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A dual amplification electrochemical immunosensor based on [email protected] NPs for carcinoembryonic antigen detection
Analytical Biochemistry 574 (2019) 23–30
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A dual amplification electrochemical immunosensor based on HRP-Au@Ag NPs for carcinoembryonic antigen detection
T
Peipei Chen, Xiaoxia Hua, Jianhui Liu, Hanbiao Liu, Fangquan Xia, Dong Tian, Changli Zhou* Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Electrochemical immunosensor Au@Ag nanoparticles Carcinoembryonic antigen Dual amplification
A sensitive sandwich-type electrochemical immunosensor based on dual amplification strategy was constructed. The dual amplification strategy has been used secondary antibody(Ab2)-horseradish peroxidase(HRP)-Au@Ag nanoparticles (Au@Ag NPs) for carcinoembryonic antigen(CEA) detection. Ab2-HRP-Au@Ag NPs as dual amplification markers triggered the disproportionation of H2O2, which could facilitate the catalytic oxidation of hydroquinone to quinone(BQ). In addition, due to their large surface area and excellent conductivity, nitrogendoped graphene were used as a platform to firmly assemble primary antibody (Ab1). Above mentioned generated amout of BQ are corresponding to trace CEA, resulting in the highly electrochemical reduction signal. Under the optimal conditions, the linear range of CEA concentration was 0.0001–100 ng mL−1, and the limit of detection (LOD) could be as low as 0.05 pg mL−1. Importantly, the immunosensor also showed acceptable stability, reproducibility and selectivity.
1. Introduction Malignant tumor has been known as the high morbidity and mortality disease that seriously endangers human health. According to the latest reports (February 2018) from Health Organization (WHO), nearly one-sixth of deaths can be caused by cancer [1]. Therefore, it is more and more crucial to sensitively determine tumor markers in advance [2–4], which could enormously increase the cure efficiency of numerous cancers. As the tumor-associated antigen, carcinoembryonic antigen (CEA) has been ecognized as a critical indicator for clinical diagnosis of ovarian carcinoma, colon tumors, breast tumors and cystadenocarcinoma [5,6]. However, the amount of CEA in serum is quite low in the early stage of diseases. Recently, diverse strategies and instruments already have been exploited for the CEA test, including radioimmunoassay [7,8], surface plasmon resonance [9] and fluorescence immunoassay [10,11]. Unfortunately, great majority of these means exhibit shortcoming, such as sophisticated instrumentation, radiation hazards or time-consuming [12]. So, alternative approaches are still urgently desired. In recent years, in virtue of the characteristics of rapid detection, portability, low-budget, and specificity, electrochemical immunoassay has trigged increasingly attention [13,14]. Fabrication of a portable electrochemical immunosensor with amplified electrochemical signals and rapid detection is of great importance. Graphene(GR), as a two-dimensional nanomaterial with special *
chemical and physical properties such as superficial area and fast carrier mobility, has been wide spread in electrochemistry [14–17]. In recently, trying to adjust the electronic characteristics of GR in many kinds of ways to achieve better properties has give rise to maximum attention. Among them, chemical doping is an effective strategy to change the nature of nanomaterials, manipulate capillary chemistry, custom-tailor electronic property, and produce the host materials of the elemental composition of local change [15,16]. Nitrogen-doped graphene(N-GR) are functionalized GR material, the electronic characteristics of which are intrinsically changed by chemical doping with foreign N atoms. Compared with graphene, N-GR has better conductivities, electrocatalytic activity, and biocompatibility [2]. Therefore, choosing N-GR as electrode platform, not only can increase surface area like graphene layers structure, but also improves electrical conductivity by the doping of nitrogen, leading to the amplification of electrochemical signal. Meanwhile, the signals are amplified by different labeled coupling antibodies, such as electroactive nanoparticles, enzymes, redox nanocomposites, and nanoelectrocatalysts [17,18]. Among them, enzyme catalysis is utilized firstly due to its high catalytic efficiency for the substrate. And nanoparticles also put up some unique merits, such as excellent stability over electrode microenvironment and large effective surface area. Among nanoparticles, noble metals nanoparticles with alloy structure exhibited outstanding mimicking-enzyme catalytic
Corresponding author. E-mail address: [email protected] (C. Zhou).
https://doi.org/10.1016/j.ab.2019.03.003 Received 25 December 2018; Received in revised form 9 March 2019; Accepted 9 March 2019 Available online 20 March 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
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solution under magnetically stirring. Finally, the solution was poured into a Teflon-lined autoclave and heated at 80 °C about 3 h. The black sediments were washed and dried to obtain N-GR. 0.5 mg N-GR was added to the 0.5 mL CS solution(0.5 wt%), and the ultrasonic dispersion for 4 h, then the CS-N-GR was obtained.
performance particularly [19]. For example, recent study found that Au@Ag NPs could provide outstanding catalytic activities for hydrogen peroxide (H2O2) reduction contrast with Au NPs or Ag NPs by oneself [20,21]. Although both enzyme and nanoparticles also could be applied as probes in the immunosensors, they still have development space through ingenious combination. Recently, enzyme and nanomaterials based dual amplification strategy has gained increased importance in achieving high sensitivity and selectivity of analytes. For example, Dong group has reported PtPd/N-GQDs@Au possessed excellent mimicking-enzyme catalytic activity towards H2O2 reduction [22]. Kuang et al. developed a simple and sensitive electrochemical immunoassay based on Au NPs and HRP for Pantoea stewartii sbusp. stewartii-NCPPB 449 (PSS) detection [23]. Lin et al. reported the sandwich-type amperometric immunosensor for cancer biomarker based on carbon nanotubes (CNTs) and Concanavalin A (ConA) for dual-amplification signal [24]. In this study, we prepared uniform and stable Au@Ag NPs that are stabilized by citrate ions without introducing any surfactants or polymers according to the published literature [25]. Then a simple and sensitive electrochemical immunoassay method for CEA detection has been developed, using chitosan functionlized nitrogen-doped graphene (CS-N-GR) as substrate material. Meanwhile, Ab2-HRP-Au@Ag NPs as dual amplification markers triggered the disproportionation of H2O2, which could facilitate the catalytic oxidation of hydroquinone (HQ) to quinone (BQ). The limit of detection (0.05 pg mL−1) fully met the requirements of clinical diagnosis for critical value (3 ng mL−1) in normal human serum for CEA. It made known that the above-mentioned immunosensor provided a potential possibility for the determination of CEA in clinical diagnosis.
2.4. Preparation of the Ab2-HRP-Au@Ag NPs Au NPs were first synthesized by means of the Turkevich method [28]. 5 mL of 0.5% trisodium citrate solution was put into 95 mL HAuCl4 boiling aqueous solution (0.5 mmol L−1). The solution was boiled for approximately 15 min and then cooled to normal atmospheric temperature. Au@Ag NPs was synthesized as follows [25]. 60 μL of 0.1 mol L−1 ascorbic acid (AA), 15 μL of AgNO3 (0.1 mol L−1) and 75 μL of NaOH (0.1 mol L−1) were introduced into 10 mL Au seed solution successively, and stirred slowly for 30 min. The solution turned yellow, in which suggested that synthesis of Au@Ag NPs was successful. 4 μL K2CO3 solution (0.1 mol L−1) was mixed with 0.5 mL of Au@Ag NPs solution, and then 0.5 mL of 10 μg mL−1 Ab2-HRP solution was added to the above-mentioned mixture at the final specific concentration of 5 μg mL−1 to react for 1 h. Successively, 50 μL BSA solution (10%) was employed to block nonspecific active sites for 1 h. The conjugates were washed with phosphate buffered solution (PBS) (0.1 mol L−1, pH 7.0). The Ab2-HRP modified conjugates were resuspended in 100 μL of PBS solution (0.1 mol L−1, pH 7.0) again. The preparation of the Ab2-HRP-Au@Ag NPs has been listed in Scheme 1A.
2.5. Fabrication of the electrochemical immunoassay sensor 2. Experimental The fabrication procedure of our immunosensor was illustrated in Scheme 1B. First, glassy carbon electrode(GCE) with a diameter of 4 mm was polished using 0.3 and 0.05 μm alumina slurry, cleaned ultrasonically repeatedly in alternative baths of ultrapure water and absolute alcohol, and dried by nitrogen. Then, 10 μL CS-N-GR suspension was dripped onto the pre-handled GCE and dehydrated at normal atmospheric temperature, recorded as CS-N-GR/GCE. Next, 10 μL of 10 μg mL−1 Ab1 solution was dropped on CS-N-GR/GCE, containing 0.1 mol L−1 N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (1:4) at 4 °C for 1 h and rinsed repeated with PBS, recorded as Ab1/CS-N-GR/GCE. Then, 10 μL of 2% BSA solution was dropped onto Ab1/CS-N-GR/GCE and incubated to hinder nonspecific active sites for 60 min at 37 °C, which was recorded as BSA/Ab1/CS-N-GR/GCE. For the detection of CEA, the BSA/Ab1/CS-N-GR/GCE was washed thoroughly with PBS, incubated with 10 μL of CEA solution with various concentrations for 60 min at 37 °C, and subsequently washed with PBS to wipe off the physical adsorption CEA, recorded as CEA/BSA/Ab1/CS-N-GR/GCE. Finally, 10 μL of Ab2-HRP-Au@Ag NPs was dripped onto the surface of CEA/BSA/ Ab1/CS-N-GR/GCE to combine with CEA at 37 °C for 60 min and the electrode was washed with pH 7.0 PBS solution (0.1 mol L−1) three times. The sandwich electrochemical immunoassay sensor was formed, recorded as Ab2-HRP-Au@Ag NPs/CEA/BSA/Ab1/CS-N-GR/GCE. The above-prepared immunosensor was stored at 4 °C and ready to be used.
2.1. Chemicals and reagents Monoclonal anti-CEA capture antibodies (Ab1), CEA, anti-CEA (Ab2) and anti-CEA/HRP labeled antibody (Ab2-HRP) were ordered from Biocell Biotechnol. Co., Ltd. Hydrogen tetrachloroaurate hydrate (HAuCl4·4H2O, 99.9%) was obtained from Sinopharm Chemical Reagent Shanghai Co., Ltd. Hydroquinone (HQ) was obtained from Tianjin branch close the chemical reagent Co., Ltd. And bovine serum albumin (BSA) was reserved from Beijing ding changsheng biotechnology Co., Ltd. All other reagents were of analytical grade and ultrapure water was utilized in the experimental process. 2.2. Apparatus Electrochemical determines were operated on CHI760E electrochemical workstation (Shanghai, China). Scanning electron microscope (SEM) image and energy dispersive X-Ray spectroscopy (EDX) image were carried out using Quanta FEG250 field emission environmental SEM (FEI, USA). Transmission electron microscope (TEM) images were recorded by a JEM-2100F microscope (JEOL, Japan). X-Ray Powder Diffraction (XRD) was operated with a D8 advance X-ray diffractometer (Bruker AXS, Germany). UV–vis was measured using a Lambda 35 UV/ Vis Spectrometer (Perkin-Elmer, USA). 2.3. Preparation of the CS-N-GR
2.6. Detection of CEA Graphene oxide (GO) was prepared from graphite powder through the improved Hummers method [26]. And N-GR was synthesized by hydrothermal reduction [27]. Firstly, the synthetic GO was suspended in water via sonication and centrifugation with specific concentration of 2 mg mL−1. Then ammonia and hydrazine hydrate were used to chemically reduce GO solution in hydrothermal environment. Specifically, the pH of the mixed solution could be adjusted to 10 by 30% NH3·H2O. Next, 2 mL hydrazine hydrate was put into 70 mL above
The electrochemical tests were performed in phosphate buffer solution (0.1 mol L−1, pH 7.0). The quantitative detection of CEA was recorded by Differential pulse voltammetry (DPV) from 0.6 to −0.5 V (pulse amplitude = 50 mV, pulse width = 50 ms and pulse period = 0.5 s). 10 mL of pH 7.0 PBS solution(0.1 mol L−1) containing the substrate of 3.5 mmol L−1 HQ as probe of transferred electron and 1.0 mmol L−1 H2O2 was put into the electrochemical cell. 24
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Scheme 1. (A) Preparation of the Ab2-HRP-Au@Ag NPs; (B) Schematic diagram of the sandwich immunoassay.
3. Results and discussion
was inset in Fig. 1A). For Fig. 1B, the difference of dark and gray contrast between Au@Ag NPs in TEM image was observed, indicating that the Ag layer was thickness coated on the Au NPs surface in the core/shell structure. The Au@Ag NPs solution was stable after storing for 15 days without significant change in the colour and precipitation. In accordance with TEM results, two types of lattice fringes with interplanar spacings of 0.145 and 0.235 nm were clearly observed (Fig. 1C), which were attributed to the (220) and (111) planes of Au/
3.1. Characterization of the N-GR and Au@Ag NPs The preparation of Au@Ag NPs was characterized by UV–vis spectra, TEM and HRTEM images. As shown in Fig. 2A, upon Ag shell formation, the absorption peak was shifted from 524 nm to 416 nm along with strong change of colour observed by naked-eye (the photo
Fig. 1. (A) UV–vis spectra of Au NPs (a), Au@Ag NPs (b); (B) TEM and (C) HRTEM images of Au@Ag NPs; (D) XRD patterns of GO (a) and N-GR (b); (E) SEM and (F) EDX images of N-GR. Inset in Fig. 1(A): the photo of Au NPs (a), Au@Ag NPs (b). 25
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Fig. 2. CV curves characterization: (a) bare GCE, (b) N-GR/GCE and (c) CS-NGR/GCE. in pH 7.0 PBS solution containing 3.5 mmol L−1 HQ. Scan rate: 100 mV s−1.
Fig. 3. EIS responses of the different modified electrodes in 0.1 M pH 7.0 PBS containing 0.1 mol L−1 KCl and 2.5 mmol L−1 Fe(CN)63−/Fe(CN)64−: (a) NGR/GCE, (b) CS-N-GR/GCE, (c) Ab1/CS-N-GR/GCE, (d) bare GCE, (e) BSA/Ab1/ CS-N-GR/GCE, (f) Ab2-HRP-Au@Ag NPs/CEA/BSA/Ab1/CS-N-GR/GCE, (g) CEA/BSA/Ab1/CS-N-GR/GCE.
Ag, respectively [29,30]. Fig. 1D showed the X-ray diffraction patterns of GO and N-GR. GO had a sharp peak at 10.64°, corresponding to an interplanar crystal spacing of 0.83 nm according to Bragg equation calculation. This peak was completely instead by a broad peak at around 25° for N-GR with interplanar crystal spacing of 0.36 nm, confirming the recovery of graphitic crystal structure [27]. As shown in Fig. 1E, the N-GR was sheet with ripples and folds. The EDX results of N-GR (Fig. 1F) confirmed the existence of N on the N-GR.
(curve b). This was due to that introduction of nitrogen atoms (N) in GR could give rise to the change of electronic characteristics. Nitrogen atoms with long electron pairs can lead to the delocalization conjugated system with the sp2-hybridized carbon framework. Thus, the conductivity of CS-N-GR/GCE was enhanced [33]. 3.3. Characterization of the immunosensor
3.2. Electrochemical behaviors of HQ on modified electrode EIS was also used to evaluate the fabricated immunosensor during step modification. Fig. 3 showed the nyquist plots of EIS for the modified electrode at diverse stages. Apparently, the NG modified electrode exhibited a smaller semicircle domain (curve a). Nevertheless, after immobilization of Ab1 on the CS-N-GR/GCE, big semicircle could be observed (curve c), indicating the enlargement of the charge transfer resistance (Rct), which could be attributed to the membrane of the protein to counteract the interfacial electron transfer and insulate the conductive support. Similarly, after seize the BSA (e) and CEA (g), the semicircle persistently increased, because that BSA protein layers and the antigen-antibody complex on the electrode blocked the electron transfer. However, after modification of Ab2-HRP-Au@Ag NPs, Rct decreased significantly (curve f), perhaps owing to the superior conductivity of Au@Ag NPs.
Cyclic voltammograms (CVs) of various electrodes were compared in pH 7.0 PBS solution(0.1 mol L−1) containing 3.5 mmol L−1 HQ. As shown in Fig. 2, redox peaks could be received on bare GCE (Epa, 0.26 V; Epc, −0.05 V) (curve a), indicating that HQ expressed a quasireversible electrochemical behavior. For N-GR/GCE (curve b), redox peaks were located at 0.13 V (Epa) and −0.06 V (Epc), respectively. The oxidation peak shifted negatively and redox peak currents increased significantly relative to bare GCE. The results make clear that N-GR/ GCE processes superior electrocatalytic performance and sensitivity toward HQ. While on CS-N-GR/GCE (c), redox peak currents decreased with more positive oxidation peak (Epa, 0.17 V) and more positive reduction peak (Epc,-0.00 V) than curve b. The above experimental phenomena can attribute to existing of CS on N-GR. In addition, compared with Fig. 2, the redox peak and current had no obvious differences in diverse modified GCE when the PBS solution containing 3.5 mmol L−1 HQ and 1.0 mmol L−1 H2O2. This phenomenon further proves that H2O2 had no direct influence on the oxidation of HQ. In 0.1 mmol L−1 Fe(CN)63− and 0.1 mol L−1 KCl solution, the effective surface area of various electrodes was evaluateded with CV on the basis of the Randles-Sevcik equation [31]:
3.4. Electrochemical behaviors of the dual amplified immunosensors Diverse labeled Ab2 bioconjugates were measured in pH 7.0 PBS solution containing 3.5 mmol L−1 HQ and 1.0 mmol L−1 H2O2 or without 1.0 mmol L−1 H2O2. The Ab2-HRP modified electrode in pH 7.0 PBS solution containing 3.5 mmol L−1 HQ exhibited a relatively weak electrochemical response(Fig. 4A, curve a). The results for Ab2-Au@Ag NPs (Fig. 4A, curve b) and Ab2-HRP-Au@Ag NPs(Fig. 4A, curve c) can be higher relative to Ab2-HRP. When diverse labeled Ab2 bioconjugates were tested in pH 7.0 PBS solution containing 3.5 mM HQ and 1.0 mmol L−1 H2O2, it could be found that the electrochemical response ascended significantly (Fig. 4A, curve a', b' and c'). Apparently, compared with Fig. 4A, curve a' and b', the oxidation peak and reduction peak were increased significantly (Fig. 4A, curve c'). These comparisons demonstrated that the Ab2-HRP-Au@Ag NPs as dual amplification markers effectively triggered the disproportionation of H2O2, which could facilitate the catalytic oxidation of HQ to BQ. The DPV results for diverse labeled Ab2 bioconjugates are consistent with CV in pH 7.0 PBS solution containing 3.5 mmol L−1 HQ and 1.0 mmol L−1 H2O2. From
Ipc = (2.69 × 105) n3/2AD01/2 ν1/2C0 where Ipc is the reduction peak current (A), C0 is the concentration of K3[Fe(CN)6] (mmol L−1) and ν is the scan rate (V s−1), D0 is the diffusion coefficient of K3[Fe(CN)6] in the solution (D0 = 6.5 × 106 cm2 s−1) [32], A is the effective surface area (cm2), n is the electron transfer number. On account of the equation, the effective surface area of GCE, N-GR/GCE and CS-N-GR/GCE was calculated as 0.106, 0.408 and 0.425 cm2, respectively. Therefore, N-GR and CS-N-GR both can increase effective surface area. Furthermore, electrochemical impedance spectroscopy (EIS) was operated to evaluate the electrical conductivity (Fig. 3). On the N-GR/GCE (curve a), the Rct value in the low frequency region was lower than GCE (curve d) and CS-N-GR/GCE 26
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Fig. 4. (A) CV curves of immunosensors with different labeled Ab2 bioconjugates: (a, a´) Ab2HRP, (b, b´) Ab2-Au@Ag NPs, (c, c´) Ab2-HRPAu@Ag NPs in pH 7.0 PBS solution containing 3.5 mM HQ and 1.0 mmol L−1 H2O2 or without 1.0 mmol L−1 H2O2. Scan rate: 100 mV s−1. (B) DPV curves of immunosensors with different labeled Ab2 bioconjugates: (a) Ab2-HRP, (b) Ab2-Au@Ag NPs, (c) Ab2-HRP-Au@Ag NPs in pH 7.0 PBS solution containing 3.5 mM HQ and 1.0 mmol L−1 H2O2.
H2O2 in the solution were changed, respectively. It was observed that the optimized concentration of HQ was 3.5 mmol L−1 (Fig. 5B), while that for H2O2 was 1.0 mmol L−1 (Fig. 5C). Moreover, the optimal incubation time was discussed as exhibited in Fig. 5D. Because it was necessary to spend time on reaching the maximum formation of the sandwich immunocomplex, Ipc increased with the raise of incubation time. At the time of 60 min, Ipc reached maximum because the preparation of sandwich-type immunoassay could be get saturated. Hence, 60 min was chosen as the optimal incubation time for the immunoassay. 3.6. Electrochemical detection of CEA Under the optimal conditions, the proposed immunosensor was applied for detecting CEA with different concentrations. As shown in Fig. 6A, Ipc increases with the increase of CEA concentration from 0.01 to 100 ng mL−1. The calibration plot in Fig. 6B expresses a good linear relationship between the concentration of CEA and Ipc. The linear regression equations in the range from 0.0001 ng mL−1 to 100 ng mL−1 for CEA is Ipc (μA) = −39.08–5.24lgcCEA (ng mL−1) with a correlation coefficient of 0.986. On the basis of triple standard deviation, the LOD is calculated to 0.05 pg mL−1. Additionally, the results were compared with that of other CEA biosensors, the detection limit and linear range were displayed in Table 1. These comparison results indicate that the proposed electrochemical immunosensor is highly sensitive for CEA detection.
Scheme 2. Dual amplification schematic diagram.
above results, we can conclude that HRP as amplification marker facilitate the catalytic oxidation of HQ in the HQ-H2O2 system (Scheme 2, eq1, eq2). In addition, and most importantly, Au@Ag NPs could be considered as one kind of mimicking-enzyme catalytic marker to catalyze the oxidation reaction of HQ to BQ in the HQ-H2O2 system (Scheme 2, eq3, eq4, eq5) [34]. Au@Ag NPs as a catalyst can transfer electrons to hydrogen peroxide, resulting in the rupture of the oxygenoxygen bond of hydrogen peroxide and producing OH•. The OH• was stabilized at the surface of Au@Ag NPs. They would further react with HQ and generate BQ. Then, BQ was reduced by obtaining electron from cathode (Scheme 2, eq6). The corresponding DPV cathodic peak current intensity of Ab2-HRP-Au@Ag NPs (Fig. 4B, curve b) enhanced about 1.6-fold and 1.3-fold than Ab2-HRP (Fig. 4B, curve a) Ab2-Au@Ag NPs (Fig. 4B, curve b), respectively. Consequently say, the electrochemical reduction signal was dual amplified by HRP and Au@Ag NPs.
3.7. Reproducibility, stability and selectivity of the immunosensor To research the repeatability, the same five immunosensors, which were incubated with 10 ng mL−1 CEA and then incubated with Au@Ag NPs and HRP labeled Ab2 bioconjugates, were measured under the same conditions. The relative standard deviation after calculate was 4.3%, indicating that the repeatability of proposed immunosensor could be satisfactory. As we can see from Fig. 7A, to evaluate the stability of the proposed immunosensor, they were keeped in reserve at 4 °C for 10 days and Ipc were put down in writing periodically. During the period, the electrochemical response didn't changed much, and only dropped less than 12% on the last day for contrast with fresh prepared. It demonstrated that the proposed immunosensor had a reasonable stability. As we can see from Fig. 7B, the selectivity of the immunosensors toward CEA (10 ng mL−1) was studied using some possible interfering substances (100 ng mL−1 each), including IgG, BSA, AFP, PSA and UA. The results showed that compared with Ipc received from 10 ng mL−1 CEA, the variations of Ipc caused by interfering substances were inferior 7%, declaring that the immunosensor had excellent selectivity.
3.5. Optimization of the experimental conditions of the immunoassay The pH of buffer solution, substrate concentration of HQ and H2O2, and incubation time played important roles in the enhancement of current response, so further explorations were performed using DPV. Fig. 5 shows that cathodic current peak (Ipc) varies with the substrate concentration of buffer solution pH, HQ, H2O2, and incubation time (CEA 1 ng mL−1). Herein, PBS of pH 7.0 was selected as the detection solution for CEA detection. Additionally, the concentrations of HQ and
3.8. Application in analysis of serum samples As we can see from Table 2, the analytical feasibility and potential clinical application of the fabricated immunosensor were assessed and 27
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Fig. 5. Current response of immunosensor in PBS solution (A) at different pH under 1.0 mmol L−1 H2O2 and 3.5 mmol L−1 HQ incubation for 45 min; (B) at different HQ concentrations under 1.0 mmol L−1 H2O2 and incubation for 45 min (pH 7.0); (C) at different H2O2 concentrations under 3.5 mmol L−1 HQ, and incubation for 45 min (pH 7.0); (D) at different incubation times under 3.5 mmol L−1 HQ, 1.0 mmol L−1 H2O2. (Error bars present the relative standard deviation detected for 3 times).
Fig. 6. (A) DPV responses of the proposed immunosensor after incubation with different concentrations of CEA, a-g, 0.0001, 0.001, 0.01, 0.1, 1, 20, 100 ng mL−1; (B) calibration curves of the immunoassay toward CEA in PBS (pH HQ, 7.0), containing 3.5 mmol L−1 −1 1.0 mmol L H2O2 (Error bars present the relative standard deviation detected for 3 times).
Table 1 Comparison of analytical properties of various immunosensor towards CEA. Electrode modification
Labels
Characteristics
Linear range (ng mL−1)
LOD (pg mL−1)
Ref.
GCE/Bi fIlm GCE/Hg fIlm SPCE/Au/Cys/Lectin Au/L-Cys CS-NG/GCE
Si/Pb/anti-CEA CdS/DNA-anti-CEA HRP-CEA CNTs/PDDA/Con/HRP-CEA Au@Ag/HRP-anti-CEA
Required harsh conditions to synthesis high-quality QDs Required enrichment and time-consuming The complex electrode preparation and low sensitivity The complex process of synthetic and low sentivity Benefited from the catalysis of HRP and Au@Ag NPs
0.05–25 0.1–100 0.5–10 5–200 0.0001–100
5.0 3.3 10 18 0.05
[35] [36] [37] [24] This work
4. Conclusions
contrasted with the reference method (ELISA method). Seven complex clinical serum samples that obtained from Shandong Provincial Hospital were evaluated. We haven't seen the significant differences among the two methods. Subsequently, recovery experiments were carried out to evaluate the accuracy of our immunosensor. Compared with the values of reference method, it's clear that our immunosensor has excellent accuracy for CEA detection from complex clinical serum samples. We could also observe that the recovery was ranging from 80.0% to 110% and the RSD varied from 2.14% to 6.23%. The satisfying results clearly offered a potential application for the determination of CEA in clinical diagnostics.
In this work, a sensitive sandwich-type electrochemical immunosensor based on dual amplification strategy for detection of CEA was successfully developed. With the disproportionation reaction of the H2O2, HRP and Au@Ag NPs acted as dual amplification markers possessed superior electrocatalytic activity toward the oxidation of HQ. Meanwhile, nitrogen-doped graphene with excellent conductivity and large surface area were used as a platform for immobilize the primary antibody. Importantly, the LOD of the immunosensor for CEA detection could be lowered to 0.05 pg mL−1. The low detection limit showed that employing suitable materials to promote the electron transfer and amplify signal could be an effective way for the fabrication of highly sensitive nanomaterials-driven electrochemical immunosensors. This 28
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Fig. 7. (A) Current response of the immunosensor in different time; (B) Current response of the immunosensor in the detection of CEA (10 ng mL−1) with interfering substances (100 ng mL−1). transfected with CEA-mRNA, Eur. J. Cancer 38 (2002) 184–193. [7] N.R. Hughes, Factors which influence the radioimmunoassay of carcinoembryonic antigen (CEA), Mol. Immunol. 17 (1980) 665–673. [8] Q. Lang, F. Wang, L. Yin, M. Liu, V.A. Petrenko, A. Liu, Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen, Anal. Chem. 86 (2014) 2767–2774. [9] R. Li, F. Feng, Z.Z. Chen, Y.F. Bai, F.F. Guo, F.Y. Wu, G. Zhou, Sensitive detection of carcinoembryonic antigen using surface plasmon resonance biosensor with gold nanoparticles signal amplification, Talanta 140 (2015) 143–149. [10] X. Yang, Y. Zhuo, S. Zhu, Y. Luo, Y. Feng, Y. Xu, Selectively assaying CEA based on a creative strategy of gold nanoparticles enhancing silver nanoclusters' fluorescence, Biosens. Bioelectron. 64 (2015) 345–351. [11] H. Miao, L. Wang, Y. Zhuo, Z. Zhou, X. Yang, Label-free fluorimetric detection of CEA using carbon dots derived from tomato juice, Biosens. Bioelectron. 86 (2016) 83–89. [12] P. Santharaman, M. Das, S.K. Singh, N.K. Sethy, K. Bhargava, J.C. Claussen, C. Karunakaran, Label-free electrochemical immunosensor for the rapid and sensitive detection of the oxidative stress marker superoxide dismutase 1 at the pointof-care, Sensor. Actuator. B Chem. 236 (2016) 546–553. [13] M. Freitas, W. Correr, J. Cancino-Bernardi, M.F. Barroso, C. Delerue-Matos, V. Zucolotto, Impedimetric immunosensors for the detection of Cry1Ab protein from genetically modified maize seeds, Sensor. Actuator. B Chem. 237 (2016) 702–709. [14] L. Yang, H. Zhao, S. Fan, S. Deng, Q. Lv, J. Lin, C.P. Li, Label-free electrochemical immunosensor based on gold-silicon carbide nanocomposites for sensitive detection of human chorionic gonadotrophin, Biosens. Bioelectron. 57 (2014) 199–206. [15] Y. Shao, J. Sui, G. Yin, Y. Gao, Nitrogen-doped carbon nanostructures and their composites as catalytic materials for proton exchange membrane fuel cell, Appl. Catal. B Environ. 79 (2008) 89–99. [16] B.G. Sumpter, V. Meunier, J.M. Romoherrera, E. Cruzsilva, D.A. Cullen, H. Terrones, D.J. Smith, M. Terrones, Nitrogen-mediated carbon nanotube growth: diameter reduction, metallicity, bundle dispersability, and bamboo-like structure formation, ACS Nano 1 (2007) 369–375. [17] L. Zhao, C. Li, H. Qi, Q. Gao, C. Zhang, Electrochemical lectin-based biosensor array for detection and discrimination of carcinoembryonic antigen using dual amplification of gold nanoparticles and horseradish peroxidase, Sensor. Actuator. B Chem. 235 (2016) 575–582. [18] L. Ma, D. Ning, H. Zhang, J. Zheng, Au@Ag nanorods based electrochemical immunoassay for immunoglobulin G with signal enhancement using carbon nanofibers-polyamidoamine dendrimer nanocomposite, Biosens. Bioelectron. 68 (2015) 175–180. [19] H. Chang, H. Zhang, J. Lv, B. Zhang, W. Wei, J. Guo, Pt NPs and DNAzyme functionalized polymer nanospheres as triple signal amplification strategy for highly sensitive electrochemical immunosensor of tumour marker, Biosens. Bioelectron. 86 (2016) 156–163. [20] Y. Wang, Y. Zhang, Y. Su, F. Li, H. Ma, H. Li, B. Du, Q. Wei, Ultrasensitive nonmediator electrochemical immunosensors using Au/Ag/Au core/double shell nanoparticles as enzyme-mimetic labels, Talanta 124 (2014) 60–66. [21] X. Yang, Y. Wang, Y. Liu, X. Jiang, A sensitive hydrogen peroxide and glucose biosensor based on gold/silver core–shell nanorods, Electrochim. Acta 108 (2013) 39–44. [22] Y. Yang, Q. Liu, Y. Liu, J. Cui, H. Liu, P. Wang, Y. Li, L. Chen, Z. Zhao, Y. Dong, A novel label-free electrochemical immunosensor based on functionalized nitrogendoped graphene quantum dots for carcinoembryonic antigen detection, Biosens. Bioelectron. 90 (2017) 31–38. [23] Y. Zhao, L. Liu, D. Kong, H. Kuang, L. Wang, C. Xu, Dual amplified electrochemical immunosensor for highly sensitive detection of Pantoea stewartii sbusp. stewartii, ACS Appl. Mater. Interfaces 6 (2014) 21178–21183. [24] P. Yang, X. Li, L. Wang, Q. Wu, Z. Chen, X. Lin, Sandwich-type amperometric immunosensor for cancer biomarker based on signal amplification strategy of multiple enzyme-linked antibodies as probes modified with carbon nanotubes and concanavalin A, J. Electroanal. Chem. 732 (2014) 38–45. [25] A.K. Samal, L. Polavarapu, S. Rodal-Cedeira, L.M. Liz-Marzan, J. Perez-Juste,
Table 2 The results of determine CEA in serum samples(n = 3). Sample
1 2 3 4 5 6 7
ELISA (ng mL−1)
The immunosensor Found (ng mL−1)
Added (ng mL−1)
Total found (ng mL−1)
Recovery (%)
RSD (%)
2.25 1.17 1.91 7.61 26.0 22.2 28.5
2.28 1.20 1.96 7.53 25.7 22.4 28.1
1 1 1 5 10 10 10
3.41 2.19 2.82 12.1 28.8 29.0 42.4
105 101 97.0 96.0 80.0 90.0 110
6.23 4.84 3.95 4.16 5.32 2.14 2.82
method has great application prospect for cancer diagnosis. Declarations The author(s) declare(s) that they have no conflicts of interest to disclose. Acknowledgements This work was supported by the Natural Science Foundation of Shandong Province (China, ZR2017MB063, ZR2013BM003) and the Science and Technology Development Plan Project of Shandong Province (China, No. 2012G0022116). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ab.2019.03.003. References [1] C. Zhang, S. Zhang, Y. Jia, Y. Li, P. Wang, Q. Liu, Z. Xu, X. Li, Y. Dong, Sandwichtype electrochemical immunosensor for sensitive detection of CEA based on the enhanced effects of Ag NPs@CS spaced Hemin/rGO, Biosens. Bioelectron. 126 (2018) 785–791. [2] S. Deng, J. Lei, Y. Huang, Y. Cheng, H. Ju, Electrochemiluminescent quenching of quantum dots for ultrasensitive immunoassay through oxygen reduction catalyzed by nitrogen-doped graphene-supported hemin, Anal. Chem. 85 (2013) 5390–5396. [3] T. Xu, X. Jia, X. Chen, Z. Ma, Simultaneous electrochemical detection of multiple tumor markers using metal ions tagged immunocolloidal gold, Biosens. Bioelectron. 56 (2014) 174–179. [4] M. Yang, S. Gong, Immunosensor for the detection of cancer biomarker based on percolated graphene thin film, Chem. Commun. 46 (2010) 5796–5798. [5] W. Zhong, Z. Yu, J. Zhan, T. Yu, Y. Lin, Z.S. Xia, Y.H. Yuan, Q.K. Chen, Association of serum levels of CEA, CA199, CA125, CYFRA21-1 and CA72-4 and disease characteristics in colorectal cancer, Pathol. Oncol. Res. 21 (2015) 83–95. [6] E. Eppler, H. Hörig, H.L. Kaufman, P. Groscurth, L. Filgueira, Carcinoembryonic antigen (CEA) presentation and specific T cell-priming by human dendritic cells
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A dual amplification electrochemical immunosensor based on HRP-Au@Ag NPs for carcinoembryonic antigen detection
T
Peipei Chen, Xiaoxia Hua, Jianhui Liu, Hanbiao Liu, Fangquan Xia, Dong Tian, Changli Zhou* Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Electrochemical immunosensor Au@Ag nanoparticles Carcinoembryonic antigen Dual amplification
A sensitive sandwich-type electrochemical immunosensor based on dual amplification strategy was constructed. The dual amplification strategy has been used secondary antibody(Ab2)-horseradish peroxidase(HRP)-Au@Ag nanoparticles (Au@Ag NPs) for carcinoembryonic antigen(CEA) detection. Ab2-HRP-Au@Ag NPs as dual amplification markers triggered the disproportionation of H2O2, which could facilitate the catalytic oxidation of hydroquinone to quinone(BQ). In addition, due to their large surface area and excellent conductivity, nitrogendoped graphene were used as a platform to firmly assemble primary antibody (Ab1). Above mentioned generated amout of BQ are corresponding to trace CEA, resulting in the highly electrochemical reduction signal. Under the optimal conditions, the linear range of CEA concentration was 0.0001–100 ng mL−1, and the limit of detection (LOD) could be as low as 0.05 pg mL−1. Importantly, the immunosensor also showed acceptable stability, reproducibility and selectivity.
1. Introduction Malignant tumor has been known as the high morbidity and mortality disease that seriously endangers human health. According to the latest reports (February 2018) from Health Organization (WHO), nearly one-sixth of deaths can be caused by cancer [1]. Therefore, it is more and more crucial to sensitively determine tumor markers in advance [2–4], which could enormously increase the cure efficiency of numerous cancers. As the tumor-associated antigen, carcinoembryonic antigen (CEA) has been ecognized as a critical indicator for clinical diagnosis of ovarian carcinoma, colon tumors, breast tumors and cystadenocarcinoma [5,6]. However, the amount of CEA in serum is quite low in the early stage of diseases. Recently, diverse strategies and instruments already have been exploited for the CEA test, including radioimmunoassay [7,8], surface plasmon resonance [9] and fluorescence immunoassay [10,11]. Unfortunately, great majority of these means exhibit shortcoming, such as sophisticated instrumentation, radiation hazards or time-consuming [12]. So, alternative approaches are still urgently desired. In recent years, in virtue of the characteristics of rapid detection, portability, low-budget, and specificity, electrochemical immunoassay has trigged increasingly attention [13,14]. Fabrication of a portable electrochemical immunosensor with amplified electrochemical signals and rapid detection is of great importance. Graphene(GR), as a two-dimensional nanomaterial with special *
chemical and physical properties such as superficial area and fast carrier mobility, has been wide spread in electrochemistry [14–17]. In recently, trying to adjust the electronic characteristics of GR in many kinds of ways to achieve better properties has give rise to maximum attention. Among them, chemical doping is an effective strategy to change the nature of nanomaterials, manipulate capillary chemistry, custom-tailor electronic property, and produce the host materials of the elemental composition of local change [15,16]. Nitrogen-doped graphene(N-GR) are functionalized GR material, the electronic characteristics of which are intrinsically changed by chemical doping with foreign N atoms. Compared with graphene, N-GR has better conductivities, electrocatalytic activity, and biocompatibility [2]. Therefore, choosing N-GR as electrode platform, not only can increase surface area like graphene layers structure, but also improves electrical conductivity by the doping of nitrogen, leading to the amplification of electrochemical signal. Meanwhile, the signals are amplified by different labeled coupling antibodies, such as electroactive nanoparticles, enzymes, redox nanocomposites, and nanoelectrocatalysts [17,18]. Among them, enzyme catalysis is utilized firstly due to its high catalytic efficiency for the substrate. And nanoparticles also put up some unique merits, such as excellent stability over electrode microenvironment and large effective surface area. Among nanoparticles, noble metals nanoparticles with alloy structure exhibited outstanding mimicking-enzyme catalytic
Corresponding author. E-mail address: [email protected] (C. Zhou).
https://doi.org/10.1016/j.ab.2019.03.003 Received 25 December 2018; Received in revised form 9 March 2019; Accepted 9 March 2019 Available online 20 March 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
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solution under magnetically stirring. Finally, the solution was poured into a Teflon-lined autoclave and heated at 80 °C about 3 h. The black sediments were washed and dried to obtain N-GR. 0.5 mg N-GR was added to the 0.5 mL CS solution(0.5 wt%), and the ultrasonic dispersion for 4 h, then the CS-N-GR was obtained.
performance particularly [19]. For example, recent study found that Au@Ag NPs could provide outstanding catalytic activities for hydrogen peroxide (H2O2) reduction contrast with Au NPs or Ag NPs by oneself [20,21]. Although both enzyme and nanoparticles also could be applied as probes in the immunosensors, they still have development space through ingenious combination. Recently, enzyme and nanomaterials based dual amplification strategy has gained increased importance in achieving high sensitivity and selectivity of analytes. For example, Dong group has reported PtPd/N-GQDs@Au possessed excellent mimicking-enzyme catalytic activity towards H2O2 reduction [22]. Kuang et al. developed a simple and sensitive electrochemical immunoassay based on Au NPs and HRP for Pantoea stewartii sbusp. stewartii-NCPPB 449 (PSS) detection [23]. Lin et al. reported the sandwich-type amperometric immunosensor for cancer biomarker based on carbon nanotubes (CNTs) and Concanavalin A (ConA) for dual-amplification signal [24]. In this study, we prepared uniform and stable Au@Ag NPs that are stabilized by citrate ions without introducing any surfactants or polymers according to the published literature [25]. Then a simple and sensitive electrochemical immunoassay method for CEA detection has been developed, using chitosan functionlized nitrogen-doped graphene (CS-N-GR) as substrate material. Meanwhile, Ab2-HRP-Au@Ag NPs as dual amplification markers triggered the disproportionation of H2O2, which could facilitate the catalytic oxidation of hydroquinone (HQ) to quinone (BQ). The limit of detection (0.05 pg mL−1) fully met the requirements of clinical diagnosis for critical value (3 ng mL−1) in normal human serum for CEA. It made known that the above-mentioned immunosensor provided a potential possibility for the determination of CEA in clinical diagnosis.
2.4. Preparation of the Ab2-HRP-Au@Ag NPs Au NPs were first synthesized by means of the Turkevich method [28]. 5 mL of 0.5% trisodium citrate solution was put into 95 mL HAuCl4 boiling aqueous solution (0.5 mmol L−1). The solution was boiled for approximately 15 min and then cooled to normal atmospheric temperature. Au@Ag NPs was synthesized as follows [25]. 60 μL of 0.1 mol L−1 ascorbic acid (AA), 15 μL of AgNO3 (0.1 mol L−1) and 75 μL of NaOH (0.1 mol L−1) were introduced into 10 mL Au seed solution successively, and stirred slowly for 30 min. The solution turned yellow, in which suggested that synthesis of Au@Ag NPs was successful. 4 μL K2CO3 solution (0.1 mol L−1) was mixed with 0.5 mL of Au@Ag NPs solution, and then 0.5 mL of 10 μg mL−1 Ab2-HRP solution was added to the above-mentioned mixture at the final specific concentration of 5 μg mL−1 to react for 1 h. Successively, 50 μL BSA solution (10%) was employed to block nonspecific active sites for 1 h. The conjugates were washed with phosphate buffered solution (PBS) (0.1 mol L−1, pH 7.0). The Ab2-HRP modified conjugates were resuspended in 100 μL of PBS solution (0.1 mol L−1, pH 7.0) again. The preparation of the Ab2-HRP-Au@Ag NPs has been listed in Scheme 1A.
2.5. Fabrication of the electrochemical immunoassay sensor 2. Experimental The fabrication procedure of our immunosensor was illustrated in Scheme 1B. First, glassy carbon electrode(GCE) with a diameter of 4 mm was polished using 0.3 and 0.05 μm alumina slurry, cleaned ultrasonically repeatedly in alternative baths of ultrapure water and absolute alcohol, and dried by nitrogen. Then, 10 μL CS-N-GR suspension was dripped onto the pre-handled GCE and dehydrated at normal atmospheric temperature, recorded as CS-N-GR/GCE. Next, 10 μL of 10 μg mL−1 Ab1 solution was dropped on CS-N-GR/GCE, containing 0.1 mol L−1 N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (1:4) at 4 °C for 1 h and rinsed repeated with PBS, recorded as Ab1/CS-N-GR/GCE. Then, 10 μL of 2% BSA solution was dropped onto Ab1/CS-N-GR/GCE and incubated to hinder nonspecific active sites for 60 min at 37 °C, which was recorded as BSA/Ab1/CS-N-GR/GCE. For the detection of CEA, the BSA/Ab1/CS-N-GR/GCE was washed thoroughly with PBS, incubated with 10 μL of CEA solution with various concentrations for 60 min at 37 °C, and subsequently washed with PBS to wipe off the physical adsorption CEA, recorded as CEA/BSA/Ab1/CS-N-GR/GCE. Finally, 10 μL of Ab2-HRP-Au@Ag NPs was dripped onto the surface of CEA/BSA/ Ab1/CS-N-GR/GCE to combine with CEA at 37 °C for 60 min and the electrode was washed with pH 7.0 PBS solution (0.1 mol L−1) three times. The sandwich electrochemical immunoassay sensor was formed, recorded as Ab2-HRP-Au@Ag NPs/CEA/BSA/Ab1/CS-N-GR/GCE. The above-prepared immunosensor was stored at 4 °C and ready to be used.
2.1. Chemicals and reagents Monoclonal anti-CEA capture antibodies (Ab1), CEA, anti-CEA (Ab2) and anti-CEA/HRP labeled antibody (Ab2-HRP) were ordered from Biocell Biotechnol. Co., Ltd. Hydrogen tetrachloroaurate hydrate (HAuCl4·4H2O, 99.9%) was obtained from Sinopharm Chemical Reagent Shanghai Co., Ltd. Hydroquinone (HQ) was obtained from Tianjin branch close the chemical reagent Co., Ltd. And bovine serum albumin (BSA) was reserved from Beijing ding changsheng biotechnology Co., Ltd. All other reagents were of analytical grade and ultrapure water was utilized in the experimental process. 2.2. Apparatus Electrochemical determines were operated on CHI760E electrochemical workstation (Shanghai, China). Scanning electron microscope (SEM) image and energy dispersive X-Ray spectroscopy (EDX) image were carried out using Quanta FEG250 field emission environmental SEM (FEI, USA). Transmission electron microscope (TEM) images were recorded by a JEM-2100F microscope (JEOL, Japan). X-Ray Powder Diffraction (XRD) was operated with a D8 advance X-ray diffractometer (Bruker AXS, Germany). UV–vis was measured using a Lambda 35 UV/ Vis Spectrometer (Perkin-Elmer, USA). 2.3. Preparation of the CS-N-GR
2.6. Detection of CEA Graphene oxide (GO) was prepared from graphite powder through the improved Hummers method [26]. And N-GR was synthesized by hydrothermal reduction [27]. Firstly, the synthetic GO was suspended in water via sonication and centrifugation with specific concentration of 2 mg mL−1. Then ammonia and hydrazine hydrate were used to chemically reduce GO solution in hydrothermal environment. Specifically, the pH of the mixed solution could be adjusted to 10 by 30% NH3·H2O. Next, 2 mL hydrazine hydrate was put into 70 mL above
The electrochemical tests were performed in phosphate buffer solution (0.1 mol L−1, pH 7.0). The quantitative detection of CEA was recorded by Differential pulse voltammetry (DPV) from 0.6 to −0.5 V (pulse amplitude = 50 mV, pulse width = 50 ms and pulse period = 0.5 s). 10 mL of pH 7.0 PBS solution(0.1 mol L−1) containing the substrate of 3.5 mmol L−1 HQ as probe of transferred electron and 1.0 mmol L−1 H2O2 was put into the electrochemical cell. 24
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Scheme 1. (A) Preparation of the Ab2-HRP-Au@Ag NPs; (B) Schematic diagram of the sandwich immunoassay.
3. Results and discussion
was inset in Fig. 1A). For Fig. 1B, the difference of dark and gray contrast between Au@Ag NPs in TEM image was observed, indicating that the Ag layer was thickness coated on the Au NPs surface in the core/shell structure. The Au@Ag NPs solution was stable after storing for 15 days without significant change in the colour and precipitation. In accordance with TEM results, two types of lattice fringes with interplanar spacings of 0.145 and 0.235 nm were clearly observed (Fig. 1C), which were attributed to the (220) and (111) planes of Au/
3.1. Characterization of the N-GR and Au@Ag NPs The preparation of Au@Ag NPs was characterized by UV–vis spectra, TEM and HRTEM images. As shown in Fig. 2A, upon Ag shell formation, the absorption peak was shifted from 524 nm to 416 nm along with strong change of colour observed by naked-eye (the photo
Fig. 1. (A) UV–vis spectra of Au NPs (a), Au@Ag NPs (b); (B) TEM and (C) HRTEM images of Au@Ag NPs; (D) XRD patterns of GO (a) and N-GR (b); (E) SEM and (F) EDX images of N-GR. Inset in Fig. 1(A): the photo of Au NPs (a), Au@Ag NPs (b). 25
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Fig. 2. CV curves characterization: (a) bare GCE, (b) N-GR/GCE and (c) CS-NGR/GCE. in pH 7.0 PBS solution containing 3.5 mmol L−1 HQ. Scan rate: 100 mV s−1.
Fig. 3. EIS responses of the different modified electrodes in 0.1 M pH 7.0 PBS containing 0.1 mol L−1 KCl and 2.5 mmol L−1 Fe(CN)63−/Fe(CN)64−: (a) NGR/GCE, (b) CS-N-GR/GCE, (c) Ab1/CS-N-GR/GCE, (d) bare GCE, (e) BSA/Ab1/ CS-N-GR/GCE, (f) Ab2-HRP-Au@Ag NPs/CEA/BSA/Ab1/CS-N-GR/GCE, (g) CEA/BSA/Ab1/CS-N-GR/GCE.
Ag, respectively [29,30]. Fig. 1D showed the X-ray diffraction patterns of GO and N-GR. GO had a sharp peak at 10.64°, corresponding to an interplanar crystal spacing of 0.83 nm according to Bragg equation calculation. This peak was completely instead by a broad peak at around 25° for N-GR with interplanar crystal spacing of 0.36 nm, confirming the recovery of graphitic crystal structure [27]. As shown in Fig. 1E, the N-GR was sheet with ripples and folds. The EDX results of N-GR (Fig. 1F) confirmed the existence of N on the N-GR.
(curve b). This was due to that introduction of nitrogen atoms (N) in GR could give rise to the change of electronic characteristics. Nitrogen atoms with long electron pairs can lead to the delocalization conjugated system with the sp2-hybridized carbon framework. Thus, the conductivity of CS-N-GR/GCE was enhanced [33]. 3.3. Characterization of the immunosensor
3.2. Electrochemical behaviors of HQ on modified electrode EIS was also used to evaluate the fabricated immunosensor during step modification. Fig. 3 showed the nyquist plots of EIS for the modified electrode at diverse stages. Apparently, the NG modified electrode exhibited a smaller semicircle domain (curve a). Nevertheless, after immobilization of Ab1 on the CS-N-GR/GCE, big semicircle could be observed (curve c), indicating the enlargement of the charge transfer resistance (Rct), which could be attributed to the membrane of the protein to counteract the interfacial electron transfer and insulate the conductive support. Similarly, after seize the BSA (e) and CEA (g), the semicircle persistently increased, because that BSA protein layers and the antigen-antibody complex on the electrode blocked the electron transfer. However, after modification of Ab2-HRP-Au@Ag NPs, Rct decreased significantly (curve f), perhaps owing to the superior conductivity of Au@Ag NPs.
Cyclic voltammograms (CVs) of various electrodes were compared in pH 7.0 PBS solution(0.1 mol L−1) containing 3.5 mmol L−1 HQ. As shown in Fig. 2, redox peaks could be received on bare GCE (Epa, 0.26 V; Epc, −0.05 V) (curve a), indicating that HQ expressed a quasireversible electrochemical behavior. For N-GR/GCE (curve b), redox peaks were located at 0.13 V (Epa) and −0.06 V (Epc), respectively. The oxidation peak shifted negatively and redox peak currents increased significantly relative to bare GCE. The results make clear that N-GR/ GCE processes superior electrocatalytic performance and sensitivity toward HQ. While on CS-N-GR/GCE (c), redox peak currents decreased with more positive oxidation peak (Epa, 0.17 V) and more positive reduction peak (Epc,-0.00 V) than curve b. The above experimental phenomena can attribute to existing of CS on N-GR. In addition, compared with Fig. 2, the redox peak and current had no obvious differences in diverse modified GCE when the PBS solution containing 3.5 mmol L−1 HQ and 1.0 mmol L−1 H2O2. This phenomenon further proves that H2O2 had no direct influence on the oxidation of HQ. In 0.1 mmol L−1 Fe(CN)63− and 0.1 mol L−1 KCl solution, the effective surface area of various electrodes was evaluateded with CV on the basis of the Randles-Sevcik equation [31]:
3.4. Electrochemical behaviors of the dual amplified immunosensors Diverse labeled Ab2 bioconjugates were measured in pH 7.0 PBS solution containing 3.5 mmol L−1 HQ and 1.0 mmol L−1 H2O2 or without 1.0 mmol L−1 H2O2. The Ab2-HRP modified electrode in pH 7.0 PBS solution containing 3.5 mmol L−1 HQ exhibited a relatively weak electrochemical response(Fig. 4A, curve a). The results for Ab2-Au@Ag NPs (Fig. 4A, curve b) and Ab2-HRP-Au@Ag NPs(Fig. 4A, curve c) can be higher relative to Ab2-HRP. When diverse labeled Ab2 bioconjugates were tested in pH 7.0 PBS solution containing 3.5 mM HQ and 1.0 mmol L−1 H2O2, it could be found that the electrochemical response ascended significantly (Fig. 4A, curve a', b' and c'). Apparently, compared with Fig. 4A, curve a' and b', the oxidation peak and reduction peak were increased significantly (Fig. 4A, curve c'). These comparisons demonstrated that the Ab2-HRP-Au@Ag NPs as dual amplification markers effectively triggered the disproportionation of H2O2, which could facilitate the catalytic oxidation of HQ to BQ. The DPV results for diverse labeled Ab2 bioconjugates are consistent with CV in pH 7.0 PBS solution containing 3.5 mmol L−1 HQ and 1.0 mmol L−1 H2O2. From
Ipc = (2.69 × 105) n3/2AD01/2 ν1/2C0 where Ipc is the reduction peak current (A), C0 is the concentration of K3[Fe(CN)6] (mmol L−1) and ν is the scan rate (V s−1), D0 is the diffusion coefficient of K3[Fe(CN)6] in the solution (D0 = 6.5 × 106 cm2 s−1) [32], A is the effective surface area (cm2), n is the electron transfer number. On account of the equation, the effective surface area of GCE, N-GR/GCE and CS-N-GR/GCE was calculated as 0.106, 0.408 and 0.425 cm2, respectively. Therefore, N-GR and CS-N-GR both can increase effective surface area. Furthermore, electrochemical impedance spectroscopy (EIS) was operated to evaluate the electrical conductivity (Fig. 3). On the N-GR/GCE (curve a), the Rct value in the low frequency region was lower than GCE (curve d) and CS-N-GR/GCE 26
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Fig. 4. (A) CV curves of immunosensors with different labeled Ab2 bioconjugates: (a, a´) Ab2HRP, (b, b´) Ab2-Au@Ag NPs, (c, c´) Ab2-HRPAu@Ag NPs in pH 7.0 PBS solution containing 3.5 mM HQ and 1.0 mmol L−1 H2O2 or without 1.0 mmol L−1 H2O2. Scan rate: 100 mV s−1. (B) DPV curves of immunosensors with different labeled Ab2 bioconjugates: (a) Ab2-HRP, (b) Ab2-Au@Ag NPs, (c) Ab2-HRP-Au@Ag NPs in pH 7.0 PBS solution containing 3.5 mM HQ and 1.0 mmol L−1 H2O2.
H2O2 in the solution were changed, respectively. It was observed that the optimized concentration of HQ was 3.5 mmol L−1 (Fig. 5B), while that for H2O2 was 1.0 mmol L−1 (Fig. 5C). Moreover, the optimal incubation time was discussed as exhibited in Fig. 5D. Because it was necessary to spend time on reaching the maximum formation of the sandwich immunocomplex, Ipc increased with the raise of incubation time. At the time of 60 min, Ipc reached maximum because the preparation of sandwich-type immunoassay could be get saturated. Hence, 60 min was chosen as the optimal incubation time for the immunoassay. 3.6. Electrochemical detection of CEA Under the optimal conditions, the proposed immunosensor was applied for detecting CEA with different concentrations. As shown in Fig. 6A, Ipc increases with the increase of CEA concentration from 0.01 to 100 ng mL−1. The calibration plot in Fig. 6B expresses a good linear relationship between the concentration of CEA and Ipc. The linear regression equations in the range from 0.0001 ng mL−1 to 100 ng mL−1 for CEA is Ipc (μA) = −39.08–5.24lgcCEA (ng mL−1) with a correlation coefficient of 0.986. On the basis of triple standard deviation, the LOD is calculated to 0.05 pg mL−1. Additionally, the results were compared with that of other CEA biosensors, the detection limit and linear range were displayed in Table 1. These comparison results indicate that the proposed electrochemical immunosensor is highly sensitive for CEA detection.
Scheme 2. Dual amplification schematic diagram.
above results, we can conclude that HRP as amplification marker facilitate the catalytic oxidation of HQ in the HQ-H2O2 system (Scheme 2, eq1, eq2). In addition, and most importantly, Au@Ag NPs could be considered as one kind of mimicking-enzyme catalytic marker to catalyze the oxidation reaction of HQ to BQ in the HQ-H2O2 system (Scheme 2, eq3, eq4, eq5) [34]. Au@Ag NPs as a catalyst can transfer electrons to hydrogen peroxide, resulting in the rupture of the oxygenoxygen bond of hydrogen peroxide and producing OH•. The OH• was stabilized at the surface of Au@Ag NPs. They would further react with HQ and generate BQ. Then, BQ was reduced by obtaining electron from cathode (Scheme 2, eq6). The corresponding DPV cathodic peak current intensity of Ab2-HRP-Au@Ag NPs (Fig. 4B, curve b) enhanced about 1.6-fold and 1.3-fold than Ab2-HRP (Fig. 4B, curve a) Ab2-Au@Ag NPs (Fig. 4B, curve b), respectively. Consequently say, the electrochemical reduction signal was dual amplified by HRP and Au@Ag NPs.
3.7. Reproducibility, stability and selectivity of the immunosensor To research the repeatability, the same five immunosensors, which were incubated with 10 ng mL−1 CEA and then incubated with Au@Ag NPs and HRP labeled Ab2 bioconjugates, were measured under the same conditions. The relative standard deviation after calculate was 4.3%, indicating that the repeatability of proposed immunosensor could be satisfactory. As we can see from Fig. 7A, to evaluate the stability of the proposed immunosensor, they were keeped in reserve at 4 °C for 10 days and Ipc were put down in writing periodically. During the period, the electrochemical response didn't changed much, and only dropped less than 12% on the last day for contrast with fresh prepared. It demonstrated that the proposed immunosensor had a reasonable stability. As we can see from Fig. 7B, the selectivity of the immunosensors toward CEA (10 ng mL−1) was studied using some possible interfering substances (100 ng mL−1 each), including IgG, BSA, AFP, PSA and UA. The results showed that compared with Ipc received from 10 ng mL−1 CEA, the variations of Ipc caused by interfering substances were inferior 7%, declaring that the immunosensor had excellent selectivity.
3.5. Optimization of the experimental conditions of the immunoassay The pH of buffer solution, substrate concentration of HQ and H2O2, and incubation time played important roles in the enhancement of current response, so further explorations were performed using DPV. Fig. 5 shows that cathodic current peak (Ipc) varies with the substrate concentration of buffer solution pH, HQ, H2O2, and incubation time (CEA 1 ng mL−1). Herein, PBS of pH 7.0 was selected as the detection solution for CEA detection. Additionally, the concentrations of HQ and
3.8. Application in analysis of serum samples As we can see from Table 2, the analytical feasibility and potential clinical application of the fabricated immunosensor were assessed and 27
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Fig. 5. Current response of immunosensor in PBS solution (A) at different pH under 1.0 mmol L−1 H2O2 and 3.5 mmol L−1 HQ incubation for 45 min; (B) at different HQ concentrations under 1.0 mmol L−1 H2O2 and incubation for 45 min (pH 7.0); (C) at different H2O2 concentrations under 3.5 mmol L−1 HQ, and incubation for 45 min (pH 7.0); (D) at different incubation times under 3.5 mmol L−1 HQ, 1.0 mmol L−1 H2O2. (Error bars present the relative standard deviation detected for 3 times).
Fig. 6. (A) DPV responses of the proposed immunosensor after incubation with different concentrations of CEA, a-g, 0.0001, 0.001, 0.01, 0.1, 1, 20, 100 ng mL−1; (B) calibration curves of the immunoassay toward CEA in PBS (pH HQ, 7.0), containing 3.5 mmol L−1 −1 1.0 mmol L H2O2 (Error bars present the relative standard deviation detected for 3 times).
Table 1 Comparison of analytical properties of various immunosensor towards CEA. Electrode modification
Labels
Characteristics
Linear range (ng mL−1)
LOD (pg mL−1)
Ref.
GCE/Bi fIlm GCE/Hg fIlm SPCE/Au/Cys/Lectin Au/L-Cys CS-NG/GCE
Si/Pb/anti-CEA CdS/DNA-anti-CEA HRP-CEA CNTs/PDDA/Con/HRP-CEA Au@Ag/HRP-anti-CEA
Required harsh conditions to synthesis high-quality QDs Required enrichment and time-consuming The complex electrode preparation and low sensitivity The complex process of synthetic and low sentivity Benefited from the catalysis of HRP and Au@Ag NPs
0.05–25 0.1–100 0.5–10 5–200 0.0001–100
5.0 3.3 10 18 0.05
[35] [36] [37] [24] This work
4. Conclusions
contrasted with the reference method (ELISA method). Seven complex clinical serum samples that obtained from Shandong Provincial Hospital were evaluated. We haven't seen the significant differences among the two methods. Subsequently, recovery experiments were carried out to evaluate the accuracy of our immunosensor. Compared with the values of reference method, it's clear that our immunosensor has excellent accuracy for CEA detection from complex clinical serum samples. We could also observe that the recovery was ranging from 80.0% to 110% and the RSD varied from 2.14% to 6.23%. The satisfying results clearly offered a potential application for the determination of CEA in clinical diagnostics.
In this work, a sensitive sandwich-type electrochemical immunosensor based on dual amplification strategy for detection of CEA was successfully developed. With the disproportionation reaction of the H2O2, HRP and Au@Ag NPs acted as dual amplification markers possessed superior electrocatalytic activity toward the oxidation of HQ. Meanwhile, nitrogen-doped graphene with excellent conductivity and large surface area were used as a platform for immobilize the primary antibody. Importantly, the LOD of the immunosensor for CEA detection could be lowered to 0.05 pg mL−1. The low detection limit showed that employing suitable materials to promote the electron transfer and amplify signal could be an effective way for the fabrication of highly sensitive nanomaterials-driven electrochemical immunosensors. This 28
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Fig. 7. (A) Current response of the immunosensor in different time; (B) Current response of the immunosensor in the detection of CEA (10 ng mL−1) with interfering substances (100 ng mL−1). transfected with CEA-mRNA, Eur. J. Cancer 38 (2002) 184–193. [7] N.R. Hughes, Factors which influence the radioimmunoassay of carcinoembryonic antigen (CEA), Mol. Immunol. 17 (1980) 665–673. [8] Q. Lang, F. Wang, L. Yin, M. Liu, V.A. Petrenko, A. Liu, Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen, Anal. Chem. 86 (2014) 2767–2774. [9] R. Li, F. Feng, Z.Z. Chen, Y.F. Bai, F.F. Guo, F.Y. Wu, G. Zhou, Sensitive detection of carcinoembryonic antigen using surface plasmon resonance biosensor with gold nanoparticles signal amplification, Talanta 140 (2015) 143–149. [10] X. Yang, Y. Zhuo, S. Zhu, Y. Luo, Y. Feng, Y. Xu, Selectively assaying CEA based on a creative strategy of gold nanoparticles enhancing silver nanoclusters' fluorescence, Biosens. Bioelectron. 64 (2015) 345–351. [11] H. Miao, L. Wang, Y. Zhuo, Z. Zhou, X. Yang, Label-free fluorimetric detection of CEA using carbon dots derived from tomato juice, Biosens. Bioelectron. 86 (2016) 83–89. [12] P. Santharaman, M. Das, S.K. Singh, N.K. Sethy, K. Bhargava, J.C. Claussen, C. Karunakaran, Label-free electrochemical immunosensor for the rapid and sensitive detection of the oxidative stress marker superoxide dismutase 1 at the pointof-care, Sensor. Actuator. B Chem. 236 (2016) 546–553. [13] M. Freitas, W. Correr, J. Cancino-Bernardi, M.F. Barroso, C. Delerue-Matos, V. Zucolotto, Impedimetric immunosensors for the detection of Cry1Ab protein from genetically modified maize seeds, Sensor. Actuator. B Chem. 237 (2016) 702–709. [14] L. Yang, H. Zhao, S. Fan, S. Deng, Q. Lv, J. Lin, C.P. Li, Label-free electrochemical immunosensor based on gold-silicon carbide nanocomposites for sensitive detection of human chorionic gonadotrophin, Biosens. Bioelectron. 57 (2014) 199–206. [15] Y. Shao, J. Sui, G. Yin, Y. Gao, Nitrogen-doped carbon nanostructures and their composites as catalytic materials for proton exchange membrane fuel cell, Appl. Catal. B Environ. 79 (2008) 89–99. [16] B.G. Sumpter, V. Meunier, J.M. Romoherrera, E. Cruzsilva, D.A. Cullen, H. Terrones, D.J. Smith, M. Terrones, Nitrogen-mediated carbon nanotube growth: diameter reduction, metallicity, bundle dispersability, and bamboo-like structure formation, ACS Nano 1 (2007) 369–375. [17] L. Zhao, C. Li, H. Qi, Q. Gao, C. Zhang, Electrochemical lectin-based biosensor array for detection and discrimination of carcinoembryonic antigen using dual amplification of gold nanoparticles and horseradish peroxidase, Sensor. Actuator. B Chem. 235 (2016) 575–582. [18] L. Ma, D. Ning, H. Zhang, J. Zheng, Au@Ag nanorods based electrochemical immunoassay for immunoglobulin G with signal enhancement using carbon nanofibers-polyamidoamine dendrimer nanocomposite, Biosens. Bioelectron. 68 (2015) 175–180. [19] H. Chang, H. Zhang, J. Lv, B. Zhang, W. Wei, J. Guo, Pt NPs and DNAzyme functionalized polymer nanospheres as triple signal amplification strategy for highly sensitive electrochemical immunosensor of tumour marker, Biosens. Bioelectron. 86 (2016) 156–163. [20] Y. Wang, Y. Zhang, Y. Su, F. Li, H. Ma, H. Li, B. Du, Q. Wei, Ultrasensitive nonmediator electrochemical immunosensors using Au/Ag/Au core/double shell nanoparticles as enzyme-mimetic labels, Talanta 124 (2014) 60–66. [21] X. Yang, Y. Wang, Y. Liu, X. Jiang, A sensitive hydrogen peroxide and glucose biosensor based on gold/silver core–shell nanorods, Electrochim. Acta 108 (2013) 39–44. [22] Y. Yang, Q. Liu, Y. Liu, J. Cui, H. Liu, P. Wang, Y. Li, L. Chen, Z. Zhao, Y. Dong, A novel label-free electrochemical immunosensor based on functionalized nitrogendoped graphene quantum dots for carcinoembryonic antigen detection, Biosens. Bioelectron. 90 (2017) 31–38. [23] Y. Zhao, L. Liu, D. Kong, H. Kuang, L. Wang, C. Xu, Dual amplified electrochemical immunosensor for highly sensitive detection of Pantoea stewartii sbusp. stewartii, ACS Appl. Mater. Interfaces 6 (2014) 21178–21183. [24] P. Yang, X. Li, L. Wang, Q. Wu, Z. Chen, X. Lin, Sandwich-type amperometric immunosensor for cancer biomarker based on signal amplification strategy of multiple enzyme-linked antibodies as probes modified with carbon nanotubes and concanavalin A, J. Electroanal. Chem. 732 (2014) 38–45. [25] A.K. Samal, L. Polavarapu, S. Rodal-Cedeira, L.M. Liz-Marzan, J. Perez-Juste,
Table 2 The results of determine CEA in serum samples(n = 3). Sample
1 2 3 4 5 6 7
ELISA (ng mL−1)
The immunosensor Found (ng mL−1)
Added (ng mL−1)
Total found (ng mL−1)
Recovery (%)
RSD (%)
2.25 1.17 1.91 7.61 26.0 22.2 28.5
2.28 1.20 1.96 7.53 25.7 22.4 28.1
1 1 1 5 10 10 10
3.41 2.19 2.82 12.1 28.8 29.0 42.4
105 101 97.0 96.0 80.0 90.0 110
6.23 4.84 3.95 4.16 5.32 2.14 2.82
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