1859
Electrophoresis 2015, 36, 1859–1861
Liang Chen Jinhong Guo School of Electronic Engineering, University of Electronic Science and Technology of China, Chengdu, P. R. China
Received February 9, 2015 Revised May 8, 2015 Accepted May 9, 2015
Short Communication
Printed interdigital electrodes on plastic film for tumor cells density monitoring Printed sensor has been introduced as a rapid and cost-effective platform for biomedical applications, especially for wearable biosensing and point-of-care diagnosis. In this paper, we reported a tumor cell density monitoring device with printed interdigital electrodes on a plastic film. Numerical simulation of electrical impedance spectroscopy shows good agreement with the experimental measurement of DI water and PBS solution. Different concentrations of cancer cells were used to demonstrate the capability of cell culture monitoring. The developed device is highly cost-effective and easy for fabrication, which may have great potentials in low-cost analysis of biological cells. In a word, this paper shows a more rapid and simple cell counting method as compared to tedious microscope cell counting. Keywords: Cell culture monitoring / Electrical impedance spectroscopy / Interdigital electrode / Printed sensor DOI 10.1002/elps.201500074
Flow cytometry has become the gold standard for clinical hematologic assay after development of half a century. Conventional flow cytometry requires specific fluorescent dyes to stain the target cells for sensitive detection and accurate counting [1, 2]. By comparing three optical signals, namely forward scatter, side scatter, and fluorescence signal, statistical information of cell type, cell size, and cell population, can be obtained in a few minutes. Although flow cytometry is a highly powerful technology, its cost is however intimidating. For example, a typically popular commercial flow cytometer, Beckman–Coulter FC500, is more than US $100 000. In addition, the fluorescent labeling procedure requires highly labor-intensive and time-consuming sample preprocessing, and therefore significantly limits its applicability for point-ofcare diagnosis. Recently, label-free methods such as capacitive sensing, electrical cell substrate impedance sensing, field-effect transistor based sensing and electrochemical sensing have been used for detection and enumeration of living cells [4, 5]. Among all the existing techniques, resistive pulse sensor originated from the famous Coulter principle has emerged as one of the most cost-effective platforms for cell analysis in a completely fluidic environment [6–8]. This technique has been widely used for size characterization of biological cells such as blood cells [9] and tumor cells [10]. The main limitation of Coulter principle is that it can only provide the cell size
Correspondence: Jinhong Guo, School of Electronic Engineering, University of Electronic Science and Technology of China, Chengdu, 610054, P. R. China E-mail: [email protected]
Abbreviation: EIS, electrical impedance spectroscopy
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
information based on DC resistance measurements. Electrical impedance spectroscopy (EIS) is a frequency-based method that is based on the circuit model of the biological cell. Upon the AC signal excitation, the cell model consists of resistor, inductor, and capacitor. Different concentrations of biological cells behave differently in equivalent circuit, which induces different impedance. EIS method has rendered as a more powerful platform to interrogate the electrical properties of intracellular structures at single cell level [10]. However, existing EIS approaches require complicated and expensive fabrication of microelectrodes. In this paper, we presented the EIS study of cancer cell density in aqueous buffers using printed electrodes on a plastic film. When cells are in contact with electrode surfaces, the electrical impedance may change significantly, which enables the characterization of cell density by monitoring the change in impedance signals (amplitude and phase). The cost-effective printed EIS sensor may be widely used in portable and wearable biosensing applications. A plastic film (500 m thickness) was purchased to pattern a set of two identical interdigital electrode based EIS sensors. Stencil patterns were custom-designed using AutoCAD (Autodesk, San Rafael, CA) and outsourced for fabrication on stainless steel masks. An Au-based ink from Ercon (Wareham, MA) was printed directly onto the plastic film to fabricate the conductive electrodes. The printed interdigital electrode is 100 m in width and 20 m in gap, as shown in Fig. 1. PDMS chamber with a punch hole was bond with the plastic film. And the sample volume is approximately 100 L. A three-dimensional (3D) numerical model was developed using a commercial finite element method based
Colour Online: See the article online to view Figs. 1–4 in colour.
www.electrophoresis-journal.com
1860
L. Chen and J. Guo
Electrophoresis 2015, 36, 1859–1861
Figure 2. (A) Image of the MCF-7 cells under a bright-field microscope. (B) Red dye labeled MCF-7 cells under a fluorescence microscope.
Figure 1. (A) Microscopic image of the printed interdigital electrode; B) photo of the fabricated device; (C) bending of the device. (D) The computational domain in the numerical model, the electrodes were set as the perfect electrical conductive layer.
simulation tool (COMSOL Multiphysics, CA, USA). The electrical field is governed by the following equation: E = −∇V,
(1)
where V is the electrical potential, E is the electrical field strength. The electric current density can be written as: J = E + j D,
(2)
where is the electrical conductivity, J is the current density, D is the electrical displacement field, is the angular frequency. An adaptive mesh scheme was used in the model. In the sensing aperture region, an ultrafine mesh set with a minimum mesh size of 0.1 m was used. For the rest of the computational domain, a fine mesh set was employed. The electrical current is calculated by the integration of current density over the electrode surface, expressed as: J • d S. (3) I= S
The electrical impedance is defined as the ratio of the applied voltage and the resulting electrical current: Z=
V . I
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(4)
Figure 3. Numerical and experimental impedance magnitude of DI water and PBS solution.
In the simulation, the voltage excitation has a magnitude of 1 V and its frequency range is from 1 kHz to 1 MHz. MCF-7 cells (American Type Culture Collection, MD) were cultured in DMEM supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM DMEM nonessential amino acids. The cells were grown at 37 C under 5% CO2 in a T75 flask. Figure 2A shows the MCF-7 cells under a brightfield microscope and Fig. 2B presents the MCF-7 cells stained with red fluorescent dyes under a fluorescence microscope. DI water and PBS solution were first used to test the fabricated device. A PDMS chamber used for sample loading was bonded on the plastic film immediately after plasma treatment. The impedance measurement was performed from 1 kHz to 1 MHz. As indicated in Fig. 3, the simulation results show good agreement with experimental results for both DI water and PBS solution. With the frequency above 100 kHz, the impedance magnitude of DI water in the device decreases sharply; while the impedance magnitude of PBS solution almost remains the same. This observation is due to the significant difference of electrical conductivity between DI water and PBS solution. Two MCF-7 cell suspensions in PBS solution with different concentrations, 2 × 104 cells/mL and 2 × 105 cells/mL, were used to test the fabricated device. Figure 4A shows the impedance magnitude of the two different cell suspensions
www.electrophoresis-journal.com
Electrophoresis 2015, 36, 1859–1861
Microfluidics and Miniaturization
1861
of the impedance study concludes that EIS measurement at a lower frequency is preferable for cell density monitoring. In conclusion, in this study we demonstrated a printed EIS sensor on a plastic film for cell culture monitoring. A finite element method based numerical model was implemented to simulate the EIS sensing, which agrees with the impedance measurement of DI water and PBS solution. MCF-7 cell suspensions with different concentrations were distinguished based on the impedance measurement. A lower frequency is recommended for more sensitive cell density monitoring. This study can significantly reduce the fabrication cost of EIS sensors and make them suitable for low-cost cell culture monitoring, as well as point-of-care diagnosis. This research is supported by a seed grant from Institute of Fundamental and Frontier Science at University of Electronic Science and Technology of China awarded to J. G. The authors have declared no conflict of interest.
References [1] Zieglschmid, V., Hollmann, C., Bocher, O., Crit. Rev. Clin. Lab. Sci. 2005, 42, 155–196. [2] Wang, X., Qian, X., Beitler, J. J., Chen, Z. G., Khuri, F. R., Lewis, M. M., Shin, H. J. C., Nie, S., Shin, D. M., Cancer Res. 2011, 71, 1526. [3] Prakash, S. B., bshire, P., Biosens. Bioelectron. 2008, 23, 1449–1457. Figure 4. Magnitude (A) and phase (B) of electrical impedance of two different cell suspensions: 2 × 104 cells/mL and 2 × 105 cells/mL, filled in the fabricated device.
[4] Xie, F., Xu, Y., Wang, L., Mitchelson, K., Xing, W., Cheng, J., Analyst 2012, 137, 1343–1350. [5] Li, T., Fan, Q., Liu, T., Zhu, X., Zhao, J., Li, G., Biosens. Bioelectron. 2010, 25, 2686–2689.
as a function of the frequency. PBS solution is more conducting than MCF-7 cells. As a result, a higher cell concentration exhibits greater impedance. As the frequency increases, the capacitance of cell membrane decreases and accordingly leads to a decrease in the impedance for both cell concentrations. Moreover, the contribution to the overall impedance from MCF-7 cells decreases with the increase in the frequency. Therefore, the difference in impedance magnitude between the two different cell concentrations decreases as the frequency increases. Figure 4B further reveals that the phase difference between different cell concentrations also depends on the operating frequency. Both the magnitude and phase
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[6] Guo, J., Pui, T. S., Rahman, A. R. R., Kang, Y., Electrophoresis 2013, 34, 417–424. [7] Guo, J., Li, H., Chen, Y., Kang, Y., IEEE Sens. J. 2014, 14, 2112–2117. [8] Guo, J., Li, C.M., Kang, Y., Biomed. Microd. 2014, 16, 681–686. [9] Choi, H., Kim, K. B., Jeon, C. S., Hwang, I., Lee, S., Kim, H. K., Kim, H. C., Chung, T. D., Lab Chip 2013, 13, 970–977 [10] Guo, J., Pui, T., Ban, Y., Rahman, A. R. A., Kang. Y., IEEE Trans. Biomed. Eng. 2013, 60, 3269–3275. [11] Pui, T. S., Agarwal, A., Ye, F., Balasubramanian, N., Chen, P., Small 2009, 5, 208–212.
www.electrophoresis-journal.com
Electrophoresis 2015, 36, 1859–1861
Liang Chen Jinhong Guo School of Electronic Engineering, University of Electronic Science and Technology of China, Chengdu, P. R. China
Received February 9, 2015 Revised May 8, 2015 Accepted May 9, 2015
Short Communication
Printed interdigital electrodes on plastic film for tumor cells density monitoring Printed sensor has been introduced as a rapid and cost-effective platform for biomedical applications, especially for wearable biosensing and point-of-care diagnosis. In this paper, we reported a tumor cell density monitoring device with printed interdigital electrodes on a plastic film. Numerical simulation of electrical impedance spectroscopy shows good agreement with the experimental measurement of DI water and PBS solution. Different concentrations of cancer cells were used to demonstrate the capability of cell culture monitoring. The developed device is highly cost-effective and easy for fabrication, which may have great potentials in low-cost analysis of biological cells. In a word, this paper shows a more rapid and simple cell counting method as compared to tedious microscope cell counting. Keywords: Cell culture monitoring / Electrical impedance spectroscopy / Interdigital electrode / Printed sensor DOI 10.1002/elps.201500074
Flow cytometry has become the gold standard for clinical hematologic assay after development of half a century. Conventional flow cytometry requires specific fluorescent dyes to stain the target cells for sensitive detection and accurate counting [1, 2]. By comparing three optical signals, namely forward scatter, side scatter, and fluorescence signal, statistical information of cell type, cell size, and cell population, can be obtained in a few minutes. Although flow cytometry is a highly powerful technology, its cost is however intimidating. For example, a typically popular commercial flow cytometer, Beckman–Coulter FC500, is more than US $100 000. In addition, the fluorescent labeling procedure requires highly labor-intensive and time-consuming sample preprocessing, and therefore significantly limits its applicability for point-ofcare diagnosis. Recently, label-free methods such as capacitive sensing, electrical cell substrate impedance sensing, field-effect transistor based sensing and electrochemical sensing have been used for detection and enumeration of living cells [4, 5]. Among all the existing techniques, resistive pulse sensor originated from the famous Coulter principle has emerged as one of the most cost-effective platforms for cell analysis in a completely fluidic environment [6–8]. This technique has been widely used for size characterization of biological cells such as blood cells [9] and tumor cells [10]. The main limitation of Coulter principle is that it can only provide the cell size
Correspondence: Jinhong Guo, School of Electronic Engineering, University of Electronic Science and Technology of China, Chengdu, 610054, P. R. China E-mail: [email protected]
Abbreviation: EIS, electrical impedance spectroscopy
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
information based on DC resistance measurements. Electrical impedance spectroscopy (EIS) is a frequency-based method that is based on the circuit model of the biological cell. Upon the AC signal excitation, the cell model consists of resistor, inductor, and capacitor. Different concentrations of biological cells behave differently in equivalent circuit, which induces different impedance. EIS method has rendered as a more powerful platform to interrogate the electrical properties of intracellular structures at single cell level [10]. However, existing EIS approaches require complicated and expensive fabrication of microelectrodes. In this paper, we presented the EIS study of cancer cell density in aqueous buffers using printed electrodes on a plastic film. When cells are in contact with electrode surfaces, the electrical impedance may change significantly, which enables the characterization of cell density by monitoring the change in impedance signals (amplitude and phase). The cost-effective printed EIS sensor may be widely used in portable and wearable biosensing applications. A plastic film (500 m thickness) was purchased to pattern a set of two identical interdigital electrode based EIS sensors. Stencil patterns were custom-designed using AutoCAD (Autodesk, San Rafael, CA) and outsourced for fabrication on stainless steel masks. An Au-based ink from Ercon (Wareham, MA) was printed directly onto the plastic film to fabricate the conductive electrodes. The printed interdigital electrode is 100 m in width and 20 m in gap, as shown in Fig. 1. PDMS chamber with a punch hole was bond with the plastic film. And the sample volume is approximately 100 L. A three-dimensional (3D) numerical model was developed using a commercial finite element method based
Colour Online: See the article online to view Figs. 1–4 in colour.
www.electrophoresis-journal.com
1860
L. Chen and J. Guo
Electrophoresis 2015, 36, 1859–1861
Figure 2. (A) Image of the MCF-7 cells under a bright-field microscope. (B) Red dye labeled MCF-7 cells under a fluorescence microscope.
Figure 1. (A) Microscopic image of the printed interdigital electrode; B) photo of the fabricated device; (C) bending of the device. (D) The computational domain in the numerical model, the electrodes were set as the perfect electrical conductive layer.
simulation tool (COMSOL Multiphysics, CA, USA). The electrical field is governed by the following equation: E = −∇V,
(1)
where V is the electrical potential, E is the electrical field strength. The electric current density can be written as: J = E + j D,
(2)
where is the electrical conductivity, J is the current density, D is the electrical displacement field, is the angular frequency. An adaptive mesh scheme was used in the model. In the sensing aperture region, an ultrafine mesh set with a minimum mesh size of 0.1 m was used. For the rest of the computational domain, a fine mesh set was employed. The electrical current is calculated by the integration of current density over the electrode surface, expressed as: J • d S. (3) I= S
The electrical impedance is defined as the ratio of the applied voltage and the resulting electrical current: Z=
V . I
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(4)
Figure 3. Numerical and experimental impedance magnitude of DI water and PBS solution.
In the simulation, the voltage excitation has a magnitude of 1 V and its frequency range is from 1 kHz to 1 MHz. MCF-7 cells (American Type Culture Collection, MD) were cultured in DMEM supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM DMEM nonessential amino acids. The cells were grown at 37 C under 5% CO2 in a T75 flask. Figure 2A shows the MCF-7 cells under a brightfield microscope and Fig. 2B presents the MCF-7 cells stained with red fluorescent dyes under a fluorescence microscope. DI water and PBS solution were first used to test the fabricated device. A PDMS chamber used for sample loading was bonded on the plastic film immediately after plasma treatment. The impedance measurement was performed from 1 kHz to 1 MHz. As indicated in Fig. 3, the simulation results show good agreement with experimental results for both DI water and PBS solution. With the frequency above 100 kHz, the impedance magnitude of DI water in the device decreases sharply; while the impedance magnitude of PBS solution almost remains the same. This observation is due to the significant difference of electrical conductivity between DI water and PBS solution. Two MCF-7 cell suspensions in PBS solution with different concentrations, 2 × 104 cells/mL and 2 × 105 cells/mL, were used to test the fabricated device. Figure 4A shows the impedance magnitude of the two different cell suspensions
www.electrophoresis-journal.com
Electrophoresis 2015, 36, 1859–1861
Microfluidics and Miniaturization
1861
of the impedance study concludes that EIS measurement at a lower frequency is preferable for cell density monitoring. In conclusion, in this study we demonstrated a printed EIS sensor on a plastic film for cell culture monitoring. A finite element method based numerical model was implemented to simulate the EIS sensing, which agrees with the impedance measurement of DI water and PBS solution. MCF-7 cell suspensions with different concentrations were distinguished based on the impedance measurement. A lower frequency is recommended for more sensitive cell density monitoring. This study can significantly reduce the fabrication cost of EIS sensors and make them suitable for low-cost cell culture monitoring, as well as point-of-care diagnosis. This research is supported by a seed grant from Institute of Fundamental and Frontier Science at University of Electronic Science and Technology of China awarded to J. G. The authors have declared no conflict of interest.
References [1] Zieglschmid, V., Hollmann, C., Bocher, O., Crit. Rev. Clin. Lab. Sci. 2005, 42, 155–196. [2] Wang, X., Qian, X., Beitler, J. J., Chen, Z. G., Khuri, F. R., Lewis, M. M., Shin, H. J. C., Nie, S., Shin, D. M., Cancer Res. 2011, 71, 1526. [3] Prakash, S. B., bshire, P., Biosens. Bioelectron. 2008, 23, 1449–1457. Figure 4. Magnitude (A) and phase (B) of electrical impedance of two different cell suspensions: 2 × 104 cells/mL and 2 × 105 cells/mL, filled in the fabricated device.
[4] Xie, F., Xu, Y., Wang, L., Mitchelson, K., Xing, W., Cheng, J., Analyst 2012, 137, 1343–1350. [5] Li, T., Fan, Q., Liu, T., Zhu, X., Zhao, J., Li, G., Biosens. Bioelectron. 2010, 25, 2686–2689.
as a function of the frequency. PBS solution is more conducting than MCF-7 cells. As a result, a higher cell concentration exhibits greater impedance. As the frequency increases, the capacitance of cell membrane decreases and accordingly leads to a decrease in the impedance for both cell concentrations. Moreover, the contribution to the overall impedance from MCF-7 cells decreases with the increase in the frequency. Therefore, the difference in impedance magnitude between the two different cell concentrations decreases as the frequency increases. Figure 4B further reveals that the phase difference between different cell concentrations also depends on the operating frequency. Both the magnitude and phase
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[6] Guo, J., Pui, T. S., Rahman, A. R. R., Kang, Y., Electrophoresis 2013, 34, 417–424. [7] Guo, J., Li, H., Chen, Y., Kang, Y., IEEE Sens. J. 2014, 14, 2112–2117. [8] Guo, J., Li, C.M., Kang, Y., Biomed. Microd. 2014, 16, 681–686. [9] Choi, H., Kim, K. B., Jeon, C. S., Hwang, I., Lee, S., Kim, H. K., Kim, H. C., Chung, T. D., Lab Chip 2013, 13, 970–977 [10] Guo, J., Pui, T., Ban, Y., Rahman, A. R. A., Kang. Y., IEEE Trans. Biomed. Eng. 2013, 60, 3269–3275. [11] Pui, T. S., Agarwal, A., Ye, F., Balasubramanian, N., Chen, P., Small 2009, 5, 208–212.
www.electrophoresis-journal.com
