Technical Note pubs.acs.org/ac
Carbon-Ag/AgCl Probes for Detection of Cell Activity in Droplets Kosuke Ino,*,† Kaoru Ono,† Toshiharu Arai,† Yasufumi Takahashi,‡ Hitoshi Shiku,† and Tomokazu Matsue*,†,‡ †
Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8579, Japan
‡
S Supporting Information *
ABSTRACT: In this study, we fabricated a probe consisting of a carbon nanoelectrode and an Ag/AgCl reference electrode for detecting the activity of cells in single droplets. HeLa cells were confined into a single droplet, and the alkaline phosphatase (ALP) activity of the cells was electrochemically measured using the probe inserted into the droplet. The ALP of the confined cells catalyzed the hydrolysis of p-aminophenyl phosphate (PAPP) to yield p-aminophenol (PAP) that gave electrochemical responses. Since the tip of the carbon-Ag/ AgCl probe is very small, it is useful for electrochemical analysis of cells using droplets.
C
phosphatase (ALP) activity in a single droplet. The ALP activity of HeLa cells confined in single droplets was successfully characterized by electrochemical measurements with the carbon-Ag/AgCl probe. The results indicate that the present probe is flexible and widely applicable to the electrochemical measurements in small environments.
ompartmentalization of individual samples in microenvironments, such as microwells, is becoming a powerful method for high throughput assays in chemistry and biology research. Among a variety of ways to confine samples into small environments, the use of microwells and/or droplets is the easiest and simplest method to create a specific compartment. Fluorescence or chemiluminescence detection has usually been used for characterization of chemical processes taking place in such small environments. Electrochemical methods have also been applied for analysis of limited microspaces, since various types of micro- or nanoelectrodes that meet the requirements and geometry of microenvironments are available. We have previously applied microelectrochemical measurements to analyze single-cell gene expression in microwells that were used for compartmenting and concentrating the target molecules in microenvironments.1−3 Reactions that proceed in droplets have also been detected with an electrochemical method.4−6 Gao et al. reported using positionable dual microelectrodes that consist of a nanometer-to-micrometerradius Au disk working electrode and an approximately 80 μmradius Ag/AgCl reference electrode for single-cell analysis.5 Spaine et al. reported using positionable voltammetric cells with a theta capillary that consist of carbon fiber microelectrode sealed in epoxy and Ag/AgCl reference electrode to make electrical contact to the analyte solution via a salt bridge at the tip.4 Since these probes have an internal reference electrode, the tip size can be small enough to insert the probe into a single droplet. We have also previously fabricated nanoelectrodes7 and multifunctional nanoprobes with a nanoelectrode and electrolyte-filled nanochannel to be used for scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM).8 In this study, we used double-barrel nanoprobes that consist of carbon nanoelectrode and reference electrode (carbon-Ag/ AgCl probe) for electrochemical measurement of alkaline © 2013 American Chemical Society
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MATERIALS AND METHODS Probe Fabrication. The fabrication process was described in our previous paper.8 Briefly, a quartz theta glass capillary (Sutter Instrument, USA) was pulled using a CO2 laser puller (model P-2000, Sutter Instrument, San Rafael, CA, USA) to make a fine double barrel tip. Both of the ends of the barrels were blocked with reusable putty-like pressure-sensitive adhesive. Then, one of the barrels was opened, and butane gas was passed through to deposit carbon inside this barrel only. The taper of the pipet was inserted into another quartz capillary, which was filled with nitrogen gas to prevent oxidation of the carbon layer and bending of the capillary by high temperature. This approach also protected the pipet aperture from closing through softening of the quartz pipet walls. To form a pyrolytic carbon layer inside the capillary, the pipet taper was then heated with a Bunsen burner. The deposited layer of carbon inside the pipet was observed by scanning electron microscopy (SEM). After fabricating the carbon electrode, a 0.1 M KCl solution was filled in another pipet using a small needle. After filling, air in the tip of the probes was removed by tapping and/or heating the tip. An Ag/ AgCl wire was inserted into the solution for a reference electrode (Figure 1). The carbon-Ag/AgCl probes were stored Received: December 9, 2012 Accepted: March 14, 2013 Published: March 14, 2013 3832
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Technical Note
Figure 1. Schematic illustration of electrochemical detection of cell activity in a droplet using a carbon-Ag/AgCl probe.
Figure 2. SEM image of a carbon-Ag/AgCl probe.
barrel. The probe has a carbon nanoelectrode as a working electrode and Ag/AgCl electrode as a reference/counter electrode. We used a 0.1 M KCl solution for the internal Ag/ AgCl electrode. Since the integrated carbon-Ag/AgCl probe was easily and rapidly fabricated,8 the probe can be useful for electrochemical detection of droplets. The cyclic voltammogram of 0.50 mM FcCH2OH using the internal reference electrode (Ag/AgCl/0.1 M KCl) is very similar to that with the external reference electrode (Ag/AgCl/ sat. KCl), while the voltammogram shifts in the negative direction (approximately 100 mV) (Figure 3). This shift is
in air. Since the solution in the carbon-Ag/AgCl probes was evaporated gradually, the probe was washed with water and a 0.1 M KCl solution was filled again before the experiments. Characterization of Carbon-Ag/AgCl Probes. To characterize the carbon-Ag/AgCl probes, cyclic voltammograms (scan rate: 100 mV/s) of bulk solutions of 0.50 mM ferrocenemethanol (FcCH2OH) in 0.1 KCl were recorded using those carbon-Ag/AgCl probes. A two-electrode system comprising the carbon electrode as the working electrode and an internal Ag/AgCl reference/counter electrode or an external Ag/AgCl reference/counter electrode was employed for voltammetry. The carbon-Ag/AgCl probes were then applied to detect ALP activity. ALP (Oriental Yeast Co., Ltd., Japan) was added into Tris−HCl buffer (pH 9.5) containing 1.0 mM p-aminophenyl phosphate (PAPP, LKT Lab Inc., USA) as a substrate and 2.0 mM MgCl2. After a 30 min incubation to allow ALP-catalyzed hydrolysis of PAPP to yield p-aminophenol (PAP),9,10 the carbon-Ag/AgCl probe was inserted into the bulk solution and cyclic voltammetry was performed. Electrochemical Detection of Cell Activity in a Droplet. Figure 1 shows a schematic illustration of electrochemical detection of cell activity in a droplet using a carbonAg/AgCl probe. HeLa cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). The HeLa cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 50 μg/mL penicillin/streptomycin at 37 °C under a 5% CO2 humidified atmosphere. The HeLa cells were harvested and suspended into Tris−HCl buffer (pH 9.5) containing 1.0 mM PAPP and 2 mM MgCl2. The droplets (0.1 μL) of the cell suspension were pushed out to the mineral oil with a pipetman. The droplets containing the HeLa cells were observed with a microscope (Nikon Diaphot 200, Nikon, Tokyo, Japan) to count the number of HeLa cells in the droplets. The droplets were trapped into a cone-shape microwell11 to control the position of the droplets correctly. After a 30 min incubation for hydrolysis of PAPP to PAP catalyzed by ALP from the HeLa cells, the carbon-Ag/AgCl probe was approached from the mineral oil phase to the droplets. The tip potential was maintained at 0.30 V vs the internal Ag/AgCl to oxidize PAP. After inserting the carbonAg/AgCl probe into the droplet, cyclic voltammetry was performed. The position of the carbon-Ag/AgCl probe was controlled with a motor-driven XYZ-positioner (Chuo-Seiki M9103, Japan).
Figure 3. Cyclic voltammograms (scan rate = 100 mV/s) of bulk solutions of 0.50 mM FcCH2OH in 0.1 M KCl with an external reference (red, Ag/AgCl/sat. KCl) and an internal reference (blue, Ag/AgCl/0.1 M KCl) electrode.
caused by the difference of Cl− concentration in the reference electrode. The radius of the carbon electrode was roughly calculated as 1.0 μm from the plateau current using 7.8 × 10−10 m2 s−1 as the diffusion coefficient of FcCH2OH.12 Since the size of the carbon electrode differed every time when carbon electrodes were fabricated, the radius of the carbon-Ag/AgCl probes were evaluated by the use of 0.50 mM FcCH2OH solution for every probe. The carbon-Ag/AgCl probe was used to evaluate the ALP activity using PAPP as a substrate. Figure 4 shows cyclic voltammograms on the probe in bulk solutions containing 1.0 mM PAPP and 0−10 ng/mL ALP. ALP catalyzes the hydrolysis of PAPP to yield PAP. The electrochemical signal for PAP increases proportionally with the ALP concentration, indicating that the carbon-Ag/AgCl probe can be used for quantitative determination of ALP. Due to irreversibility of PAP oxidation,13 steady-state current for PAP was not observed. The CVs of PAP inside the droplet were similar to those in bulk solution (Figure S1, Supporting Information). On the basis of the above results, we applied the carbon-Ag/ AgCl probe to evaluate the ALP activity of HeLa cells trapped
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RESULTS AND DISCUSSION Figure 2 shows the SEM image of the tip of the carbon-Ag/ AgCl probe. One barrel was filled with the carbon, and the radius of the carbon at the tip was determined to be approximately 200 nm. Another empty barrel was filled with an electrolyte, and Ag/AgCl electrode was inserted into the 3833
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Figure 4. Cyclic voltammograms (scan rate = 100 mV/s) of bulk solutions of 1.0 mM PAPP in Tris−HCl (pH9.5) and 0−10 ng/mL ALP. The voltammetry was performed after a 30 min incubation.
Figure 6. Electrochemical detection of cell activity in a droplet using carbon-Ag/AgCl probes. Cyclic voltammetry was performed after inserting the carbon-Ag/AgCl probes into the droplet. The scan rate was 100 mV/s. The potential was scanned from −0.20 to 0.30 V. The currents at 0.30 V were plotted onto a graph. Blue (9, 56, 59, 60, and 95 cells), yellow (0, 10, 44, and 55 cells), and red (1, 22, 29, 34, 36, and 46 cells) symbols indicate the electrochemical signals using three difeferent carbon-Ag/AgCl probes (plateau current in CVs of 0.50 mM FcCH2OH: 0.57, 0.80, and 1.15 nA for blue, yellow, and red, respectively).
in a single droplet. However, because of the small size, it was impossible to guide the tip of the carbon-Ag/AgCl probe with the optical microscopes. Thus, the insertion of the tip of the carbon-Ag/AgCl probe into the droplet was confirmed by the use of the approach curves. Figure 5 shows the approach curve
estimated at 2.6 μM. We have previously reported the heterogeneity of single cells by detecting ALP activity of individual HeLa cells.10 The scattered relationship between number of cells and the current in Figure 6 might be caused by differences in activity of individual cells. Variation of droplet size and/or electrode fouling may also result in the scatter in the data in Figure 6. Partition of PAPP and PAP between water and oil phases was investigated. A 500 μL solution of Tris−HCl buffer containing 1.0 mM PAPP or PAP was mixed with the same volume of mineral oil for 15 min. The concentration of remaining PAPP in the Tris−HCl buffer was determined by cyclic voltammetry and found to decrease by 17%. Although these results show the PAPP was slightly partitioned from water to oil phase or denatured during the incubation, most of PAPP remained in the droplets since PAPP concentration in oil phase was very low due to its phosphate group. In this study, we did not optimize PAPP concentration. For more sensitive detection, the optimization will be necessary. The PAP concentration dropped by about 40% after the partition, indicating that accumulation of PAP after ALP-catalyzed reaction was not significant. In this study, droplets were used to compartment cells and to show the utility of the carbon-Ag/AgCl probes in small environments. Since ALP has been frequently used as a reporter protein, detection of ALP at single cell level in a small environment has attracted much interest.14 We have previously reported electrochemical chip device for cell analyses through ALP detection for bioassays,15 such as reporter gene assay16 and evaluation of embryonic stem (ES) cells.11,17,18 Compared to the chip device-based cell analysis, the present droplet-based cell analysis is flexible and the size of the microenvironment is controllable, although it is not a high throughput analysis. Small droplets are used for analysis of mass transfer of redox compounds through water/oil phase.19,20 Since the tip of the carbon-Ag/AgCl probe is very small, the carbon-Ag/AgCl probe can also be applied for the analysis of mass transfer of redox compounds through water/oil phase.
Figure 5. Approach curve from the bulk of the volume of mineral oil to the water droplets containing 1.0 mM PAP. The tip potential was 0.30 V vs the internal Ag/AgCl reference electrode. The scan rate was 2.0 μm/s.
when the carbon-Ag/AgCl probe was moved from the bulk of mineral oil to the water droplet containing 1.0 mM PAP. The current increased dramatically when the tip was inserted completely into the droplet because PAP was oxidized at the carbon electrode. The approach curve shows an initial jump in current, followed by a slow increase in current, and then a second rapid rise. At the initial jump, the probe might be in contact with the droplet. At the second jump, the probe must be inserted into the droplet completely and PAP is efficiently oxidized. The tip was further inserted by 20−30 μm, and the ALP activity in the droplet was evaluated by cyclic voltammetry. Figure 6 shows the relationship between the concentration of PAP and the number of HeLa cells trapped in a single droplet. The concentration of PAP in a droplet was estimated from the voltammograms by the use of the empirical equation
C = 0.6(iPAP/iFc) where C is the concentration of PAP in mM, iPAP is the oxidation current (pA) at 0.30 V in a droplet, and iFc is the plateau current (pA) of 0.50 mM FcCH2OH using the same probe. The concentration increased with the number of the cells, which indicates that the present method can be applied for detection of ALP activity in cells in a droplet. The PAP concentration in a droplet (0.1 μL) containing a single cell was 3834
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Technical Note
(17) Ino, K.; Nishijo, T.; Arai, T.; Kanno, Y.; Takahashi, Y.; Shiku, H.; Matsue, T. Angew. Chem., Int. Ed. 2012, 51, 6648−6652. (18) Ino, K.; Kanno, Y.; Nishijo, T.; Goto, T.; Arai, T.; Takahashi, Y.; Shiku, H.; Matsue, T. Chem. Commun. 2012, 48, 8505−8507. (19) Ino, K.; Kanno, Y.; Arai, T.; Inoue, K. Y.; Takahashi, Y.; Shiku, H.; Matsue, T. Anal. Chem. 2012, 84, 7593−7598. (20) Nakatani, K.; Sudo, M.; Kitamura, N. J. Phys. Chem. B 1998, 102, 2908−2913.
CONCLUSIONS ALP activity of cells in single droplets was electrochemically detected by the probe that consists of the carbon electrode and the Ag/AgCl reference electrode. PAP was produced from ALP-catalyzed hydrolysis on the HeLa cells compartmented in the droplets, and the electrochemical signals from PAP oxidation on the electrode were successfully detected by the use of the carbon-Ag/AgCl probes. The carbon-Ag/AgCl probe can be widely applicable for electrochemical cell analyses using droplets.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (K.I.); matsue@ bioinfo.che.tohoku.ac.jp (T.M.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (A) (No. 22245011) and a Grant-in-Aid for Young Scientists (B) (No. 23760745) from the Japan Society for the Promotion of Science (JSPS). This work is partly supported by the Cabinet Office, Government of Japan, through its “Funding Program for Next Generation World-Leading Researchers”.
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REFERENCES
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