Improving the Electrochemical Imaging Sensitivity of Scanning

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Improving the electrochemical imaging sensitivity of scanning electrochemical microscopy-scanning ion conductance microscopy by using electrochemical Pt deposition Mustafa #en, Yasufumi Takahashi, Yoshiharu Matsumae, Yoshiko Horiguchi, Akichika Kumatani, Kosuke Ino, Hitoshi Shiku, and Tomokazu Matsue Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00027 • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Analytical Chemistry

Improving the electrochemical imaging sensitivity of scanning electrochemical microscopymicroscopy-scanning ion conductance microscopy by using electrochemical Pt deposition Mustafa Şena,b*, Yasufumi Takahashia,c,d**, Yoshiharu Matsumaea, Yoshiko Horiguchia, Akichika Kumatanic, Kosuke Inoa, Hitoshi Shikua, and Tomokazu Matsuea,c*** a

Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579,

Japan b

Department of Biomedical Engineering, Faculty of Engineering and Architecture,

Izmir 35620, Turkey c Advanced

Institute for Materials Research (AIMR), Tohoku University, Sendai

980-8577, Japan d

PRESTO, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

*Corresponding author: Mustafa Şen E-mail: [email protected] ** Corresponding author: Yasufumi Takahashi E-mail: [email protected] ***Corresponding author: Tomokazu Matsue E-mail: [email protected]

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ABSTRACT We fabricated a platinum-based double barrel probe for scanning electrochemical microscopy-scanning ion conductance microscopy (SECM-SICM) by electrodepositing platinum onto the carbon nanoelectrode of the double barrel probe. The deposition conditions were optimized to attain highly-sensitive electrochemical measurements and imaging. Simultaneous SECM-SICM imaging of electrochemical features and noncontact topography by using the optimized probe afforded high-resolution images of epidermal growth factor receptors (EGFR) on the membrane surface of A431 cells.

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Introduction Scanning electrochemical microscopy (SECM) is a powerful tool to investigate localized electrochemical reactions. Because it can be used under physiological conditions, it has long been applied to cells to detect the activity of some proteins1-2. Using microelectrodes for SECM in electrochemical studies offers a few advantages including low ohmic drop, low charging current, and enhanced mass transport because of their critical dimensions in the micro/submicroscale3-5. The benefits of using such small electrodes are expected to be achieved to a greater extent with nanoelectrodes, in which the primary benefit is due to enhanced mass transport6. Although SECM imaging with nanoelectrode probes provides high spatial resolution,1,4,7-9 chemical sensitivity is usually lost. The amperometric detection of a few µM of electroactive species by using a nanoelectrode with a diameter of approximately 250 nm produces a subpicoampere current level, which is below the noise level in a conventional experimental atmosphere. Among various noises arising from the background current and measurement system, inevitable electronic noise, referred to as Johnson noise, is the most crucial. Although, the external interfering signals can be eliminated by means of proper shielding and grounding, noise can be reduced to a certain level which is defined mostly by Johnson noise. Therefore, it limits the capability of nanoprobes in electrochemical bioimaging, causing poor sensitivity, particularly in the case of imaging cell proteins with high resolution. Here, we report the sensitivity improvement of conventional SECM-SICM with double barrel carbon nanoprobes. The electrochemical response was amplified by Pt deposition10,11 on the carbon nanoelectrode, which enlarged the total flux of electroactive species at the electrode surface12-14. The improved double barrel probe with a Pt-deposited electrode was used as the probe for the constant-distance1,9,15-24 mode SECM-SICM

in

order

to

obtain

the

electrochemical

images

of

immunocytochemically-stained EGFR proteins on A431 cells (Figure 1a). This is the first study of using a sphere type electrode for high-resolution electrochemical imaging with SECM.

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Experimental Section Chemicals Chemicals and materials. RPMI-1640 medium (Gibco Invitrogen, Japan), fetal bovine serum (FBS; Gibco), 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES; Dojindo Laboratories, Japan), p-aminophenyl phosphate monosodium salt (PAPP; LKT Laboratory Inc., USA), penicillin/streptomycin (Gibco), phosphate buffered saline (PBS(−); Wako Pure Chemical Industries, Japan), albumin, from bovine serum, crystallized (PBS(−); Wako Pure Chemical Industries, Japan), 97 % ferrocenemethanol (FcCH2OH: Aldrich, USA), choloroplatinic acid solution (H2PtCl6: Sigma-Aldrich, USA), 4 % paraformaldehyde phosphate buffer solution (PBS(−); Wako Pure Chemical Industries, Japan), mouse monoclonal epidermal growth factor receptor (EGFR) Ab-2 (clone 225) (Thermo Scientific, USA), goat polyclonal alkaline phosphatase conjugate secondary antibodies to mouse IgG-H&L (Invitrogen, USA). All solutions were prepared using distilled and deionized water (Direct-Q, Millipore). Electrode fabrication For fabrication of double barrel carbon nanoprobes, a quartz theta pipette (O.D.: 1.2 mm, I.D.: 0.90 mm, 7.5 cm length, Sutter Instruments, USA) was pulled in a laser puller (P-2000, Sutter Instruments, USA). Fabrication method is illustrated with a schematic in Figure S1. A wide range of probe size up to a few µm in diameter can be fabricated by changing the pulling parameters. First, both of the ends of the barrels were closed with a reusable past and then one of the barrels was opened and pressurized with butane for pyrolytical deposition of carbon inside this barrel only. The tapper of the pipette was inserted inside another quartz capillary (O.D.: 1 mm, I.D.: 0.7 mm, 7.5 cm length, Sutter Instruments, USA) through which nitrogen gas was injected to prevent both oxidation of deposited carbon and bending of the probe tip by high temperature. Lastly, a pyrolytic carbon layer was formed inside the probe by heating the pipette tapper for a time ranging from 10 to 20 sec. Following the bare carbon double barrel probe fabrication, platinum was electrochemically deposited at surface of the SECM side of the barrel, for which the nanoprobe was immersed in 2 mM H2PtCl6 in 0.1 M hydrochloric acid, and electrochemical deposition was performed at +0.0 V vs. Ag/AgCl. Prior to electrochemical imaging, Pt deposition was performed under surveillance of SICM current, for which through the SICM barrel a potential of -1 V vs. Ag/AgCl was applied during the Pt deposition at 0 V vs. Ag/AgCl. Afterward, the modified probes were not allowed to dry as some solution gets into the SICM side of the probe that might result in clogging of the opening. Therefore, the probes were either kept in MiliQ water at 4°C or used immediately after modification. Probes with good hopping mode behavior 4


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were used for imaging applications. Characterization Characterization of Pt deposition regarding electrode size and sensitivity improvement To elaborate the effect of Pt deposition on the electrode size, electrodes with final Pt deposition currents of −1 nA, −2.5 nA, −5 nA, −10 nA, and −20 nA were fabricated, respectively. Prior to the electrochemical deposition, all electrodes were subjected to a mild electrochemical pretreatment, for which a potential of -1 V vs Ag/AgCl was applied through the carbon side of the probe in a PBS solution to activate the electrodes for an efficient Pt deposition. Subsequently, the Pt deposited electrodes were immersed into a PBS solution containing 1 mM FcCH2OH at +0.3 V vs. Ag/AgCl to obtain oxidation currents for size determination. All electrode tips were washed with and kept in MiliQ water. To investigate the effect of Pt deposition on sensitivity improvement, electrodes with final Pt deposition currents of −1 nA, −10 nA, -20 nA and −50 nA were fabricated. The probes were immersed in a PBS solution at +0.3 V vs Ag/AgCl, respectively. After the current response reached to a steady state, concentrated FcCH2OH solutions were added into the PBS solution to reach FcCH2OH concentration levels of 100 nM, 500 nM, 1 µM, and 10 µM, respectively. Simulation for theoretical SECM curve Theoretical approach curves of a sphere electrode were simulated using COMSOL Multiphysics software (ver. 4.4, Comsol, Inc., USA). As mentioned in the text, the size of Pt deposited probe was estimated from the SEM image (Figure 1b) and used to construct 2-D models (Figure S2), for which insulating and conductive layers were placed under the electrodes, respectively. The radius of the sphere electrode was set at 1.25 µm. A reversible one-electron reaction was used to simulate electrochemical behavior of the sphere electrode in approaching mode. The diffusion coefficient of FcCH2OH was set to 7.1×10-6 cm2 s-1. Initial concentration of FcCH2OH in the bulk was set at 1.0 mM, while it was 0 mM on the electrode surface. Initially the probe tip was placed far away from the substrate of interest (conductive or insulating) to calculate the steady state current and then, the probe tip was brought close to the substrate gradually. In the case of insulating substrate, the diffusion flux of FcCH2OH was set at 0 mol/m2s. The diffusion flux of FcCH2OH on the sphere electrode surface was used for evaluation of the FcCH2OH oxidation currents.

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SECMSECM-SICM instrumentation The details of SECM-SICM system regarding both instrumentation and operation mode were described elsewhere5,6. Briefly, the SECM-SICM system was operated in hopping mode and the current decrease of SICM for the distance regulation was set at in the range of 0.4-1 %. Both faradaic and ionic currents were constantly measured with a dual channel MultiClamp700B patch-clamp amplifier (Axon Instruments) and filtered using low-pass filter at 1kHz. A continuous data acquisition hardware and software (Axon Digidata 1440, Axon instruments) were used to digitize and analyze the data. The control of the probe on x, y and z axis was done by a piezoelectric scanner (Physik Instrumente, 621.2CL and 621.ZCL). The system was controlled and data were acquired using a program written with LabVIEW (National Instruments). Cell culture and immunocytochemical immunocytochemical staining of EGFR A431 cells (CCL-2.2) were purchased from ATCC and cultured in RPMI-1640 medium containing 10% FBS and 50 µg/mL penicillin/streptomycin at 37 °C under a 5% CO2 humidified atmosphere. For immunochemical staining, the A431 cells were seeded into a 35 mm dish (Falcon, USA) at a density of 2-4×105 cells in 2 mL of RPMI-1640 medium (10% FBS + 50 µg/mL penicillin/streptomycin) and incubated at 37 °C. After three days of culture, A431 cells were washed twice with PBS and fixed using 4 % paraformaldehyde for 15 min. Following the cell fixation, the cells were incubated with 4 % BSA+PBS solution for 1 h at room temperature to block unspecific bindings of antibodies. Subsequently, the cells were washed twice using PBS containing 0.2% tween for 5 min. Then the cells were incubated with anti-EGFR primary antibodies in 4 % BSA+PBS at 4 ºC overnight. The next day, cells were once again washed with PBS containing 0.2% tween for 5 min to remove excessive antibodies and incubated with alkaline phosphatase (ALP)-conjugate secondary antibodies in 4 % BSA+PBS for 1 h at room temperature. Electrochemical imaging Following the immunocytochemical staining of EGFR proteins, the cells were washed three times with HEPES solution (10 mM HEPES, 150 mM NaCl, 4.20 mM KCl, 11.20 mM glucose, 2.30 mM MgCl2, 1 mM Na2HPO4). Afterward, the detection solution containing 4.7 mM PAPP in a HEPES was introduced (pH 9.5, optimum pH for ALP reaction detection25) for electrochemical and topographical imaging using SECM-SICM. ALP labeled in the secondary antibodies catalyzes the hydrolysis of PAPP into

p-aminophenol, which is electrochemically oxidized at the Pt-deposited SECM electrode 6


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of the double barrel probe set at +0.3 V vs. Ag/AgCl for electrochemical imaging of EGFR. The potential of the SICM electrode of the double barrel probe was set at +0.2 V vs Ag/AgCl in order to monitor the ionic current as a feedback signal to obtain the topography of the cell. Prior to the imaging, SICM barrel was filled with 4.7 mM PAPP in a HEPES (pH 9.5) to avoid salt concentration gradient cell potentials and liquid junction potentials. ALP label of secondary antibodies catalyzes the hydrolysis of PAPP into PAP, which is electrochemically oxidized at 0.3 V vs. Ag/AgCl by the Pt deposited electrode of the SECM-SICM double barrel probe.

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Results and Discussion The detailed fabrication process of the SECM-SICM double barrel carbon probe and the operation of the system were described in detail elsewhere8,22 . Probes with a size ranging from a few tens of nm to a few micrometer can be easily fabricated by changing the capillary pulling parameters. In this study, probes with a tip diameter of approximately 500 nm were used (Figure 1b). Following the double barrel carbon probe fabrication, Pt was electrochemically deposited on the carbon nanoelectrode. Pt was spherically grown on the carbon surface. During the process, deposition current was continuously monitored to estimate the size of the deposited Pt sphere. We halted the deposition when the deposition current (final deposition current) increased to −1 nA, −2.5 nA, −5 nA, −10 nA, and −20 nA. The electrode was then transferred to a PBS solution containing 1 mM FcCH2OH, and the oxidation currents were measured at +0.5 V vs. Ag/AgCl. Figure 2aii shows the correlation between the final deposition current and the FcCH2OH oxidation currents, indicating a clearly liner relationship. Therefore, Pt deposition current can be used as a relative measure to fabricate Pt-deposited electrodes with a desired size ranging from nanometer to micrometer. Even though, Pt deposition has long been used to modify electrode surfaces for various purposes, it has never been applied for spherical electrode fabrication. Since spherical electrode construction with desired diameter is technically difficult, Pt deposition at the surface of nano/ultra-micro probe electrode can offer an easy solution for such a challenge. To investigate the effect of electrochemical deposition on sensitivity, electrodes with various final Pt deposition currents (up to −1 nA, −10 nA, −20 nA, −50 nA; Figure 2ai) were prepared, and the detection limits of these electrodes for FcCH2OH were determined. In brief, the probes were immersed in PBS solution with no FcCH2OH at +0.5 V vs. Ag/AgCl. Once the oxidation current attained a steady state, concentrated FcCH2OH solutions were added to obtain final concentrations of 100 nM, 500 nM, 1 µM, and 10 µM (Figure S3). Current was plotted against the respective FcCH2OH concentration level after subtraction of the background current (PBS current data were subtracted from the corresponding FcCH2OH oxidation current). Figure 2b shows that both the bare carbon electrodes and the Pt-deposited electrode with a final deposition current of −1 nA did not show any significant response up to 10 µM of FcCH2OH+PBS. In other words, the oxidation current response for these two probes was below the noise level making it difficult to measure. This is mostly because small currents lead to small signal to noise ratio (S/N), in comparison with external noise and hereby affecting the sensitivity of the probes in a negative manner. The other three electrodes showed 8


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appreciable responses in 500 nM FcCH2OH+PBS with a S/N ratio of higher than three. In the case of a sphere electrode, all electrode surface is uniformly accessible resulting in uniform current density all across the surface. In addition, rough surface of Pt deposited probe has a large surface causing high level of current and hence high S/N ratio. Although larger electrodes provide a larger response, the increased electrode area lowers the spatial resolution and increases the capacitive current. Therefore, the Pt-deposited electrode with a final deposition current of −10 nA was chosen for the electrochemical imaging of EGFR with SECM-SICM. From the SEM and optical microscope images (Figures 1b and 2ai), the deposited Pt shows a spherical shape with a diameter of 2.5 µm. Prior to SECM-SICM imaging by using the double barrel probe with the Pt-deposited electrode, it was confirmed that the Pt deposition did not clog the SICM barrel because the ionic current flowing through the barrel opening is used as a feedback signal for noncontact imaging26-33. The electrochemical Pt deposition at +0.0 V vs. Ag/AgCl was performed under surveillance of the SICM ion current at −1.0 V vs. Ag/AgCl (Figure S4). The electrochemical behavior of the Pt-deposited probe (radius: 1.25 µm) was then analyzed by assuming the shape of the electrode tip to be spherical. The theoretical SECM curves of a spherical electrode were simulated for both negative and positive substrates (Figure S2) for comparison with the experimental SECM curves. A relatively good fit was observed between the theoretical and experimental curves of both SECM (Figure 3ai-bi) and SICM (Figure 3aii-bii). Therefore, the Pt-deposited electrode can be considered to have a relatively spherical shape and used for electrochemical imaging with quantitative analysis. We applied this system to the high sensitivity imaging of EGFR—a cell surface protein with a crucial role in the promotion or inhibition of certain cells. A431 is a commonly used cell type for studying the interaction between epidermal growth factor (EGF) and receptors as they have extremely high numbers of EGFR (1–3 ×106 per cell)34. For electrochemical imaging, EGFR proteins were labeled with primary and ALP-conjugate secondary antibodies by using a standard protocol35. The electrochemical and topographical imaging with the SECM-SICM system was conducted in HEPES buffer (pH 9.5) containing 4.7 mM PAPP. The cyclic voltammograms of 1 mM FcCH2OH clearly indicate that Pt deposition results in a 50-fold increase in current response (Figure 4ai-ii). The increase in current response also improves the topographic and electrochemical images (Figure 4ci-ii) of the EGFR proteins of A431 compared with the images obtained using a bare carbon probe (Figure 4bi-ii). These results clearly show 9



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that the Pt-deposited probe allows sensitive electrochemical imaging with a high-resolution. The electrochemical image taken with a bare carbon tip is not clear because the current resolution is not good as it is only 2.7 pA of total variation of current on the current axis (Figure 4bii). The SECM image taken with the Pt-deposited probe has a current scale spanning over 16.3 pA on the current axis (Figure cii), almost 6 times larger than that for the bare carbon probe. The better current resolution simply leads to a better quality of image and because the EGFR protein is distributed throughout the cell36, the protein at the edges of the cell can be easily seen in the electrochemical image by using the Pt-deposited probe, while it is blurry in the image from the bare carbon probe. Conclusion Remarks In summary, the size of the sphere-shaped electrodes were precisely controlled using electrochemical deposition current (or time) allowing easy fabrication of electrodes with a desired size ranging from nanometer to micrometer. Double barrel SECM-SICM probes with deposited Pt showed higher electrochemical sensitivity than the bare nanosized carbon electrodes primarily due to enhanced faradaic current, which happened as a result of electrochemical Pt deposition; therefore, they led to electrochemically sensitive high-resolution imaging of EGFR proteins on A431 cells. We intend to use these electrodes for the electrochemical mapping of heterogeneously distributed colocalized surface proteins at cell surfaces. Acknowledgements This work was supported by a Grant-in-Aid for Development of Systems and Technology for Advanced Measurement and Analysis from the Japan Science and Technology Agency (JST). This work was supported by JST, PRESTO. This work was supported by a Grant-in-Aid for Scientific Research (A) (No. 22245011) from the Japan Society for the Promotion of Science (JSPS). Supporting Information Available Additional data and observations. This information is available free of charge via the Internet at http://pubs.acs.org/.

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Figure 1. Schematic illustration for the sensitive electrochemical imaging of EGFR proteins labeled with ALP conjugate antibodies using SECM-SICM system in hopping mode (a). Bare carbon (bi) and Pt modified (bii) SECM-SICM double barrel probes shown in respective SEM images.

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Figure 2. Images and current responses of the SECM-SICM probes with different levels of Pt deposition (ai-ii) and calibration curves showing oxidation current responses of corresponding nano/microelectrodes up to 1.0 µM of FcCH2OH in PBS (b) (n= 3). To correlate the final Pt deposition current and oxidation current response, electrodes with various final Pt deposition currents were immersed in a PBS solution containing 1.0 mM FcCH2OH at +0.5 V vs. Ag/AgCl (n = 3). The potential of the Pt-deposited electrode of the probes were held at +0.5 V vs. Ag/AgCl to oxidize FcCH2OH into FcCH2OH+. Currents shown in Fig. 2 b were background subtracted. Blue square, bare carbon; data indicated by red triangle, orange circle, green triangle and dark red circle indicate, respectively, the currents observed at the Pt deposited electrodes with final deposition current of -50 nA, -20 nA, -10 nA, and -1 nA.

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Figure 3. Approach curves for comparison of theoretical and experimental SECM currents at insulating (ai) and conductive substrates (bi). During the approach, a constant potential of +0.5 V vs. Ag/AgCl was applied through the SECM side of the probe for oxidation of 1 mM of FcCH2OH. Respective SICM currents during approach at +0.2 V vs. Ag/AgCl were shown in (aii-bii).

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Figure 4. Electrochemical imaging of immunocytochemically stained EGFR proteins on A431 cells using SECM-SICM system. Cyclic voltammetry of bare carbon (ai (red line)-aii) and a Pt deposited electrode probe (-10 nA of final deposition current) (ai (blue line)) in 1 mM of FcCH2OH+PBS. Topographic and electrochemical images of A431 cells in 4.7 mM PAPP using bare carbon (bi-bii) and Pt deposited (ci-cii) probes. SECM and SICM electrodes were held at +0.3 and +0.2 V vs. Ag/AgCl, respectively (The scanned areas with bare (bi-bii) and Pt deposited electrodes (ci-cii) are 80 × 80 µm and 75 × 75 µm, respectively. The scanned area for the magnified images in (ci-cii) is 50 × 50 µm.).

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17


Improving the Electrochemical Imaging Sensitivity of Scanning

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