrochemical workstation with a conventional three-electrode system comprising a platinum wire as the auxiliary electrode, a saturated calomel electrode as the reference electrode and the modified GCE as the working electrode.
The immunoassay was carried out as follows: the immunosensor was incubated with various concentrations of AFP antigen for 40 min at 37 °C to form the antigen–antibody immunocomplex on the modified electrode. After washing with PBS (pH 7.2) and dried with high-purity nitrogen steam, the electrochemical properties of the modified electrode were characterized by differential pulse voltammetry (DPV) in 0.01 M PBS (pH 7.2) containing 0.1 mM [Ru(NH3)6]3 þ and 0.1 M KCl. In DPVs, the potential was cycled from 0 to -0.6 V with scan rate of 50 mV s 1. All DPV experiments were performed at room temperature.
3. Results and discussion 3.1. Characterization of SnO2/Au nanocomposite Fig. 1 shows the XRD characterization of SnO2/Au nanocomposite prepared by the proposed method. The diffraction peaks at around 38.3°, 44.5°, 64.6°, 77.8° could be indexed to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of a pure face-centered crystalline structure of Au, respectively, confirming that the prepared Au nanoparticles were well crystallized. The peaks at 25.7°, 33.8° and 51.9° were due to the diffraction at the (1 1 0), (1 0 1) and (2 1 1) planes of SnO2, respectively, indicating the successful formation of metallic SnO2. HR-TEM image (inset of Fig. 1) of SnO2/Au nanocomposite showed that Au nanoparticles had uniform particle size, and smaller size of SnO2 nanoparticles distributed among Au nanoparticles. 3.2. Characterization of GO and RGO The GO and RGO was monitored by UV–vis analysis. As shown in Fig. 2A, the UV–vis absorption spectrum of the GO solution showed two bands: a strong band centered at about 230 nm,
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Fig. 1. XRD pattern and HR-TEM image (inset) of SnO2/Au nanocomposite.
which can be assigned to the π-πn transition of aromatic C ¼ C bonds, and a shoulder peak at about 300 nm corresponding to nπn transition of C ¼O bonds. After reduction, the absorption peak corresponding to the aromatic C ¼C bond red-shifts to 265 nm while the shoulder peak at 300 nm decreases in intensity, indicating the reduction of GO and the restoration of a p-conjugation network in the RGO (Zhang et al., 2012; Zhao et al., 2010). Fig. 2B showed the FT-IR spectra of GO and RGO. The FT-IR spectrum of GO confirmed the successful oxidation of graphite. In details, the broad and intense peak at 3225 cm 1 and the absorption bands at 1386 cm 1 originate from the stretching vibration and deformation vibration of O–H. The C ¼O stretching vibration of the COOH groups situated at the edges of GO was observed at 1720 cm 1. The peaks at 1055 cm 1 were due to the stretching vibration of C–O (Ji et al., 2012). The peak at 1623 cm 1 was assigned to the vibrations of the adsorbed water molecules and also the contributions of the skeletal vibrations of unoxidized graphitic domains (Y. Xu et al., 2008). After being reduced to RGO, peaks for oxygen-containing functional groups of GO were significantly reduced in the FT-IR spectrum and two peaks at
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approximately 1564 and 1200 cm 1 were found. The peak at 1564 cm 1 could be attributed to the skeletal vibration of the aromatic C ¼C, and the peak at 1200 cm 1 could be assigned to the C–O stretch (Park et al., 2009). The above results showed that GO was successfully synthesized and effectively reduced to RGO. Raman spectroscopy has been widely used to characterize crystal structure, disorder and defects in graphene-based materials (Sobon et al., 2012). The reduction of GO can be seen from Raman spectra by the changes in relative intensity of two main peaks: D and G. As shown in Fig. 2C, The D-band (at around 1352 cm 1 for GO and 1350 cm 1 for RGO) was correlated with sp3 hybridized carbon atoms as it requires a defect for its activation by double resonance. The G-band (at around 1600 cm 1 for GO and 1596 cm-1 for RGO) was associated with sp2-hybridized carbon atoms and originates from the doubly degenerate zone center E2g mode. Changes in the relative intensities of the D and G bands (D/ G) indicated the changes of the electronic conjugation state of the GO during reduction (Guo et al., 2012). As compared with GO, the D/G intensity ratio of RGO increases, indicating a decrease in the average domain size of the sp2-hybridized carbon atoms, further demonstrating the effective reduction of GO. Besides, the 2D band originates from a two phonon double resonance Raman process associated with the band structure of graphene, which could be used to determine the number of layers of graphene (Yang et al., 2013). A 2D band observed in different regions indicates that the graphene has only a few layers. From the TEM image (Fig. 2D), the RGO synthesized by this method had several layers with a large size of several micrometers. It could be also observed in the image that there were some surface wrinkles in some places, which was the characteristic of RGO, further indicating the successful synthesis of RGO. 3.3. Characterization of the immunosensor To probe the feature of the modified electrode surface, the
Fig. 2. UV–vis spectra (A), FT-IR (B), Raman spectra (C) of GO (a) and RGO (b). (D) TEM image of RGO.
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Fig. 3. (A) Peak currents and DPVs (inset) of bare GCE (a), RGO/GCE (b), SnO2/Au/RGO/GCE (c), anti-AFP/SnO2/Au/RGO/GCE (d), BSA/anti-AFP/SnO2/Au/RGO/GCE (e) and AFP/BSA/anti-AFP/SnO2/Au/RGO/GCE (f). SEM of RGO (B), SnO2/Au/RGO (C) and anti-AFP/SnO2/Au/RGO (D).
electrochemical behaviors of [Ru(NH3)6]3 þ were investigated after each assemble step. DPVs of [Ru(NH3)6]3 þ on the different modified electrodes were shown in Fig. 3A. Compared to the bare GCE, the peak current increased dramatically after RGO being modified onto the GCE surface, suggesting that RGO had significantly improved the electrical conductivity and accelerated electron transfer on the electrode. After modified with SnO2/Au nanocomposite, the peak current was further increased. However, the peak current decreased distinctly after modified with anti-AFP, indicating the successful immobilization of anti-AFP on the modified electrode. Then the electrode was modified with the blocking agent BSA to prevent the nonspecific binding of AFP. The DPV current was further decreased after the antibody–antigen reaction, demonstrating the immunosensor has captured the AFP. SEM was also used to investigate the successful fabrication of the immunosensor. It was clearly observed that the surface of RGO has thin wrinkling paper-like structure (Fig. 3B). Fig. 3C showed that SnO2/Au nanocomposite was firmly scattered on the surface of RGO. Fig. 3D indicated that anti-AFP was successively immobilized on the surface of SnO2/Au/RGO.
3.4. Performance of the immunosensor Under the optimal conditions, DPVs for AFP detection were obtained. Fig. 4A shows the peak currents decreased with the increase concentrations of AFP. The oxidation peak current of the immunosensor was found to be proportional to the AFP concentration in range from 0.02 to 50 ng mL-1. The linear regression equation was ipa (μA)¼ 11.96 log CAFP / ng mL 1 ‒ 28.26 with a correlation coefficient of 0.9959. The detection limit was 0.01 ng mL 1 (S/N ¼3). Additionally, the analytical performance of this method has been compared with those of other nanomaterials-based immunosensor for the detection of AFP (Table S1). As can be seen, the proposed immunosensor exhibited a lower detection limit. 3.5. Selectivity and reproducibility In order to assess the selectivity of the immunosensor to AFP, the interferences that potentially co-exist with AFP in biological systems were investigated. These included amino acids (e.g. L-Cys and L-Arg), polypeptides (e.g. GSH), proteins (e.g. Casein and BSA)
Fig. 4. (A) DPV responses of the proposed immunosensor to different concentrations of AFP (a–j: 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 10, 50 ng mL 1). Inset: plots of peak currents versus logarithm of AFP concentration. (B) Study of the interferences with the proposed immunosensor. All the concentrations of L-Arg, L-Cys, GSH, BSA and Casein were 10 ng mL 1. The concentration of AFP was 1 ng mL 1. Error bars represent standard deviation, n¼ 3.
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Table 1 Determination of AFP added in human blood serum (n ¼3) with the proposed immunosensor. Samples
Added (ng mL 1)
Found (ng mL 1)
RSD (%)
Recovery (%)
1 2 3 4
0.1 0.5 2.0 10
0.106 0.492 2.013 9.617
4.0 1.3 2.0 2.5
106.0 98.4 100.7 96.2
and their mixture with AFP. As seen from Fig. 4B, a strong decrease in the current with the existence of the target AFP compared to the other interferences. Even though the high concentrations of these interferences were coexisted in detecting of AFP, the signal had no apparent difference. These tests revealed the high specificity of the electrochemical immunoassay. The reproducibility of the immunoassay system was evaluated at the AFP concentration of 0.2 and 1 ng mL 1, and the relative standard deviation for three times were 3.52% and 1.50%, respectively. Meanwhile, three freshly prepared modified electrodes had been used for the detection of 0.2 and 1 ng mL 1 AFP. All electrodes exhibited similar electrochemical response and the relative standard deviation were 3.38% and 4.49%, respectively, demonstrating that the proposed AFP immunosensor has good reproducibility. 3.6. Application of the immunosensor The feasibility of the immunosensor for real sample analysis was assessed by standard addition methods in serum samples. The experimental results were shown in Table 1. The recovery was in the range from 96.2% to 106.0%, which indicated that the developed immunosensor might be applied for the detection of AFP in human serum for routine clinical diagnosis.
4. Conclusions In this paper, a novel approach for fabrication of the immunosensor for AFP was proposed based on the immobilization of anti-AFP on graphene/SnO2/Au nanocomposite modified glassy carbon electrode. The oxidation peak current of the immunosensor was found to be proportional to the AFP concentration in the range from 0.02 to 50 ng mL 1 with a linear coefficient of 0.9959. This electrochemical immunoassay has a low detection limit of 0.01 ng mL 1 (S/N ¼3). The proposed strategy had several attractive advantages, such as easy preparation, simple operation, low cost, high sensitivity, selectivity and reproducibility. Moreover, this novel method was expected great potential for antigen detection in clinical and biomedical applications.
Acknowledgments This work was supported by a Grant from the Major National Scientific Research Plan of China (973 Program) (Grant no. 2011CB933202) and the National Natural Science Foundation of China (Grant no. 21205132).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.012.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Sensitive electrochemical immunosensor for α-fetoprotein based on graphene/SnO2/Au nanocomposite Junfeng Liu a, Guanhua Lin b, Can Xiao a, Ying Xue a, Ankang Yang a, Hongxuan Ren c,n, Wensheng Lu b,n, Hong Zhao a,n, Xiangjun Li a, Zhuobin Yuan a a
School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, 19A YuQuan Road, Beijing 100049, China Beijing National Laboratory for Molecular Sciences, Key Laboratory of Colloids and Surfaces, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China c National Center for Nanoscience and Technology of China, Beijing 100080, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 7 January 2015 Received in revised form 19 March 2015 Accepted 5 April 2015 Available online 8 April 2015
A label-free electrochemical immunosensor for sensitive detection of α-fetoprotein (AFP) was developed based on graphene/SnO2/Au nanocomposite. The graphene/SnO2/Au nanocomposite modified glassy carbon electrode was used to immobilize α-fetoprotein antibody (anti-AFP) and to construct the immunosensor. Results demonstrated that the peak currents of [Ru(NH3)6]3 þ decreased due to the interaction between antibody and antigen on the modified electrode. Thus, a label-free immunosensor for the detection of AFP was realized by monitoring the peak current change of [Ru(NH3)6]3 þ . The factors influencing the performance of the immunosensor were investigated in details. Under optimal conditions, the peak currents obtained by DPV decreased linearly with the increasing AFP concentrations in the range from 0.02 to 50 ng mL 1 with a linear coefficient of 0.9959. This electrochemical immunoassay has a low detection limit of 0.01 ng mL 1 (S/N¼ 3) and was successfully applied to the determination of AFP in serum samples. & 2015 Elsevier B.V. All rights reserved.
Keywords: Electrochemical immunosensor α-Fetoprotein Graphene Nanocomposite
1. Introduction
α-Fetoprotein (AFP), a plasma protein produced by the yolk sac and the liver during fetal life (Du et al., 2010), is one of the most extensively used clinical cancer biomarkers. An elevated AFP concentration in adult plasma is widely accepted as an early indicator for diagnosis and prognostics of some cancerous diseases such as hepatocellular cancer, yolk sac cancer, liver metastasis from gastric cancer, testicular cancer, and nasopharyngeal cancer (Alpert et al., 1971; Stefanova et al., 1988; Tomasi, 1977; Yamagata et al., 1998). Hence, it is very important to detect trace amount of AFP for early discovery, early diagnosis and early treatment. Several conventional methods have been reported for the detection of AFP, such as atomic absorption spectrometry (Wang et al., 2001), quartz crystal microbalance (Chou et al., 2002), surface plasmon resonance (Chang et al., 2009), inductively coupled plasma mass spectrometry (Zhang et al., 2004), chemiluminescence assay (Bi et al., 2009; Yang et al., 2009), electrochemiluminescence (Cao et al., 2012; Yuan et al., 2010) and n
Corresponding authors. E-mail addresses: [email protected] (H. Ren), [email protected] (W. Lu), [email protected] (H. Zhao). http://dx.doi.org/10.1016/j.bios.2015.04.012 0956-5663/& 2015 Elsevier B.V. All rights reserved.
electrochemical immunosensors (Gao et al., 2013; Liang et al., 2012; Liu and Ma, 2014; Liu et al., 2011; Peng et al., 2014; Shen et al., 2014; Tang et al., 2011; Wang et al., 2009; Wang and Xue, 2013; Xu et al., 2014). Among these methods, electrochemical immunosensors possessed of significant advantages, such as simple pretreatment procedure, instrument simplicity, small analytical volumes, high sensitivity and selectivity. Compared with the labeled immunosensors, the electrochemical label-free immunosensors have been attracted increasing attention due to its simple, low cost, ease of preparation and no need of secondary antibody. Graphene, a two-dimensional sheet of sp2 conjugated atomic carbon with one-atom thickness (Meyer et al., 2007), has captured worldwide interest in the fields of material science, physics, chemistry and nanotechnology during recent years due to its unique electronic (Neto et al., 2009), thermal (Balandin, 2011), mechanical (Ovidko, 2013) and optical (Falkovsky, 2008a, 2008b) properties. Owing to its high electrical conductivity, high surfaceto-volume ratio, high electron transfer rate and exceptional thermal stability, it has been widely applied for the fabrication of electrochemical sensors as modified material on the surface of glassy carbon and graphite electrodes (Hu et al., 2011; Kang et al., 2010; Shao et al., 2010). One of the most common methods to obtain graphene was chemical reduction of exfoliated graphite
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oxide in the presence of stabilizers such as sodium dodecyl sulfate (SDS) and sodium dodecyl benzene sulfonate (SDBS), which could prevent the aggregation of graphene sheet via coulomb repulsion between the charged hydrophilic parts of the ion surfactants and graphene nanosheets (Yang et al., 2013). Although the reduced graphene oxide (RGO) obtained by this method has high electric current intensity in electrochemical analysis, many of its unique properties and more extensive practical application might be realized after it is integrated into more complex assemblies (Fu et al., 2009). For example, more sensitive current response, good biocompatibility and electrocatalytic activity were obtained when assembled with noble metal or metal oxide nanoparticles (C. Xu et al., 2008; Zhou et al., 2010). Noble metal nanoparticles have been commonly utilized as intermediator to immobilize antibody with efficiently retaining its activity and to enhance current response in the sensitive amperometric immunosensor (Wang et al., 2014; Yang et al., 2014; Zhu et al., 2013). The metal-oxide nanoparticles have excellent stability in acidic and oxidative environments, and also have a remarkable promotional effect on electrochemical properties of materials. Thus, many kinds of graphene/metal nanocomposite and graphene/metal-oxide nanocomposite such as graphene/Au, graphene/ZnO, graphene/Fe3O4 and graphene/SnO2 have been widely used for the fabrication of electrochemical sensors (Kavitha et al., 2012; Wang et al., 2013; Yang et al., 2013; Zhang et al., 2013). However, the synthesis process of these materials was usually time-consuming, complicated and strict conditions need to be controlled. In this work, a simple synthesis method of graphene/SnO2/Au nanocomposite was developed for the detection of AFP. In room temperature, SnO2/Au nanocomposite was synthesized by reduction of HAuCl4 using SnCl2 as a reductant and a single-chain surfactant with multi-amine headgroups, bis(amidoethyl-carbamoylethyl) octadecylamine (C18N3) (Fig. S1) as a stabilizer. The good water solubility and enhanced bonding ability with metal nanoparticles made C18N3 wide application for synthesis of various size of nanoparticles (Jia et al., 2013, 2011). Besides, the excellent characteristics of amine-terminated surfactants such as biocompatible, nonimmunogenic and low mammalian toxicity (Kukowska-Latallo et al., 2005) made C18N3 attractive for biological applications. Based on the unique characteristics of RGO and SnO2/Au nanocomposite synthesized by this method, a novel electrochemical immunosensor was designed through layer-bylayer self-assembly. After immobilization of anti-AFP on the modified electrode, the detection of AFP was realized by monitoring the peak current change of [Ru(NH3)6]3 þ resulting from the antigen–antibody reaction. Compared to other reported methods, there were some great advantages of this proposed method: (1) the synthesis method of SnO2/Au nanocomposite was simple. (2) The good biocompatible of graphene/SnO2/Au (Cao et al., 2012; Kuila et al., 2011; Wu et al., 2012; Yuan et al., 2010; Zhu et al., 2011) provide a suitable condition for the interaction between antibody and antigen. (3) The synergistic effect among graphene, SnO2 and Au (Yang et al., 2015) made the sensitive detection of AFP be easily realized. Particularly, the proposed method could be applied to the detection of AFP in real human serum samples.
2. Experimental 2.1. Materials and reagents AFP and anti-AFP were purchased from Biocell Co. (ZhengZhou, China). Graphite powder, chloroauric acid (HAuCl4) and hydrazine hydrate (40 wt%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). KMnO4, H2O2 (30%), H2SO4, HCl, KCl, NaH2PO4 and Na2HPO4 were bought from Beijing Chemical
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Reagent Company (Beijing, China). Sodium dodecyl sulfate (SDS) was purchased from Jing Wei Chemical Co., Ltd (Shanghai, China). L-Arginine (L-Arg) and SnCl2 were obtained from J&K scientific Co., Ltd (Shanghai, China). L-Cysteine (L-Cys) was from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). Glutathione (GSH) and bovine serum albumin (BSA) were brought from Beijing CellChip Biotechnology Co., Ltd (Beijing, China). Casein was purchased from Acros Organics (New Jersey, USA). Hexaammineruthenium chloride was bought from Johnson Matthey (Ward Hill, MA). C18N3 was synthesized according to the method reported by Wang et al., (2008). All chemicals and solvent used were of analytical grade and were used as received without further purification. Aqueous solutions were prepared with doubly distilled water. Phosphate buffer saline (PBS) was prepared by mixing the stock solutions of 0.01 M NaH2PO4 and 0.01 M Na2HPO4. 2.2. Apparatus Electrochemical measurements were conducted on a CHI 760 C Electrochemical Workstation (Shanghai, China). The UV–vis spectra were measured with a UV-2550 spectrophotometer (Shimadzu, Japan) at room temperature. Fourier Transform-Infrared spectra (FT-IR) were measured on a FT-IR Bruker Vertex 70 spectrophotometer in transmission mode (Karlsruhe, Germany). X-ray diffraction (XRD) data were collected on MSALXD2 using Cu Kα (1.5406 Å ) radiation with a 2θ range from 20° to 80° at a step width of 0.01°. The Raman measurements were performed at room temperature using a Raman spectrometer (DXR, USA) with a 514 nm laser excitation source. For a morphological characterization of the modified electrode, a field-emission scanning electron microscope (SEM) Hitachi S-4800 (Tokyo, Japan) was used. TEM images were obtained using a Philips EM-400 transmission electron microscopy at 80 kV (Eindhoven, Netherlands). TEM samples were prepared by placing a drop of solution on carbon-coated copper grid and dried at room temperature. The pH measurements were made with a PB-10 precision pH meter (Sartorius, Germany), and sonication was carried out with a KuDos Ultrasonic Cleaner (Shanghai, China). All electrochemical experiments were carried out using a three-electrode system consisting of a working electrode (3 mm in diameter, Tianjin Aidahengsheng Technology Co., Ltd., China), a counter electrode (platinum wire electrode), and a reference electrode (saturated calomel electrode). 2.3. Synthesis of SnO2/Au nanocomposite The SnO2/Au nanocomposite was synthetized as follows: 50 μL of HAuCl4 (0.02 M) was added to 1875 μL of C18N3 aqueous solution (0.1 mM). The mixture was shaken gently about 20 min, then 75 μL of SnCl2 (0.02 M) was added to the mixture and shaken slightly for 30 min. The reaction was performed at room temperature. The prepared sample was storage at 5 °C for further use. The process of the synthesis was shown in Scheme 1A. 2.4. Preparation of GO and RGO Graphene oxide (GO) was prepared from purified natural graphite according to a modified Hummers’ method (Hummers and Offeman, 1958). First, about 1 g of graphite powder and 0.5 g of sodium nitrate were added to 70 mL of concentrated H2SO4 (in an ice bath). Then 3 g of KMnO4 was gradually added in the mixture. After continually stirred for 2 h, the mixture was diluted with de-ionized (DI) water. Then 5% H2O2 was added into the solution until the color of the mixture changed to brilliant yellow, indicating the fully oxidization of graphite. The mixture was filtered and washed with dilute HCl solution and DI water respectively to remove metal ions and the acid. Finally, the obtained
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Scheme 1. The schematic diagrams of (A) the synthesis of SnO2/Au nanocomposite and (B) the fabrication of the proposed immunosensor.
graphene oxide slurry was re-dispersed in DI water and then the yellow brown GO were obtained after exfoliated by ultra-sonication. The RGO were synthesized by reducing graphene oxide based on our previous reports (Yang et al., 2013), and the black product was storage at 5 °C for electrode modification. 2.5. Fabrication of proposed immunosensor The glassy carbon electrode was polished with a piece of diamond paper and 0.05 mm alumina slurry sequentially and then cleaned by ultrasonic in ethanol and water for 2 min, respectively. The cleaned GCE was dried with high-purity nitrogen steam for further modification. After the cleaning procedure, 6 μL of the RGO was first dropped on the pretreated GCE surface and dried under an infrared lamp. Then 4 μL of the prepared SnO2/Au nanocomposite was dropped on RGO/GCE surface and dried slowly in the air. Next, 6 μL of anti-AFP (100 μg mL 1) was evenly distributed on the modified electrode surface and kept in 37 °C for 2 h. Following that, 4 μL of 0.25% BSA was added on the surface for 30 min at 37 °C to block the possible remaining active sites and avoid the non-specific adsorption. After every modificatory step, the modified electrode was cleaned with PBS (pH 7.2) to remove the physically absorbed species and dried with high-purity nitrogen steam. The proposed immunosensor was stored at 4 °C when not in use. The stepwise assembly of the proposed immunosensor was shown in Scheme 1B. 2.6. Experimental measurements Electrochemical measurements were conducted by coupling the CHI 760C electrochemical workstation with a conventional three-electrode system comprising a platinum wire as the auxiliary electrode, a saturated calomel electrode as the reference electrode and the modified GCE as the working electrode.
The immunoassay was carried out as follows: the immunosensor was incubated with various concentrations of AFP antigen for 40 min at 37 °C to form the antigen–antibody immunocomplex on the modified electrode. After washing with PBS (pH 7.2) and dried with high-purity nitrogen steam, the electrochemical properties of the modified electrode were characterized by differential pulse voltammetry (DPV) in 0.01 M PBS (pH 7.2) containing 0.1 mM [Ru(NH3)6]3 þ and 0.1 M KCl. In DPVs, the potential was cycled from 0 to -0.6 V with scan rate of 50 mV s 1. All DPV experiments were performed at room temperature.
3. Results and discussion 3.1. Characterization of SnO2/Au nanocomposite Fig. 1 shows the XRD characterization of SnO2/Au nanocomposite prepared by the proposed method. The diffraction peaks at around 38.3°, 44.5°, 64.6°, 77.8° could be indexed to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of a pure face-centered crystalline structure of Au, respectively, confirming that the prepared Au nanoparticles were well crystallized. The peaks at 25.7°, 33.8° and 51.9° were due to the diffraction at the (1 1 0), (1 0 1) and (2 1 1) planes of SnO2, respectively, indicating the successful formation of metallic SnO2. HR-TEM image (inset of Fig. 1) of SnO2/Au nanocomposite showed that Au nanoparticles had uniform particle size, and smaller size of SnO2 nanoparticles distributed among Au nanoparticles. 3.2. Characterization of GO and RGO The GO and RGO was monitored by UV–vis analysis. As shown in Fig. 2A, the UV–vis absorption spectrum of the GO solution showed two bands: a strong band centered at about 230 nm,
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Fig. 1. XRD pattern and HR-TEM image (inset) of SnO2/Au nanocomposite.
which can be assigned to the π-πn transition of aromatic C ¼ C bonds, and a shoulder peak at about 300 nm corresponding to nπn transition of C ¼O bonds. After reduction, the absorption peak corresponding to the aromatic C ¼C bond red-shifts to 265 nm while the shoulder peak at 300 nm decreases in intensity, indicating the reduction of GO and the restoration of a p-conjugation network in the RGO (Zhang et al., 2012; Zhao et al., 2010). Fig. 2B showed the FT-IR spectra of GO and RGO. The FT-IR spectrum of GO confirmed the successful oxidation of graphite. In details, the broad and intense peak at 3225 cm 1 and the absorption bands at 1386 cm 1 originate from the stretching vibration and deformation vibration of O–H. The C ¼O stretching vibration of the COOH groups situated at the edges of GO was observed at 1720 cm 1. The peaks at 1055 cm 1 were due to the stretching vibration of C–O (Ji et al., 2012). The peak at 1623 cm 1 was assigned to the vibrations of the adsorbed water molecules and also the contributions of the skeletal vibrations of unoxidized graphitic domains (Y. Xu et al., 2008). After being reduced to RGO, peaks for oxygen-containing functional groups of GO were significantly reduced in the FT-IR spectrum and two peaks at
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approximately 1564 and 1200 cm 1 were found. The peak at 1564 cm 1 could be attributed to the skeletal vibration of the aromatic C ¼C, and the peak at 1200 cm 1 could be assigned to the C–O stretch (Park et al., 2009). The above results showed that GO was successfully synthesized and effectively reduced to RGO. Raman spectroscopy has been widely used to characterize crystal structure, disorder and defects in graphene-based materials (Sobon et al., 2012). The reduction of GO can be seen from Raman spectra by the changes in relative intensity of two main peaks: D and G. As shown in Fig. 2C, The D-band (at around 1352 cm 1 for GO and 1350 cm 1 for RGO) was correlated with sp3 hybridized carbon atoms as it requires a defect for its activation by double resonance. The G-band (at around 1600 cm 1 for GO and 1596 cm-1 for RGO) was associated with sp2-hybridized carbon atoms and originates from the doubly degenerate zone center E2g mode. Changes in the relative intensities of the D and G bands (D/ G) indicated the changes of the electronic conjugation state of the GO during reduction (Guo et al., 2012). As compared with GO, the D/G intensity ratio of RGO increases, indicating a decrease in the average domain size of the sp2-hybridized carbon atoms, further demonstrating the effective reduction of GO. Besides, the 2D band originates from a two phonon double resonance Raman process associated with the band structure of graphene, which could be used to determine the number of layers of graphene (Yang et al., 2013). A 2D band observed in different regions indicates that the graphene has only a few layers. From the TEM image (Fig. 2D), the RGO synthesized by this method had several layers with a large size of several micrometers. It could be also observed in the image that there were some surface wrinkles in some places, which was the characteristic of RGO, further indicating the successful synthesis of RGO. 3.3. Characterization of the immunosensor To probe the feature of the modified electrode surface, the
Fig. 2. UV–vis spectra (A), FT-IR (B), Raman spectra (C) of GO (a) and RGO (b). (D) TEM image of RGO.
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Fig. 3. (A) Peak currents and DPVs (inset) of bare GCE (a), RGO/GCE (b), SnO2/Au/RGO/GCE (c), anti-AFP/SnO2/Au/RGO/GCE (d), BSA/anti-AFP/SnO2/Au/RGO/GCE (e) and AFP/BSA/anti-AFP/SnO2/Au/RGO/GCE (f). SEM of RGO (B), SnO2/Au/RGO (C) and anti-AFP/SnO2/Au/RGO (D).
electrochemical behaviors of [Ru(NH3)6]3 þ were investigated after each assemble step. DPVs of [Ru(NH3)6]3 þ on the different modified electrodes were shown in Fig. 3A. Compared to the bare GCE, the peak current increased dramatically after RGO being modified onto the GCE surface, suggesting that RGO had significantly improved the electrical conductivity and accelerated electron transfer on the electrode. After modified with SnO2/Au nanocomposite, the peak current was further increased. However, the peak current decreased distinctly after modified with anti-AFP, indicating the successful immobilization of anti-AFP on the modified electrode. Then the electrode was modified with the blocking agent BSA to prevent the nonspecific binding of AFP. The DPV current was further decreased after the antibody–antigen reaction, demonstrating the immunosensor has captured the AFP. SEM was also used to investigate the successful fabrication of the immunosensor. It was clearly observed that the surface of RGO has thin wrinkling paper-like structure (Fig. 3B). Fig. 3C showed that SnO2/Au nanocomposite was firmly scattered on the surface of RGO. Fig. 3D indicated that anti-AFP was successively immobilized on the surface of SnO2/Au/RGO.
3.4. Performance of the immunosensor Under the optimal conditions, DPVs for AFP detection were obtained. Fig. 4A shows the peak currents decreased with the increase concentrations of AFP. The oxidation peak current of the immunosensor was found to be proportional to the AFP concentration in range from 0.02 to 50 ng mL-1. The linear regression equation was ipa (μA)¼ 11.96 log CAFP / ng mL 1 ‒ 28.26 with a correlation coefficient of 0.9959. The detection limit was 0.01 ng mL 1 (S/N ¼3). Additionally, the analytical performance of this method has been compared with those of other nanomaterials-based immunosensor for the detection of AFP (Table S1). As can be seen, the proposed immunosensor exhibited a lower detection limit. 3.5. Selectivity and reproducibility In order to assess the selectivity of the immunosensor to AFP, the interferences that potentially co-exist with AFP in biological systems were investigated. These included amino acids (e.g. L-Cys and L-Arg), polypeptides (e.g. GSH), proteins (e.g. Casein and BSA)
Fig. 4. (A) DPV responses of the proposed immunosensor to different concentrations of AFP (a–j: 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 10, 50 ng mL 1). Inset: plots of peak currents versus logarithm of AFP concentration. (B) Study of the interferences with the proposed immunosensor. All the concentrations of L-Arg, L-Cys, GSH, BSA and Casein were 10 ng mL 1. The concentration of AFP was 1 ng mL 1. Error bars represent standard deviation, n¼ 3.
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Table 1 Determination of AFP added in human blood serum (n ¼3) with the proposed immunosensor. Samples
Added (ng mL 1)
Found (ng mL 1)
RSD (%)
Recovery (%)
1 2 3 4
0.1 0.5 2.0 10
0.106 0.492 2.013 9.617
4.0 1.3 2.0 2.5
106.0 98.4 100.7 96.2
and their mixture with AFP. As seen from Fig. 4B, a strong decrease in the current with the existence of the target AFP compared to the other interferences. Even though the high concentrations of these interferences were coexisted in detecting of AFP, the signal had no apparent difference. These tests revealed the high specificity of the electrochemical immunoassay. The reproducibility of the immunoassay system was evaluated at the AFP concentration of 0.2 and 1 ng mL 1, and the relative standard deviation for three times were 3.52% and 1.50%, respectively. Meanwhile, three freshly prepared modified electrodes had been used for the detection of 0.2 and 1 ng mL 1 AFP. All electrodes exhibited similar electrochemical response and the relative standard deviation were 3.38% and 4.49%, respectively, demonstrating that the proposed AFP immunosensor has good reproducibility. 3.6. Application of the immunosensor The feasibility of the immunosensor for real sample analysis was assessed by standard addition methods in serum samples. The experimental results were shown in Table 1. The recovery was in the range from 96.2% to 106.0%, which indicated that the developed immunosensor might be applied for the detection of AFP in human serum for routine clinical diagnosis.
4. Conclusions In this paper, a novel approach for fabrication of the immunosensor for AFP was proposed based on the immobilization of anti-AFP on graphene/SnO2/Au nanocomposite modified glassy carbon electrode. The oxidation peak current of the immunosensor was found to be proportional to the AFP concentration in the range from 0.02 to 50 ng mL 1 with a linear coefficient of 0.9959. This electrochemical immunoassay has a low detection limit of 0.01 ng mL 1 (S/N ¼3). The proposed strategy had several attractive advantages, such as easy preparation, simple operation, low cost, high sensitivity, selectivity and reproducibility. Moreover, this novel method was expected great potential for antigen detection in clinical and biomedical applications.
Acknowledgments This work was supported by a Grant from the Major National Scientific Research Plan of China (973 Program) (Grant no. 2011CB933202) and the National Natural Science Foundation of China (Grant no. 21205132).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.012.
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