Electrochimica Acta 147 (2014) 650–656
Contents lists available at ScienceDirect
Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Paper-based electrochemical immunosensor for carcinoembryonic antigen based on three dimensional ﬂower-like gold electrode and gold-silver bimetallic nanoparticles Guoqiang Sun a , Ya-nan Ding a , Chao Ma a , Yan Zhang a , Shenguang Ge b , Jinghua Yu a, *, Xianrang Song c 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, University of Jinan, Jinan 250022, PR China c Cancer Research Center, Shandong Tumor Hospital, Jinan 250012, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 July 2014 Received in revised form 26 September 2014 Accepted 29 September 2014 Available online xxx
An enzyme-free electrochemical immunoassay is introduced into microﬂuidic paper-based analytical devices for the ﬁrst time using novel three dimensional ﬂower-like Au nanoparticles modiﬁed paper working electrode (FLAuNPs-PWE) as sensor platform and gold (Au)-silver (Ag) bimetallic nanoparticles (Au-Ag BMNPs) as tracer. FLAuNPs with large surface area serve as an effective matrix for primary antibodies. Au-Ag BMNPs with good biological activity, such as mimicking natural peroxidases, are used for binding of signal antibodies (Ab2). Then the Ab2 labeled BMNPs is linked to the FLAuNPs-PWE via sandwich immunoreactions. Enhanced sensitivity is achieved by efﬁcient catalysis of the Au-Ag BMNPs toward hydrogen peroxide. Under the optimized experimental conditions, the proposed paper-based electrochemical immunosensor exhibits excellent analytical performance for enzyme-free detection of carcinoembryonic antigen, ranging from 0.001-50 ng mL 1 with a low detection limit of 0.3 pg mL 1. The immunosensor shows good precision, acceptable stability and reproducibility. The proposed method provides a new promising platform for the design of the highly sensitive detection method, showing great promise for clinical application. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Enzyme-free Electrochemical immunosensor paper-based devices Hydrogen peroxide
1. Introduction Paper, as one of the most important inventions, was already utilized extensively as a platform in analytical and clinical chemistry . Since the ﬁrst microﬂuidic paper-based analytical devices (m-PADs) were proposed by Whitesides et al. [2,3], paper has never attracted as much attention as it does now. This system combine the simplicity, portability, disposability and low-cost of paper strip tests and the multiplex analysis and complex function of the conventional lab-on-a-chip devices. m-PADs have been recently introduced as promising devices for point-of-care testing (POCT) because they have attractive features including small size, low cost, easy-to-use and low reagent and sample consumption [4–6]. Much effort has been directed toward the development of
* Corresponding author. Tel.: +86 531 82767161. E-mail address: [email protected]
(J. Yu). http://dx.doi.org/10.1016/j.electacta.2014.09.149 0013-4686/ ã 2014 Elsevier Ltd. All rights reserved.
m-PADs, including fabrication methods [7–9], functionalizations [10–12] and analytical methods [13,14]. Recently, many immunoassay techniques, such as colorimetric immunoassay , localized surface plasmon resonance immunoassay , ﬂuorescent immunoassay , electrochemical immunoassay [18–20], chemluminescent immunoassay  and photoelectrochemical immunoassay , have been widely employed as analytical methods on m-PADs. Owing to its simple instrumentation and operation, fast analysis, high sensitivity and selectivity, electrochemical (EC) method has traditionally received the major share of the attention in development of quantitative methods for m-PADs. The advantages of EC method would make EC immunoassays an innately exciting strategy for building a new generation of paper-based POCT devices. To further perform sensitive-enhanced sandwich-type EC immunoassay, attention has been focused on popular strategies that couple bio-component immobilization  and signal ampliﬁcation . Au nanoparticles (AuNPs) have attracted much attention due to their large surface area, good biocompatibility,
G. Sun et al. / Electrochimica Acta 147 (2014) 650–656
Carbon ink (ED423 ss) and Ag/AgCl ink (CNC-01) were purchased from Acheson. Whatman chromatography paper #1 was obtained from GE Healthcare Worldwide (Pudong Shanghai, China) and used with further adjustment of size (A4 size). H2O2 (30%, w/v) was obtained from Chemical Reagent Co. (Tianjin, China). Tetrachloroauric acid (HAuCl44H2O, 1%, w/w), bovine serum albumin (BSA), AgNO3 and ascorbic acid (AA) were obtained from Sigma-Aldrich Chemical Co. (USA). Polyvinyl pyrrolidone (PVP) was purchased from Alfa Aesar China Ltd. Ammonia hydroxide (28 wt% NH3 in water) was obtained from Sinopharm Chemical Reagent Co. Ltd. NaBH4 was products from Shanghai Chemical Reagent Co. Ultrapure water obtained from a Millipore water puriﬁcation system (resistivity 18.2 MV cm, Milli-Q, Millipore) was used in all assays and solutions. Phosphate buffer saline (PBS) was prepared by mixing stock solutions of 0.1 M KH2PO4 and 0.1 M Na2HPO4. All other reagents were of analytical grade and used without further puriﬁcation.
and superior conductivity. Three dimensional (3D) hierarchical architectures with porous structures have triggered more and more research enthusiasm in recent years for their high surface-tovolume ratio and showed distinctive physicochemical properties in comparison with conventional nanocrystallites [25–27]. Taking into consideration the above advantages, a 3D ﬂower-like AuNPs layer was grown on the surfaces of cellulose ﬁbers from AuNPs seeds in the paper sample zone to fabricate novel ﬂower-like AuNPs modiﬁed paper working electrode (FLAuNPs-PWE). Nowadays, bimetallic nanoparticles have been demonstrated to exhibit improved catalytic performance, because of the synergistic effect and the electronic effect. Their properties are not only related to size and shape, but also to composition and structure . Gold (Au)-silver (Ag) bimetallic nanoparticles (Au-Ag BMNPs) were chosen as mimicking natural peroxidases in this work, and provided a high electrocatalytic activity toward hydrogen peroxide (H2O2) reduction . Furthermore, Au-Ag BMNPs were recognized as ideal nanocarriers because of their superior electronic conductivity and good biocompatibility. Integrating the dualsignal ampliﬁcation strategy, an enzyme-free biosensor was constructed for ultrasensitive and selective detection. In this work, a novel sandwich-type enzyme-free EC immunosensor on a microﬂuidic origami device was developed. This novel FLAuNPs-PWE was prepared by a seed-mediated growth approach. The introduction of FLAuNPs-PWE accelerated the electron transfer rate to amplify the EC signal as well as provided a biocompatible microenvironment for the immobilization of capture antibodies. Meanwhile, Au-Ag BMNPs were successfully synthesized through a simple kinetic controlled co-reduction route. The Au-Ag BMNPs were selected as nanocarriers for loading numerous signal antibodies (Ab2) to obtain the Au-Ag-Ab2 bioconjugates, which could produce an electrocatalytic response by reduction of H2O2 and further amplify EC signals. By coupling with the sandwich-type immunoassay and EC analytical technique, the proposed strategy exhibited an excellent analytical performance for detection of carcinoembryonic antigen (CEA).
EC measurements were carried out with a CHI 660D electrochemistry workstation (Shanghai CH Instruments Co., China) with a three-electrode system, whereas the modiﬁed PWE with a diameter of 6.0 mm was used as the working electrode, screenprinted carbon electrode and Ag/AgCl electrode were used as the counter electrode and the reference electrode, respectively. Scanning electron microscope (SEM) images were obtained using a QUANTA FEG 250 thermal ﬁeld emission SEM (FEI Co., USA), and the microscope was equipped with an Oxford X-MAX50 energy dispersive spectrometer (EDS) (Oxford, Britain). Transmission electron microscope (TEM) images were obtained from a JEOL JEM-1400 microscope (JEOL, Japan). Electrochemical impedance spectroscopy (EIS) was performed on IM6x electrochemical station (Zahner, Germany). Ultraviolet-visible (UV-vis) absorption spectra were recorded on a UV-2550 spectrophotometer (Shimadzu, Japan).
2.3. Preparation of the Au-Ag BMNPs
The Au-Ag BMNPs were prepared through a simple kinetic controlled co-reduction route at room temperature according to the reported literature . Brieﬂy, 0.2 g of PVP was ﬁrstly added into 10 mL of AgNO3 (2 mM) aqueous solution and stirred for 5 min, when the solution became clear, 2 mL of 2.8% NH3H2O aqueous solution
Primary antibodies (Ab1), Ab2 and CEA standards solutions were purchased from Linc-Bio Science Co. Ltd. (Shanghai, China). The clinical serum samples were from Shandong Tumor Hospital.
Scheme 1. The fabrication process of the EC origami immunodevice.
G. Sun et al. / Electrochimica Acta 147 (2014) 650–656
was injected into the above mixture and stirred for another 2 min at room temperature. Subsequently, 0.5 mL of HAuCl4 aqueous solution was added. After 2 min, 1 mL of AA solution (100 mM) was added under stirring. The solution color changed within 10 s. The products were washed with water and ethanol several times by centrifugation at 8000 rpm. Finally, the products were re-dispersed in 5 mL 0.1 M PBS (pH 7.4) for further use. 2.4. Bioconjugation of Au-Ag BMNPs with Ab2 The preparation procedure of the Au-Ag-Ab2 bioconjugate was as follows: 200 mL of as-prepared Au-Ag BMNPs were diluted to 1 mL with PBS (pH 7.4). Then 10 mL of Ab2 (20 mg mL 1) was added into the solution, followed by gentle shaking at room temperature for 3 h. During this process, Ab2 could be loaded onto the Au-Ag BMNPs through physical adsorption and the interactions between the mercapto or amino groups of Ab2 and the Au-Ag BMNPs. The mixture was washed with PBS, then centrifuged at 10,000 rpm for 5 min, repeatedly washed and centrifuged ﬁve times. Finally, the obtained Au-Ag-Ab2 were re-dispersed in 0.5 mL of PBS and stored at 4 C before use. 2.5. Fabrication of the EC immunosensor on the origami device The fabrication process of the paper-based EC immunosensor (the fabrication details of the origami device could be found in the supporting information) was shown in Scheme 1. This novel FLAuNPs-PWE was fabricated through a direct chemical reduction of HAuCl4 by AA. Firstly, the suspension of AuNPs seeds was prepared by using NaBH4 as the reductant and stabilized with sodium citrate according to the previous reported literature . Then, 20.0 mL of as-prepared AuNPs seeds solution were dropped into the paper sample zone of bare PWE. Then the origami device was equilibrated at room temperature for 1 h to optimize the surface immobilization of AuNPs seeds on cellulose ﬁbers. After rinsing with water thoroughly according to the method in our previous work  to remove loosely bound AuNPs seeds, 12 mL of HAuCl4 solution was applied to paper working zone on the back of a screen-printed carbon working electrode, followed immediately by the addition of 12 mL of AA solution (200 mM), and incubated at room temperature for 5 min. Subsequently, the resulting FLAuNPsPWE was washed with water thoroughly. Thus a layer of
interconnected FLAuNPs on cellulose ﬁbers with good conductivity were obtained, which were dried at room temperature for 20 min. The EC origami immunodevice was constructed by immobilizing Ab1 in paper working zone of FLAuNPs-PWE. In brief, 5 mL of Ab1 (20 mg mL 1) was applied to the FLAuNPs-PWE, and incubated for 30 min. Then, the electrode surface was washed with PBS and incubated with 1% BSA for 30 min to block possible remaining active sites against nonspeciﬁc adsorption. Finally, the immunosensor was rinsed with PBS and stored at 4 C in dry air in the dark prior to use. 2.6. EC immunoassay To carry out the EC detection, 5.0 mL sample solution containing different concentrations of CEA in PBS was added to electrode and allowed to incubation for 200 s at room temperature, and then the electrode was washed extensively to remove unbound CEA molecules. Finally, the prepared Au-Ag-Ab2 was dropped onto the electrode surface. After another 200 s, the electrode was washed and ready for measurement. Finally, as shown in Scheme S5, the auxiliary tab was folded down below the sample tab and clamped with a home-made device-holder similar to our previous work , which was comprised of two circuit boards (named Board-A and Board-B respectively) with conductive pads on them, to ﬁx and connect this origami device to the electrochemical workstation. For amperometric measurement of the immunosensor, a detection potential of -0.5 V was selected. After the background current was at a state with PBS (10 mL, 0.1 M) only, H2O2 was added to the cell (the ﬁnal concentration of H2O2 was 5 mM) and the current change was recorded. The current was collected and registered as the signal of the immunosensor which was relative to the concentration of CEA samples. 3. Results and discussion 3.1. Structure characterization The novel FLAuNPs-PWE was constructed through the growth of an interconnected FLAuNPs layer on the surfaces of cellulose ﬁbers in paper sample zone (Scheme S1). Different size and sharp of AuNPs layer were obtained when changing the concentration of
Fig. 1. Growth of FLAuNPs layer on the surface of cellulose in paper sample zone of PWE with different concentrations of AA under different magniﬁcation: (A), (B) and (C) with 100 mM AA; (D), (E) and (F) with 200 mM AA.
G. Sun et al. / Electrochimica Acta 147 (2014) 650–656
AA. The AuNPs seeds were rapidly enlarged by the addition of 12 mL of 100 mM AA solution under the self-catalytic reduction mechanism of Au NPs growth (Fig. 1A-C). However, and the size and shape of the result AuNPs were irregular (Fig. 1C). When 12 mL of 200 mM AA was used, a continuous and dense conducting FLAuNPs layer was obtained completely on the cellulose ﬁber surfaces after 5 min of growth (Fig. 1D-F). As shown in Fig. 1F, the obtained AuNPs with obvious 3D hierarchical structure. High surface-to-volume ratio of this structure could provide much more multidimensional spaces for increasing the load of Ab1 and beneﬁt the analytical application. The Au-Ag BMNPs were synthesized through a simple kinetic controlled co-reduction route at room temperature. The SEM image of the Au-Ag BMNPs was shown in Fig. 2A, the surfaces of the BMNPs were unsmooth and uneven, and the size of the BMNPs was about 150 nm. The corresponding EDS was shown in Fig. 2B, the sample consisted of Au, Ag elements, indicating the successfully synthesis of Au-Ag BMNPs, and the atomic percentage of Au in Au-Ag BMNPs was 8.63% while Ag was 91.37%. Fig. 2C was a TEM image of the Au-Ag BMNPs which exhibited a coarse surface. UV-vis absorption spectroscopy was used to monitor the formation of Au-Ag-Ab2 (Fig. 2D). A broad absorption peak at 611 nm was observed for the synthesized Au-Ag BMNPs due to surface plasmon resonances in metallic nanoparticles (curve a). When Ab2 molecules were conjugated to the surface of Au-Ag BMNPs, another absorption peak at 278 nm was achieved (curve b), which was mainly derived from that of Ab2 molecules (curve c). These results indicated that the bionanolabels were successfully prepared. 3.2. Characterization of the immunosensor fabrication EIS was an effective method to monitor the changes of interfacial properties, allowing the understanding of chemical transformation
and processes associated with the conductive electrode surface. The EIS was used to get information of the impedance changes of the sensor interface in the modiﬁcation process and its spectra were obtained in 5.0 mmol L 1 [Fe(CN)6]3 /4 solution containing 0.1 mol L 1 KCl. In EIS, the semicircle diameter of EIS spectra equals the electron-transfer resistance, Ret. As seen in the EIS spectra (Fig. 3A), a large resistance was obtained at bare PWE (curve a). After FLAuNPs layer was coated on the bare PWE, a remarkable decrease in the resistance value was observed (curve b), implying that the AuNPs was an excellent electric conducting material and accelerated the electron transfer. Subsequently, when the Ab1 was loaded on the surface of FLAuNPs-PWE, the Ret increased (curve c) which suggested that the Ab1 molecules were successfully immobilized on the surface and formed an additional barrier and further prevented the redox probe to the electrode surface. Similarly, the immobilization of BSA and CEA all independently slowed down the electron-transfer kinetics of the redox probe at the FLAuNPs-PWE interface, thereby resulting in the increasing impedance of the FLAuNPs-PWE (curve d, curve e), which testiﬁed to the immobilization of these substances. At last, when the resulting electrode reacted with Au-Ag-Ab2, interestingly the resistance decreased (curve f), indicating that the synthesized Au-Ag-Ab2 possessed high conductivity and good electron transfer efﬁciency, although the protein adsorption layer acted as barrier to the interfacial electron transfer. 3.3. Electrocatalytic activity of Au-Ag BMNPs toward H2O2 reduction As a demonstration of application of such Au-Ag BMNPs, they were deposited on bare FLAuNPs-PWE surface to test their catalytical performance for H2O2 reduction. Fig. 3B presents the cyclic voltammograms (CVs) of the FLAuNPs-PWE and Au-Ag BMNPs modiﬁed FLAuNPs-PWE toward the reduction of H2O2 in N2-saturated PBS at pH 7.4 in the absence or presence of 5 mM
Fig. 2. (A) SEM image of the Au-Ag BMNPs. (B) EDS of the Au-Ag BMNPs. (C) TEM image of the Au-Ag BMNPs. (D) UV-vis absorption spectrum of (a) Au-Ag BMNPs, (b) Au-AgAb2, (c) Ab2.
G. Sun et al. / Electrochimica Acta 147 (2014) 650–656
Fig. 3. (A) EIS spectra of (a) bare PWE, (b) FLAuNPs-PWE, (c) Ab1/FLAuNPs-PWE, (d) BSA/Ab1/FLAuNPs-PWE, (e) CEA/BSA/Ab1/FLAuNPs-PWE and (f) Au-Ag-Ab2 CEA/BSA/Ab1/ FLAuNPs-PWE in 5.0 mmol L 1 [Fe(CN)6]3 /4 solution containing 0.1 mol L 1 KCl. (B) CVs of different electrodes in N2-saturated PBS solution (pH 7.4) of 0 ((b) Au-Ag BMNPs modiﬁed FLAuNPs-PWE) and 5 mM H2O2 ((a) bare FLAuNPs-PWE, (c) Au-Ag BMNPs modiﬁed FLAuNPs-PWE).
H2O2 at a scan rate of 50 mV s 1. It was obviously seen that the response of the bare FLAuNPs-PWE toward the reduction of H2O2 was pretty weak (curve a). In comparison with the CVs response of the Au-Ag BMNPs modiﬁed FLAuNPs-PWE in the absence of H2O2 (curve b), an obvious reduction current was observed at the Au-Ag BMNPs modiﬁed FLAuNPs-PWE in the presence of 5 mM H2O2 (curve c). These observations indicated that the Au-Ag BMNPs exhibit the ability to promote the reduction of H2O2 and therefore resulted in the generation of EC signals. In addition, the peroxidase-like activity of Au-Ag BMNPs was similar to horseradish peroxidase (HRP) (Fig. S1). 3.4. Optimization of experimental conditions The experimental conditions, which can affect the amperometric determination of CEA, including the pH of supporting electrolyte, time of immunoreaction and the incubation temperature of antigen-antibody were optimized. A series of experiments were conducted to select optimal analytical conditions using 1 ng mL 1 CEA. The effect of pH on the amperometric responses of the immunosensor has been investigated from 4.0 to 8.5. As shown in Fig. S2A, an optimal amperometric response was achieved at pH 7.4. Highly acidic or alkaline surroundings would damage the immobilized protein, especially in alkalinity. Hence, PBS (pH 7.4) was selected as the electrolyte for CEA detection. The incubation time was an important parameter for both capturing CEA on the electrode surface and speciﬁcally recognizing Au-Ag-Ab2. At room temperature, the amperometric responses increased with the increasing incubation time of CEA used in sandwich-type immunoassay and then leveled off, which indicated a saturated binding in the immunoreaction. The optimal
incubation time of CEA immunocomplex was 200 s (Fig. S2B). In the second immunoassay incubation step, the catalytic current showed the same changing tendency, and the response current reached a plateau at about 200 s (Fig. S2C). Therefore, an incubation time of 200 s was selected for the sandwich-type immunoassay. As shown in Fig. S2D, the current response increased with increasing temperature up to 37 C, which was attributed to the increasing immunoreaction rate between antigens and antibodies. When temperature was over 37 C, the current response decreased. The reason was that high temperature caused an irreversible denaturation of proteins. However, the temperature at 25 C (room temperature) showed acceptable sensitivity for the detection of CEA. Meanwhile, considering the practical feasibility in real life, all the experiments in this study were carried out at room temperature. The concentration of labeled antibody affected the extent and speed of immunoreaction, different concentrations of Ab2 was used to label Au-Ag BMNPs. As shown in Fig. S3A, with the increasing concentration of Ab2, the current increased and reached the maximum values at 20 mg mL 1. Thus, 20 mg mL 1 Ab2 was used to label Au-Ag BMNPs. As shown in Fig. S3B, the optimal amperometric response was achieved at the incubation time of 3 h, indicating the saturated binding of Ab2 onto Au-Ag BMNPs. Subsequently, 3 h was chosen as the optimal incubation time between Au-Ag BMNPs and Ab2. 3.5. Analytical performance The analytical performance of this method was veriﬁed by using samples of 5.0 mL of standard human CEA solutions at various concentrations in PBS under the optimal conditions. The current
Fig. 4. (A) Current signals of this immunosensor incubated with different concentrations of CEA (0, 0.001, 0.005, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50 ng mL 1, from bottom to top); (B) Logarithmic calibration curve for CEA. 0.1 M PBS solution (pH 7.4) containing 5 mM H2O2 as supporting electrolyte. n = 11 for each point, error bars represent standard deviation (SD).
G. Sun et al. / Electrochimica Acta 147 (2014) 650–656
responses for different concentrations of CEA were shown in Fig. 4A whereas Fig. 4B showed the derived calibration curve correspondingly. With an increasing concentration of CEA, good correlation between the increased current intensity and variable concentration of CEA was observed, with a wide dynamic range (0.001-50 ng mL 1) that covered most of the levels in human plasmas and serums. The equation of the calibration curve was DCurrent = 40.69 + 15.58 lg [CEA] (ng mL 1) with a correlation coefﬁcient of 0.9965 (n = 11). The detection limit for CEA concentration was estimated to be 0.3 pg mL 1 at a signal-tonoise ratio of 3. Compared with other CEA assays reported in the literatures [33–37](Table 1), the proposed EC immunosensor had a relative large linear range and low detection limit. Thus, on the basis of this standard curve, the EC approach should be useful for the determination of CEA in real serum samples, because the cutoff value of CEA in clinical diagnosis is 5 ng mL 1 . 3.6. Reproducibility, stability, speciﬁcity and application in real sample. The precision and reproducibility of this EC immunoassay was evaluated by intra-assay and inter-assay relative standard deviation (RSD). The intra-assay precision was evaluated by assaying one CEA level (1 ng mL 1) for four replicate measurements. Analyzed from the experimental results, the intra-assay RSD were 4.3%, whereas the inter-assay RSD of 5.1% was obtained by measuring the same samples with four electrodes prepared independently at the identical experimental conditions. These results suggested an acceptable precision and reproducibility of the proposed protocol. Stability of the immunosensors was also a key factor in their application and development. The stability of the immunosensor was examined by storing the immunosensor in PBS (pH 7.4) at 4 C for different periods. The current response of the as-prepared immunosensor decreased 3.2% after one week storage in pH 7.4 PBS. Four weeks later, the current response of the immunosensor using Au-Ag BMNPs as labels decreased to about 92% of its initial value. The slow decrease in the current response may be due to the gradual denaturation of antibody. Usually, nonspeciﬁc adsorption was a major problem in immunosensing, since it cannot be distinguished from speciﬁc adsorption and consequently inﬂuences the sensitivity. The speciﬁc analyte has to be measured in the presence of a relatively high amount of nonspeciﬁc species in diagnostic applications. In this study, three kinds of interfering agents, including cancer antigen 125 (CA 125), alpha-fetoprotein (AFP) and prostatespeciﬁc antigen (PSA) were used to evaluate the selectivity of the immunosensor. The signal was compared by assaying CEA (1 ng mL 1) with interfering agents (10 ng mL 1). As indicated from Fig. S4A, signiﬁcantly higher current response was observed with the target CEA than with other biomarkers. When CEA coexisted with these sample interfering agents, no apparent signal change took place in comparison with that of only CEA, which indicated that the proposed immunosensor revealed sufﬁciently selective for the detection of CEA. We also used three kinds of other signal antibodies (CA 125-Ab2, AFP-Ab2, PSA-Ab2) to replace Ab2 to test Table 1 Comparison of analytical properties of different immunoassays toward CEA. Immunoassay format
Linear range (ng mL 1)
Detection limit (pg mL 1)
EC Chemiluminescence Flow injection EC Electrochemiluminescence Photoelectrochemical EC
2.0-20 1.0-70 0.5-25 0.01-50 0.05-20 0.001-50
1000 650 220 6 10 0.3
33 34 35 36 37 This work
the current response. Due to these antibodies could not speciﬁcally bind CEA, thus, the Au-Ag BMNPs could not introduce into the immunosensor, and a low current response was obtained (Fig. S4B). The results suggested that the CEA could speciﬁcally bind to its antibodies (CEA-Ab2). The feasibility of the immunoassay system for clinical applications was investigated by analyzing several real samples in comparison with the reference values (results provided by the Shandong Tumor Hospital, China) obtained by a commercially available Electrochemiluminescent Analyzer (ROCHE E601, Switzerland). Human serum samples were diluted to different concentrations with a PBS solution of pH 7.4, and each sample was analysed for six times. Parts of the results were shown in Table S1. There was no signiﬁcant difference between the two methods. Hence, the developed immunoassay methodology could be reasonably applied in the clinical determination of CEA levels in human serum. 4. Conclusions In this work, we have designed an enzyme-free EC origami immunodevice for the detection of CEA based on FLAuNPs-PWE as sensor platform and Au-Ag BMNPs as signal labels. It was the ﬁrst direct modiﬁcation of PWE on m-PADs through the growth of a FLAuNPs layer. This novel FLAuNPs-PWE possessed much larger surface areas and much more active sites to immobilize large amount of capture antibodies. In addition, the Au-Ag BMNPs tracing tag kept good biological activity to antibodies and exhibited efﬁcient catalysis towards H2O2 reduction. With a sandwich-type immunoassay format, the proposed immunosensor showed excellent analytical performance for detection of CEA with a wide linear range and low detection limit and acceptable stability, reproducibility, and accuracy. This work could contribute to further expand the detection mode of m-PADs and provided POCT applications in remote regions and developing countries. 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). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.electacta.2014.09.149. References  J.P. Comer, Semiquantitative speciﬁc test paper for glucose in urine, Anal. Chem. 28 (1956) 1478.  A.W. Martinez, S.T. Phillips, M.J. Butte, G.M. Whitesides, Patterned paper as a platform for inexpensive, low-volume, portable bioassays, Angew. Chem. Int. Ed. 46 (2007) 1318.  A.W. Martinez, S.T. Phillips, B.J. Wiley, M. Gupta, G.M. Whitesides, Flash: a rapid method for prototyping paper-based microﬂuidic devices, Lab Chip 8 (2008) 2146.  R. Mukhopadhyay, Cheap, handheld colorimeter to read paper-based diagnostic devices, Anal. Chem. 81 (2009) 8659.  S.K. Sia, L.J. Kricka, Microﬂuidics and point-of-care testing, Lab Chip 8 (2008) 1982.  R. Mukhopadhyay, Medical diagnostics with paper and camera phones, Anal. Chem. 80 (2008) 3949.  Y. Lu, W.W. Shi, J.H. Qin, B.C. Lin, Fabrication and characterization of paperbased microﬂuidics prepared in nitrocellulose membrane by wax printing, Anal. Chem. 82 (2009) 329.  K. Abe, K. Suzuki, D. Citterio, Inkjet-printed microﬂuidic multianalyte chemical sensing paper, Anal. Chem. 80 (2008) 6928.
G. Sun et al. / Electrochimica Acta 147 (2014) 650–656
 B. Yoon, D.Y. Ham, O. Yarimaga, H. An, C.W. Lee, J.M. Kim, Inkjet printing of conjugated polymer precursors on paper substrates for colorimetric sensing and ﬂexible electrothermochromic display, Adv. Mater. 23 (2011) 5492.  H. Hwang, S.H. Kim, T.H. Kim, J.K. Park, Y.K. Cho, Paper on a disc: balancing the capillary-driven ﬂow with a centrifugal force, Lab Chip 11 (2011) 3404.  E. Carrilho, S.T. Phillips, S.J. Vella, A.W. Martinez, G.M. Whitesides, Paper microzone plates, Anal. Chem. 81 (2009) 5990.  R.F. Carvalhal, M.S. Kfouri, M.H. de Oliveira Piazetta, A.L. Gobbi, L.T. Kubota, Electrochemical detection in a paper-based separation device, Anal. Chem. 82 (2010) 1162.  W. Dungchai, O. Chailapakul, C.S. Henry, Electrochemical detection for paperbased microﬂuidics, Anal. Chem. 81 (2009) 5821.  J.L. Delaney, C.F. Hogan, J. Tian, W. Shen, Electrogenerated chemiluminescence detection in paper-based microﬂuidic sensors, Anal. Chem. 83 (2011) 1300.  A. Apilux, Y. Ukita, M. Chikae, O. Chilapakul, Y. Takamura, Development of automated paper-based devices for sequential multistep sandwich enzymelinked immunosorbent assays using inkjet printing, Lab Chip 13 (2013) 126.  L.M. Tian, J.J. Morrissey, R. Kattumenu, N. Gandra, E.D. Kharasch, S. Singamaneni, Bioplasmonic paper as a platform for detection of kidney cancer biomarkers, Anal. Chem. 84 (2012) 9928.  B.S. Miranda, E.M. Linares, S. Thalhammer, L.T. Kubota, Development of a disposable and highly sensitive paper-based immunosensor for early diagnosis of asian soybean rust, Biosens. Bioelectron. 45 (2013) 123.  M. Santhiago, C.S. Henry, L.T. Kubota, Low cost, simple three dimensional electrochemical paper-based analytical device for determination of p-nitrophenol, Electrochim. Acta 130 (2014) 771.  D.J. Zang, L. Ge, M. Yan, X.R. Song, J.H. Yu, Electrochemical immunoassay on a 3D microﬂuidic paper-based device, Chem. Commun. 48 (2012) 4683.  X.L. Wu, H. Kuang, C.L. Hao, C.R. Xing, L.B. Wang, C.L. Xu, Paper supported immunosensor for detection of antibiotics, Biosens. Bioelectron. 33 (2012) 309.  Z.X. Gao, H.F. Li, J.J. Liu, J.M. Lin, A simple microﬂuidic chlorine gas sensor based on gas-liquid chemiluminescence of luminol-chlorine system, Anal. Chim. Acta 622 (2008) 143.  P.P. Wang, L. Ge, S.G. Ge, J.H. Yu, M. Yan, J.D. Huang, A paper-based photoelectrochemical immunoassay for low-cost and multiplexed point-ofcare testing, Chem. Commun. 49 (2013) 3294.  L. Ge, J.X. Yan, X.R. Song, M. Yan, S.G. Ge, J.H. Yu, Three-dimensional paperbased electrochemiluminescence immunodevice for multiplexed measurement of biomarker s and point-of-care testing, Biomaterials 33 (2012) 1024.  E. Fu, T. Liang, J. Houghtaling, S. Ramachandran, S.A. Ramsey, B. Lutz, P. Yager, Enhanced sensitivity of lateral ﬂow tests using a two-dimensional paper network format, Anal. Chem. 83 (2011) 7941.
 Y. Li, T. Sasaki, Y. Shimizu, N. Koshizaki, Hexagonal-close-packed, hierarchical amorphous TiO2 nanocolumn arrays: transferability, enhanced photocatalytic activity, and superamphiphilicity without UV irradiation, J. Am. Chem. Soc. 130 (2008) 14755.  X.J. Dai, Y.S. Luo, W.D. Zhang, S.Y. Fu, Facile hydrothermal synthesis and photocatalytic activity of bismuth tungstate hierarchical hollow spheres with an ultrahigh surface area, Dalton Trans. 39 (2010) 3426.  C.X. Wang, L.W. Yin, L.Y. Zhang, Y.X. Qi, N. Lun, N.N. Liu, Large scale synthesis and gas-sensing properties of anatase TiO2 three-dimensional hierarchical nanostructures, Langmuir 26 (2010) 12841.  X. Zhang, Y. Cao, S. Yu, F.C. Yang, P.X. Xi, An electrochemical biosensor for ascorbic acid based on carbon-supported PdNi nanoparticles, Biosens. Bioelectron. 44 (2013) 183.  W.Z. Li, L. Kuai, Q. Qin, B.Y. Geng, Ag-Au bimetallic nanostructures: coreduction synthesis and their component-dependent performance for enzyme-free H2O2 sensing, J. Mater. Chem. A 1 (2013) 7111.  B.D. Busbee, S.O. Obare, C.J. Murphy, An improved synthesis of high-aspectratio gold nanorods, Adv. Mater. 15 (2003) 414.  J.X. Yan, L. Ge, X.R. Song, M. Yan, S.G. Ge, J.H. Yu, Paper-based electrochemiluminescent 3D immunodevice for lab-on-paper, speciﬁc, and sensitive point-of-care testing, Chem. Eur. J. 18 (2012) 4938.  J.J. Lu, S.G. Ge, L. Ge, M. Yan, J.H. Yu, Electrochemical DNA sensor based on three-dimensional folding paper device for speciﬁc and sensitive point-of-care testing, Electrochim. Acta 80 (2012) 334.  J.H. Lin, W. Qu, S.S. Zhang, Electrochemical immunosensor for carcinoembryonic antigen based on antigen immobilization in gold nanoparticles modiﬁed chitosan membrane, Anal. Sci. 27 (2007) 1059.  Z.F. Fu, Z.J. Yang, J.H. Tang, H. Liu, F. Yan, H.X. Ju, Channel and substrate zone two-dimensional resolution for chemiluminescent multiplex immunoassay, Anal. Chem. 79 (2007) 7376.  J. Wu, J.H. Tang, Z. Dai, F. Yan, H.X. Ju, N.E. Murr, A disposable electrochemical immunosensor for ﬂow injection immunoassay of carcinoembryonic antigen, Biosens. Bioelectron. 22 (2006) 102.  Y. Zhang, W.Y. Liu, S.G. Ge, M. Yan, S.W. Wang, J.H. Yu, N.Q. Li, X.R. Song, Multiplexed sandwich immunoassays using ﬂow-injection electrochemiluminescence with designed substrate spatial-resolved technique for detection of tumor markers, Biosens. Bioelectron. 41 (2013) 684.  W.W. Tu, W.J. Wang, J.P. Li, S.Y. Deng, H.X. Ju, Chemiluminescence excited photoelectrochemistry using graphene-quantum dots nanocomposite for biosensing, Chem. Commun. 48 (2012) 6535.  T.X. Li, Modern clinical immunoassay, Military Medical Science Press, Beijing, 2007, pp. 178.