Author’s Accepted Manuscript An amplified electrochemical immunosensor based o n in situ-produced 1-naphthol as electroactive substance and graphene oxide and Pt nanoparticles functionalized CeO2 nanocomposites as signal enhancer Zhe-Han Yang, Ying Zhuo, Ruo Yuan, Ya-Qin Chai
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S0956-5663(15)00036-6 http://dx.doi.org/10.1016/j.bios.2015.01.035 BIOS7410
To appear in: Biosensors and Bioelectronic Received date: 14 October 2014 Revised date: 5 January 2015 Accepted date: 16 January 2015 Cite this article as: Zhe-Han Yang, Ying Zhuo, Ruo Yuan and Ya-Qin Chai, An amplified electrochemical immunosensor based on in situ-produced 1-naphthol as electroactive substance and graphene oxide and Pt nanoparticles functionalized CeO2 nanocomposites as signal enhancer, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.01.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
An amplified electrochemical immunosensor based on in situ-produced 1-naphthol as electroactive substance and graphene oxide and Pt nanoparticles functionalized CeO2 nanocomposites as signal enhancer Zhe-Han Yang, Ying Zhuo, Ruo Yuan*, Ya-Qin Chai*1 Key Laboratory of Luminescence and Real-Time Analytic chemistry,（southwest university） Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
Abstract In this work, an amplified electrochemical immunosensor based on 1-naphthol as electroactive substance and Pt/CeO2/GO composites as catalytic amplifier was constructed for sensitive detection influenza. Through “sandwich” reaction, the Pt/CeO2/GO functionalized bioconjugates were captured on electrode surface and the electrochemical signal directly originated from 1-naphthol, which was in situ produced with high local concentration though the hydrolysis of 1-naphthyl phosphate catalyzed by ALP. Then, 1-naphthol as new reactant was oxidized by Pt/CeO2/GO composites with outstanding catalytic performance, resulting in detection signal amplification. In addition, as compared to label electroactive substance to antibodies, a simplified preparative step of immunosensor could be achieved because the signal probe get rid of introducation other electroactive substances. The proposed immunosensor achieved a linear range of 1.0×10-3 to 1.0 ng mL-1 and 5.0 to 1.0×102 ng mL-1 with a detection limit of 0.43 pg mL-1 (defined as S/N=3). *
Corresponding author. Tel.: +86-23-68252277; fax: +86 23 68252277. E-mail address: [email protected] (Y.Q. Chai), [email protected] (R. Yuan) 1
Key words: In situ produce; Amplification; Graphene oxide and Pt nanoparticles functionalized CeO2 nanocomposites; electrochemical immunosensor; Influenza. 1. Introduction Influenza is a worldwide significant human and animal health problem (Waldmann et al., 2014). Once influenza virus emerges in human body, it may cause all known pandemics and most epidemics because the virus is easily transmitted and spread (Magid et al., 2013). Especially, in recent years, influenza causes outbreaks globally and, less commonly, pandemics (Louie et al., 2010). Indeed, influenza has seriously threatened human health (Li et al., 2011). However, in clinical diagnosis, as a consequence of the features of influenza infection overlap with other respiratory pathogens and the limited use of specific diagnostic tests, the diagnosis is often delayed, causing many people died of influenza annually (Lynch et al., 2007). Thus, many methods for influenza assay have been explored including aptamer-based biosensors (Le et al., 2014), nucleic acid-based electrochemical biosensor (Aydinlik et al., 2011) and nanoparticle-based biosensor (Kamikawa et al., 2010). Electrochemical immunosensors based on highly biospecific recognition interaction between the antigen and antibody, have recently aroused much interest for fast, sensitively detect target antigen (Chen et al., 2007; Kanso et al., 2014; Ojeda et al., 2014). Among them, sandwich-type immunosensor has been received much attention in recent years because of its improved sensitivity originated from different signal amplified strategies (Dutta et al., 2014; Liu et al., 2013; Lin et al., 2013; Cao et al., 2013). However, the detectable signal of conventional sandwich-type immunosensor must be obtained from the labeled electroactive substance such as 2
metal nanoparticles (ZnS, PbS, CdS) (Dai et al., 2011;) and redox-active small molecules (thionine, toluidine blue and prussian blue) (Han et al., 2013; Song et al., 2010; Wang et al., 2014). Compared with labeling macromolecular, the small molecules labeled in these immunosensors exhibit more heterogeneity and easily peel off from electrode surface to the solution, resulting in unstable output signal, poor reproducibility in electrochemical performance and increase of the analytical time (Liu et al., 2004). Therefore, it is necessary to pursue new mode to propose immunosensor for precise detection of target antigen. Alternatively, in situ-produced electroactive substance by enzyme-catalyzed reaction may be an excellent choice in sensitive electrochemical immunosensor construction. Recently, nanomaterial-based signal amplification has gained increased importance in achieving high sensitivity and selectivity of analytes (Chen et al., 2013). Among the various types of nanoparticulate systems, cerium oxide nanoparticles (nanoCeO2) have attained significant attention because of their catalytic and free radical scavenging properties (Wang et al., 2013; Paun et al., 2012; Zhao et al., 2012). Owing to its outstanding catalytic performance, it has been widely applied in the detection of dopamine (Njagi et al., 2008), alkaline phosphatase (ALP) (Hayat et al., 2013) and carcino-embryonic antigen (CEA) (Tang et al., 2013). However, nanoCeO2 suffer from the low electron conductivity at the cost of catalytic performance, which seriously affect their catalytic efficiency (Chu et al., 2011). To overcome this shortcoming, graphene oxide (GO) with superior electron conductivity and large specific surface area was adopted as a large matrix for loading nanoCeO2 and Pt 3
nanoparticles with the final formation of functionalized nanoCeO2 (Pt/CeO2/GO), which possess more enhanced catalytic activities and stabilities than nanoCeO2. However, to our best knowledge, Pt/CeO2/GO-based signal amplification has not been reported in the field of electrochemical immunosensor. Herein, a novel amplification immunosensor based on in situ producing 1-naphthol as an electroactive substance and Pt/CeO2/GO composites as a catalytic amplifier for sensitive detection of influenza was constructed. Firstly, the labeled ALP catalyzed p-NPP hydrolysis to produce a new reactant, 1-naphthol, which was further oxidized by nanoCeO2 and Pt NPs, resulting in amplification of electrochemical signal (as shown in scheme B). The electrochemical signal originated from 1-naphthol without introduction other electroactives substance. Notably, in situ-produced electroactive substance could generate high local concentrations on the surface of Pt/CeO2/GO composites leading to kinetic enhancement. Moreover, Owing to the high surface area of Pt/CeO2/GO composites, amounts of bioconjugates featuring ALP and secondary antibody (Ab2) could be linked to Pt/CeO2/GO composites to form signal probe. This method will provide a new mode for proposing sandwich-type immunosensor.
2 Experiments 2.1 Chemicals and Biochemicals Antigen influenza, monoclonal primary antibody anti-influenza and tracer secondary antibody anti-influenza were from Ye Xiang Bio. Co. Ltd (Hangzhou, China). Chloroplatinic acid (H2PtCl6), gold chloride tetra hydrate (HAuCl4·4H2O), 4
potassium palladium chloride (K2PdCl6), BSA and ALP (activity 6500 units/mg) were purchased from Sigma-Aldrich. 1-Naphthyl acid phosphate (p-NPP) was from Jiang Lai Bio.Co. Ltd (Shanghai, China). Cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and lysine were from Ke Long Bio. Co. Ltd (Chengdu, China). Graphite oxide (GO) was from Nanjing Xian Feng nano Co. (Nanjing, China). 0.1 M phosphate buffered solutions (PBS) with different pH value was used in experimental process. All chemicals were analytical grade and used without further purification. 2.2 Instrumentation A conventional three-compartment electrochemical cell including a platinum wire auxiliary electrode, working electrode (Φ=4 mm) and a saturated calomel reference electrode (SCE) were used. The pH measurements were made with a digital ionanalyzer (Model PHS-3C, Da zhong Instruments, Shanghai, China) and a pH meter (MP 230, Mettler-Toledo Switzerland). Cyclic voltammetric (CV) and differential pulse voltammetry (DPV) measurements were carried out with a CHI 852C electrochemistry workstation (Shanghai CH Instruments, China). The morphology of nanoparticles was estimated from scanning electron microscopy (SEM, S-4800, Hitachi, Japan). 2.3 Synthesis of Pt/CeO2/GO composites The CeO2/GO was synthesized by the method described previously with slight modification (Wang et al., 2012). Firstly, 10 mg GO was dispersed in 5 mL double-distilled water and ultrasoniced to obtain homogeneous GO dispersions. Then 15 mL of Ce(NO3)3·6H2O (25 mg mL-1) and 40 uL of NaOH (2 M) solution was 5
added into the as-prepared GO dispersions while stirring. Subsequently, the mixture solution was sealed in a Teflon-lined stainless steel autoclave and maintained at 100 °C for 24 h. After cooling to room temperature, the stable black dispersion was centrifuged and washed with double-distilled water. Finally, the obtained CeO2/GO composites were redispersed in double-distilled water before further use. 3 mM of L-lysine was added into the above CeO2/GO solution and sonicated for 1 h, and then the solution was centrifuged and redispersed in 10 mL double-distilled water. Subsequently, 2 mL of K2PtCl6 (1% w/w) aqueous solution was successively added into the as-obtained L-lysine-stabilized CeO2/GO solution while stirring, and then 100 μL of NaBH4（80 mg mL-1） was added in the solution. After stirred for 2 h at room temperature, the obtained Pt/CeO2/GO was centrifuged and washed with double-distilled water. 2.4 Preparation of ALP and antibody multilabeled Pt/CeO2/GO bioconjugates (Ab2 bioconjugates) Firstly, 100 μL of antibody was added into 2 mL as-prepared Pt/CeO2/GO composites and gently stirred for 12 h at 4 °C. Then the antibody coated Pt/CeO2/GO (Ab2/Pt/CeO2/GO) bioconjugates were collected by centrifugation and redispersed in PBS (pH 7.4). Then, 80 μL of ALP was dissolved in Ab2/Pt/CeO2/GO solution and incubated at 4 °C for 4 h. Finally, after centrifugation, the ALP/Ab2/Pt/CeO2/GO was dispersed in PBS (pH 7.4) and stored at 4 °C for further use. The preparation process of the Ab2 bioconjugates was shown in Scheme 1A. 2.5. Fabrication of the immunosensor 6
A GCE was polished carefully with 0.05 and 0.3 μm alumina powder on fine abrasive paper sequentially. Then the GCE was ultrasoniced in double-distilled water for 1 min and repeated the procedure twice to obtain a mirror-like surface. The pretreated electrode was immersed into a mixed solution containing 10 mM HAuCl4·4H2O, 10 mM K2PdCl6 as well as 2 M Na2SO4 for electrochemical deposition under a constant potential of −0.2 V for 50 s to obtain AuPd NP film. Subsequently, 15 μL primary antibody (Ab1) was attached onto the formed AuPd/GCE for 12 h at 4 °C. Successively, the resulting immunosensor was blocked with 1 mg mL-1 BSA for 1 h at room temperature. After every step, the modified electrode was thoroughly cleaned with double-distilled water to remove the physically adsorbed species. Based on the sandwich format, the proposed immunosensor was incubated with influenza samples with different concentration for 30 min at 37 °C, subsequently 15 μL of the prepared Ab2 bioconjugates solution was dropped for immune reaction at 37 °C, followed by washing with PBS (pH 7.4) to remove the unbound Ab2 bioconjugates. The electrochemical signal was carried out after adding 30 mM p-NPP, which gave the quantitative criteria for electrochemical detection of influenza. 2.6. Experimental measurements Electrochemical experiments were carried out in a standard three-electrode system. The electrodepositing of AuPd NPs was accomplished in a solution containing 10 mM HAuCl4·4H2O, 10 mM K2PdCl6 as well as 2 M Na2SO4 by applying a constant potential of −0.2 V for 50 s. DPV were performed in 2 mL 0.1 M PBS (pH 7.5) at room temperature with 30 mM 1-naphthyl acid phosphate salt. The DPV parameters 7
applied were 50 mV s-1 sweeping rate, 20 mV pulse amplitude, 50 ms pulse width, 2 s pulse period and voltage range from 0.2 to 0.6 V. CV of the electrode fabrication were performed in 5.0 mM [Fe(CN)6]3−/4− with a scanning potential from −0.2 to 0.6 V at a scan rate of 50 mV s-1.
3 Results and Discussion 3.1 Characteristics of the nanomaterials The morphology of the CeO2/GO and Pt/CeO2/GO composites was investigated by scanning electron microscopy (SEM). Fig. 1A showed SEM image of grapheme oxide (GO). The restacked parts and wrinkles could be observed, which was attributed to electronic repulsion between the soft and flexible layers. After reacted with Ce(NO3)3·6H2O, as show in Fig. 1B, the as-obtained nanoCeO2 were well dispersed on graphene nanosheet with a uniform small size. When Pt NPs were in situ reduced on CeO2/GO, more intensive highlights could be observed on the surface of GO showed in Fig. 1C. It was interesting to note that the size of these highlights increased, indicating that the L-lysine was preferred to adsorb on the hydrophilic surface of nanoCeO2 and then induced the in situ growth of Pt NPs around nanoCeO2. Typical SEM images revealed Pt/CeO2/GO composites were successfully prepared. X-ray photoelectron spectroscopy (XPS) was employed to further analyze the chemical composition of the Pt/CeO2/GO composites as shown in Fig. 1D. The characteristic peaks for C1s, O1s, N1s, Ce3d and Pt core level regions could be obviously observed at the Pt/CeO2/GO composites. The C1s, O1s signal indicated in present of GO and the Ce3d signal suggested the successful growth of nanoCeO2 on 8
GO. The N1s core level mainly derived from the L-lysine, which proved that the L-lysine with full of –NH2 groups was preferred to adsorb on the surface of CeO2 NPs. In addition, the characteristic peak at Pt was observed, implying the successful loading Pt NPs onto nanoCeO2 (more detail information supported in EIS, Fig 1S). 3.2 Electrochemical characterization of the immunosensor CV measurement was performed to monitor the surface features of electrode in the presence of 5.0 mM [Fe(CN)6]3−/4−. As shown in Fig. 2, a pair of well-defined redox peaks of [Fe(CN)6]3−/4− was obtained at bare GCE (curve a). When AuPd NPs were electrodeposited on the electrode surface, the redox peak currents increased significantly (curve b), which ascribed to the conductivity of AuPd NPs promoted the electron transfer. After primary antibody was immobilized on AuPd/GCE, an obvious decrease of peak current was observed (curve c). Subsequently, when the modified electrode was blocked with BSA, a further decrease peak current was obtained (curve d). Finally, the peak current decreased apparently after incubated with antigen (curve e). The reason for that the primary antibody, BSA and antigen could obstruct electron transfer on the surface of electrode. 3.3 Optimization of immunosensor preparation conditions In order to maximize the immunosensor response, the experimental conditions including AuPd NPs electrodepositing time, concentration of Ab1 and the effect of pH on the ALP activity were optimized. The electrodepositing time of AuPd NPs played an important role in electron transport on the surface of electrode, therefore it was necessary to investigate the effect of AuPd NPs electrodepositing time. The electrode 9
was investigated in PBS with 5.0 mM [Fe(CN)6]3−/4− using CV measurement after electrodeposited for 20 s, 30 s, 40 s, 50 s, 60 s in mixture solution of HAuCl4 and K2PdCl6 respectively. As show in Fig. 3A, the peak current (oxide peak current as an example) increased in 20 s to 50 s, when electrodeposited for 60 s, the peak current decreased. Thus, we chose 50 s as optimal electrodepositing time. To obtain the optimal Ab1 concentration, four concentrations of Ab1 (0.44, 4.4, 44, 440 μg mL-1) were immobilized on AuPd NPs modified electrode respectively, and CVs of Ab1/AuPd/GCE was conducted in the presence of 5.0 mM [Fe(CN)6]3−/4−. Because Ab1 as one kind of protein obstructed electron transfer on the electrode surface, as seen from Fig. 3B, the peak current (oxide peak current as an example) decreased with the increasing concentration of Ab1 and then tended to level off after more than 44 μg mL-1. Therefore, 44 μg mL-1 was chosen as the optimal Ab1 concentration. The ALP activity and surface state of nanoCeO2 would depend on the pH of cell. Thus, further test was designed to evaluate the effect of buffer conditions on the hydrolysis of 1-naphthyl phosphate substrate by ALP. Therefore, different pH condition range from 7.0 to 9.0 containing 30 mg mL-1 1-naphthyl phosphate was used as detection substrate. As shown in Fig. 3C, the optimum pH value was obtained at a pH 7.5, indicating that optimal activity of ALP could be achieved at pH 7.5. 3.4 Comparison of different labeled Ab2 bioconjugates To investigate the effect of signal amplification with Pt/CeO2/GO composites, the contrast experiment was conducted by comparing their current responses of different 10
probes under the same conditions. Three kinds of Ab2-functionalized probes (1) ALP/Ab2/GO, (2) ALP/Ab2/CeO2/GO, (3) ALP/Ab2/Pt/CeO2/GO were prepared. And the same batch of immunosensors incubated with the 10 ng mL-1 influenza, then incubated with different labeled Ab2 probes solutions respectively. As shown in Fig. 4, the current response of immunosensor with ALP/Ab2/CeO2/GO (Fig. 4B) was improved in comparison with the immunosensor with ALP/Ab2/GO (Fig. 4A). This may ascribe to the catalytic activity of CeO2 toward 1-naphthol. When compared with ALP/Ab2/Pt/CeO2/GO probe (Fig. 4C), the change of the current response further increased. This confirmed that Pt NPs also can be used for catalytic oxidation of 1-naphthol. The current response of the immunosensor with ALP/Ab2/Pt/CeO2/GO probe (Fig. 4C) exhibited greater change than the immunosensor with ALP/Ab2/GO probe (Fig. 4A). This result confirmed the cooperative catalytic activity of Pt NPs and nanoCeO2 toward 1-naphthol, and demonstrated promising characteristics in signal amplification system. Therefore, we chose ALP/Ab2/Pt/CeO2/GO as Ab2 probe throughout experiment. 3.5 Analytical performance of the electrochemical immunosensor The analytical performance of the proposed immunosensor was evaluated by measuring the current generated from the oxidation of 1-naphthol using DPV. The immunosensor was employed to detect a series of influenza standard solution with various concentrations to assess the sensitivity and quantitative range of the immunoassay. It could be seen that the peak current increased with the increasing influenza concentration and the calibration plots was constituted from two linear 11
segments with different slopes：the first segment ranged from 1.0×10-3 to 1.0 ng mL-1 with linear equation I = 0.3137logc + 4.349, and the second segment ranged from 5.0 to 1.0×102 ng mL-1 with linear equation I = 0.05092c + 4.721 (Fig. 5). The decrease of sensitivity (slope) in the second linear range (higher influenza antigen concentrations), can likely dues to a kinetic limitation. Moreover, the detection limit of 0.34 pg mL-1 was evaluated using a signal three-fold the background noise. Such low detection limit was obviously lower than influence biosensor recently reported (Table 1), suggesting that the proposed electrochemical immunosensor possess a superior sensitivity and completely met the requirement of influenza assay. The results demonstrated that the proposed method could be used to detect influenza concentration quantitatively. 3.6 Specificity, cross-sensitivity and stability of the immunosensor for influenza detection In this study, to investigate the differences in response of the immunoassay to cross recognition
level,
carcinoembryonic
antigen
(CEA),
α-fetoprotein
(AFP),
procalcitonin (PCT), L-cysine and BSA were used as interfering substances. The immunosensor incubated with 10 ng mL-1 of influenza solution which was contained 100 ng mL-1 and 200 ng mL-1 of interfering substances respectively. The immunosensor were assessed by DPV response under the same experimental conditions. The experimental results were shown in Table 2, the selectivity of the proposed immunosensor was acceptable. Besides, five immunosensors incubated with 10 ng mL-1 of influenza were chosed to challenge the stability of immunosensor. All 12
five immunosensors evaluated intermittently (every 5 days) by DPV measurement and stored at 4 °C when not use. After 20 days, the electrochemical signal retained 92.5 %, 94.2%, 90.1%, 90.9% and 91.2% of its initial current, which indicated the immunosensor had good stability. 3.7 Clinical application In order to assess the reliability of the proposed immunosensor for influenza detection, the proposed immunosensor for clinical applications was investigated by adding different concentrations of influenza into human real serum samples which were obtained from the Da Ping Hospital (Chongqing, China). The results were shown in Table 3. These data showed that the recovery (between 96.5 % and 104 %) was acceptable, and the result demonstrated the excellent promise for determining influenza in real biological samples.
4 Conclusions In summary, the present study has successfully developed an amplified immunosensor for the detection of influenza using ALP to catalyze 1-naphthyl phosphate to in situ produce 1-naphthol as electroactive substance. Firstly, the electrochemical signal originated from 1-naphthol without introduction other electroactive substance. Secondly, in situ produce 1-naphthol could generate high local concentrations on the surface of Pt/CeO2/GO composites, leading to a kinetic enhancement in catalysis oxide of 1-naphthol. Moreover, 1-naphthol was oxidized by Pt/CeO2/GO composites resulting in signal amplification. In the view of these advantages, we anticipate that this method will provide a new mode for proposing 13
sandwich-type immunosensor. Acknowledgements The authors are grateful for the National Natural Science Foundation of China (21275119, 51473136, 21075100,) the Fundamental Research Funds for the Central Universities (XDJK2013A027, XDJK2014A012), China.
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Fig. 1 SEM images of GO (A), CeO2/GO (B) and Pt/CeO2/GO (C); XPS image of Pt/CeO2/GO (D) Fig. 2 CV responses of different modified electrodes in pH 7.4 PBS containing 5.0 mM [Fe(CN)6]3-/4- as redox probe at scan rate of 50 mV s-1: (a) bare GCE, (b) AuPd/GCE, (c) Ab1/ AuPd/GCE, (d) BSA/Ab1/ AuPd/GCE, (e) Ag/BSA/Ab1/ AuPd/GCE. Fig. 3 Optimization of experimental parameters: (A) effect of AuPd NPs electrodepositing time on electrochemical response； (B) influence of the concentrations of Ab1 on the signal response of immunosensor; (C) effect of the pH on the activity of ALP. Fig. 4 the CV responses after the sandwich format immunoreaction of the proposed immunosensor in the absence of (a) and in the presence of 30 mM 1-naphthyl acid phosphate (b) in PBS (pH 7.5) by using various labels: (A) ALP/Ab2/GO, (B) ALP/Ab2/CeO2/GO and (C) ALP/Ab2/Pt/CeO2/GO. Fig. 5 (A) the DPV responses of the target immunosensor after incubation with nine concentrations (1.0×10-3, 1.0×10-2, 1.0×10-1, 1.0, 5.0, 10, 30, 60, 1.0×102 ng mL-1) of influenza under optimal conditions. (B) and (c) Calibration curve of the anodic peak current changes of the immusensor for 1.0×10-3 to 1.0 ng mL-1 and 5.0 to 1.0×102 ng mL-1 influenza, respectively.
17
Scheme 1.
Fig.1
18
Fig.2
Fig.3
19
Fig.4
Fig.5
Tabel 1 Comparisons of proposed immunosensor with other reported methods influenza detection. Analytical method
Detection limit -1
EIS
2.1 pg mL
EIS
2.2 pg mL-1
fluorescence
13.9 pg mL-1
Linear range
4-20 pg mL
Ref. -1
(Jarocka et al., 2014)
4.0-20.0 pg mL-1
(Jarocka et al., 2014)
5-50 ng mL-1
(Chang et al., 2010)
20
DPV
1 ng mL-1
DPV
0.43 pg mL-1
0.05-2 μg mL-1
(Zhou et al., 2013)
0.0010-0.10 ng mL-1,
Our work
5.0- 1.0×102 ng mL-1
Tabel 2 Interference degree/crossing recognition level of the developed immunoassays Corssing reagents
0
100(ng mL-1)
200(ng mL-1)
Influenza +CEA
5.25
5.19
5.16
Influenza +PCT
5.24
5.17
5.10
Influenza +AFP
5.31
5.22
5.11
Influenza +BSA
5.28
5.21
5.09
Influenza +L-Cys
5.23
5.16
5.13
“+” mean that influenza was mixted with interfering substances.
Tabel 3
Recovery results of the proposed immunosensor in human serum Sample no.
Added (ng mL-1)
Found (ng mL−1)
Recovery (%)
1
10
9.6
96
2
20
21
105
3
30
29
97
4
40
41
103
5
50
49
98
6
60
61
101
Research Highlights
Alkaline phosphatase catalyzed 1-naphthyl phosphate hydrolysis and in situ produce 1-naphthol as electroactive substance. 21
Graphene oxide and Pt nanoparticles functionalized CeO2 nanocomposites (Pt/CeO2/GO) were used to oxide 1-naphthol, resulting in detection signal amplification.
Graphene oxide and Pt nanoparticles was used to functionalized CeO2 nanoparticles to improve electron conductivity, catalytic activities and stabilities of CeO2 nanoparticles.
Pt/CeO2/GO nanocomposites with a large of area increased the immobilized amount of alkaline phosphatase and secondary antibody.
22
PII: DOI: Reference:
www.elsevier.com/locate/bios
S0956-5663(15)00036-6 http://dx.doi.org/10.1016/j.bios.2015.01.035 BIOS7410
To appear in: Biosensors and Bioelectronic Received date: 14 October 2014 Revised date: 5 January 2015 Accepted date: 16 January 2015 Cite this article as: Zhe-Han Yang, Ying Zhuo, Ruo Yuan and Ya-Qin Chai, An amplified electrochemical immunosensor based on in situ-produced 1-naphthol as electroactive substance and graphene oxide and Pt nanoparticles functionalized CeO2 nanocomposites as signal enhancer, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.01.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
An amplified electrochemical immunosensor based on in situ-produced 1-naphthol as electroactive substance and graphene oxide and Pt nanoparticles functionalized CeO2 nanocomposites as signal enhancer Zhe-Han Yang, Ying Zhuo, Ruo Yuan*, Ya-Qin Chai*1 Key Laboratory of Luminescence and Real-Time Analytic chemistry,（southwest university） Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
Abstract In this work, an amplified electrochemical immunosensor based on 1-naphthol as electroactive substance and Pt/CeO2/GO composites as catalytic amplifier was constructed for sensitive detection influenza. Through “sandwich” reaction, the Pt/CeO2/GO functionalized bioconjugates were captured on electrode surface and the electrochemical signal directly originated from 1-naphthol, which was in situ produced with high local concentration though the hydrolysis of 1-naphthyl phosphate catalyzed by ALP. Then, 1-naphthol as new reactant was oxidized by Pt/CeO2/GO composites with outstanding catalytic performance, resulting in detection signal amplification. In addition, as compared to label electroactive substance to antibodies, a simplified preparative step of immunosensor could be achieved because the signal probe get rid of introducation other electroactive substances. The proposed immunosensor achieved a linear range of 1.0×10-3 to 1.0 ng mL-1 and 5.0 to 1.0×102 ng mL-1 with a detection limit of 0.43 pg mL-1 (defined as S/N=3). *
Corresponding author. Tel.: +86-23-68252277; fax: +86 23 68252277. E-mail address: [email protected] (Y.Q. Chai), [email protected] (R. Yuan) 1
Key words: In situ produce; Amplification; Graphene oxide and Pt nanoparticles functionalized CeO2 nanocomposites; electrochemical immunosensor; Influenza. 1. Introduction Influenza is a worldwide significant human and animal health problem (Waldmann et al., 2014). Once influenza virus emerges in human body, it may cause all known pandemics and most epidemics because the virus is easily transmitted and spread (Magid et al., 2013). Especially, in recent years, influenza causes outbreaks globally and, less commonly, pandemics (Louie et al., 2010). Indeed, influenza has seriously threatened human health (Li et al., 2011). However, in clinical diagnosis, as a consequence of the features of influenza infection overlap with other respiratory pathogens and the limited use of specific diagnostic tests, the diagnosis is often delayed, causing many people died of influenza annually (Lynch et al., 2007). Thus, many methods for influenza assay have been explored including aptamer-based biosensors (Le et al., 2014), nucleic acid-based electrochemical biosensor (Aydinlik et al., 2011) and nanoparticle-based biosensor (Kamikawa et al., 2010). Electrochemical immunosensors based on highly biospecific recognition interaction between the antigen and antibody, have recently aroused much interest for fast, sensitively detect target antigen (Chen et al., 2007; Kanso et al., 2014; Ojeda et al., 2014). Among them, sandwich-type immunosensor has been received much attention in recent years because of its improved sensitivity originated from different signal amplified strategies (Dutta et al., 2014; Liu et al., 2013; Lin et al., 2013; Cao et al., 2013). However, the detectable signal of conventional sandwich-type immunosensor must be obtained from the labeled electroactive substance such as 2
metal nanoparticles (ZnS, PbS, CdS) (Dai et al., 2011;) and redox-active small molecules (thionine, toluidine blue and prussian blue) (Han et al., 2013; Song et al., 2010; Wang et al., 2014). Compared with labeling macromolecular, the small molecules labeled in these immunosensors exhibit more heterogeneity and easily peel off from electrode surface to the solution, resulting in unstable output signal, poor reproducibility in electrochemical performance and increase of the analytical time (Liu et al., 2004). Therefore, it is necessary to pursue new mode to propose immunosensor for precise detection of target antigen. Alternatively, in situ-produced electroactive substance by enzyme-catalyzed reaction may be an excellent choice in sensitive electrochemical immunosensor construction. Recently, nanomaterial-based signal amplification has gained increased importance in achieving high sensitivity and selectivity of analytes (Chen et al., 2013). Among the various types of nanoparticulate systems, cerium oxide nanoparticles (nanoCeO2) have attained significant attention because of their catalytic and free radical scavenging properties (Wang et al., 2013; Paun et al., 2012; Zhao et al., 2012). Owing to its outstanding catalytic performance, it has been widely applied in the detection of dopamine (Njagi et al., 2008), alkaline phosphatase (ALP) (Hayat et al., 2013) and carcino-embryonic antigen (CEA) (Tang et al., 2013). However, nanoCeO2 suffer from the low electron conductivity at the cost of catalytic performance, which seriously affect their catalytic efficiency (Chu et al., 2011). To overcome this shortcoming, graphene oxide (GO) with superior electron conductivity and large specific surface area was adopted as a large matrix for loading nanoCeO2 and Pt 3
nanoparticles with the final formation of functionalized nanoCeO2 (Pt/CeO2/GO), which possess more enhanced catalytic activities and stabilities than nanoCeO2. However, to our best knowledge, Pt/CeO2/GO-based signal amplification has not been reported in the field of electrochemical immunosensor. Herein, a novel amplification immunosensor based on in situ producing 1-naphthol as an electroactive substance and Pt/CeO2/GO composites as a catalytic amplifier for sensitive detection of influenza was constructed. Firstly, the labeled ALP catalyzed p-NPP hydrolysis to produce a new reactant, 1-naphthol, which was further oxidized by nanoCeO2 and Pt NPs, resulting in amplification of electrochemical signal (as shown in scheme B). The electrochemical signal originated from 1-naphthol without introduction other electroactives substance. Notably, in situ-produced electroactive substance could generate high local concentrations on the surface of Pt/CeO2/GO composites leading to kinetic enhancement. Moreover, Owing to the high surface area of Pt/CeO2/GO composites, amounts of bioconjugates featuring ALP and secondary antibody (Ab2) could be linked to Pt/CeO2/GO composites to form signal probe. This method will provide a new mode for proposing sandwich-type immunosensor.
2 Experiments 2.1 Chemicals and Biochemicals Antigen influenza, monoclonal primary antibody anti-influenza and tracer secondary antibody anti-influenza were from Ye Xiang Bio. Co. Ltd (Hangzhou, China). Chloroplatinic acid (H2PtCl6), gold chloride tetra hydrate (HAuCl4·4H2O), 4
potassium palladium chloride (K2PdCl6), BSA and ALP (activity 6500 units/mg) were purchased from Sigma-Aldrich. 1-Naphthyl acid phosphate (p-NPP) was from Jiang Lai Bio.Co. Ltd (Shanghai, China). Cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and lysine were from Ke Long Bio. Co. Ltd (Chengdu, China). Graphite oxide (GO) was from Nanjing Xian Feng nano Co. (Nanjing, China). 0.1 M phosphate buffered solutions (PBS) with different pH value was used in experimental process. All chemicals were analytical grade and used without further purification. 2.2 Instrumentation A conventional three-compartment electrochemical cell including a platinum wire auxiliary electrode, working electrode (Φ=4 mm) and a saturated calomel reference electrode (SCE) were used. The pH measurements were made with a digital ionanalyzer (Model PHS-3C, Da zhong Instruments, Shanghai, China) and a pH meter (MP 230, Mettler-Toledo Switzerland). Cyclic voltammetric (CV) and differential pulse voltammetry (DPV) measurements were carried out with a CHI 852C electrochemistry workstation (Shanghai CH Instruments, China). The morphology of nanoparticles was estimated from scanning electron microscopy (SEM, S-4800, Hitachi, Japan). 2.3 Synthesis of Pt/CeO2/GO composites The CeO2/GO was synthesized by the method described previously with slight modification (Wang et al., 2012). Firstly, 10 mg GO was dispersed in 5 mL double-distilled water and ultrasoniced to obtain homogeneous GO dispersions. Then 15 mL of Ce(NO3)3·6H2O (25 mg mL-1) and 40 uL of NaOH (2 M) solution was 5
added into the as-prepared GO dispersions while stirring. Subsequently, the mixture solution was sealed in a Teflon-lined stainless steel autoclave and maintained at 100 °C for 24 h. After cooling to room temperature, the stable black dispersion was centrifuged and washed with double-distilled water. Finally, the obtained CeO2/GO composites were redispersed in double-distilled water before further use. 3 mM of L-lysine was added into the above CeO2/GO solution and sonicated for 1 h, and then the solution was centrifuged and redispersed in 10 mL double-distilled water. Subsequently, 2 mL of K2PtCl6 (1% w/w) aqueous solution was successively added into the as-obtained L-lysine-stabilized CeO2/GO solution while stirring, and then 100 μL of NaBH4（80 mg mL-1） was added in the solution. After stirred for 2 h at room temperature, the obtained Pt/CeO2/GO was centrifuged and washed with double-distilled water. 2.4 Preparation of ALP and antibody multilabeled Pt/CeO2/GO bioconjugates (Ab2 bioconjugates) Firstly, 100 μL of antibody was added into 2 mL as-prepared Pt/CeO2/GO composites and gently stirred for 12 h at 4 °C. Then the antibody coated Pt/CeO2/GO (Ab2/Pt/CeO2/GO) bioconjugates were collected by centrifugation and redispersed in PBS (pH 7.4). Then, 80 μL of ALP was dissolved in Ab2/Pt/CeO2/GO solution and incubated at 4 °C for 4 h. Finally, after centrifugation, the ALP/Ab2/Pt/CeO2/GO was dispersed in PBS (pH 7.4) and stored at 4 °C for further use. The preparation process of the Ab2 bioconjugates was shown in Scheme 1A. 2.5. Fabrication of the immunosensor 6
A GCE was polished carefully with 0.05 and 0.3 μm alumina powder on fine abrasive paper sequentially. Then the GCE was ultrasoniced in double-distilled water for 1 min and repeated the procedure twice to obtain a mirror-like surface. The pretreated electrode was immersed into a mixed solution containing 10 mM HAuCl4·4H2O, 10 mM K2PdCl6 as well as 2 M Na2SO4 for electrochemical deposition under a constant potential of −0.2 V for 50 s to obtain AuPd NP film. Subsequently, 15 μL primary antibody (Ab1) was attached onto the formed AuPd/GCE for 12 h at 4 °C. Successively, the resulting immunosensor was blocked with 1 mg mL-1 BSA for 1 h at room temperature. After every step, the modified electrode was thoroughly cleaned with double-distilled water to remove the physically adsorbed species. Based on the sandwich format, the proposed immunosensor was incubated with influenza samples with different concentration for 30 min at 37 °C, subsequently 15 μL of the prepared Ab2 bioconjugates solution was dropped for immune reaction at 37 °C, followed by washing with PBS (pH 7.4) to remove the unbound Ab2 bioconjugates. The electrochemical signal was carried out after adding 30 mM p-NPP, which gave the quantitative criteria for electrochemical detection of influenza. 2.6. Experimental measurements Electrochemical experiments were carried out in a standard three-electrode system. The electrodepositing of AuPd NPs was accomplished in a solution containing 10 mM HAuCl4·4H2O, 10 mM K2PdCl6 as well as 2 M Na2SO4 by applying a constant potential of −0.2 V for 50 s. DPV were performed in 2 mL 0.1 M PBS (pH 7.5) at room temperature with 30 mM 1-naphthyl acid phosphate salt. The DPV parameters 7
applied were 50 mV s-1 sweeping rate, 20 mV pulse amplitude, 50 ms pulse width, 2 s pulse period and voltage range from 0.2 to 0.6 V. CV of the electrode fabrication were performed in 5.0 mM [Fe(CN)6]3−/4− with a scanning potential from −0.2 to 0.6 V at a scan rate of 50 mV s-1.
3 Results and Discussion 3.1 Characteristics of the nanomaterials The morphology of the CeO2/GO and Pt/CeO2/GO composites was investigated by scanning electron microscopy (SEM). Fig. 1A showed SEM image of grapheme oxide (GO). The restacked parts and wrinkles could be observed, which was attributed to electronic repulsion between the soft and flexible layers. After reacted with Ce(NO3)3·6H2O, as show in Fig. 1B, the as-obtained nanoCeO2 were well dispersed on graphene nanosheet with a uniform small size. When Pt NPs were in situ reduced on CeO2/GO, more intensive highlights could be observed on the surface of GO showed in Fig. 1C. It was interesting to note that the size of these highlights increased, indicating that the L-lysine was preferred to adsorb on the hydrophilic surface of nanoCeO2 and then induced the in situ growth of Pt NPs around nanoCeO2. Typical SEM images revealed Pt/CeO2/GO composites were successfully prepared. X-ray photoelectron spectroscopy (XPS) was employed to further analyze the chemical composition of the Pt/CeO2/GO composites as shown in Fig. 1D. The characteristic peaks for C1s, O1s, N1s, Ce3d and Pt core level regions could be obviously observed at the Pt/CeO2/GO composites. The C1s, O1s signal indicated in present of GO and the Ce3d signal suggested the successful growth of nanoCeO2 on 8
GO. The N1s core level mainly derived from the L-lysine, which proved that the L-lysine with full of –NH2 groups was preferred to adsorb on the surface of CeO2 NPs. In addition, the characteristic peak at Pt was observed, implying the successful loading Pt NPs onto nanoCeO2 (more detail information supported in EIS, Fig 1S). 3.2 Electrochemical characterization of the immunosensor CV measurement was performed to monitor the surface features of electrode in the presence of 5.0 mM [Fe(CN)6]3−/4−. As shown in Fig. 2, a pair of well-defined redox peaks of [Fe(CN)6]3−/4− was obtained at bare GCE (curve a). When AuPd NPs were electrodeposited on the electrode surface, the redox peak currents increased significantly (curve b), which ascribed to the conductivity of AuPd NPs promoted the electron transfer. After primary antibody was immobilized on AuPd/GCE, an obvious decrease of peak current was observed (curve c). Subsequently, when the modified electrode was blocked with BSA, a further decrease peak current was obtained (curve d). Finally, the peak current decreased apparently after incubated with antigen (curve e). The reason for that the primary antibody, BSA and antigen could obstruct electron transfer on the surface of electrode. 3.3 Optimization of immunosensor preparation conditions In order to maximize the immunosensor response, the experimental conditions including AuPd NPs electrodepositing time, concentration of Ab1 and the effect of pH on the ALP activity were optimized. The electrodepositing time of AuPd NPs played an important role in electron transport on the surface of electrode, therefore it was necessary to investigate the effect of AuPd NPs electrodepositing time. The electrode 9
was investigated in PBS with 5.0 mM [Fe(CN)6]3−/4− using CV measurement after electrodeposited for 20 s, 30 s, 40 s, 50 s, 60 s in mixture solution of HAuCl4 and K2PdCl6 respectively. As show in Fig. 3A, the peak current (oxide peak current as an example) increased in 20 s to 50 s, when electrodeposited for 60 s, the peak current decreased. Thus, we chose 50 s as optimal electrodepositing time. To obtain the optimal Ab1 concentration, four concentrations of Ab1 (0.44, 4.4, 44, 440 μg mL-1) were immobilized on AuPd NPs modified electrode respectively, and CVs of Ab1/AuPd/GCE was conducted in the presence of 5.0 mM [Fe(CN)6]3−/4−. Because Ab1 as one kind of protein obstructed electron transfer on the electrode surface, as seen from Fig. 3B, the peak current (oxide peak current as an example) decreased with the increasing concentration of Ab1 and then tended to level off after more than 44 μg mL-1. Therefore, 44 μg mL-1 was chosen as the optimal Ab1 concentration. The ALP activity and surface state of nanoCeO2 would depend on the pH of cell. Thus, further test was designed to evaluate the effect of buffer conditions on the hydrolysis of 1-naphthyl phosphate substrate by ALP. Therefore, different pH condition range from 7.0 to 9.0 containing 30 mg mL-1 1-naphthyl phosphate was used as detection substrate. As shown in Fig. 3C, the optimum pH value was obtained at a pH 7.5, indicating that optimal activity of ALP could be achieved at pH 7.5. 3.4 Comparison of different labeled Ab2 bioconjugates To investigate the effect of signal amplification with Pt/CeO2/GO composites, the contrast experiment was conducted by comparing their current responses of different 10
probes under the same conditions. Three kinds of Ab2-functionalized probes (1) ALP/Ab2/GO, (2) ALP/Ab2/CeO2/GO, (3) ALP/Ab2/Pt/CeO2/GO were prepared. And the same batch of immunosensors incubated with the 10 ng mL-1 influenza, then incubated with different labeled Ab2 probes solutions respectively. As shown in Fig. 4, the current response of immunosensor with ALP/Ab2/CeO2/GO (Fig. 4B) was improved in comparison with the immunosensor with ALP/Ab2/GO (Fig. 4A). This may ascribe to the catalytic activity of CeO2 toward 1-naphthol. When compared with ALP/Ab2/Pt/CeO2/GO probe (Fig. 4C), the change of the current response further increased. This confirmed that Pt NPs also can be used for catalytic oxidation of 1-naphthol. The current response of the immunosensor with ALP/Ab2/Pt/CeO2/GO probe (Fig. 4C) exhibited greater change than the immunosensor with ALP/Ab2/GO probe (Fig. 4A). This result confirmed the cooperative catalytic activity of Pt NPs and nanoCeO2 toward 1-naphthol, and demonstrated promising characteristics in signal amplification system. Therefore, we chose ALP/Ab2/Pt/CeO2/GO as Ab2 probe throughout experiment. 3.5 Analytical performance of the electrochemical immunosensor The analytical performance of the proposed immunosensor was evaluated by measuring the current generated from the oxidation of 1-naphthol using DPV. The immunosensor was employed to detect a series of influenza standard solution with various concentrations to assess the sensitivity and quantitative range of the immunoassay. It could be seen that the peak current increased with the increasing influenza concentration and the calibration plots was constituted from two linear 11
segments with different slopes：the first segment ranged from 1.0×10-3 to 1.0 ng mL-1 with linear equation I = 0.3137logc + 4.349, and the second segment ranged from 5.0 to 1.0×102 ng mL-1 with linear equation I = 0.05092c + 4.721 (Fig. 5). The decrease of sensitivity (slope) in the second linear range (higher influenza antigen concentrations), can likely dues to a kinetic limitation. Moreover, the detection limit of 0.34 pg mL-1 was evaluated using a signal three-fold the background noise. Such low detection limit was obviously lower than influence biosensor recently reported (Table 1), suggesting that the proposed electrochemical immunosensor possess a superior sensitivity and completely met the requirement of influenza assay. The results demonstrated that the proposed method could be used to detect influenza concentration quantitatively. 3.6 Specificity, cross-sensitivity and stability of the immunosensor for influenza detection In this study, to investigate the differences in response of the immunoassay to cross recognition
level,
carcinoembryonic
antigen
(CEA),
α-fetoprotein
(AFP),
procalcitonin (PCT), L-cysine and BSA were used as interfering substances. The immunosensor incubated with 10 ng mL-1 of influenza solution which was contained 100 ng mL-1 and 200 ng mL-1 of interfering substances respectively. The immunosensor were assessed by DPV response under the same experimental conditions. The experimental results were shown in Table 2, the selectivity of the proposed immunosensor was acceptable. Besides, five immunosensors incubated with 10 ng mL-1 of influenza were chosed to challenge the stability of immunosensor. All 12
five immunosensors evaluated intermittently (every 5 days) by DPV measurement and stored at 4 °C when not use. After 20 days, the electrochemical signal retained 92.5 %, 94.2%, 90.1%, 90.9% and 91.2% of its initial current, which indicated the immunosensor had good stability. 3.7 Clinical application In order to assess the reliability of the proposed immunosensor for influenza detection, the proposed immunosensor for clinical applications was investigated by adding different concentrations of influenza into human real serum samples which were obtained from the Da Ping Hospital (Chongqing, China). The results were shown in Table 3. These data showed that the recovery (between 96.5 % and 104 %) was acceptable, and the result demonstrated the excellent promise for determining influenza in real biological samples.
4 Conclusions In summary, the present study has successfully developed an amplified immunosensor for the detection of influenza using ALP to catalyze 1-naphthyl phosphate to in situ produce 1-naphthol as electroactive substance. Firstly, the electrochemical signal originated from 1-naphthol without introduction other electroactive substance. Secondly, in situ produce 1-naphthol could generate high local concentrations on the surface of Pt/CeO2/GO composites, leading to a kinetic enhancement in catalysis oxide of 1-naphthol. Moreover, 1-naphthol was oxidized by Pt/CeO2/GO composites resulting in signal amplification. In the view of these advantages, we anticipate that this method will provide a new mode for proposing 13
sandwich-type immunosensor. Acknowledgements The authors are grateful for the National Natural Science Foundation of China (21275119, 51473136, 21075100,) the Fundamental Research Funds for the Central Universities (XDJK2013A027, XDJK2014A012), China.
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Kamikawa, T.L., Mikolajczyk, M.G., Kennedy, M., Zhang, P., Wang, W., Scottb, D. E., Alocilja, E. C., 2010. Biosens. Bioelectron. 26, 1346-1352. Paun, C., Safonova, O. V., Szlachetko, J., Abdala, P. M., Nachtegaal, M., Sa, J., Kleymenov, E., Cervellino, A., Krumeich, F., van Bokhoven, J. A., 2012. J. Phys. Chem. C, 116, 7312−7317. Song, Z., Yuan, R., Chai, Y., Zhuo, Y., Jiang, W., Su, H., Che, X. Li, J., Chem. 2010. Commun., 46, 6750–6752. Tang, J., Zhou, J., Li, Q., Tang, D., Chen, G. H. Yang, 2013. Chem. Commun., 49, 1530-1532. Wang, X., Li, X., Liu, D., Song, S., Zhang, H., 2012. Chem. Commun., 48, 2885–2887. Wang, X., Liu, D., Song, S. Zhang, H., 2013. J. Am. Chem. Soc. 135, 15864−15872. Wang, Y., Li, X., Cao, W., Li, Y., Li, H., Du, B., Wei, Q., 2014. Biosens. Bioelectron., 61, 618–624. Waldmann, M., Jirmann, R., Hoelscher, K., Wienke, M., Niemeyer, F.C., Rehders, D., Meyer, B., 2014. J. Am. Chem. Soc., 136, 783−788. Zhao, L., Peng, B., Hernandez-Viezcas, J.A., Rico. C., Sun, Y., Peralta-Videa, J.R., 2012. ACS Nano, 6, 9615–9622. Zhou, C.H., Long, Y.M., Qi, B.P., Pang, D.W., Zhang Z.L., 2013. Electrochem. Commu. 31,129-132. Scheme 1. Schematic illustration of the stepwise preparation procedure of signal probe (A). And the amplification mechanism of detection signal (B). 16
Fig. 1 SEM images of GO (A), CeO2/GO (B) and Pt/CeO2/GO (C); XPS image of Pt/CeO2/GO (D) Fig. 2 CV responses of different modified electrodes in pH 7.4 PBS containing 5.0 mM [Fe(CN)6]3-/4- as redox probe at scan rate of 50 mV s-1: (a) bare GCE, (b) AuPd/GCE, (c) Ab1/ AuPd/GCE, (d) BSA/Ab1/ AuPd/GCE, (e) Ag/BSA/Ab1/ AuPd/GCE. Fig. 3 Optimization of experimental parameters: (A) effect of AuPd NPs electrodepositing time on electrochemical response； (B) influence of the concentrations of Ab1 on the signal response of immunosensor; (C) effect of the pH on the activity of ALP. Fig. 4 the CV responses after the sandwich format immunoreaction of the proposed immunosensor in the absence of (a) and in the presence of 30 mM 1-naphthyl acid phosphate (b) in PBS (pH 7.5) by using various labels: (A) ALP/Ab2/GO, (B) ALP/Ab2/CeO2/GO and (C) ALP/Ab2/Pt/CeO2/GO. Fig. 5 (A) the DPV responses of the target immunosensor after incubation with nine concentrations (1.0×10-3, 1.0×10-2, 1.0×10-1, 1.0, 5.0, 10, 30, 60, 1.0×102 ng mL-1) of influenza under optimal conditions. (B) and (c) Calibration curve of the anodic peak current changes of the immusensor for 1.0×10-3 to 1.0 ng mL-1 and 5.0 to 1.0×102 ng mL-1 influenza, respectively.
17
Scheme 1.
Fig.1
18
Fig.2
Fig.3
19
Fig.4
Fig.5
Tabel 1 Comparisons of proposed immunosensor with other reported methods influenza detection. Analytical method
Detection limit -1
EIS
2.1 pg mL
EIS
2.2 pg mL-1
fluorescence
13.9 pg mL-1
Linear range
4-20 pg mL
Ref. -1
(Jarocka et al., 2014)
4.0-20.0 pg mL-1
(Jarocka et al., 2014)
5-50 ng mL-1
(Chang et al., 2010)
20
DPV
1 ng mL-1
DPV
0.43 pg mL-1
0.05-2 μg mL-1
(Zhou et al., 2013)
0.0010-0.10 ng mL-1,
Our work
5.0- 1.0×102 ng mL-1
Tabel 2 Interference degree/crossing recognition level of the developed immunoassays Corssing reagents
0
100(ng mL-1)
200(ng mL-1)
Influenza +CEA
5.25
5.19
5.16
Influenza +PCT
5.24
5.17
5.10
Influenza +AFP
5.31
5.22
5.11
Influenza +BSA
5.28
5.21
5.09
Influenza +L-Cys
5.23
5.16
5.13
“+” mean that influenza was mixted with interfering substances.
Tabel 3
Recovery results of the proposed immunosensor in human serum Sample no.
Added (ng mL-1)
Found (ng mL−1)
Recovery (%)
1
10
9.6
96
2
20
21
105
3
30
29
97
4
40
41
103
5
50
49
98
6
60
61
101
Research Highlights
Alkaline phosphatase catalyzed 1-naphthyl phosphate hydrolysis and in situ produce 1-naphthol as electroactive substance. 21
Graphene oxide and Pt nanoparticles functionalized CeO2 nanocomposites (Pt/CeO2/GO) were used to oxide 1-naphthol, resulting in detection signal amplification.
Graphene oxide and Pt nanoparticles was used to functionalized CeO2 nanoparticles to improve electron conductivity, catalytic activities and stabilities of CeO2 nanoparticles.
Pt/CeO2/GO nanocomposites with a large of area increased the immobilized amount of alkaline phosphatase and secondary antibody.
22
