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A Temporal Sensing Platform Based on Bipolar Electrode for the Ultra-sensitive Detection of Cancer Cell Hai-Wei Shi, Wei Zhao, Zhen Liu, Xi-Cheng Liu, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02204 • Publication Date (Web): 10 Aug 2016 Downloaded from http://pubs.acs.org on August 12, 2016

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Analytical Chemistry

A Temporal Sensing Platform Based on Bipolar Electrode for the Ultra-sensitive Detection of Cancer Cell Hai-Wei Shi, Wei Zhao*, Zhen Liu, Xi-Cheng Liu, Jing-Juan Xu*, and Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China. Corresponding authors *Tel/fax: +86-25-89687294. E-mail: [email protected]. [email protected].

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ABSTRACT: We report a bipolar electrode (BPE) sensing platform for the temporal detection of cancer cells. Combining the advantages of anodic dissolution and electrochemiluminescence (ECL), this strategy shows an ultra-low detection limit down to 5 cells/cm2. At the anode worked as the reporting pole, Au NPs were assembled through DNA double strand, which served as both catalyzer for the ECL reaction of luminol/H2O2 and seeds for the chemical reduction of Ag, the anodic dissolution probe. The duration of Ag layer dissolution was positively correlation with the amount of Ag, but negatively relation to the controlled potential and the conductivity of the circuit. Therefore, it was possible to amplify slight conductivity change through tuning the other two factors. As the formation of Ag@Au completely quenched the ECL emission of luminol, the ECL emission recovery reflected the extent of anodic dissolution. Through monitoring the ECL recovery time before and after the incubation of cells on the cathode, a few number of cells could be quantified due to slight difference of the conductivity. This method shows several merits. Firstly, the combination of anodic dissolution and ECL significantly increases the detection sensitivity of BPE device. In addition, this strategy broadens the application of BPE for the ultra-sensitive monitoring of cancer cells, which was applied to investigate the capture efficiencies of antibodies and aptamers towards MCF-7 and A549.

KEYWORDS: Bipolar electrode; electrochemiluminescence; anodic dissolution; temporal sensing; cell detection


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INTRODUCTION A bipolar electrode (BPE) system is formed by positioning a conductor in a uniform electric field, when the interfacial potential difference at the poles of BPE is sufficiently high, oxidation and reduction reactions occur at the extremities.1-5 The development of bipolar electrochemistry shows several advantages compared to conventional electrochemical methods, including making direct electrical contact to nanoscale electrodes, maintaining control of large arrays of electrodes simultaneously, and controlling electrodes which are mobile in the solution, etc.6,7 During the past 15 years, BPEs have been used as electroanalytical sensors relying on electrical coupling between the two poles named as the sensing and the reporting poles. Since the current passing through the two poles must be equal (icat=-ian), the reporting pole reflects the state of the sensing one. Most reporting poles utilized optical readouts include electrochemiluminescence (ECL), fluorescence and anodic dissolution.4,7-10 Among these methods, ECL became the most popular one since it is highly sensitive, with near zero-background signal.11,12 The combination of ECL and BPE opened the door to BPE sensing of a wide variety of analytes.2,5,8,13,14 In 2012, Crooks’ group reported an intuitive method of comparing the dissolution amount of Ag microbands at the anode of BPE through optical micrograph for rapid screening of electrocatalysts of the oxygen reduction reaction (ORR).6 The approach was based on simultaneous activation of ORR and Ag electrodissolution at the cathodic and anodic poles, respectively. The electrochemical activities of the two poles were directly couple via the BPE, hence the extent of Ag electrodissolution was directly related to the ORR activity. At current stage, BPE sensors with optical readout have been mostly adopted for bioanalysis and screening of electrocatalysts.1,15 In this work, we broaden the applications of BPE for the cellular analysis. When a cell attaches on the surface of the electrode, on one hand, as a substance with low conductivity, the impedance of 3 ACS Paragon Plus Environment


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electrode/solution interface increases, on the other hand, cell membrane hinders the electron-transfer of electroactive species at the electrode surface.16-18 Along with the increasing amount of cells, the electrode surface conductivity decreases. Electrical cell-substrate impedance sensing (ECIS) was generally applied for monitoring of cell attachment and behavior.19-24 For the quantification of different types of cancer cells, the limit of detection (LOD) of ECIS was from 103 to 106 cells/cm2. Here in this work, we built a temporal sensing platform combining BPE-ECL sensing and anodic dissolution, which greatly enhanced the sensitivity for the cellular analysis. We firstly assumed after the incubation of cells (MCF-7) on one pole of the BPE, the decrease of electron-transfer could be sensitively monitored at the other end of BPE via ECL. Taking the cathode of BPE (4mm×5mm) as the sensing pole, after capturing a dozen of MCF-7 cells, the ECL emission change of luminol at the gold nanoparticles (AuNPs) modified anode could be identified. The detection limit was around 55 cells/cm2, which was significantly lower than LOD obtained by ECIS. For further improving the sensitivity, anodic dissolution was introduced and the detection mode was extended to time dimension. Using AuNPs as the seeds, Ag nanoparticles was fast deposited on the anode.25,26 Due to the decrease of the electrode conductivity and resonance energy transfer (RET) from luminol to Ag@Au, ECL emission of luminol was quenched to zero. When a sufficient potential was applied on the BPE, Ag started to dissolve and finally the ECL emission recovered. The duration time of anodic dissolution indicated by the ECL recovery was negatively related to the conductivity of the BPE. The number of cells incubated on the BPE was quantified by the ECL recovery time instead of intensity. Through tuning the amount of Ag and the applied potential, the ECL recovery time was able to be modulated. Therefore, slight difference of BPE conductivity could be amplified and validated via time scale analysis. Under optimal conditions, a single cell on 0.2 cm2 cathode was identified (5 cells/cm2). The temporal detection of cancer cells combined ECL and anodic dissolution achieved 10 4 ACS Paragon Plus Environment

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times lower detection limit compared with conventional ECL-BPE strategy. Using this BPE sensor, the affinity capture efficiencies of different antibodies and aptamer towards MCF-7 and A549 cells were evaluated, which provided guidance for the enrichment and capture of cancer cells from peripheral blood. As a much more sensitive approach, the BPE based strategy covers the shortage of ECIS for monitoring small number of cells in cell attachment and behaviors associated with processes such as cell micromotion, cell cytotoxicity, wound-healing as well as cellular responses to physical and chemical stimulations. EXPERIMENTAL SECTION Chemicals and Regents. Bovine serum albumin (BSA), 3-aminopropyl triethoxysilane (APTES), thionine and silver enhancer kit (solution A and solution B) were purchased from Sigma-Aldrich (USA). Polyclonal antibody to carcinoembryonic antigen (Anti-CEA IgG) and mouse IgG monoclonal antibody for AFP (anti-AFP) were got from Cloud-Clone Corp. 5’ end amino-modified mucin-1 (MUC1) aptamer was synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). 0.01 M sterilization phosphate buffer saline (PBS) and fetal bovine serum were purchased from Jiangsu KeyGEN Bio-TECH Corp (China). Glutaraldehyde (GA) was obtained from sinopharm chemical reagent company (Shanghai, China). 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 from Dow Corning (Midland, MI, USA). ECL detection solution was 0.10 M PBS (pH 7.4) involving 0.06 mM luminol and 0.2 M H2O2. All solutions were prepared with Millipore (model milli-Q) purified water. Instruments. ECL signals were measured with MPI-E electrochemiluminescence analyser from Xi’An Remax Electronic Science &Technology Co. Ltd (Xi’An, China, 350 nm~650 nm). Original cell concentrations were achieved by CountessⅡFL type cell counting machine from Invitrogen 5 ACS Paragon Plus Environment


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Corporation and specific cell numbers were counted under a Leica Dmire 2 type microscope with an Olympus DP71 CCD. Biological processes were performed in Thermo Forma CO2 incubator. Scanning electron microscopic (SEM) images were acquired from a Hitachi S4800 scanning electron microscope (Hitachi Co., Japan). Fabrication of the ECL Device. The pattern of the closed BPE was designed according to our previous work utilizing a fixed PDMS mold to ensure the unity of the size and distance of the electrode. Specifically, the closed BPE system consisted of three separate rectangular ITO electrodes lined with a 0.6 cm gap. Two side electrodes were the same size of 1.5 cm long and 0.5 cm wide, while the intermediate electrode was 2 cm long and 0.5 cm wide. The electrode group was achieved from etching on the base of a single ITO electrode.27 The whole etched BPE was first immersed in 1 M NaOH for 4 hours at room temperature to ensure the presence of hydroxyl groups on the electrode surface. Followed by immersed in a 5% (v/ v) ethanol solution of APTES at 4 °C for 12 hours, the hydroxyl-modified microchip was functionalized with –NH2. Rinsed three times with ethanol and purified water successively, excess and loose bounded silane was removed, after which, two PDMS slices each containing a specific reservoir (1.6 cm long, 0.6 cm wide) were adjacently affixed on the electrode intervals at labelled positions. 50 µL glutaraldehyde (2.5%) aqueous solution was then carefully pipetted to both anode and cathode of the BPE surface and kept at 37 °C for 1 h to bring in aldehyde on both ends. Rinsing with 0.1 M PBS (pH 7.4) thoroughly, the anode and cathode were immediately treated separately. For the cathode, 50 µL of 10 µg/ mL anti-CEA IgG was pipetted to the reservoir to cover the cathode and incubated in carbon dioxide incubator at 37 °C for 1 h. For the anode, 50 µL of 1 µM capture DNA (Cp-DNA) with 5’ end modified with amino was added to the anodic reservoir and after 1.5 h incubation, the anode was then blocked by 2 wt % BSA solution at 37 °C for 1 h and carefully rinsed with PBS (pH 7.4). Then, 50µL 1 µM Au-DNA composed of a 5 6 ACS Paragon Plus Environment

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nm gold nanoparticle and target DNA which was complementary to former Cp-DNA was pipetted in to react with the Cp-DNA to bring Au NPs to the anode. At last, the anode was immersed in freshly prepared silver enhancer solution for 1 min. Then the reaction solution was carefully removed with a straw and the anode was rinsed immediately with PBS (pH 7.4) to get rid of the unreacted silver ions.5 Quantification of Cancerous Cells. After adding 100 µL 0.1 M PBS (pH 7.4) containing 0.1 M NaCl to the cathodic reservoir and 98 µL 0.2 M H2O2 together with 2 µL 3 mM luminol to the anodic reservoir, ECL measurements were performed on a MPI-E system with the PMT set at -500 V. The two driving electrodes were connected to two separate silver conductive tapes for the easy grip of the clips of the apparatus. At a constant applied voltage of 3.4 V, the Au-DNA improved ECL signal was recorded as a reference. Besides, the recovery processes of the BPEs were also recorded after silver reduction without incubation of cells on the cathode. Based on the same former BPE, certain amount of MCF-7 cells was incubated on the cathode to increase the electron transfer resistance, and PBS (pH 7.4 containing 0.1 M NaCl) was gently added to the reservoir and pipetted out to get rid of the uncaptured cells. The specific amount of cells was counted manually under a Leica optical microscope. Then, silver was in situ reduced on anode to coat the Au NPs for 60 s. At the same voltage of 3.4 V, ECL measurements were performed and the recovery times with different cell amounts on the cathode were recorded. Evaluation of the affinity capture efficiencies of antibodies and aptamer towards MCF-7 and A549 cells. The configuration was then utilized to compare the capture rate of different antibodies or aptamer towards the antigens on MCF-7 and A549 cell membrane. CEA, AFP and mucin1 were selected as the target antigens and their antibodies anti-CEA IgG, anti-AFP antibody and mucin-1 aptamer were respectively modified to separate cathodes with similar concentrations. In detail, 50 7 ACS Paragon Plus Environment


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µL of anti-CEA IgG, anti-AFP and mucin-1 aptamer with approximately equivalent amount of solute were pipetted to the three independent reservoirs to cover the cathode and incubated in carbon dioxide incubator at 37 °C for 1 h with a cover. Then, the three modified cathodes were carefully rinsed with PBS to get rid of the unreacted antibodies and aptamer. Freshly digested MCF-7 or and A549 cells were then quickly diluted to a concentration of 2×103 cells/ mL which was counted with an automatic counter and 30µL of dilution was added to each reservoir with different modifications for incubation. The silver reduction durations were compared between different antibodies and the capture rate to each antibody or antigen could be calculated based on the total amount. Besides, the capture rate variations of certain antibodies against time could be investigated. RESULTS AND DISSCUSSION Principle of the Temporal Resolution ECL-BPE Biosensor. The fabrication process of ECL-BPE biosensor and the mechanism of determination of MCF-7 cells are described in detail in Scheme 1. The anti-CEA IgG modified cathode served as sensing pole for the analysis of certain amount of MCF-7 cells. At the anode side, luminol-H2O2 pair was added in the anode reservoir as the ECL reporting probe. As efficient catalyzer of luminol-H2O2, 5 nm AuNPs were assembled to the surface of the anode through DNA double strand according to our previous report.28-30 Here the modification of Au NPs provides higher electron transfer rate (Figure 1B) meanwhile maintains the transmission of ITO. As shown in Figure 1A, the ECL intensity increased more than 2 times from 5000 a.u. to 11000 a.u. at 3.8 V driving voltage, which was ascribed to the catalytic activity and higher electron transfer rate of Au NPs. After the incubation of MCF-7 cells on the cathode, the ECL intensities of luminol slightly decreased (Figure 2A), which was ascribed to the low conductivity of the cell and the steric hindrance of the cell membrane to the electron-transfer of electroactive species at the electrode surface. However, since the dimension of a cell is extremely small compared with 8 ACS Paragon Plus Environment

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the area of cathode (1 : 1.3×104), although the BPE-ECL sensing platform is highly sensitive, as shown in Figure 2B, the difference of ECL signal was hard to be distinguished when the number of cells was lower than 55 cells/cm2. To promote the performance of the BPE cellular sensor, we designed another strategy which combined anodic dissolution with ECL. At the anode, Ag was catalytically deposited on the surface of 5 nm AuNPs to form an Ag@Au layer with ca. 1µm thickness (Figure S2), which significantly quenched the ECL emission of luminol (Figure 1A). Then under a certain potential, Ag should dissolve from the surface and the ECL signal recovered. The duration of the anodic dissolution indicated by ECL emission recovery could be used for the determination of the amount of cells. After 60s deposition, the ECL emission of luminol decreased to zero (Figure 3B). Such sharp quenching effect was explained from the following aspects. On one hand, as thick silver layer formed on the surface of ITO, the catalytic effect from AuNPs to luminol was blocked and the electron transfer resistance of the BPE increased (Figure 1B). On the other hand, as shown in Figure S1, the Ag@Au layer show absorption peak at ~400 nm. The spectral overlap between ECL spectrum of luminol (centered at ~420 nm) and the absorption spectrum of Ag@Au resulted in the resonance energy transfer (RET) from luminol to Ag@Au layer and greatly quenched the anodic ECL emission. Theoretically, the duration of the electrodissolution is related to three factors, which are the amount of Ag, the controlled potential and the conductivity of the circuit. Through increasing the amount of Ag and decreasing the driving potential, we assume slight conductivity change induced by the incubation of cells could be amplified and indicated by the ECL recovery time. As shown in Figure 3A, from deposition time of 15 s to 120 s, longer time resulted in more amount of Ag, hence longer recovery time was observed under the driving potential of 3.4 V. The driving voltage for Ag 9 ACS Paragon Plus Environment


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electrodissolution was also studied. As shown in Figure 3C, voltages from 3.0 V to 3.8 V were examined and the ECL recovery time decreased along with the increase of driving voltage. At potential higher than 3.4 V, ITO electrode couldn’t last a long time because of the surface reduction. Longer deposition time and lower driving potential resulted in longer duration of anodic dissolution, hence slight change of the conductivity of the BPE reflected by ECL recovery time could be correspondingly amplified and detected. Considering the ECL background and acceptable sensing duration, deposition time of 60 s and driving potential of 3.4 V were chosen to measure 5 MCF-7 cells incubated at the cathode (Figure 3D). The results were satisfactory. Without any incubation on the cathode, the ECL signal quickly reached 90% of original signal within 125s (black curve). While with 5 cells on the cathode, it takes ca. 300 s (red curve) to reach the platform. Without introducing anodic dissolution, this number of cells couldn’t be identified. But here a large time difference of 175 s was observed. To verify the performance of this temporal resolution BPE sensor, a number of MCF-7 cells were measured based on 60 s deposition of Ag and 3.4 V driving voltage. Quantification of MCF-7 Cells on cathode. The influence of several particular numbers (1, 2, 3, 4, 5, 8, 11, 28, 50 and 96) of MCF-7 cells on the recovery process was measured. Before each measurement, the specific number of MCF-7 cells on the electrode was manually counted under an optical microscope. The results showed that the more MCF-7 cells were incubated, the more slowly ECL signal recovered. The cell amount had a great impact on the ECL recovery. The relationship between the differences of ECL recovery time with and without cell incubation, and the amount of MCF-7 cells was established and shown in Figure 4A. The quantitative detection of ultra-low number of cells from 1-11 cells was achieved (Figure 4B), with a detection limit down to 5 cells/cm2. The BPE device could be re-used after simple surface treatments. The anode could be easily 10 ACS Paragon Plus Environment

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recovered by adding silver enhancer for 60 seconds after each detection process. The cathode was ready to use after ultrasonic treatment in pure water for 15 min to break the cells and get rid of the residues. After 7 times detections, the ECL intensity decreased to 84% of original signal, but the duration of anodic dissolution reflecting by the ECL recovery time was barely changed. The ultra-sensitive and convenient BPE cellular sensor is suitable to in-vitro monitor the cell attachment and behavior. The status of cell shows no obvious morphology change under a continuous constant voltage of 3.4 V within 20 min (Figure S3). Therefore, it shows potential in applications such as cellular responses to physical and chemical changes, as well as cell attachment and spreading. Evaluation of affinity capture efficiency of antibodies and aptamers towards MCF-7 and A549 cells. Antibodies and aptamers serve as capture agents for the isolation of cancer cells from complex cellular fluids. Investigating their capture efficiencies helps building multivalent adhesive domain and incorporating into microfluidic devices for clinical disease diagnosis, therapy monitoring and biological studies. Using the BPE cellular sensing platform, the affinity capture efficiencies of different agents could be simply characterized. Anti-CEA IgG, anti-AFP antibodies (10 µg/mL) and mucin-1 aptamers (0.1 µM) with similar densities on the ITO surfaces were applied to capture 60 MCF-7 and A549 cells (2×103 cells/mL, 30µL), respectively. The capture rates were calculated according to the standard curves of recovery time towards different amount of cells on the cathode. For the capture of MCF-7 cells, as shown in Figure 5A, CEA and AFP antibodies exhibited similar capture rates under the same modification and incubation conditions, which turned stable after 60 min incubation of MCF-7 cells. The mucin1 aptamer appeared with an overall lower capture rate of around 20% after 90 min incubation of MCF-7 cells. Although aptamers were reported with higher affinity to free antigens, the amount of aptamers should be over abundance compared with 11 ACS Paragon Plus Environment


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antibodies for the highly efficient capture of cells. For example, Fan’s group compared the capture efficiencies of anti-PTK7 antibodies and sgc8 aptamers to CEM and Ramos cells. Results indicated that 300-fold density aptamers (10 µM) showed equivalent capture efficiencies compared with antibodies (5 µg/mL). Therefore, it was reasonable that with close densities, the capture efficiency of antibody was higher than that of aptamer. Figure 5B shows the comparison of the capture efficiencies of these three antibodies and aptamer with 60 min cell incubation duration, which was in good agreement with the observation results with microscope. Similar operations were also carried out with A549 cells, which were displayed in Figure 5C and 5D. The capture rates of antibodies to A549 were relatively lower, which could be related to the lower expressions of relevant antigens on A549 membrane than on MCF-7 cells. The high sensitive BPE strategy was proved as a useful tool to investigate cells with ultra-low amount. CONCLUSION In this paper, we proposed a novel BPE based device for cell detection via the combination of anodic dissolution of Ag and ECL sensing, and successfully realized an ultra-sensitive determination of single cell at 0.2 cm2 ITO electrode. Compared with ECIS and ECL-BPE methods, the temporal BPE sensing platform for the quantification of cells showed much lower detection limit down to 5 cells/cm2. It was applied to evaluate the affinity capture rates of ITO modified with different antibodies and aptamers towards MCF-7 and A549 cells. This work suggested BPE could be used not only for the biological detection of electroactive species, but also in vitro monitoring of cell behavior through the change of conductivity at electrode/solution interface.

AUTHOR INFORMATION Corresponding Authors 12 ACS Paragon Plus Environment

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*Tel/Fax: +86-25-89687294. E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We appreciate the financial support of this project from the 973 Program (Grant 2012CB932600), National Natural Science Foundation of China (No. 21475058, 21535003). This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Supporting Information Available. UV curves of Au NPs and Au@Ag NPs. This material is available free of charge via the Internet at http://pubs.acs.org.

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Scheme 1. Schematic diagram and detecting mechanism of the time-resolution ECL biosensor based on closed bipolar electrode.



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Figure 1. (A) Typical ECL curves using cyclic voltammetry method respectively with ssDNA (black), dsDNA after the hybridization process with Au NPs modified thrombin aptamer (red), and Ag@Au (green) on the anode. Inset was the typical curves of ECL intensity versus time. (B) Corresponding current variations after each step of modification described above.

Figure 2. (A) Typical curves of ECL intensity versus time after the hybridization process with Au NPs with (red) and without (black) 28 MCF-7 cells incubated on the cathode. (B) The difference of ECL intensities after incubation of cells versus the number of cells.


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Figure 3. (A) Recovery process with different silver reduction durations of 15 s, 30 s, 45 s, 60 s, 90 s and 120 s, respectively, under voltage of 3.4 V; (B) the initial ECL intensities with different silver reduction durations. (C) Recovery time at different applied voltages of 3.0 V, 3.2 V, 3.3 V, 3.4 V, 3.5 V, 3.6 V and 3.8 V, respectively, with 60 s in situ sliver reduction. (D) Typical ECL recovery curves without MCF-7 cells (black) and with 5 MCF-7 cells (red) on the cathode at a constant voltage of 3.4 V. The processes were recorded with a 0.2 M H2O2 and 0.06 mM luminol detecting solution.

Figure 4. (A) Corresponding relationships between the amount of MCF-7 cells and the delay time of 17 ACS Paragon Plus Environment


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recovery process; (B) Linear fitting with less than 11 cells.

Figure 5. (A) Capture efficiencies of MCF-7 incubation durations (15 min, 30 min, 45 min, 60 min, 75 min and 90 min, respectively) with different antibodies on electrode surface; (B) The capture efficiency comparison via BPE sensor and microscope at 60 min incubation under CEA, AFP and MUC1 recognition；(C) Capture efficiencies s of A549 incubation durations (15 min, 30 min, 45 min, 60 min, 75 min and 90 min, respectively) with different antibodies on electrode surface; (D) The capture efficiency comparison via BPE sensor and microscope at 60 min A549 incubation under CEA, AFP and MUC1 recognition.


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