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graphene nanocomposite
Sensors and Actuators B 210 (2015) 460–467
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Application of nanoporous Pd as catalytically promoted nanolabels for ultrasensitive electrochemiluminescence immunosensor based on Ag/graphene nanocomposite Shuai Li a , Fang Liu a , Shenguang Ge b , Jinghua Yu a,∗ , Mei Yan a a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, School of Material Science and Engineering, University of Jinan, Jinan 250022, PR China
a r t i c l e
i n f o
Article history: Received 24 June 2014 Received in revised form 20 December 2014 Accepted 5 January 2015 Available online 12 January 2015 Keywords: Electrochemiluminescence Ag/graphene nanocomposite Nanoporous Pd Immunosensor
a b s t r a c t An ultrasensitive sandwich-type electrochemiluminescence (ECL) immunosensor was designed for carcinoembryonic antigen (CEA) detection. This immunosensor was developed using nanoporous Pd as excellent labels and Ag/graphene (Ag–G) nanocomposite as biosensing substrates. Ag–G nanocomposite was prepared by simultaneous reduction of graphene oxide and Ag ions with a rapid and efficient onestep approach and served as an effective matrix for primary antibodies attachment. The nanoporous Pd obtained from dealloying Pd/Al alloy is an excellent catalyst for the oxidation of H2 O2 to generate O2 due to its large specific surface area and high catalytic activity. O2 could improve the ECL performance of peroxydisulfate through reducing the strong oxidant SO4 •− . The proposed immunosensor successfully fulfilled the ultrasensitive detection of CEA in the range from 1 pg mL−1 to 500 ng mL−1 with a detection limit of 0.6 pg mL−1 . Finally, our ECL immunosensor has advantages of high sensitivity, specificity and stability, and could be used to monitor CEA level in human serum with satisfactory results. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Recently, developing low-cost, portable, reliable and ultrasensitive immunosensors for the detection of disease-related biomarker proteins in complex biological matrixes has been an area of extensive research interest, and it has attracted considerable attention from many fields for its promising potential in early disease screening and diagnosis [1,2]. Some assays have enabled labelfree, simple, and even high-throughput detection of target proteins [3–5]. However, increasing demands for early diagnosis of diseases and understanding of fundamental biological processes are pushing the need for sensitive detection of biomarkers, especially at low levels [6,7]. Until now, some immunoassays have been used for clinical serum sample measurements, for example, fluorescence immunoassay [8], enzyme-linked immunosorbent assay [9], electrophoretic immunoassay [10], chemiluminescence immunoassay [11] and immune polymerase chain reaction assay [12]. However
∗ Corresponding author. Tel.: +86 531 82767161. E-mail address: [email protected] (J. Yu). http://dx.doi.org/10.1016/j.snb.2015.01.015 0925-4005/© 2015 Elsevier B.V. All rights reserved.
there is still a critical demand for simple, rapid, sensitive and low-cost detection techniques for the earlier profiling of cancer biomarkers. Electrochemiluminescence (ECL) is a light emission process in a redox reaction of electrogenerated reactants [13,14], which has advantages of simple instrumentation, high sensitivity, low cost, wide dynamic concentration response range and potential- and spatial-control [15]. The ECL immunosensor has become a powerful device for ultrasensitive biomolecule detection and quantification by combining the selectivity of the biological recognition elements and the sensitivity of the ECL technique [16]. However, the bioanalysis based on those conventional ECL reagents possesses some limitations. For instance, ruthenium labels may result in the loss of biological activity of the molecules [17]; luminol ECL signal is weak in neutral solution [18]; the release of Cd2+ from CdTe quantum dots can result in a cytotoxic effect and is a potential environmental hazard [19]. As a result, it is urgent to search for environmental-friendly ECL reagents. Koval’chuk and coworkers observed the ECL behavior of peroxydisulfate in aqueous solution at magnesium, silver and platinum electrodes, and proposed that the interaction of water with oxidant SO4 − formed lightemitting species containing 1 (O2 )2 and 3 (O2 )2 [20,21], in which oxidant SO4 − , a strongly oxidizing intermediate, were generated
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during the electrochemical reduction of peroxydisulfate,. Researchers also found that in situ generated O2 is an especially promising method to improve the ECL applications of peroxydisulfate [22]. The novel strategy of in situ generated coreactant has successfully resolved the defect of peroxydisulfate system with advantages of simplicity, sensitivity, economy and low toxicity. However, using dissolved oxygen as coreactant in the detection solution has the disadvantages of difficulty to label and low concentration. To the best of our knowledge, Pd could catalyze H2 O2 to generate O2 due to its high catalytic activity [23]. Great efforts have been dedicated towards the construction of functional Pd nanostructures with various morphologies in order to achieve higher catalytic activity and utilization. Recently, nanoporous Pd with three-dimensional pore-ligament structure have attracted great attention due to their special physicochemical properties [24,25]. When used as catalyst, nanoporous Pd have some advantages compared with Pd nanoparticles (NPs): (1) it is free of particle agglomeration and can be easily employed because the size of nanoporous Pd is larger than Pd NPs; (2) the surface of the nanoporous Pd is extremely clean, by being prepared in concentrated acidic or alkaline solution (without any surfactants) and subsequently washed with water; (3) this method can achieve a nearly 100% yield with essentially no Pd loss [26–28] when prepared by simple dealloying instead of Pd salts reduction. Therefore, it is valuable to explore the ECL performance of K2 S2 O8 system by nanoporous Pd labeled antibody. To perform as an excellent sandwich-type ECL immunosensor, stability and activity of the immobilized captured antibody on platform have been a longstanding goal. Various nanomaterials have been employed to modify the biosensing platform, aiming at improving the sensing performance. Recently, graphene (G) has attracted enormous interests in constructing ECL biosensors due to its novel properties such as large specific surface area, outstanding conductivity, high thermal and chemical stability [29,30]. Moreover, the surface properties of graphene can be adjusted by chemical modification, which facilitate its utilization in NPs/graphene nanocomposites materials [31,32]. NPs/graphene nanocomposites have high electrolyte contact area, structural stability and enhanced electron transport rate, much attention has been paid to the field. Among the different kinds of metal NPs, Ag NPs are of great importance because of their catalytic, electrical and antibacterial properties. Therefore, considerable efforts have been made to synthesize Ag/graphene (Ag–G) composite materials as a biosensing platform to immobilized captured antibody [33–35]. However, it remains a challenge to develop a rapid approach for large-scale growth of NPs on graphene under mild conditions. Here we demonstrate a rapid, efficient and one-step method for growth of Ag NPs on graphene by using poly(N-vinyl2-pyrrolidone) (PVP) as the reducing agent and surface modifier under mild conditions. PVP with unique biocompatibility and high watersolubility is used to prevent the aggregation/re-stacking of graphene sheets and Ag NPs. PVP can also adsorb silver ion so that Ag NPs can grow on the G, and protect the Ag NPs from oxidation [36,37]. In this contribution, we have successfully designed a sensitive sandwich-type ECL immunosensor for ultrasensitive CEA detection by employing Ag–G nanocomposite and nanoporous Pd for dual amplification. The introduction of Ag–G nanocomposite accelerated the electron transfer rate to amplify the ECL signal as well as provided a biocompatible microenvironment for the immobilization of antibody. Another efficient amplification strategy was proposed by making the effective use of the catalysis of prepared nanoporous Pd to in situ produce O2 as coreactant, which was commendable to enhance the sensitivity of peroxydisulfate system. We expect the nanoporous Pd to perform as excellent ECL labels as
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well as the Ag–G nanocomposite modified electrode to open new perspectives in the development of tools for analytical chemistry. 2. Experimental 2.1. Reagents Carcino-embryonic antigen (CEA), the primary anti-CEA (Ab1 ) and the secondary anti-CEA (Ab2 ) were gotten from Shanghai LincBio Science Co. Ltd. (Shanghai, China). Bovine serum albumin (BSA, 96–99%) was obtained from Sigma (St. Louis, MO, USA). PVP with an average molecular weight of 29,000 g mol−1 and a trade name of K30 was provided by Beijing Yili Fine chemical Co. (Beijing, China). Natural graphite flakes with an average diameter of 48 m were supplied from Huadong Graphite Factory (Pingdu, China). Silver nitrate (AgNO3 ), chloroplatinic acid (H2 PtCl6 ), potassium chlorate (KClO3 ), glutaraldehyde (GA), ethylenediamine, sodium hydroxide (NaOH) and ethanol were purchased from Beijing Chemical Factory (Beijing, China). Potassium persulfate (K2 S2 O8 ), hydrogen peroxide (H2 O2 ) solution (30%), sulfuric acid (H2 SO4 ) and nitric acid (HNO3 ) were purchased from Shanghai Chemical Reagent Company (Shanghai, China). All chemicals and solvents used were analytical grade or the highest purity available and were used as received without purification. All solutions were prepared using Milli-Q water (Millipore) as a solvent. Phosphate buffered solutions (PBS, pH 7.4) were prepared using 0.01 mol L−1 KH2 PO4 and 0.01 mol L−1 Na2 HPO4 . The CEA was stored at 4 ◦ C, and its standard solution was prepared daily with PBS solution as in use. The clinical serum samples were provided by Shandong Tumor Hospital. 2.2. Apparatus The ECL experiments were carried out on a MPI-B multipleparameter chemiluminescence analytical testing system equipped with a MPI-A/B multifunction chemiluminescence detector (Xi’An Remax Electronic Science & Technology Co. Ltd. Xi’an, Changchun Institute of Applied Chemistry Chinese Academy Sciences, China) at room temperature with the voltage of the photomultiplier tube (PMT) set at 800 V. Cyclic voltammetric measurements (CVs) were performed with a CHI 760D electrochemical workstation (Shanghai CH Instruments, China). Electrochemical impedance spectroscopy (EIS) was carried out on an IM6x electrochemical station (Zahner, Germany) in the solution of 0.1 mol L−1 KCl containing 5 mmol L−1 K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ]. Scanning electron microscope (SEM) and energy dispersive spectrometer (EDS) images were obtained using a JSM-6700F microscope (Japan). Transmission electron microscopy (TEM) images were obtained using a Hitachi H-800 microscope (Japan). UV–vis absorption spectra were recorded on a UV-3101 spectrophotometer (Shimadzu, Japan). All experiments were carried out with a conventional threeelectrode system with the modified glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode (WE), a platinum counter electrode (CE) and an Ag/AgCl (sat. KCl) reference electrode (RE). 2.3. Preparation of nanoporous Pd Xu et al. reported that Pd20 Al80 (atomic ratio) alloy foils were prepared by refining high-purity (>99.9%) Pd and Al in an arc furnace, followed by melt-spinning under argon-protected atmosphere. These foils were typically 20–50 m in thickness, 2–4 mm in width, and several centimeters in length. The dealloying was performed in 1.0 mol L−1 NaOH solution for 24 h. After the dealloying, the foils became brittle, and they were crushed to uniformed grain (micrometer scale) by a mortar prior to characterization [38]. Then
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the nanoporous Pd was washed with ultrapure water several times until the pH of washing water ≈7.
3. Results and discussion 3.1. Characterization of Ag–G nanocomposite and nanoporous Pd
2.4. Preparation of nanoporous Pd labeled Ab2 First, nanoporous Pd was dealt with ethylenediamine to obtained amino modified nanoporous Pd. Then 1 mL of the amination nanoporous Pd suspension was mixed with 1 mL of Ab2 solution (20 g mL−1 , in 0.01 mol L−1 pH 7.4 PBS) and 1 mL of GA (20 mmol L−1 ). After incubation at 4 ◦ C for 24 h, the residual antibody was removed by centrifugation and washing with 0.01 mol L−1 PBS several times. After that, the nanoporous Pd labeled Ab2 was redispersed in 2 mL of 1% BSA solution for 2 h, under stirring, to block the nonspecific binding sites of the nanoporous Pd labeled Ab2 . After being centrifuged and washed with PBS, the resultant nanoporous Pd labeled Ab2 was dispersed with 0.01 mol L−1 of PBS (pH 7.4) to a final volume of 2 mL and stored at 4 ◦ C for later usage [39]. 2.5. Fabrication of the ECL immunosensor via sandwich mode The whole process for constructing the modified electrode is shown schematically in Scheme 1. Prior to each measurement, the GCE with a diameter of 3 mm was successively polished using 1, 0.3, and 0.05 m alumina slurry and then washed ultrasonically in ethanol and water for 5 min, respectively. The cleaned GCE was dried with high-purity nitrogen steam for the next modification. Graphene oxide (GO) and Ag–G nanocomposite were prepared using the reported methods (details in the Supplemental information) [40,41]. 5 L of Ag–G nanocomposite solution was dropped on the center of the pretreated GCE and allowed to dry at room temperature for over 30 min, following by dropping 5 L 20 g mL−1 Ab1 (10 mmol L−1 PBS, pH 7.4) onto it. Next, they were rinsed with PBS (pH 7.4) to remove physically absorbed Ab1 and dropped with 5 L 1% BSA solution for 1 h at 37 ◦ C to block possible remaining active sites against nonspecific adsorption. Then the modified electrode was washed with PBS (pH 7.4) to remove unabsorbed materials. Subsequently, the electrode was incubated with a varying concentration of CEA solution. Finally, it was washed and the prepared nanoporous Pd labeled Ab2 was dropped on the electrode surface, followed by washing, and used for the ECL signal measuring. 2.6. ECL detection ECL measurements were done at room temperature and the potential swept from −0.5 to −1.8 V with scan rate of 100 mV s−1 in a solution of 10 mmol L−1 PBS buffer (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant with a PMT voltage of 800 V. The ECL signals related to the CEA concentrations could be measured.
The representative SEM images of G before (Fig. 1A) and after (Fig. 1B) with the deposition of Ag NPs also clearly demonstrated the formation of the nanocomposites. From an overlook image, it was observed that the G with some corrugation has been decorated with Ag NPs with negligible nanoparticle agglomeration. Additionally, the representative TEM images exhibited numerous, individual, dark dots on G (Fig. 1B, inset), which indicated the Ag NPs were distributed homogeneously on the G surface. The as-prepared G (black) and Ag–G nanocomposites (red) were confirmed by UV–vis absorption spectrum as displayed in Fig. 1C. The characteristic peak of pure G was observed at 267 nm. After the Ag NPs were deposited, the characteristic peak of Ag NPs was observed in Ag–G nanocomposite at 408 nm which indicated the efficient adsorption of Ag NPs onto the G surface. The SEM images of nanoporous Pd was showed in Fig. 1D. As observed, the obtained nanoporous Pd have similar 3D bicontinuous structures with ligament size of 6 nm. nanoporous Pd with ultrafine ligaments (less than 10 nm) are usually formed during the dealloying due to the slow diffusion rate of Pd. Furthermore, the TEM images of nanoporous Pd (inset of Fig. 1D) showed an obvious porous structure.
3.2. Characterization of the immunosensor Previous studies have revealed that the detailed information about the immunosensor preparation and the immuno-binding on the immunosensor could be investigated by EIS [42], and the result was shown in Fig. 2A. The impedance spectra include a semicircle portion and a linear portion. The semicircle diameter at higher frequencies corresponds to the electron-transfer resistance (Ret ), and the linear part at lower frequencies corresponds to the diffusion process. In the section of EIS study, the frequency ranges from 0.01 to 100 000 Hz with signal amplitude of 5 mV in a background solution of 5.0 mmol L−1 [Fe(CN)6 ]3−/4− solution containing 0.1 mol L−1 KCl. It was observed that the bare GCE revealed a very small semicircular domain (curve a). After the Ag–G was deposited onto the electrode, the electrode showed a lower resistance (curve b), implying that Ag–G was an excellent electric conducting material and accelerated the electron transfer. Subsequently, the immobilization of Ab1 generated an insulating protein layer (curve c), which increased the resistance. Similarly, BSA, CEA-Ag and nanoporous Pd-Ab2 could both resist the electron-transfer kinetics of the redox probe at the electrode interface, resulting in the increasing impedance of the electrode (curves d–f). These results were consistent with the fact that the electrode was fabricated as expected.
Scheme 1. Schematic representation of the fabrication and ECL detection procedures of the ECL immunosensor.
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Fig. 1. (A) Representative SEM images of G. (B) Representative SEM images of Ag–G nanocomposite. Inset: TEM images of Ag–G nanocomposite. (C) UV–vis absorption spectra of G (black) and Ag–G nanocomposite (red). (D) Representative SEM images of nanoporous Pd. Inset: TEM images of nanoporous Pd. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. ECL behaviors of the nanoporous Pd The sandwich-type immunoassay was also supported by ECL data which were scanned from −0.5 to −1.8 V in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant. As shown in Fig. 2B, no obvious increase of ECL emission could be observed on bare GCE (curve a), Ab1 /Ag–G/GCE (curve b), CEA-Ag/BSA/Ab1 /Ag–G/GCE (curve c), nanoporous Pd-Ab2 /BSA/Ab1 /Ag–G/GCE (curve d), Ab2 /CEAAg/BSA/Ab1 /Ag–G/GCE (curve e), whereas a ECL signal appeared from non-porous Pd NPs-Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE (curve
f, the details of preparation of non-porous Pd NPs in the Supplemental information) at the same CEA concentration (1 ng mL−1 ). However, the ECL signal appeared from nanoporous Pd-Ab2 /CEAAg/BSA/Ab1 /Ag–G/GCE (curve g) was even more stronger at the same CEA concentration (1 ng mL−1 ). It shows that the nanoporous Pd plays a crucial role for the enhancement of the ECL signal. In a control experiment, there was no ECL emission from the nanoporous Pd-Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE without the presence of 0.1 mol L−1 K2 S2 O8 (data not shown). It should be noticed that the slight ECL emission (curves a–e in Fig. 2B) was attributed to the reduction of S2 O8 2− ions to the strong oxidant
Fig. 2. (A) EIS spectra of bare GCE (a), Ag–G/GCE (b), Ab1 /Ag–G/GCE (c), BSA/Ab1 /Ag–G/GCE (d), CEA-Ag/BSA/Ab1 /Ag–G/GCE (e) and Pd-Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE (f) in a background solution of 5.0 mmol L−1 [Fe(CN)6 ]3−/4− solution containing 0.1 mol L−1 KCl. The frequency range is between 0.01 and 100 000 Hz with signal amplitude of 5 mV. (B) ECL-potential curves obtained at bare GCE (a), Ab1 /Ag–G/GCE (b), CEA-Ag/BSA/Ab1 /Ag–G/GCE (c), nanoporous Pd-Ab2 /BSA/Ab1 /Ag–G/GCE (d), Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE (e), non-porous Pd NPs-Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE (f) and nanoporous Pd-Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE (g) at the CEA concentration of 1 ng mL−1 . The ECL intensity of all experiments was recorded under continuous cyclic scans between −0.5 and −1.8 V in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant. Scan rate: 100 mV s−1 . The voltage of the PMT was 800 V.
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Fig. 3. ECL profiles of the immunosensor in the presence (a–j) of different concentrations of CEA in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant. CEA concentration (ng mL−1 ): (a) 0, (b) 0.001, (c) 0.005, (d) 0.01, (e) 0.1, (f) 1, (g) 10, (h) 50, (i) 100, (j) 500 (the potential was continuous cyclic scans between −0.5 and −1.8 V and the voltage of the PMT was set at 800 V). (B) Relationship between ECL and CEA concentration, each point was the average of six measurements. Inset: logarithmic calibration curve of the immunosensor.
SO4 •− on the electrode surface. All these results suggested that the enhanced ECL emission was produced by the reaction of the captured nanoporous Pd-Ab2 labels and S2 O8 2− . 3.4. Analytical performance To assess the sensitivity and the quantitative range of the proposed immunoassay, the analytical performance of the prepared biosensor was explored by quantifying the CEA solution with different concentrations including a blank one as a reference under optimized conditions (the details of optimization of experimental conditions in the Supplemental information). The ECL intensity was recorded under continuous cyclic scans between −0.5 and −1.8 V in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant. As expected, the ECL intensity in the presence of CEA (curves b–j) was higher than that in the absence of CEA (curve a), and increased gradually with increasing concentrations of CEA (Fig. 3A). The standard calibration curves for CEA detection was shown in Fig. 3B, exhibiting a good linear relationship with the logarithm of CEA concentration from 0.001 to 500 ng mL−1 . The linear regression equation was adjusted to ECL = 6368.17 + 1969.57 lg(cCEA , pg mL−1 ) with the correlation coefficient of 0.9982. The limit of detection for CEA was 0.6 pg mL−1 . The relative standard deviation of eleven replicate determinations of CEA was 3.4%, 3.0% and 2.7% at 0.001, 0.01 and 0.1 ng mL−1 (n = 11), respectively. The results demonstrated that the proposed method could be used for the determination of CEA. Moreover, Table 1 shows the linear range and detection limit of immunosensors with previous reports [43–47]. Compared with other ECL immunosensors, the proposed immunosensor has a relatively large linear range and low detection limit.
3.5. Specificity, stability and reproducibility of the immunosensor To investigate the specificity of the proposed ECL immunosensor, several biomolecules including prostate protein antigen (PSA), ␣-fetoprotein (AFP), and human chorionic gonadotrophin (HCG), which were abundant in serum or similar to CEA, were studied with the same experimental procedures in this work (Fig. 4A). When 1 ng mL−1 PSA, ATP, and HCG were incubated respectively, the ECL signals were almost the same as the background signal. When 1 ng mL−1 CEA coexisted with 1 ng mL−1 PSA, 1 ng mL−1 AFP or 1 ng mL−1 HCG, no apparent signal change took place in comparison with only CEA in the buffer. However, the ECL signal of buffer with CEA was tremendously higher than buffer without CEA. These results indicated that the compounds coexisting in the sample matrix did not interfere with the determination of CEA. Hence, the proposed immunosensor revealed sufficient selectivity for the detection of CEA. After the immunosensor was stored in pH 7.4 PBS at 4 ◦ C over 30 days, it was used to detect the same CEA concentration. The ECL intensity was recorded under continuous cyclic scans between −0.5 and −1.8 V for 20 cycles in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant. The ECL signal–time curve under continuous potential scanning for 20 cycles was shown in Fig. 4B. The ECL intensity of the immunosensor using nanoporous Pd labeled Ab2 decreased to about 91% of its initial value, demonstrating that the immunosensor had good stability. Since reproducibility was a very important feature for an immunosensor, it was necessary to investigate this feature of the developed immunosensor. The reproducibility of the proposed immunosensor was investigated with intra-assay and interassay precision. Intra-assay precision of the immunosensor was
Table 1 The results of comparing with other immunoassay biosensing systems. Detection methods
Biomarker proteins
Substrates
Linear range (ng mL−1 )
Detection limit (pg mL−1 )
References
Electrochemiluminescence Electrochemiluminescence
CEA CEA
0.02–80 0.01–40
6.8 1.67
[43] [44]
Electrochemiluminescence
CEA
0.005–50
0.7
[45]
Electrochemiluminescence
CEA
0.001–250
0.72
[46]
Electrochemiluminescence Electrochemiluminescence
CEA CEA
g-C3 N4 -Au Multi-walled carbon nanotubesquantum dot (MWNTs-QDs) Nanoporous gold-Au nanoparticle (NPG-Au) Nanoporous silver (NPS) G-Au G-Ag
10–80 0.001–500
3.3 0.6
[47] Proposed method
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Fig. 4. (A) The specificity of the immunosensor to CEA, PSA, AFP, and HCG. The concentration of all biomolecules was 1 ng mL−1 . (B) ECL emissions in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant under continuous cyclic scans between −0.5 and −1.8 V for 20 cycles. The CEA concentration was 1 ng mL−1 . Scan rate: 100 mV s−1 . The voltage of the PMT was 800 V.
Table 2 Assay results of clinical serum samples using the proposed and reference methods. Samples
1
2
3
4
5
Proposed method (ng mL−1 )a Reference method (ng mL−1 )a Relative error (%)
1.31 1.27 3.15
1.54 1.58 −2.53
1.98 2.05 −3.41
2.77 2.69 2.97
3.53 3.42 3.22
a
The average value of six successive determinations.
evaluated by assaying one level of CEA for six similar measurements. Inter-assay precision was estimated by determining one CEA level with six immunosensors. The intra-assay and inter-assay variation coefficients (CVs) obtained for 0.5 ng mL−1 CEA were 4.9% and 6.8% for the ECL assay. Obviously, the inter-assay CV showed a good electrode-to-electrode reproducibility of the fabrication protocol, while the low value of intra-assay CV indicated that the immunosensor could be used repeatedly. Both intra-assay and inter-assay applications demonstrated acceptable reproducibility. This could be ascribed to the simplicity of the label and the immunosensor preparing method.
same time, S2 O8 2− was reduced to the strong oxidant SO4 •− , then obtained OOH• could react with SO4 •− to forming 1 (O2 )2 * which can produce light when they return back to ground states. The possible ECL mechanisms could be inferred as the following equations:
3.6. Application of ECL immunosensor in human serum samples
H2 O2 −→O2 + H2 O
(4)
To evaluate the feasibility and application potential of the immunosensor, the assay results of clinical serum samples (provided by Shandong Cancer Hospital) using the proposed method were compared with reference values obtained by commercial ECL single-analyte tests. When the levels of tumor markers were over calibration ranges, serum samples were appropriately diluted with 0.01 mol L−1 PBS (pH 7.4) prior to the assay. The results were listed in Table 2 with the relative errors ranged from −3.41% to 3.22%. It was obviously suggested that there was no significant difference between the results obtained by two methods. Therefore, the immunoarray system constructed in this work could be reasonably applied in the clinical determination of CEA in human plasma.
O2 + H2 O + e− → HOO• + HO−
(5)
SO4 •− + HOO• → HSO4 − + 1 (O2 )2 ∗
(6)
S2 O8 2− + e− → SO4 •− + SO4 2−
(1)
SO4 •− + H2 O → HO• + HSO4 −
(2)
HO• → HOO• + H2 O
(3)
Pd
3.7. Possible mechanism for the ECL behavior of the system ECL is a method of converting electrical energy to radiative energy. It involves the production of reactive intermediates from stable precursors at the surface of an electrode. These intermediates then react under a variety of conditions to form excited state that emit light. For such ECL system of S2 O8 2− , the possible mechanism could be inferred according to previous reported literatures [48]. First, nanoporous Pd could catalyzed H2 O2 to generate O2 in situ. Upon the potential scan with an initial negative direction, the obtained O2 was reacted with water and reduced to OOH• , at the
1
(O2 )2 ∗ → 23 O2 + hv
(7)
4. Conclusions A novel ECL immunosensor for the sensitive detection of CEA was first developed using nanoporous Pd as excellent ECL labels. The main advantages of the present immunosensor can be attributed to two aspects. First, the obtained Ag–G could be an ideal substrate for antibody immobilization with good stability and bioactivity. Second, a novel ECL labels of nanoporous Pd was achieved with excellent ECL activity with S2 O8 2− . It is evidenced that the uniform pore size, large surface area and good catalysis in the nanoporous Pd plays a crucial role for the enhancement of the ECL signal. As a result, the as-proposed ECL immunosensor exhibited excellent performances, such as good stability, excellent sensitivity, high selectivity, and satisfactory sample analytical performance, etc. In principle, the proposed strategy could be applicable for the determination of other biologically important compounds based on the formation of an antibody–antigen immunocomplex.
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Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21277058, 21175058) and the Natural Science Foundation of Shandong Province, China (ZR2012BZ002).
[26]
[27]
Appendix A. Supplementary data
[28]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2015.01.015.
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Biographies Shuai Li studies in School of Chemistry and Chemical Engineering, University of Jinan as postgraduate student. Fang Liu studies in School of Chemistry and Chemical Engineering, University of Jinan as postgraduate student. Shenguang Ge received his B.Sc. in applied chemistry from University of Jinan in 2003 and obtained his Ph.D. degree from Shandong University in 2013. He then joined University of Jinan, as an associate professor working on the biosensor and chemsensor.
S. Li et al. / Sensors and Actuators B 210 (2015) 460–467 Jinghua Yu received her Ph.D. degree in analytical chemistry in 2003 from Lanzhou Institute of Chemical Physics, China. She is currently positioned as a professor at University of Jinan. She spends most of her time investigating biomedical engineering, especially for the development of biosensor devices and analytical tools.
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Mei Yan received her B.Sc. in applied chemistry from University of Jinan in 1999 and obtained her Ph.D. in 2005 from Institute of chemistry Chinese Academy of Sciences. She then joined University of Jinan, as a professor working on the synthesis and performance of advanced functional materials.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Application of nanoporous Pd as catalytically promoted nanolabels for ultrasensitive electrochemiluminescence immunosensor based on Ag/graphene nanocomposite Shuai Li a , Fang Liu a , Shenguang Ge b , Jinghua Yu a,∗ , Mei Yan a a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b Shandong Provincial Key Laboratory of Preparation and Measurement of Building Materials, School of Material Science and Engineering, University of Jinan, Jinan 250022, PR China
a r t i c l e
i n f o
Article history: Received 24 June 2014 Received in revised form 20 December 2014 Accepted 5 January 2015 Available online 12 January 2015 Keywords: Electrochemiluminescence Ag/graphene nanocomposite Nanoporous Pd Immunosensor
a b s t r a c t An ultrasensitive sandwich-type electrochemiluminescence (ECL) immunosensor was designed for carcinoembryonic antigen (CEA) detection. This immunosensor was developed using nanoporous Pd as excellent labels and Ag/graphene (Ag–G) nanocomposite as biosensing substrates. Ag–G nanocomposite was prepared by simultaneous reduction of graphene oxide and Ag ions with a rapid and efficient onestep approach and served as an effective matrix for primary antibodies attachment. The nanoporous Pd obtained from dealloying Pd/Al alloy is an excellent catalyst for the oxidation of H2 O2 to generate O2 due to its large specific surface area and high catalytic activity. O2 could improve the ECL performance of peroxydisulfate through reducing the strong oxidant SO4 •− . The proposed immunosensor successfully fulfilled the ultrasensitive detection of CEA in the range from 1 pg mL−1 to 500 ng mL−1 with a detection limit of 0.6 pg mL−1 . Finally, our ECL immunosensor has advantages of high sensitivity, specificity and stability, and could be used to monitor CEA level in human serum with satisfactory results. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Recently, developing low-cost, portable, reliable and ultrasensitive immunosensors for the detection of disease-related biomarker proteins in complex biological matrixes has been an area of extensive research interest, and it has attracted considerable attention from many fields for its promising potential in early disease screening and diagnosis [1,2]. Some assays have enabled labelfree, simple, and even high-throughput detection of target proteins [3–5]. However, increasing demands for early diagnosis of diseases and understanding of fundamental biological processes are pushing the need for sensitive detection of biomarkers, especially at low levels [6,7]. Until now, some immunoassays have been used for clinical serum sample measurements, for example, fluorescence immunoassay [8], enzyme-linked immunosorbent assay [9], electrophoretic immunoassay [10], chemiluminescence immunoassay [11] and immune polymerase chain reaction assay [12]. However
∗ Corresponding author. Tel.: +86 531 82767161. E-mail address: [email protected] (J. Yu). http://dx.doi.org/10.1016/j.snb.2015.01.015 0925-4005/© 2015 Elsevier B.V. All rights reserved.
there is still a critical demand for simple, rapid, sensitive and low-cost detection techniques for the earlier profiling of cancer biomarkers. Electrochemiluminescence (ECL) is a light emission process in a redox reaction of electrogenerated reactants [13,14], which has advantages of simple instrumentation, high sensitivity, low cost, wide dynamic concentration response range and potential- and spatial-control [15]. The ECL immunosensor has become a powerful device for ultrasensitive biomolecule detection and quantification by combining the selectivity of the biological recognition elements and the sensitivity of the ECL technique [16]. However, the bioanalysis based on those conventional ECL reagents possesses some limitations. For instance, ruthenium labels may result in the loss of biological activity of the molecules [17]; luminol ECL signal is weak in neutral solution [18]; the release of Cd2+ from CdTe quantum dots can result in a cytotoxic effect and is a potential environmental hazard [19]. As a result, it is urgent to search for environmental-friendly ECL reagents. Koval’chuk and coworkers observed the ECL behavior of peroxydisulfate in aqueous solution at magnesium, silver and platinum electrodes, and proposed that the interaction of water with oxidant SO4 − formed lightemitting species containing 1 (O2 )2 and 3 (O2 )2 [20,21], in which oxidant SO4 − , a strongly oxidizing intermediate, were generated
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during the electrochemical reduction of peroxydisulfate,. Researchers also found that in situ generated O2 is an especially promising method to improve the ECL applications of peroxydisulfate [22]. The novel strategy of in situ generated coreactant has successfully resolved the defect of peroxydisulfate system with advantages of simplicity, sensitivity, economy and low toxicity. However, using dissolved oxygen as coreactant in the detection solution has the disadvantages of difficulty to label and low concentration. To the best of our knowledge, Pd could catalyze H2 O2 to generate O2 due to its high catalytic activity [23]. Great efforts have been dedicated towards the construction of functional Pd nanostructures with various morphologies in order to achieve higher catalytic activity and utilization. Recently, nanoporous Pd with three-dimensional pore-ligament structure have attracted great attention due to their special physicochemical properties [24,25]. When used as catalyst, nanoporous Pd have some advantages compared with Pd nanoparticles (NPs): (1) it is free of particle agglomeration and can be easily employed because the size of nanoporous Pd is larger than Pd NPs; (2) the surface of the nanoporous Pd is extremely clean, by being prepared in concentrated acidic or alkaline solution (without any surfactants) and subsequently washed with water; (3) this method can achieve a nearly 100% yield with essentially no Pd loss [26–28] when prepared by simple dealloying instead of Pd salts reduction. Therefore, it is valuable to explore the ECL performance of K2 S2 O8 system by nanoporous Pd labeled antibody. To perform as an excellent sandwich-type ECL immunosensor, stability and activity of the immobilized captured antibody on platform have been a longstanding goal. Various nanomaterials have been employed to modify the biosensing platform, aiming at improving the sensing performance. Recently, graphene (G) has attracted enormous interests in constructing ECL biosensors due to its novel properties such as large specific surface area, outstanding conductivity, high thermal and chemical stability [29,30]. Moreover, the surface properties of graphene can be adjusted by chemical modification, which facilitate its utilization in NPs/graphene nanocomposites materials [31,32]. NPs/graphene nanocomposites have high electrolyte contact area, structural stability and enhanced electron transport rate, much attention has been paid to the field. Among the different kinds of metal NPs, Ag NPs are of great importance because of their catalytic, electrical and antibacterial properties. Therefore, considerable efforts have been made to synthesize Ag/graphene (Ag–G) composite materials as a biosensing platform to immobilized captured antibody [33–35]. However, it remains a challenge to develop a rapid approach for large-scale growth of NPs on graphene under mild conditions. Here we demonstrate a rapid, efficient and one-step method for growth of Ag NPs on graphene by using poly(N-vinyl2-pyrrolidone) (PVP) as the reducing agent and surface modifier under mild conditions. PVP with unique biocompatibility and high watersolubility is used to prevent the aggregation/re-stacking of graphene sheets and Ag NPs. PVP can also adsorb silver ion so that Ag NPs can grow on the G, and protect the Ag NPs from oxidation [36,37]. In this contribution, we have successfully designed a sensitive sandwich-type ECL immunosensor for ultrasensitive CEA detection by employing Ag–G nanocomposite and nanoporous Pd for dual amplification. The introduction of Ag–G nanocomposite accelerated the electron transfer rate to amplify the ECL signal as well as provided a biocompatible microenvironment for the immobilization of antibody. Another efficient amplification strategy was proposed by making the effective use of the catalysis of prepared nanoporous Pd to in situ produce O2 as coreactant, which was commendable to enhance the sensitivity of peroxydisulfate system. We expect the nanoporous Pd to perform as excellent ECL labels as
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well as the Ag–G nanocomposite modified electrode to open new perspectives in the development of tools for analytical chemistry. 2. Experimental 2.1. Reagents Carcino-embryonic antigen (CEA), the primary anti-CEA (Ab1 ) and the secondary anti-CEA (Ab2 ) were gotten from Shanghai LincBio Science Co. Ltd. (Shanghai, China). Bovine serum albumin (BSA, 96–99%) was obtained from Sigma (St. Louis, MO, USA). PVP with an average molecular weight of 29,000 g mol−1 and a trade name of K30 was provided by Beijing Yili Fine chemical Co. (Beijing, China). Natural graphite flakes with an average diameter of 48 m were supplied from Huadong Graphite Factory (Pingdu, China). Silver nitrate (AgNO3 ), chloroplatinic acid (H2 PtCl6 ), potassium chlorate (KClO3 ), glutaraldehyde (GA), ethylenediamine, sodium hydroxide (NaOH) and ethanol were purchased from Beijing Chemical Factory (Beijing, China). Potassium persulfate (K2 S2 O8 ), hydrogen peroxide (H2 O2 ) solution (30%), sulfuric acid (H2 SO4 ) and nitric acid (HNO3 ) were purchased from Shanghai Chemical Reagent Company (Shanghai, China). All chemicals and solvents used were analytical grade or the highest purity available and were used as received without purification. All solutions were prepared using Milli-Q water (Millipore) as a solvent. Phosphate buffered solutions (PBS, pH 7.4) were prepared using 0.01 mol L−1 KH2 PO4 and 0.01 mol L−1 Na2 HPO4 . The CEA was stored at 4 ◦ C, and its standard solution was prepared daily with PBS solution as in use. The clinical serum samples were provided by Shandong Tumor Hospital. 2.2. Apparatus The ECL experiments were carried out on a MPI-B multipleparameter chemiluminescence analytical testing system equipped with a MPI-A/B multifunction chemiluminescence detector (Xi’An Remax Electronic Science & Technology Co. Ltd. Xi’an, Changchun Institute of Applied Chemistry Chinese Academy Sciences, China) at room temperature with the voltage of the photomultiplier tube (PMT) set at 800 V. Cyclic voltammetric measurements (CVs) were performed with a CHI 760D electrochemical workstation (Shanghai CH Instruments, China). Electrochemical impedance spectroscopy (EIS) was carried out on an IM6x electrochemical station (Zahner, Germany) in the solution of 0.1 mol L−1 KCl containing 5 mmol L−1 K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ]. Scanning electron microscope (SEM) and energy dispersive spectrometer (EDS) images were obtained using a JSM-6700F microscope (Japan). Transmission electron microscopy (TEM) images were obtained using a Hitachi H-800 microscope (Japan). UV–vis absorption spectra were recorded on a UV-3101 spectrophotometer (Shimadzu, Japan). All experiments were carried out with a conventional threeelectrode system with the modified glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode (WE), a platinum counter electrode (CE) and an Ag/AgCl (sat. KCl) reference electrode (RE). 2.3. Preparation of nanoporous Pd Xu et al. reported that Pd20 Al80 (atomic ratio) alloy foils were prepared by refining high-purity (>99.9%) Pd and Al in an arc furnace, followed by melt-spinning under argon-protected atmosphere. These foils were typically 20–50 m in thickness, 2–4 mm in width, and several centimeters in length. The dealloying was performed in 1.0 mol L−1 NaOH solution for 24 h. After the dealloying, the foils became brittle, and they were crushed to uniformed grain (micrometer scale) by a mortar prior to characterization [38]. Then
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the nanoporous Pd was washed with ultrapure water several times until the pH of washing water ≈7.
3. Results and discussion 3.1. Characterization of Ag–G nanocomposite and nanoporous Pd
2.4. Preparation of nanoporous Pd labeled Ab2 First, nanoporous Pd was dealt with ethylenediamine to obtained amino modified nanoporous Pd. Then 1 mL of the amination nanoporous Pd suspension was mixed with 1 mL of Ab2 solution (20 g mL−1 , in 0.01 mol L−1 pH 7.4 PBS) and 1 mL of GA (20 mmol L−1 ). After incubation at 4 ◦ C for 24 h, the residual antibody was removed by centrifugation and washing with 0.01 mol L−1 PBS several times. After that, the nanoporous Pd labeled Ab2 was redispersed in 2 mL of 1% BSA solution for 2 h, under stirring, to block the nonspecific binding sites of the nanoporous Pd labeled Ab2 . After being centrifuged and washed with PBS, the resultant nanoporous Pd labeled Ab2 was dispersed with 0.01 mol L−1 of PBS (pH 7.4) to a final volume of 2 mL and stored at 4 ◦ C for later usage [39]. 2.5. Fabrication of the ECL immunosensor via sandwich mode The whole process for constructing the modified electrode is shown schematically in Scheme 1. Prior to each measurement, the GCE with a diameter of 3 mm was successively polished using 1, 0.3, and 0.05 m alumina slurry and then washed ultrasonically in ethanol and water for 5 min, respectively. The cleaned GCE was dried with high-purity nitrogen steam for the next modification. Graphene oxide (GO) and Ag–G nanocomposite were prepared using the reported methods (details in the Supplemental information) [40,41]. 5 L of Ag–G nanocomposite solution was dropped on the center of the pretreated GCE and allowed to dry at room temperature for over 30 min, following by dropping 5 L 20 g mL−1 Ab1 (10 mmol L−1 PBS, pH 7.4) onto it. Next, they were rinsed with PBS (pH 7.4) to remove physically absorbed Ab1 and dropped with 5 L 1% BSA solution for 1 h at 37 ◦ C to block possible remaining active sites against nonspecific adsorption. Then the modified electrode was washed with PBS (pH 7.4) to remove unabsorbed materials. Subsequently, the electrode was incubated with a varying concentration of CEA solution. Finally, it was washed and the prepared nanoporous Pd labeled Ab2 was dropped on the electrode surface, followed by washing, and used for the ECL signal measuring. 2.6. ECL detection ECL measurements were done at room temperature and the potential swept from −0.5 to −1.8 V with scan rate of 100 mV s−1 in a solution of 10 mmol L−1 PBS buffer (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant with a PMT voltage of 800 V. The ECL signals related to the CEA concentrations could be measured.
The representative SEM images of G before (Fig. 1A) and after (Fig. 1B) with the deposition of Ag NPs also clearly demonstrated the formation of the nanocomposites. From an overlook image, it was observed that the G with some corrugation has been decorated with Ag NPs with negligible nanoparticle agglomeration. Additionally, the representative TEM images exhibited numerous, individual, dark dots on G (Fig. 1B, inset), which indicated the Ag NPs were distributed homogeneously on the G surface. The as-prepared G (black) and Ag–G nanocomposites (red) were confirmed by UV–vis absorption spectrum as displayed in Fig. 1C. The characteristic peak of pure G was observed at 267 nm. After the Ag NPs were deposited, the characteristic peak of Ag NPs was observed in Ag–G nanocomposite at 408 nm which indicated the efficient adsorption of Ag NPs onto the G surface. The SEM images of nanoporous Pd was showed in Fig. 1D. As observed, the obtained nanoporous Pd have similar 3D bicontinuous structures with ligament size of 6 nm. nanoporous Pd with ultrafine ligaments (less than 10 nm) are usually formed during the dealloying due to the slow diffusion rate of Pd. Furthermore, the TEM images of nanoporous Pd (inset of Fig. 1D) showed an obvious porous structure.
3.2. Characterization of the immunosensor Previous studies have revealed that the detailed information about the immunosensor preparation and the immuno-binding on the immunosensor could be investigated by EIS [42], and the result was shown in Fig. 2A. The impedance spectra include a semicircle portion and a linear portion. The semicircle diameter at higher frequencies corresponds to the electron-transfer resistance (Ret ), and the linear part at lower frequencies corresponds to the diffusion process. In the section of EIS study, the frequency ranges from 0.01 to 100 000 Hz with signal amplitude of 5 mV in a background solution of 5.0 mmol L−1 [Fe(CN)6 ]3−/4− solution containing 0.1 mol L−1 KCl. It was observed that the bare GCE revealed a very small semicircular domain (curve a). After the Ag–G was deposited onto the electrode, the electrode showed a lower resistance (curve b), implying that Ag–G was an excellent electric conducting material and accelerated the electron transfer. Subsequently, the immobilization of Ab1 generated an insulating protein layer (curve c), which increased the resistance. Similarly, BSA, CEA-Ag and nanoporous Pd-Ab2 could both resist the electron-transfer kinetics of the redox probe at the electrode interface, resulting in the increasing impedance of the electrode (curves d–f). These results were consistent with the fact that the electrode was fabricated as expected.
Scheme 1. Schematic representation of the fabrication and ECL detection procedures of the ECL immunosensor.
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Fig. 1. (A) Representative SEM images of G. (B) Representative SEM images of Ag–G nanocomposite. Inset: TEM images of Ag–G nanocomposite. (C) UV–vis absorption spectra of G (black) and Ag–G nanocomposite (red). (D) Representative SEM images of nanoporous Pd. Inset: TEM images of nanoporous Pd. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. ECL behaviors of the nanoporous Pd The sandwich-type immunoassay was also supported by ECL data which were scanned from −0.5 to −1.8 V in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant. As shown in Fig. 2B, no obvious increase of ECL emission could be observed on bare GCE (curve a), Ab1 /Ag–G/GCE (curve b), CEA-Ag/BSA/Ab1 /Ag–G/GCE (curve c), nanoporous Pd-Ab2 /BSA/Ab1 /Ag–G/GCE (curve d), Ab2 /CEAAg/BSA/Ab1 /Ag–G/GCE (curve e), whereas a ECL signal appeared from non-porous Pd NPs-Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE (curve
f, the details of preparation of non-porous Pd NPs in the Supplemental information) at the same CEA concentration (1 ng mL−1 ). However, the ECL signal appeared from nanoporous Pd-Ab2 /CEAAg/BSA/Ab1 /Ag–G/GCE (curve g) was even more stronger at the same CEA concentration (1 ng mL−1 ). It shows that the nanoporous Pd plays a crucial role for the enhancement of the ECL signal. In a control experiment, there was no ECL emission from the nanoporous Pd-Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE without the presence of 0.1 mol L−1 K2 S2 O8 (data not shown). It should be noticed that the slight ECL emission (curves a–e in Fig. 2B) was attributed to the reduction of S2 O8 2− ions to the strong oxidant
Fig. 2. (A) EIS spectra of bare GCE (a), Ag–G/GCE (b), Ab1 /Ag–G/GCE (c), BSA/Ab1 /Ag–G/GCE (d), CEA-Ag/BSA/Ab1 /Ag–G/GCE (e) and Pd-Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE (f) in a background solution of 5.0 mmol L−1 [Fe(CN)6 ]3−/4− solution containing 0.1 mol L−1 KCl. The frequency range is between 0.01 and 100 000 Hz with signal amplitude of 5 mV. (B) ECL-potential curves obtained at bare GCE (a), Ab1 /Ag–G/GCE (b), CEA-Ag/BSA/Ab1 /Ag–G/GCE (c), nanoporous Pd-Ab2 /BSA/Ab1 /Ag–G/GCE (d), Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE (e), non-porous Pd NPs-Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE (f) and nanoporous Pd-Ab2 /CEA-Ag/BSA/Ab1 /Ag–G/GCE (g) at the CEA concentration of 1 ng mL−1 . The ECL intensity of all experiments was recorded under continuous cyclic scans between −0.5 and −1.8 V in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant. Scan rate: 100 mV s−1 . The voltage of the PMT was 800 V.
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Fig. 3. ECL profiles of the immunosensor in the presence (a–j) of different concentrations of CEA in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant. CEA concentration (ng mL−1 ): (a) 0, (b) 0.001, (c) 0.005, (d) 0.01, (e) 0.1, (f) 1, (g) 10, (h) 50, (i) 100, (j) 500 (the potential was continuous cyclic scans between −0.5 and −1.8 V and the voltage of the PMT was set at 800 V). (B) Relationship between ECL and CEA concentration, each point was the average of six measurements. Inset: logarithmic calibration curve of the immunosensor.
SO4 •− on the electrode surface. All these results suggested that the enhanced ECL emission was produced by the reaction of the captured nanoporous Pd-Ab2 labels and S2 O8 2− . 3.4. Analytical performance To assess the sensitivity and the quantitative range of the proposed immunoassay, the analytical performance of the prepared biosensor was explored by quantifying the CEA solution with different concentrations including a blank one as a reference under optimized conditions (the details of optimization of experimental conditions in the Supplemental information). The ECL intensity was recorded under continuous cyclic scans between −0.5 and −1.8 V in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant. As expected, the ECL intensity in the presence of CEA (curves b–j) was higher than that in the absence of CEA (curve a), and increased gradually with increasing concentrations of CEA (Fig. 3A). The standard calibration curves for CEA detection was shown in Fig. 3B, exhibiting a good linear relationship with the logarithm of CEA concentration from 0.001 to 500 ng mL−1 . The linear regression equation was adjusted to ECL = 6368.17 + 1969.57 lg(cCEA , pg mL−1 ) with the correlation coefficient of 0.9982. The limit of detection for CEA was 0.6 pg mL−1 . The relative standard deviation of eleven replicate determinations of CEA was 3.4%, 3.0% and 2.7% at 0.001, 0.01 and 0.1 ng mL−1 (n = 11), respectively. The results demonstrated that the proposed method could be used for the determination of CEA. Moreover, Table 1 shows the linear range and detection limit of immunosensors with previous reports [43–47]. Compared with other ECL immunosensors, the proposed immunosensor has a relatively large linear range and low detection limit.
3.5. Specificity, stability and reproducibility of the immunosensor To investigate the specificity of the proposed ECL immunosensor, several biomolecules including prostate protein antigen (PSA), ␣-fetoprotein (AFP), and human chorionic gonadotrophin (HCG), which were abundant in serum or similar to CEA, were studied with the same experimental procedures in this work (Fig. 4A). When 1 ng mL−1 PSA, ATP, and HCG were incubated respectively, the ECL signals were almost the same as the background signal. When 1 ng mL−1 CEA coexisted with 1 ng mL−1 PSA, 1 ng mL−1 AFP or 1 ng mL−1 HCG, no apparent signal change took place in comparison with only CEA in the buffer. However, the ECL signal of buffer with CEA was tremendously higher than buffer without CEA. These results indicated that the compounds coexisting in the sample matrix did not interfere with the determination of CEA. Hence, the proposed immunosensor revealed sufficient selectivity for the detection of CEA. After the immunosensor was stored in pH 7.4 PBS at 4 ◦ C over 30 days, it was used to detect the same CEA concentration. The ECL intensity was recorded under continuous cyclic scans between −0.5 and −1.8 V for 20 cycles in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant. The ECL signal–time curve under continuous potential scanning for 20 cycles was shown in Fig. 4B. The ECL intensity of the immunosensor using nanoporous Pd labeled Ab2 decreased to about 91% of its initial value, demonstrating that the immunosensor had good stability. Since reproducibility was a very important feature for an immunosensor, it was necessary to investigate this feature of the developed immunosensor. The reproducibility of the proposed immunosensor was investigated with intra-assay and interassay precision. Intra-assay precision of the immunosensor was
Table 1 The results of comparing with other immunoassay biosensing systems. Detection methods
Biomarker proteins
Substrates
Linear range (ng mL−1 )
Detection limit (pg mL−1 )
References
Electrochemiluminescence Electrochemiluminescence
CEA CEA
0.02–80 0.01–40
6.8 1.67
[43] [44]
Electrochemiluminescence
CEA
0.005–50
0.7
[45]
Electrochemiluminescence
CEA
0.001–250
0.72
[46]
Electrochemiluminescence Electrochemiluminescence
CEA CEA
g-C3 N4 -Au Multi-walled carbon nanotubesquantum dot (MWNTs-QDs) Nanoporous gold-Au nanoparticle (NPG-Au) Nanoporous silver (NPS) G-Au G-Ag
10–80 0.001–500
3.3 0.6
[47] Proposed method
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465
Fig. 4. (A) The specificity of the immunosensor to CEA, PSA, AFP, and HCG. The concentration of all biomolecules was 1 ng mL−1 . (B) ECL emissions in 0.01 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 K2 S2 O8 and H2 O2 as the coreactant under continuous cyclic scans between −0.5 and −1.8 V for 20 cycles. The CEA concentration was 1 ng mL−1 . Scan rate: 100 mV s−1 . The voltage of the PMT was 800 V.
Table 2 Assay results of clinical serum samples using the proposed and reference methods. Samples
1
2
3
4
5
Proposed method (ng mL−1 )a Reference method (ng mL−1 )a Relative error (%)
1.31 1.27 3.15
1.54 1.58 −2.53
1.98 2.05 −3.41
2.77 2.69 2.97
3.53 3.42 3.22
a
The average value of six successive determinations.
evaluated by assaying one level of CEA for six similar measurements. Inter-assay precision was estimated by determining one CEA level with six immunosensors. The intra-assay and inter-assay variation coefficients (CVs) obtained for 0.5 ng mL−1 CEA were 4.9% and 6.8% for the ECL assay. Obviously, the inter-assay CV showed a good electrode-to-electrode reproducibility of the fabrication protocol, while the low value of intra-assay CV indicated that the immunosensor could be used repeatedly. Both intra-assay and inter-assay applications demonstrated acceptable reproducibility. This could be ascribed to the simplicity of the label and the immunosensor preparing method.
same time, S2 O8 2− was reduced to the strong oxidant SO4 •− , then obtained OOH• could react with SO4 •− to forming 1 (O2 )2 * which can produce light when they return back to ground states. The possible ECL mechanisms could be inferred as the following equations:
3.6. Application of ECL immunosensor in human serum samples
H2 O2 −→O2 + H2 O
(4)
To evaluate the feasibility and application potential of the immunosensor, the assay results of clinical serum samples (provided by Shandong Cancer Hospital) using the proposed method were compared with reference values obtained by commercial ECL single-analyte tests. When the levels of tumor markers were over calibration ranges, serum samples were appropriately diluted with 0.01 mol L−1 PBS (pH 7.4) prior to the assay. The results were listed in Table 2 with the relative errors ranged from −3.41% to 3.22%. It was obviously suggested that there was no significant difference between the results obtained by two methods. Therefore, the immunoarray system constructed in this work could be reasonably applied in the clinical determination of CEA in human plasma.
O2 + H2 O + e− → HOO• + HO−
(5)
SO4 •− + HOO• → HSO4 − + 1 (O2 )2 ∗
(6)
S2 O8 2− + e− → SO4 •− + SO4 2−
(1)
SO4 •− + H2 O → HO• + HSO4 −
(2)
HO• → HOO• + H2 O
(3)
Pd
3.7. Possible mechanism for the ECL behavior of the system ECL is a method of converting electrical energy to radiative energy. It involves the production of reactive intermediates from stable precursors at the surface of an electrode. These intermediates then react under a variety of conditions to form excited state that emit light. For such ECL system of S2 O8 2− , the possible mechanism could be inferred according to previous reported literatures [48]. First, nanoporous Pd could catalyzed H2 O2 to generate O2 in situ. Upon the potential scan with an initial negative direction, the obtained O2 was reacted with water and reduced to OOH• , at the
1
(O2 )2 ∗ → 23 O2 + hv
(7)
4. Conclusions A novel ECL immunosensor for the sensitive detection of CEA was first developed using nanoporous Pd as excellent ECL labels. The main advantages of the present immunosensor can be attributed to two aspects. First, the obtained Ag–G could be an ideal substrate for antibody immobilization with good stability and bioactivity. Second, a novel ECL labels of nanoporous Pd was achieved with excellent ECL activity with S2 O8 2− . It is evidenced that the uniform pore size, large surface area and good catalysis in the nanoporous Pd plays a crucial role for the enhancement of the ECL signal. As a result, the as-proposed ECL immunosensor exhibited excellent performances, such as good stability, excellent sensitivity, high selectivity, and satisfactory sample analytical performance, etc. In principle, the proposed strategy could be applicable for the determination of other biologically important compounds based on the formation of an antibody–antigen immunocomplex.
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Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21277058, 21175058) and the Natural Science Foundation of Shandong Province, China (ZR2012BZ002).
[26]
[27]
Appendix A. Supplementary data
[28]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2015.01.015.
[29]
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Biographies Shuai Li studies in School of Chemistry and Chemical Engineering, University of Jinan as postgraduate student. Fang Liu studies in School of Chemistry and Chemical Engineering, University of Jinan as postgraduate student. Shenguang Ge received his B.Sc. in applied chemistry from University of Jinan in 2003 and obtained his Ph.D. degree from Shandong University in 2013. He then joined University of Jinan, as an associate professor working on the biosensor and chemsensor.
S. Li et al. / Sensors and Actuators B 210 (2015) 460–467 Jinghua Yu received her Ph.D. degree in analytical chemistry in 2003 from Lanzhou Institute of Chemical Physics, China. She is currently positioned as a professor at University of Jinan. She spends most of her time investigating biomedical engineering, especially for the development of biosensor devices and analytical tools.
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Mei Yan received her B.Sc. in applied chemistry from University of Jinan in 1999 and obtained her Ph.D. in 2005 from Institute of chemistry Chinese Academy of Sciences. She then joined University of Jinan, as a professor working on the synthesis and performance of advanced functional materials.