Biosensors and Bioelectronics 68 (2015) 757–762
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy using highly loaded palladium nanoparticles/carbon decorated magnetic microspheres as signal labels Lei Ji, Zhankui Guo, Tao Yan, Hongmin Ma, Bin Du n, Yueyun Li nn, Qin Wei Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 12 November 2014 Received in revised form 31 January 2015 Accepted 6 February 2015 Available online 7 February 2015
An ultrasensitive sandwich-type electrochemical immunosensor for quantitative detection of alpha fetoprotein (AFP) was proposed based on a novel signal amplification strategy in this work. Carbon decorated Fe3O4 magnetic microspheres (Fe3O4@C) with large specific surface area and good adsorption property were used as labels to anchor palladium nanoparticles (Pd NPs) and the secondary antibodies (Ab2). Pd NPs were loaded on Fe3O4@C to obtain Fe3O4@C@Pd with core–shell structure by electrostatic attraction, which were further used to immobilize Ab2 due to the bonding of Pd-NH2. A signal amplification strategy was the noble metal nanoparticles, such as Pd NPs, exhibiting high electrocatalytic activities toward hydrogen peroxide (H2O2) reduction. This signal amplification was novel not only because of the great capacity, but also the ease of magnetic separation from the sample solution based on their magnetic property. Moreover, carboxyl-functionalized multi-walled carbon nanotubes (MWCNTs-COOH) were used for the immobilization of primary antibodies (Ab1). Therefore, high sensitivity could be realized by the designed immunosensor based on this novel signal amplification strategy. Under optimal conditions, the immunosensor exhibited a wide linear range of 0.5 pg/mL to 10 ng/mL toward AFP with a detection limit of 0.16 pg/mL (S/N¼ 3). Moreover, it revealed good selectivity, acceptable reproducibility and stability, indicating a potential application in clinical monitoring of tumor biomarkers. & 2015 Elsevier B.V. All rights reserved.
Keywords: Core–shell Multi-walled carbon nanotubes Magnetic microspheres Alpha fetoprotein Immunosensor
1. Introduction Tumor biomarkers could be found with formation of tumor in the body. The detection of tumor biomarkers is of great significance in early clinical diagnosis and disease prevention (Sakamoto et al., 2014). The sensitive and reliable detection of tumor marker is currently the subject of intensive studies (Fan et al., 2013; Li et al., 2013). Alpha fetoprotein (AFP) is a fetal serum protein which is primarily produced in the yolk sac and endodermal organ of the embryo (Matsunou et al., 1994). In adults, the content of AFP is physiologically diminished, but can be reproduced under some pathological conditions, including hepatic regeneration, carcinogenesis of germinal tumors, hepatocellular carcinoma, and a subset of extrahepatic adenocarcinoma referred to as AFP-producing adenocarcinoma (Ishikura et al., 1985). AFPproducing adenocarcinoma is a highly malignant subtype of n
Corresponding author. Fax: þ 86 531 82765969. Corresponding author. Tel.: +86 533 2781203; fax: +86 533 2781664. E-mail addresses: [email protected] (B. Du), [email protected] (Y. Li).
nn
http://dx.doi.org/10.1016/j.bios.2015.02.010 0956-5663/& 2015 Elsevier B.V. All rights reserved.
adenocarcinoma, which accounts for 2.7–5.4% of primary gastric adenocarcinoma (Chang et al., 1992; Kono et al., 2008). It has been considered that the production of AFP in adenocarcinoma suggests enteroblastic differentiation or hepatoid differentiation of tumor cells (Furuya et al., 2011; Ishikura et al., 1985; Matsunou et al., 1994). Enzyme-linked immunosorbent assay (Bi and Liu, 2013), high performance liquid chromatography (Huang et al., 2003), and single-strand conformation polymorphism assay (Bosari et al., 1995) have been developed to detect tumor markers, which are time-consuming, expensive and with low sensitivity. Recently, an electrochemical immunosensor based on the principle of highly biospecific recognition interactions between antigens and the corresponding antibodies (Jung et al., 2010) has been developed for detection of tumor markers in clinical diagnosis because of its high sensitivity, low cost, fast response and ease of handling and miniaturization (Wang et al., 2014). For a sandwich-type electrochemical immunosensor, what kind of labels was used to be conjugated to secondary antibodies (Ab2) for signal amplification always attracts extensive attentions.
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In this study, an ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy was proposed for the quantitative of alpha fetoprotein (AFP), where high loaded Fe3O4@C@Pd magnetic microspheres were used as signal labels. Thus, magnetic microspheres have been widely used in sensor fabrication (Teymourian et al., 2013; Yang et al., 2014) and protein separation (Chen et al., 2009; Deng et al., 2009; Qi et al., 2009; Xu et al., 2006). Carbon decorated Fe3O4 magnetic microspheres (Fe3O4@C) with large specific surface area were used as a label matrix to capture palladium nanoparticles (Pd NPs) and the secondary antibodies (Ab2). Meanwhile, carbon acted as electron mediator to accelerate the electron transfer. The noble metal nanoparticles like Pd NPs, exhibited high electrocatalytic activity toward hydrogen peroxide (H2O2) reduction (Kim et al., 2014). The high load Pd NPs would not only improve the catalytic current, but also capture a mass of Ab2 by the bonding of Pd-NH2. The immobilization of primary antibodies (Ab1) is another point for the signal amplification and ultrasensitive detection of a sandwich-type electrochemical immunosensor (Jeong et al., 2013). Multi-walled carbon nanotubes (MWCNTs) have attracted great interests due to their unique properties including fast electron transportation, high thermal conductivity (Shahrokhian et al., 2009). In this work, the obtained carboxyl-functionalized multiwalled carbon nanotubes (MWCNTs-COOH) exhibited several superiorities: (1) MWCNTs were shortened and produced carboxyl groups, which could introduce negative charges on the MWCNTs and improved their dispersion in water; (2) the functional groups on the outer walls of MWCNTs-COOH could capture more Ab1, while retaining intact inner walls for highly conducting network (Liang et al., 2012). Therefore, signal amplification and ultrasensitive detection could be achieved by this designed sandwichtype electrochemical immunosensor for the quantitative of AFP in human serum.
2. Experimental section 2.1. Materials and reagents Human AFP, antibody to human AFP (anti-AFP) and bovine serum albumin (BSA, 96–99%) were obtained from Shanghai LincBio Science Co., Ltd., China. Multi-walled carbon nanotubes were purchased from TIMESNARO Co. Ltd (Chengdu, China). 1-(3-(Dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC, 98.5%), N-hydroxysuccinimide (NHS, 98%) and sodium tetrachloropalladate (II) (Na2PdCl4) were purchased from Shanghai Aladdin Chemistry Co., Ltd., China. Ethylene glycol, hydrogen peroxide, FeCl3 6H2O and NaBH4 were acquired from Sinopharm Chemical Regent Co. Ltd. Phosphate buffered solutions (PBS) were prepared by compounding the solution of KH2PO4 (1/15 mol/L) and Na2HPO4 (1/15 mol/L) to appropriate pH values. PBS was used as electrolyte for all electrochemistry measurements. Ultrapure water was used in all runs. All other chemicals were of analytical grade and used without further purification. 2.2. Apparatus All electrochemical measurements were performed on a CHI760D electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd., China). A conventional three-electrode configuration was used: a glassy carbon electrode (GCE, 4 mm diameter) as working electrode, a saturated calomel electrode (SCE) as the reference electrode and a Pt wire as the counter electrode. Scanning electron microscope (SEM) and Energy Dispersive X-Ray Spectroscopy (EDS) were recorded by JEOL JSM-6700 F microscope
(Japan). Transmission electron microscope (TEM) images were obtained from a JEOL-2100 microscope (Japan). Fourier transform infrared spectroscopy (FTIR) spectrum was obtained from VERTEX 70 spectrometer (Bruker, Germany). 2.3. Synthesis of palladium nanoparticles Firstly, 20 mL of aqueous solution containing Na2PdCl4 (73.6 mg) and trisodium citrate (73.5 mg) was prepared where trisodium citrate acted as a capping agent to restrict the particle growth. Then, 0.6 mL of NaBH4 solution (0.01 M) was added at once into the palladium solution under constant stirring and lasted for another 30 s. Finally the solution would turn brown black color, which indicated the successful formation of Pd nanoparticles (Pd NPs). 2.4. Synthesis of Fe3O4@C@Pd magnetic microspheres The Fe3O4@C magnetic nanoparticles were synthesized through hydrothermal reaction according to the published reference (Qi et al., 2010). Fe3O4@C was dispersed into an aqueous solution of poly(diallyldimethylammonium chloride) (PDDA) (0.20%) that contained 2.0 10 2 M Tris and 2.0 10 2 M NaCl and the resulting dispersion was stirred for 20 min. Residual PDDA was removed by using a magnet and the PDDA electrostatic adsorbed microspheres were rinsed with water for three times. The obtained magnetic microspheres (20 mg) were redispersed in 30 mL of the as-synthesized solution of Pd NPs and the mixture was stirred for another 20 min. The presence of a layer of adsorbed positively charged PDDA on the Fe3O4@C magnetic microspheres ensured the efficient adsorption of negatively charged Pd NPs. After the magnetic separation, the obtained Fe3O4@C@Pd was washed with ultrapure water three times and dried under vacuum at 50 °C for 12 h. 2.5. Synthesis of carboxyl-functionalized Multi-walled carbon nanotubes MWCNTs-COOH were synthesized according to the previous literature (Dong et al., 2012). Briefly, MWCNTs were dispersed in HNO3 (30%) and then refluxed for 24 h at 140 °C to shorten the nanotubes and to produce carboxylic groups focusing on the open ends as well as the sidewalls, which introduced negative charges on the MWCNTs and improved water dispersibility. 2.6. Preparation of the Fe3O4@C@Pd-Ab2 labels The preparation process of Fe3O4@C@Pd-Ab2 is presented in Scheme 1A. 2 mg of Fe3O4@C@Pd magnetic microspheres were dispersed into 20 mL of ultrapure water, and then ultrasonic treatment lasted for 30 min. The supernatant was removed with the help of a magnet. Fe3O4@C@Pd (2 mg) was added to Ab2 dispersion (10 μg/mL, 1 mL) and incubated for 12 h at 4 °C. After the oscillation, it was centrifuged for 15 min at 4 °C with 6500 rpm in refrigerated centrifuge and the supernatant was removed afterwards. Then the obtained immunocomplex was stored in 1 mL of PBS solution (pH ¼7.0) at 4 °C before use. 2.7. Fabrication of the immunosensor The preparation process of the immunosensor is displayed in Scheme 1B. In the current experiment, the glassy carbon electrode (GCE) was polished with alumina powders (0.05 μm). Afterwards, 6 μL of MWCNTs-COOH solutions were dropped onto the surface of GCE. The carboxyl group of MWCNTs-COOH was activated with EDC/NHS (Liu et al., 2013) for 20 min. Subsequently, 6 μL of Ab1
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Scheme 1. (A) Preparation process of Fe3O4@C@ Pd-Ab2 and (B) schematic presentation of the immunosensor fabrication.
was incubated on the former layer for 1 h and washed to remove the unbounded ones, followed by coating with BSA solution (3 μL, 0.1 wt%) to block non-specific binding sites. Then 6 μL of the AFP
500 nm
200 nm
with various concentrations was incubated onto the electrode for 1 h. Finally, Fe3O4@C@Pd-Ab2 was captured by the former layer. The resulting electrodes were cleansed, and stored at 4 °C prior to use.
500 nm
500 nm
200 nm
Fig. 1. SEM images of the Fe3O4 (A), Fe3O4@C (B) and Fe3O4@C@Pd (C); TEM images of the Fe3O4 (D), Fe3O4@C (E) and Fe3O4@C@Pd (F).
200 nm
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2.8. Electrochemical measurements For amperometric measurement of the immunosensor, 0.4 V was selected as detection potential (Ren et al., 2014) because such a low potential would be beneficial to decrease the background current and minimize the responses of common interference species. After the background current was stabilized, 10 μL of H2O2 (5.0 M) was injected into 10 mL PBS (pH ¼6.5) to form a uniform solution under mild stirring, and the current change was recorded. The cyclic voltammetry (CV) was scanned in the solution containing 2.5 mmol/L K3[Fe(CN)6] and 0.1 mol/L KCl. All measurements were performed at room temperature. The current change in response to the immunological reaction versus the varying concentrations of AFP was taken as the analytical signal.
3. Result and discussion 3.1. Characterization of Fe3O4, Fe3O4@C and Fe3O4@C@Pd Fig. 1A and D shows respectively the SEM and TEM images of the Fe3O4. It could be seen that Fe3O4 has a ball-like structure. A transparent voile-like structure covering on the surface of the ball could be seen from Fig. 1B and E, which indicates that Fe3O4@C magnetic microspheres were synthesized successfully. The voilelike structure did not only provide the high surface to anchor particles, but also owned the capacity of highly conducting. As shown in Fig. 1C or F, small nanoparticles, which represented Pd NPs, emerged on the surface of the shell-like structure. This phenomenon revealed that Pd NPs were loaded on the surface of Fe3O4@C as expected. The EDX spectrum (Fig. S1) of the Fe3O4 @C@Pd further demonstrated that Pd NPs was immobilized on a thin gray shell. In a word, the result indicated that the core–shell Fe3O4@C@Pd was obtained. 3.2. Characterization of carboxyl-functionalized multi-walled carbon nanotubes According to the literature (Dong et al., 2012), specific absorption peaks of hydroxyl (3425 cm 1, 930 cm 1), carbonyl (1660 cm–1) and epoxy (1080 cm 1) (maybe water loss between two carboxyl groups which were close) groups appeared on the FTIR spectrum of MWCNTs-COOH (Fig. 2), indicating the carboxyl group existed on the surface of MWCNTs.
Fig. 3. Electrochemical impedance spectra recorded in the solution containing 2.5 mmol/L K3[Fe(CN)6], 2.5 mmol/L K2[Fe(CN)6], and 0.1 mol/L KCl; (a) GCE, (b) GCE/ MWCNTs-COOH, (c) GCE/MWCNTs-COOH/Ab1, (d) GCE/MWCNTs-COOH/Ab1/BSA, (e) GCE/MWCNTs-COOH/Ab1/BSA/AFP, and (f) GCE/MWCNTs-COOH/Ab1/BSA/AFP/ Fe3O4@C@Pd-Ab2.
the interface properties of surface-modified electrodes and the electron-transfer resistance at the electrode surface was an important parameter (Chang and Park, 2010; Katz and Willner, 2003). Therefore, layer-by-layer modification process of the immunosensor was characterized by electrochemical impedance spectroscopy (EIS). It was known that the high frequency region of the impedance plot showed a semicircle related to the redox probe [Fe(CN)6]3 /4 , and the diameter was corresponded to the electron-transferring resistance. The low frequency displayed a Warburg line that corresponded to the diffusion step of the overall process (Guo et al., 2011; Panagopoulou et al., 2010). As shown in the Nyquist curves (Fig. 3), the GCE (curve a) appeared to be linear. After the electrode was modified with MWCNTs-COOH, the EIS exhibited a very low interfacial resistance, indicating the MWCNTs-COOH greatly enhanced the electron transfer of the redox probe (curve b). Coated with anti-AFP (curve c), the resistance of electrode increased clearly due to its bad electrical conductivity. Then the electrode was incubated with BSA and AFP consecutively (curves d and e), the diameter of the semicircle became large, demonstrating the protein could hinder the electronic conductivity and each layer of the substance was modified successfully. Lastly, Fe3O4@C@Pd-Ab2 was dropped on the electrode (curve f), which displayed a larger semicircle than before. Clearly, the assembly of these materials and biomolecules onto the surface of the electrode was successful.
3.3. Characterization of assembly and fabrication 3.4. Optimization of the experimental conditions Electrochemical characteristics were effective tools for probing
Fig. 2. The FTIR spectrum of MWCNTs-COOH.
For the sake of obtaining best analytical performance of AFP, the conditions of the experiment have been optimized. Since the alkaline or acid solutions may break the antigen– antibody linkage and inactivate the biomolecules (Yuan et al., 2004), the pH of the PBS has a great influence on the immunosensor. To make it optimized, various pH of the PBS have been prepared to detect the immunosensor. Fig. S2A shows that pH ¼6.5 PBS was appropriate for the test. The function of MWCNTs-COOH is to enhance the specific area to connect more biomolecules with little resistance increasement. Thus, the effect of MWCNTs-COOH concentration should be investigated. As seen in Fig. S2B, the MWCNTs-COOH concentration is lower than 2.0 mg/mL, leading to the rapid increase of the current response; the MWCNTs-COOH concentration is higher than 2.0 mg/mL, leading to the slow decrease of the current change. MWCNTs-COOH, as the moderate enhancer, can contribute to the electrical conductivity that may be attributed to the increase of specific surface area and electroactive materials. While, over-much
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methods for the detection of AFP in Table S1, which also illustrated that the designed immunosensor had a lower detection limit than what was previously reported. 3.7. Selectivity, stability, and reproducibility of the immunosensor
Fig. 4. Amperometric i–t curve of Fe3O4 (a), Fe3O4@C (b), and Fe3O4@C@Pd (c).
The specificity of the immunosensor played an important role in analyzing biological samples in situ without separation. To evaluate the specificity of the electrochemical immunosensor, we tested the system with other biomarkers, e.g. human IgG (HIgG), BSA, carcino-embryonic antigen (CEA), melanoma adhesion molecule antigen (CD146), prostate specific antigen (PSA). The signal was compared by assaying AFP (1 ng/mL) with interfering agents (100 ng/mL). As indicated from Fig. S3A, significantly higher current response was observed with the target AFP than with other biomarkers. When AFP coexisted with these sample interfering agents, no apparent signal change took place in comparison with that of only AFP, which indicated that the proposed immunosensor revealed sufficiently selectivity for the detection of AFP. In the following experiment, five working electrodes were prepared under same conditions were further measured to test the regeneration in Fig. S3B. The standard deviation achieved from the measurement suggested a satisfactory reproductivity within 1.5%. A further study on the stability showed that the obvious current change could hardly exist after 30 days at 4 °C in Fig. S4, indicating the stability of the immunosensor was acceptable. 3.8. Real sample analysis
Fig. 5. Calibration curve of the immunosensor toward different concentrations of AFP. Error bar¼ RSD (n ¼5).
MWCNTs-COOH may decrease the electrical conductivity. The concentration of MWCNTs-COOH may reach a top value to increase the conductivity, but over the concentration, the resistance of MWCNTs-COOH may exceed the conductivity. Therefore, there exists an optimal MWCNTs-COOH concentration of 2.0 mg/mL to conduct the electrochemical tests.
In order to test the precision and accuracy of this designed immunosensor, it was used to detect the recoveries of different concentrations of AFP in human serum samples by standard addition methods (Table S2) (Saxberg and Kowalski, 1979). In this work, the real human serum was diluted appropriately times before detected by this immunosensor. So the real concentration of human serum can be obtained indirectly after a calculation process. The RSD ranged from 1.35% to 2.20% and the recovery was in the range from 98.2% to 102.0%. Thus, the designed immunosensor could be effectively applied into the quantitative detection of AFP in human serum.
3.5. Comparison of different signal labels In order to demonstrate the significance of Fe3O4@C@Pd in the designed immunosensor for signal amplification, GCE (Fig. 4) was decorated by using three different signal labels (1 ng/mL) including Fe3O4 (curve a), Fe3O4@C (curve b), Fe3O4@C@Pd (curve c). It could be observed that using Fe3O4@C@Pd as signal labels exhibited much greater electrochemical response in comparison with others, which might be ascribed to the following reasons: first, Fe3O4@C with high superficial area could increase the load of Pd; second, Pd NPs owned high electrocatalytic activity toward H2O2 reduction; third, carbon with high electrical conductivity and Pd NPs could facilitate electron transfer.
4. Conclusion This work has developed an ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy using Fe3O4@C@Pd magnetic as signal and MWCNTs-COOH as conducting materials for the quantitative detection of AFP. Due to the great surface area of the core–shell nanomaterial and the high conductivity of MWCNTs-COOH, this fabricated immunosensor displayed a low detection limit with a broad linear range, which showed a promising application in clinical diagnosis.
3.6. Analysis and detection Acknowledgements Under the optimum conditions, immunosensors based on MWCNTs-COOH were used to detect different concentrations of AFP. Fig. 5 presents the analysis and detection of AFP with the prepared immunosensors. It worked well over a broad liner range of 0.5 pg/mL to 10 ng/mL with a low detection limit of 0.16 pg/mL (S/N ¼ 3). The equation of the calibration curve was △I ¼45.195 logc þ0.877, R2 ¼0.981. The linear range and detection limit of the designed immunosensor were compared with previously reported
This study was supported by the National Natural Science Foundation of China (Nos. 21175057, 21375047, 21377046), the Science and Technology Development Plan of Shandong Province (No. 2014GSF120004), the Science and Technology Plan Project of Jinan (No. 201307010) and Qin Wei thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and UJN (No. ts20130937).
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Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.02.010.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy using highly loaded palladium nanoparticles/carbon decorated magnetic microspheres as signal labels Lei Ji, Zhankui Guo, Tao Yan, Hongmin Ma, Bin Du n, Yueyun Li nn, Qin Wei Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 12 November 2014 Received in revised form 31 January 2015 Accepted 6 February 2015 Available online 7 February 2015
An ultrasensitive sandwich-type electrochemical immunosensor for quantitative detection of alpha fetoprotein (AFP) was proposed based on a novel signal amplification strategy in this work. Carbon decorated Fe3O4 magnetic microspheres (Fe3O4@C) with large specific surface area and good adsorption property were used as labels to anchor palladium nanoparticles (Pd NPs) and the secondary antibodies (Ab2). Pd NPs were loaded on Fe3O4@C to obtain Fe3O4@C@Pd with core–shell structure by electrostatic attraction, which were further used to immobilize Ab2 due to the bonding of Pd-NH2. A signal amplification strategy was the noble metal nanoparticles, such as Pd NPs, exhibiting high electrocatalytic activities toward hydrogen peroxide (H2O2) reduction. This signal amplification was novel not only because of the great capacity, but also the ease of magnetic separation from the sample solution based on their magnetic property. Moreover, carboxyl-functionalized multi-walled carbon nanotubes (MWCNTs-COOH) were used for the immobilization of primary antibodies (Ab1). Therefore, high sensitivity could be realized by the designed immunosensor based on this novel signal amplification strategy. Under optimal conditions, the immunosensor exhibited a wide linear range of 0.5 pg/mL to 10 ng/mL toward AFP with a detection limit of 0.16 pg/mL (S/N¼ 3). Moreover, it revealed good selectivity, acceptable reproducibility and stability, indicating a potential application in clinical monitoring of tumor biomarkers. & 2015 Elsevier B.V. All rights reserved.
Keywords: Core–shell Multi-walled carbon nanotubes Magnetic microspheres Alpha fetoprotein Immunosensor
1. Introduction Tumor biomarkers could be found with formation of tumor in the body. The detection of tumor biomarkers is of great significance in early clinical diagnosis and disease prevention (Sakamoto et al., 2014). The sensitive and reliable detection of tumor marker is currently the subject of intensive studies (Fan et al., 2013; Li et al., 2013). Alpha fetoprotein (AFP) is a fetal serum protein which is primarily produced in the yolk sac and endodermal organ of the embryo (Matsunou et al., 1994). In adults, the content of AFP is physiologically diminished, but can be reproduced under some pathological conditions, including hepatic regeneration, carcinogenesis of germinal tumors, hepatocellular carcinoma, and a subset of extrahepatic adenocarcinoma referred to as AFP-producing adenocarcinoma (Ishikura et al., 1985). AFPproducing adenocarcinoma is a highly malignant subtype of n
Corresponding author. Fax: þ 86 531 82765969. Corresponding author. Tel.: +86 533 2781203; fax: +86 533 2781664. E-mail addresses: [email protected] (B. Du), [email protected] (Y. Li).
nn
http://dx.doi.org/10.1016/j.bios.2015.02.010 0956-5663/& 2015 Elsevier B.V. All rights reserved.
adenocarcinoma, which accounts for 2.7–5.4% of primary gastric adenocarcinoma (Chang et al., 1992; Kono et al., 2008). It has been considered that the production of AFP in adenocarcinoma suggests enteroblastic differentiation or hepatoid differentiation of tumor cells (Furuya et al., 2011; Ishikura et al., 1985; Matsunou et al., 1994). Enzyme-linked immunosorbent assay (Bi and Liu, 2013), high performance liquid chromatography (Huang et al., 2003), and single-strand conformation polymorphism assay (Bosari et al., 1995) have been developed to detect tumor markers, which are time-consuming, expensive and with low sensitivity. Recently, an electrochemical immunosensor based on the principle of highly biospecific recognition interactions between antigens and the corresponding antibodies (Jung et al., 2010) has been developed for detection of tumor markers in clinical diagnosis because of its high sensitivity, low cost, fast response and ease of handling and miniaturization (Wang et al., 2014). For a sandwich-type electrochemical immunosensor, what kind of labels was used to be conjugated to secondary antibodies (Ab2) for signal amplification always attracts extensive attentions.
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In this study, an ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy was proposed for the quantitative of alpha fetoprotein (AFP), where high loaded Fe3O4@C@Pd magnetic microspheres were used as signal labels. Thus, magnetic microspheres have been widely used in sensor fabrication (Teymourian et al., 2013; Yang et al., 2014) and protein separation (Chen et al., 2009; Deng et al., 2009; Qi et al., 2009; Xu et al., 2006). Carbon decorated Fe3O4 magnetic microspheres (Fe3O4@C) with large specific surface area were used as a label matrix to capture palladium nanoparticles (Pd NPs) and the secondary antibodies (Ab2). Meanwhile, carbon acted as electron mediator to accelerate the electron transfer. The noble metal nanoparticles like Pd NPs, exhibited high electrocatalytic activity toward hydrogen peroxide (H2O2) reduction (Kim et al., 2014). The high load Pd NPs would not only improve the catalytic current, but also capture a mass of Ab2 by the bonding of Pd-NH2. The immobilization of primary antibodies (Ab1) is another point for the signal amplification and ultrasensitive detection of a sandwich-type electrochemical immunosensor (Jeong et al., 2013). Multi-walled carbon nanotubes (MWCNTs) have attracted great interests due to their unique properties including fast electron transportation, high thermal conductivity (Shahrokhian et al., 2009). In this work, the obtained carboxyl-functionalized multiwalled carbon nanotubes (MWCNTs-COOH) exhibited several superiorities: (1) MWCNTs were shortened and produced carboxyl groups, which could introduce negative charges on the MWCNTs and improved their dispersion in water; (2) the functional groups on the outer walls of MWCNTs-COOH could capture more Ab1, while retaining intact inner walls for highly conducting network (Liang et al., 2012). Therefore, signal amplification and ultrasensitive detection could be achieved by this designed sandwichtype electrochemical immunosensor for the quantitative of AFP in human serum.
2. Experimental section 2.1. Materials and reagents Human AFP, antibody to human AFP (anti-AFP) and bovine serum albumin (BSA, 96–99%) were obtained from Shanghai LincBio Science Co., Ltd., China. Multi-walled carbon nanotubes were purchased from TIMESNARO Co. Ltd (Chengdu, China). 1-(3-(Dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC, 98.5%), N-hydroxysuccinimide (NHS, 98%) and sodium tetrachloropalladate (II) (Na2PdCl4) were purchased from Shanghai Aladdin Chemistry Co., Ltd., China. Ethylene glycol, hydrogen peroxide, FeCl3 6H2O and NaBH4 were acquired from Sinopharm Chemical Regent Co. Ltd. Phosphate buffered solutions (PBS) were prepared by compounding the solution of KH2PO4 (1/15 mol/L) and Na2HPO4 (1/15 mol/L) to appropriate pH values. PBS was used as electrolyte for all electrochemistry measurements. Ultrapure water was used in all runs. All other chemicals were of analytical grade and used without further purification. 2.2. Apparatus All electrochemical measurements were performed on a CHI760D electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd., China). A conventional three-electrode configuration was used: a glassy carbon electrode (GCE, 4 mm diameter) as working electrode, a saturated calomel electrode (SCE) as the reference electrode and a Pt wire as the counter electrode. Scanning electron microscope (SEM) and Energy Dispersive X-Ray Spectroscopy (EDS) were recorded by JEOL JSM-6700 F microscope
(Japan). Transmission electron microscope (TEM) images were obtained from a JEOL-2100 microscope (Japan). Fourier transform infrared spectroscopy (FTIR) spectrum was obtained from VERTEX 70 spectrometer (Bruker, Germany). 2.3. Synthesis of palladium nanoparticles Firstly, 20 mL of aqueous solution containing Na2PdCl4 (73.6 mg) and trisodium citrate (73.5 mg) was prepared where trisodium citrate acted as a capping agent to restrict the particle growth. Then, 0.6 mL of NaBH4 solution (0.01 M) was added at once into the palladium solution under constant stirring and lasted for another 30 s. Finally the solution would turn brown black color, which indicated the successful formation of Pd nanoparticles (Pd NPs). 2.4. Synthesis of Fe3O4@C@Pd magnetic microspheres The Fe3O4@C magnetic nanoparticles were synthesized through hydrothermal reaction according to the published reference (Qi et al., 2010). Fe3O4@C was dispersed into an aqueous solution of poly(diallyldimethylammonium chloride) (PDDA) (0.20%) that contained 2.0 10 2 M Tris and 2.0 10 2 M NaCl and the resulting dispersion was stirred for 20 min. Residual PDDA was removed by using a magnet and the PDDA electrostatic adsorbed microspheres were rinsed with water for three times. The obtained magnetic microspheres (20 mg) were redispersed in 30 mL of the as-synthesized solution of Pd NPs and the mixture was stirred for another 20 min. The presence of a layer of adsorbed positively charged PDDA on the Fe3O4@C magnetic microspheres ensured the efficient adsorption of negatively charged Pd NPs. After the magnetic separation, the obtained Fe3O4@C@Pd was washed with ultrapure water three times and dried under vacuum at 50 °C for 12 h. 2.5. Synthesis of carboxyl-functionalized Multi-walled carbon nanotubes MWCNTs-COOH were synthesized according to the previous literature (Dong et al., 2012). Briefly, MWCNTs were dispersed in HNO3 (30%) and then refluxed for 24 h at 140 °C to shorten the nanotubes and to produce carboxylic groups focusing on the open ends as well as the sidewalls, which introduced negative charges on the MWCNTs and improved water dispersibility. 2.6. Preparation of the Fe3O4@C@Pd-Ab2 labels The preparation process of Fe3O4@C@Pd-Ab2 is presented in Scheme 1A. 2 mg of Fe3O4@C@Pd magnetic microspheres were dispersed into 20 mL of ultrapure water, and then ultrasonic treatment lasted for 30 min. The supernatant was removed with the help of a magnet. Fe3O4@C@Pd (2 mg) was added to Ab2 dispersion (10 μg/mL, 1 mL) and incubated for 12 h at 4 °C. After the oscillation, it was centrifuged for 15 min at 4 °C with 6500 rpm in refrigerated centrifuge and the supernatant was removed afterwards. Then the obtained immunocomplex was stored in 1 mL of PBS solution (pH ¼7.0) at 4 °C before use. 2.7. Fabrication of the immunosensor The preparation process of the immunosensor is displayed in Scheme 1B. In the current experiment, the glassy carbon electrode (GCE) was polished with alumina powders (0.05 μm). Afterwards, 6 μL of MWCNTs-COOH solutions were dropped onto the surface of GCE. The carboxyl group of MWCNTs-COOH was activated with EDC/NHS (Liu et al., 2013) for 20 min. Subsequently, 6 μL of Ab1
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Scheme 1. (A) Preparation process of Fe3O4@C@ Pd-Ab2 and (B) schematic presentation of the immunosensor fabrication.
was incubated on the former layer for 1 h and washed to remove the unbounded ones, followed by coating with BSA solution (3 μL, 0.1 wt%) to block non-specific binding sites. Then 6 μL of the AFP
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with various concentrations was incubated onto the electrode for 1 h. Finally, Fe3O4@C@Pd-Ab2 was captured by the former layer. The resulting electrodes were cleansed, and stored at 4 °C prior to use.
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200 nm
Fig. 1. SEM images of the Fe3O4 (A), Fe3O4@C (B) and Fe3O4@C@Pd (C); TEM images of the Fe3O4 (D), Fe3O4@C (E) and Fe3O4@C@Pd (F).
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2.8. Electrochemical measurements For amperometric measurement of the immunosensor, 0.4 V was selected as detection potential (Ren et al., 2014) because such a low potential would be beneficial to decrease the background current and minimize the responses of common interference species. After the background current was stabilized, 10 μL of H2O2 (5.0 M) was injected into 10 mL PBS (pH ¼6.5) to form a uniform solution under mild stirring, and the current change was recorded. The cyclic voltammetry (CV) was scanned in the solution containing 2.5 mmol/L K3[Fe(CN)6] and 0.1 mol/L KCl. All measurements were performed at room temperature. The current change in response to the immunological reaction versus the varying concentrations of AFP was taken as the analytical signal.
3. Result and discussion 3.1. Characterization of Fe3O4, Fe3O4@C and Fe3O4@C@Pd Fig. 1A and D shows respectively the SEM and TEM images of the Fe3O4. It could be seen that Fe3O4 has a ball-like structure. A transparent voile-like structure covering on the surface of the ball could be seen from Fig. 1B and E, which indicates that Fe3O4@C magnetic microspheres were synthesized successfully. The voilelike structure did not only provide the high surface to anchor particles, but also owned the capacity of highly conducting. As shown in Fig. 1C or F, small nanoparticles, which represented Pd NPs, emerged on the surface of the shell-like structure. This phenomenon revealed that Pd NPs were loaded on the surface of Fe3O4@C as expected. The EDX spectrum (Fig. S1) of the Fe3O4 @C@Pd further demonstrated that Pd NPs was immobilized on a thin gray shell. In a word, the result indicated that the core–shell Fe3O4@C@Pd was obtained. 3.2. Characterization of carboxyl-functionalized multi-walled carbon nanotubes According to the literature (Dong et al., 2012), specific absorption peaks of hydroxyl (3425 cm 1, 930 cm 1), carbonyl (1660 cm–1) and epoxy (1080 cm 1) (maybe water loss between two carboxyl groups which were close) groups appeared on the FTIR spectrum of MWCNTs-COOH (Fig. 2), indicating the carboxyl group existed on the surface of MWCNTs.
Fig. 3. Electrochemical impedance spectra recorded in the solution containing 2.5 mmol/L K3[Fe(CN)6], 2.5 mmol/L K2[Fe(CN)6], and 0.1 mol/L KCl; (a) GCE, (b) GCE/ MWCNTs-COOH, (c) GCE/MWCNTs-COOH/Ab1, (d) GCE/MWCNTs-COOH/Ab1/BSA, (e) GCE/MWCNTs-COOH/Ab1/BSA/AFP, and (f) GCE/MWCNTs-COOH/Ab1/BSA/AFP/ Fe3O4@C@Pd-Ab2.
the interface properties of surface-modified electrodes and the electron-transfer resistance at the electrode surface was an important parameter (Chang and Park, 2010; Katz and Willner, 2003). Therefore, layer-by-layer modification process of the immunosensor was characterized by electrochemical impedance spectroscopy (EIS). It was known that the high frequency region of the impedance plot showed a semicircle related to the redox probe [Fe(CN)6]3 /4 , and the diameter was corresponded to the electron-transferring resistance. The low frequency displayed a Warburg line that corresponded to the diffusion step of the overall process (Guo et al., 2011; Panagopoulou et al., 2010). As shown in the Nyquist curves (Fig. 3), the GCE (curve a) appeared to be linear. After the electrode was modified with MWCNTs-COOH, the EIS exhibited a very low interfacial resistance, indicating the MWCNTs-COOH greatly enhanced the electron transfer of the redox probe (curve b). Coated with anti-AFP (curve c), the resistance of electrode increased clearly due to its bad electrical conductivity. Then the electrode was incubated with BSA and AFP consecutively (curves d and e), the diameter of the semicircle became large, demonstrating the protein could hinder the electronic conductivity and each layer of the substance was modified successfully. Lastly, Fe3O4@C@Pd-Ab2 was dropped on the electrode (curve f), which displayed a larger semicircle than before. Clearly, the assembly of these materials and biomolecules onto the surface of the electrode was successful.
3.3. Characterization of assembly and fabrication 3.4. Optimization of the experimental conditions Electrochemical characteristics were effective tools for probing
Fig. 2. The FTIR spectrum of MWCNTs-COOH.
For the sake of obtaining best analytical performance of AFP, the conditions of the experiment have been optimized. Since the alkaline or acid solutions may break the antigen– antibody linkage and inactivate the biomolecules (Yuan et al., 2004), the pH of the PBS has a great influence on the immunosensor. To make it optimized, various pH of the PBS have been prepared to detect the immunosensor. Fig. S2A shows that pH ¼6.5 PBS was appropriate for the test. The function of MWCNTs-COOH is to enhance the specific area to connect more biomolecules with little resistance increasement. Thus, the effect of MWCNTs-COOH concentration should be investigated. As seen in Fig. S2B, the MWCNTs-COOH concentration is lower than 2.0 mg/mL, leading to the rapid increase of the current response; the MWCNTs-COOH concentration is higher than 2.0 mg/mL, leading to the slow decrease of the current change. MWCNTs-COOH, as the moderate enhancer, can contribute to the electrical conductivity that may be attributed to the increase of specific surface area and electroactive materials. While, over-much
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methods for the detection of AFP in Table S1, which also illustrated that the designed immunosensor had a lower detection limit than what was previously reported. 3.7. Selectivity, stability, and reproducibility of the immunosensor
Fig. 4. Amperometric i–t curve of Fe3O4 (a), Fe3O4@C (b), and Fe3O4@C@Pd (c).
The specificity of the immunosensor played an important role in analyzing biological samples in situ without separation. To evaluate the specificity of the electrochemical immunosensor, we tested the system with other biomarkers, e.g. human IgG (HIgG), BSA, carcino-embryonic antigen (CEA), melanoma adhesion molecule antigen (CD146), prostate specific antigen (PSA). The signal was compared by assaying AFP (1 ng/mL) with interfering agents (100 ng/mL). As indicated from Fig. S3A, significantly higher current response was observed with the target AFP than with other biomarkers. When AFP coexisted with these sample interfering agents, no apparent signal change took place in comparison with that of only AFP, which indicated that the proposed immunosensor revealed sufficiently selectivity for the detection of AFP. In the following experiment, five working electrodes were prepared under same conditions were further measured to test the regeneration in Fig. S3B. The standard deviation achieved from the measurement suggested a satisfactory reproductivity within 1.5%. A further study on the stability showed that the obvious current change could hardly exist after 30 days at 4 °C in Fig. S4, indicating the stability of the immunosensor was acceptable. 3.8. Real sample analysis
Fig. 5. Calibration curve of the immunosensor toward different concentrations of AFP. Error bar¼ RSD (n ¼5).
MWCNTs-COOH may decrease the electrical conductivity. The concentration of MWCNTs-COOH may reach a top value to increase the conductivity, but over the concentration, the resistance of MWCNTs-COOH may exceed the conductivity. Therefore, there exists an optimal MWCNTs-COOH concentration of 2.0 mg/mL to conduct the electrochemical tests.
In order to test the precision and accuracy of this designed immunosensor, it was used to detect the recoveries of different concentrations of AFP in human serum samples by standard addition methods (Table S2) (Saxberg and Kowalski, 1979). In this work, the real human serum was diluted appropriately times before detected by this immunosensor. So the real concentration of human serum can be obtained indirectly after a calculation process. The RSD ranged from 1.35% to 2.20% and the recovery was in the range from 98.2% to 102.0%. Thus, the designed immunosensor could be effectively applied into the quantitative detection of AFP in human serum.
3.5. Comparison of different signal labels In order to demonstrate the significance of Fe3O4@C@Pd in the designed immunosensor for signal amplification, GCE (Fig. 4) was decorated by using three different signal labels (1 ng/mL) including Fe3O4 (curve a), Fe3O4@C (curve b), Fe3O4@C@Pd (curve c). It could be observed that using Fe3O4@C@Pd as signal labels exhibited much greater electrochemical response in comparison with others, which might be ascribed to the following reasons: first, Fe3O4@C with high superficial area could increase the load of Pd; second, Pd NPs owned high electrocatalytic activity toward H2O2 reduction; third, carbon with high electrical conductivity and Pd NPs could facilitate electron transfer.
4. Conclusion This work has developed an ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy using Fe3O4@C@Pd magnetic as signal and MWCNTs-COOH as conducting materials for the quantitative detection of AFP. Due to the great surface area of the core–shell nanomaterial and the high conductivity of MWCNTs-COOH, this fabricated immunosensor displayed a low detection limit with a broad linear range, which showed a promising application in clinical diagnosis.
3.6. Analysis and detection Acknowledgements Under the optimum conditions, immunosensors based on MWCNTs-COOH were used to detect different concentrations of AFP. Fig. 5 presents the analysis and detection of AFP with the prepared immunosensors. It worked well over a broad liner range of 0.5 pg/mL to 10 ng/mL with a low detection limit of 0.16 pg/mL (S/N ¼ 3). The equation of the calibration curve was △I ¼45.195 logc þ0.877, R2 ¼0.981. The linear range and detection limit of the designed immunosensor were compared with previously reported
This study was supported by the National Natural Science Foundation of China (Nos. 21175057, 21375047, 21377046), the Science and Technology Development Plan of Shandong Province (No. 2014GSF120004), the Science and Technology Plan Project of Jinan (No. 201307010) and Qin Wei thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and UJN (No. ts20130937).
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Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.02.010.
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