Biosensors and Bioelectronics 54 (2014) 20–26
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Electrochemiluminescence of luminol enhanced by the synergetic catalysis of hemin and silver nanoparticles for sensitive protein detection Xinya Jiang, Yaqin Chai n, Haijun Wang, Ruo Yuan n Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People's Republic of China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 July 2013 Received in revised form 27 September 2013 Accepted 7 October 2013 Available online 23 October 2013
A novel and ultrasensitive electrochemiluminescence (ECL) immunosensor, which was based on the amplifying ECL of luminol by hemin-reduced graphene oxide (hemin-rGO) and Ag nanoparticles (AgNPs) decorated reduced graphene oxide (Ag-rGO), was constructed for the detection of carcinoembryonic antigen (CEA). For this proposed sandwich-type ECL immunosensor, Au nanoparticles electrodeposited (DpAu) onto hemin-rGO (DpAu/hemin-rGO) constructed the base of the immunosensor. DpAu had outstanding electrical conductivity to promote the electron transfer at the electrode interface and had good biocompatibility to load large amounts of primary antibody (Ab1), which provided an excellent platform for this immunosensor. Moreover, AgNPs and glucose oxidase (GOD) functionalized graphene labeled secondary antibody (Ag-rGO–Ab2–GOD) was designed as the signal probe for the sandwiched immunosensor. Not only did the hemin-rGO improve the electron transfer of the electrode surface, but hemin also further amplified the ECL signal of luminol in the presence of hydrogen peroxide (H2O2). With the aid of Ag-rGO–Ab2–GOD, enhanced signal was obtained by in situ generation of H2O2 and catalysis of AgNPs to ECL reaction of the luminol–H2O2 system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of CEA in the range from 0.1 pg mL 1 to 160 ng mL 1 with a detection limit of 0.03 pg mL 1 (SN 1 ¼ 3). & 2013 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Luminol Hemin Graphene Nanoparticles Glucose oxidase
1. Introduction Recently, electrochemiluminescence (ECL) has become an extremely attractive method because of its simplified set-up, low background signal, high sensitivity, rapidity and controllability (Jiang and Ju, 2007; Cao et al., 2006; Jie et al., 2010). As one of the most commonly used efficient ECL luminophores, luminol has some excellent inherent properties such as inexpensive, nontoxic and high light-emitting quantum yield (Qin et al., 1998; Lin and Chen, 2006), so special attention had focused on the ECL studies concerning luminol for food testing (Haghighi and Bozorgzadeh, 2011; Guo et al., 2011b), clinical diagnostics (Lv et al., 2004; Shen et al., 2011) and DNA analysis (Zhang et al., 2009; Chai et al., 2010). In order to amplify the ECL signal of luminol, several efforts have been devoted to accelerate the oxidization of luminol, through hastening the decomposition of H2O2 to form reactive oxygen species (ROSs) such as superoxide anion O2d and hydroxyl radical OHd, because these free radicals are the important intermediates
n
Corresponding authors. Tel.: þ 86 23 68252277; fax: þ 86 23 68253172. E-mail addresses: [email protected] (Y. Chai), [email protected] (R. Yuan).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.006
leading to the ECL reaction (Vitt et al., 1991; Cai et al., 2010). To the best of our knowledge, nanoparticles, which have virtues of good biocompatibility, large surface area, excellent electrocatalytic activity and fascinating conductivity, not only facilitate the immobilization of enzyme and proteins but also catalyze H2O2 to generate a great amount of ROSs. For instance, our group reported an ECL immunosensor based on the luminol–H2O2 system using Pd nanoparticles as catalysis for ultrasensitive immunoassay (Niu et al., 2011). Cheng et al. (2012) developed an ultrasensitive luminol ECL immunosensor for CEA detection based on ZnO nanoparticles. Among the wide variety of metal nanoparticles, AgNPs show excellent catalytic and electrocatalytic activities (Zhang et al., 2005; Jana et al., 1999). In addition, Guo et al. (2008) had demonstrated that AgNPs exhibited better catalysis activity than gold and platinum nanoparticles. Therefore, inspired by the superior property, AgNPs can be selected as an efficient catalyst in ECL sensors for its low oxidation potential when oxidized by H2O2 (Guo et al., 2008). Hemin, with high electrocatalytic activity due to its reversible redox potential of FeIII/FeII, can catalyze a variety of oxidation reactions like peroxidase enzymes (Genfa and Dasgupta, 1992). However, direct application of hemin as an oxidation catalyst is of
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a significant challenge because of its molecular aggregation, which causes passivation of its catalytic activity (Bruice, 1991). In order to improve the stability or activity, the hemin decorated on materials with high surface area is becoming an alternative approach. For example, G-quadruplex DNA oligomers, graphene or carbon nanotube have been considered as excellent carriers for hemin (Jiang et al., 2013; Yuan et al., 2012; Xie et al., 2012; Guo et al., 2011a, 2011b; Tu et al., 2010; Liu et al., 2007). Based on the functionalized hemin, many electrochemical (Wang et al., 2013; Bai et al., 2011) or chemiluminescence (CL) (Pavlov et al., 2004; Liu et al., 2011) sensors have been successfully constructed and exciting results have been obtained. However, until now, researchers have concentrated little attention on the study of the application of hemin in the ECL sensor. In the present research, we constructed a novel and simple sandwich-type immunosensor for CEA detection via amplifying the ECL signal of luminol by the catalysis of hemin-rGO and Ag-rGO. In brief, DpAu obtained by electrodeposition on the hemin-rGO nanocomposites was served as the immunosensor platform, which played two main roles. On one hand, DpAu amplified the ECL signal of luminol for its fascinating conductivity, on the other hand, DpAu served as carriers to immobilize primary antibody (Ab1) for its favorable biocompatibility. Moreover, AgNPs-rGO nanocomposites were used to load secondary antibody (Ab2) and GOD. In the presence of oxygen, these loaded GOD immediately catalyzed the oxidation of glucose in the detection to in situ generate H2O2, which could subsequently promote the oxidation of luminol with an amplified ECL signal. Additionally, hemin and AgNPs could further enhance the ECL signal of luminol owing to the decomposable catalysis of H2O2 to produce increased amounts of ROSs. With excellent sensitivity, selectivity and stability, the as-proposed ECL immunosensor based on hemin-rGO and AgNPs-rGO provided great potential in clinical applications.
2. Experimental 2.1. Reagents and material Graphene oxide (GO) was obtained from Nanjing Xianfeng Nano Co. (Nanjing, China). Hemin, luminol (98%), glucose oxidase (GOD), AgNO3 and bovine serum albumin (BSA, 96–99%) were bought from Sigma-Aldrich Co. (St. Louis, MO, USA). Gold chloride tetrahydrate (HAuCl4 4H2O) and sodium borohydride (NaBH4) were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). Carcinoembryonic antibody (Anti-CEA), carcinoembryonic antigen (CEA), fetoprotein antigen (AFP), prostate specific antigen (PSA) and human chorionic gonadotropin (HCG) standard solutions were obtained from Biocell (Zhengzhou, China). The serum samples were provided by South-west Hospital (Chongqing, China). 1 10 2 M Stock solution of luminol was prepared by dissolving luminol in 0.1 M NaOH solution and storing it at 4 1C when not in use. Phosphated buffered solution (PBS) (pH 7.4, 0.1 M) was prepared using 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl. The standard CEA, AFP, PSA and HCG stock solutions were prepared with PBS (pH 7.4) and stored at 4 1C. All other chemicals and solvents used were of analytical grade and were used as received. Double-distilled water was used throughout this study. 2.2. Apparatus The ECL signals were monitored with a model MPI-A ECL analyzer (Xi'an Remax Electronic Science & Technology Co. Ltd., Xi'an, China) with the voltage of the photomultiplier tube (PMT) set at 800 V in the process of detection and the potential scan from
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0.2 to 0.7 V. Cyclic voltammetric (CV) measurements and depositions were carried out on a CHI 610A electrochemistry workstation (Shanghai CH Instruments, China). All experiments were performed with a conventional three-electrode system containing a platinum wire as counter electrode, an Ag/AgCl (sat. KCl) as reference electrode and the modified glassy carbon electrode (GCE Φ ¼4 mm) as working electrode. The scanning electron micrograph was taken by using scanning electron microscopy (SEM, S-4800, Hitachi, Japan). UV–vis absorption spectra were performed on a Lambda 17 UV–vis 8500 spectrometer (PE Co., USA) added to a quartz cuvette. Transmission electron microscope (TEM) images were acquired by a transmission electron microscope (TEM, TECNAI 10, Philips Fei Co., Hillsboro, OR, Japan).
2.3. The preparation of hemin-rGO and Ag-rGO nanocomposites Hemin-rGO nanocomposites were synthesized with a simple wet-chemical strategy through the π–π interactions by following the procedure according to the literature (Zhang et al., 2012) with a minor modification. Briefly, 20.0 mL of the homogeneous graphene oxide dispersion (0.5 mg mL 1) was mixed with 20.0 mL of hemin aqueous solution (0.5 mg mL 1) and 200.0 μL of ammonia solution. Then 30 μL hydrazine solution was added into the resulting mixture. After vigorously stirring for a few minutes, the vial was put in a water bath (60 1C) for 3.5 h. Next, the product was obtained by filtration and washed several times with doubledistilled water. The obtained hemin-rGO nanocomposites can be redispersed readily in water by ultrasonication. Moreover, the preparation of rGO was similar to that of hemin-rGO without the addition of hemin. The Ag-rGO nanocomposites were prepared according to the reference (Li and Liu, 2010) with a slight modification by the reduction of silver ions in the GO dispersion solution. First, 2 mL AgNO3 solution (0.01 M) was added to 20 mL GO solution (0.25 mg mL 1) with stirring for 30 min. Subsequently, an alkaline solution of sodium borohydride (10 mL, 0.8 mg mL 1) was added to the resulting homogeneous solution. Then the mixture was vigorously stirred at room temperature for another 2 h. Finally, the color of the dispersion solution changed from brown to black. Then the product was collected by centrifugation and washed several times with double-distilled water. The obtained compounds were stored in the refrigerator at 4 1C when not in use. Additionally, the preparation of Pt-rGO and Pd-rGO nanocomposites were similar to that of Ag-rGO. 2.4. Preparation of GOD and Ab2 labeled Ag-rGO (Ag-rGO–Ab2–GOD) bioconjugate The Ag-rGO–Ab2–GOD bioconjugates were prepared according to the following steps. Briefly, 1 mL of the above Ag-rGO suspension was mixed with 0.2 mL anti-CEA, allowing them to react under soft stirring at 4 1C for 6 h. Then, Ag-rGO–Ab2 bioconjuagtes were obtained by centrifugation at 10,000 rpm for 15 min at 4 1C to remove excess anti-CEA. Subsequently, 1 mL GOD (1 mg mL 1) was added into the obtained Ag-rGO–Ab2 bioconjuagtes and incubated for 4 h at 4 1C to block the remaining active sites of the AgNPs surface. At last, the Ag-rGO–Ab2–GOD bioconjugates were collected by centrifugation and redispersed in 1 mL PBS (pH 7.4) and stored at 4 1C until use. 2.5. Fabrication of the ECL immunosensor Scheme 1 shows the schematic illustration of the ECL immunosensor fabrication process. The glassy carbon electrode (GCE,
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Scheme 1. The fabrication process of the proposed ECL immunosensor and the mechanism for the multiple signal amplification strategy.
Ф ¼4 mm) was firstly polished on a polishing cloth with 0.3 and 0.05 μm alumina powder respectively to obtain a mirror-like surface, and was sonicated with ethanol and double-distilled water. 8 μL of the hemin-rGO nanocomposite was firstly dropped onto the pretreated GCE (abbreviated as hemin-rGO/GCE) to increase the effective area of the electrode. After drying at room temperature, the electrode was immersed into HAuCl4 solution (1%) to form DpAu layer (abbreviated as DpAu/hemin-rGO/GCE) via potentiostatic electrodeposition for 30 s at 0.2 V. Then 20 μL of anti-CEA (Ab1) solution was dropped onto the surface of the DpAu layer (abbreviated as Ab1/DpAu/hemin-rGO/GCE) with 6 h incubation at 4 1C. Subsequently, 15 μL of BSA solution (1%) was placed onto the electrode for 30 min to block the non-specific binding sites (abbreviated as BSA/Ab1/DpAu/hemin-rGO/GCE). Followed by washing thoroughly with pH 7.4 PBS, the modified GCE was incubated in CEA solution for 40 min at room temperature (abbreviated as CEA/BSA/Ab1/DpAu/hemin-rGO/GCE). At last, as a sandwich format, the resultant electrode was immersed in the Ag-rGO–Ab2–GOD solution for immunereaction.
3. Results and discussion
confirm that hemin molecules were attached to graphene. Fig. 1B (inset of Fig. 1A) displays a loose and homogeneous surface morphology indicating that hemin and graphene were welldispersed. After Au nanoparticles were electrodeposited onto the hemin-rGO membranes, a porous and pultaceous morphology was observed with many globular features (Fig. 1C), which provided a significant increase in the effective electrode surface for the loading of biomolecules. To verify the successful synthesis of AgNPs-rGO, UV–vis absorption spectrum was employed. Fig. 1D showed the UV–vis spectra of GO and Ag-rGO. As expected, the UV–vis spectrum of GO (curve a) exhibited a maximum absorption at 226 nm, which corresponds to the π–πn transitions of aromatic CQC bonds. Compared with the UV–vis spectrum obtained from the Ag-rGO (curve b), the peak at 226 nm is noticeablely redshifted to 262 nm because of the formation of rGO. Simultaneously, a distinctive absorption peak at around 399 nm is also observed, suggesting the formation of AgNPs. Additionally, Fig. 1E (inset of Fig. 1D) displays the TEM image after the rGO was modified with AgNPs, from which we can see that AgNPs were distributed uniformly over the lamellate mono-layer sheets of rGO. The result further indicated that the AgNPs-rGO nanocomposites were successfully prepared.
3.1. Characteristics of the different nanocomposites 3.2. The characterization of the ECL immunosensor The successful synthesis of hemin-rGO was characterized by a UV–vis method. As shown in Fig. 1A, graphene oxide (GO) dispersion (curve a) exhibited a maximum absorption at 226 nm which ascribed to the π πn transition of aromatic CQC bonds. The spectrum of hemin solution (curve b) included a typical Soret band absorption at 386 nm and a group of weak absorptions (Q-bands) between longer wavelengths (500–700 nm) (Xue et al., 2012). While the hemin-rGO (curve c) not only displayed a broad absorption at 267 nm which corresponds to the rGO, but also contained a peak at 417 nm which corresponded to the Soret band of hemin with a large bathochromic shift (31 nm). These results clearly
3.2.1. CV characterization of the ECL immunosensor CVs at each immobilization steps were recorded to monitor the fabrication of the proposed ECL immunosensor and the analysis was performed in 0.10 M PBS (pH 7.4) from 0.6 to 0 V at a scan rate of 100 mV s 1. As shown in Fig. 2A, a pair of well-defined redox peaks were obviously observed when the hemin-rGO were presented on the electrode surface (curve b) compared with bare GCE (curve a). The redox peaks should be attributed only to the direct electrochemistry of hemin, which is the characteristic of a single electron transfer process of iron at the core of hemin
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Fig. 1. (A) UV–vis spectra of (a) GO suspension, (b) hemin solution and (c) hemin-rGO suspension. (B) SEM (inset A) image of hemin-rGO. (C) SEM image of DpAu/heminrGO. (D) UV–vis absorption spectra of GO suspension (a) and Ag-rGO nanocomposites (b), and (E) TEM (inset D) image of Ag-rGO.
(Guo et al., 2011a). From the image, we can see that the current of DpAu/hemin-rGO modified electrode increased (curve c), which was attributed to the excellent conductivity of DpAu. Peak current decreased clearly after anti-CEA was assembled on the DpAu layer (curve d), indicating that the capture antibody protein can severely hinder the electron transfer. Subsequently, when the nonelectroactive BSA was used to block nonspecific sites, the peak current further decreased (curve e). After the immunosensor was incubated with CEA antigen, a dramatically decrease in current was observed (curve f), which incriminated to the antigen–antibody immunocomplex blocking layer.
3.2.2. ECL characterization of the immunosensor The corresponding ECL intensity–potential curves of the modified electrode were recorded step by step to characterize the fabrication process of the ECL immunosensor. The detection process was executed in 2 mL PBS (pH 7.4) with 1 10 4 M luminol and 1 10 2 M glucose. As shown in Fig. 2B, a very weak ECL signal (curve a) was obtained for the bare GCE. After the bare electrode was modified with hemin-rGO nanocomposites (curve b), the ECL signal increased for the reasons that hemin and graphene can both facilitate electron transfer to the electrode surface. When Au nanoparticles were electrodeposited onto the modified electrode, the ECL signal was further enhanced (curve c) for the excellent conductivity of the metal nanoparticle. Then the ECL signals declined in succession after immobilization of anti-CEA (curve d), blocking with BSA (curve e) and incubation with CEA (curve f). That was attributed to the formation of non-conductive layers which slowed down the electron transfer speed and led to the decrease of signals. However, when the GOD–Ab2–Ag-rGO bioconjugates were immobilized onto the modified electrode surface and proper amounts of glucose were added into the detection solution, a great enhancement of the ECL
signal (curve g) appeared and the possible mechanism is shown in Scheme 1. 3.3. The probable mechanism of the immunosensor To the best of our knowledge, H2O2 is a kind of an efficient coreactant for luminol ECL reactions. In the case of the luminol– H2O2 system, ROSs such as superoxide anion (O2d ) and hydroxyl radical (OHd), which are supposed to be generated from H2O2 (Li et al., 2009), are the important intermediates leading to the ECL reaction of luminol. But in the absence of a catalyst, the rate of H2O2 changing to ROSs is very slow with a relatively weak ECL signal, so in order to amplify the ECL signal of luminol, metal nanoparticles are usually used to hasten the decomposition of H2O2. According to Fig. 3A, the ECL signal of rGO modified electrode (curve a) is similar with that of hemin-rGO (curve b) modified electrode when they are detected in 10 4 M luminol solution. After we added 4 mL H2O2 (0.01 M) in 10 4 M luminol in PBS (pH 7.4) solution, it could be seen that the ECL signals increased, apparently for the reason that H2O2 acted as a coreactant of luminol. However, the curve d (hemin-rGO modified electrode) is stronger than curve c (rGO modified electrode), which may be attributed to the effect of hemin. From Fig. 3B, in the absence of GOD, the ECL signal of Ag-rGO–BSA–Ab2/CEA/BSA/Ab1/DpAu/rGO was relatively weak (curve a), while the ECL signal could be significantly enhanced in the presence of GOD (curve c) for the GOD catalyzed glucose to in situ produce H2O2, which was subsequently catalyzed by AgNPs. However, the ECL signal was lower due to the absence of AgNPs (curve b). These results indicated that hemin, GOD and AgNPs were necessary for the strong ECL signal. According to the literatures (Chen et al., 2000; Cao et al., 2013), the probable mechanism is shown as follows: (LH is the deprotonated luminol and Ap2 n is 3-aminophthalate)
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LH e -LH -L þ H þ GOx
Glucose þ O2 - Gluconic acid þ H 2 O2 H2 O2 þ L U
AgNPs; hemin
-
Ap2 n-Ap2 þhυ
Ap2 n þ products
(1)
3.4. Performance of the proposed ECL immunosensor for CEA detection
ð2Þ ð3Þ
(4)
3.4.1. Calibration curve Under the above optimum conditions, we investigated the quantitative range of the proposed ECL immunosensor for the CEA detection and found that the ECL intensity of the immunosensor increased with the increasing antigen concentrations by the sandwich immunoreaction format. The relationship between the ECL intensity and the concentration of CEA is shown in Fig. 4. A linear relationship between the ECL intensity and the concentrations of CEA antigen was obtained (inset of Fig. 4) in the range from 0.1 pg mL 1 to 160 ng mL 1. The detection limit of CEA was 0.03 pg mL 1 (SN 1 ¼3). The linear relationship can be represented as IECL ¼5856.86 þ1077.38 log (cCEA) with the correlation coefficient of R¼0.9947. Compared with other methods of CEA immunoassay (Table S1), the proposed ECL immunosensor exhibited higher sensitivity with a lower detection limit. The results demonstrated that the proposed immunosensor could be employed to determine CEA quantitatively. 3.4.2. Selectivity, stability and reproducibility of the ECL immunosensor The stability of the ECL intensity of the proposed immunosensor to various concentrations of CEA antigen was also investigated and the results are presented in Fig. 5(A). It shows that the ECL intensity increased with the increasing concentration of CEA, and a relatively stable curve at every concentration could be obtained. From the practical point, selectivity is an important criterion for immunosensors. To investigate the selectivity of the immunosensor,
Fig. 2. (A) CVs of (a) bare GCE, (b) hemin-rGO, (c) DpAu/hemin-rGO, (d) Ab1/DpAu/ hemin-rGO, (e) BSA/Ab1/DpAu/hemin-rGO, and (f) CEA/BSA/Ab1/DpAu/hemin-rGO modified GCE in 0.1 M PBS (pH 7. 4), scan rate: 100 mV s 1. (B) ECL profiles of different modified electrodes: (a) bare GCE, (b) hemin-rGO, (c) DpAu/hemin-rGO, (d) Ab1/DpAu/hemin-rGO, (e) BSA/Ab1/DpAu/hemin-rGO, (f) CEA/BSA/Ab1/DpAu/ hemin-rGO and (g) Ab2/CEA/BSA/Ab1/DpAu/hemin-rGO.
Fig. 4. ECL–potential curves of the immunosensor with different concentrations of CEA. CEA concentration (ng mL 1): (a) 0.0001, (b) 0.001, (c) 0.01, (d) 0.1, (e) 1, (f) 10, (g) 40, (h) 80, and (i) 160.
Fig. 3. (A) ECL signals for rGO (a), hemin-rGO (b), modified electrodes in luminol (10 4 M) and ECL signals for rGO (c), and hemin-rGO (d) modified electrodes in luminol (10 4 M) and H2O2 (0.01 M). (B) ECL signals for Ag-rGO–BSA–Ab2/CEA/BSA/Ab1/DpAu/rGO (a), rGO-GOD-Ab2/CEA/BSA/Ab1/DpAu/rGO (b) and Ag-rGO–GOD–Ab2/CEA/BSA/ Ab1/DpAu/rGO (c), which were detected in 2 mL PBS (pH 7.4) with 1 10 4 M luminol containing 1 10 2 M glucose.
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Fig. 5. (A) The ECL stability of proposed immunosensor to various concentrations of CEA antigen. (B) The specificity of the proposed ECL immunosensor: blank (without CEA), AFP (20 ng mL 1), PSA (20 ng mL 1), HCG (100 MIU mL 1), CEA (1 ng mL 1) and mixture (1 ng mL 1 CEA, 20 ng mL 1 AFP, 20 ng mL 1 PSA and 100 MIU mL 1 HCG).
some interference experiments were accomplished by detecting the ECL signals of the proposed immunosensor by incubating with interference samples under the same experimental conditions. As shown in Fig. 5(B), compared with blank solution, no remarkable changes were observed from the pure interference solution of AFP (20 ng mL 1), PSA (20 ng mL 1) and HCG (100 MIU mL 1). However, the ECL signal obtained from the mixed incubating solution (1 ng mL 1 CEA, 100 MIU mL 1 HCG, 20 ng mL 1 AFP and 20 ng mL 1 PSA) was in concordance with that obtained from the pure CEA solution. The experimental results suggested that the immunosensor displayed good selectivity for the determination of CEA. The reproducibility of the proposed immunosensor was examined by determining 10 ng mL 1 of CEA antigen for three reduplicate measurements in the same batch (intra-assay) and various batches (inter-assay). The relative standard deviations (R.S.D.) of the intra- and inter-assay were less than 5%. This demonstrated that the reproducibility of the proposed immunosensor for detecting CEA was acceptable.
potential in the determination of tumor markers for clinical diagnostics.
Acknowledgments This work was financially supported by the NNSF of China (21075100 and 21275119), State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), Ministry of Education of China (708073), Natural Science Foundation of Chongqing City (CSTC2010BB4121 and CSTC-2011BA7003), Specialized Research Fund for the Doctoral Program of Higher Education (20100182110015) and the Postgraduate Science and Technology Innovation Program of Southwest China University (Grant no. KB2010006).
Appendix A. Supplementary material Supporting information associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios. 2013.10.006.
3.5. Preliminary analysis of real samples References The feasibility of the ECL immunoassay system for clinical applications was investigated by the standard addition methods in human serum. As shown in Table S2, the recovery (between 93.7% and 106.5%) was acceptable, which provided a promising tool for determining AFP in real biological samples.
4. Conclusions In conclusion, we have successfully developed a novel sandwichtype luminol ECL immunosensor for ultrasensitive detection of CEA based on hemin-rGO and Ag-rGO. In the assay protocol, the amplification of the luminol ECL signal could be obtained by in situ generated H2O2 as a coreactant with high-content GOD and the catalysis of hemin and AgNPs for the decomposition of H2O2 to produce increased amounts of ROSs. Three main advantages could be ascribed to the high sensitivity: Firstly, the hemin not only had excellent catalytic performance to decompose H2O2, but could also facilitate electron transfer to the electrode surface. Secondly, DpAu had outstanding electrical conductivity to promote the electron transfer and increase the ECL intensity. Lastly, AgNPs loaded large amounts of GOD which could efficiently catalyze glucose to in situ generate H2O2 and simultaneously catalyze H2O2 to produce various ROSs. Due to the multiple signal amplification strategies, the luminol ECL signal of the fabricated immunosensor was greatly enhanced. Moreover, the as-proposed immunosenosr exhibited outstanding sensitivity, acceptable reproducibility and specificity, thus this strategy could provide a promising
Bai, L.J., Yuan, R., Chai, Y.Q., Yuan, Y.L., Zhuo, Y., Mao, L., 2011. Biosens. Bioelectron. 26, 4331–4336. Bruice, T.C., 1991. Acc. Chem. Res. 24, 243–249. Cai, X., Yan, J.L., Chu, H.H., Wu, M.S., Tu, Y.F., 2010. Sens. Actuators B: Chem. 143, 655–659. Cao, W.D., Ferrance, J.P., Demas, J., Landers, J.P., 2006. J. Am. Chem. Soc. 128, 7572–7578. Cao, Y.L., Yuan, R., Chai, Y.Q., Liu, H.J., Liao, Y.H., Zhuo, Y., 2013. Talanta 113, 106–112. Chai, Y., Tian, D.Y., Wang, W., Cui, H., 2010. Chem. Commun. 46, 7560–7562. Chen, G.N., Zhang, L., Lin, R.E., Yang, Z.C., Duan, J.P., Chen, H.Q., 2000. Talanta 50, 1275–1281. Cheng, Y.F., Yuan, R., Chai, Y.Q., Niu, H., Cao, Y.L., Liu, H.J., Bai, L.J., Yuan, Y.L., 2012. Anal. Chim. Acta 745, 137–142. Genfa, Z., Dasgupta, P.K., 1992. Anal. Chem. 64, 517–522. Guo, J.Z., Cui, H., Zhou, W., Wang, W., 2008. J. Photochem. Photobiol. A: Chem. 193, 89–96. Guo, Y.J., Deng, L., Li, J., Guo, S.J., Wang, E.K., Dong, S.J., 2011a. ACS Nano 5, 1282–1290. Guo, Z.Y., Gai, P.P., Hao, T.T., Duan, J., 2011b. J. Agric. Food Chem. 59, 5257–5262. Haghighi, B., Bozorgzadeh, S., 2011. Talanta 85, 2189–2193. Jana, N.R., Sau, T.K., Pal, T., 1999. J. Phys. Chem. B 103, 115–121. Jiang, H., Ju, H.X., 2007. Chem. Commun. 4, 404–406. Jiang, L.P., Yuan, R., Chai, Y.Q., Yuan, Y.L., Bai, L.J., Wang, Y., 2013. Analyst 138, 1497–1503. Jie, G.F., Liu, P., Zhang, S.S., 2010. Chem. Commun. 46, 1323–1325. Li, J., Liu, C.Y., 2010. Eur. J. Inorg. Chem. 8, 1244–1248. Li, S.F., Zhang, X.M., Du, W.X., Ni, Y.H., Wei, X.W., 2009. J. Phys. Chem. C 113, 1046–1051. Lin, K.C., Chen, S.M., 2006. J. Electroanal. Chem. 589, 52–59. Liu, X.Q., Freeman, R., Golub, E., Willner, I., 2011. ACS Nano 5, 7648–7655. Liu, Y., Yan, Y.L., Lei, J.P., Wu, F., Ju, H.X., 2007. Electrochem. Commun. 9, 2564–2570. Lv, J., Luo, L.R., Zhang, Z.J., 2004. Anal. Chim. Acta 510, 35–39. Niu, H., Yuan, R., Chai, Y.Q., Mao, L., Yuan, Y.L., Cao, Y.L., Zhuo, Y., 2011. Chem. Commun. 47, 8397–8399.
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Pavlov, V., Xiao, Y., Gill, R., Dishon, A., Kotler, M., Willner, I., 2004. Anal. Chem. 76, 2152–2156. Qin, W., Zhang, Z.J., Liu, H.J., 1998. Anal. Chem. 70, 3579–3584. Shen, W., Tian, D.Y., Cui, H., Yang, D., Bian, Z.P., 2011. Biosens. Bioelectron. 27, 18–24. Tu, W.W., Lei, J.P., Zhang, S.Y., Ju, H.X., 2010. Chem.-Eur. J. 16, 10771–10777. Vitt, J.E., Johnson, D.C., Engstrom, R.C., 1991. J. Electrochem. Soc. 138, 1637–1643. Wang, Q.B., Lei, J.P., Deng, S.Y., Zhang, L., Ju, H.X., 2013. Chem. Commun. 49, 916–918. Xie, S.B., Chai, Y.Q., Yuan, R., Bai, L.J., Yuan, Y.L., Wang, Y., 2012. Anal. Chim. Acta 755, 46–53.
Xue, T., Jiang, S., Qu, Y.Q., Su, Q., Cheng, R., Dubin, S., Chiu, C.Y., Kaner, R., Huang, Y., Duan, X.F., 2012. Angew. Chem. Int. Ed. 51, 3822–3825. Yuan, Y.L., Yuan, R., Chai, Y.Q., Zhuo, Y., Ye, X.Y., Gan, X.X., Bai, L.J., 2012. Chem. Commun. 48, 4621–4623. Zhang, L.Y., Li, D., Meng, W.L., Huang, Q., Su, Y., Wang, L.H., Song, S.P., Fan, C.H., 2009. Biosens. Bioelectron. 25, 368–372. Zhang, M.H., Yuan, R., Chai, Y.Q., Chen, S.H., Zhong, X., Zhong, X.A., Wang, C., 2012. RSC Adv. 2, 4639–4641. Zhang, Z.J., Liu, C.Y., Sun, L.W., 2005. J. Phys. Chem. B 109, 1730–1735.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Electrochemiluminescence of luminol enhanced by the synergetic catalysis of hemin and silver nanoparticles for sensitive protein detection Xinya Jiang, Yaqin Chai n, Haijun Wang, Ruo Yuan n Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People's Republic of China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 July 2013 Received in revised form 27 September 2013 Accepted 7 October 2013 Available online 23 October 2013
A novel and ultrasensitive electrochemiluminescence (ECL) immunosensor, which was based on the amplifying ECL of luminol by hemin-reduced graphene oxide (hemin-rGO) and Ag nanoparticles (AgNPs) decorated reduced graphene oxide (Ag-rGO), was constructed for the detection of carcinoembryonic antigen (CEA). For this proposed sandwich-type ECL immunosensor, Au nanoparticles electrodeposited (DpAu) onto hemin-rGO (DpAu/hemin-rGO) constructed the base of the immunosensor. DpAu had outstanding electrical conductivity to promote the electron transfer at the electrode interface and had good biocompatibility to load large amounts of primary antibody (Ab1), which provided an excellent platform for this immunosensor. Moreover, AgNPs and glucose oxidase (GOD) functionalized graphene labeled secondary antibody (Ag-rGO–Ab2–GOD) was designed as the signal probe for the sandwiched immunosensor. Not only did the hemin-rGO improve the electron transfer of the electrode surface, but hemin also further amplified the ECL signal of luminol in the presence of hydrogen peroxide (H2O2). With the aid of Ag-rGO–Ab2–GOD, enhanced signal was obtained by in situ generation of H2O2 and catalysis of AgNPs to ECL reaction of the luminol–H2O2 system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of CEA in the range from 0.1 pg mL 1 to 160 ng mL 1 with a detection limit of 0.03 pg mL 1 (SN 1 ¼ 3). & 2013 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Luminol Hemin Graphene Nanoparticles Glucose oxidase
1. Introduction Recently, electrochemiluminescence (ECL) has become an extremely attractive method because of its simplified set-up, low background signal, high sensitivity, rapidity and controllability (Jiang and Ju, 2007; Cao et al., 2006; Jie et al., 2010). As one of the most commonly used efficient ECL luminophores, luminol has some excellent inherent properties such as inexpensive, nontoxic and high light-emitting quantum yield (Qin et al., 1998; Lin and Chen, 2006), so special attention had focused on the ECL studies concerning luminol for food testing (Haghighi and Bozorgzadeh, 2011; Guo et al., 2011b), clinical diagnostics (Lv et al., 2004; Shen et al., 2011) and DNA analysis (Zhang et al., 2009; Chai et al., 2010). In order to amplify the ECL signal of luminol, several efforts have been devoted to accelerate the oxidization of luminol, through hastening the decomposition of H2O2 to form reactive oxygen species (ROSs) such as superoxide anion O2d and hydroxyl radical OHd, because these free radicals are the important intermediates
n
Corresponding authors. Tel.: þ 86 23 68252277; fax: þ 86 23 68253172. E-mail addresses: [email protected] (Y. Chai), [email protected] (R. Yuan).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.006
leading to the ECL reaction (Vitt et al., 1991; Cai et al., 2010). To the best of our knowledge, nanoparticles, which have virtues of good biocompatibility, large surface area, excellent electrocatalytic activity and fascinating conductivity, not only facilitate the immobilization of enzyme and proteins but also catalyze H2O2 to generate a great amount of ROSs. For instance, our group reported an ECL immunosensor based on the luminol–H2O2 system using Pd nanoparticles as catalysis for ultrasensitive immunoassay (Niu et al., 2011). Cheng et al. (2012) developed an ultrasensitive luminol ECL immunosensor for CEA detection based on ZnO nanoparticles. Among the wide variety of metal nanoparticles, AgNPs show excellent catalytic and electrocatalytic activities (Zhang et al., 2005; Jana et al., 1999). In addition, Guo et al. (2008) had demonstrated that AgNPs exhibited better catalysis activity than gold and platinum nanoparticles. Therefore, inspired by the superior property, AgNPs can be selected as an efficient catalyst in ECL sensors for its low oxidation potential when oxidized by H2O2 (Guo et al., 2008). Hemin, with high electrocatalytic activity due to its reversible redox potential of FeIII/FeII, can catalyze a variety of oxidation reactions like peroxidase enzymes (Genfa and Dasgupta, 1992). However, direct application of hemin as an oxidation catalyst is of
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a significant challenge because of its molecular aggregation, which causes passivation of its catalytic activity (Bruice, 1991). In order to improve the stability or activity, the hemin decorated on materials with high surface area is becoming an alternative approach. For example, G-quadruplex DNA oligomers, graphene or carbon nanotube have been considered as excellent carriers for hemin (Jiang et al., 2013; Yuan et al., 2012; Xie et al., 2012; Guo et al., 2011a, 2011b; Tu et al., 2010; Liu et al., 2007). Based on the functionalized hemin, many electrochemical (Wang et al., 2013; Bai et al., 2011) or chemiluminescence (CL) (Pavlov et al., 2004; Liu et al., 2011) sensors have been successfully constructed and exciting results have been obtained. However, until now, researchers have concentrated little attention on the study of the application of hemin in the ECL sensor. In the present research, we constructed a novel and simple sandwich-type immunosensor for CEA detection via amplifying the ECL signal of luminol by the catalysis of hemin-rGO and Ag-rGO. In brief, DpAu obtained by electrodeposition on the hemin-rGO nanocomposites was served as the immunosensor platform, which played two main roles. On one hand, DpAu amplified the ECL signal of luminol for its fascinating conductivity, on the other hand, DpAu served as carriers to immobilize primary antibody (Ab1) for its favorable biocompatibility. Moreover, AgNPs-rGO nanocomposites were used to load secondary antibody (Ab2) and GOD. In the presence of oxygen, these loaded GOD immediately catalyzed the oxidation of glucose in the detection to in situ generate H2O2, which could subsequently promote the oxidation of luminol with an amplified ECL signal. Additionally, hemin and AgNPs could further enhance the ECL signal of luminol owing to the decomposable catalysis of H2O2 to produce increased amounts of ROSs. With excellent sensitivity, selectivity and stability, the as-proposed ECL immunosensor based on hemin-rGO and AgNPs-rGO provided great potential in clinical applications.
2. Experimental 2.1. Reagents and material Graphene oxide (GO) was obtained from Nanjing Xianfeng Nano Co. (Nanjing, China). Hemin, luminol (98%), glucose oxidase (GOD), AgNO3 and bovine serum albumin (BSA, 96–99%) were bought from Sigma-Aldrich Co. (St. Louis, MO, USA). Gold chloride tetrahydrate (HAuCl4 4H2O) and sodium borohydride (NaBH4) were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). Carcinoembryonic antibody (Anti-CEA), carcinoembryonic antigen (CEA), fetoprotein antigen (AFP), prostate specific antigen (PSA) and human chorionic gonadotropin (HCG) standard solutions were obtained from Biocell (Zhengzhou, China). The serum samples were provided by South-west Hospital (Chongqing, China). 1 10 2 M Stock solution of luminol was prepared by dissolving luminol in 0.1 M NaOH solution and storing it at 4 1C when not in use. Phosphated buffered solution (PBS) (pH 7.4, 0.1 M) was prepared using 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl. The standard CEA, AFP, PSA and HCG stock solutions were prepared with PBS (pH 7.4) and stored at 4 1C. All other chemicals and solvents used were of analytical grade and were used as received. Double-distilled water was used throughout this study. 2.2. Apparatus The ECL signals were monitored with a model MPI-A ECL analyzer (Xi'an Remax Electronic Science & Technology Co. Ltd., Xi'an, China) with the voltage of the photomultiplier tube (PMT) set at 800 V in the process of detection and the potential scan from
21
0.2 to 0.7 V. Cyclic voltammetric (CV) measurements and depositions were carried out on a CHI 610A electrochemistry workstation (Shanghai CH Instruments, China). All experiments were performed with a conventional three-electrode system containing a platinum wire as counter electrode, an Ag/AgCl (sat. KCl) as reference electrode and the modified glassy carbon electrode (GCE Φ ¼4 mm) as working electrode. The scanning electron micrograph was taken by using scanning electron microscopy (SEM, S-4800, Hitachi, Japan). UV–vis absorption spectra were performed on a Lambda 17 UV–vis 8500 spectrometer (PE Co., USA) added to a quartz cuvette. Transmission electron microscope (TEM) images were acquired by a transmission electron microscope (TEM, TECNAI 10, Philips Fei Co., Hillsboro, OR, Japan).
2.3. The preparation of hemin-rGO and Ag-rGO nanocomposites Hemin-rGO nanocomposites were synthesized with a simple wet-chemical strategy through the π–π interactions by following the procedure according to the literature (Zhang et al., 2012) with a minor modification. Briefly, 20.0 mL of the homogeneous graphene oxide dispersion (0.5 mg mL 1) was mixed with 20.0 mL of hemin aqueous solution (0.5 mg mL 1) and 200.0 μL of ammonia solution. Then 30 μL hydrazine solution was added into the resulting mixture. After vigorously stirring for a few minutes, the vial was put in a water bath (60 1C) for 3.5 h. Next, the product was obtained by filtration and washed several times with doubledistilled water. The obtained hemin-rGO nanocomposites can be redispersed readily in water by ultrasonication. Moreover, the preparation of rGO was similar to that of hemin-rGO without the addition of hemin. The Ag-rGO nanocomposites were prepared according to the reference (Li and Liu, 2010) with a slight modification by the reduction of silver ions in the GO dispersion solution. First, 2 mL AgNO3 solution (0.01 M) was added to 20 mL GO solution (0.25 mg mL 1) with stirring for 30 min. Subsequently, an alkaline solution of sodium borohydride (10 mL, 0.8 mg mL 1) was added to the resulting homogeneous solution. Then the mixture was vigorously stirred at room temperature for another 2 h. Finally, the color of the dispersion solution changed from brown to black. Then the product was collected by centrifugation and washed several times with double-distilled water. The obtained compounds were stored in the refrigerator at 4 1C when not in use. Additionally, the preparation of Pt-rGO and Pd-rGO nanocomposites were similar to that of Ag-rGO. 2.4. Preparation of GOD and Ab2 labeled Ag-rGO (Ag-rGO–Ab2–GOD) bioconjugate The Ag-rGO–Ab2–GOD bioconjugates were prepared according to the following steps. Briefly, 1 mL of the above Ag-rGO suspension was mixed with 0.2 mL anti-CEA, allowing them to react under soft stirring at 4 1C for 6 h. Then, Ag-rGO–Ab2 bioconjuagtes were obtained by centrifugation at 10,000 rpm for 15 min at 4 1C to remove excess anti-CEA. Subsequently, 1 mL GOD (1 mg mL 1) was added into the obtained Ag-rGO–Ab2 bioconjuagtes and incubated for 4 h at 4 1C to block the remaining active sites of the AgNPs surface. At last, the Ag-rGO–Ab2–GOD bioconjugates were collected by centrifugation and redispersed in 1 mL PBS (pH 7.4) and stored at 4 1C until use. 2.5. Fabrication of the ECL immunosensor Scheme 1 shows the schematic illustration of the ECL immunosensor fabrication process. The glassy carbon electrode (GCE,
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Scheme 1. The fabrication process of the proposed ECL immunosensor and the mechanism for the multiple signal amplification strategy.
Ф ¼4 mm) was firstly polished on a polishing cloth with 0.3 and 0.05 μm alumina powder respectively to obtain a mirror-like surface, and was sonicated with ethanol and double-distilled water. 8 μL of the hemin-rGO nanocomposite was firstly dropped onto the pretreated GCE (abbreviated as hemin-rGO/GCE) to increase the effective area of the electrode. After drying at room temperature, the electrode was immersed into HAuCl4 solution (1%) to form DpAu layer (abbreviated as DpAu/hemin-rGO/GCE) via potentiostatic electrodeposition for 30 s at 0.2 V. Then 20 μL of anti-CEA (Ab1) solution was dropped onto the surface of the DpAu layer (abbreviated as Ab1/DpAu/hemin-rGO/GCE) with 6 h incubation at 4 1C. Subsequently, 15 μL of BSA solution (1%) was placed onto the electrode for 30 min to block the non-specific binding sites (abbreviated as BSA/Ab1/DpAu/hemin-rGO/GCE). Followed by washing thoroughly with pH 7.4 PBS, the modified GCE was incubated in CEA solution for 40 min at room temperature (abbreviated as CEA/BSA/Ab1/DpAu/hemin-rGO/GCE). At last, as a sandwich format, the resultant electrode was immersed in the Ag-rGO–Ab2–GOD solution for immunereaction.
3. Results and discussion
confirm that hemin molecules were attached to graphene. Fig. 1B (inset of Fig. 1A) displays a loose and homogeneous surface morphology indicating that hemin and graphene were welldispersed. After Au nanoparticles were electrodeposited onto the hemin-rGO membranes, a porous and pultaceous morphology was observed with many globular features (Fig. 1C), which provided a significant increase in the effective electrode surface for the loading of biomolecules. To verify the successful synthesis of AgNPs-rGO, UV–vis absorption spectrum was employed. Fig. 1D showed the UV–vis spectra of GO and Ag-rGO. As expected, the UV–vis spectrum of GO (curve a) exhibited a maximum absorption at 226 nm, which corresponds to the π–πn transitions of aromatic CQC bonds. Compared with the UV–vis spectrum obtained from the Ag-rGO (curve b), the peak at 226 nm is noticeablely redshifted to 262 nm because of the formation of rGO. Simultaneously, a distinctive absorption peak at around 399 nm is also observed, suggesting the formation of AgNPs. Additionally, Fig. 1E (inset of Fig. 1D) displays the TEM image after the rGO was modified with AgNPs, from which we can see that AgNPs were distributed uniformly over the lamellate mono-layer sheets of rGO. The result further indicated that the AgNPs-rGO nanocomposites were successfully prepared.
3.1. Characteristics of the different nanocomposites 3.2. The characterization of the ECL immunosensor The successful synthesis of hemin-rGO was characterized by a UV–vis method. As shown in Fig. 1A, graphene oxide (GO) dispersion (curve a) exhibited a maximum absorption at 226 nm which ascribed to the π πn transition of aromatic CQC bonds. The spectrum of hemin solution (curve b) included a typical Soret band absorption at 386 nm and a group of weak absorptions (Q-bands) between longer wavelengths (500–700 nm) (Xue et al., 2012). While the hemin-rGO (curve c) not only displayed a broad absorption at 267 nm which corresponds to the rGO, but also contained a peak at 417 nm which corresponded to the Soret band of hemin with a large bathochromic shift (31 nm). These results clearly
3.2.1. CV characterization of the ECL immunosensor CVs at each immobilization steps were recorded to monitor the fabrication of the proposed ECL immunosensor and the analysis was performed in 0.10 M PBS (pH 7.4) from 0.6 to 0 V at a scan rate of 100 mV s 1. As shown in Fig. 2A, a pair of well-defined redox peaks were obviously observed when the hemin-rGO were presented on the electrode surface (curve b) compared with bare GCE (curve a). The redox peaks should be attributed only to the direct electrochemistry of hemin, which is the characteristic of a single electron transfer process of iron at the core of hemin
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Fig. 1. (A) UV–vis spectra of (a) GO suspension, (b) hemin solution and (c) hemin-rGO suspension. (B) SEM (inset A) image of hemin-rGO. (C) SEM image of DpAu/heminrGO. (D) UV–vis absorption spectra of GO suspension (a) and Ag-rGO nanocomposites (b), and (E) TEM (inset D) image of Ag-rGO.
(Guo et al., 2011a). From the image, we can see that the current of DpAu/hemin-rGO modified electrode increased (curve c), which was attributed to the excellent conductivity of DpAu. Peak current decreased clearly after anti-CEA was assembled on the DpAu layer (curve d), indicating that the capture antibody protein can severely hinder the electron transfer. Subsequently, when the nonelectroactive BSA was used to block nonspecific sites, the peak current further decreased (curve e). After the immunosensor was incubated with CEA antigen, a dramatically decrease in current was observed (curve f), which incriminated to the antigen–antibody immunocomplex blocking layer.
3.2.2. ECL characterization of the immunosensor The corresponding ECL intensity–potential curves of the modified electrode were recorded step by step to characterize the fabrication process of the ECL immunosensor. The detection process was executed in 2 mL PBS (pH 7.4) with 1 10 4 M luminol and 1 10 2 M glucose. As shown in Fig. 2B, a very weak ECL signal (curve a) was obtained for the bare GCE. After the bare electrode was modified with hemin-rGO nanocomposites (curve b), the ECL signal increased for the reasons that hemin and graphene can both facilitate electron transfer to the electrode surface. When Au nanoparticles were electrodeposited onto the modified electrode, the ECL signal was further enhanced (curve c) for the excellent conductivity of the metal nanoparticle. Then the ECL signals declined in succession after immobilization of anti-CEA (curve d), blocking with BSA (curve e) and incubation with CEA (curve f). That was attributed to the formation of non-conductive layers which slowed down the electron transfer speed and led to the decrease of signals. However, when the GOD–Ab2–Ag-rGO bioconjugates were immobilized onto the modified electrode surface and proper amounts of glucose were added into the detection solution, a great enhancement of the ECL
signal (curve g) appeared and the possible mechanism is shown in Scheme 1. 3.3. The probable mechanism of the immunosensor To the best of our knowledge, H2O2 is a kind of an efficient coreactant for luminol ECL reactions. In the case of the luminol– H2O2 system, ROSs such as superoxide anion (O2d ) and hydroxyl radical (OHd), which are supposed to be generated from H2O2 (Li et al., 2009), are the important intermediates leading to the ECL reaction of luminol. But in the absence of a catalyst, the rate of H2O2 changing to ROSs is very slow with a relatively weak ECL signal, so in order to amplify the ECL signal of luminol, metal nanoparticles are usually used to hasten the decomposition of H2O2. According to Fig. 3A, the ECL signal of rGO modified electrode (curve a) is similar with that of hemin-rGO (curve b) modified electrode when they are detected in 10 4 M luminol solution. After we added 4 mL H2O2 (0.01 M) in 10 4 M luminol in PBS (pH 7.4) solution, it could be seen that the ECL signals increased, apparently for the reason that H2O2 acted as a coreactant of luminol. However, the curve d (hemin-rGO modified electrode) is stronger than curve c (rGO modified electrode), which may be attributed to the effect of hemin. From Fig. 3B, in the absence of GOD, the ECL signal of Ag-rGO–BSA–Ab2/CEA/BSA/Ab1/DpAu/rGO was relatively weak (curve a), while the ECL signal could be significantly enhanced in the presence of GOD (curve c) for the GOD catalyzed glucose to in situ produce H2O2, which was subsequently catalyzed by AgNPs. However, the ECL signal was lower due to the absence of AgNPs (curve b). These results indicated that hemin, GOD and AgNPs were necessary for the strong ECL signal. According to the literatures (Chen et al., 2000; Cao et al., 2013), the probable mechanism is shown as follows: (LH is the deprotonated luminol and Ap2 n is 3-aminophthalate)
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LH e -LH -L þ H þ GOx
Glucose þ O2 - Gluconic acid þ H 2 O2 H2 O2 þ L U
AgNPs; hemin
-
Ap2 n-Ap2 þhυ
Ap2 n þ products
(1)
3.4. Performance of the proposed ECL immunosensor for CEA detection
ð2Þ ð3Þ
(4)
3.4.1. Calibration curve Under the above optimum conditions, we investigated the quantitative range of the proposed ECL immunosensor for the CEA detection and found that the ECL intensity of the immunosensor increased with the increasing antigen concentrations by the sandwich immunoreaction format. The relationship between the ECL intensity and the concentration of CEA is shown in Fig. 4. A linear relationship between the ECL intensity and the concentrations of CEA antigen was obtained (inset of Fig. 4) in the range from 0.1 pg mL 1 to 160 ng mL 1. The detection limit of CEA was 0.03 pg mL 1 (SN 1 ¼3). The linear relationship can be represented as IECL ¼5856.86 þ1077.38 log (cCEA) with the correlation coefficient of R¼0.9947. Compared with other methods of CEA immunoassay (Table S1), the proposed ECL immunosensor exhibited higher sensitivity with a lower detection limit. The results demonstrated that the proposed immunosensor could be employed to determine CEA quantitatively. 3.4.2. Selectivity, stability and reproducibility of the ECL immunosensor The stability of the ECL intensity of the proposed immunosensor to various concentrations of CEA antigen was also investigated and the results are presented in Fig. 5(A). It shows that the ECL intensity increased with the increasing concentration of CEA, and a relatively stable curve at every concentration could be obtained. From the practical point, selectivity is an important criterion for immunosensors. To investigate the selectivity of the immunosensor,
Fig. 2. (A) CVs of (a) bare GCE, (b) hemin-rGO, (c) DpAu/hemin-rGO, (d) Ab1/DpAu/ hemin-rGO, (e) BSA/Ab1/DpAu/hemin-rGO, and (f) CEA/BSA/Ab1/DpAu/hemin-rGO modified GCE in 0.1 M PBS (pH 7. 4), scan rate: 100 mV s 1. (B) ECL profiles of different modified electrodes: (a) bare GCE, (b) hemin-rGO, (c) DpAu/hemin-rGO, (d) Ab1/DpAu/hemin-rGO, (e) BSA/Ab1/DpAu/hemin-rGO, (f) CEA/BSA/Ab1/DpAu/ hemin-rGO and (g) Ab2/CEA/BSA/Ab1/DpAu/hemin-rGO.
Fig. 4. ECL–potential curves of the immunosensor with different concentrations of CEA. CEA concentration (ng mL 1): (a) 0.0001, (b) 0.001, (c) 0.01, (d) 0.1, (e) 1, (f) 10, (g) 40, (h) 80, and (i) 160.
Fig. 3. (A) ECL signals for rGO (a), hemin-rGO (b), modified electrodes in luminol (10 4 M) and ECL signals for rGO (c), and hemin-rGO (d) modified electrodes in luminol (10 4 M) and H2O2 (0.01 M). (B) ECL signals for Ag-rGO–BSA–Ab2/CEA/BSA/Ab1/DpAu/rGO (a), rGO-GOD-Ab2/CEA/BSA/Ab1/DpAu/rGO (b) and Ag-rGO–GOD–Ab2/CEA/BSA/ Ab1/DpAu/rGO (c), which were detected in 2 mL PBS (pH 7.4) with 1 10 4 M luminol containing 1 10 2 M glucose.
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Fig. 5. (A) The ECL stability of proposed immunosensor to various concentrations of CEA antigen. (B) The specificity of the proposed ECL immunosensor: blank (without CEA), AFP (20 ng mL 1), PSA (20 ng mL 1), HCG (100 MIU mL 1), CEA (1 ng mL 1) and mixture (1 ng mL 1 CEA, 20 ng mL 1 AFP, 20 ng mL 1 PSA and 100 MIU mL 1 HCG).
some interference experiments were accomplished by detecting the ECL signals of the proposed immunosensor by incubating with interference samples under the same experimental conditions. As shown in Fig. 5(B), compared with blank solution, no remarkable changes were observed from the pure interference solution of AFP (20 ng mL 1), PSA (20 ng mL 1) and HCG (100 MIU mL 1). However, the ECL signal obtained from the mixed incubating solution (1 ng mL 1 CEA, 100 MIU mL 1 HCG, 20 ng mL 1 AFP and 20 ng mL 1 PSA) was in concordance with that obtained from the pure CEA solution. The experimental results suggested that the immunosensor displayed good selectivity for the determination of CEA. The reproducibility of the proposed immunosensor was examined by determining 10 ng mL 1 of CEA antigen for three reduplicate measurements in the same batch (intra-assay) and various batches (inter-assay). The relative standard deviations (R.S.D.) of the intra- and inter-assay were less than 5%. This demonstrated that the reproducibility of the proposed immunosensor for detecting CEA was acceptable.
potential in the determination of tumor markers for clinical diagnostics.
Acknowledgments This work was financially supported by the NNSF of China (21075100 and 21275119), State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), Ministry of Education of China (708073), Natural Science Foundation of Chongqing City (CSTC2010BB4121 and CSTC-2011BA7003), Specialized Research Fund for the Doctoral Program of Higher Education (20100182110015) and the Postgraduate Science and Technology Innovation Program of Southwest China University (Grant no. KB2010006).
Appendix A. Supplementary material Supporting information associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios. 2013.10.006.
3.5. Preliminary analysis of real samples References The feasibility of the ECL immunoassay system for clinical applications was investigated by the standard addition methods in human serum. As shown in Table S2, the recovery (between 93.7% and 106.5%) was acceptable, which provided a promising tool for determining AFP in real biological samples.
4. Conclusions In conclusion, we have successfully developed a novel sandwichtype luminol ECL immunosensor for ultrasensitive detection of CEA based on hemin-rGO and Ag-rGO. In the assay protocol, the amplification of the luminol ECL signal could be obtained by in situ generated H2O2 as a coreactant with high-content GOD and the catalysis of hemin and AgNPs for the decomposition of H2O2 to produce increased amounts of ROSs. Three main advantages could be ascribed to the high sensitivity: Firstly, the hemin not only had excellent catalytic performance to decompose H2O2, but could also facilitate electron transfer to the electrode surface. Secondly, DpAu had outstanding electrical conductivity to promote the electron transfer and increase the ECL intensity. Lastly, AgNPs loaded large amounts of GOD which could efficiently catalyze glucose to in situ generate H2O2 and simultaneously catalyze H2O2 to produce various ROSs. Due to the multiple signal amplification strategies, the luminol ECL signal of the fabricated immunosensor was greatly enhanced. Moreover, the as-proposed immunosenosr exhibited outstanding sensitivity, acceptable reproducibility and specificity, thus this strategy could provide a promising
Bai, L.J., Yuan, R., Chai, Y.Q., Yuan, Y.L., Zhuo, Y., Mao, L., 2011. Biosens. Bioelectron. 26, 4331–4336. Bruice, T.C., 1991. Acc. Chem. Res. 24, 243–249. Cai, X., Yan, J.L., Chu, H.H., Wu, M.S., Tu, Y.F., 2010. Sens. Actuators B: Chem. 143, 655–659. Cao, W.D., Ferrance, J.P., Demas, J., Landers, J.P., 2006. J. Am. Chem. Soc. 128, 7572–7578. Cao, Y.L., Yuan, R., Chai, Y.Q., Liu, H.J., Liao, Y.H., Zhuo, Y., 2013. Talanta 113, 106–112. Chai, Y., Tian, D.Y., Wang, W., Cui, H., 2010. Chem. Commun. 46, 7560–7562. Chen, G.N., Zhang, L., Lin, R.E., Yang, Z.C., Duan, J.P., Chen, H.Q., 2000. Talanta 50, 1275–1281. Cheng, Y.F., Yuan, R., Chai, Y.Q., Niu, H., Cao, Y.L., Liu, H.J., Bai, L.J., Yuan, Y.L., 2012. Anal. Chim. Acta 745, 137–142. Genfa, Z., Dasgupta, P.K., 1992. Anal. Chem. 64, 517–522. Guo, J.Z., Cui, H., Zhou, W., Wang, W., 2008. J. Photochem. Photobiol. A: Chem. 193, 89–96. Guo, Y.J., Deng, L., Li, J., Guo, S.J., Wang, E.K., Dong, S.J., 2011a. ACS Nano 5, 1282–1290. Guo, Z.Y., Gai, P.P., Hao, T.T., Duan, J., 2011b. J. Agric. Food Chem. 59, 5257–5262. Haghighi, B., Bozorgzadeh, S., 2011. Talanta 85, 2189–2193. Jana, N.R., Sau, T.K., Pal, T., 1999. J. Phys. Chem. B 103, 115–121. Jiang, H., Ju, H.X., 2007. Chem. Commun. 4, 404–406. Jiang, L.P., Yuan, R., Chai, Y.Q., Yuan, Y.L., Bai, L.J., Wang, Y., 2013. Analyst 138, 1497–1503. Jie, G.F., Liu, P., Zhang, S.S., 2010. Chem. Commun. 46, 1323–1325. Li, J., Liu, C.Y., 2010. Eur. J. Inorg. Chem. 8, 1244–1248. Li, S.F., Zhang, X.M., Du, W.X., Ni, Y.H., Wei, X.W., 2009. J. Phys. Chem. C 113, 1046–1051. Lin, K.C., Chen, S.M., 2006. J. Electroanal. Chem. 589, 52–59. Liu, X.Q., Freeman, R., Golub, E., Willner, I., 2011. ACS Nano 5, 7648–7655. Liu, Y., Yan, Y.L., Lei, J.P., Wu, F., Ju, H.X., 2007. Electrochem. Commun. 9, 2564–2570. Lv, J., Luo, L.R., Zhang, Z.J., 2004. Anal. Chim. Acta 510, 35–39. Niu, H., Yuan, R., Chai, Y.Q., Mao, L., Yuan, Y.L., Cao, Y.L., Zhuo, Y., 2011. Chem. Commun. 47, 8397–8399.
26
X. Jiang et al. / Biosensors and Bioelectronics 54 (2014) 20–26
Pavlov, V., Xiao, Y., Gill, R., Dishon, A., Kotler, M., Willner, I., 2004. Anal. Chem. 76, 2152–2156. Qin, W., Zhang, Z.J., Liu, H.J., 1998. Anal. Chem. 70, 3579–3584. Shen, W., Tian, D.Y., Cui, H., Yang, D., Bian, Z.P., 2011. Biosens. Bioelectron. 27, 18–24. Tu, W.W., Lei, J.P., Zhang, S.Y., Ju, H.X., 2010. Chem.-Eur. J. 16, 10771–10777. Vitt, J.E., Johnson, D.C., Engstrom, R.C., 1991. J. Electrochem. Soc. 138, 1637–1643. Wang, Q.B., Lei, J.P., Deng, S.Y., Zhang, L., Ju, H.X., 2013. Chem. Commun. 49, 916–918. Xie, S.B., Chai, Y.Q., Yuan, R., Bai, L.J., Yuan, Y.L., Wang, Y., 2012. Anal. Chim. Acta 755, 46–53.
Xue, T., Jiang, S., Qu, Y.Q., Su, Q., Cheng, R., Dubin, S., Chiu, C.Y., Kaner, R., Huang, Y., Duan, X.F., 2012. Angew. Chem. Int. Ed. 51, 3822–3825. Yuan, Y.L., Yuan, R., Chai, Y.Q., Zhuo, Y., Ye, X.Y., Gan, X.X., Bai, L.J., 2012. Chem. Commun. 48, 4621–4623. Zhang, L.Y., Li, D., Meng, W.L., Huang, Q., Su, Y., Wang, L.H., Song, S.P., Fan, C.H., 2009. Biosens. Bioelectron. 25, 368–372. Zhang, M.H., Yuan, R., Chai, Y.Q., Chen, S.H., Zhong, X., Zhong, X.A., Wang, C., 2012. RSC Adv. 2, 4639–4641. Zhang, Z.J., Liu, C.Y., Sun, L.W., 2005. J. Phys. Chem. B 109, 1730–1735.
