Visual Electrochemiluminescence Detection of Cancer Biomarkers on

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Visual Electrochemiluminescence Detection of Cancer Biomarkers on a Closed Bipolar Electrode Array Chip Mei-Sheng Wu, Zhen Liu, Hai-Wei Shi, Hong-Yuan Chen, and Jing-Juan Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac502989f • Publication Date (Web): 02 Dec 2014 Downloaded from http://pubs.acs.org on December 6, 2014

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Visual Electrochemiluminescence Detection of Cancer Biomarkers on a Closed Bipolar Electrode Array Chip Mei-Sheng Wu,†,‡ Zhen Liu, † Hai-Wei Shi, † Hong-Yuan Chen, † JingJuan Xu*,† †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China ‡ Department of Chemistry, College of Science, Nanjing Agricultural University, Nanjing 210095, China * To whom correspondence should be addressed: [email protected] (J.J. Xu) Tel: +86-25-83597294

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ABSTRACT: This paper describes a novel electrochemiluminescence (ECL) imaging platform for simultaneous detection of cancer biomarkers based on closed bipolar electrode (BPE) array. It consists of two separated channel arrays: detection channel array and sensing channel array, which are connected by a group of parallel ITO BPEs on glass substrate. Besides, two parallel ITO strips are fabricated at the two sides of BPE array and employed as driving electrodes. After Au film are electrochemically deposited on the cathodes of BPE array, nanobioprobes including biorecognition elements (aptamer or antibody) and a novel electrochemical tag which is synthesized by doping thionine in silica nanoparticles (Th@SiO2 NPs), are introduced into the cathodes by immunoreaction or DNA hybridization. The Th@SiO2 coupled nanobioprobes as both recognition probes and signal amplification indicators could mediate the ECL signals of Ru(bpy)32+/tripropylamine (TPA) on the anodes of BPE array through faradaic reaction due to the charge neutrality of BPE. Thus, multiplex detection of cancer biomarkers (adenosine tripho-sphate (ATP), prostate-specific antigen (PSA), α-fetoprotein (AFP) and thrombin.) is realized by forming specific sensing interface onto the cathodic poles of BPEs in different sensing channels and reported by the ECL images of Ru(bpy)32+/TPA system on the anodic poles of BPEs in detection channels. Results demonstrates that this visual ECL platform enables sensitive detection with excellent reproducibility, which may open a new door towards the development of simple, sensitive, cost-effective, and high throughput detection methods on biochips.

quantitative detection methods based on imaging the ECL signals at BPE array for bioanalysis. Thionine, as a good electron transfer mediator, has been widely used in electrochemical immunoassay because of its excellent electroactivity.13-15 Its reduction potential is about 0.064 V (versus NHE)16 which is much more negative than that of dissolved oxygen in water (O2 /H2O, 1.23 versus NHE at standard conditions). In the absence of thionine, oxygen is reduced at the cathode and Ru(bpy)32+/TPA are oxidized at the anode at the same time when ΔEelec (interfacial potential difference between the solution and BPE) exceeds the difference between the formal potentials of the redox processes occurring at the two ends of BPE. After the immersion of cathode of BPE in thionine solution, ΔEelec will be decreased efficiently. Therefore, it is expected that thionine could be used as electrochemical indicator to enhance the ECL signal at the anode through its reduction reaction at the cathode. In this paper, to achieve sensitive visual detection, large number of thionine molecules are successfully doped into silica nanoparticles (Th@SiO2 NPs) using water-in-oil (W/O) microemulsion method. Th@SiO2 NPs shows great promise in bioanalysis due to their improved stability, water solubility, low toxicity, and easy surface modification. They are then labeled with various recognition probes for simultaneous detection of cancer biomarkers through closed BPE-ECL imaging array. Au-ITO hybrid BPE was used to increase the electrode conductivity and facilitate the further immobilization of biomolecules similarly as our previous work.11 After that, Th@SiO2 NPs –recognition probes are patterned onto the cathode of BPE array surface to enhance the ECL signals. By transferring the electric signal to optical signal of Ru(bpy)32+ ECL, a tiny change of sample level can be distinguished by observing the brightness of the ECL. With this design, we have achieved the rapid detection of adenosine tripho-sphate (ATP), total prostate-specic antigen (PSA), α-fetoprotein (AFP), and thrombin. It can detect 8 samples simultaneously without the use of multi-channel electrochemical workstation. The proposed potable ECL-BPE array design shows great promise for array sensor development.

INTRODUCTION Electrochemiluminescence (ECL) approach based on bipolar electrode (BPE), as a useful tool, has recently attracted considerable attention in bioanalysis.1-6 BPE is a conductor which is immersed into a solution (open BPE) or two separated solutions (closed BPE). 7 An external voltage (∆Etot) is applied across the solution in order to induce the faradaic reactions at the anode and cathode of BPE, respectively. Therefore, there is no direct electrical connection between it and external power which facilitates the fabrication of portable device. Besides, the attractive properties of BPE-ECL platform, such as that it doesn’t require a light source and is free from the effects of scattered light in samples compared with fluorescence, are ideally suited for miniaturized multiplexed detection through simultaneous imaging the ECL signals at BPE array. However, signal collection in most BPE-ECL bioanalysis system is using a photomultiplier tube (PMT) because of its high sensitivity. In order to achieve visual detection, specific strategies or signal amplification approaches should be developed. Recently, we reported a BPE-ECL strategy for visual analysis of prostate-specific antigen (PSA) on the basis of electrical switch control of ECL generation on BPEs. The target-guided electrical switch make two neighbouring BPEs behave like a continuous H-shaped BPE, which increased the length of BPE and decreased the driving voltage greatly.8 Moreover, a number of attempts have been made to improve the sensitivity, mainly including anode and cathode amplification technologies. The former amplification approach was mainly concerned with encapsulating large number of Ru(bpy)32+ molecules into silica nanoparticle and modified at the anode surface to improve the sensitivity.9 In comparison with anode amplification strategy, faradaic reactions at the cathode of BPE modulated ECL emission is much more facile and versatile. For example, noble nanoparticles have been introduced into the cathode of BPE to catalyze the reduction reactions of dissolved oxygen1,10,11 by conjugating them at the termini of recognition probes or electrochemically depositing approach. Some electroactive species (K3Fe(CN)35, ferrocene,12 benzyl viologen4) have also been introduced into the cell or modified at the cathode surface to decrease the external voltage for driving the faradaic reactions occurred at the two ends of BPE. Some of them have been used to visually monitor the presence of glycated hemoglobin5 and DNA1, but no quantitative data were provided. Thus there is a broad space to develop visual

EXPERIMENTAL SECTION Materials. N-(3-dimethylaminopropyl)-N’-ethyl-carbodiimide hydrochloride (EDC), Ru(bpy)32+, tripropylamine (TPA), thionine acetate, (3-aminopropyl)triethoxysilane (APTES) and

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thrombin were obtained from Sigma-Aldrich (USA). Adenosine tripho-sphate (ATP) was purchased from Sangon Biotech (Shanghai) Co., LTD. L-Cysteine was purchased from Sinopharm chemical reagent company (Shanghai, China). Total prostate-specific antigen (PSA), α-fetoprotein (AFP), mouse monoclonal capture and signal PSA antibodies and AFP antibodies were purchased from Linc-Bio Science (China). ECL detection solution was 0.1 M phosphate buffered saline (PBS, pH 7.4) containing 10 mM Ru(bpy)32+ and 50 mM TPA. All solutions were prepared using Millipore (model milli-Q) purified water and stored at 4 ºC in a refrigerator. All the other chemicals were of analytical grade. All of the DNA probes were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China) and the sequences are shown in Supporting Information. Indium tin oxide (ITO) -coated (thickness, ~100 nm; resistance, ~10 Ω/square) aluminosilicate glass slides were purchased from CSG (Shenzhen, China). Sylgard 184 (including PDMS monomer and curing agent) was purchased from Dow Corning (Midland, MI, USA). SG-2506 borosilicate glass (with 145 nm thick chrome film and 570 nm thick positive S-1805 type photoresist) was from Changsha Shaoguang Chrome Blank Co. Ltd. Instruments. ECL images were captured by Olympus DP71 cooled CCD camera and analyzed by the Image-Pro Plus (IPP) 6.0 software on the basis of integrated optical density. ECL signals were measured with MPI-E electrochemiluminescence analyzer (Xi’An Remax Electronic Science &Technology Co. Ltd., Xi’An, China, 350 nm~650 nm). The UV − vis absorption spectra were obtained on a Shimadzu UV-3600 UV − vis − NIR photo − spectrometer (Shimadzu Co.) and NanoDrop 2000c UV-Vis spectrophotometer. The X-ray photoelectron spectroscopy (XPS) data were obtained using a commercial XPS system (PHI 5000 Versa Probe) equipped with a hemispherical electron analyser and monochromatic Al Kα X-ray excitation source. Preparation of Th@SiO2 and Th@SiO2-recognition probe (Th@SiO2-aptamer, Th@SiO2-Ab2) complex. 1.77 mL of Triton X-100, 7.5 mL of cyclohexane, 1.8 mL of 1-hexanol, and 340 µL of thionine acetate (50 mM) were mixed with constant magnetic stirring to form the water-in-oil microemulsion. After addition of 100 µL of tetraethyl orthosilicate (TEOS) and 60 µL of NH3•H2O, the hydrolysis reaction was allowed to continue for 24 h. Acetone was then added to destroy the emulsion, followed by centrifuging and washing with ethanol and water. The obtained nanoparticles were treated with 10% APTES alcoholic solution (2 mL) at 30 ºC for 2 h and then rinsed with ethanol to remove loosely bound APTES. Th@SiO2 NPs were obtained after being collected by centrifugation and washed several times with PBS. The amino functionalized Th@SiO2 NPs were dispersed in 2 mL PBS. 50 µL of 25% (v/v) glutaraldehyde (GA) was added into amino functionalized Th@SiO2 NPs (0.5 mL) in PBS for incubating 3 h at room temperature. After careful washing with 0.1 M PBS (containing 0.1 M NaCl), the GA modified Th@SiO2 NPs were employed for incubating with ATP aptamer (100 µM, 50 µL), thrombin aptamer (100 µM, 50 µL), mouse monoclonal signal PSA antibody (2 mg/mL, 25µL), and AFP antibody (2 mg/mL, 25µL) at 4 ºC for 12 h, respectively. The precipitates were centrifuged and washed with 0.1 M PBS (containing 0.1 M NaCl). The resulting Th@SiO2 NPs-

recognition probes were dispersed in 0.1 M PBS (pH 7.4, 0.1 M NaCl, 5 mL) and stored at 4 ºC. Microfluidic Chip Design and Fabrication. The configuration of the microfluidic chip was illustrated in Scheme 1. The PDMS slice was composed of one main channel connected with eight fingers as reporting channels (1.3 cm length, 1.0 mm width, 45 µm depth) and eight parallel sensing channels (1.3 cm length, 1.0 mm width, 45 µm depth). 3 mm diameter holes were punched at each end of the channel to serve as reservoirs. The separated sensing channel and reporting channel were connected by ITO BPEs (1.8 cm length, 0.9 mm width) on glass substrate in order to provide spatial resolution analysis of multiplex analytes simultaneously. Besides, a pair of ITO strips (5.2 cm length, 12 mm width, 2.6 cm gap) were patterned at the two sides of BPE array which were served as driving electrodes. The PDMS chip and glass chip were fabricated by a previously reported procedure.17 In brief, the glass plate was exposed to UV radiation under a mask with designed micropatterns, followed by developing with 0.5% NaOH solution, the Cr layer was then removed by a 0.2 M ammonium cerium (IV) nitrate solution. Etching of this glass chip was carried out in a water bath with 1 M HF/NH4F solution for 30 min. After that, glass chip with corresponding patterns was obtained. PDMS base and curing agent were mixed thoroughly (10:1), degassed and then poured on this patterned glass, after 30 min in 80 °C, the patterned PDMS was done.

Scheme 1 The schematic diagram (A) and the photograph (B) of multiplexed ECL imaging-based screening platform for biosensing applications.

The PDMS mold predesigned with BPE pattern was attached to the ITO-coated glass slice. The channels were filled with ink and allowed to dry in thermostat at 30 ºC for 30 min. Then, the PDMS mold was peeled off and the exposed ITO surface was etched with an aqueous acid solution (FeCl3: HNO3: HCl = 0.5 M: 1 M: 1 M). The remaining ink was removed with ethanol, forming both rectangle substrate electrodes and parallel electrode array. Another PDMS mold containing detection channel array and sample sensing channel array was fabricated with the same technique and then covered on ITO BPE chip. Fabrication of Sensing Interfaces. Au film was deposited on the cathode of BPE according to our previous report with slight modification.7 1 % HAuCl4 was introduced into all of

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the detection channel array and sensing channel array. Then a voltage of 2.5 V was applied on the driving electrodes for 180 s using CHI 660C electrochemical workstation. The color of the cathode changed from blue (ITO) to yellow (Au) due to the formation of Au film. The as-prepared Au-ITO hybrid BPE was then washed with ultrapure water. After that, 0.175 M LCysteine was added into the sensing channel array and incubated for approximately 10 h allowing the formation of carboxyl group on Au surface. After rinsing with PBS for twice, a mixture solution containing 20 mg/mL EDC and 10 mg/mL NHS was added to this channel and kept for 1 h. After being rinsed with 0.1 M PBS (containing 0.1 M NaCl), capture probes (0.1 µM ATP capture DNA, 0.1 µM thrombin capture DNA, 2 µg/mL capture PSA antibody, and 2 µg/mL capture AFP antibody) were added into different sensing channels, respectively. Then the microfluidic chip was kept overnight followed by blocking with tris-HCl (pH 7.4) and thoroughly rinsed with 0.1 M PBS (containing 0.1 M NaCl). ECL Visual Detection of Multiplex Biomarkers. The principle for multiplex detection was illustrated in Scheme 2. Sample solutions with different concentration and Th@SiO2recognition probes were introduced into sensing channel array and incubated at 37 °C for 1 h, respectively. The sensing channels were then washed twice with PBS. The detection channels were filled with 0.1 M PBS buffer solution (pH 7.4) containing 10 mM Ru(bpy)32+ and 50 mM TPA. A constant potential was applied on the driving electrodes, and the ECL imaging were acquired with CCD camera at an exposure time of 10 s. For investigating all of the ECL images at each BPE simultaneously, the distance between the microfluidic chip and objective lens was fixed at 16.2 cm. For further study the ECL enhancing mechanism, the ECL-potential experiments were performed using MPI-E electrochemical and electrochemiluminescence analyzer by applying a linearly increasing voltage (from 0 V to 6.0 V) at the two ends of microchannel with the scan rate of 0.1 V/s. Extraction of adenosine from cancer cells. K562 leukemia cells were cultured in RPMI 1640 medium (GIBCO) supplemented with fetal bovine serum (10%) (FBS, GIBCO), penicillin (100 µg/mL) and streptomycin (100 µg/mL) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were collected by centrifugation at 1000 rpm for 5 min and then washed twice with sterile PBS. The sediment was resuspended in PBS to obtain a homogeneous cell suspension. The cells were disrupted by sonication for 20 min at 0 °C. After that, the lysate was centrifuged at 15,000 rpm for 20 min and the obtained suspension was diluted step by step.

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resistance (R) is approximately equal to Rs. Therefore, the potential difference obtained at the two ends of BPE/solution interface (∆Eelec) is mainly governed by the electric field across the channel and the length of BPE (lBPE). While in a “closed” BPE device, the driving electrodes are immersed into two separated solutions and the only current path in the latter device is through BPE (eq.2). 18 Since the total resistance of a parallel circuit (R) is smaller than any of the resistor in the parallel network, the fraction of voltage dropped at the BPE/solution interface in closed BPE system is larger than that in open BPE system.

Etot = iRchannel + iR + iRchannel + Esys Etot = iRchannel + iRBPE + iRchannel + Esys

1 1 1 （ = + ） R Rs RBPE

(1) (2)

In order to illustrate the difference between open BPE electrochemistry and closed BPE electrochemistry, ITO substrate (Scheme 1) was embedded in PDMS slice with one microchannel (Figure 1B-a) and two separated microchannels (Figure 1B, b, c and d). From the ECL images recorded in Figure 1B, a bright and weak spots (a) could be seen at the anodes of driving electrode and BPE in the open BPE strategy. In closed BPE approach, two obvious ECL spots (b) can also be seen when the sensing channel and the reporting channel were filled with Ru(bpy)32+/TPA (Figure 1B-b). If the sensing channel in the closed BPE mode was filled with PBS (Figure 1B-c), only one ECL signal can be seen at the anode of BPE (Figure 1B-c). It should be noted that the ECL signal obtained at the anode of BPE in the closed BPE is approximately 9(Figure 1B-c) to 15-fold (Figure 1B-b) of brighter than that in the open BPE (Figure 1B-a), confirming the enhanced potential difference between solution/BPE interface in the closed BPE. Besides, this design enables the selective fabrication of biosensing interface at two ends of BPE and avoids the chemical interference between sensing reactions and reporting reactions. Thus, the excellent spatial resolution and specific surface functionalization of closed BPE-ECL approach made it suitable for constructing multiplex imaging platform with high sensitivity.

RESULTS AND DISCUSSION Characterization of Open and Closed BPE Based Detection Mode Although BPE-ECL has attracted more attention for bioanalysis, there are only a few examples of closed BPE-ECL. Figure 1A is a schematic diagram and the analogous electrical circuit of open bipolar cell (a) and closed bipolar cell (b). In an “open” BPE platform, the driving electrodes are immersed into the same solution and the current can flow through both the solution and the BPE (eq.1). Here, Esys is related to the charge transfer resistance at driving electrode/solution interface and the system resistance.8 If the resistance of solution is much lower than BPE (Rs