Cite this: Chem. Commun., 2014, 50, 10949 Received 26th April 2014, Accepted 14th May 2014

Disposable paper-based bipolar electrode for sensitive electrochemiluminescence detection of a cancer biomarker† Qiu-Mei Feng,‡ Jian-Bin Pan,‡ Huai-Rong Zhang, Jing-Juan Xu* and Hong-Yuan Chen

DOI: 10.1039/c4cc03102d

A disposable paper-based bipolar electrode (BPE) was reported for the first time for the sensitive electrochemiluminescence detection of a prostate specific antigen (PSA).

Since microfluidic paper-based analytical devices (mPADs) were proposed by Whitesides’ group,1 they have attracted increasing attention and interest especially in recent year. Due to the portability, disposability and low cost of paper-based devices, which have the complex functions of conventional lab-on-a-chip devices, mPADs are becoming an efficient approach for biological/ chemical analysis.2 Various detection methods such as electrochemistry,2 colorimetry,3 fluorescence,4 electrochemiluminescence (ECL)5 and chemiluminescence6 have been used in mPADs. Among these, ECL is one of the most promising methods due to its low background noise, simple optical setup and good sensitivity. Bipolar electrodes (BPEs), as electronic conductors without any direct electrical connection, have been demonstrated to be powerful tools for chemical and biological analysis. Manz’s group were the first to apply Ru(bpy)32+ ECL for signal reporting in a bipolar system. Based on this method, they successfully detected co-reactants and quenchers in the ECL reactions.7 Later, Crook’s group expanded the applications of bipolar electrochemistry to measure concentration, separation and electrocatalysis by making full use of both poles of BPE.8 Xu’s group effectively applied BPEs in bioanalysis, such as for the detection of ATP, cell surface proteins and c-Myc mRNA in cancer cells.9 Recently, dual-channel bipolar devices, which can separate analytes, and ECL probes have been adopted and these are able to successfully eliminate the background signal from the driving electrode.10 Moreover, ECL imaging has been introduced into a bipolar system for visual bioanalysis in the detection of PSA.11 State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China. E-mail: [email protected]; Fax: +86-25-83597924; Tel: +86-25-83597924 † Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c4cc03102d ‡ These authors contributed equally to this work.

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In this paper, combining the advantages of paper-based analytical devices and dual-channel bipolar device for the first time, we have fabricated a paper-based BPE device for the quantitative analysis of the cancer biomarker PSA. This paperbased BPE device consists of two hydrophilic cells patterned using hydrophobic wax. These two cells are connected by a carbon ink BPE as an electronic conductor. Another carbon ink electrode is used as the driving anode, while silver/silver chloride ink is used as the driving cathode and these are screen printed on the other ends of the two cells. By adding H2O2 into the sensing cell, the ECL intensity of Ru(bpy)32+ and tripropylamine (TPrA) is enhanced at the reporting cell. At the same time, the ECL responses could be significantly improved after modification of the BPE cathode with multi-walled carbon nanotubes (MWCNTs). A PSA sensor was then designed by the assembly of an immuno-sandwich structure on the cathode, in which the PSA antibody Ab1 was at first immobilized onto the MWCNT-modified cathode. Glucose oxidase (GOD) could be attached to cathode surface by coimmobilizing GOD and PSA antibody Ab2 onto the surface of silica nanoparticles in the presence of PSA. GOD catalyzes glucose to generate H2O2 in situ and electrochemical reduction of H2O2 at the cathode can initiate ECL light output due to the charge balance between the anode and cathode of BPE. The fabrication procedures for this paper-based BPE device and the mechanism of the ECL-based strategy are illustrated in Scheme 1. Further details can be found in ESI.† The principle behind the sensing for this BPE is displayed in Scheme 2. When a potential Etot is applied across the driving electrodes in the reservoirs, the majority of Etot is lost in the reservoir along the BPE, because of the high solution resistance. If the resistance of the bipolar electrode is neglected, the electric field (V0) in the solution across the BPE can be represented as V0 = Etot/(l1 + l2) and the potential difference (DEelec) across the bipolar electrode can be expressed using the following equation: DE elec ¼ V0  ðxa þ xc Þ ¼

Etot  ðxa þ xc Þ ðl 1 þ l 2 Þ

Here, (l1 + l2) represents the total length between the two driving electrodes except the part of BPE on which no solution

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Fig. 1 (A) ECL curves for the paper-based BPE in different concentrations of H2O2. ECL measurements were carried out in both the reporting cell (pH 7.4 PBS containing 1.0 mM Ru(bpy)32+ and 50 mM TPrA) and the sensing cell (pH 7.4 PBS containing 0, 0.1 nM, 0.5 nM H2O2). (B) ECL signals for the paperbased BPE with different modified cathodes: (a) bare BPE; (b) CS-MWCNTs/ BPE; (c) Ab1/CS-MWCNTs/BPE; (d) PSA/Ab1/CS-MWCNTs/BPE and (e) SiO2GOD-Ab2/PSA/Ab1/CS-MWCNTs/BPE. ECL measurements were carried out in both the reporting cell (pH 7.4 PBS containing 1.0 mM Ru(bpy)32+ and 50 mM TPrA) and the sensing cell (pH 7.4 PBS containing 10 mM glucose). Scheme 1 Preparation of the ECL biosensor for PSA assay based on a paper-based BPE: (A) fabrication procedure for the paper-based BPE; (B) preparation of nanobioprobes through coimmobilization of GOD and Ab2 onto silica nanoparticles. (C) Preparation procedure for the immunosensing surface.

Scheme 2

The sensing principle of paper-based BPEs.

exists, while xa and xc represent the length of the anode and cathode of the BPE, respectively. If DEelec is high enough, the oxidation–reduction reaction is triggered at both poles situated in the reporting cell and sensing cell. Then, Ru(bpy)32+/TPrA in the reporting cell is oxidized and an ECL signal appears. O2 or H2O2 in the sensing cell is reduced. The electron transfer processes occurring at the BPE are electronically coupled to the ECL reaction due to the connection between cathode and anode. Thus, the charge balance permits the ECL light output to be quantitatively correlated to electrochemical reductions at the cathode. This paper-based BPE system was used to detect H2O2 in the sensing cell via the Ru(bpy)32+ ECL generated from the anode in the reporting cell. As shown in Fig. 1A, with an increase of H2O2 concentration from 0 to 0.5 nM, the ECL intensity increased. It is easier for H2O2 to obtain electrons at the cathode than oxygen and so the corresponding current density will be enhanced. So the ECL signals strengthen. These results illustrated that H2O2, which is produced from the oxidase reaction can be

10950 | Chem. Commun., 2014, 50, 10949--10951

quantitatively detected. Thus, a PSA sensor was designed by assembling a GOD labelled immuno-sandwich structure on the cathode. The ECL performance during the step-by-step fabrication process was characterized and the corresponding ECL intensity curves are shown in Fig. 1B. Signal amplification was achieved by using MWCNTs to modify the cathode of BPE, thus accelerating electron transfer and by using silica nanoparticles as a carrier to label antibodies.12 As a result, the ECL intensity of the CS-MWCNTs modified BPE (Fig. 1B, curve b) was higher than that of bare BPE (Fig. 1B, curve a). After the immobilization of Ab1 (Fig. 1B, curve c) onto the CS-MWCNTs modified BPE, the ECL intensity decreased. Subsequently, the surface incubated with PSA (Fig. 1B, curve d) led to a further decrease in the ECL intensity. These phenomena can primarily be attributed to the formation of less conductive layers on the cathode of BPE. However, the ECL signal was improved considerably after the inclusion of SiO2-GOD-Ab2 onto the PSA/Ab1/CS-MWCNTs modified BPE in the presence of glucose (Fig. 1B, curve e). This increasing ECL effect was mainly due to the presence of H2O2, which was a product of the oxidation of glucose catalyzed by GOD in situ on the cathode of BPE. The surface of the modified cathode was also investigated by SEM. In contrast to the pure cellulose paper (Fig. S1, ESI†), the morphology of the CS-MWCNTs modified paper (Fig. 2B)

Fig. 2 SEM images of the CS-MWCNTs modified pure cellulose paper (A) and a magnified version (B), the Ab1/CS-MWCNTs modified pure cellulose paper (C) and the SiO2-GOD-Ab2/PSA/Ab1/CS-MWCNTs modified pure cellulose paper (D).

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Fig. 3 (A) Effect of BPE width on the ECL intensity. (B) The calibration curve for the driving voltage (Etot) assay from 3.0 to 3.5 V.

showed good dispersion, which was advantageous for improving the conductivity. Upon immobilization of Ab1 onto the surface of the CS-MWCNTs bioconjugate, obvious aggregation of the trapped biomolecules could be observed and the MWCNTs appeared larger in size, indicating the successful immobilization of Ab1 (Fig. 2C). After PSA and SiO2-GOD-Ab2 were assembled onto the surface of Ab1/CS-MWCNTs using sandwich immuno-reactions, the SEM image showed that MWCNTs became ‘‘fatter’’ and the surface was much rougher and richer. In addition, several particles with diameters of approximately 50 nm were observed (Fig. 2D), which indicated the successful binding of SiO2-GOD-Ab2 onto the surface of MWCNTs. The UV-vis absorbance spectra shown in Fig. S2 (ESI†) confirm that the antibodies and GOD were successfully assembled onto the silica nanospheres. The size of BPE and the driving voltage are two of the main factors that influence the ECL measurements.9c,10b Fig. 3A illustrates the influence of the BPE area on the ECL response. The ECL intensity increases with an increase of the BPE width from 2 mm to 4 mm. However, for the 4 mm width, the ECL signal was not stable due to excessive consumption of the electroactive species. Further increasing the width of BPE to 6 mm and 8 mm lead to a quick decrease in ECL intensity. Considering these results, the 2 mm width of BPE was chosen in the following investigations. As depicted in Fig. S3, ESI† and Fig. 3B, the ECL intensity increased with an increase in the driving voltage from 3.0 V to 3.3 V. However, when the driving voltage was higher than 3.3 V, the ECL signal decreased. It has been reported that a high driving voltage can initiate background reactions, such as the oxidation of water on the anode of BPEs, and this in turn leads to the formation of oxygen that both chemically and physically (via bubble formation on the BPE) interferes with ECL emission.10b In order to evaluate the sensitivity and potential application of the paper-based BPE, various concentrations of PSA were assayed. The ECL responses and calibration curves for different concentrations of PSA are shown in Fig. 4. With the increase in PSA concentration the ECL response increased, exhibiting a good linear relationship with logarithmic PSA concentration from 1.0 pg mL1 to 100 ng mL1 (correlation coefficient, 0.9909). The detection limit of PSA was estimated to be 1 pg mL1 based on a signal to noise ratio of 3. The stability of the immunosensor was evaluated over 30 consecutive cyclic potential scans by monitoring the ECL response of 100 ng mL1 PSA. There did not appear to be any obvious changes in the ECL intensity (Fig. S4, ESI†). Furthermore, the assay precision of the immunosensor was also evaluated using five different paper-based BPEs fabricated independently.

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Fig. 4 (A) ECL response of the paper-based BPE biosensor incubated with different concentrations of PSA (from bottom to top: 0 to 100 ng mL1). (B) The linear curve for the PSA assay. ECL measurements were carried out in the reporting cell (pH 7.4 PBS containing 1.0 mM Ru(bpy)32+ and 50 mM TPrA) and the sensing cell (pH 7.4 PBS containing 10 mM glucose). Voltage of the photomultiplier tube: 400 V; scan rate: 0.2 V s1. Etot = 3.3 V.

The relative standard deviation was 8.7% for 100 ng mL1 of PSA, indicating acceptable precision and fabrication reproducibility. In summary, this work is the first demonstration of the integration of a disposable paper-based platform with a bipolar electrochemical device for the determination of the cancer marker PSA with a wide linear range, low detection limit and acceptable accuracy. This paper-based BPE analytical device not only retains the simplicity, low cost, portability and disposability of a paperbased device, but also provides new opportunities and directions in bipolar analytical chemistry. The mass production, reproducibility and disposability of this paper-based bipolar sensing platform may open more opportunities for clinical diagnostics. We gratefully acknowledge the 973 Program (Grant 2012CB932600), the National Natural Science Foundation (Grants 21025522, 21327902), the National Natural Science Funds for Creative Research Groups (Grant 21121091) and the Science and technology support project (BE2013073) of Jiangsu Province, China.

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Chem. Commun., 2014, 50, 10949--10951 | 10951

Disposable paper-based bipolar electrode for sensitive electrochemiluminescence detection of a cancer biomarker.

A disposable paper-based bipolar electrode (BPE) was reported for the first time for the sensitive electrochemiluminescence detection of a prostate sp...
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