Biosensors and Bioelectronics 63 (2015) 39–46
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An electrochemical immunosensor for ultrasensitive detection of carbohydrate antigen 199 based on Au@CuxOS yolk–shell nanostructures with porous shells as labels Aiping Guo a, Yueyun Li b, Wei Cao a, Xianchao Meng a, Dan Wu a, Qin Wei a,n, Bin Du a a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b School of Chemical Engineering, Shandong University of Technology, Zibo 255049, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 18 June 2014 Accepted 8 July 2014 Available online 11 July 2014
A novel and sensitive electrochemical immunosensor for ultrasensitive detection of pancreatic cancer biomarker carbohydrate antigen 199 (CA199) was proposed by using Au@CuxOS yolk–shell nanostructures with porous shells as labels for signal amplification. Au@CuxOS yolk–shell nanostructures exhibit high electrocatalytic activity toward the reduction of hydrogen peroxide (H2O2) as analytical signal. Moreover, secondary antibody (Ab2) can adsorb on the surface of Au@CuxOS with porous shells which has large surface area and could greatly increase the probability of Ab2–antigen interactions thereby leading to higher sensitivity. Reduced graphene oxide–tetraethylene pentamine (rGO–TEPA), containing abundant amine groups, was supported Au nanoparticles as a support platform to immobilize the primary antibody (Ab1). The resulting sensing interface of rGO–TEPA/AuNPs could provide a large electroconductive surface area, allowing high loadings of the biological recognition elements as well as the occurrence of electrocatalytic and electron-transfer processes. Under optimal conditions, the immunosensor exhibited a wide linear response to CA199 ranging from 0.001 to 12 U/mL with a low detection limit of 0.0005 U/mL. The designed immunosensor displayed good precision, high sensitivity, acceptable stability and reproducibility, and has been applied to the analysis of serum with satisfactory results. The proposed method provides a new promising platform of clinical immunoassay for other biomolecules. & 2014 Elsevier B.V. All rights reserved.
Keywords: Pancreatic cancer biomarker CA199 Au@CuxOS yolk–shell nanostructures with porous shells Reduced graphene oxide–tetraethylene pentamine (rGO–TEPA) Au nanoparticles
1. Introduction Sensitive and accurate detection of disease-related biomarkers is critical to many areas of biomedical research and diagnosis (Kitano, 2002). In particular, the clinical measurement of cancer biomarkers shows great promise for early diagnosis, disease monitoring, and highly reliable prediction (Chen et al., 2013; Srinivas et al., 2001; Wang et al., 2011). Carbohydrate antigen 199 (CA199) is a preferred label for pancreatic cancer, which is a highly lethiferous sarcomata and elevates in the peripheral blood of the majority of pancreatic cancer patients but does not achieve the performance required for either early detection or diagnosis, due to both false positive and false negative readings (Goonetilleke and Siriwardena, 2007). It is reported to be frequently found out at the patients of pancreatic cancer with a high score of over 79% (Del Villano et al., 1983; Wild, 2001). Thus, the detection of CA199 level n
Corresponding author. Tel.: þ 86 531 82767872; fax: þ86 531 82765969. E-mail address: [email protected] (Q. Wei).
http://dx.doi.org/10.1016/j.bios.2014.07.017 0956-5663/& 2014 Elsevier B.V. All rights reserved.
in human serum plays an important role in the diagnosis and management of pancreatic cancer. Various methods have been employed to detect CA199, such as electric field-driven assay (Wu et al., 2008), immobilized horseradish peroxidase assay (Du et al., 2007), and chemiluminescent multiplex assay (Fu et al., 2007). While highly accurate, some of these techniques involve disadvantages such as relatively sophisticated instruments, significant sample volume, limited sensitivity, and clinically unrealistic expense and time. Therefore, there is a real need to develop operationally simple, highly sensitive, and inexpensive methods to detect levels of the biomarkers in both normal and cancer patient sera. Electrochemical immunosensors are bioanalytical tools that feature simplicity of construction, the possibility to be massproduced, cost-effectiveness, ease of use, feasible miniaturization, and subsequent portability. These advantages make it attractive for use as a high-performance screening tool for the detection of biomolecules (Tang et al., 2010; Chen et al., 2010). Signal amplification and noise reduction are crucial for obtaining low detection limits in clinical immunoassays (Fu et al., 2006; Long et al., 2005).
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Sandwich-type immunoassay has the advantages of high specificity and sensitivity because of the use of a couple of match antibodies (Knopp, 2006), which is a widely used protocol for ultrasensitive detection due to the high selectivity, low cost, and ease of miniaturization (Ahirwal and Mitra, 2010; Wei et al., 2010). One key research area of electrochemical immunosensors is development of biosensors with various nanomaterials of physical and chemical properties, because they can provide a large surface area and improve the biocompatibility and stability of the system (K.J. Huang et al., 2013; T.Y. Huang et al. 2013; Zhou et al., 2009). Reduced graphene oxide (rGO) is often used in electrochemical sensors (Gan et al., 2011; K.J. Huang et al., 2013; T.Y. Huang et al. 2013; Kaur et al., 2013; Sun et al., 2013; Teymourian et al., 2013; Zhang et al., 2013), because its abundant defects and chemical groups facilitate charge transfer and thus ensure high electrochemical activity. Moreover, the populated chemical moieties on the rGO surface offer the convenience and flexibility for various functionalizations to enhance the sensor performance. More importantly, rGO may also be functionalized through covalent or non-covalent methods in order to further enhance its sensitivity, specificity, loading capacity, biocompatibility, etc. Reduced graphene oxide–tetraethylene pentamine (rGO–TEPA) is a novel material which is a combination of rGO and tetraethylene pentamine through covalent bonding. The structure of rGO–TEPA is shown in Fig. S1. It not only keeps the original property of rGO but also promotes water solubility. Additionally, AuNPs are extremely attractive materials for fabricating sensors because of its unique properties, including simple preparation, good biocompatibility, excellent conductivity, and easy stabilization of the colloidal dispersion by using covalent or noncovalent surface coatings (Ding et al., 2004; Li et al., 2011). Then, based on these properties, rGO–TEPA/AuNPs could be an excellent immunosensor platform because of the combination of physical and chemical properties of rGO–TEPA and the surface reactivity of AuNPs for immobilizing a large amount of antibodies, and the application of rGO–TEPA/ AuNPs as a sensor platform not only increases the amount of immobilized antibodies, but also accelerates the electron transfer process assisted by AuNPs for signal enhancement, which further enhance the sensitivity of the immunosensor. In the design and fabrication of highly sensitive electrochemical immunosensors, signal amplification and antibody immobilization are the crucial steps (Sánchez et al., 2008). Recently, yolk– shell nanostructures have attracted considerable attention because of their various applications in catalysis, batteries, and biomedical fields (Kim et al., 2008; Liu et al., 2013, 2010; Sanles-Sobrido et al., 2009). The yolk–shell nanostructures possess the unique structure of a hollow shell and an encapsulated single spherical core. The difference between yolk–shell and core–shell nanostructure is the space between core and shell. This space can act as a nanoreactor and enhance the catalytic performance (Park and Song, 2011; J.J. Zhang et al., 2010; Q. Zhang et al., 2010). The yolk–shell nanostructures have some excellent properties, such as low density, good flow ability, and its ease of functionalization (Kamata et al., 2003). In this paper, a metal/semiconductor yolk–shell nanostructure Au@CuxOS with porous shells was prepared and used as labels to adsorb secondary antibodies (Huang et al., 2010). Au@CuxOS yolk–shell nanostructures show high electrocatalytic activity toward hydrogen peroxide (H2O2) reduction, which could produce an electrocatalytic response by reduction of H2O2 and significantly amplify electrochemical signals. Herein, we demonstrated a unique nonenzymatic sandwichtype immunosensor based on rGO–TEPA/AuNPs modified the electrode as a supporting interface and Au@CuxOS yolk–shell nanostructures with porous shells as labels for the highly sensitive detection of CA199. The proposed rGO–TEPA/AuNPs can be used as an immunosensing platform because it exhibits excellent
electrically conductivity property, hydrophilicity and large specific surface area. Meanwhile, we used Au@CuxOS yolk–shell nanostructures with porous shells for the immobilization of secondary antibody (Ab2) to obtain the Au@CuxOS–Ab2 bio-conjugates, which could produce an electrocatalytic response by reduction of H2O2 and significantly amplify electrochemical signals. The results of electrochemical studies suggested that the developed immunosensor possesses great performance for CA199 determination and has a great potential application available for the analysis of other low-abundant proteins.
2. Experimental section 2.1. Materials and apparatus Reduced graphene oxide–tetraethylene pentamine (rGO–TEPA) were purchased from Nanjing XFNANO Materials TECH Co., Ltd. (China). Carbohydrate antigen 199 (CA199) and anti-CA199 antibody (Ab) were purchased from Shanghai Linc-Bio Science Co. Ltd. (China). K3Fe(CN)6 was purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (China). Bovine serum albumin (BSA, 96–99%) was purchased from Sigma (USA) and used as received. Hydrazine hydrate solution (H4N2 H2O, 80%), copper (II) nitrate trihydrate (Cu(NO3)2 3H2O), and sodium sulfide (Na2S 9H2O) were purchased from Shanghai Reagents Limited Corporation (Shanghai, China). HAuCl4 3H2O were obtained from Sigma-Aldrich (Beijing, China). Phosphate buffered saline (PBS, 0.1 mol/L containing 0.1 mol/L NaCl, pH 7.4) was used as an electrolyte for all electrochemical measurements. Doubly distilled water was used throughout the experiments. All electrochemical measurements were performed on a CHI760D electrochemical workstation (Shanghai CH Instruments Co., China). Transmission electron microscope (TEM) images were obtained from a Hitachi H-800 microscope (Japan). Scanning electron microscope (SEM) images were obtained using field emission SEM (ZEISS, Germany). Electrochemical impedance spectroscopy (EIS) was performed in [Fe(CN)6]3 /4 working solution in the frequency range of 0.1–105 Hz. A conventional threeelectrode system was used for all electrochemical measurements: the modified 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. 2.2. Synthesis of Au nanoparticles (AuNPs) The AuNPs were prepared by the following procedures: 0.50 g of PVP was added into 10 mL of deionized water and then 15 mL of 0.1 M ascorbic acid and 2.5 mL of 9.5 mM HAuCl4 were added into the above solution under continuous stirring for 30 min at room temperature. The products were centrifuged and washed with deionized water and ethanol 3 times in turn. The obtained AuNPs were dispersed in 10 mL of deionized water for further use. 2.3. Synthesis of Au@CuxOS yolk–shell nanostructures Au@CuxOS yolk–shell nanostructures were synthesized by a modified literature procedure (Zhou et al., 2012). In a typical procedure, Au@Cu2O core–shell nanostructures were firstly prepared as follows. 0.20 g of PVP and 1.0 mL of AuNPs solution were added into 10 mL of 10 mM Cu(NO3)2 3H2O solution. After that, 10 μL of H4N2 H2O was added into the solution, the mixture was stirred vigorously for 2 min. The products were then collected by centrifugation, washed with deionized water and ethanol 3 times, and dried at 60 °C in a vacuum.
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Scheme 1. Schematic illustration of the stepwise preparation of the Au@CuxOS––Ab2 (A) and immunosensor (B).
The obtained Au@Cu2O core–shell nanostructures were dissolved into 10 mL of water, and then 0.12 g of Na2S powder was added under vigorous stirring for 1 h to ensure they were sufficiently sulfurized, resulting in the formation of Au@CuxOS yolk–shell nanostructures with porous shells. The formation of yolk–shell nanostructures is because O2 diffuses quicker than S2 because of the larger ionic radius of S2 than O2 . The products were collected by centrifugation, and washed with deionized water and absolute ethanol two times to remove the impurities. 2.4. Preparation of Au@CuxOS–Ab2 The preparation procedure of Au@CuxOS–Ab2 is illustrated in Scheme 1A, The Au@CuxOS (2 mg) was dispersed in 1.0 mL of pH 7.4 PBS. This dispersion was then mixed with 1.0 mL of 10 mg/mL of anti-CA199. The mixture was allowed to react at room temperature under stirring for 60 min, followed by centrifugation. The resulting Au@CuxOS–Ab2 was washed with PBS and then redispersed in 1.0 mL of PBS and stored at 4 °C before use. 2.5. Fabrication of the immunosensor Scheme 1B shows the schematic representation of the preparation of the immunosensor. GCE was polished with 1, 0.3, and 0.05 μm alumina powder sequentially, and then washed ultrasonically in ethanol for a few minutes and dried in air at room
temperature. Afterwards, 6 μL of rGO–TEPA solution was initially dropped on the electrode surface and dried at room temperature. Then, 6 μL of AuNPs was added onto electrode surface. After 1 h of reaction and washing, 6 μL of prepared excessive anti-CA199 solution (10 μg/mL) was added onto the electrode surface and incubated for 1 h. The electrode was then washed and incubated in 1 wt% BSA solution to block nonspecific binding sites. Subsequently, CA199 buffer solution with a varying concentration was added onto the electrode surface and incubated for 1 h, and then the electrode was washed extensively to remove unbound CA199 molecules. Finally, the prepared Au@CuxOS–Ab2 was dropped onto the electrode surface. After another 1 h, the electrode was washed and ready for measurement.
3. Results and discussion 3.1. Characterization of Au@CuxOS, rGO–TEPA and AuNPs As shown in Fig. 1a and b, the rGO–TEPA is observed to have a wrinkled, paper-like structure, and is transparent with irregular size. It has the large surface area in favor of electron transportation. Au nanoparticles are well monodispersed and uniformly spherical in shape (Fig. 1c). The mean size of Au particles was approximately 35 nm. The morphology of Au@CuxOS was yolk– shell nanostructures in the size range 240 nm with hollow porous shell (Fig. 1d), which gives rise to a large specific surface area and
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Fig. 1. SEM and TEM image of rGO–TEPA (a and b); TEM image of AuNPs (c); SEM and TEM image of Au@CuxOS (d and e); EDX image of Au@CuxOS (f).
will be especially effective for the further adsorption of biomolecules. A detail surface structure of Au@CuxOS yolk–shell nanostructures is shown in Fig. 1e, the SEM displayed a type of spherical morphology and dispersed homogeneously. EDX spectrum was carried out to reveal the components of the as-prepared yolk–shell nanostructure. As shown in Fig. 1f, obvious Au, Cu, S, and O
elements were observed, in which the signal of Au is assigned to the Au core, and that of Cu and S elements are belonged to the shell. The Au@CuxOS yolk–shell nanostructure has different valence states of Cu ions containing CuS and Cu2O, which is the reason that defined as “x”. Thus, the final product is called Au@Cux OS yolk–shell nanostructure.
Fig. 2. (A) Amperometric response of the immunosensors for the detection of 8 U/mL CA199 with different labels at 0.5 V vs. Ag/AgCl toward successive addition of 5 mmol/L H2O2 in N2-saturated PBS: (a) Au–Ab2; (b) Au@Cu2O–Ab2; (c) Au@CuxOS–Ab2. (B) Cyclic voltammograms of the immunosensor for the detection of 8 U/mL CA199 using Au@CuxOS–Ab2 without (a) and with 5 mmol/L H2O2 (b).
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3.2. Characterization of the immunosensor using Au@CuxOS as labels For sandwich-type immunosensors, the sensitivity is mainly determined by the label. Herein, the electrochemical signal of the immunosensor is based on the high electrocatalytic activity of Au@CuxOS for the reduction of H2O2. For comparison purposes, the catalytic performances of Au–Ab2, Au@Cu2O–Ab2, and Au@CuxOS–Ab2 toward H2O2 were investigated. The amperometric responses of the immunosensors prepared with different labels for the detection of 8 U/mL of CA199 toward H2O2 at 0.5 V are shown in Fig. 2. For the immunosensor using Au–Ab2, there was a little current response (curve a). For the immunosensor using Au@Cu2O–Ab2, there is a much larger current response (curve b). As expected, the immunosensor using Au@CuxOS–Ab2 as labels displayed the highest current change (curve c). These results show that the Au@CuxOS yolk–shell nanostructures are superior to Au or Au@Cu2O as labels in the fabrication of the immunosensor, it was necessary to use the label Au@CuxOS–Ab2 for the following experiments. Meanwhile, as shown in Fig. S1B, the cyclic voltammetrys (CVs) of Au@CuxOS–Ab2 modified electrode in pH 7.4 PBS (a) containing 5 mmol/L H2O2 (b) further proved the conclusion. In order to demonstrate the successful immobilization of Ab2 onto Au@CuxOS yolk–shell nanostructures, the Au@CuxOS–Ab2 was characterized using UV–vis spectroscopy (Fig. S2). According to the UV–vis spectral analysis, no absorption peak was observed for the Au@CuxOS yolk–shell nanostructures (curve c). A distinct absorption peak from the antibody was observed in the spectra of Au@CuxOS–Ab2 (curve b), which may be attributed to the absorption peak from the antibody itself at 282 nm (curve a). 3.3. Characterization of the fabrication of the immunosensor The stepwise construction process of the immunosensor was characterized by electrochemical impedance spectroscopy (EIS). EIS is an effective method for probing the features of a modified electrode surface (J.J. Zhang et al., 2010; Q. Zhang et al., 2010). The impedance spectra consist of a semicircle portion and a linear portion. The semicircle portion at higher frequencies corresponds to the electron-transfer-limited process, and the linear portion at lower frequencies represents the diffusion-limited process. The semicircle diameter equals the electron-transfer resistance (Ret). Fig. 3 illustrates the EIS of different electrodes in the presence of 2.5 mmol/L [Fe(CN)6]3 /4 containing 0.1 mol/L KCl. The potentiostatic condition of the EIS measurement was at 0.181 V. It can be seen that the bare GCE exhibited a very small semicircle domain (curve a), which is characteristic of a diffusion-limiting step in the electrochemical process. When the rGO–TEPA was modified onto the electrode (curve b), the resistance is much smaller than that of bare GCE, the reason for this observation was that rGO–TEPA is an excellent electrically conducting material, which makes electron transfer easier. After AuNPs was modified onto the rGO–TEPA/GCE (curve c), the resistance decreased is a little compared with the rGO–TEPA/GCE, which indicates that AuNPs is beneficial to the electron transfer. Then, after incubation with Ab1, the resistance was significantly enlarged, which indicates Ab1 was immobilized on the electrode successfully and blocked electron transfer (curve d). Additionally, a larger semicircle diameter of curve e can be found, clarifying the high resistance of the electrode interface as the prepared immunosensor is blocked with BSA (curve e). Subsequently, the value of resistance increased again (curve f), which indicates the successful capture of CA199 and the formation of immunocomplex layer blocking the electron transfer. When Au@CuxOS–Ab2 nanoparticles were immobilized, the resistance increased to the maximum (curve g), which indicates that the electrode was well-modified.
Fig. 3. EIS obtained for different modified electrodes in 0.1 mol/L KCl containing 2.5 mmol/L [Fe(CN)6]3 /4 (a) GCE; (b) rGO–TEPA/GCE; (c) AuNPs/rGO–TEPA/GCE; (d) Ab1/AuNPs/rGO–TEPA/GCE; (e) BSA/Ab1/AuNPs/rGO–TEPA/GCE; (f) CA199/ BSA/Ab1/AuNPs/rGO–TEPA/GCE; (g) Au@CuxOS–Ab2/CA199/BSA/Ab1/AuNPs/rGO– TEPA/GCE.
SEM images were also used for investigation of the surface structure and morphology of the modified electrode. The SEM image shown in Fig. S4a provided more detailed structure of rGO– TEPA/GCE, on which a wrinkled and paper-like structure can be observed, leading to increased surface area and thus electrochemical sensitivity of the modified electrode. It also can be seen that there were plenty of Au nanoparticles uniformly dispersed on the surface of rGO–TEPA/GCE (Fig. S4b), which indicated that Au nanoparticles had been successfully modified on the surface of rGO–TEPA/GCE. Fig. S4c shows that CA199/BSA/Ab1 had been immobilized on the surface of rGO–TEPA/GCE. Fig. S4d clearly shows that Au@CuxOS yolk–shell nanostructures with porous shells for the adsorption of biomolecules are globular structural morphology, indicating the successful immobilization of Au@Cux OS–Ab2 on the surface of modified electrode. 3.4. Optimization of experimental conditions The electrochemical performance of the CA199 immunosensor would be influenced by many factors. The pH of the working buffer has a great effect on the electrochemical behavior of immunosensor. During the immunosensor preparation process, the same concentration of CA199 (8 U/mL) was used to fabricate the immunosensor. To achieve an optimal electrochemical signaling, the pH value of the substrate solution is an important factor for the current response. As shown in Fig. 4A, the current response increases with the variation of pH from 4.5 to 7.4, and then decreases with the variation of pH from 7.4 to 9.1. The experimental results show that the optimal amperometric response was achieved at pH 7.4. It is likely because highly acidic or alkaline surroundings could damage the immobilized protein (Yuan et al., 2004). Thus, PBS at pH 7.4 was used as the electrolyte for all electrochemical measurement. The concentration of rGO–TEPA in the electrode modification was investigated on the electrochemical response of the immunosensor. As seen in Fig. 4B, with the increasing of the concentration of Au@CuxOS yolk–shell nanostructures solution, the current response of the electrode to 8 U/mL of CA199 increased highest at 2 mg/mL. Therefore, the optimal concentration of Au@CuxOS yolk– shell nanostructures was 2 mg/mL.
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Fig. 4. Effect of the pH (A); the concentration of Au@CuxOS (B); incubation time (C) and temperature (D) on the response of the immunosensor for the detection of 8 U/mL CA199.
The influence of incubation time was an important parameter for immunoreaction. The immunosensor was incubated in the 8 U/mL CA199 for different time periods. As shown in Fig. 4C, it was found that the amperometric response increased linearly with the reaction time increased from 15 to 60 min, and reached a maximum value at 60 min. Then it decreased from 60 to 90 min. As a result, an incubation time of 60 min was chosen in our work. The effect of incubation temperature for the antibody–antigen reaction was examined at the range from 15 to 55 °C. As can be seen in Fig. 4D, the amperometric response was increased with increasing temperature values from 15 to 37 °C, indicating that the formation of antibody–antigen immunocomplex was gradually promoted in the sufficient immunoreaction time. Meanwhile, the amperometric response was reduced with increasing higher temperatures (37–55 °C), which may be attributed to the fact that the higher temperature might cause irreversible denaturation of antibody and result in dissociation reaction of antibody–antigen conjugate. Therefore, the temperature of 37 °C was chosen to be a suitable incubation temperature.
3.5. Analytical performance of the immunosensor Under the optimum conditions, the immunosensors using Au@CuxOS–Ab2 as labels were used to detect different concentrations of CA199 in pH 7.4 PBS at 0.5 V. The relationship between the current response toward 5 mmol/L H2O2 and CA199 concentration is shown in Fig. 5. As can be seen, the catalytic current change was linear with the concentration of CA199 in the range from 0.001 to 12 U/mL with a regression equation of the calibration curve: Y¼18.93 þ9.562X and correlation coefficient of 0.9984 with a detection limit of 0.0005 U/mL. The low detection limit may be attributed to several factors: First, rGO–TEPA, a novel material, is a combination of rGO and tetraethylene pentamine through covalent bonding, which has large surface area, high conductivity and electronic property to transfer electrons thereby leading to higher sensitivity. Meanwhile, AuNPs possess good biocompatibility which was used to capture more Ab1 and amplify the signal. In addition, Au@CuxOS yolk–shell nanostructures have large specific surface area and show high electroactivity toward H2O2, which could greatly increase the probability of Ab2–antigen
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that might interfere with the response of immunosensor was investigated. Interference studies were performed using BSA, carbohydrate antigen 125 (CA125), carbohydrate antigen 724 (CA724) and squamous cell carcinoma antigen (SCCA). The 2 U/mL of CA199 solution containing 200 U/mL or 100 ng/mL of interfering substances was measured by the immunosensor and the results are shown in Fig. S3. The current variation due to the interfering substances was less than 5.0% of that without interferences, indicating that the selectivity of the immunosensor was acceptable. Stability of the immunosensors is also a key factor in their application and development. The stability of the immunosensor was examined by checking their current responses periodically. When the immunosensor was not in use, it was stored in pH 7.4 PBS at 4 °C, after 6 days, the current response of the immunosensor decreased 3.2%. After 3 weeks, the catalytic current of the immunosensor using Au@CuxOS yolk–shell nanostructures as labels decreased to about 87% of its initial value, suggesting the stability of the immunosensors was also acceptable. The slow decrease in the current response may be due to the gradual denaturation of antibody. 3.7. Application of the immunosensor in serum sample In order to evaluate the feasibility of the developed immunoassay for real sample analysis, the proposed immunosensor was used for the determination of CA199 in serum samples using the standard addition methods. Then, 1 U/mL, 3 U/mL and 5 U/mL of CA199 solution were added into serum samples. As shown in Table S2, the recovery of CA199 was from 98.7% to 102.4% and the relative standard deviation (RSD) was in the range of 2.09–3.54%. The fact showed that the developed immunoassay methodology could be preliminarily applied to the clinical determination of the CA199 levels in serum samples.
Fig. 5. (A) Amperometric response of the immunosensor for the varied concentration of CA199 at 0.5 V vs. Ag/AgCl in nitrogen-saturated PBS (5 mmol/L H2O2), with AFP concentration of (a) 0.001, (b) 0.005, (c) 0.05, (d) 0.1, (e) 0.3, (f) 0.5, (g) 1, (h) 3, (i) 5, (j) 8, (k) 10 and (l) 12; and (B) calibration curve of the immunosensor toward different concentrations of CA199. Error bar ¼ RSD (n¼ 5).
interactions thereby leading to higher sensitivity. Hence, the proposed strategy could provide a stable immobilization and sensitized recognition platform for analytes as micromolecules and possesses promising applications in clinical serum samples. The performance of the immunosensor was compared with previously described electrochemical immunosensors for the detection of CA199. As can be seen in Table S1, the immunosensor described here has a lower detection limit than previously described immunosensors. Therefore, the proposed electrochemical immunosensor showed higher sensitivity. 3.6. Reproducibility, selectivity, and stability of the immunosensor To evaluate the reproducibility of the immunosensor, a series of five electrodes were prepared for the detection of 5 U/mL CA199. The relative standard deviation (RSD) of the measurements for the five electrodes was 2.5%. The results indicated acceptable reproducibility and precision of the proposed immunoassay. The selectivity of the immunosensor was also investigated. Selective determination of target analytes plays an important role in analyzing biological samples. The effect of possible inhibitors
4. Conclusions A novel electrochemical immunosensor for sensitive detection of CA199 has been developed based on Au@CuxOS yolk–shell nanostructures with porous shells as labels for signal amplification. To construct high-performance electrochemical immunosensor, rGO–TEPA and AuNPs were immobilized on the electrode, which can increase the surface area to capture a large amount of primary antibodies (Ab1) as well as improve the electronic transmission rate. With a sandwich-type assay format, the signal was monitored through labeling the high electrocatalytic activity of Au@CuxOS yolk–shell nanostructures toward catalytic reduction of H2O2. The proposed immunosensor showed good reproducibility and excellent sensitivity, which could display low detection limit and high sensitivity. This developed strategy provides a new promising platform of clinical immunoassay for other biomolecules.
Acknowledgments This study was supported by the National Natural Science Foundation of China (Nos. 21175057, 21375047, and 21377046), the Science and Technology Plan Project of Jinan (No. 201307010) and QW thank the Special Foundation for Taishan Scholar Professorship of Shandong Province and UJN (No. ts20130937).
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Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.017.
References Ahirwal, G.K., Mitra, C.K., 2010. Biosens. Bioelectron. 25, 2016–2020. Chen, H.F., Gao, Z.Q., Cui, Y.L., Chen, G.N., Tang, D.P., 2013. Biosens. Bioelectron. 44, 108–114. Chen, X.J., Zhang, K., Zhou, J.J., Xuan, J., Yan, W., Jiang, L.P., Zhu, J.J., 2010. Biosens. Bioelectron. 25, 1130–1136. Del Villano, B.C., Brennan, S., Brock, P., Bucher, C., Liu, V., McClure, M., Rake, B., Space, S., Westrick, B., Schoemaker, H., Zurawski Jr., V.R., 1983. Clin. Chem. 29, 549–552. Ding, Y., Chen, M.W., Erlebacher, J., 2004. J. Am. Chem. Soc. 126, 6876–6877. Du, D., Xu, X.X., Wang, S.F., Zhang, A.D., 2007. Talanta 71, 1257–1262. Fu, Z.F., Liu, H., Ju, H.X., 2006. Anal. Chem. 78, 6999–7005. Fu, Z.F., Yang, Z.J., Tang, J.H., Liu, H., Yan, F., Ju, H.X., 2007. Anal. Chem. 79, 7376– 7382. Goonetilleke, K.S., Siriwardena, A.K., 2007. Eur. J. Surg. Oncol. 33, 266–270. Gan, T., Hu, C.G., Chen, Z.L., Hu, S.S., 2011. Talanta 85, 310–316. Huang, J.L., Yang, G.G., Meng, W.J., Wu, L.P., Zhu, A.P., Jiao, X.A., 2010. Biosens. Bioelectron. 25, 1204–1211. Huang, K.J., Li, J., Wu, Y.Y., Liu, Y.M., 2013. Bioelectrochemistry 90, 18–23. Huang, T.Y., Huang, J.H., Wei, H.Y., Ho, K.C., Chu, C.W., 2013. Biosens. Bioelectron. 43, 173–179. Kamata, K., Lu, Y., Xia, Y.N., 2003. J. Am. Chem. Soc. 125, 2384–2385. Kaur, B., Pandiyan, T., Satpati, B., Srivastava, R., 2013. Colloid Surf. B. 111, 97–106. Kim, J., Kim, H.S., Lee, N., Kim, T., Kim, H., Yu, T., Song, I.C., Moon, W.K., Hyeon, T., 2008. Angew. Chem. Int. Ed. 47, 8438–8441.
Kitano, H., 2002. Science 295, 1662–1664. Knopp, D., 2006. Anal. Bioanal. Chem. 385, 425–427. Li, F., Feng, Y., Dong, P.J., Yang, L.M., Tang, B., 2011. Biosens. Bioelectron. 26, 1947– 1952. Liu, J.W., Cheng, J., Che, R.C., Xu, J.J., Liu, M.M., Liu, Z.W., 2013. J. Phys. Chem. C 117, 489–495. Long, S.B., Campbell, E.B., Mackinnon, R., 2005. Science 309, 903–908. Lu, Y., Zhao, Y., Yu, L., Dong, L., Shi, C., Hu, M.J., Xu, Y.J., Wen, L.P., Yu, S.H., 2010. Adv. Mater. 22, 1407–1411. Park, J.C., Song, H., 2011. Nano Res. 4, 33–49. Sanles-Sobrido, M., Exner, W., Rodriguez-Lorenzo, L., Rodriguez-Gonzalez, B., Correa-Duarte, M.A., Alvarez-Puebla, R.A., Liz-Marzan, L.M., 2009. J. Am. Chem. Soc. 131, 2699–2705. Sánchez, S., Roldán, M., Pérez, S., Fàbregas, E., 2008. Anal. Chem. 80, 6508–6514. Srinivas, P.R., Kramer, B.S., Srivastava, S., 2001. Lancet Oncol. 2, 698–704. Sun, B., Zhang, K., Chen, L.J., Guo, L.T., Ai, S.Y., 2013. Biosens. Bioelectron. 44 48–51 Tang, D.P., Tang, J., Su, B.L., Ren, J.J., Chen, G.N., 2010. Biosens. Bioelectron. 25, 1658– 1662. Teymourian, H., Salimi, A., Khezrian, S., 2013. Biosens. Bioelectron. 49, 1–8. Wang, F., Chen, Y.H., Liu, C.Y., Ma, D.G., 2011. Chem. Commun. 47, 3502–3504. Wei, Q., Li, T., Wang, G.L., Li, H., Qian, Z.Y., Yang, M.H., 2010. Biomaterials 31, 7332– 7339. Wild, D., 2001. The Immunoassay Handbook. Nature Publishing Group, London, UK, pp. 643–644. Wu, J., Yan, Y.T., Yan, F., Ju, H.X., 2008. Anal. Chem. 80, 6072–6077. Yuan, R., Tang, D.P., Chai, Y.Q., Zhong, X., Liu, Y., Dai, J.Y., 2004. Langmuir 20, 7240– 7245. Zhang, J.J., Cheng, F.F., Zheng, T.T., Zhu, J.J., 2010. Anal. Chem. 82, 3547–3555. Zhang, Q., Lee, I., Ge, J.P., Zaera, F., Yin, Y.D., 2010. Adv. Funct. Mater. 20, 2201–2214. Zhang, Y.X., Cheng, Y.X., Zhou, Y.Y., Li, B.G., Gu, W., Shi, X.H., Xian, Y.Z., 2013. Talanta 107, 211–218. Zhou, L.Z., Kuai, L., Li, W.Z., Geng, B.Y., 2012. Appl. Mater. Interfaces 4, 6463–6467. Zhou, M., Zhai, Y.M., Dong, S.J., 2009. Anal. Chem. 81, 5603–5613.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
An electrochemical immunosensor for ultrasensitive detection of carbohydrate antigen 199 based on Au@CuxOS yolk–shell nanostructures with porous shells as labels Aiping Guo a, Yueyun Li b, Wei Cao a, Xianchao Meng a, Dan Wu a, Qin Wei a,n, Bin Du a a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b School of Chemical Engineering, Shandong University of Technology, Zibo 255049, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 18 June 2014 Accepted 8 July 2014 Available online 11 July 2014
A novel and sensitive electrochemical immunosensor for ultrasensitive detection of pancreatic cancer biomarker carbohydrate antigen 199 (CA199) was proposed by using Au@CuxOS yolk–shell nanostructures with porous shells as labels for signal amplification. Au@CuxOS yolk–shell nanostructures exhibit high electrocatalytic activity toward the reduction of hydrogen peroxide (H2O2) as analytical signal. Moreover, secondary antibody (Ab2) can adsorb on the surface of Au@CuxOS with porous shells which has large surface area and could greatly increase the probability of Ab2–antigen interactions thereby leading to higher sensitivity. Reduced graphene oxide–tetraethylene pentamine (rGO–TEPA), containing abundant amine groups, was supported Au nanoparticles as a support platform to immobilize the primary antibody (Ab1). The resulting sensing interface of rGO–TEPA/AuNPs could provide a large electroconductive surface area, allowing high loadings of the biological recognition elements as well as the occurrence of electrocatalytic and electron-transfer processes. Under optimal conditions, the immunosensor exhibited a wide linear response to CA199 ranging from 0.001 to 12 U/mL with a low detection limit of 0.0005 U/mL. The designed immunosensor displayed good precision, high sensitivity, acceptable stability and reproducibility, and has been applied to the analysis of serum with satisfactory results. The proposed method provides a new promising platform of clinical immunoassay for other biomolecules. & 2014 Elsevier B.V. All rights reserved.
Keywords: Pancreatic cancer biomarker CA199 Au@CuxOS yolk–shell nanostructures with porous shells Reduced graphene oxide–tetraethylene pentamine (rGO–TEPA) Au nanoparticles
1. Introduction Sensitive and accurate detection of disease-related biomarkers is critical to many areas of biomedical research and diagnosis (Kitano, 2002). In particular, the clinical measurement of cancer biomarkers shows great promise for early diagnosis, disease monitoring, and highly reliable prediction (Chen et al., 2013; Srinivas et al., 2001; Wang et al., 2011). Carbohydrate antigen 199 (CA199) is a preferred label for pancreatic cancer, which is a highly lethiferous sarcomata and elevates in the peripheral blood of the majority of pancreatic cancer patients but does not achieve the performance required for either early detection or diagnosis, due to both false positive and false negative readings (Goonetilleke and Siriwardena, 2007). It is reported to be frequently found out at the patients of pancreatic cancer with a high score of over 79% (Del Villano et al., 1983; Wild, 2001). Thus, the detection of CA199 level n
Corresponding author. Tel.: þ 86 531 82767872; fax: þ86 531 82765969. E-mail address: [email protected] (Q. Wei).
http://dx.doi.org/10.1016/j.bios.2014.07.017 0956-5663/& 2014 Elsevier B.V. All rights reserved.
in human serum plays an important role in the diagnosis and management of pancreatic cancer. Various methods have been employed to detect CA199, such as electric field-driven assay (Wu et al., 2008), immobilized horseradish peroxidase assay (Du et al., 2007), and chemiluminescent multiplex assay (Fu et al., 2007). While highly accurate, some of these techniques involve disadvantages such as relatively sophisticated instruments, significant sample volume, limited sensitivity, and clinically unrealistic expense and time. Therefore, there is a real need to develop operationally simple, highly sensitive, and inexpensive methods to detect levels of the biomarkers in both normal and cancer patient sera. Electrochemical immunosensors are bioanalytical tools that feature simplicity of construction, the possibility to be massproduced, cost-effectiveness, ease of use, feasible miniaturization, and subsequent portability. These advantages make it attractive for use as a high-performance screening tool for the detection of biomolecules (Tang et al., 2010; Chen et al., 2010). Signal amplification and noise reduction are crucial for obtaining low detection limits in clinical immunoassays (Fu et al., 2006; Long et al., 2005).
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Sandwich-type immunoassay has the advantages of high specificity and sensitivity because of the use of a couple of match antibodies (Knopp, 2006), which is a widely used protocol for ultrasensitive detection due to the high selectivity, low cost, and ease of miniaturization (Ahirwal and Mitra, 2010; Wei et al., 2010). One key research area of electrochemical immunosensors is development of biosensors with various nanomaterials of physical and chemical properties, because they can provide a large surface area and improve the biocompatibility and stability of the system (K.J. Huang et al., 2013; T.Y. Huang et al. 2013; Zhou et al., 2009). Reduced graphene oxide (rGO) is often used in electrochemical sensors (Gan et al., 2011; K.J. Huang et al., 2013; T.Y. Huang et al. 2013; Kaur et al., 2013; Sun et al., 2013; Teymourian et al., 2013; Zhang et al., 2013), because its abundant defects and chemical groups facilitate charge transfer and thus ensure high electrochemical activity. Moreover, the populated chemical moieties on the rGO surface offer the convenience and flexibility for various functionalizations to enhance the sensor performance. More importantly, rGO may also be functionalized through covalent or non-covalent methods in order to further enhance its sensitivity, specificity, loading capacity, biocompatibility, etc. Reduced graphene oxide–tetraethylene pentamine (rGO–TEPA) is a novel material which is a combination of rGO and tetraethylene pentamine through covalent bonding. The structure of rGO–TEPA is shown in Fig. S1. It not only keeps the original property of rGO but also promotes water solubility. Additionally, AuNPs are extremely attractive materials for fabricating sensors because of its unique properties, including simple preparation, good biocompatibility, excellent conductivity, and easy stabilization of the colloidal dispersion by using covalent or noncovalent surface coatings (Ding et al., 2004; Li et al., 2011). Then, based on these properties, rGO–TEPA/AuNPs could be an excellent immunosensor platform because of the combination of physical and chemical properties of rGO–TEPA and the surface reactivity of AuNPs for immobilizing a large amount of antibodies, and the application of rGO–TEPA/ AuNPs as a sensor platform not only increases the amount of immobilized antibodies, but also accelerates the electron transfer process assisted by AuNPs for signal enhancement, which further enhance the sensitivity of the immunosensor. In the design and fabrication of highly sensitive electrochemical immunosensors, signal amplification and antibody immobilization are the crucial steps (Sánchez et al., 2008). Recently, yolk– shell nanostructures have attracted considerable attention because of their various applications in catalysis, batteries, and biomedical fields (Kim et al., 2008; Liu et al., 2013, 2010; Sanles-Sobrido et al., 2009). The yolk–shell nanostructures possess the unique structure of a hollow shell and an encapsulated single spherical core. The difference between yolk–shell and core–shell nanostructure is the space between core and shell. This space can act as a nanoreactor and enhance the catalytic performance (Park and Song, 2011; J.J. Zhang et al., 2010; Q. Zhang et al., 2010). The yolk–shell nanostructures have some excellent properties, such as low density, good flow ability, and its ease of functionalization (Kamata et al., 2003). In this paper, a metal/semiconductor yolk–shell nanostructure Au@CuxOS with porous shells was prepared and used as labels to adsorb secondary antibodies (Huang et al., 2010). Au@CuxOS yolk–shell nanostructures show high electrocatalytic activity toward hydrogen peroxide (H2O2) reduction, which could produce an electrocatalytic response by reduction of H2O2 and significantly amplify electrochemical signals. Herein, we demonstrated a unique nonenzymatic sandwichtype immunosensor based on rGO–TEPA/AuNPs modified the electrode as a supporting interface and Au@CuxOS yolk–shell nanostructures with porous shells as labels for the highly sensitive detection of CA199. The proposed rGO–TEPA/AuNPs can be used as an immunosensing platform because it exhibits excellent
electrically conductivity property, hydrophilicity and large specific surface area. Meanwhile, we used Au@CuxOS yolk–shell nanostructures with porous shells for the immobilization of secondary antibody (Ab2) to obtain the Au@CuxOS–Ab2 bio-conjugates, which could produce an electrocatalytic response by reduction of H2O2 and significantly amplify electrochemical signals. The results of electrochemical studies suggested that the developed immunosensor possesses great performance for CA199 determination and has a great potential application available for the analysis of other low-abundant proteins.
2. Experimental section 2.1. Materials and apparatus Reduced graphene oxide–tetraethylene pentamine (rGO–TEPA) were purchased from Nanjing XFNANO Materials TECH Co., Ltd. (China). Carbohydrate antigen 199 (CA199) and anti-CA199 antibody (Ab) were purchased from Shanghai Linc-Bio Science Co. Ltd. (China). K3Fe(CN)6 was purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (China). Bovine serum albumin (BSA, 96–99%) was purchased from Sigma (USA) and used as received. Hydrazine hydrate solution (H4N2 H2O, 80%), copper (II) nitrate trihydrate (Cu(NO3)2 3H2O), and sodium sulfide (Na2S 9H2O) were purchased from Shanghai Reagents Limited Corporation (Shanghai, China). HAuCl4 3H2O were obtained from Sigma-Aldrich (Beijing, China). Phosphate buffered saline (PBS, 0.1 mol/L containing 0.1 mol/L NaCl, pH 7.4) was used as an electrolyte for all electrochemical measurements. Doubly distilled water was used throughout the experiments. All electrochemical measurements were performed on a CHI760D electrochemical workstation (Shanghai CH Instruments Co., China). Transmission electron microscope (TEM) images were obtained from a Hitachi H-800 microscope (Japan). Scanning electron microscope (SEM) images were obtained using field emission SEM (ZEISS, Germany). Electrochemical impedance spectroscopy (EIS) was performed in [Fe(CN)6]3 /4 working solution in the frequency range of 0.1–105 Hz. A conventional threeelectrode system was used for all electrochemical measurements: the modified 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. 2.2. Synthesis of Au nanoparticles (AuNPs) The AuNPs were prepared by the following procedures: 0.50 g of PVP was added into 10 mL of deionized water and then 15 mL of 0.1 M ascorbic acid and 2.5 mL of 9.5 mM HAuCl4 were added into the above solution under continuous stirring for 30 min at room temperature. The products were centrifuged and washed with deionized water and ethanol 3 times in turn. The obtained AuNPs were dispersed in 10 mL of deionized water for further use. 2.3. Synthesis of Au@CuxOS yolk–shell nanostructures Au@CuxOS yolk–shell nanostructures were synthesized by a modified literature procedure (Zhou et al., 2012). In a typical procedure, Au@Cu2O core–shell nanostructures were firstly prepared as follows. 0.20 g of PVP and 1.0 mL of AuNPs solution were added into 10 mL of 10 mM Cu(NO3)2 3H2O solution. After that, 10 μL of H4N2 H2O was added into the solution, the mixture was stirred vigorously for 2 min. The products were then collected by centrifugation, washed with deionized water and ethanol 3 times, and dried at 60 °C in a vacuum.
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Scheme 1. Schematic illustration of the stepwise preparation of the Au@CuxOS––Ab2 (A) and immunosensor (B).
The obtained Au@Cu2O core–shell nanostructures were dissolved into 10 mL of water, and then 0.12 g of Na2S powder was added under vigorous stirring for 1 h to ensure they were sufficiently sulfurized, resulting in the formation of Au@CuxOS yolk–shell nanostructures with porous shells. The formation of yolk–shell nanostructures is because O2 diffuses quicker than S2 because of the larger ionic radius of S2 than O2 . The products were collected by centrifugation, and washed with deionized water and absolute ethanol two times to remove the impurities. 2.4. Preparation of Au@CuxOS–Ab2 The preparation procedure of Au@CuxOS–Ab2 is illustrated in Scheme 1A, The Au@CuxOS (2 mg) was dispersed in 1.0 mL of pH 7.4 PBS. This dispersion was then mixed with 1.0 mL of 10 mg/mL of anti-CA199. The mixture was allowed to react at room temperature under stirring for 60 min, followed by centrifugation. The resulting Au@CuxOS–Ab2 was washed with PBS and then redispersed in 1.0 mL of PBS and stored at 4 °C before use. 2.5. Fabrication of the immunosensor Scheme 1B shows the schematic representation of the preparation of the immunosensor. GCE was polished with 1, 0.3, and 0.05 μm alumina powder sequentially, and then washed ultrasonically in ethanol for a few minutes and dried in air at room
temperature. Afterwards, 6 μL of rGO–TEPA solution was initially dropped on the electrode surface and dried at room temperature. Then, 6 μL of AuNPs was added onto electrode surface. After 1 h of reaction and washing, 6 μL of prepared excessive anti-CA199 solution (10 μg/mL) was added onto the electrode surface and incubated for 1 h. The electrode was then washed and incubated in 1 wt% BSA solution to block nonspecific binding sites. Subsequently, CA199 buffer solution with a varying concentration was added onto the electrode surface and incubated for 1 h, and then the electrode was washed extensively to remove unbound CA199 molecules. Finally, the prepared Au@CuxOS–Ab2 was dropped onto the electrode surface. After another 1 h, the electrode was washed and ready for measurement.
3. Results and discussion 3.1. Characterization of Au@CuxOS, rGO–TEPA and AuNPs As shown in Fig. 1a and b, the rGO–TEPA is observed to have a wrinkled, paper-like structure, and is transparent with irregular size. It has the large surface area in favor of electron transportation. Au nanoparticles are well monodispersed and uniformly spherical in shape (Fig. 1c). The mean size of Au particles was approximately 35 nm. The morphology of Au@CuxOS was yolk– shell nanostructures in the size range 240 nm with hollow porous shell (Fig. 1d), which gives rise to a large specific surface area and
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Fig. 1. SEM and TEM image of rGO–TEPA (a and b); TEM image of AuNPs (c); SEM and TEM image of Au@CuxOS (d and e); EDX image of Au@CuxOS (f).
will be especially effective for the further adsorption of biomolecules. A detail surface structure of Au@CuxOS yolk–shell nanostructures is shown in Fig. 1e, the SEM displayed a type of spherical morphology and dispersed homogeneously. EDX spectrum was carried out to reveal the components of the as-prepared yolk–shell nanostructure. As shown in Fig. 1f, obvious Au, Cu, S, and O
elements were observed, in which the signal of Au is assigned to the Au core, and that of Cu and S elements are belonged to the shell. The Au@CuxOS yolk–shell nanostructure has different valence states of Cu ions containing CuS and Cu2O, which is the reason that defined as “x”. Thus, the final product is called Au@Cux OS yolk–shell nanostructure.
Fig. 2. (A) Amperometric response of the immunosensors for the detection of 8 U/mL CA199 with different labels at 0.5 V vs. Ag/AgCl toward successive addition of 5 mmol/L H2O2 in N2-saturated PBS: (a) Au–Ab2; (b) Au@Cu2O–Ab2; (c) Au@CuxOS–Ab2. (B) Cyclic voltammograms of the immunosensor for the detection of 8 U/mL CA199 using Au@CuxOS–Ab2 without (a) and with 5 mmol/L H2O2 (b).
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3.2. Characterization of the immunosensor using Au@CuxOS as labels For sandwich-type immunosensors, the sensitivity is mainly determined by the label. Herein, the electrochemical signal of the immunosensor is based on the high electrocatalytic activity of Au@CuxOS for the reduction of H2O2. For comparison purposes, the catalytic performances of Au–Ab2, Au@Cu2O–Ab2, and Au@CuxOS–Ab2 toward H2O2 were investigated. The amperometric responses of the immunosensors prepared with different labels for the detection of 8 U/mL of CA199 toward H2O2 at 0.5 V are shown in Fig. 2. For the immunosensor using Au–Ab2, there was a little current response (curve a). For the immunosensor using Au@Cu2O–Ab2, there is a much larger current response (curve b). As expected, the immunosensor using Au@CuxOS–Ab2 as labels displayed the highest current change (curve c). These results show that the Au@CuxOS yolk–shell nanostructures are superior to Au or Au@Cu2O as labels in the fabrication of the immunosensor, it was necessary to use the label Au@CuxOS–Ab2 for the following experiments. Meanwhile, as shown in Fig. S1B, the cyclic voltammetrys (CVs) of Au@CuxOS–Ab2 modified electrode in pH 7.4 PBS (a) containing 5 mmol/L H2O2 (b) further proved the conclusion. In order to demonstrate the successful immobilization of Ab2 onto Au@CuxOS yolk–shell nanostructures, the Au@CuxOS–Ab2 was characterized using UV–vis spectroscopy (Fig. S2). According to the UV–vis spectral analysis, no absorption peak was observed for the Au@CuxOS yolk–shell nanostructures (curve c). A distinct absorption peak from the antibody was observed in the spectra of Au@CuxOS–Ab2 (curve b), which may be attributed to the absorption peak from the antibody itself at 282 nm (curve a). 3.3. Characterization of the fabrication of the immunosensor The stepwise construction process of the immunosensor was characterized by electrochemical impedance spectroscopy (EIS). EIS is an effective method for probing the features of a modified electrode surface (J.J. Zhang et al., 2010; Q. Zhang et al., 2010). The impedance spectra consist of a semicircle portion and a linear portion. The semicircle portion at higher frequencies corresponds to the electron-transfer-limited process, and the linear portion at lower frequencies represents the diffusion-limited process. The semicircle diameter equals the electron-transfer resistance (Ret). Fig. 3 illustrates the EIS of different electrodes in the presence of 2.5 mmol/L [Fe(CN)6]3 /4 containing 0.1 mol/L KCl. The potentiostatic condition of the EIS measurement was at 0.181 V. It can be seen that the bare GCE exhibited a very small semicircle domain (curve a), which is characteristic of a diffusion-limiting step in the electrochemical process. When the rGO–TEPA was modified onto the electrode (curve b), the resistance is much smaller than that of bare GCE, the reason for this observation was that rGO–TEPA is an excellent electrically conducting material, which makes electron transfer easier. After AuNPs was modified onto the rGO–TEPA/GCE (curve c), the resistance decreased is a little compared with the rGO–TEPA/GCE, which indicates that AuNPs is beneficial to the electron transfer. Then, after incubation with Ab1, the resistance was significantly enlarged, which indicates Ab1 was immobilized on the electrode successfully and blocked electron transfer (curve d). Additionally, a larger semicircle diameter of curve e can be found, clarifying the high resistance of the electrode interface as the prepared immunosensor is blocked with BSA (curve e). Subsequently, the value of resistance increased again (curve f), which indicates the successful capture of CA199 and the formation of immunocomplex layer blocking the electron transfer. When Au@CuxOS–Ab2 nanoparticles were immobilized, the resistance increased to the maximum (curve g), which indicates that the electrode was well-modified.
Fig. 3. EIS obtained for different modified electrodes in 0.1 mol/L KCl containing 2.5 mmol/L [Fe(CN)6]3 /4 (a) GCE; (b) rGO–TEPA/GCE; (c) AuNPs/rGO–TEPA/GCE; (d) Ab1/AuNPs/rGO–TEPA/GCE; (e) BSA/Ab1/AuNPs/rGO–TEPA/GCE; (f) CA199/ BSA/Ab1/AuNPs/rGO–TEPA/GCE; (g) Au@CuxOS–Ab2/CA199/BSA/Ab1/AuNPs/rGO– TEPA/GCE.
SEM images were also used for investigation of the surface structure and morphology of the modified electrode. The SEM image shown in Fig. S4a provided more detailed structure of rGO– TEPA/GCE, on which a wrinkled and paper-like structure can be observed, leading to increased surface area and thus electrochemical sensitivity of the modified electrode. It also can be seen that there were plenty of Au nanoparticles uniformly dispersed on the surface of rGO–TEPA/GCE (Fig. S4b), which indicated that Au nanoparticles had been successfully modified on the surface of rGO–TEPA/GCE. Fig. S4c shows that CA199/BSA/Ab1 had been immobilized on the surface of rGO–TEPA/GCE. Fig. S4d clearly shows that Au@CuxOS yolk–shell nanostructures with porous shells for the adsorption of biomolecules are globular structural morphology, indicating the successful immobilization of Au@Cux OS–Ab2 on the surface of modified electrode. 3.4. Optimization of experimental conditions The electrochemical performance of the CA199 immunosensor would be influenced by many factors. The pH of the working buffer has a great effect on the electrochemical behavior of immunosensor. During the immunosensor preparation process, the same concentration of CA199 (8 U/mL) was used to fabricate the immunosensor. To achieve an optimal electrochemical signaling, the pH value of the substrate solution is an important factor for the current response. As shown in Fig. 4A, the current response increases with the variation of pH from 4.5 to 7.4, and then decreases with the variation of pH from 7.4 to 9.1. The experimental results show that the optimal amperometric response was achieved at pH 7.4. It is likely because highly acidic or alkaline surroundings could damage the immobilized protein (Yuan et al., 2004). Thus, PBS at pH 7.4 was used as the electrolyte for all electrochemical measurement. The concentration of rGO–TEPA in the electrode modification was investigated on the electrochemical response of the immunosensor. As seen in Fig. 4B, with the increasing of the concentration of Au@CuxOS yolk–shell nanostructures solution, the current response of the electrode to 8 U/mL of CA199 increased highest at 2 mg/mL. Therefore, the optimal concentration of Au@CuxOS yolk– shell nanostructures was 2 mg/mL.
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Fig. 4. Effect of the pH (A); the concentration of Au@CuxOS (B); incubation time (C) and temperature (D) on the response of the immunosensor for the detection of 8 U/mL CA199.
The influence of incubation time was an important parameter for immunoreaction. The immunosensor was incubated in the 8 U/mL CA199 for different time periods. As shown in Fig. 4C, it was found that the amperometric response increased linearly with the reaction time increased from 15 to 60 min, and reached a maximum value at 60 min. Then it decreased from 60 to 90 min. As a result, an incubation time of 60 min was chosen in our work. The effect of incubation temperature for the antibody–antigen reaction was examined at the range from 15 to 55 °C. As can be seen in Fig. 4D, the amperometric response was increased with increasing temperature values from 15 to 37 °C, indicating that the formation of antibody–antigen immunocomplex was gradually promoted in the sufficient immunoreaction time. Meanwhile, the amperometric response was reduced with increasing higher temperatures (37–55 °C), which may be attributed to the fact that the higher temperature might cause irreversible denaturation of antibody and result in dissociation reaction of antibody–antigen conjugate. Therefore, the temperature of 37 °C was chosen to be a suitable incubation temperature.
3.5. Analytical performance of the immunosensor Under the optimum conditions, the immunosensors using Au@CuxOS–Ab2 as labels were used to detect different concentrations of CA199 in pH 7.4 PBS at 0.5 V. The relationship between the current response toward 5 mmol/L H2O2 and CA199 concentration is shown in Fig. 5. As can be seen, the catalytic current change was linear with the concentration of CA199 in the range from 0.001 to 12 U/mL with a regression equation of the calibration curve: Y¼18.93 þ9.562X and correlation coefficient of 0.9984 with a detection limit of 0.0005 U/mL. The low detection limit may be attributed to several factors: First, rGO–TEPA, a novel material, is a combination of rGO and tetraethylene pentamine through covalent bonding, which has large surface area, high conductivity and electronic property to transfer electrons thereby leading to higher sensitivity. Meanwhile, AuNPs possess good biocompatibility which was used to capture more Ab1 and amplify the signal. In addition, Au@CuxOS yolk–shell nanostructures have large specific surface area and show high electroactivity toward H2O2, which could greatly increase the probability of Ab2–antigen
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that might interfere with the response of immunosensor was investigated. Interference studies were performed using BSA, carbohydrate antigen 125 (CA125), carbohydrate antigen 724 (CA724) and squamous cell carcinoma antigen (SCCA). The 2 U/mL of CA199 solution containing 200 U/mL or 100 ng/mL of interfering substances was measured by the immunosensor and the results are shown in Fig. S3. The current variation due to the interfering substances was less than 5.0% of that without interferences, indicating that the selectivity of the immunosensor was acceptable. Stability of the immunosensors is also a key factor in their application and development. The stability of the immunosensor was examined by checking their current responses periodically. When the immunosensor was not in use, it was stored in pH 7.4 PBS at 4 °C, after 6 days, the current response of the immunosensor decreased 3.2%. After 3 weeks, the catalytic current of the immunosensor using Au@CuxOS yolk–shell nanostructures as labels decreased to about 87% of its initial value, suggesting the stability of the immunosensors was also acceptable. The slow decrease in the current response may be due to the gradual denaturation of antibody. 3.7. Application of the immunosensor in serum sample In order to evaluate the feasibility of the developed immunoassay for real sample analysis, the proposed immunosensor was used for the determination of CA199 in serum samples using the standard addition methods. Then, 1 U/mL, 3 U/mL and 5 U/mL of CA199 solution were added into serum samples. As shown in Table S2, the recovery of CA199 was from 98.7% to 102.4% and the relative standard deviation (RSD) was in the range of 2.09–3.54%. The fact showed that the developed immunoassay methodology could be preliminarily applied to the clinical determination of the CA199 levels in serum samples.
Fig. 5. (A) Amperometric response of the immunosensor for the varied concentration of CA199 at 0.5 V vs. Ag/AgCl in nitrogen-saturated PBS (5 mmol/L H2O2), with AFP concentration of (a) 0.001, (b) 0.005, (c) 0.05, (d) 0.1, (e) 0.3, (f) 0.5, (g) 1, (h) 3, (i) 5, (j) 8, (k) 10 and (l) 12; and (B) calibration curve of the immunosensor toward different concentrations of CA199. Error bar ¼ RSD (n¼ 5).
interactions thereby leading to higher sensitivity. Hence, the proposed strategy could provide a stable immobilization and sensitized recognition platform for analytes as micromolecules and possesses promising applications in clinical serum samples. The performance of the immunosensor was compared with previously described electrochemical immunosensors for the detection of CA199. As can be seen in Table S1, the immunosensor described here has a lower detection limit than previously described immunosensors. Therefore, the proposed electrochemical immunosensor showed higher sensitivity. 3.6. Reproducibility, selectivity, and stability of the immunosensor To evaluate the reproducibility of the immunosensor, a series of five electrodes were prepared for the detection of 5 U/mL CA199. The relative standard deviation (RSD) of the measurements for the five electrodes was 2.5%. The results indicated acceptable reproducibility and precision of the proposed immunoassay. The selectivity of the immunosensor was also investigated. Selective determination of target analytes plays an important role in analyzing biological samples. The effect of possible inhibitors
4. Conclusions A novel electrochemical immunosensor for sensitive detection of CA199 has been developed based on Au@CuxOS yolk–shell nanostructures with porous shells as labels for signal amplification. To construct high-performance electrochemical immunosensor, rGO–TEPA and AuNPs were immobilized on the electrode, which can increase the surface area to capture a large amount of primary antibodies (Ab1) as well as improve the electronic transmission rate. With a sandwich-type assay format, the signal was monitored through labeling the high electrocatalytic activity of Au@CuxOS yolk–shell nanostructures toward catalytic reduction of H2O2. The proposed immunosensor showed good reproducibility and excellent sensitivity, which could display low detection limit and high sensitivity. This developed strategy provides a new promising platform of clinical immunoassay for other biomolecules.
Acknowledgments This study was supported by the National Natural Science Foundation of China (Nos. 21175057, 21375047, and 21377046), the Science and Technology Plan Project of Jinan (No. 201307010) and QW thank the Special Foundation for Taishan Scholar Professorship of Shandong Province and UJN (No. ts20130937).
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Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.017.
References Ahirwal, G.K., Mitra, C.K., 2010. Biosens. Bioelectron. 25, 2016–2020. Chen, H.F., Gao, Z.Q., Cui, Y.L., Chen, G.N., Tang, D.P., 2013. Biosens. Bioelectron. 44, 108–114. Chen, X.J., Zhang, K., Zhou, J.J., Xuan, J., Yan, W., Jiang, L.P., Zhu, J.J., 2010. Biosens. Bioelectron. 25, 1130–1136. Del Villano, B.C., Brennan, S., Brock, P., Bucher, C., Liu, V., McClure, M., Rake, B., Space, S., Westrick, B., Schoemaker, H., Zurawski Jr., V.R., 1983. Clin. Chem. 29, 549–552. Ding, Y., Chen, M.W., Erlebacher, J., 2004. J. Am. Chem. Soc. 126, 6876–6877. Du, D., Xu, X.X., Wang, S.F., Zhang, A.D., 2007. Talanta 71, 1257–1262. Fu, Z.F., Liu, H., Ju, H.X., 2006. Anal. Chem. 78, 6999–7005. Fu, Z.F., Yang, Z.J., Tang, J.H., Liu, H., Yan, F., Ju, H.X., 2007. Anal. Chem. 79, 7376– 7382. Goonetilleke, K.S., Siriwardena, A.K., 2007. Eur. J. Surg. Oncol. 33, 266–270. Gan, T., Hu, C.G., Chen, Z.L., Hu, S.S., 2011. Talanta 85, 310–316. Huang, J.L., Yang, G.G., Meng, W.J., Wu, L.P., Zhu, A.P., Jiao, X.A., 2010. Biosens. Bioelectron. 25, 1204–1211. Huang, K.J., Li, J., Wu, Y.Y., Liu, Y.M., 2013. Bioelectrochemistry 90, 18–23. Huang, T.Y., Huang, J.H., Wei, H.Y., Ho, K.C., Chu, C.W., 2013. Biosens. Bioelectron. 43, 173–179. Kamata, K., Lu, Y., Xia, Y.N., 2003. J. Am. Chem. Soc. 125, 2384–2385. Kaur, B., Pandiyan, T., Satpati, B., Srivastava, R., 2013. Colloid Surf. B. 111, 97–106. Kim, J., Kim, H.S., Lee, N., Kim, T., Kim, H., Yu, T., Song, I.C., Moon, W.K., Hyeon, T., 2008. Angew. Chem. Int. Ed. 47, 8438–8441.
Kitano, H., 2002. Science 295, 1662–1664. Knopp, D., 2006. Anal. Bioanal. Chem. 385, 425–427. Li, F., Feng, Y., Dong, P.J., Yang, L.M., Tang, B., 2011. Biosens. Bioelectron. 26, 1947– 1952. Liu, J.W., Cheng, J., Che, R.C., Xu, J.J., Liu, M.M., Liu, Z.W., 2013. J. Phys. Chem. C 117, 489–495. Long, S.B., Campbell, E.B., Mackinnon, R., 2005. Science 309, 903–908. Lu, Y., Zhao, Y., Yu, L., Dong, L., Shi, C., Hu, M.J., Xu, Y.J., Wen, L.P., Yu, S.H., 2010. Adv. Mater. 22, 1407–1411. Park, J.C., Song, H., 2011. Nano Res. 4, 33–49. Sanles-Sobrido, M., Exner, W., Rodriguez-Lorenzo, L., Rodriguez-Gonzalez, B., Correa-Duarte, M.A., Alvarez-Puebla, R.A., Liz-Marzan, L.M., 2009. J. Am. Chem. Soc. 131, 2699–2705. Sánchez, S., Roldán, M., Pérez, S., Fàbregas, E., 2008. Anal. Chem. 80, 6508–6514. Srinivas, P.R., Kramer, B.S., Srivastava, S., 2001. Lancet Oncol. 2, 698–704. Sun, B., Zhang, K., Chen, L.J., Guo, L.T., Ai, S.Y., 2013. Biosens. Bioelectron. 44 48–51 Tang, D.P., Tang, J., Su, B.L., Ren, J.J., Chen, G.N., 2010. Biosens. Bioelectron. 25, 1658– 1662. Teymourian, H., Salimi, A., Khezrian, S., 2013. Biosens. Bioelectron. 49, 1–8. Wang, F., Chen, Y.H., Liu, C.Y., Ma, D.G., 2011. Chem. Commun. 47, 3502–3504. Wei, Q., Li, T., Wang, G.L., Li, H., Qian, Z.Y., Yang, M.H., 2010. Biomaterials 31, 7332– 7339. Wild, D., 2001. The Immunoassay Handbook. Nature Publishing Group, London, UK, pp. 643–644. Wu, J., Yan, Y.T., Yan, F., Ju, H.X., 2008. Anal. Chem. 80, 6072–6077. Yuan, R., Tang, D.P., Chai, Y.Q., Zhong, X., Liu, Y., Dai, J.Y., 2004. Langmuir 20, 7240– 7245. Zhang, J.J., Cheng, F.F., Zheng, T.T., Zhu, J.J., 2010. Anal. Chem. 82, 3547–3555. Zhang, Q., Lee, I., Ge, J.P., Zaera, F., Yin, Y.D., 2010. Adv. Funct. Mater. 20, 2201–2214. Zhang, Y.X., Cheng, Y.X., Zhou, Y.Y., Li, B.G., Gu, W., Shi, X.H., Xian, Y.Z., 2013. Talanta 107, 211–218. Zhou, L.Z., Kuai, L., Li, W.Z., Geng, B.Y., 2012. Appl. Mater. Interfaces 4, 6463–6467. Zhou, M., Zhai, Y.M., Dong, S.J., 2009. Anal. Chem. 81, 5603–5613.
