Biosensors and Bioelectronics 63 (2015) 7–13

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3D origami electrochemical immunodevice for sensitive point-of-care testing based on dual-signal amplification strategy Chao Ma a, Weiping Li a, Qingkun Kong a, Hongmei Yang a, Zhaoquan Bian a, Xianrang Song b, Jinghua Yu a, Mei Yan a,n 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 Cancer Research Center, Shandong Tumor Hospital, Jinan 250117, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 6 May 2014 Received in revised form 23 June 2014 Accepted 6 July 2014 Available online 11 July 2014

A dual signal amplification immunosensing strategy that offers high sensitivity and specificity for the detection of low-abundance biomarkers was designed on a 3D origami electrochemical device. High sensitivity was achieved by using novel Au nanorods modified paper working electrode (AuNRs-PWE) as sensor platform and metal ion-coated Au/bovine serum albumin (Au/BSA) nanospheres as tracing tags. High specificity was further obtained by the simultaneous measurement of two cancer markers on AuNRs-PWE surface using different metal ion-coated Au/BSA tracers. The metal ions could be detected directly through differential pulse voltammetry (DPV) without metal preconcentration, and the distinct voltammetric peaks had a close relationship with each sandwich-type immunoreaction. The position and size of the peaks reflected the identity and level of the corresponding antigen. Integrating the dual-signal amplification strategy, a novel 3D origami electrochemical immunodevice for simultaneous detecting carcinoembryonic antigen (CEA) and cancer antigen 125 (CA125) with linear ranges of over 4 orders of magnitude with detection limits down to 0.08 pg mL  1 and 0.06 mU mL  1 was successfully developed. This strategy exhibits high sensitivity and specificity with excellent performance in real human serum assay. The AuNRs-PWE and the designed tracer on this immunodevice provided a new platform for lowcost, high-throughput and multiplex immunoassay and point-of-care testing in remote regions, developing or developed countries. & 2014 Published by Elsevier B.V.

Keywords: Electrochemical Multiplex immunoassay 3D origami device Point-of-care testing

1. Introduction Development of microfluidic devices for sensitive point-of-care testing (POCT) of cancer markers for early cancer diagnosis and treatment is a crucial need, primarily in resource-limited settings, because they require low volumes of reagents, easy to use, store and transport (Martinez et al., 2010; Liu and Crooks, 2011). Since the first patterned paper was proposed by Martinez et al. (2007), microfluidic paper-based analytical devices (m-PADs) have recently emerged as ideal platforms for POCT due to their excellent advantages, including low-cost of fabrication and minimal equipment requirements (Chen et al., 2012; Pelton, 2009). The m-PADs are easily fabricated by wax-printing (Carrilho et al., 2009) because this method is among the cheapest and most easily implemented means of mass production available. The last five years have witnessed a fast progress in the field of m-PADs (Bruzewicz et al., 2008; Tao et al., 2011; Cheng et al., 2010; Ge n

Corresponding author. Tel.: þ 86 531 82767161; fax: þ86 531 82765956. E-mail address: [email protected] (M. Yan).

http://dx.doi.org/10.1016/j.bios.2014.07.014 0956-5663/& 2014 Published by Elsevier B.V.

et al., 2013a, 2013b), which represent new and outstanding approaches to truly simple, portable, and low-cost devices for disease diagnoses, environmental detection, and biological assay. Recently, electrochemical and luminescent methods alone as well as their combination have been widely employed as analytical methods on m-PADs, such as chemiluminescence (Ge et al., 2012a, 2012b), fluorescence (Ellerbee et al., 2009), and electrochemiluminescence (Wang et al., 2012a, 2012b). Electrochemical (EC) methods (Kimmel et al., 2012), particularly amperometric methods (Feng et al., 2012), which distinguish themselves by convenient miniaturization and integration, easy signal quantification, simple instrumentation, and low cost of the entire assay, can be ideal alternatives. Meanwhile, due to the limited specificity of cancer markers to a particular disease, multiplex immunoassay methods for simultaneous detection of panel of biomarkers have been developed for clinical application (Stoeva et al., 2006; Zong et al., 2012). Signal amplification is the most popular strategy that has been extensively used for the development of paper-based assay (Ge et al., 2012a, 2012b; Wang et al., 2012a, 2012b). By introducing

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multiple electroactive species per binding event is a versatile signal amplification technique widely used in EC-based bioassay. Recently, metal ions were used as electroactive labels for the assay of proteins (Feng et al., 2012; Wu et al., 2013). Bovine serum albumin (BSA), a natural biocompatible protein, could provide abundant amino functional groups, which was largely used as a chelating agent for the absorption of metal ions. Herein, 3D Au/ BSA nanospheres, equipped with convenient and green synthesis route, good biocompatibility, and excellent conductivity, exchanged with different metal ions such as Pb2 þ and Cd2 þ fabrication of versatile labels. To further increase the detection sensitivity, nanomaterial-based sensor platform with large surface area and superior conductivity can be used. An Au nanorods (NRs) layer modified paper working electrode (PWE) to fabricate novel AuNRs-PWE on 3D origami EC device has not been reported yet. The combination of AuNRs-PWE as sensor platform and Au/BSA– metal ion as tracing tag can be a promising amplification strategy for constructing sensitive EC biosensor. In this work, we first design and report a 3D origami metal ionbased EC immunodevice for highly sensitive cancer markers detection by employing AuNRs-PWE and Au/BSA nanospheres for dual amplification. The introduction of AuNRs-PWE accelerated the electron transfer rate to amplify the electrochemical signal as well as provided a biocompatible microenvironment for the immobilization of antibody. Au/BSA nanospheres were selected as excellent nanocarriers for loading numerous metal ions such as Pb2 þ and Cd2 þ to form Au/BSA–metal ion tracers. Carcinoembryonic antigen (CEA) antibodies and cancer antigen 125 (CA125) antibodies were conjugated with Au/BSA–Pb2 þ and Au/BSA–Cd2 þ to fabricate anti-CEA–Au/BSA–Pb2 þ and anti-CA125–Au/BSA–Cd 2þ bioconjugates and were brought onto the AuNRs-PWE surface upon the completion of sandwich immunoreactions. On the basis of AuNRs-PWE as sensor platform and Au/BSA–metal ion as tracers, a novel and facile strategy for simultaneous electrochemical detection of CEA and CA125 was developed. This proposed strategy exhibited good stability, precision, and reproducibility, indicating its wide range of potential applications in POCT.

2. Materials and methods 2.1. Materials and reagents All reagents were of analytical grade and used as received. Ultrapure water obtained from a Millipore water purification system ( 418.2 MΩ, Milli-Q, Millipore) was used in all assays and solutions. Antigens (CEA and CA125), mouse monoclonal capture antibody (Ab1), and signal antibody (Ab2) were purchased from Linc-Bio Science Co. Ltd. (Shanghai, China). The clinical serum samples were from Shandong Tumor Hospital. Bovine serum albumin (BSA), glutaraldehyde solution 25% (GA), and ascorbic acid (AA) were obtained from Sigma-Aldrich Chemical Co. (USA). Tetrachloroauric acid (HAuCl4), hydroxylammonium chloride (NH2 OH∙HCl), NaBH4, and trisodium citrate was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Phosphate buffered solutions (PBS, 10.0 mM) with different pH values were prepared with KH2PO4 and Na2HPO4. Carbon ink (ELECTRODEDAGPF-407C) and Ag/AgCl ink (ELECTRODAG7019 (18DB19C)) were purchased from Acheson (Germany). Whatman chromatography paper #1 (58.0 cm  68.0 cm) (pure cellulose paper) was obtained from GE Healthcare Worldwide (Pudong, Shanghai, China) and used with further adjustment of size (A4 size).

2.2. Apparatus All electrochemical immunoassay measurements were performed on a CHI 760D workstation (Chenhua, Shanghai, China). Transmission electron microscopy (TEM) investigations were performed using JEOL 4000 EX microscope. Scanning electron microscopy (SEM) images and energy-dispersed spectrum (EDS) experiment were recorded on a QUANTA FEG 250 thermal field emission SEM (FEI Co., USA). UV–vis absorption spectra were recorded on a UV-3101 spectrophotometer (Shimadzu, Japan). X-ray photoelectron spectra (XPS) were measured using an ESCALAB 250 spectrometer (Thermo Fisher Scientific) with monochromatized Al-Kα X-ray radiation (1486.6 eV) in ultrahigh vacuum ( o10  7 Pa). 2.3. Preparation of Ab2–Au/BSA–metal ion Firstly, Au/BSA nanospheres were synthesized according to the literature (Hu et al., 2013). Briefly, 50 mg of BSA was dissolved into 10 mL of Milli-Q water under magnetic stirring. Then, 10 mL of HAuCl4 solution (10 mM) was added to the BSA solution stirred for 5 min at room temperature. Finally, 50 mg of AA was added to the above mixture solution fleetly and reacted for 5 min. The solid product was washed with ethanol and Milli-Q water for several times and stored at 4 °C. For absorption of metal ion, 0.5 mL of Au/BSA (40 mg mL  1) was dispersed in 10 mL of 10 mM Pb(NO3)2 and Cd(NO3)2 solution, respectively, and stirred for 24 h. After washing with Milli-Q water for several times, the Au/BSA–metal ion products were dispersed in 2 mL of Milli-Q water. For immobilization of Ab2, the tracers were dispersed in 1 mL of GA solution (2.5%) and sonicated for 10 min. After washing with PBS solution (pH 7.4), 1.0 mL of anti-CEA (10 mg mL  1) solution was added into the Au/BSA–Pb2 þ tracer and 1.0 mL of anti-CA125 (10 mg mL  1) solution was added into the Au/BSA–Cd2 þ tracer, respectively, and shaken for 10 h. After centrifugation, the obtained bioconjugates were further washed with PBS (pH 7.4) and re-dispersed in 4 mL of PBS (pH 7.4) as the assay solution. 2.4. Preparation of this novel AuNRs-PWE Prior to the AuNRs-PWE preparation, as shown in Scheme 1, this 3D origami EC device, which contains one auxiliary pad surrounded by one sample tab with the same size (12.0 mm  20.0 mm  0.18 mm), was fabricated in bulk by waxprinting (details in Supporting information). Then the wax-penetrated paper sheet was ready for screen-printing of electrodes on their corresponding paper zones. Finally, the paper sheet was cut into individual origami devices for further modification. The AuNRs-PWE with enhanced conductivity and enlarged surface area was fabricated through a seed-mediated growth approach. Briefly, as-prepared Au nanoparticle (AuNP) seeds solution (15 mL) (Busbee et al., 2003) was dropped into one paper working zone. Then the origami device was equilibrated at room temperature for 2 h to optimize the surface immobilization of AuNP seeds on cellulose fibers, followed by washing with Milli-Q water (Yan et al., 2012a, 2012b). Subsequently, freshly prepared growth aqueous solution (15 mL) containing HAuCl4 (1.0 mM) and NH2OH∙HCl (10.0 mM) was applied into the AuNP seeded PWE, and incubated for 10 min. Finally, the resulting AuNRs-PWE was washed with Milli-Q water thoroughly, and dried at room temperature for 30 min. 2.5. Preparation of this 3D origami EC immunodevice As shown in Scheme 2, in brief, 4.0 mL of CEA and CA125 Ab1s (20 mg mL  1) were dropped onto the AuNRs-PWE through the

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Scheme 1. Schematic representation of process to fabricate 3D origami device. (A) Paper sheet was firstly patterned in bulk using a wax printer. (B) On each wax-patterned origami device, carbon working electrode was screen-printed on paper working zone; carbon counter-electrode and Ag/AgCl reference electrode were screen-printed on paper auxiliary zone respectively. (C) One origami device.

strong effect of Au–NH2 bonding, and incubated for 40 min. After washing with PBS (pH 7.4), 4.0 mL of 1% BSA solution was then applied to the AuNRs-PWE and incubated for 30 min at room temperature to block nonspecific active sites. After washing with PBS (pH 7.4) containing 0.5% BSA, 2.0 mL of sample solution containing different concentrations of CEA and CA125 in PBS was added to the AuNRs-PWE and incubated for 210 s at room temperature, followed by washing with PBS (pH 7.4). Then, 4.0 mL of the designed bioconjugates were added to the AuNRsPWE, and incubated for 210 s at room temperature, followed by washing with PBS (pH 7.4). After a folding procedure, with the aids of a simple home-made device-holder, this 3D origami EC immunodevice was fixed and connected to the electrochemical workstation. A differential pulse voltammetry (DPV) scan from 0.9 V to 0.3 V in HAc/NaAc solution (pH 4.5, 40 mL) was performed to record the amperometric responses for simultaneous detection of CEA and CA125.

3. Results and discussion 3.1. Characterization of Ab2–Au/BSA–metal ion Fig. 1 displayed the typical SEM images of the designed tracers. The Au/BSA nanospheres showed spherical morphologies with an average size of 200 nm in diameter and a good monodispersity (Figs. 1A and S1), which clearly revealed the core–shell structure and a thin BSA layer wrapped outside. The morphologies of Au/ BSA–metal ion tracers did not change after loading with Pb2 þ and Cd2 þ (Fig. S2A and B). Meanwhile, the presence of lead and cadmium in the Au/BSA–metal ion tracers were confirmed with

EDS (Fig. S2C and D). Subsequently, the Au/BSA–metal ion tracers could be further used as labels in bioassay. Antibodies were obviously trapped on the surface of Au/BSA–Pb2 þ and Au/BSA–Cd2 þ , respectively (Fig. 1B and C). To further confirm the successful preparation of the designed bioconjugate, UV–vis spectroscopy was employed. As shown in Fig. 1D, one absorption peak at 525 nm attributed to AuNP collective electron oscillations or localized surface plasma resonance was observed for the Au/BSA–Pb2 þ tracer (curve a) (Strozyk et al., 2012). After the immobilization of the anti-CEA Ab2, another distinct adsorption peak was observed on the spectra of anti-CEA Ab2–Au/BSA–Pb2 þ (curve c), which could be attributed to the adsorption peak from the anti-CEA Ab2 itself at 276 nm (curve b) (Yan et al., 2012a, 2012b). 3.2. Characterizations of AuNRs-PWE As shown in SEM image (Fig. S3), the bare paper working zone with porous structure could provide an excellent interface for the AuNP seeds. As the growth time increased (0–10 min), the AuNP seeds were rapidly enlarged by incubating in the growth solution under the self-catalytic reduction mechanism of AuNP growth (Zayats et al., 2004). After 10 min of growth, a dense AuNRs conducting layer was observed in the paper working zone (Fig. 2A–C). In addition, the successful preparation of the AuNRsPWE was confirmed by XPS (Fig. 2D) and the peaks observed at 82.6 eV and 86.4 eV were ascribed to metallic gold. The novel AuNRs-PWE would benefit the further antibodies modification and analytical application. Herein, Ab2–Au/BSA–metal ion was assembled onto the AuNRs-PWE surface upon the completion of sandwich immunoreactions (Fig. S4A and B).

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Scheme 2. Schematic representation of the fabrication and assay procedures of 3D origami EC immunodevice. (A) AuNRs-PWE: growth of an interconnected AuNRs layer on the surfaces of cellulose fibers in bare PWE. (B) After immobilization with Ab1s, BSA, CEA and CA125. (C) After incubating with the designed tracers. (D) After modification, this immunodevice was integrated with a transparent device-holder, the device-holder was clamped closely and 40 mL supporting electrolyte was added for electrochemical assay.

Fig. 1. SEM images of (A) Au/BSA nanospheres. (B) Anti-CEA Ab2–Au/BSA–Pb2 þ . (C) Anti-CA125 Ab2–Au/BSA–Cd2 þ . (D) UV–vis spectra: Au/BSA–Pb2 þ tracer (curve a), antiCEA Ab2 (curve b), anti-CEA Ab2–Au/BSA–Pb2 þ (curve c).

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Fig. 2. (A–C) Enlarged AuNRs conducting layer on the surfaces of cellulose fibers in paper sample zone at 10 min growth time under different magnification. (D) XPS of (a) PWE and (b) AuNRs-PWE.

3.3. Characterization of this 3D origami EC immunodevice The cyclic voltammetry (CV) of [Fe(CN)6]3  /4  on the AuNRsPWE was investigated (Fig. S5A). The bare PWE exhibited one set of well defined redox peaks (curve a). After 5 min of growth, there was an increase of the peak current (curve b). After 10 min of growth, an obvious increase of the peak current was observed (curve c), indicating that the AuNRs-PWE had a significant higher peak current and larger CV area compared with the bare one (unmodified PWE). As an available approach to monitor the interfacial properties of the modified PWE, EIS has been generally employed to characterize the layer-by-layer assembly steps (Fig. S5B) (Ge et al., 2013a, 2013b). For a bare PWE, a small semicircle domain was observed (curve a). After the growth of an AuNR conducting layer, the AuNRs-PWE showed a lower resistance (curve b). However, the resistance increased markedly after Ab1 were stably immobilized onto the AuNRs-PWE (curve c). Similarly, the immobilization of BSA also generated an insulating protein layer, which increased resistance of the PWE (curve d).

stripping step, Pb2 þ and Cd2 þ could be released, thus generating the detection signal (Liu et al., 2013). Compared with Cd2 þ –Ab2/ CA125/Ab1/PWE (curve a, Cd2 þ ion bound directly with Ab2 and used as the tracer), the EC signal of Cd2 þ –Ab2/CA125/Ab1/AuNRsPWE (curve b) and Au/BSA–Cd2 þ –Ab2/CA125/Ab1-/AuNRs-PWE (curve c) was highly enhanced 6.4-fold and 12.4-fold, respectively. The reasons may be attributed to combining the advantages of high-binding capability of the AuNRs-PWE with an increase of metal ion loading per immunoassay event. 3.5. Influence of cross-reactivity The cross-reactivity was evaluated by comparing the EC responses of two analytes to those containing only one analyte. As expected, the EC responses showed minimal difference when the incubation solution contained one or two kinds of analytes (Fig. S6B). Obviously, the multiplex detection of CEA and CA125 would not cause interference with each other, and the cross reactivity between CEA and CA125 could be negligible. 3.6. Analytical performance

3.4. EC detection using dual-signal amplification The great amplification of the EC signal with AuNRs-PWE and Au/BSA–metal ion tracer was demonstrated in Fig. S6A. The principle of the EC detection was shown below:

Mn + + ne− → M

(1)

M → Mn + + ne−

(2) 2þ



In the deposition step, Pb and Cd ions could be reduced to metallic Pb and Cd (Nikelly and Cooke, 1957). Subsequently, in the

The EC performance of the 3D origami immunodevice would be influenced by many factors. Herein, we investigated the dependence of the DPV peak current under different experimental parameters such as detection solution pH and incubation time (Fig. S7). As a result, 0.2 M HAc/NaAc (pH 4.5) and incubation time with 210 s was selected for the EC immunoassay. Under the optimized conditions, the peak currents of DPV increased with increasing concentration of CEA and CA125 (Fig. 3A). The striping peaks at  0.46 V and  0.74 V were ascribed to the oxidation of lead and cadmium, respectively. The position of

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Fig. 3. (A) Typical DPV of 3D origami EC immunodevice for CEA and CA125 concentrations (from a to g: 0.0001, 0.001, 0.01, 0.1, 1.0, 10, 50 ng mL  1/U mL  1, respectively). Logarithmic calibration curves for CEA (B) and CA125 (C) in 0.2 M HAc/NaAc (pH 4.5) where the error bars were the standard deviations for 10 times of parallel detection. All the tests are under the optimal conditions.

Table 1 Assay results of human serum samples by the proposed and reference methods. Samples

Sample-1 Sample-2 Sample-3 Sample-4 Sample-5 a

CEA concentration (ng mL  1)

CA125 concentration (U mL  1)

Proposed methoda

Reference methoda

Relative error (%)

Proposed methoda

Reference methoda

Relative error (%)

12.3 26.4 38.5 40.8 47.2

12.5 27.1 37.8 39.9 46.7

 1.6  2.6 1.9 2.3 1.1

18.2 24.1 31.4 37.6 44.7

17.7 25.0 30.8 38.4 44.1

2.8  3.6 1.9  2.1 1.4

Average of 10 measurements.

two peaks could reflect the identity of corresponding antigens. Two calibration plots showed good linear relationships between the peak currents and the logarithm of the analyte concentrations in the range from 0.1 pg mL  1 to 50 ng mL  1 and 0.1 mU mL  1 to 50 U mL  1, respectively. The linear regression equations with CEA were I (mA)¼3.48þ 0.70 lg cCEA (ng mL  1, R ¼0.9976) (Fig. 3B) and the standard deviations (SDs) of the slope and the intercept were 0.017 and 0.051 by 10 replicate determinations of 1.0 ng mL  1 respectively. The linear regression equations with CA125 were I (mA)¼ 11.0þ2.21 lg cCA125 (U mL  1, R ¼0.9980) (Fig. 3C) and the SDs of the slope and the intercept were 0.045 and 0.11 by 10 replicate determinations of 1.0 U mL  1 respectively. The limits of detection for the two tumor markers were 0.08 pg mL  1 and 0.06 mU mL  1 with a signal-to-noise ratio of 3, respectively, which were not only much lower than those previously reported by different methods with different amplification (Table S1). The low detection limits might be attributed to the enormous loading of metal ions, which greatly amplified the peak signals. The wide linear ranges for two analytes were also very significant for POCT. The inter-assay precision of this immunodevice was examined by detecting 0.1 ng mL  1/U mL  1 CEA and CA125. The relative standard deviation (RSD) for parallel detections of CEA and CA125 with 10 immunodevices was 2.67% and 2.82%, respectively, indicating good precision of the immunoassay method and acceptable fabrication reproducibility of the immunodevices. In addition, no obvious change was observed after storage for 14 days, but a 4% decrease of its initial EC response was noticed after 30 days (Fig. S8). 3.7. Application in human serum samples

by commercial electrochemiluminescent single-analyte tests (Tables 1, S2 and S3). When the levels of cancer markers were over the calibration range, the serum sample was appropriately diluted with 10 mM pH 7.4 PBS, prior to assaying. These results showed acceptable feasibility to detect CEA and CA125 in human serum.

4. Conclusion In summary, a novel 3D origami multiplex EC immunodevice of cancer biomarkers was successfully developed based on dualsignal amplification. It was the first direct modification of PWE on m-PADs through the growth of an AuNR layer. Both the signal amplification of Au/BSA–metal ion tracer and the electron transfer acceleration of AuNRs-PWE greatly improve the sensitivity of the immunoassay method. The resulting immunodevice for quantitative detection of CEA and CA125 possesses high sensitivity, good reproducibility, and long-term stability. This proposed method can be expanded readily for detecting other cancer biomarkers and has the potential for reliable diagnosis of cancer and other diseases.

Acknowledgments This work was financially supported by Natural Science Research Foundation of China (21277058 and 51273048), Natural Science Foundation of Shandong Province, China (ZR2012BZ002), and Technology Development Plan of Shandong Province, China (Grant no. 2012GGB01181). Appendix A. Supplementary information

The analytical reliability and application potential of the proposed method were evaluated by comparing the assay results of five human serum samples with the reference values obtained

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.014.

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3D origami electrochemical immunodevice for sensitive point-of-care testing based on dual-signal amplification strategy.

A dual signal amplification immunosensing strategy that offers high sensitivity and specificity for the detection of low-abundance biomarkers was desi...
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