1466 Sarah Burgarella1 Marco Di Bari2 1 Advanced
System Technology, STMicroelectronics Srl, Milan, Italy 2 Department of Electronics, Information Technology and Bioengineering, Politecnico di Milano, Milan, Italy
Received October 15, 2014 Revised February 19, 2015 Accepted February 20, 2015
Electrophoresis 2015, 36, 1466–1470
Short Communication
A portable and integrated instrument for cell manipulation by dielectrophoresis The physical manipulation of biological cells is a key point in the development of miniaturized systems for point-of-care analyses. Dielectrophoresis (DEP) has been reported by several laboratories as a promising method in biomedical research for label-free cell manipulation without physical contact, by exploiting the dielectric properties of cells suspended in a microfluidic sample, under the action of high-gradient electric fields. In view of a more extended use of DEP phenomena in lab-on-chip devices for point-of-care settings, we have developed a portable instrument, integrating on the same device the microfluidic biochip for cell manipulation and all the laboratory functions (i.e., DEP electric signal generation, microscopic observation of the biological sample under test and image acquisition) that are normally obtained by combining different nonportable standard laboratory instruments. The nonuniform electric field for cell manipulation on the biochip is generated by microelectrodes, patterned on the silicon substrate of microfluidic channels, using standard microfabrication techniques. Numerical modeling was performed to simulate the electric field distribution, quantify the DEP force, and optimize the geometry of the microelectrodes. The developed instrument includes an electronic board, which allows the control of the electric signal applied to electrodes necessary for DEP, and a miniaturized optical microscope system that allows visual inspection and eventually cell counting, as well as image and video recording. The system also includes the control software. The portable and integrated platform described in this work therefore represents a complete and innovative solution of applied research, suitable for many biological applications. Keywords: Cell manipulation / Dielectrophoresis / Lab-on-chip / Portable instrument DOI 10.1002/elps.201400481
In the last years, several laboratories have studied dielectrophoretic techniques, appearing to be a very interesting tool for solving different problems in medicine, biology, biophysics, and engineering. Dielectrophoresis (DEP) [1–3], known since the 1950s, exploits the dielectric properties of cells, suspended in a buffer medium, undergoing the action of high-gradient electric fields. The spatially nonuniform electric field induces particle polarization [1–3]: particles with higher polarizability than that of the surrounding medium experience positive DEP (pDEP), moving toward regions with high electric field, whereas particles less polarized than the surrounding medium experience negative DEP (nDEP), moving toward regions of low electric field [4,5]. DEP phenomena have been extensively studied as a label-free technique for cell separation [6]. In recent years, a more general interest in integrated biological analysis systems has been growing in the field of Correspondence: Sarah Burgarella, STMicroelectronics Srl, Via Camillo Olivetti 2, 20864 Agrate Brianza (MB), Italy E-mail: [email protected]
Abbreviations: DEP, dielectrophoresis; LOC, lab-on-chip; MEMS, microelectromechanical systems; nDEP, negative DEP; pDEP, positive DEP C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
medical diagnostics, environmental monitoring, agrofood field, and biomedical research. In particular, a progressive miniaturization of devices has been observed, which permitted mobility, automation, and convenience in mass production. Device miniaturization is obtainable, today, using MEMS (microelectromechanical systems) technology. An MEMS device is a highly miniaturized microsystem that can integrate functions of electric, mechanical, optical, and fluidic nature on the same semiconductor substrate. All components of an MEMS device, even those that are not strictly electronic, are made using standard microelectronics processes, making the manufacturing process cheap and easily implementable by integrated circuits producers. Recently, a second level of integration has been introduced, in order to integrate different laboratory functions that are normally obtainable by only using different macroscopic instruments, on the same device. Such devices are named lab-on-chip (LOC) and their development was possible thanks to the recent progress in the microfluidic field. The use of LOC allows many benefits: costs reduction, an increase in sensibility, efficiency of the processes and rapidity, and the use of a limited quantity of samples and reagents. Miniaturized systems for cellular analysis have received an increased attention in the areas of point-of-care diagnostics and biomedical research. The target consists of low-cost, www.electrophoresis-journal.com
Electrophoresis 2015, 36, 1466–1470
simple, automatized, and portable solutions for the physical manipulation of biological cells. Precise cell handling is fundamental in microcytometry and cell-counting applications. Moreover, cell manipulation and sorting are essential in LOC devices designed for molecular diagnostic applications, as they represent the preliminary stage of sample preparation, interfacing the clinical sample to the molecular domain. LOC devices based on DEP phenomena could represent the miniaturized solution for carrying on complex experimental biological experiments. The work described in this short communication has been oriented to the development of a portable instrument, integrating on the same device the microfluidic LOC for cell manipulation and all the laboratory functions (i.e., DEP electric signal generation, microscopic observation of the biological sample under test, and image acquisition) that are normally obtained by combining different nonportable standard laboratory instruments (i.e., function generator, optical microscope, and camera imaging system). Experimental tests have been performed with the purpose to confirm the predicted behavior of the cells in the various electrodes configurations designed and to demonstrate the efficacy of the system in sorting applications. In particular, viable and nonviable Saccharomyces cerevisiae yeast cells were used. The developed instrument includes an electronic board, which allows the control of the electric signal applied to electrodes necessary for DEP, and a miniaturized optical microscope system that allows visual inspection and eventually cell counting, as well as image and video recording. The system also includes the control software. The electronic board acting as function generator allows the control in frequency and amplitude of the electric signal applied to the DEP biochip. The sinusoidal, triangular and square wave outputs have amplitude adjustable between 2 and 36 Vpp and frequency adjustable up to 25 MHz. The integrated optical microscope operates in epiillumination setup at 8× magnification, allowing the visual inspection of the sample under analysis: light generated by a white LED source and reflected from the surface of the specimen reenters the objective and is directed to a complementary metal–oxide–semiconductor image sensor (Fig. 1). Thanks to the high-reflective surface of the silicon biochip, the epi-illumination setup does not require any fluorescent labeling of the cells: the images shown in Fig. 3 have been acquired from unlabeled yeast cell populations, illuminated with the white LED source. The DEP instrument is equipped with a first motorized stage allowing the regulation of the position of the biochip in the field of view of the microscope, and of a second motorized stage allowing the regulation of the focus of the image acquisition system. The functioning of the instrument is controlled by dedicated software running on a personal computer: the software allows the user to select the type of electric signal (i.e., sinusoidal, triangular or square wave output), its frequency (up to 25 MHz), and its amplitude (between 2 and 36 Vpp). A “start” button sends the DEP excitation to the electrodes of C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Microfluidics and Miniaturization
1467
Figure 1. The DEP instrument with complementary metal–oxide– semiconductor camera, LED light source, epi-illumination microscope, signal generator, and DEP biochip.
the biochip, while a “stop” button ends it. The software also allows the user to adjust the position of the biochip in the field of view by controlling the motorized stages and to manage image acquisition and video recording from the complementary metal–oxide–semiconductor camera. The DEP instrument works with dedicated LOCs connected to the electronics using a flex cable. The nonuniform electric field for cell manipulation is generated by gold microelectrodes, patterned on the silicon substrate of microfluidic channels, using standard microfabrication techniques (Fig. 2). Different geometric configurations of microelectrodes have been used, fabricating different interchangeable chips. Numerical modeling was performed to simulate the electric field distribution, quantify the DEP force, and optimize the geometry of the microelectrodes. Regarding the standard microfabrication techniques, a layer of photoresist was deposited by spin coating on the silicon substrate and patterned using a dedicated mask for each electrode configuration. A 250-nm-thick gold layer was deposited by evaporation on the patterned substrate, so that gold remained www.electrophoresis-journal.com
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S. Burgarella and M. Di Bari
Figure 2. The DEP biochip with DEP electrodes, microfluidic channel, and contact pads.
attached to silicon to form electrodes, whereas gold on photoresist was then removed with acetone. A 6 nm Cr seed layer was used to ensure better adhesion between Au and Si. The electrode width and separation were 30 m for the multibar array shown in Fig. 3. For the realization of the microfluidic channel and the insulating hexagonal structures shown in Fig. 3B and C, the negative resist SiNR 3570 was used. For the realization of the ring of the channel, the SiNR has been deposited by lamination, and the resulting channel is 50 m in height. For the realization of the insulating hexagonal pillars (Fig. 3B and C), the SiNR has been deposited by spin coating at a speed of 1200 rpm, and the resulting pillars are 3 m in height. After the soft-bake, maskless lithography was performed, utilizing a laser UV diode operating at a wavelength of 405 nm and with a resolution of 1 m. The SiNR being a negative photoresist, the material crosslinks where it is exposed to UV rays. The development is achieved by immersing the chip in isopropyl alcohol for 3 min, then rinsing and drying the chips on the spin coater. A predrilled glass slide, with thickness of 0.5 mm, was bonded directly on the ring in SiNR, using UV-activated glue, in order to obtain the ceiling of the micro channel. Using a tower drill, two openings of 1 mm in diameter were obtained at the two opposite ends of the slide, and serving as inlet and outlet of the micro channel. A pair of polycarbonate Luer connectors, used as microfluidic interfaces, is bonded on the glass in correspondence of the inlet and outlet holes, using UV-activated glue. In the experiments described in this work, the microfluidic channel is filled manually by simple pipetting directly into the inlet of the biochip a sample of 5 l of cell suspension. The microfluidic LOC can be filled also automatically, with control of the fluid’s flow, by using an external syringe pump connected through the chip’s fluidic standard Luers connections (Fig. 2).
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2015, 36, 1466–1470
Experiments on the DEP instrument have been successfully performed for the separation of viable and nonviable S. cerevisiae yeast cells on the DEP biochip. S. cerevisiae is a yeast with round to ovoid cells, about 8 m in diameter [7], and was selected for the preliminary experiments since, in general, yeast cells are widely used in dielectrophoretic applications [8, 9]. Nonviable yeast cells were obtained by heat treatment and were stained with methylene blue in order to make them better visible in the microscope observation. Two different samples were prepared with viable and nonviable stained S. cerevisiae yeast cells suspended in an aqueous solution with electric conductivity of 435 S/cm, obtained by dilution of commercially available PBS solution with DI water. Known mixture of viable and nonviable cells, with a concentration of 2 × 106 cells/mL estimated using a B¨urker cell counting chamber, was separated and selectively isolated using positive and negative dielectrophoretic forces. The suspending medium conductivity of 435 S/cm has been chosen because it enhances the differences in the dielectrophoretic behavior of the two cells populations: nonviable cells become less polarizable than the surrounding medium and can be manipulated only by nDEP (the real component of the Clausius–Mossotti factor is negative for the applied electric field frequency ranging from 100 kHz to 2MHz), while viable cells still exhibit pDEP and can be manipulated by pDEP (the crossover frequency of the real component of the Clausius–Mossotti factor is set at about 200 kHz, with nDEP for frequencies of the electric field below this value and with pDEP for frequencies of the electric field above this value). Due to their different dielectric properties [10], viable yeast cells are more polarizable than the suspending medium and are attracted at the electrodes edges by pDEP, while nonviable cells are less polarizable than the suspending medium and are repelled from the electrodes by nDEP and accumulate in the low-gradient electric field zones (Fig. 3A): nonviable cells are repelled from the electrodes by nDEP and gathered in the low-field areas (empty zones far from the electrodes, and along the axis of the electrode gaps, above the electrodes plane), while viable cells are attracted at the electrode edges by pDEP. The electric excitation applied in this experiment is a sinusoidal wave with frequency of 1 MHz and voltage amplitude of 20 Vpp. The image shown in Fig. 3A is acquired 10 s after the activation of the DEP excitation. By changing the geometry of the electrodes configuration, accumulating hexagonal structure made by insulating material (SiNR) can be introduced to gather a selected cell type (Fig. 3B and C): viable yeast cells suspended in an aqueous solution with electric conductivity of 435 S/cm are initially randomly distributed in the microchannel and gather in the insulating hexagonal structures under the action of the nDEP excitation. The electric excitation applied in this experiment is a sinusoidal wave with frequency of 100 kHz and voltage amplitude of 20 Vpp. Figure 3B shows the initial random distribution of cells in the microchannel. The image shown in Fig. 3C is acquired 25 s after the activation of the DEP excitation.
www.electrophoresis-journal.com
Electrophoresis 2015, 36, 1466–1470
Microfluidics and Miniaturization
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Figure 3. Results of the experiments on Saccharomyces cerevisiae yeast cells. (A) Separation of viable and nonviable S. cerevisiae yeast cells in aqueous solution with conductivity at 435 S/cm and sinusoidal electrical excitation with frequency of 1 MHz and voltage amplitude of 20 Vpp. (B) Viable S. cerevisiae yeast cells in aqueous solution with conductivity at 435 S/cm are randomly distributed in the microchannel. (C) Cells gather in the accumulating hexagonal structures after the nDEP excitation (sinusoidal wave with frequency of 100 kHz and voltage amplitude of 20 Vpp).
As conclusions, the results of this work demonstrate that the integration of different laboratory functions in the miniaturized DEP instrument allows the portability of DEP in point-of-care settings and opens up new possibilities for
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cell analysis in a variety of environments. The miniaturization of the DEP equipment can represent the solution for carrying on dielectrophoretic experiments with biological samples directly on field.
www.electrophoresis-journal.com
1470
S. Burgarella and M. Di Bari
The authors have declared no conflict of interest.
References [1] Pohl, H. A., J. Appl. Phys. 1951, 22, 869–871. [2] Pohl, H. A., J. Appl. Phys. 1958, 29, 1182–1188. [3] Pohl, H. A., Dielectrophoresis: The Behavior of Neutral Matter in Nonuniform Electric Fields, Cambridge, New York, Cambridge University Press, 1978. [4] Ghallab, Y., Badawy, W., IEEE Circuits and Systems Magazine 2004, 4, 5–15. [5] Wu, J., Nanobiotechnol. IET 2008, 2, 14–27.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2015, 36, 1466–1470
[6] Li, H., Bashir, R., Sens. Actuat. B Chem. 2002, 86, 215–221. [7] Urdaneta, M., Smela, E., Electrophoresis 2007, 28, 3145–3155. [8] Kriegmaier, M., Zimmermann, M., Wolf, K., Zimmermann, U., Sukhorukov, V. L., Biochim. Biophys. Acta 2001, 1568, 135–146. [9] Fatoyinbo, H. O., Hoettges, K. F., Hughes, M. P., Electrophoresis 2008, 29, 3–10. [10] Braschler, T., Demierre, N., Nascimento, E., Silva, T., Oliva, A. G., Renaud, P., Lab Chip 2008, 8, 280– 286.
www.electrophoresis-journal.com
System Technology, STMicroelectronics Srl, Milan, Italy 2 Department of Electronics, Information Technology and Bioengineering, Politecnico di Milano, Milan, Italy
Received October 15, 2014 Revised February 19, 2015 Accepted February 20, 2015
Electrophoresis 2015, 36, 1466–1470
Short Communication
A portable and integrated instrument for cell manipulation by dielectrophoresis The physical manipulation of biological cells is a key point in the development of miniaturized systems for point-of-care analyses. Dielectrophoresis (DEP) has been reported by several laboratories as a promising method in biomedical research for label-free cell manipulation without physical contact, by exploiting the dielectric properties of cells suspended in a microfluidic sample, under the action of high-gradient electric fields. In view of a more extended use of DEP phenomena in lab-on-chip devices for point-of-care settings, we have developed a portable instrument, integrating on the same device the microfluidic biochip for cell manipulation and all the laboratory functions (i.e., DEP electric signal generation, microscopic observation of the biological sample under test and image acquisition) that are normally obtained by combining different nonportable standard laboratory instruments. The nonuniform electric field for cell manipulation on the biochip is generated by microelectrodes, patterned on the silicon substrate of microfluidic channels, using standard microfabrication techniques. Numerical modeling was performed to simulate the electric field distribution, quantify the DEP force, and optimize the geometry of the microelectrodes. The developed instrument includes an electronic board, which allows the control of the electric signal applied to electrodes necessary for DEP, and a miniaturized optical microscope system that allows visual inspection and eventually cell counting, as well as image and video recording. The system also includes the control software. The portable and integrated platform described in this work therefore represents a complete and innovative solution of applied research, suitable for many biological applications. Keywords: Cell manipulation / Dielectrophoresis / Lab-on-chip / Portable instrument DOI 10.1002/elps.201400481
In the last years, several laboratories have studied dielectrophoretic techniques, appearing to be a very interesting tool for solving different problems in medicine, biology, biophysics, and engineering. Dielectrophoresis (DEP) [1–3], known since the 1950s, exploits the dielectric properties of cells, suspended in a buffer medium, undergoing the action of high-gradient electric fields. The spatially nonuniform electric field induces particle polarization [1–3]: particles with higher polarizability than that of the surrounding medium experience positive DEP (pDEP), moving toward regions with high electric field, whereas particles less polarized than the surrounding medium experience negative DEP (nDEP), moving toward regions of low electric field [4,5]. DEP phenomena have been extensively studied as a label-free technique for cell separation [6]. In recent years, a more general interest in integrated biological analysis systems has been growing in the field of Correspondence: Sarah Burgarella, STMicroelectronics Srl, Via Camillo Olivetti 2, 20864 Agrate Brianza (MB), Italy E-mail: [email protected]
Abbreviations: DEP, dielectrophoresis; LOC, lab-on-chip; MEMS, microelectromechanical systems; nDEP, negative DEP; pDEP, positive DEP C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
medical diagnostics, environmental monitoring, agrofood field, and biomedical research. In particular, a progressive miniaturization of devices has been observed, which permitted mobility, automation, and convenience in mass production. Device miniaturization is obtainable, today, using MEMS (microelectromechanical systems) technology. An MEMS device is a highly miniaturized microsystem that can integrate functions of electric, mechanical, optical, and fluidic nature on the same semiconductor substrate. All components of an MEMS device, even those that are not strictly electronic, are made using standard microelectronics processes, making the manufacturing process cheap and easily implementable by integrated circuits producers. Recently, a second level of integration has been introduced, in order to integrate different laboratory functions that are normally obtainable by only using different macroscopic instruments, on the same device. Such devices are named lab-on-chip (LOC) and their development was possible thanks to the recent progress in the microfluidic field. The use of LOC allows many benefits: costs reduction, an increase in sensibility, efficiency of the processes and rapidity, and the use of a limited quantity of samples and reagents. Miniaturized systems for cellular analysis have received an increased attention in the areas of point-of-care diagnostics and biomedical research. The target consists of low-cost, www.electrophoresis-journal.com
Electrophoresis 2015, 36, 1466–1470
simple, automatized, and portable solutions for the physical manipulation of biological cells. Precise cell handling is fundamental in microcytometry and cell-counting applications. Moreover, cell manipulation and sorting are essential in LOC devices designed for molecular diagnostic applications, as they represent the preliminary stage of sample preparation, interfacing the clinical sample to the molecular domain. LOC devices based on DEP phenomena could represent the miniaturized solution for carrying on complex experimental biological experiments. The work described in this short communication has been oriented to the development of a portable instrument, integrating on the same device the microfluidic LOC for cell manipulation and all the laboratory functions (i.e., DEP electric signal generation, microscopic observation of the biological sample under test, and image acquisition) that are normally obtained by combining different nonportable standard laboratory instruments (i.e., function generator, optical microscope, and camera imaging system). Experimental tests have been performed with the purpose to confirm the predicted behavior of the cells in the various electrodes configurations designed and to demonstrate the efficacy of the system in sorting applications. In particular, viable and nonviable Saccharomyces cerevisiae yeast cells were used. The developed instrument includes an electronic board, which allows the control of the electric signal applied to electrodes necessary for DEP, and a miniaturized optical microscope system that allows visual inspection and eventually cell counting, as well as image and video recording. The system also includes the control software. The electronic board acting as function generator allows the control in frequency and amplitude of the electric signal applied to the DEP biochip. The sinusoidal, triangular and square wave outputs have amplitude adjustable between 2 and 36 Vpp and frequency adjustable up to 25 MHz. The integrated optical microscope operates in epiillumination setup at 8× magnification, allowing the visual inspection of the sample under analysis: light generated by a white LED source and reflected from the surface of the specimen reenters the objective and is directed to a complementary metal–oxide–semiconductor image sensor (Fig. 1). Thanks to the high-reflective surface of the silicon biochip, the epi-illumination setup does not require any fluorescent labeling of the cells: the images shown in Fig. 3 have been acquired from unlabeled yeast cell populations, illuminated with the white LED source. The DEP instrument is equipped with a first motorized stage allowing the regulation of the position of the biochip in the field of view of the microscope, and of a second motorized stage allowing the regulation of the focus of the image acquisition system. The functioning of the instrument is controlled by dedicated software running on a personal computer: the software allows the user to select the type of electric signal (i.e., sinusoidal, triangular or square wave output), its frequency (up to 25 MHz), and its amplitude (between 2 and 36 Vpp). A “start” button sends the DEP excitation to the electrodes of C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Microfluidics and Miniaturization
1467
Figure 1. The DEP instrument with complementary metal–oxide– semiconductor camera, LED light source, epi-illumination microscope, signal generator, and DEP biochip.
the biochip, while a “stop” button ends it. The software also allows the user to adjust the position of the biochip in the field of view by controlling the motorized stages and to manage image acquisition and video recording from the complementary metal–oxide–semiconductor camera. The DEP instrument works with dedicated LOCs connected to the electronics using a flex cable. The nonuniform electric field for cell manipulation is generated by gold microelectrodes, patterned on the silicon substrate of microfluidic channels, using standard microfabrication techniques (Fig. 2). Different geometric configurations of microelectrodes have been used, fabricating different interchangeable chips. Numerical modeling was performed to simulate the electric field distribution, quantify the DEP force, and optimize the geometry of the microelectrodes. Regarding the standard microfabrication techniques, a layer of photoresist was deposited by spin coating on the silicon substrate and patterned using a dedicated mask for each electrode configuration. A 250-nm-thick gold layer was deposited by evaporation on the patterned substrate, so that gold remained www.electrophoresis-journal.com
1468
S. Burgarella and M. Di Bari
Figure 2. The DEP biochip with DEP electrodes, microfluidic channel, and contact pads.
attached to silicon to form electrodes, whereas gold on photoresist was then removed with acetone. A 6 nm Cr seed layer was used to ensure better adhesion between Au and Si. The electrode width and separation were 30 m for the multibar array shown in Fig. 3. For the realization of the microfluidic channel and the insulating hexagonal structures shown in Fig. 3B and C, the negative resist SiNR 3570 was used. For the realization of the ring of the channel, the SiNR has been deposited by lamination, and the resulting channel is 50 m in height. For the realization of the insulating hexagonal pillars (Fig. 3B and C), the SiNR has been deposited by spin coating at a speed of 1200 rpm, and the resulting pillars are 3 m in height. After the soft-bake, maskless lithography was performed, utilizing a laser UV diode operating at a wavelength of 405 nm and with a resolution of 1 m. The SiNR being a negative photoresist, the material crosslinks where it is exposed to UV rays. The development is achieved by immersing the chip in isopropyl alcohol for 3 min, then rinsing and drying the chips on the spin coater. A predrilled glass slide, with thickness of 0.5 mm, was bonded directly on the ring in SiNR, using UV-activated glue, in order to obtain the ceiling of the micro channel. Using a tower drill, two openings of 1 mm in diameter were obtained at the two opposite ends of the slide, and serving as inlet and outlet of the micro channel. A pair of polycarbonate Luer connectors, used as microfluidic interfaces, is bonded on the glass in correspondence of the inlet and outlet holes, using UV-activated glue. In the experiments described in this work, the microfluidic channel is filled manually by simple pipetting directly into the inlet of the biochip a sample of 5 l of cell suspension. The microfluidic LOC can be filled also automatically, with control of the fluid’s flow, by using an external syringe pump connected through the chip’s fluidic standard Luers connections (Fig. 2).
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2015, 36, 1466–1470
Experiments on the DEP instrument have been successfully performed for the separation of viable and nonviable S. cerevisiae yeast cells on the DEP biochip. S. cerevisiae is a yeast with round to ovoid cells, about 8 m in diameter [7], and was selected for the preliminary experiments since, in general, yeast cells are widely used in dielectrophoretic applications [8, 9]. Nonviable yeast cells were obtained by heat treatment and were stained with methylene blue in order to make them better visible in the microscope observation. Two different samples were prepared with viable and nonviable stained S. cerevisiae yeast cells suspended in an aqueous solution with electric conductivity of 435 S/cm, obtained by dilution of commercially available PBS solution with DI water. Known mixture of viable and nonviable cells, with a concentration of 2 × 106 cells/mL estimated using a B¨urker cell counting chamber, was separated and selectively isolated using positive and negative dielectrophoretic forces. The suspending medium conductivity of 435 S/cm has been chosen because it enhances the differences in the dielectrophoretic behavior of the two cells populations: nonviable cells become less polarizable than the surrounding medium and can be manipulated only by nDEP (the real component of the Clausius–Mossotti factor is negative for the applied electric field frequency ranging from 100 kHz to 2MHz), while viable cells still exhibit pDEP and can be manipulated by pDEP (the crossover frequency of the real component of the Clausius–Mossotti factor is set at about 200 kHz, with nDEP for frequencies of the electric field below this value and with pDEP for frequencies of the electric field above this value). Due to their different dielectric properties [10], viable yeast cells are more polarizable than the suspending medium and are attracted at the electrodes edges by pDEP, while nonviable cells are less polarizable than the suspending medium and are repelled from the electrodes by nDEP and accumulate in the low-gradient electric field zones (Fig. 3A): nonviable cells are repelled from the electrodes by nDEP and gathered in the low-field areas (empty zones far from the electrodes, and along the axis of the electrode gaps, above the electrodes plane), while viable cells are attracted at the electrode edges by pDEP. The electric excitation applied in this experiment is a sinusoidal wave with frequency of 1 MHz and voltage amplitude of 20 Vpp. The image shown in Fig. 3A is acquired 10 s after the activation of the DEP excitation. By changing the geometry of the electrodes configuration, accumulating hexagonal structure made by insulating material (SiNR) can be introduced to gather a selected cell type (Fig. 3B and C): viable yeast cells suspended in an aqueous solution with electric conductivity of 435 S/cm are initially randomly distributed in the microchannel and gather in the insulating hexagonal structures under the action of the nDEP excitation. The electric excitation applied in this experiment is a sinusoidal wave with frequency of 100 kHz and voltage amplitude of 20 Vpp. Figure 3B shows the initial random distribution of cells in the microchannel. The image shown in Fig. 3C is acquired 25 s after the activation of the DEP excitation.
www.electrophoresis-journal.com
Electrophoresis 2015, 36, 1466–1470
Microfluidics and Miniaturization
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Figure 3. Results of the experiments on Saccharomyces cerevisiae yeast cells. (A) Separation of viable and nonviable S. cerevisiae yeast cells in aqueous solution with conductivity at 435 S/cm and sinusoidal electrical excitation with frequency of 1 MHz and voltage amplitude of 20 Vpp. (B) Viable S. cerevisiae yeast cells in aqueous solution with conductivity at 435 S/cm are randomly distributed in the microchannel. (C) Cells gather in the accumulating hexagonal structures after the nDEP excitation (sinusoidal wave with frequency of 100 kHz and voltage amplitude of 20 Vpp).
As conclusions, the results of this work demonstrate that the integration of different laboratory functions in the miniaturized DEP instrument allows the portability of DEP in point-of-care settings and opens up new possibilities for
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cell analysis in a variety of environments. The miniaturization of the DEP equipment can represent the solution for carrying on dielectrophoretic experiments with biological samples directly on field.
www.electrophoresis-journal.com
1470
S. Burgarella and M. Di Bari
The authors have declared no conflict of interest.
References [1] Pohl, H. A., J. Appl. Phys. 1951, 22, 869–871. [2] Pohl, H. A., J. Appl. Phys. 1958, 29, 1182–1188. [3] Pohl, H. A., Dielectrophoresis: The Behavior of Neutral Matter in Nonuniform Electric Fields, Cambridge, New York, Cambridge University Press, 1978. [4] Ghallab, Y., Badawy, W., IEEE Circuits and Systems Magazine 2004, 4, 5–15. [5] Wu, J., Nanobiotechnol. IET 2008, 2, 14–27.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2015, 36, 1466–1470
[6] Li, H., Bashir, R., Sens. Actuat. B Chem. 2002, 86, 215–221. [7] Urdaneta, M., Smela, E., Electrophoresis 2007, 28, 3145–3155. [8] Kriegmaier, M., Zimmermann, M., Wolf, K., Zimmermann, U., Sukhorukov, V. L., Biochim. Biophys. Acta 2001, 1568, 135–146. [9] Fatoyinbo, H. O., Hoettges, K. F., Hughes, M. P., Electrophoresis 2008, 29, 3–10. [10] Braschler, T., Demierre, N., Nascimento, E., Silva, T., Oliva, A. G., Renaud, P., Lab Chip 2008, 8, 280– 286.
www.electrophoresis-journal.com
