Biosensors and Bioelectronics 74 (2015) 59–65
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Novel signal amplification strategy for ultrasensitive sandwich-type electrochemical immunosensor employing Pd–Fe3O4-GS as the matrix and SiO2 as the label Yulan Wang, Hongmin Ma, Xiaodong Wang, Xuehui Pang, Dan Wu, Bin Du, Qin Wei n 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 27 March 2015 Received in revised form 21 May 2015 Accepted 15 June 2015 Available online 20 June 2015
An ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy was developed for the quantitative determination of human immunoglobulin G (IgG). Pd nanocubes functionalized magnetic graphene sheet (Pd–Fe3O4-GS) was employed as the matrix to immobilize the primary antibodies (Ab1). Owing to the synergetic effect between Pd nanocubes and magnetic graphene sheet (Fe3O4-GS), Pd–Fe3O4-GS can provide an obviously increasing electrochemical signal by electrochemical catalysis towards hydrogen peroxide (H2O2). Silicon dioxide (SiO2) was functionalized as the label to conjugate with the secondary antibodies (Ab2). Due to the larger steric hindrance of the obtained conjugate (SiO2@Ab2), the sensitive decrease of the electrochemical signal can be achieved after the specific recognition between antibodies and antigens. In this sense, this proposed immunosensor can achieve a high sensitivity, especially in the presence of low concentrations of IgG. Under optimum conditions, the proposed immunosensor offered an ultrasensitive and specific determination of IgG down to 3.2 fg/mL. This immunoassay method would open up a new promising platform to detect various tumor markers at ultralow levels for early diagnoses of different cancers. & 2015 Elsevier B.V. All rights reserved.
Keywords: Pd–Fe3O4-GS SiO2 Sandwich-type electrochemical immunosensor Human immunoglobulin G Tumor markers
1. Introduction Electrochemical immunosensor based on specific recognition between antigens and antibodies can achieve the determination of biomarkers by building a relationship between the concentration of antigen and the electrochemical signal (Li et al., 2011; Liu et al., 2014). In recent decades, electrochemical immunosensor has owned a unique place in the determination of biological and environmental analyte (Beitollahi et al., 2008; Mazloum-Ardakani et al., 2011a, 2012b) due to its simplicity, sensitivity, portability and ease of operation (Bahadir and Sezginturk, 2015). Hence, it has provided comprehensive application in various fields like the biochemistry, clinical chemistry and environmental monitoring (Chen et al., 2012; Han et al., 2011; Hou et al., 2014; Li et al., 2014; Mazloum-Ardakani et al., 2010, 2011b, 2012a; Zhang et al., 2015, 2014a). As the most used and classic analytical model, the sandwich-type structure has been widely employed for the study of electrochemical immunosensor (Liu et al., 2013; Wang et al., 2014b; Xia et al., 2013; Yang et al., 2013; Zhou et al., 2014). In n
Corresponding author. Fax: þ 86 531 82765969. E-mail address: [email protected] (Q. Wei).
http://dx.doi.org/10.1016/j.bios.2015.06.033 0956-5663/& 2015 Elsevier B.V. All rights reserved.
traditional sandwich-type electrochemical immunosensors, most of the signal amplification strategies are based on employing multifunctional nanomaterials with good electrochemical characteristics as the label (Han et al., 2013; Wang et al., 2013, 2014d). In these strategies, with the increase of the concentration of antigen, the number of the label conjugated with secondary antibodies (Ab2) will increase accordingly, resulting in the enhancement of electrochemical signal. Obviously, the label acts as the key factor to decide the sensitivity of the traditional sandwich-type immunosensor. However, the sensitivity may also be limited because the specific recognition between antibodies and antigens can generate a hydrophobic and insulating layer on the modified electrode and hinder the electron transfer (Fan et al., 2014). Herein, a sandwich-type electrochemical immunosensor based on a novel signal amplification strategy by unitizing multifunctional nanomaterials with good electrochemical characteristics as the matrix is developed. In this strategy, the electrochemical signal will decrease with the increase of the concentration of antigen because the steric hindrance of antigen can depress the electron transfer. In this case, the matrix becomes the key factor of sensitivity of the designed immunosensor. The hydrophobic and insulating layer still hinder the electron transfer but
60
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
will have a positive effect on the sensitivity. In this work, Pd nanocubes functionalized magnetic graphene sheet (Pd–Fe3O4-GS) was employed as the matrix. The synergic effect between Pd nanocubes and magnetic graphene sheet (Fe3O4-GS) can increase the electrocatalytic activity of Pd–Fe3O4-GS towards hydrogen peroxide (H2O2), leading to a high sensitivity of the designed immunosensor. What's more, the large specific surface area and good biocompatibility of Pd–Fe3O4-GS are beneficial to the immobilization of primary antibodies (Ab1) (Chen et al., 2008; Guo et al., 2009). In order to achieve the determination of low concentrations of antigen, the label with bad electron transfer ability and electrocatalytic activity can be employed to increase the change of electrochemical signal. To date, silicon dioxide (SiO2) has attracted wide attention in biological fields owing to its exciting features such as the uniform size, large surface area, low toxicity, high biocompatibility, good stability and low cost (Yang et al., 2010). Apart from that, SiO2 also possesses the large steric hindrance, bad electron transfer ability and bad electrocatalytic activity. Therefore, SiO2 was employed as the label to conjugate with Ab2 in this work. Both the obtained conjugate (SiO2@Ab2) and antigen can facilitate the decrease of electrocatalytic current response and contribute to the excellent sensitivity for this designed immunosensor.
2. Materials and methods 2.1. Apparatus and reagents All electrochemical measurements were performed on a CHI760E electrochemical workstation (Huakeputian Technology Beijing Co., Ltd., China). A conventional three-electrode system was used for all electrochemical measurements: a glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire electrode as the counter electrode. Scanning electron microscope (SEM) images and energy dispersive X-ray spectral data (EDX) were obtained by using Quanta FEG250 field emission environmental SEM (FEI, United States) operated at 4 KV. UV–vis measurements were carried out by using a Lambda 35 UV/ vis Spectrometer (Perkin-Elmer, United States). Transmission electron microscope (TEM) images were obtained from a Hitachi H-600 microscope (Japan). Fourier transform infrared spectroscopy (FTIR) spectrum was obtained from VERTEX 70 spectrometer (Bruker, Germany). Human immunoglobulin G (IgG) and Goat Anti-Human IgG (anti-IgG, Ab1 and Ab2) were purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd., China. Bovine serum albumin (BSA), L-ascorbic acid (AA) and tetraethyl orthosilicate (TEOS) were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China. 1-(3-(Dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 3-aminopropyltriethoxysilane (APTES), sodium tetrachloropalladate (Na2PdCl4), potassium bromide (KBr) and sodium acetate trihydrate (NaAc 3H2O) were purchased from Shanghai Aladdin Chemistry Co., Ltd, China. Ammonium hydroxide solution (NH3 H2O), ethanol, ethylene glycol and ethanediamine were purchased from Tianjin Fuyu Fine Chemical Co., Ltd., China. Ferric chloride hexahydrate (FeCl3 6H2O) was purchased from Tianjin Damao Chemical Reagent Co., Ltd., China. Polyvinylpyrrolidone (PVP, MW ¼55,000) was purchased from Beijing Yili Fine Chemical Co., Ltd., China. Phosphate buffered saline (PBS, 1/15 M Na2HPO4 and KH2PO4) was used as an electrolyte for all electrochemistry measurement. The concentration of IgG stock solution is 1 mg/mL, which was diluted stepwise to the dynamic detection range of the
immunosensor by the PBS at pH ¼7.4. All other reagents were of analytical grade and ultrapure water was used throughout the study. 2.2. Synthesis of the Pd nanocubes The Pd nanocubes were synthesized by adding the Na2PdCl4 solution into a mixture of AA and KBr according to the reference (Jin et al., 2010). In a typical synthesis the Pd nanocubes, an aqueous solution (8.0 mL) containing PVP (105 mg), AA (60 mg) and KBr (600 mg) was placed in a vial and preheated to 80 °C in an oil bath under magnetic stirring for 10 min. Subsequently, an aqueous solution (3.0 mL) containing Na2PdCl4 (57 mg) was added with a pipet. After the vial had been capped, the reaction was maintained at 80 °C for 3 h. The product was collected by centrifugation, washed three times with water to remove excess PVP, and redispersed in water (11 mL). 2.3. Synthesis of the NH2–Fe3O4-GS Graphene oxide (GO) was synthesized by an improved Hummers method (Marcano et al., 2010). In brief, a mixture of concentrated H2SO4 (36 mL) and H3PO4 (4 mL) was added into a mixture of graphite flakes (0.3 g) and KMnO4 (1.8 g), producing a slight exotherm to 35–40 °C. The reaction was then heated to 50 °C and stirred for 12 h. After that, the reaction was cooled to room temperature and poured onto ice (40 mL) with 30% H2O2 (0.3 mL), and the mixture was centrifuged and the supernatant was decanted away. For workup, the remaining solid material was washed in succession with water, 30% HCl, ethanol and ether. The obtained solid was dried in vacuum overnight. In a typical synthesis of Fe3O4-GS (Guo et al., 2014), FeCl3 6H2O (0.5 g) was dissolved in ethylene glycol (10 mL) to form a clear solution, followed by the addition of NaAc (1.5 g), ethanediamine (5 mL) and GO (0.5 g). The mixture was stirred vigorously for 30 min and then sealed in a teflon-lined stainless steel autoclave. The autoclave was heated to 200 °C and maintained for 8 h, and then was cooled to room temperature. The resulting black powder was washed several times and dried at 35 °C under high vacuum overnight. It should be noted that the GO was translated into the graphene sheet (GS) in the process of reaction. The 3-aminopropyl-functionalized Fe3O4-GS (NH2–Fe3O4-GS) was synthesized by a modified method according to references (Li et al., 2013; Yamaura et al., 2004). In brief, Fe3O4-GS (0.1 g) was dispersed in a solution of ethanol (10 mL) containing APTES (0.1 mL), which was placed in a vial and preheated to 70 °C in an oil bath under magnetic stirring for 1.5 h. The NH2–Fe3O4-GS was obtained by magnetic separation process and dried at 35 °C under high vacuum overnight. 2.4. Preparation of the Pd–Fe3O4-GS The prepared Pd nanocubes solution (400 μL) was added into a solution of NH2–Fe3O4-GS (2 mg/mL, 2 mL) and shaked at room temperature for 24 h. Following the magnetic separation process, the final product was obtained after it was washed three times with water to remove excess Pd nanocubes, and redispersed in water (2 mL). 2.5. Synthesis of the NH2–SiO2 Monodisperse SiO2 nanospheres were prepared by a slightly modified Stober process (Zhang et al., 2009). In a typical synthesis of SiO2, TEOS (4.5 mL) was rapidly added into a mixture solution of ethanol (61.75 mL), NH3 H2O (9.0 mL) and water (24.75 mL). The obtained mixture solution was stirred vigorously for 2 h at room
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
61
temperature. The product was collected by centrifugation, washed three times with ethanol and dried at 35 °C under high vacuum overnight. The 3-aminopropyl-functionalized SiO2 (NH2–SiO2) were synthesized by a modified method according to the literature (Cauvel et al., 1997). Briefly, SiO2 (0.1 g) were dispersed in a solution of ethanol (10 mL) containing APTES (0.1 mL), which was placed in a vial and preheated to 70 °C in an oil bath under magnetic stirring for 1.5 h. A free-flowing powdery material was obtained by mild centrifugation and dried at 35 °C under high vacuum overnight. 2.6. Preparation of the SiO2@Ab2 A mixture solution of NH2–SiO2 (2 mg/mL, 2 mL), Ab2 dispersion (10 μg/mL, 1 mL) and EDC/NHS (10 mM/2 mM, 1 mL) was stirred for 12 h at 4 °C. After centrifugation, BSA solution (10 mg/mL, 1 mL) was added into the obtained precipitate, and then stirred for 12 h at 4 °C. Following further centrifugation, the resulting SiO2@Ab2 label was redispersed in 2 mL of water and stored at 4 °C. 2.7. Fabrication of the immunosensor Fig. 1 shows the schematic diagram of the proposed sandwichtype electrochemical immunosensor. A GCE was polished to a mirror-like finish with 1.0, 0.3 and 0.05 μm alumina powder and then thoroughly cleaned before use. First, an aqueous solution of Pd–Fe3O4-GS (2 mg/mL, 6 μL) was added onto the surface of GCE and then dried. After washing, Ab1 dispersion (10 μg/mL, 6 μL) was added onto the Pd–Fe3O4-GS/GCE. After incubated at 4 °C for 1 h and washed, BSA solution (10 mg/mL, 3 μL) was added onto the Ab1/Pd–Fe3O4-GS/GCE to eliminate nonspecific binding sites. After incubated for another 1 h at 4 °C, the BSA/Ab1/Pd–Fe3O4-GS/ GCE was washed and incubated with a varying concentration of IgG (5 10 6–5 ng/mL, 6 μL) for 1 h at room temperature, and then the IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE was washed extensively to remove unbounded IgG molecules. Finally, the prepared SiO2@Ab2 label solution (2 mg/mL, 6 μL) was added onto the modified electrode surface for 1 h at room temperature, and the SiO2@Ab2/IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE was washed thoroughly for measurement. For amperometric i–t curve to record the amperometric response, a detection potential of 0.4 V was selected. 5 mM H2O2 was added into the PBS after the back ground current was stabilized.
3. Results and discussion 3.1. Characterization of Pd–Fe3O4-GS and SiO2 SEM images of the synthesized Pd–Fe3O4-GS are shown in Fig. 2A and B. It can be observed that the GS exhibits lamellar folds structure and is loaded with massive Fe3O4 NPs. The Fe3O4 NPs own an average grain diameter of 270 nm. Lots of Pd nanocubes are successfully linked on the surfaces of both Fe3O4 NPs and GS. The Pd nanocubes own an average size of 18 nm is confirmed by the TEM image (Fig. 2C). EDX spectrum of Pd–Fe3O4-GS (Fig. 2D) was used to further confirm what kinds of elements were contained in the Pd–Fe3O4-GS. Al element can be observed because the sample was fixed on the aluminum foil to test. As the Fig. 2E shows, the prepared aqueous solution of Pd–Fe3O4-GS can be well dispersed in water and easily separated from the dispersion by a magnet. Fig. 2F and G shows the SEM image and the EDX spectrum of the SiO2 respectively, which indicates the SiO2 with an average size of 400 nm was synthesized successfully. The FTIR spectrum of
Fig. 1. The schematic immunosensor.
illustration
of
the
sandwich-type
electrochemical
NH2–SiO2 is shown in Fig. 2H. The absorption bands at 3140 cm 1 and 1630 cm 1 are respectively caused by N–H bond stretching vibration and bending vibration, which corresponds to –NH2 on the surface of SiO2. The absorption bands at 1100 cm 1 and 475 cm 1 are caused by typical vibration of Si–O–Si and the absorbance band at 1400 cm 1 are caused by C-H bending vibration in the methylene group from APTES. These results suggest that NH2-SiO2 was successfully synthesized (Zou et al., 2014). In order to identify that Ab2 could be conjugated onto the surface of NH2– SiO2, UV–vis spectrums of Ab2, SiO2 and SiO2@Ab2 are shown in Fig. 2I. An absorption peak at 278 nm is observed for Ab2 dispersion (curve a). The aqueous solution of SiO2 (curve b) has no obvious absorption peak. Therefore, the absorption peak at 278 nm is mainly ascribed to the presence of Ab2 in aqueous solution of SiO2@Ab2 (curve c). So it can be concluded that Ab2 was excessively conjugated onto the surface of NH2–SiO2 (Tang et al., 2008). 3.2. The mechanism of signal amplification strategy In this work, the electrocatalytic activity of the matrix is key factor of sensitivity of the designed immunosensor. In order to investigate the signal amplification mechanism of the matrix, control experiments of different nanomaterials for the reduction towards 5 mM H2O2 were carried out at 0.4 V in PBS at pH ¼ 6.8. As shown in Fig. 3A, different electrocatalytic current response curves were obtained when bare GCE was modified with 1 mg/mL of Fe3O4-GS (curve a), Pd nanocubes (curve b) and Pd–Fe3O4-GS (curve c). It can be observed that 1 mg/mL of Fe3O4-GS (curve a) has an electrocatalytic current response of about 3 μA and 1 mg/mL of Pd nanocubes has an electrocatalytic current response of about 6 μA (curve b). Interestingly, the Pd–Fe3O4-GS exhibits much greater electrocatalytic current response of about 13 μA (curve c) which is even larger than the summation of that produced by Fe3O4-GS and Pd nanocubes. It demonstrates that there exists the synergetic effect between Pd nanocubes and Fe3O4-GS, which can increase the electrocatalytic activity of Pd–Fe3O4-GS towards the reduction of H2O2. The sensitivity of the proposed immunosensor finally depends on the electrocatalytic current response change produced by IgG and SiO2@Ab2. In order to demonstrate the signal amplification
62
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
Fig. 2. SEM images of Pd–Fe3O4-GS (A and B) and SiO2 (F); TEM image of Pd nanocubes (C); EDX spectrums of Pd–Fe3O4-GS (D) and SiO2 (G); Digital photograph of the aqueous solution of Pd–Fe3O4-GS without and with an additional magnetic field (E); FTIR spectrum of NH2–SiO2 (H); UV–vis spectrums(I) of Ab2 (a), SiO2 (b) and SiO2@Ab2 (c).
mechanism of IgG and SiO2@Ab2, control experiments of different modified electrodes (2 mg/mL of Pd–Fe3O4-GS) for the reduction of 5 mM H2O2 were carried out at 0.4 V in PBS at pH ¼ 6.8. Fig. 3B shows different electrocatalytic current response curves of BSA/ Ab1/Pd–Fe3O4-GS/GCE (curve a), IgG/BSA/Ab1/Pd–Fe3O4-GS/ GCE (curve b) and SiO2@Ab2/IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE (curve c). When 5 ng/mL of IgG was recognized by the modified electrode, the electrocatalytic current response only decreased about 5 μA. But after it was further recognized with the SiO2@Ab2, the electrocatalytic current response could decrease about 14 μA. It can be concluded that in the presence of low concentrations of IgG, SiO2@Ab2 can provide a larger steric hindrance and depress the electron transfer more effectively, producing a more sensitive
influence towards the electrocatalytic signal response. 3.3. Optimization of experimental conditions In order to achieve an optimal electrocatalytic current response, the optimization of experimental conditions is necessary. The value of pH mainly influences the electrocatalytic process of the matrix towards the reduction of H2O2 and the activity of biological proteins. Fig. S1A shows the electrocatalytic current responses of Pd–Fe3O4-GS (2 mg/mL) in different pH values of PBS. As shown in this figure, the optimal electrocatalytic current response was achieved at pH ¼6.8. Moreover, the antibodies and antigens can maintain their biological activity in this approximate
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
63
Fig. 3. (A) Electrocatalytic current responses towards 5 mM H2O2 of 1 mg/mL of Fe3O4-GS (curve a), Pd nanocubes (curve b) and Pd–Fe3O4-GS (curve c); (B) Electrocatalytic current responses towards 5 mM H2O2 of BSA/Ab1/Pd–Fe3O4-GS/GCE (curve a), IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE (curve b) and SiO2@Ab2/IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE (curve c).
neutral condition. Therefore, PBS at pH ¼6.8 was selected for the test throughout this study. The concentration of Pd–Fe3O4-GS is an important factor influencing the value of electrocatalytic current response. Fig. S1B shows the electrocatalytic current responses of different concentrations of Pd–Fe3O4-GS in PBS at pH ¼6.8. As shown in this figure, the optimal electrocatalytic current response was achieved at a concentration of 2 mg/mL. Therefore, 2 mg/mL of Pd–Fe3O4-GS was selected as the matrix throughout this study. The concentration of NH2-SiO2 is an important factor influencing the value of electrocatalytic current response change. Fig. S1C shows the electrocatalytic current responses of different concentrations of NH2–SiO2 as the label for the determination of 5 ng/mL of IgG in PBS at pH ¼ 6.8. As shown in this figure, the optimal electrocatalytic current response change could be achieved at a concentration of 2 mg/mL. Therefore, 2 mg/mL of NH2–SiO2 was selected as the label throughout this study. In addition, the other experimental conditions were controlled strictly. For example, the modified sample value was 6 μL, the incubation time was 1 h, the incubation temperature was the room temperature, and the concentration of antibodies was 10 mg/mL. All these above factors would make sure antibodies and antigens could be effectively and specifically recognized with each other on the GCE with a diameter of 4 mm. Under the optimal conditions, the designed immunosensor could exhibit an optimal electrochemical signal for the quantitative determination of IgG.
3.4. Electrochemical characterization of the immunosensor The fabrication process of this sandwich-type electrochemical immunosensor can be monitored by amperometric i–t curves, as shown in Fig. 4A. The bare GCE (curve a) had no electrocatalytic current response towards the reduction of H2O2. After modified with the Pd–Fe3O4-GS (curve b), the electrocatalytic current response increased to about 30 mA. Following that, Ab1 (curve c), BSA (curve d) and IgG (curve e) were modified layer by layer on the electrode, the electrocatalytic current response gradually decreased because of the non-conductive bioactive substances without any electrocatalytic properties can hinder the electron transfer. The electrocatalytic current response decreased significantly when the SiO2@Ab2 (curve f) was modified on the electrode, which was mainly ascribed to the obviously increasing steric hindrance of the SiO2@Ab2. Thus, the electrocatalytic current responses confirmed the successful formation of the SiO2@Ab2 and the successful fabrication of the proposed immunosensor. A.C. impedance is another suitable method for monitoring the changes of the surface features during the fabrication process of this sandwich-type electrochemical immunosensor (Lu et al., 2014). In this study, A.C. impedance method was also used to characterize fabrication process of the proposed immunosensor. Fig. 4B shows the Nyquist plots of A.C. impedance spectroscopy in the process of modifying electrode, which were recorded from 1 to 105 Hz at 0.2 V in a solution containing 0.1 M KCl and 2.5 mM Fe(CN)63 − /Fe(CN)64 −. Nyquist plots consist of two portions.
Fig. 4. Electrocatalytic current responses towards 5 mM H2O2 in PBS at pH ¼6.8 (A) and Nyquist plots of the A.C. impedance method in a solution containing 0.1 M KCl and 2.5 mM Fe(CN)63 −/Fe(CN)64 − (B): GCE (a), Pd–Fe3O4-GS/GCE (b), Ab1/Pd–Fe3O4-GS/GCE (c), BSA/Ab1/Pd–Fe3O4-GS/GCE (d), IgG/BSA/Ab1/ Pd–Fe3O4-GS/GCE (e) and SiO2@Ab2/ IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE (f).
64
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
The linear portion at low frequencies is associated with electrochemical behavior limited by diffusion. The semicircle portion at high frequencies is associated with the electrochemical process subject to electron transfer, where the diameter corresponds to the resistance. Simply speaking, resistance change can be judged by observing the diameter change of semicircle portion. It could be observed that the bare GCE exhibited a very small resistance (curve a). After modified with the Pd–Fe3O4-GS, a smaller resistance (curve b) was observed due to the good electron transfer ability of Pd–Fe3O4-GS. Gradually increasing resistance indicated the successful modification of the non-conductive bioactive substances when Ab1 (curve c), BSA (curve d) and IgG (curve e) were modified layer by layer on the electrode. When the SiO2@Ab2 (curve f) was modified on the electrode, an obvious increase of resistance could be observed, suggesting that SiO2@Ab2 can depress the electron transfer effectively by the specific recognition between antibodies and antigens.
prepared for the determination of 5 ng/mL of IgG. The relative standard deviation (RSD) of the measurements for five electrodes was 3.2%, suggesting the precision and reproducibility of the proposed electrochemical sandwich-type immunosensor was acceptable. To investigate the specificity of the fabricated immunosensor, study was performed by using AFP, CEA and BSA as the interference. 50 ng/mL of interference solution with 5 ng/mL of IgG was measured by the immunosensor. The electrocatalytic current response variation due to the interference was less than 5% of that without the interference, indicating the selectivity of the proposed immunosensor was acceptable. To test the stability of the immunosensor, the sensor was stored at 4 °C when not in use. After one month, no apparent change for the determination of the same concentration of IgG was found. The good stability could be ascribed to the good biocompatibility of the matrix and the label. The reproducibility, selectivity and stability of this proposed immunosensor were all acceptable, thus it was suitable for the quantitative determination of IgG in real human samples.
3.5. Calibration curves of the immunosensor 3.7. Real sample analysis Under optimum conditions, the proposed sandwich-type electrochemical immunosensor was used to detect different concentrations of IgG. Fig. 5A shows the electrocatalytic current responses of the proposed immunosensor for the determination of IgG covering the concentration range from 5 10 6 (curve a) to 5 ng/mL (curve g). As the concentration of IgG increased, a greater amount of SiO2@Ab2 was specifically bound on the electrode surface to hinder the electron transfer, leading to the gradually decreasing electrocatalytic current response. A linear relationship between electrocatalytic current responses and the logarithmic values of IgG concentration was obtained (Fig. 5B). And the linear regression equation of the calibration curve being I ¼4.60 2.12 logC with correlation coefficient of 0.990 and an extremely low limit of detection (LOD, S/N ¼3) of 3.2 fg/mL. The good sensitivity was ascribed to the novel signal amplification strategy of the designed sandwich-type electrochemical immunosensor. The linear range and LOD of the proposed immunosensor was compared with sandwich-type electrochemical immunosensors previously reported for the determination of other biomarkers in Table 1. It can be found that the proposed immunosensor shows a satisfying linear range and LOD. 3.6. Reproducibility, selectivity and stability To evaluate the reproducibility of the immunosensor, a series of immunosensors fabricated on five different electrodes were
In order to test the precision and accuracy of the proposed electrochemical sandwich-type immunosensor, it was used to detect the recoveries of different concentrations of IgG in human serum samples by recovery tests (Table S1). The RSD was in the range from 1.4% to 4.8% and the recovery was in the range from 99.6% to 100.1%. Thus, the immunosensor can be effectively applied in the quantitative determination of IgG in human serums. In order to further validate the proposed immunosensor, a comparison with the commercialized available enzyme-linked immunosorbent assay (ELISA) method is shown in Table S2. The relative error between the two methods was in the range from 2.3% to 2.2%. These data revealed a good agreement between the two analytical methods, further indicating the feasibility of the proposed immunosensor for clinical application.
4. Conclusions This work has developed an ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy by employing Pd–Fe3O4-GS as the matrix and SiO2 as the label for the quantitative determination of IgG. This designed immunosensor displayed an extremely low LOD, a wide detection range, good selectivity, acceptable reproducibility and stability. The novel fabrication procedure and the ultrasensitivity
Fig. 5. (A) Electrocatalytic current responses of the immunosensor for the determination of different concentrations of IgG: 5 10 6 ng/mL (a), 5 10 5 ng/mL (b), 5 10 4 ng/mL (c), 0.005 ng/mL (d), 0.05 ng/mL (e), 0.5 ng/mL (f) and 5 ng/mL (g); (B) Calibration curve of the immunosensor for the determination of different concentrations of IgG. Error bar ¼RSD (n¼ 5, 4.9%, 4.7%, 4.9%, 3.9%, 4.3%, 2.6%, and 3.2%).
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
65
Table 1 Comparison of different sandwich-type electrochemical immunosensors for the determination of biomarkers. Tumor marker
Linear range
LOD
Reference
Ara h 1 (a major peanut allergen) Carcinoembryonic antigen (CEA) Squamous cell carcinoma antigen (SCCA) Prostate-specific antigen (PSA) Alpha fetoprotein (AFP) IgG CEA SCCA Human immunodeficiency virus p24 IgG IgG
12.6–2000 ng/mL 0.05–200 ng/mL 0.05–18 ng/mL 0.01–10 ng/mL 0.01–25 ng/mL 0.01–200 ng/mL 1 pg/mL–50 ng/mL 0.5 pg/mL–40 ng/mL 0.5 pg/mL–8.5 ng/mL 0.01 pg/mL–100 ng/mL 0.005 pg/mL–5 ng/mL
3.8 ng/mL 18 pg/mL 15.3 pg/mL 5 pg/mL 4 pg/mL 4 pg/mL 0.27 pg/mL 0.18 pg/mL 0.15 pg/mL 4.3 fg/mL 3.2 fg/mL
(Alves et al., 2015) (Yang et al., 2014) (Wu et al., 2013) (Wei et al., 2010) (Wang et al., 2014a) (Cao et al., 2013) (Wang et al., 2015) (Wang et al., 2014c) (Fang et al., 2015) (Zhang et al., 2014b) This work
of this designed immunosensor might provide wide potential applications for the quantitative determination of other biomarkers in clinical diagnosis.
Acknowledgments This study was supported by the National Natural Science Foundation of China (Nos. 21175057, 21375047, 21377046 and 21405059), the Science and Technology Plan Project of Jinan (No. 201307010), the Science and Technology Development Plan of Shandong Province (No. 2014GSF120004), the Special Project for Independent Innovation and Achievements Transformation of Shandong Province (No. 2014ZZCX05101), and QW thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province (No. ts20130937) and UJN.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bios.2015.06.033.
References Alves, R.C., Pimentel, F.B., Nouws, H.P.A., Marques, R.C.B., González-García, M.B., Oliveira, M.B.P.P., Delerue-Matos, C., 2015. Biosens. Bioelectron. 64, 19–24. Bahadir, E.B., Sezginturk, M.K., 2015. Biosens. Bioelectron. 68C, 62–71. Beitollahi, H., Ardakani, M.M., Ganjipour, B., Naeimi, H., 2008. Biosens. Bioelectron. 24, 362–368. Cao, X., Liu, S., Feng, Q., Wang, N., 2013. Biosens. Bioelectron. 49, 256–262. Cauvel, A., Renard, G., Brunel, D., 1997. J. Org. Chem 62, 749–751. Chen, H., Müller, M.B., Gilmore, K.J., Wallace, G.G., Li, D., 2008. Adv. Mater. 20, 3557–3561. Chen, H., Tang, D., Zhang, B., Liu, B., Cui, Y., Chen, G., 2012. Talanta 91, 95–102. Fan, G.-C., Han, L., Zhu, H., Zhang, J.-R., Zhu, J.-J., 2014. Anal. Chem. 86, 12398–12405. Fang, Y.-S., Huang, X.-J., Wang, L.-S., Wang, J.-F., 2015. Biosens. Bioelectron. 64, 324–332. Guo, S., Li, D., Zhang, L., Li, J., Wang, E., 2009. Biomaterials 30, 1881–1889. Guo, X., Du, B., Wei, Q., Yang, J., Hu, L., Yan, L., Xu, W., 2014. J. Hazard. Mater. 278, 211–220. Han, J., Zhuo, Y., Chai, Y.-Q., Mao, L., Yuan, Y.-L., Yuan, R., 2011. Talanta 85, 130–135. Han, J., Zhuo, Y., Chai, Y., Xiang, Y., Yuan, R., Yuan, Y., Liao, N., 2013. Biosens. Bioelectron. 41, 116–122. Hou, L., Tang, Y., Xu, M., Gao, Z., Tang, D., 2014. Anal. Chem. 86, 8352–8358. Jin, M., Liu, H., Zhang, H., Xie, Z., Liu, J., Xia, Y., 2010. Nano Res. 4, 83–91. Li, H., Wei, Q., He, J., Li, T., Zhao, Y., Cai, Y., Du, B., Qian, Z., Yang, M., 2011. Biosens. Bioelectron. 26, 3590–3595.
Li, P.F., Xu, Y., Cheng, X.-H., 2013. Surf. Coat. Technol. 232, 331–339. Li, R., Wu, K., Liu, C., Huang, Y., Wang, Y., Fang, H., Zhang, H., Li, C., 2014. Anal. Chem. 86, 5300–5307. Liu, F., Xiang, G., Yuan, R., Chen, X., Luo, F., Jiang, D., Huang, S., Li, Y., Pu, X., 2014. Biosens. Bioelectron. 60, 210–217. Liu, J., Lu, C.-Y., Zhou, H., Xu, J.-J., Wang, Z.-H., Chen, H.-Y., 2013. Chem. Commun. 49, 6602–6604. Lu, W., Cao, X., Tao, L., Ge, J., Dong, J., Qian, W., 2014. Biosens. Bioelectron. 57, 219–225. Marcano, D.C., Kosynkin, D.V., Berlin, J.M., Sinitskii, A., Sun, Z., Slesarev, A., Alemany, L.B., Lu, W., Tour, J.M., 2010. ACS Nano 4, 4806–4814. Mazloum-Ardakani, M., Beitollahi, H., Amini, M.K., Mirkhalaf, F., Abdollahi-Alibeik, M., 2010. Sens. Actuators B 151, 243–249. Mazloum-Ardakani, M., Beitollahi, H., Amini, M.K., Mirkhalaf, F., Mirjalili, B.-F., 2011a. Biosens. Bioelectron. 26, 2102–2106. Mazloum-Ardakani, M., Ganjipour, B., Beitollahi, H., Amini, M.K., Mirkhalaf, F., Naeimi, H., Nejati-Barzoki, M., 2011b. Electrochim. Acta 56, 9113–9120. Mazloum-Ardakani, M., Sheikh-Mohseni, M.A., Abdollahi-Alibeik, M., Benvidi, A., 2012a. Sens. Actuators B 171–172, 380–386. Mazloum-Ardakani, M., Taleat, Z., Khoshroo, A., Beitollahi, H., Dehghani, H., 2012b. Biosens. Bioelectron. 35, 75–81. Tang, D., Yuan, R., Chai, Y., 2008. Anal. Chem. 80, 1582–1588. Wang, H., Li, X., Mao, K., Li, Y., Du, B., Zhang, Y., Wei, Q., 2014a. Anal. Biochem. 465, 121–126. Wang, Q., Song, Y., Chai, Y., Pan, G., Li, T., Yuan, Y., Yuan, R., 2014b. Biosens. Bioelectron. 60, 118–123. Wang, X., Chen, L., Su, X., Ai, S., 2013. Biosens. Bioelectron. 47, 171–177. Wang, X., Chu, C., Shen, L., Deng, W., Yan, M., Ge, S., Yu, J., Song, X., 2015. Sens. Actuators B 206, 30–36. Wang, Y., Li, X., Cao, W., Li, Y., Li, H., Du, B., Wei, Q., 2014c. Talanta 129, 411–416. Wang, Y., Li, X., Cao, W., Li, Y., Li, H., Du, B., Wei, Q., 2014d. Biosens. Bioelectron. 61, 618–624. Wei, Q., Xiang, Z., He, J., Wang, G., Li, H., Qian, Z., Yang, M., 2010. Biosens. Bioelectron. 26, 627–631. Wu, D., Fan, H., Li, Y., Zhang, Y., Liang, H., Wei, Q., 2013. Biosens. Bioelectron. 46, 91–96. Xia, N., Deng, D., Zhang, L., Yuan, B., Jing, M., Du, J., Liu, L., 2013. Biosens. Bioelectron. 43, 155–159. Yamaura, M., Camilo, R.L., Sampaio, L.C., Macêdo, M.A., Nakamura, M., Toma, H.E., 2004. J. Magn. Magn. Mater. 279, 210–217. Yang, G.-H., Shi, J.-J., Wang, S., Xiong, W.-W., Jiang, L.-P., Burda, C., Zhu, J.-J., 2013. Chem. Commun. 49, 10757–10759. Yang, M., Li, H., Javadi, A., Gong, S., 2010. Biomaterials 31, 3281–3286. Yang, P., Li, X., Wang, L., Wu, Q., Chen, Z., Lin, X., 2014. J. Electroanal. Chem. 732, 38–45. Zhang, B., Liu, B., Chen, G., Tang, D., 2015. Biosens. Bioelectron. 64, 6–12. Zhang, S., Du, B., Li, H., Xin, X., Ma, H., Wu, D., Yan, L., Wei, Q., 2014a. Biosens. Bioelectron. 52, 225–231. Zhang, S., Ma, H., Yan, L., Cao, W., Yan, T., Wei, Q., Du, B., 2014b. Biosens. Bioelectron. 59, 335–341. Zhang, T., Zhang, Q., Ge, J., Goebl, J., Sun, M., Yan, Y., Liu, Y.-s, Chang, C., Guo, J., Yin, Y., 2009. J. Phys. Chem. C 113, 3168–3175. Zhou, J., Tang, J., Chen, G., Tang, D., 2014. Biosens. Bioelectron. 54, 323–328. Zou, B., Hu, Y., Cui, F., Jiang, L., Yu, D., Huang, H., 2014. J. Colloid. Interf. Sci. 417, 210–216.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Novel signal amplification strategy for ultrasensitive sandwich-type electrochemical immunosensor employing Pd–Fe3O4-GS as the matrix and SiO2 as the label Yulan Wang, Hongmin Ma, Xiaodong Wang, Xuehui Pang, Dan Wu, Bin Du, Qin Wei n 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 27 March 2015 Received in revised form 21 May 2015 Accepted 15 June 2015 Available online 20 June 2015
An ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy was developed for the quantitative determination of human immunoglobulin G (IgG). Pd nanocubes functionalized magnetic graphene sheet (Pd–Fe3O4-GS) was employed as the matrix to immobilize the primary antibodies (Ab1). Owing to the synergetic effect between Pd nanocubes and magnetic graphene sheet (Fe3O4-GS), Pd–Fe3O4-GS can provide an obviously increasing electrochemical signal by electrochemical catalysis towards hydrogen peroxide (H2O2). Silicon dioxide (SiO2) was functionalized as the label to conjugate with the secondary antibodies (Ab2). Due to the larger steric hindrance of the obtained conjugate (SiO2@Ab2), the sensitive decrease of the electrochemical signal can be achieved after the specific recognition between antibodies and antigens. In this sense, this proposed immunosensor can achieve a high sensitivity, especially in the presence of low concentrations of IgG. Under optimum conditions, the proposed immunosensor offered an ultrasensitive and specific determination of IgG down to 3.2 fg/mL. This immunoassay method would open up a new promising platform to detect various tumor markers at ultralow levels for early diagnoses of different cancers. & 2015 Elsevier B.V. All rights reserved.
Keywords: Pd–Fe3O4-GS SiO2 Sandwich-type electrochemical immunosensor Human immunoglobulin G Tumor markers
1. Introduction Electrochemical immunosensor based on specific recognition between antigens and antibodies can achieve the determination of biomarkers by building a relationship between the concentration of antigen and the electrochemical signal (Li et al., 2011; Liu et al., 2014). In recent decades, electrochemical immunosensor has owned a unique place in the determination of biological and environmental analyte (Beitollahi et al., 2008; Mazloum-Ardakani et al., 2011a, 2012b) due to its simplicity, sensitivity, portability and ease of operation (Bahadir and Sezginturk, 2015). Hence, it has provided comprehensive application in various fields like the biochemistry, clinical chemistry and environmental monitoring (Chen et al., 2012; Han et al., 2011; Hou et al., 2014; Li et al., 2014; Mazloum-Ardakani et al., 2010, 2011b, 2012a; Zhang et al., 2015, 2014a). As the most used and classic analytical model, the sandwich-type structure has been widely employed for the study of electrochemical immunosensor (Liu et al., 2013; Wang et al., 2014b; Xia et al., 2013; Yang et al., 2013; Zhou et al., 2014). In n
Corresponding author. Fax: þ 86 531 82765969. E-mail address: [email protected] (Q. Wei).
http://dx.doi.org/10.1016/j.bios.2015.06.033 0956-5663/& 2015 Elsevier B.V. All rights reserved.
traditional sandwich-type electrochemical immunosensors, most of the signal amplification strategies are based on employing multifunctional nanomaterials with good electrochemical characteristics as the label (Han et al., 2013; Wang et al., 2013, 2014d). In these strategies, with the increase of the concentration of antigen, the number of the label conjugated with secondary antibodies (Ab2) will increase accordingly, resulting in the enhancement of electrochemical signal. Obviously, the label acts as the key factor to decide the sensitivity of the traditional sandwich-type immunosensor. However, the sensitivity may also be limited because the specific recognition between antibodies and antigens can generate a hydrophobic and insulating layer on the modified electrode and hinder the electron transfer (Fan et al., 2014). Herein, a sandwich-type electrochemical immunosensor based on a novel signal amplification strategy by unitizing multifunctional nanomaterials with good electrochemical characteristics as the matrix is developed. In this strategy, the electrochemical signal will decrease with the increase of the concentration of antigen because the steric hindrance of antigen can depress the electron transfer. In this case, the matrix becomes the key factor of sensitivity of the designed immunosensor. The hydrophobic and insulating layer still hinder the electron transfer but
60
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
will have a positive effect on the sensitivity. In this work, Pd nanocubes functionalized magnetic graphene sheet (Pd–Fe3O4-GS) was employed as the matrix. The synergic effect between Pd nanocubes and magnetic graphene sheet (Fe3O4-GS) can increase the electrocatalytic activity of Pd–Fe3O4-GS towards hydrogen peroxide (H2O2), leading to a high sensitivity of the designed immunosensor. What's more, the large specific surface area and good biocompatibility of Pd–Fe3O4-GS are beneficial to the immobilization of primary antibodies (Ab1) (Chen et al., 2008; Guo et al., 2009). In order to achieve the determination of low concentrations of antigen, the label with bad electron transfer ability and electrocatalytic activity can be employed to increase the change of electrochemical signal. To date, silicon dioxide (SiO2) has attracted wide attention in biological fields owing to its exciting features such as the uniform size, large surface area, low toxicity, high biocompatibility, good stability and low cost (Yang et al., 2010). Apart from that, SiO2 also possesses the large steric hindrance, bad electron transfer ability and bad electrocatalytic activity. Therefore, SiO2 was employed as the label to conjugate with Ab2 in this work. Both the obtained conjugate (SiO2@Ab2) and antigen can facilitate the decrease of electrocatalytic current response and contribute to the excellent sensitivity for this designed immunosensor.
2. Materials and methods 2.1. Apparatus and reagents All electrochemical measurements were performed on a CHI760E electrochemical workstation (Huakeputian Technology Beijing Co., Ltd., China). A conventional three-electrode system was used for all electrochemical measurements: a glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire electrode as the counter electrode. Scanning electron microscope (SEM) images and energy dispersive X-ray spectral data (EDX) were obtained by using Quanta FEG250 field emission environmental SEM (FEI, United States) operated at 4 KV. UV–vis measurements were carried out by using a Lambda 35 UV/ vis Spectrometer (Perkin-Elmer, United States). Transmission electron microscope (TEM) images were obtained from a Hitachi H-600 microscope (Japan). Fourier transform infrared spectroscopy (FTIR) spectrum was obtained from VERTEX 70 spectrometer (Bruker, Germany). Human immunoglobulin G (IgG) and Goat Anti-Human IgG (anti-IgG, Ab1 and Ab2) were purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd., China. Bovine serum albumin (BSA), L-ascorbic acid (AA) and tetraethyl orthosilicate (TEOS) were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China. 1-(3-(Dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 3-aminopropyltriethoxysilane (APTES), sodium tetrachloropalladate (Na2PdCl4), potassium bromide (KBr) and sodium acetate trihydrate (NaAc 3H2O) were purchased from Shanghai Aladdin Chemistry Co., Ltd, China. Ammonium hydroxide solution (NH3 H2O), ethanol, ethylene glycol and ethanediamine were purchased from Tianjin Fuyu Fine Chemical Co., Ltd., China. Ferric chloride hexahydrate (FeCl3 6H2O) was purchased from Tianjin Damao Chemical Reagent Co., Ltd., China. Polyvinylpyrrolidone (PVP, MW ¼55,000) was purchased from Beijing Yili Fine Chemical Co., Ltd., China. Phosphate buffered saline (PBS, 1/15 M Na2HPO4 and KH2PO4) was used as an electrolyte for all electrochemistry measurement. The concentration of IgG stock solution is 1 mg/mL, which was diluted stepwise to the dynamic detection range of the
immunosensor by the PBS at pH ¼7.4. All other reagents were of analytical grade and ultrapure water was used throughout the study. 2.2. Synthesis of the Pd nanocubes The Pd nanocubes were synthesized by adding the Na2PdCl4 solution into a mixture of AA and KBr according to the reference (Jin et al., 2010). In a typical synthesis the Pd nanocubes, an aqueous solution (8.0 mL) containing PVP (105 mg), AA (60 mg) and KBr (600 mg) was placed in a vial and preheated to 80 °C in an oil bath under magnetic stirring for 10 min. Subsequently, an aqueous solution (3.0 mL) containing Na2PdCl4 (57 mg) was added with a pipet. After the vial had been capped, the reaction was maintained at 80 °C for 3 h. The product was collected by centrifugation, washed three times with water to remove excess PVP, and redispersed in water (11 mL). 2.3. Synthesis of the NH2–Fe3O4-GS Graphene oxide (GO) was synthesized by an improved Hummers method (Marcano et al., 2010). In brief, a mixture of concentrated H2SO4 (36 mL) and H3PO4 (4 mL) was added into a mixture of graphite flakes (0.3 g) and KMnO4 (1.8 g), producing a slight exotherm to 35–40 °C. The reaction was then heated to 50 °C and stirred for 12 h. After that, the reaction was cooled to room temperature and poured onto ice (40 mL) with 30% H2O2 (0.3 mL), and the mixture was centrifuged and the supernatant was decanted away. For workup, the remaining solid material was washed in succession with water, 30% HCl, ethanol and ether. The obtained solid was dried in vacuum overnight. In a typical synthesis of Fe3O4-GS (Guo et al., 2014), FeCl3 6H2O (0.5 g) was dissolved in ethylene glycol (10 mL) to form a clear solution, followed by the addition of NaAc (1.5 g), ethanediamine (5 mL) and GO (0.5 g). The mixture was stirred vigorously for 30 min and then sealed in a teflon-lined stainless steel autoclave. The autoclave was heated to 200 °C and maintained for 8 h, and then was cooled to room temperature. The resulting black powder was washed several times and dried at 35 °C under high vacuum overnight. It should be noted that the GO was translated into the graphene sheet (GS) in the process of reaction. The 3-aminopropyl-functionalized Fe3O4-GS (NH2–Fe3O4-GS) was synthesized by a modified method according to references (Li et al., 2013; Yamaura et al., 2004). In brief, Fe3O4-GS (0.1 g) was dispersed in a solution of ethanol (10 mL) containing APTES (0.1 mL), which was placed in a vial and preheated to 70 °C in an oil bath under magnetic stirring for 1.5 h. The NH2–Fe3O4-GS was obtained by magnetic separation process and dried at 35 °C under high vacuum overnight. 2.4. Preparation of the Pd–Fe3O4-GS The prepared Pd nanocubes solution (400 μL) was added into a solution of NH2–Fe3O4-GS (2 mg/mL, 2 mL) and shaked at room temperature for 24 h. Following the magnetic separation process, the final product was obtained after it was washed three times with water to remove excess Pd nanocubes, and redispersed in water (2 mL). 2.5. Synthesis of the NH2–SiO2 Monodisperse SiO2 nanospheres were prepared by a slightly modified Stober process (Zhang et al., 2009). In a typical synthesis of SiO2, TEOS (4.5 mL) was rapidly added into a mixture solution of ethanol (61.75 mL), NH3 H2O (9.0 mL) and water (24.75 mL). The obtained mixture solution was stirred vigorously for 2 h at room
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
61
temperature. The product was collected by centrifugation, washed three times with ethanol and dried at 35 °C under high vacuum overnight. The 3-aminopropyl-functionalized SiO2 (NH2–SiO2) were synthesized by a modified method according to the literature (Cauvel et al., 1997). Briefly, SiO2 (0.1 g) were dispersed in a solution of ethanol (10 mL) containing APTES (0.1 mL), which was placed in a vial and preheated to 70 °C in an oil bath under magnetic stirring for 1.5 h. A free-flowing powdery material was obtained by mild centrifugation and dried at 35 °C under high vacuum overnight. 2.6. Preparation of the SiO2@Ab2 A mixture solution of NH2–SiO2 (2 mg/mL, 2 mL), Ab2 dispersion (10 μg/mL, 1 mL) and EDC/NHS (10 mM/2 mM, 1 mL) was stirred for 12 h at 4 °C. After centrifugation, BSA solution (10 mg/mL, 1 mL) was added into the obtained precipitate, and then stirred for 12 h at 4 °C. Following further centrifugation, the resulting SiO2@Ab2 label was redispersed in 2 mL of water and stored at 4 °C. 2.7. Fabrication of the immunosensor Fig. 1 shows the schematic diagram of the proposed sandwichtype electrochemical immunosensor. A GCE was polished to a mirror-like finish with 1.0, 0.3 and 0.05 μm alumina powder and then thoroughly cleaned before use. First, an aqueous solution of Pd–Fe3O4-GS (2 mg/mL, 6 μL) was added onto the surface of GCE and then dried. After washing, Ab1 dispersion (10 μg/mL, 6 μL) was added onto the Pd–Fe3O4-GS/GCE. After incubated at 4 °C for 1 h and washed, BSA solution (10 mg/mL, 3 μL) was added onto the Ab1/Pd–Fe3O4-GS/GCE to eliminate nonspecific binding sites. After incubated for another 1 h at 4 °C, the BSA/Ab1/Pd–Fe3O4-GS/ GCE was washed and incubated with a varying concentration of IgG (5 10 6–5 ng/mL, 6 μL) for 1 h at room temperature, and then the IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE was washed extensively to remove unbounded IgG molecules. Finally, the prepared SiO2@Ab2 label solution (2 mg/mL, 6 μL) was added onto the modified electrode surface for 1 h at room temperature, and the SiO2@Ab2/IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE was washed thoroughly for measurement. For amperometric i–t curve to record the amperometric response, a detection potential of 0.4 V was selected. 5 mM H2O2 was added into the PBS after the back ground current was stabilized.
3. Results and discussion 3.1. Characterization of Pd–Fe3O4-GS and SiO2 SEM images of the synthesized Pd–Fe3O4-GS are shown in Fig. 2A and B. It can be observed that the GS exhibits lamellar folds structure and is loaded with massive Fe3O4 NPs. The Fe3O4 NPs own an average grain diameter of 270 nm. Lots of Pd nanocubes are successfully linked on the surfaces of both Fe3O4 NPs and GS. The Pd nanocubes own an average size of 18 nm is confirmed by the TEM image (Fig. 2C). EDX spectrum of Pd–Fe3O4-GS (Fig. 2D) was used to further confirm what kinds of elements were contained in the Pd–Fe3O4-GS. Al element can be observed because the sample was fixed on the aluminum foil to test. As the Fig. 2E shows, the prepared aqueous solution of Pd–Fe3O4-GS can be well dispersed in water and easily separated from the dispersion by a magnet. Fig. 2F and G shows the SEM image and the EDX spectrum of the SiO2 respectively, which indicates the SiO2 with an average size of 400 nm was synthesized successfully. The FTIR spectrum of
Fig. 1. The schematic immunosensor.
illustration
of
the
sandwich-type
electrochemical
NH2–SiO2 is shown in Fig. 2H. The absorption bands at 3140 cm 1 and 1630 cm 1 are respectively caused by N–H bond stretching vibration and bending vibration, which corresponds to –NH2 on the surface of SiO2. The absorption bands at 1100 cm 1 and 475 cm 1 are caused by typical vibration of Si–O–Si and the absorbance band at 1400 cm 1 are caused by C-H bending vibration in the methylene group from APTES. These results suggest that NH2-SiO2 was successfully synthesized (Zou et al., 2014). In order to identify that Ab2 could be conjugated onto the surface of NH2– SiO2, UV–vis spectrums of Ab2, SiO2 and SiO2@Ab2 are shown in Fig. 2I. An absorption peak at 278 nm is observed for Ab2 dispersion (curve a). The aqueous solution of SiO2 (curve b) has no obvious absorption peak. Therefore, the absorption peak at 278 nm is mainly ascribed to the presence of Ab2 in aqueous solution of SiO2@Ab2 (curve c). So it can be concluded that Ab2 was excessively conjugated onto the surface of NH2–SiO2 (Tang et al., 2008). 3.2. The mechanism of signal amplification strategy In this work, the electrocatalytic activity of the matrix is key factor of sensitivity of the designed immunosensor. In order to investigate the signal amplification mechanism of the matrix, control experiments of different nanomaterials for the reduction towards 5 mM H2O2 were carried out at 0.4 V in PBS at pH ¼ 6.8. As shown in Fig. 3A, different electrocatalytic current response curves were obtained when bare GCE was modified with 1 mg/mL of Fe3O4-GS (curve a), Pd nanocubes (curve b) and Pd–Fe3O4-GS (curve c). It can be observed that 1 mg/mL of Fe3O4-GS (curve a) has an electrocatalytic current response of about 3 μA and 1 mg/mL of Pd nanocubes has an electrocatalytic current response of about 6 μA (curve b). Interestingly, the Pd–Fe3O4-GS exhibits much greater electrocatalytic current response of about 13 μA (curve c) which is even larger than the summation of that produced by Fe3O4-GS and Pd nanocubes. It demonstrates that there exists the synergetic effect between Pd nanocubes and Fe3O4-GS, which can increase the electrocatalytic activity of Pd–Fe3O4-GS towards the reduction of H2O2. The sensitivity of the proposed immunosensor finally depends on the electrocatalytic current response change produced by IgG and SiO2@Ab2. In order to demonstrate the signal amplification
62
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
Fig. 2. SEM images of Pd–Fe3O4-GS (A and B) and SiO2 (F); TEM image of Pd nanocubes (C); EDX spectrums of Pd–Fe3O4-GS (D) and SiO2 (G); Digital photograph of the aqueous solution of Pd–Fe3O4-GS without and with an additional magnetic field (E); FTIR spectrum of NH2–SiO2 (H); UV–vis spectrums(I) of Ab2 (a), SiO2 (b) and SiO2@Ab2 (c).
mechanism of IgG and SiO2@Ab2, control experiments of different modified electrodes (2 mg/mL of Pd–Fe3O4-GS) for the reduction of 5 mM H2O2 were carried out at 0.4 V in PBS at pH ¼ 6.8. Fig. 3B shows different electrocatalytic current response curves of BSA/ Ab1/Pd–Fe3O4-GS/GCE (curve a), IgG/BSA/Ab1/Pd–Fe3O4-GS/ GCE (curve b) and SiO2@Ab2/IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE (curve c). When 5 ng/mL of IgG was recognized by the modified electrode, the electrocatalytic current response only decreased about 5 μA. But after it was further recognized with the SiO2@Ab2, the electrocatalytic current response could decrease about 14 μA. It can be concluded that in the presence of low concentrations of IgG, SiO2@Ab2 can provide a larger steric hindrance and depress the electron transfer more effectively, producing a more sensitive
influence towards the electrocatalytic signal response. 3.3. Optimization of experimental conditions In order to achieve an optimal electrocatalytic current response, the optimization of experimental conditions is necessary. The value of pH mainly influences the electrocatalytic process of the matrix towards the reduction of H2O2 and the activity of biological proteins. Fig. S1A shows the electrocatalytic current responses of Pd–Fe3O4-GS (2 mg/mL) in different pH values of PBS. As shown in this figure, the optimal electrocatalytic current response was achieved at pH ¼6.8. Moreover, the antibodies and antigens can maintain their biological activity in this approximate
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
63
Fig. 3. (A) Electrocatalytic current responses towards 5 mM H2O2 of 1 mg/mL of Fe3O4-GS (curve a), Pd nanocubes (curve b) and Pd–Fe3O4-GS (curve c); (B) Electrocatalytic current responses towards 5 mM H2O2 of BSA/Ab1/Pd–Fe3O4-GS/GCE (curve a), IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE (curve b) and SiO2@Ab2/IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE (curve c).
neutral condition. Therefore, PBS at pH ¼6.8 was selected for the test throughout this study. The concentration of Pd–Fe3O4-GS is an important factor influencing the value of electrocatalytic current response. Fig. S1B shows the electrocatalytic current responses of different concentrations of Pd–Fe3O4-GS in PBS at pH ¼6.8. As shown in this figure, the optimal electrocatalytic current response was achieved at a concentration of 2 mg/mL. Therefore, 2 mg/mL of Pd–Fe3O4-GS was selected as the matrix throughout this study. The concentration of NH2-SiO2 is an important factor influencing the value of electrocatalytic current response change. Fig. S1C shows the electrocatalytic current responses of different concentrations of NH2–SiO2 as the label for the determination of 5 ng/mL of IgG in PBS at pH ¼ 6.8. As shown in this figure, the optimal electrocatalytic current response change could be achieved at a concentration of 2 mg/mL. Therefore, 2 mg/mL of NH2–SiO2 was selected as the label throughout this study. In addition, the other experimental conditions were controlled strictly. For example, the modified sample value was 6 μL, the incubation time was 1 h, the incubation temperature was the room temperature, and the concentration of antibodies was 10 mg/mL. All these above factors would make sure antibodies and antigens could be effectively and specifically recognized with each other on the GCE with a diameter of 4 mm. Under the optimal conditions, the designed immunosensor could exhibit an optimal electrochemical signal for the quantitative determination of IgG.
3.4. Electrochemical characterization of the immunosensor The fabrication process of this sandwich-type electrochemical immunosensor can be monitored by amperometric i–t curves, as shown in Fig. 4A. The bare GCE (curve a) had no electrocatalytic current response towards the reduction of H2O2. After modified with the Pd–Fe3O4-GS (curve b), the electrocatalytic current response increased to about 30 mA. Following that, Ab1 (curve c), BSA (curve d) and IgG (curve e) were modified layer by layer on the electrode, the electrocatalytic current response gradually decreased because of the non-conductive bioactive substances without any electrocatalytic properties can hinder the electron transfer. The electrocatalytic current response decreased significantly when the SiO2@Ab2 (curve f) was modified on the electrode, which was mainly ascribed to the obviously increasing steric hindrance of the SiO2@Ab2. Thus, the electrocatalytic current responses confirmed the successful formation of the SiO2@Ab2 and the successful fabrication of the proposed immunosensor. A.C. impedance is another suitable method for monitoring the changes of the surface features during the fabrication process of this sandwich-type electrochemical immunosensor (Lu et al., 2014). In this study, A.C. impedance method was also used to characterize fabrication process of the proposed immunosensor. Fig. 4B shows the Nyquist plots of A.C. impedance spectroscopy in the process of modifying electrode, which were recorded from 1 to 105 Hz at 0.2 V in a solution containing 0.1 M KCl and 2.5 mM Fe(CN)63 − /Fe(CN)64 −. Nyquist plots consist of two portions.
Fig. 4. Electrocatalytic current responses towards 5 mM H2O2 in PBS at pH ¼6.8 (A) and Nyquist plots of the A.C. impedance method in a solution containing 0.1 M KCl and 2.5 mM Fe(CN)63 −/Fe(CN)64 − (B): GCE (a), Pd–Fe3O4-GS/GCE (b), Ab1/Pd–Fe3O4-GS/GCE (c), BSA/Ab1/Pd–Fe3O4-GS/GCE (d), IgG/BSA/Ab1/ Pd–Fe3O4-GS/GCE (e) and SiO2@Ab2/ IgG/BSA/Ab1/Pd–Fe3O4-GS/GCE (f).
64
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
The linear portion at low frequencies is associated with electrochemical behavior limited by diffusion. The semicircle portion at high frequencies is associated with the electrochemical process subject to electron transfer, where the diameter corresponds to the resistance. Simply speaking, resistance change can be judged by observing the diameter change of semicircle portion. It could be observed that the bare GCE exhibited a very small resistance (curve a). After modified with the Pd–Fe3O4-GS, a smaller resistance (curve b) was observed due to the good electron transfer ability of Pd–Fe3O4-GS. Gradually increasing resistance indicated the successful modification of the non-conductive bioactive substances when Ab1 (curve c), BSA (curve d) and IgG (curve e) were modified layer by layer on the electrode. When the SiO2@Ab2 (curve f) was modified on the electrode, an obvious increase of resistance could be observed, suggesting that SiO2@Ab2 can depress the electron transfer effectively by the specific recognition between antibodies and antigens.
prepared for the determination of 5 ng/mL of IgG. The relative standard deviation (RSD) of the measurements for five electrodes was 3.2%, suggesting the precision and reproducibility of the proposed electrochemical sandwich-type immunosensor was acceptable. To investigate the specificity of the fabricated immunosensor, study was performed by using AFP, CEA and BSA as the interference. 50 ng/mL of interference solution with 5 ng/mL of IgG was measured by the immunosensor. The electrocatalytic current response variation due to the interference was less than 5% of that without the interference, indicating the selectivity of the proposed immunosensor was acceptable. To test the stability of the immunosensor, the sensor was stored at 4 °C when not in use. After one month, no apparent change for the determination of the same concentration of IgG was found. The good stability could be ascribed to the good biocompatibility of the matrix and the label. The reproducibility, selectivity and stability of this proposed immunosensor were all acceptable, thus it was suitable for the quantitative determination of IgG in real human samples.
3.5. Calibration curves of the immunosensor 3.7. Real sample analysis Under optimum conditions, the proposed sandwich-type electrochemical immunosensor was used to detect different concentrations of IgG. Fig. 5A shows the electrocatalytic current responses of the proposed immunosensor for the determination of IgG covering the concentration range from 5 10 6 (curve a) to 5 ng/mL (curve g). As the concentration of IgG increased, a greater amount of SiO2@Ab2 was specifically bound on the electrode surface to hinder the electron transfer, leading to the gradually decreasing electrocatalytic current response. A linear relationship between electrocatalytic current responses and the logarithmic values of IgG concentration was obtained (Fig. 5B). And the linear regression equation of the calibration curve being I ¼4.60 2.12 logC with correlation coefficient of 0.990 and an extremely low limit of detection (LOD, S/N ¼3) of 3.2 fg/mL. The good sensitivity was ascribed to the novel signal amplification strategy of the designed sandwich-type electrochemical immunosensor. The linear range and LOD of the proposed immunosensor was compared with sandwich-type electrochemical immunosensors previously reported for the determination of other biomarkers in Table 1. It can be found that the proposed immunosensor shows a satisfying linear range and LOD. 3.6. Reproducibility, selectivity and stability To evaluate the reproducibility of the immunosensor, a series of immunosensors fabricated on five different electrodes were
In order to test the precision and accuracy of the proposed electrochemical sandwich-type immunosensor, it was used to detect the recoveries of different concentrations of IgG in human serum samples by recovery tests (Table S1). The RSD was in the range from 1.4% to 4.8% and the recovery was in the range from 99.6% to 100.1%. Thus, the immunosensor can be effectively applied in the quantitative determination of IgG in human serums. In order to further validate the proposed immunosensor, a comparison with the commercialized available enzyme-linked immunosorbent assay (ELISA) method is shown in Table S2. The relative error between the two methods was in the range from 2.3% to 2.2%. These data revealed a good agreement between the two analytical methods, further indicating the feasibility of the proposed immunosensor for clinical application.
4. Conclusions This work has developed an ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy by employing Pd–Fe3O4-GS as the matrix and SiO2 as the label for the quantitative determination of IgG. This designed immunosensor displayed an extremely low LOD, a wide detection range, good selectivity, acceptable reproducibility and stability. The novel fabrication procedure and the ultrasensitivity
Fig. 5. (A) Electrocatalytic current responses of the immunosensor for the determination of different concentrations of IgG: 5 10 6 ng/mL (a), 5 10 5 ng/mL (b), 5 10 4 ng/mL (c), 0.005 ng/mL (d), 0.05 ng/mL (e), 0.5 ng/mL (f) and 5 ng/mL (g); (B) Calibration curve of the immunosensor for the determination of different concentrations of IgG. Error bar ¼RSD (n¼ 5, 4.9%, 4.7%, 4.9%, 3.9%, 4.3%, 2.6%, and 3.2%).
Y. Wang et al. / Biosensors and Bioelectronics 74 (2015) 59–65
65
Table 1 Comparison of different sandwich-type electrochemical immunosensors for the determination of biomarkers. Tumor marker
Linear range
LOD
Reference
Ara h 1 (a major peanut allergen) Carcinoembryonic antigen (CEA) Squamous cell carcinoma antigen (SCCA) Prostate-specific antigen (PSA) Alpha fetoprotein (AFP) IgG CEA SCCA Human immunodeficiency virus p24 IgG IgG
12.6–2000 ng/mL 0.05–200 ng/mL 0.05–18 ng/mL 0.01–10 ng/mL 0.01–25 ng/mL 0.01–200 ng/mL 1 pg/mL–50 ng/mL 0.5 pg/mL–40 ng/mL 0.5 pg/mL–8.5 ng/mL 0.01 pg/mL–100 ng/mL 0.005 pg/mL–5 ng/mL
3.8 ng/mL 18 pg/mL 15.3 pg/mL 5 pg/mL 4 pg/mL 4 pg/mL 0.27 pg/mL 0.18 pg/mL 0.15 pg/mL 4.3 fg/mL 3.2 fg/mL
(Alves et al., 2015) (Yang et al., 2014) (Wu et al., 2013) (Wei et al., 2010) (Wang et al., 2014a) (Cao et al., 2013) (Wang et al., 2015) (Wang et al., 2014c) (Fang et al., 2015) (Zhang et al., 2014b) This work
of this designed immunosensor might provide wide potential applications for the quantitative determination of other biomarkers in clinical diagnosis.
Acknowledgments This study was supported by the National Natural Science Foundation of China (Nos. 21175057, 21375047, 21377046 and 21405059), the Science and Technology Plan Project of Jinan (No. 201307010), the Science and Technology Development Plan of Shandong Province (No. 2014GSF120004), the Special Project for Independent Innovation and Achievements Transformation of Shandong Province (No. 2014ZZCX05101), and QW thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province (No. ts20130937) and UJN.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bios.2015.06.033.
References Alves, R.C., Pimentel, F.B., Nouws, H.P.A., Marques, R.C.B., González-García, M.B., Oliveira, M.B.P.P., Delerue-Matos, C., 2015. Biosens. Bioelectron. 64, 19–24. Bahadir, E.B., Sezginturk, M.K., 2015. Biosens. Bioelectron. 68C, 62–71. Beitollahi, H., Ardakani, M.M., Ganjipour, B., Naeimi, H., 2008. Biosens. Bioelectron. 24, 362–368. Cao, X., Liu, S., Feng, Q., Wang, N., 2013. Biosens. Bioelectron. 49, 256–262. Cauvel, A., Renard, G., Brunel, D., 1997. J. Org. Chem 62, 749–751. Chen, H., Müller, M.B., Gilmore, K.J., Wallace, G.G., Li, D., 2008. Adv. Mater. 20, 3557–3561. Chen, H., Tang, D., Zhang, B., Liu, B., Cui, Y., Chen, G., 2012. Talanta 91, 95–102. Fan, G.-C., Han, L., Zhu, H., Zhang, J.-R., Zhu, J.-J., 2014. Anal. Chem. 86, 12398–12405. Fang, Y.-S., Huang, X.-J., Wang, L.-S., Wang, J.-F., 2015. Biosens. Bioelectron. 64, 324–332. Guo, S., Li, D., Zhang, L., Li, J., Wang, E., 2009. Biomaterials 30, 1881–1889. Guo, X., Du, B., Wei, Q., Yang, J., Hu, L., Yan, L., Xu, W., 2014. J. Hazard. Mater. 278, 211–220. Han, J., Zhuo, Y., Chai, Y.-Q., Mao, L., Yuan, Y.-L., Yuan, R., 2011. Talanta 85, 130–135. Han, J., Zhuo, Y., Chai, Y., Xiang, Y., Yuan, R., Yuan, Y., Liao, N., 2013. Biosens. Bioelectron. 41, 116–122. Hou, L., Tang, Y., Xu, M., Gao, Z., Tang, D., 2014. Anal. Chem. 86, 8352–8358. Jin, M., Liu, H., Zhang, H., Xie, Z., Liu, J., Xia, Y., 2010. Nano Res. 4, 83–91. Li, H., Wei, Q., He, J., Li, T., Zhao, Y., Cai, Y., Du, B., Qian, Z., Yang, M., 2011. Biosens. Bioelectron. 26, 3590–3595.
Li, P.F., Xu, Y., Cheng, X.-H., 2013. Surf. Coat. Technol. 232, 331–339. Li, R., Wu, K., Liu, C., Huang, Y., Wang, Y., Fang, H., Zhang, H., Li, C., 2014. Anal. Chem. 86, 5300–5307. Liu, F., Xiang, G., Yuan, R., Chen, X., Luo, F., Jiang, D., Huang, S., Li, Y., Pu, X., 2014. Biosens. Bioelectron. 60, 210–217. Liu, J., Lu, C.-Y., Zhou, H., Xu, J.-J., Wang, Z.-H., Chen, H.-Y., 2013. Chem. Commun. 49, 6602–6604. Lu, W., Cao, X., Tao, L., Ge, J., Dong, J., Qian, W., 2014. Biosens. Bioelectron. 57, 219–225. Marcano, D.C., Kosynkin, D.V., Berlin, J.M., Sinitskii, A., Sun, Z., Slesarev, A., Alemany, L.B., Lu, W., Tour, J.M., 2010. ACS Nano 4, 4806–4814. Mazloum-Ardakani, M., Beitollahi, H., Amini, M.K., Mirkhalaf, F., Abdollahi-Alibeik, M., 2010. Sens. Actuators B 151, 243–249. Mazloum-Ardakani, M., Beitollahi, H., Amini, M.K., Mirkhalaf, F., Mirjalili, B.-F., 2011a. Biosens. Bioelectron. 26, 2102–2106. Mazloum-Ardakani, M., Ganjipour, B., Beitollahi, H., Amini, M.K., Mirkhalaf, F., Naeimi, H., Nejati-Barzoki, M., 2011b. Electrochim. Acta 56, 9113–9120. Mazloum-Ardakani, M., Sheikh-Mohseni, M.A., Abdollahi-Alibeik, M., Benvidi, A., 2012a. Sens. Actuators B 171–172, 380–386. Mazloum-Ardakani, M., Taleat, Z., Khoshroo, A., Beitollahi, H., Dehghani, H., 2012b. Biosens. Bioelectron. 35, 75–81. Tang, D., Yuan, R., Chai, Y., 2008. Anal. Chem. 80, 1582–1588. Wang, H., Li, X., Mao, K., Li, Y., Du, B., Zhang, Y., Wei, Q., 2014a. Anal. Biochem. 465, 121–126. Wang, Q., Song, Y., Chai, Y., Pan, G., Li, T., Yuan, Y., Yuan, R., 2014b. Biosens. Bioelectron. 60, 118–123. Wang, X., Chen, L., Su, X., Ai, S., 2013. Biosens. Bioelectron. 47, 171–177. Wang, X., Chu, C., Shen, L., Deng, W., Yan, M., Ge, S., Yu, J., Song, X., 2015. Sens. Actuators B 206, 30–36. Wang, Y., Li, X., Cao, W., Li, Y., Li, H., Du, B., Wei, Q., 2014c. Talanta 129, 411–416. Wang, Y., Li, X., Cao, W., Li, Y., Li, H., Du, B., Wei, Q., 2014d. Biosens. Bioelectron. 61, 618–624. Wei, Q., Xiang, Z., He, J., Wang, G., Li, H., Qian, Z., Yang, M., 2010. Biosens. Bioelectron. 26, 627–631. Wu, D., Fan, H., Li, Y., Zhang, Y., Liang, H., Wei, Q., 2013. Biosens. Bioelectron. 46, 91–96. Xia, N., Deng, D., Zhang, L., Yuan, B., Jing, M., Du, J., Liu, L., 2013. Biosens. Bioelectron. 43, 155–159. Yamaura, M., Camilo, R.L., Sampaio, L.C., Macêdo, M.A., Nakamura, M., Toma, H.E., 2004. J. Magn. Magn. Mater. 279, 210–217. Yang, G.-H., Shi, J.-J., Wang, S., Xiong, W.-W., Jiang, L.-P., Burda, C., Zhu, J.-J., 2013. Chem. Commun. 49, 10757–10759. Yang, M., Li, H., Javadi, A., Gong, S., 2010. Biomaterials 31, 3281–3286. Yang, P., Li, X., Wang, L., Wu, Q., Chen, Z., Lin, X., 2014. J. Electroanal. Chem. 732, 38–45. Zhang, B., Liu, B., Chen, G., Tang, D., 2015. Biosens. Bioelectron. 64, 6–12. Zhang, S., Du, B., Li, H., Xin, X., Ma, H., Wu, D., Yan, L., Wei, Q., 2014a. Biosens. Bioelectron. 52, 225–231. Zhang, S., Ma, H., Yan, L., Cao, W., Yan, T., Wei, Q., Du, B., 2014b. Biosens. Bioelectron. 59, 335–341. Zhang, T., Zhang, Q., Ge, J., Goebl, J., Sun, M., Yan, Y., Liu, Y.-s, Chang, C., Guo, J., Yin, Y., 2009. J. Phys. Chem. C 113, 3168–3175. Zhou, J., Tang, J., Chen, G., Tang, D., 2014. Biosens. Bioelectron. 54, 323–328. Zou, B., Hu, Y., Cui, F., Jiang, L., Yu, D., Huang, H., 2014. J. Colloid. Interf. Sci. 417, 210–216.
