Visual Color-Switch Electrochemiluminescence Biosensing of Cancer

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Visual Color-Switch Electrochemiluminescence Biosensing of Cancer Cell Based on Multichannel Bipolar Electrode Chip Huai-Rong Zhang, Yinzhu Wang, Wei Zhao, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04716 • Publication Date (Web): 02 Feb 2016 Downloaded from http://pubs.acs.org on February 4, 2016

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Analytical Chemistry

Visual Color-Switch Electrochemiluminescence Biosensing of Cancer Cell Based on Multichannel Bipolar Electrode Chip Huai-Rong Zhang1,2†, Yin-Zhu Wang1†, Wei Zhao1*, Jing-Juan Xu1*, Hong-Yuan Chen1

1：State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China

2：Shandong Province Key Laboratory of Detection Technology for Tumor Makers, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, China.

* Corresponding author. Tel/Fax: +86-25-89687294; E-mail address: [email protected] (W. Zhao) [email protected] (J.J. Xu)

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ABSTRACT:

In this work, we developed a visual electrochemiluminescence (ECL) sensing platform based on a dual-bipolar electrode (D-BPE) array chip. The chip was composed of two arrays of BPEs and three separated arrays of reservoirs filled with buffer, Ru(bpy)32+-TPrA and luminol solutions, respectively. Both BPEs served as ECL reporting platforms. By applying 6.0 V voltage, an array of orange electrochemiluminescence (ECL) signals belong to Ru(bpy)32+ turned on. After adding DNAzyme and H2O2 in Ru(bpy)32+ and luminol reservoirs, the orange Ru(bpy)32+ signals decreased till vanished due to the quenching effect, meanwhile, a new array of blue ECL signals turned on because of the luminol-H2O2 ECL reaction. The designed D-BPE owns superior properties compared with three-electrode system benefited from the quantitative relation of bipolar systems, which greatly enhanced the ECL detection sensitivity. Meanwhile, the visual color-switch ECL and the ratiometric detecting principle made the results easier to evaluate and more accurate. Quantitative detection of HL-60 cancer cells from 320 cells/mL to 2.5×105 cells/mL with two linear ranges was achieved. The detection limit was down to 80 cells/mL. Finally, this D-BPE chip could distinguish the tumor cells from normal cells and provide a prospect for cancer diagnosis in a high-throughput, visual way. KEYWORDS: Electrochemiluminescence, Dual-Bipolar electrode, Ru(bpy)32+, Luminol, Extracellular H2O2, DNAzyme, Cancer cells. INTRODUCTION Bipolar electrode (BPE) serves as both anode and cathode in the electrolyte without direct electrical connection via a power supply1~3. Compared with two-electrode system, the reaction


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mechanism at the pole of BPE is more complicated since it is not only controlled by the electric field, but also influenced from the reaction at the other pole through BPE4~10. Since it is not easy to directly measure the current through BPEs, electrochemiluminescence (ECL) based BPEs has been developed by transforming electrical signal to optical signal10~13. As firstly reported by Manz’s group in 2001, a microfluidic system was configured to detect the presence of Ru(bpy)32+ and related light-emitting compounds at a bipolar electrode via ECL emission12. In their work, analytes participating in the ECL reaction at the anode were measured. And they indicated the current was expected to be limited by the limited availability of reducible substances. It was ascribed to the electroneutrality across the BPE which made the reactions on each pole of BPE electrically coupled. Utilizing this principle, Crooks group reported a dual sensing/reporting platform13. The electrochemical sensing reaction didn’t participate directly in the ECL process, but electrically coupled with ECL reactions through the BPE. The system was used to electrochemically detect benzyl viologen in solution and report its presence via Ru(bpy)32+ luminescence. Wang’s group found the changing of conductivity of sensing solutions could also be measured using BPE-ECL system, which extended its application to the detection of non-electroactive materials14. Recently, we developed a BPE-ECL biosensor for the measurement of cancer cell surface protein using ferrocence (Fc) labeled aptamer as the signal recognition and amplification probe15. Compared with three-electrode system, the quenching effect of Fc labeled aptamer to Ru(bpy)32+-TPrA on BPE was much more strengthened, which should be attributed to the electron-transfer mechanism of BPE. Limited by the cathode reaction, the oxidation of Fc on anode competed with that of Ru(bpy)32+, which amplified the quenching effect thus enhanced the detecting sensitivity.



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Since there is no need for a direct external connection to the BPE, it is easy to make miniature arrays of electrode by a single DC power supply. Crooks group reported a BPE array composed of 1000 (10×100) individual bipolar electrodes with just two driving electrodes16. The faradaic processes occurring at the cathode end of each electrode were correlated to ECL emission at the anode end, which made it possible to read out the state of each electrode simultaneously. Our group published a work for detection of cancer biomarkers based on a BPE array with 8 parallel ITO BPE. Various concentrations of target proteins incubated on the cathodic poles of BPEs could be simultaneously detected by ECL signals on the anodic poles17. Recently, Wang’s group reported a multichannel closed bipolar system with double-bipolar electrodes (D-BPE) which were series connected through the reporting reservoir containing Ru(bpy)32+-TPrA18. Both oxidants and reductants could be detected through the two BPEs in anodic reservoir and cathodic reservoir, respectively. In brief, the BPE array enables high throughput detection via ECL reporting. However, to the best of our knowledge, all BPE miniature arrays chosen one ECL reaction for reporting. If two or more ECL reporting reservoirs were introduced, the mechanism should be more complicated, but worthy to investigate. Here, we developed a D-BPE based multichannel device with two arrays of ECL reporting reservoirs with Ru(bpy)32+ and luminol, respectively. The ECL reactions happened on both BPEs. Driving by 6.0 V voltage, we observed orange ECL signals belonging to Ru(bpy)32+ at the anode of one BPEs array. By adding H2O2 and DNAzyme into the reservoirs, the orange ECL signal of Ru(bpy)32+ turned darker, conversely, a blue ECL signal belonging to luminol turned brighter at the anode of the other BPEs array. Compared with traditional three-electrode system, the ECL intensity of luminol-H2O2 was much higher because of electron transfer facilitation through one BPE to the other. And the quenching effect of H2O2 to Ru(bpy)32+ was also enhanced due to the


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competition reaction at the anode of BPEs. The mechanism was studied in detail. Since H2O2 could be produced by stimulating HL-60 cancer cells using PMA and DNAzyme could be formed from extended primer DNA by telomerase which could also be extracted from HL-60 cancer cells, based on this principle, HL-60 cancer cells were quantitatively detected and easily distinguished from normal cells. It was fascinating to observe the color changing during the detection process and the possibility of false positive result was limited due to the ratiometric assay. EXPERIMENTAL SECTION Materials 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS), phenylmethylsulfonyl fluoride (PMSF), ethylene glycol bis (-aminoethyl ether)-N, N, N, N tetraacetic acid (EGTA), glycerol, and Tween 20 were purchased from Biosharp Biotechnology. Hemin was purchased from Aladdin (Shanghai, China) and used without further purification. 2 mM Hemin solution was prepared in DMSO and stored in the dark at -20 ˚C. Sylgard 184 (including poly (dimethylsiloxane) (PDMS) monomer and curing agent) was from Dow Corning (Midland, MI). Oligonucleotides of telomerase primer, G-quadruplexes DNA, FITC-annexin V and Propidium iodide were purchased from Sangon Biotech Co. Ltd (Shanghai, China), and the DNA sequences were listed in Table 1. Deoxynucleotide mixture solution (dNTPs), Phorbol 12myristate 13-acetate (PMA, 99%), tri (2-carboxyethyl) phosphine hydrochloride (TCEP) and luminol, Ru(bpy)32+, tripropylamine(TPrA) were purchased from Sigma-Aldrich (St. Louis, MO). PBS (0.1 M, pH 7.4) buffer containing K2HPO4 and KH2PO4 was used to wash cancer cells. PBS buffer (0.1 M, pH 7.4) containing 0.1 M NaCl and 5 mM MgCl2 was employed for preparation of DNA stock solutions. All the chemicals were used as received without further purification. All other reagents were of analytical grade and used as received. All the water used in the work was



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RNAase-free. Magnetic beads were purchased from Xi'an GoldMag Nanobiotech Co., Ltd. ITOcoated (thickness ~100 nm, resistance ~10 Ω/square) aluminosilicate glass slides were purchased from CSG (Shenzhen, China). Table 1. The DNA Sequences used in this work: Name

Sequences (5’ to 3’)

Telomerase primer

5’-NH-(CH2)6-TTT TTT AAT CCG TCG AGC AGA GTT3’

G-quadruplexes DNA

5’-CTA ACC CTA ACC CTA ACC TTT TTT TTG GGT AGG GCG GGT TGG GTT TTT TTT TT-3’

Instruments. An Olympus DP71 cooled CCD camera was applied for chip imaging, results of which were analyzed by Image-Pro Plus (IPP) 6.0 software. D-BPE ITO electrodes were used as sensing platform. The apoptosis assay for HL-60 cells was performed using a FACS Calibur flow cytometer (BD Science, USA). The electrochemical and ECL emission measurements were conducted on a MPI-E electrochemiluminescence analyzer (Remax Electronic Instrument Limited Co., Xi’an, China). The spectral width of the photomultiplier tube (PMT) was 350-650 nm, and the voltage of the PMT was set at−400 V in the process of detection. Microfluidic Chip Design and Fabrication. A piece of ITO was first cut into small slices (6 cm × 6.5 cm) and those ITO slices were fabricated on a silk-screen. For screen-printing, oil ink (essential ingredient of acrylic resin) was transmitted through the silk-screen mode onto the ITO layer by brush. Once the planar electrodes were completed, a wet chemical etching procedure was carried out with chemical solution (HF: NH4F: HNO3=1: 0.5: 0.5), which produced the desired ITO electrodes. Those ITO electrode slices were cleaned by immersing in a boiling


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solution of 2 M KOH with 2-propanol for 20 min, followed by washing with water. To obtain the PDMS molds with one or more predesigned channels, SG-2506 borosilicate glass plates were fabricated by traditional photolithography and wet chemical etching techniques. They were exposed to UV radiation under a mask with the designed patterns, followed by developing with a 0.5% NaOH solution, the Cr layer was then removed by a 0.2 M ammonium cerium (IV) nitrate solution. Etching of these glass plates was carried out in 1 M HF–NH4F solution (40 ˚C) with a water bath for 28 min. After that, designed channel structure molds were obtained. Then degassed PDMS was casted on these glass masks for 1 h in 80 ˚C. After cooling at room temperature, PDMS was stripped from the masks, producing the PDMS molds. The PDMS molds were drilled with rectangular holes as reservoirs and attached to the ITO-coated glass slice. The BPE array was finally with two 15 mm × 60 mm driving electrodes and twelve 10 mm × 4 mm BPEs. Electrodeposition of Au. The deposition conditions were optimized in order to obtain a stable Au layer on the cathode of BPE. The applied driving voltage was from 4.0 V to 6.0 V and the best deposition voltage as 5.0 V was chosen. The deposition concentration and time were also optimized under this voltage using the same method. Finally, the reservoirs were filled with 30 µL HAuCl4 (2.4 mM), then a driving voltage of 5.0 V was applied for 300 s and the electrode was rinsed with water and dried in the air at last. Preparation of Telomerase Primer Modified Magnetic Beads. 100 µL Magnetic Beads (MB) were treated with 20 mg/mL EDC and 20 mg/mL NHS solution for 2 h at 37 °C, then 1×10-5 M (200 µL) telomerase primer was added for 1 h at 37 °C. The obtained DNA-MB bioconjugates were rinsed with 20 µL of 0.1 M PBS buffer to remove the unspecified telomerase primer. Finally, the DNA-MB bioconjugates were dispersed in PBS solution and stored at 4 °C.



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Cell Apoptosis Assay. HL-60 cancer cells collected in the exponential phase of growth were cultured in DMEM medium supplemented with 10% fetal calf serum, and maintained at 37 °C in a humidified atmosphere (95% air and 5% CO2). 1 mL 1×107 cells/ mL HL 60 cells was equally divided into two groups, one group was cultured with the fresh culture medium, the other was cultured with the fresh culture medium containing 0.6 ng/mL PMA, respectively, and then maintained at 37 ℃ in a humidified atmosphere (95% air and 5% CO2) for 24 h. According to the manufacturer’s instruction, these cells were washed by PBS solution for the cytotoxicity and apoptosis assay19. Cells were incubated with 10 µL FITC-annexin V and 10 µL propidium iodide (PI) for 30 min at 37 ℃ in order to stain. Finally, we analyzed cells using multi-function meter reading and flow cytometry analyzer. Hybridization of the DNAzyme-MB Label and Extraction of H2O2. HL-60 cancer cells were collected in the exponential phase of growth and washed twice with ice-cold sterile PBS, then 1×107 HL-60 cells/mL were re-suspended in 1 mL of ice-cold CHAPS lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM PMSF, 5 mM mercaptoethanol, 0.5% CHAPS, 10% glycerol). The lysate was incubated for 30 min on ice and centrifuged for 15 min at 16000 rpm at 4 °C to pellet insoluble material. Then the cleaned lysate was carefully transferred into a 1.5 mL EP tube. The lysate was used immediately for telomerase assay or frozen at -80 °C. 1 mL telomerase reaction solution which contained 100 µL telomerase extracts from different number of HL-60 cells, and 200 µL dNTPs, 700 µL telomerase reaction buffer (20 mM Tris-HCl buffer, pH 8.3, 1.5 mM MgCl2, 0.63 mM KCl, 0.05% Tween 20, 1 mM EGTA) was added into the DNA-MB bioconjugates solution, respectively, at 37 °C for 1 h. Then the solution of bioconjugates was incubated in a buffer solution, pH 7.4, composed of 25 mM


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HEPES, 20 mM KCl, 200 mM NaCl, 0.05% Triton X-100 and 1% DMSO including 0.2 mM hemin for 1 h to form G-4 DNAzyme-MB labels. HL-60 cancer cells were centrifuged and washed with 1 mL ice-cold PBS and treated with 0.6 ng/mL PMA and then centrifuged. The supernate contained H2O2 was obtained for the further use. ECL measurement at D-BPE. 0.1M PBS, 0.1M PBS with 2.5 mM Ru(bpy)32+ and 25 mM TPrA, and 0.1M PBS with 0.1M luminol were added in the buffer reservoirs, Ru(bpy)32+ reservoirs and luminol reservoirs of the D-BPE chip, respectively. 6.0 V voltage was applied on the D-BPE electrode. For mechanism study, standard H2O2 and DNAzyme solutions were added in the Ru(bpy)32+ reservoirs and luminol reservoirs. For the detection of HL-60 cells, H2O2 and DNAzyme were extracted from the cells. Quantitative calculation was based on the ECL emission intensity recorded by electrochemiluminescence analyzer. All the visual ECL signals were recorded by Olympus DP71 cooled CCD camera. ECL measurement at three-electrode system. ECL detection experiments were performed with a conventional three-electrode system containing a platinum wire as counter electrode, an Ag/AgCl as a reference electrode and a bare ITO electrode as working electrode. All the concentrations of chemicals used were the same as those in D-BPE chip.

RESULTS AND DISSCUSSION Detection principle. The structure of the designed multichannel D-BPE chip is shown in Scheme 1. The chip is composed of two driving electrodes, three separated arrays of reservoirs which are connected by two arrays of ITO BPEs indicated as BPE1 and BPE2. Each array includes 6 parallel BPEs. Au NPs were electrodeposited onto the D-BPE cathodes before



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detection because of the following reasons. The electrodeposition of Au NPs made the cathode of BPE show better electrical conductivity and lower overpotential for oxygen reduction compared with traditional ITO material. The oxygen was reduced at the cathode at same rate with ECL reaction at the anode. Therefore, the introduction of Au NPs to the cathode of BPE improved the reduction of oxygen, hence the ECL response enhanced correspondingly.15 In addition, without covering Au NPs, SnO2 at ITO was easily reduced and the cathode turned brown under the driving voltage of 6.0 V. The electrodeposition of Au NPs eliminated the selfreduction problem of ITO. Since the structure of each row of D-BPE is exactly the same, we take one of them for example to demonstrate the principle. The operation procedures of D-BPE-ECL are schematically depicted in Scheme 2, and the corresponding ECL image and intensities vs. potential are shown in Figure 1. At beginning, the three reservoirs were added with PBS buffer solution, 0.1 M luminol and 2.5 mM Ru(bpy)32+/ 25 mM TPrA, from left to right. Driving by 6.0 V, we observed orange light band on BPE2, which was attributed to the ECL reaction of Ru(bpy)32+-TPrA at the anode of BPE2. The ECL peak intensity was ca. 6000 a.u. (Figure 1, A2), and the consistency of the array with 6 D-BPEs was perfect (Figure S1). On the other hand, without coreactant, the ECL reaction of luminol was too weak to see the light band. Adding 0.02 M H2O2 in luminol reservoirs, dual color light bands were observed on both BPE1 and BPE2. The blue band was ascribed to ECL reaction of luminol-H2O2, which generated ECL peak intensity up to 5500 a.u. (Figure 1, B1). Interestingly, the orange light band turned brighter, and the ECL peak intensities increased more than 2 times to 14000 a.u. (Figure 1, B2). This phenomenon could be explained by the quantitative relation of bipolar systems: (i) the molar quantities of anodic and cathodic reactions in a reservoir should be equal; (ii) the amount of reactions occurred at both poles of a


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BPE should be in strict accordance. Therefore, the luminol-H2O2 reaction on the anode of BPE1 increased the cathode reaction of oxygen reduction on BPE2; to maintain the electroneutrality of BPE2, the reaction of Ru(bpy)32+-TPrA on the anode of BPE2 enhanced. When 0.02 M H2O2 was added in the right reservoir, the intensity of orange light band of Ru(bpy)32+-TPrA decreased intensively to ca. 2200 a.u. (Figure 1, C2). It can be explained from two aspects: firstly, since H2O2 has a lower oxidation potential (ca. 0.92 V vs Ag/AgCl) than Ru(bpy)32+ (ca. 1.2 V vs Ag/AgCl), limited by the cathode reaction, the reaction of H2O2 decomposing to O2 competed with Ru(bpy)32+-TPrA reaction at the anode, which inhibited its ECL intensity; secondly, both H2O2 and O2 could react with free radicals of TPrA and then quench the Ru(bpy)32+ signal. Therefore, we found the ECL peak intensity of Ru(bpy)32+ decreased. At the same time, the blue band of luminol became brighter, which could be attributed to the increasing electron transfer influenced by the anode reaction from H2O2 oxidation in Ru(bpy)32+ reservoirs. At last, with 0.02 M H2O2 and 0.2 µM hemin-G-quadruplex deoxyribozymes (G-4 DNAzyme) in both luminol and Ru(bpy)32+ reservoirs, the blue bands turned much brighter and the orange bands vanished. This phenomenon is attributed to the peroxidase-like catalytic activity of G-4 DNAzyme20. In luminol reservoirs, it effectively catalyzed the ECL reaction of luminol-H2O2, which greatly enhanced the ECL signal, and the corresponding ECL intensity increased from ca. 9000 a.u. to 17000 a.u. (Figure 1, C1, D1). In Ru(bpy)32+ reservoirs, the catalysis of G-4 DNAzyme to H2O2 produced more oxidation species which reacted with free radicals of TPrA and furtherly quenched the ECL light emission of Ru(bpy)32+. The ECL intensity decreased down to 1000 a.u. (Figure 1, D2) and can’t be visually observed. It is impressed to see the color switch through direct observation during the detection.



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To understand more about the sensing principle of D-BPE, we considered each BPE individually and compared the experiment results with traditional three-electrode system. As shown in Figure 2A, in three-electrode system with luminol solution, during a potential sweep from 0.0-0.6 V, the ECL peak intensity of luminol-H2O2 was around 5500 a. u. When 0.2 µM G4 DNAzyme was added, this number increased to 12000 a. u. The electrical signal followed the optical signal. In D-BPE system, when the Ru(bpy)32+ reservoirs contained 2.5 mM Ru(bpy)32+ and 25 mM TPrA, the ECL current of luminol were close to those obtained in three-electrode system. However, when 0.2 µM G-4 DNAzyme and 0.02 M H2O2 were added in Ru(bpy)32+ reservoirs, ECL peak intensity of luminol-H2O2 increased to 17000 a.u (Figure 2B). This signal enhancement could be ascribed to the quantitative relation of bipolar systems, which means the addition of G-4 DNAzyme and H2O2 in Ru(bpy)32+ reservoirs facilitated the electron transfer rate of BPE2, thus promoted the luminol-H2O2 reaction at the anode of BPE1. Quenching effect of H2O2 to Ru(bpy)32+-TPrA was also studied in three-electrode system. By adding H2O2 and G-4 DNAzyme in Ru(bpy)32+-TPrA solution, the ECL intensities decreased 36% and 63%, respectively (Figure 2C). However, in D-BPE system, those numbers were much larger as 69% and 84%(Figure 2D). It can also be explained by the electron transfer mechanism of the BPE, which means the reaction at the anode of BPE is limited by the availability of reducible substances (O2). Therefore, H2O2 with lower oxidation potential (ca. 0.92 V vs Ag/AgCl) than Ru(bpy)32+ (ca. 1.2 V vs Ag/AgCl) would be oxidized at the anode of BPE2 and compete with Ru(bpy)32+-TPrA reaction, which inhibited its ECL intensity. By adding G-4 DNAzyme and H2O2 in the luminol reservoirs, the ECL intensity of Ru(bpy)32+ increased a little (Figure 2D, curve f), which was attributed to the increasing electron transfer on BPE1.


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Compared with traditional three-electrode system, the D-BPE sensing platform exhibited higher sensitivities on both BPEs. Optimization of driving voltage and concentration of ECL reagents. Before cancer cells detection, the experimental parameters were optimized for better ECL performance. Figure S2 illustrates the influence of the driving voltage on the ECL response. The ECL intensity increased with the driving voltage increasing from 4.0 V to 6.5 V. When the driving voltage was higher than 6.0 V, the transparent dual-bipolar electrode cathodes turned brown after the experiment, indicating that the reduction of SnO2 occurred at the D-BPE cathodes. Therefore, 6.0 V was selected as the driving voltage for all the experiments. The concentration effects were also investigated. For luminol concentration study, with 0.02 M H2O2 in luminol reservoirs and 2.5 mM Ru(bpy)32+ and 25 mM TPrA in Ru(bpy)32+ reservoirs, increasing luminol concentration from 0.01 M to 0.1 M, the blue ECL emission intensity enhanced accordingly. Furtherly increasing the luminol concentration caused a slight decreaseof ECL intensity due to the self-quenching effect21 (Figure 3A). Therefore, 0.1 M luminol was chosen as the optimal concentration. The concentrations of Ru(bpy)32+ and TPrA were also optimized, and finally 2.5 mM Ru(bpy)32+ and 25 mM TPrA were used throughout the experiments in this work (Figure 3B, 3C). Determination of HL-60 Cancer Cell. It is well known that telomerase shows no activity in normal cells, which makes it impossible to form G-4 DNAzyme as the catalyst22. In addition, the amount of H2O2 formed by normal cells is less than cancer cells. Therefore, it is possible to use this D-BPE sensing platform for tumor cell diagnosis. H2O2 solution was first extracted by treating cells with PMA after centrifugation to get the supernate, and the sedimental HL-60 cancer cells were then lysed to obtain telomerase which could extend the primer and produce G-



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4 DNAzyme. The discriminating experiments were performed by the analysis of 1×106 cells/mL LO-2 cells and 1×106 cells/mL HL-60 cancer cells. As shown in Figure 4A, without adding the cell extracts in luminol and Ru(bpy)32+ reservoirs, the D-BPE chip strong orange ECL bands but no blue bands. By adding extract of LO-2 cells, orange Ru(bpy)32+ ECL emission decreased a little, and in luminol reservoirs, weak blue signals could be distinguished. However, by adding extract from HL-60 cancer cells (Figure 4B), it shows weak orange bands but remarkable blue bands. The phenomenon confirmed this method could be applied for the discrimination of cancer cells from normal cells. The ECL signals of Ru(bpy)32+ and luminol are related to the concentration of H2O2 and G-4 DNAzyme, which are proportional to the number of HL-60 cancer cells. Therefore, it is possible to quantitatively detect cancer cells based on the change of ECL intensities. Here we choose varieties concentrations of HL-60 cancer cells (0, 3.2×102, 1.6×103, 8×103, 4×104, 2×105, 1×106, 5×106 cells/mL) to make standard curve. The ECL image of D-BPE was shown in Figure 5A. The ECL of Ru(bpy)32+ turned darker and that of luminol became brighter with increasing number of HL-60 cancer cells, the trends were shown in Figure 5B. Here we applied ratiometric method for the quantitative analysis, since it is an ideal strategy to limit the interference factors via normalizing environmental variation by self-calibration and decrease the possibility of false positive. The linear relationship between the ratio of ECL intensity of Ru(bpy)32+ to luminol ECL response and the number of cells were established. There are two linear ranges in the range of 320 to 2.5×105 cells/mL. From 320 to 6×104 cells/mL, the linear fitting equation is y=13.141.54x, with R2=0.991. From 7×104 to 2.5×105 cells/mL, the equation is y=47.4-8.74x, with R2=0.986. The detection of cells reaches 5 orders. The detection limit of 80 cells/mL was calculated by S/N=3.


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To evaluate the variance of the measurements, we detected HL-60 cells on different days from 5 different batches of cells, and calculated the RSD of detection. We chose the numbers of cells at 3 levels of 320, 8000 and 1.5×105 cells/mL, and the corresponding RSD of detections are 2.7%, 2.3% and 1.4%, respectively, which showed a good reproducibility of the measurements for cells. CONCLUSION A novel D-BPE ITO chip which could give different colors of ECL signals was designed and fabricated to detect the HL-60 cancer cells. The sensing mechanism of the chip was studied in detail and compared with traditional three-electrode system. The two BPEs were coupled with each other electrically, which facilitated the ECL detecting sensitivity. By adding H2O2 and DNAzym, the orange ECL signal from Ru(bpy)32+ on BPE2 turned off and blue luminol signal on BPE1 turned on. The process is visual like a color blink, which is not only interesting but also sensitive. More importantly, this protocol can be used for tumor recognition. It is possible to distinguish cancer cells from normal cells through direct observation, since the normal cells produced much less H2O2 and G-4 DNAzyme than cancer cells. This “shining” protocol has a prospect for tumor diagnosis in a high-throughput way. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (W. Zhao); [email protected] (J.J. Xu) Tel: +86-25-89687294 Author Contributions 1: these authors contributed equally. Notes



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The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the 973 Program (Grant 2012CB932600, 2013CB933802), the National Natural Science Foundation (21327902, 21535003) of China. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

ASSOCIATED CONTENT Supporting Information ECL image of D-BPE chip without sample addition, optimization of driving voltage, cell apoptosis assay, and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.


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(22) Zhang, H.R.; Wang, Y.Z.; Wu, M.S.; Feng, Q.M.; Shi, H.W.; Chen, H.Y.; Xu, J.J. Chem.Commun. 2014, 50, 12575-12577.


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Scheme 1. Configuration of bipolar chip used in detection.

Scheme 2. Structure of D-BPE and operation procedures of BPE-ECL sensing platform.



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Figure 1. ECL image of D-BPE. The left array of reservoirs containing 0.1 M luminol while the right array reservoirs containing 2.5 mM Ru(bpy)32+ and 25 mM TPrA with a 6.0 V driving voltage (A), with 0.02 M H2O2 in luminol reservoirs (B), with 0.02 M H2O2 in both luminol and Ru(bpy)32+ reservoirs (C), and with 0.02 M H2O2 and 0.2 µM G-4 DNAzyme in both luminol


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and Ru(bpy)32+ reservoirs (D). The corresponding ECL curves of luminol and Ru(bpy)32+ are shown in A1-D1, and A2-D2, respectively.

Figure 2. (A) ECL curves of 0.1 M luminol and 0.02 M H2O2 in the absence (a) and presence (b) of 0.2 µM G-4 DNAzyme on macroscopic ITO electrode with three-electrode cell. Inset: the corresponding CV curves; (B) ECL curves of 0.1 M luminol and 0.02 M H2O2 (a); with 0.2 µM G-4 DNAzyme and 0.02 M H2O2 when Ru(bpy)32+ reservoirs containing 2.5 mM Ru(bpy)32+ and 25 mM TPrA in the absence (b) and presence (c) of 0.2 µM G-4 DNAzyme and 0.02 M H2O2 on D-BPE (The voltage applied is 6.0 V.) Inset: the corresponding CV curves. (C) ECL curves of 2.5 mM Ru(bpy)32+ and 25 mM TPrA (a); with 0.02 M H2O2 (b); with 0.2 µM G-4 DNAzyme



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and 0.02 M H2O2 (c) on macroscopic ITO electrode with three-electrode cell. Inset: corresponding CV curves. (D) ECL curves of 2.5 mM Ru(bpy)32+ and 25 mM TPrA (a), with 0.02 M H2O2 (b), with 0.02 µM G-4 DNAzyme and 0.02 M H2O2 when the luminol reservoirs containing 0.1 M luminol in the absence (c) and presence (d) of 0.2 µM G-4 DNAzyme and 0.02 M H2O2 on D-BPE (The voltage applied is 6.0 V). Inset: corresponding CV curves.

Figure 3. (A) ECL image of BPE1, the concentrations of luminol are 0.01 M, 0.05 M, 0.1 M, 0.2 M, 0.5 M, from top to bottom; respectively. (B) ECL images of BPE2, the concentrations of Ru(bpy)32+ are 0.1 mM, 0.25 mM, 0.5 mM, 2.5 mM, 5 mM, from top to bottom, with 50 mM TPrA. (C) ECL images of BPE2, the concentrations of TPrA are 1 mM, 2.5 mM, 5mM, 10 mM, 25 mM, 50 mM, from top to bottom, with 2.5 mM Ru(bpy)32+. The voltage applied is 6.0 V.


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Figure 4. ECL image of D-BPE in the absence of cells (A), and presence of 1×106 cells/mL LO2 cells (B) and 1×106 cells/mL HL-60 cancer cells (C). The luminol reservoirs containing 0.1 M luminol and Ru(bpy)32+ reservoirs containing 2.5 mM Ru(bpy)32+ and 25 mM TPrA. The voltage applied is 6.0 V.

Figure 5. (A) ECL image of D-BPE for different number of HL-60 cancer cells detection. The number of HL-60 cancer cells is 0; 3.2×102; 1.6×103; 8×103; 4×104; 2×105; 1×106; 5×106 cells/mL from left to right, respectively. (B) The voltage applied is 6.0 V. Plot of the ECL intensities vs. number of HL-60 cancer cells. The error bars represent the standard deviation. (C) The linear relationship between the number of HL-60 cancer cells and the ratio of ECL intensity of Ru(bpy)32+ to luminol.



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For TOC only


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Visual Color-Switch Electrochemiluminescence Biosensing of Cancer

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