50 IEEE JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, VOL. 8, NO. 1, MARCH 1999 Electric Impedance Spectroscopy Using Microchannels with Integrated Metal Electrodes H. Edward Ayliffe, A. Bruno Frazier, Member, IEEE, and R. D. Rabbitt Abstract—Microelectric impedance measurement systems containing microchannels with integrated gold electrodes were fabricated to enable EI measurements of femtoliter (10015 ) volumes of liquid or gas. The microinstruments were characterized using samples of air, partially deionized water, and saline solutions with various ionic concentrations over the frequency range of 100 Hz to 2 MHz. Resulting spectral patterns varied systemically as a function of ionic concentration. In addition to industrial sensing applications, this technology may prove to be beneficial in monitoring microsystems utilizing on-chip fluid chemistry, measuring the dielectric dispersion of polymer solutions, and determining the electrical properties of isolated biological materials. [296] Index Terms— Biological cells, electric impedance, micromachine, spectroscopy. I. INTRODUCTION E LECTRIC impedance (EI) measured in a onedimensional (1-D) electric field is an established method for interrogating the electromagnetic behavior of isolated materials and composite systems. Microfabrication techniques offer a low-power means of applying traditional EI concepts to the investigation of complex-valued dielectric properties of small structures and material samples. Reducing the overall size of an EI measurement device allows for increased spatial resolution while limiting the possibility of dielectric breakdown by minimizing the strength of the required electromagnetic field. In addition, microscale EI devices can be fabricated to interrogate femtoliter subdomains within nanoliters of total sample solution. In previous works, microfabricated EI sensors have demonstrated the ability to sense variations in solution temperatures, ionic concentrations, hydrogen peroxide concentrations, and even antigen-antibody binding (immunosensors) [1], [2]. These systems typically interrogate solutions using an array of surface-mounted metal electrodes with an active surface area of 1 mm . To further reduce the required sample size, we fabricated EI systems containing microchannels lined with metal electrodes. The devices allow materials to be positioned between interrogating electrodes using rapid and efficient fluid transport methods [3]–[5]. In addition to the numerous Manuscript received August 22, 1997; revised September 14, 1998. This work was supported in part by the Whitaker Foundation and the University of Utah. Subject Editor, K. Petersen. H. E. Ayliffe and R. D. Rabbitt are with the Department of Bioengineering, University of Utah, Salt Lake City, UT 84112-9202 USA (e-mail: ted.ayliffe@m.cc.utah.edu). A. B. Frazier is with the Department of Electrical Engineering, University of Utah, Salt Lake City, UT 84112-9202 USA. Publisher Item Identifier S 1057-7157(99)01184-1. applications microscale EI devices have in basic science and engineering, their size makes them especially suitable for biological studies. An investigation into the dielectric properties of proteins is one of many possible applications for an integrated microchannel/EI measurement system. The electrical characteristics of protein solutions are believed to play an important role in physiologic functions that involve protein–protein and charged ligand interactions [6]–[8]. In these studies, the dielectric dispersions of protein solutions were described in terms of the summation of distributed charges within the protein (polarization vector) and proton fluctuations. Microscale EI devices can also be applied to measure the dielectric properties of individual cells and cell aggregates. Cellular electrical properties are typically expressed as voltage/chemically dependent membrane capacitance, membrane resistance, and cytoplasmic resistance—properties which are critical to the physiology of all living cells. The three most common techniques that have previously been applied to measure cellular electrical properties include the EI cell suspension technique [9]–[11], whole-cell patch clamping [12]–[14], and electrophoresis/electrorotation [15]–[26]. All of these methods rely on estimates of cell geometry for the accurate calculation of the electrical parameters and are generally limited to wholecell resolution. Microchannels outfitted with EI measurement electrodes have the potential to supplement these traditional methods by providing increased spatial resolution (by using electrodes smaller than a single cell and by constraining the cell within the microchannel) and extending the frequency range [27]. This paper describes the fabrication of a device suitable for EI measurements of femtoliters of fluids, solutions, suspended particles, and single cells. Fig. 1 is a schematic of the microdevice in which two fluid reservoirs are connected by a single microchannel that narrows to a width of 10 m in the recording zone (channels are 4 m high). The microchannels were constructed from epoxy-based photoresist on quartz glass wafers. Full-depth gold measurement electrodes were integrated into the narrowest portion of the microchannels. Through holes were wet etched in glass coverslips and bonded to the microstructures to form the top surface of the microchannels. The resulting microchannels and electrodes are sandwiched between planar glass substrates exhibiting excellent optical qualities and allowing for direct sample observation using transmission light microscopy. Parasitic capacitance between the electrodes is minimized by isolating the electrodes on all sides with materials with high dielectric 1057–7157/99$10.00 1999 IEEE AYLIFFE et al.: ELECTRIC IMPEDANCE SPECTROSCOPY USING MICROCHANNELS 51 Fig. 1. Schematic of microelectric impedance measurement device with gold electrodes integrated into a microchannel. constants (glass and epoxy-based photoresist). The microEI measurement system was characterized using biological concentrations of ionic salt solutions, air, and deionized (DI) water over the frequency range of 100 Hz to 2 MHz. In addition, preliminary EI data from human polymorphoneuclear leukocytes (PMN’s) and teleost fish red blood cells (RBC’s) positioned within the recording zone demonstrate the potential to use EI spectral patterns to distinguish cell types. II. METHODS A metal seed layer (250 Å Ti and 750 Å Au) was sputtered on 3-in-diameter quartz glass (Pyrex 7740) substrates, patterned, and etched to form the approximate electrode dimensions, electrical connecting pads, and the conducting grid for electroplating [Fig. 2(1)]. A 4.5- m-thick layer of epoxybased photoresist SU-8 (Microlithography Chemical Corp., Newton, MA) was spun over the metal seed layer and patterned to form the fluid reservoirs and the wider portions of the connecting channel [Fig. 2(2)]. Gold microelectrodes were slowly electroplated using a low-current density (1 mA/cm ) until the electrodes were 0.5 m below the top surface of the photoresist [Fig. 2(3)]. Following electroplating, the photoresist was cured on a hot plate at 125 C for 15 min to reduce cracking during subsequent fabrication procedures. A 6000-Å-thick layer of aluminum was then sputtered on the entire wafer, patterned, and etched to act as a mask for the microchannel etching procedure [Fig. 2(4)]. An oxygen plasma etch was used to form the narrowest portion of the microchannel ( 10 m wide), which lies between the gold electrodes [Fig. 2(5)]. The unwanted photoresist and aluminum were then wet etched from the surface of the wafer and thoroughly rinsed in DI water in preparation for coverslip bonding [Fig. 2(6)]. Coverslips were fabricated to enclose the microchannels by providing the top surface of the channels and simultaneously add depth to the fluid reservoirs. Baxter Scientific no. 1.5 Fig. 2. Micromachining steps for fabrication of a microelectric impedance device. Fabrication of microelectric impedance device. (1) Sputter and pattern metal (Ti and Au) electroplating seed layer. (2) Spin on SU-8 photoresist and pattern for electroplating and fluid reservoirs. (3) Electroplate gold electrodes. (4) Sputter and pattern Al mask layer for microchannel etching. (5) O2 plasma etch microchannels and remove Al layer. (6) Bond glass coverslips. coverslips (McGaw Park, IL) were sputtered on both sides with a layer of chromium and gold. The metal layers were patterned and etched to expose clean glass in the areas forming the reservoirs and a border to separate each coverslip from neighboring glass. Additional photoresist was spun on and patterned on both sides to act as an additional mask during the glass etching. The exposed glass areas were wet etched in a 50% HF acid and DI water solution at 30 C. The square reservoir holes were etched completely through the 200- m-thick coverslips after approximately 15 min in the acid etchant. Once the individual coverslips were formed, the masking materials (Cr, Au, and photoresist) were stripped, and the coverslips were cleaned using a 70 : 30 solution of H SO and H O in preparation for bonding. 52 IEEE JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, VOL. 8, NO. 1, MARCH 1999 The fabricated coverslips were aligned over selected microchannel/electrode structures using a micromanipulator and an inverted light microscope (Olympus IMT2, Lake Success, NY). Once in position, pressure was applied to the center of the coverslip to prevent slippage and reduce the air gap between the glass and underlying microstructures. Small ( 0.5 l) drops of UV curable PVC adhesive (Loctite 3301, Rocky Hill, CT) were applied to the coverslip edge and allowed to wick under the glass by capillary forces. The compressive load from the micromanipulator slowed the flow of adhesive and greatly delayed the adhesive from flowing into the microchannels. The adhesive flow was monitored by light microscopy and was cured in place by switching on an ADAC Technologies UV source (Bantan, CT). Glass pipettes were bonded over the coverslip holes both to provide a simple and reliable means for connecting the microchannels to the external reservoirs and to help prevent unwanted evaporation of the small aliquots of test samples. The ends of 2-mm-square glass tubing (Friedrick and Dimmock, Millville, NJ) were cut at approximately 45 and ground smooth using a hand-held rotary tool (Craftsman, Sears Roebuck Co, Chicago, IL) to a length of 10 mm. The glass tubes were aligned and held in place over the fluid reservoirs using a micromanipulator and an Olympus dissecting microscope. The same UV curable bonding technique used to bond the coverslips was used to adhere the pipettes to the coverslips. Following bonding, the devices were tested for leaks using DI water under light microscopic observation. With the glass tubes in place, soft Silastic tubing (Dow Corning, Midland, MI) could be connected to the glass tubes to manipulate bulk flow with the application of positive or negative pressure. Six devices were electrically characterized over a frequency range of 100 Hz to 2 MHz using varying concentrations of phosphate buffered saline (PBS) solutions (0.5, 1, 5, and 10 times the physiologic concentration of 300 mOsm), DI water, and air. The gold electrodes were connected to a computercoupled (IEEE 488, GPIB) impedance analyzer (HP4194A) using a minimal length ( 18 in) of 22-gauge RF lead wire (Channel Master, Smithfield, NC) outfitted with gold tips. Prior to performing the impedance measurements on each device, the impedance analyzer with the cable connected was set to the open circuit configuration. The gold-tipped cable was brought into contact with the bonding pads on the microdevice using micromanipulators. Three consecutive impedance sweeps were recorded on the empty device (air measurements). DI water was added to the open fluid reservoir and negative pressure was applied to the opposite reservoir via flexible tubing to help the water flow completely into the microchannel. To assure that the fluid had filled the channel, it was continuously monitored with a Nikon Diaphot 200 confocal microscope (Melville, NY). After the DI water filled the channel, three consecutive impedance sweeps were performed. The four PBS solutions were measured from low to high salt concentration using the same method. Between each PBS solution measurement, water was flushed through the device for approximately 5 min using negative pressure and allowed to air dry. PMN’s and RBC’s were selected for the initial wholecell studies using the micro-EI devices. The PMN’s were obtained from healthy human donors and supplied by the Cardiovascular Research and Training Institute (CVRTI) at the University of Utah. Blood was stored at 4 C for no longer than 8 h. Leukocytes were isolated following methods previously reported by Keller et al. (1983) [28]. The RBC’s were obtained from a teleost fish, Opsanus tau. Unlike mammalian erythrocytes, these RBC’s are nucleated, have dimensions similar to human PMN’s, and hence are appropriate for EI spectral comparison. An aliquot of cell suspension was then added to the cell reservoir of the microdevice. The temperature of the device and cells was maintained at 19 C. Microbore silicone tubing was secured to the fluid reservoir opposite the cells to control the channel flow using suction. Cell-loaded devices were positioned on the stage of an inverted Nikon confocal microscope. The RF lead wires, configured with rigid gold contacts, were lowered under visual observation to mate with the gold contact pads on the surface of the device. The network analyzer was appropriately calibrated to account for RF transmission to the remote recording site. Cells were positioned between the electrodes via control of the bulk flow with pressure. EI spectra were measured between electrode contact pads over the frequency range from 100 Hz to 2 MHz. For each cell, the EI spectra were recorded with bathing solution in the recording zone and with a stationary cell in the recording zone. Raw spectral data, collected in this way, are therefore sensitive to dielectric properties of the microdevice, the material sample, and the geometrical configuration. III. RESULTS A scanning electron micrograph (SEM) of one of the microchannels with the integrated gold electrodes is shown prior to coverslip bonding in Fig. 3. The microchannel in the figure measures 10 m wide and 4.3 m deep with the gold electrodes measuring 8.0 m wide and 4.0 m thick. The microchannel width was designed slightly wider than the electrode gap to ensure solution/electrode contact. For this reason, the gold electrodes typically extended into the channel 1–2 m on each side. The resulting average distance between the tips of the electrodes, or electrode gap, measured 7.1 m. Prior to coverslip bonding, the depth of the channels was measured using a Dektak IIA profilometer (Sloan Technologies, Santa Barbara, CA) and found to vary by only 0.11 m with a mean value of 4.31 m. Devices with a total of eight different microchannel and electrode geometries were designed and fabricated on each wafer. An SEM of a microchannel with a bonded coverslip is shown overlaid on an image of an electrode/microchannel device prior to bonding (see Fig. 4). Square holes were wet etched completely through the 200- m-thick coverslip glass in addition to etching each coverslip free from the adjacent neighbors. The gold bonding pads are shown in Fig. 4 extending from the edges of the bonded glass coverslip. Fig. 5 shows the magnitude and phase of the EI of one device as a function of frequency when filled with air and the five different solutions between the electrodes. Individual curves are averages of three frequency sweeps. Resolution was limited to magnitudes 100 M . For this reason, the AYLIFFE et al.: ELECTRIC IMPEDANCE SPECTROSCOPY USING MICROCHANNELS 53 Fig. 3. SEM of microchannels with integrated gold electrodes prior to coverslip bonding. Fig. 4. SEM of a coverslip bonded to a microstructure can be seen (right) overlaid on an SEM of an electrode/microchannel device prior to bonding. impedance of the air-filled device could not be measured at frequencies below 100 kHz. Resulting EI data reflect properties of both the material sample in the microchannel and the current path around the sample (i.e., dielectric properties of the microdevice). Differences between the magnitude of the impedance for the five salt solutions were statistically tested at 10, 100, 500 k, significant (student -test, and 1 MHz). The average magnitude of impedance for the solutions demonstrated a nearly linear response with a constant slope (on a log scale) over the frequency range of 1 kHz to 2 MHz. Larger magnitudes correspond to the solutions having lower ionic concentrations. The phase angle generally becomes flatter for solutions with lower ionic concentrations (lower conductivity). Variations between devices were also examined. Eight different EI devices were designed and six were tested. Due to fluctuations in the design and fabrication process, the resulting microstructures have varying microchannel widths, 54 IEEE JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, VOL. 8, NO. 1, MARCH 1999 Fig. 5. (a) Magnitude and (b) phase of the device impedance with air, DI water, and varying concentrations of PBS between the electrodes. electrode shapes, and electrode gaps. This variability requires that each device be calibrated using materials of known dielectric properties for applications requiring measurement of the absolute constitutive properties of the sample. Fig. 6 shows the magnitude and phase of impedance versus frequency of six devices filled for the 300 mOsm concentration of PBS. All of the devices tested using this solution displayed impedance curves with similar characteristics. Although the magnitude of the impedance can be seen to decrease with increasing frequency for all of the devices, the rate of decrease and starting magnitude varied by up to one order of magnitude. In addition, the corresponding phase angles were considerably different between devices. Differences are believed to be due to variability in channel/electrode dimensions and geometry (see Table I). The presence of biological cells between the electrodes influences the EI of the device. Fig. 7(a) shows the magnitude of the impedance at each frequency for the PMN’s and RBC’s . Fig. 7(b) represents the corresponding phase information. Error bars on both graphs depict one standard deviation. In these recordings, relatively small channels were used such that PMN’s and RBC’s positioned between the electrodes completely filled the lumen of the channel. Differences between the device EI for the two cell types are statistically significant. The resulting confidence for the magnitude interval was greater than 99.5% and phase for all four of the frequencies depicted. IV. DISCUSSION Fabrication of the gold and epoxy-based photoresist structures on glass substrates used techniques similar to standard Fig. 6. (a) Magnitude and (b) phase of the impedance for six microelectric impedance devices when filled with a physiologic concentration of PBS (300 mOsm). TABLE I MICROCHANNEL/ELECTRODE DIMENSIONS OF THE SIX DEVICES TESTED Device Number 1 2 3 4 5 6 Channel Width (m) 10.5 10.5 10.5 10.1 10.5 10.5 Electrode Width (m) 7.7 7.7 8.8 8.5 8.2 9.0 Electrode Gap (m) 5.1 9.5 6.2 7.7 8.9 5.4 microlithography procedures using silicon wafers. Utilizing the epoxy-based photoresist SU-8 enabled the fabrication of microchannels for the gold electroplating with nearly perpendicular sideways and aspect ratios of 1 : 1 for photoresist depths of 5 m. The resulting micro-EI devices have much smaller electrode surface areas ( 32 m ) compared to recently reported efforts with planar devices and electrodes [1], [2], [29]. This reduction in surface area allows for much greater spatial resolution but also introduces electric field fringe effects. By integrating the gold electrodes into the sides of the microchannel, the limitations of planar technologies are avoided and the resulting micro-EI devices can be incorporated into numerous applications involving microfluidics. The threedimensional microchannels provide a repeatable geometry and an efficient method for measuring EI differences of extremely small material samples. The volume of fluid required to fill 150 m length of the microchannel is 6 pl, while the volume required to fill the recording zone between the electrodes is only 120 fl. The microchannel designs that gradually tapered to 10 m in width (as seen in Fig. 3) demonstrated AYLIFFE et al.: ELECTRIC IMPEDANCE SPECTROSCOPY USING MICROCHANNELS (a) (b) Fig. 7. The (a) magnitude and (b) phase of the impedance for PMN’s and RBC’s are shown at four selected frequencies. Error bars depict one standard deviation. Differences between the two cell types in both magnitude and phase are statistically significant (student t-test, < 0:005) at all frequencies tested. 6 more predictable flow characteristics compared to channels with square ends when small particles or cells were being manipulated using pressure gradients. Preliminary experiments manipulating and measuring the impedance of isolated erythrocytes and leukocytes have demonstrated applicability of this technology to be used in future cellular studies such as cell sorting, counting, or membrane biophysical characterization. The magnitude and phase of EI versus frequency when the recording zone is filled with varying concentrations of PBS, DI water, and air (as seen in Fig. 5) demonstrate interesting lowfrequency characteristics. At the lowest frequency measured (100 Hz), one might expect the resistive characteristics of the PBS solutions to dominate, resulting in a phase angle of 0 . All of the solutions measured, however, showed large capacitive properties with negative phase angles up to 75 . The possibility of artifact is highly remote in that impedance sweeps were conducted with various circuit elements of similar magnitude (resistors and capacitors) and measurements of nonionic solutions generated expected results. These negative phase 55 angles are believed to be primarily due to the electrochemical kinetics of ionic solutions with metal electrodes. The interface between the metal electrodes and ionic solution is referred to as the double layer and is often modeled using serial RC circuit elements [30]–[32]. In some cases, the RC circuit models can be tuned to closely approximate the double layer kinetics of capacitors with large area-to-separation-distance ratios for frequencies below 100 kHz [31], but parameter values depend upon specific electrode and solution composition. At higher frequencies, simple RC models cannot predict the frequency dispersion that arises due to the relaxation time required for adsorption-desorption of ions to the metal electrodes. The RC values for the double layer are known to change by a factor of three for every order of magnitude change in frequency [32]. The apparent resistive and capacitive effects of the double layer heavily influence both the low frequency magnitude and phase. As the frequency of the interrogating signal begins to exceed 1 kHz, the solution resistance begins to dominate the impedance magnitude and differences in ionic concentrations can be easily measured. The differences in the magnitude between the measured EI spectra when the recording zone contained air, water, and the four different concentrations of PBS were statistically significant (Fig. 5). The calculated confidence intervals using student -tests at four selected frequencies (10, 100, 500 k, and 1 MHz) show that these microdevices have the capability to discern a minimum of factor-of-two solution concentration differences, near physiologic concentrations, of femtoliter volumes. The impedance measurements for DI water indicate that the water may have absorbed some residual ions from the microfabrication processes when it was added to the device. The differences in magnitude between the solutions appeared consistent between 1 kHz and 2 MHz. These data suggest that optimization of the electrode material or surface coating should improve the low frequency characteristics and possibly the resolution capability of the developed micro-EI system. The small, irregular shape of the gold electrodes prevented the use of parallel-plate models to predict the behavior of the overall device and may be attributing to the complexity of the impedance curves. The significance of changes in geometry is demonstrated by the variations in measured impedance of physiologic concentrations of PBS for the six devices tested (Fig. 6). The variability in device geometry should be greatly reduced in a manufacturing environment. The accurate determination of the dielectric properties of materials measured in the micro-EI device currently requires more sophisticated modeling to include the device geometry and the electrochemical, double-layer effects. The average whole-cell EI data presented in Fig. 7 demonstrates the potential to use micro-EI spectra to differentiate between different cell types. The differences in both the magnitude and phase of impedance were significant at all four Because the raw EI spectra frequencies tested are comprised of numerous constituents, these data reflect factors in addition to the electrical properties of the individual cells. For example, the raw EI spectra are influenced by the electrode/electrolyte interface impedance and the shunt path impedance formed by the saline layer surrounding the cell. 56 IEEE JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, VOL. 8, NO. 1, MARCH 1999 Additional experimental and/or computational methods will be required for applications interested in dielectric properties of various cellular components. V. CONCLUSION Micromachining technologies have enabled the development of a micro-EI measurement system that can distinguish l) quantities of impedance differences in femtoliter (10 ionic solutions and single biological cells. The microdevices were constructed from materials having excellent dielectric and conductive properties to ensure highly sensitive impedance measurements at frequencies up to 2 MHz. Constructing the devices on planar glass substrates provided good optical properties and allowed continuous observation with light microscopy. The overall EI of the resulting microstructures were characterized with varying concentrations of phosphate buffered saline solutions, air, and DI water placed between the electrodes. Resulting impedance measurements demonstrated the ability to readily distinguish between solutions with factor-of-two differences in ionic concentration over the entire frequency range. In addition, the micro-EI system demonstrated the potential for cell-sorting and counting. ACKNOWLEDGMENT The authors gratefully acknowledge the staff of HEDCO Microfabrication Engineering Laboratory, Dr. V. Hlady, and Dr. P. Tresco. REFERENCES [1] P. V. Gerwen, W. Laureys, G. Huyberechts, M. O. D. Beeck, and K. Baert, “Nanoscaled interdigitated electrode arrays for biochemical sensors,” presented at Transducers ’97, Chicago, IL. [2] G. R. Langereis, W. Olthuis, and P. Bergveld, “Measuring conductivity, temperature and hydrogen peroxide concentration using a single sensor structure,” presented at Transducers ’97, Chicago, IL. [3] A. Manz and H. Becker, “Parallel capillaries for high throughput in electrophoretic separations and electroosmotic drug discovery systems,” presented at Transducers ’97, Chicago, IL. [4] J. M. Ramsey, A. G. Hadd, and S. C. Jacobson, “Applications of precise fluid control on microchips,” presented at Transducers ’97, Chicago, IL. [5] L. Bousse, A. Kopf-Sill, and J. W. Parce, “An electrophoretic serial to parallel converter,” presented at Transducers ’97, Chicago, IL. [6] C. T. O’Konski, “Electric properties of macromolecules. V. Theory of ionic polarization in polyelectrolytes,” J. Phys. Chem., vol. 64, pp. 605–619, 1960. [7] D. J. Barlow and J. M. Thornton, “The distribution of charged groups in proteins,” Biopolymers, vol. 25, pp. 1717–1733, 1986. [8] S. Takashima and K. Asami, “Calculation and measurement of the dipole moment of small proteins: Use of protein data base,” Biopolymers, vol. 33, pp. 59–68, 1993. [9] T. Hanai, K. Asami, and N. Koizumi, “Dielectric theory of concentrated suspensions of shell-spheres in particular reference to the analysis of biological cell suspensions,” Bull. Inst. Chem. Res., vol. 57, pp. 297–305, 1979. [10] H. P. Schwan, “Electrical properties of tissue and cell suspensions,” Adv. Med. Biol. Phys., vol. 5, pp. 147–209, 1957. [11] H. P. Schwan, Determination of Biological Impedance. New York: Academic, vol. 6, 1963. [12] I. Tasaki, Physiology and Electrochemistry of Nerve Fibers. New York: Academic, 1982. [13] S. Takashima, K. Asami, and Y. Takahashi, “Frequency domain studies of impedance characteristics of biological cells using micropipette technique,” Biophys. J., vol. 54, pp. 995–1000, 1988. [14] B. Hill and D. T. Campbell, “An improved vaseline gap voltage clamp for skeletal muscle fibers,” J. Gen. Physiol., vol. 67, pp. 265–293, 1976. [15] W. M. Arnold and U. Zimmerman, “Rotating-field-induced rotation and measurement of the membrane capacitance of single mesophyll cells of Avena sativa,” Z. Naturforsch., vol. 37c, pp. 908–915, 1982. [16] V. P. Pastushenko, P. I. Kuzmin, and Y. A. Chizmadshev, “Dielectrophoresis and electrorotation: A unified theory of spherically symmetrical cells,” Stud. Biophys., vol. 110, pp. 51–57, 1985. [17] R. Georgiewa, E. Donath, and R. Glaser, “On the determination of human erythrocyte intracellular conductivity by means of electrorotationinfluence of osmotic pressure,” Stud. Biophys., vol. 133, pp. 185–197, 1989. [18] P. Marszalek, J. J. Zielinsky, M. Fikus, and T. Y. Tsong, “Determination of electric parameters of cell membranes by a dielectrophoresis method,” Biophys. J., vol. 59, pp. 982–987, 1991. [19] Y. Huang, R. Holzel, R. Pethig, and X. B. Wang, “Differences in AC electordynamics of viable and nonviable yeast cells determined through combined dielectrophoresis and electrorotation studies,” Phys. Med. Biol., vol. 37, pp. 1449–1517, 1992. [20] K. V. I. S. Kaler, J. P. Xie, T. B. Jones, and R. Paul, “Dual-frequency dielectrophoretic levitation of canola protoplasts,” Biophys. J., vol. 63, pp. 58–69, 1992. [21] A. V. Sokirko, “The electrorotation of axisymmetrical cell,” Biol. Mem., vol. 6, pp. 587–600, 1992. [22] P. R. C. Gascoyne, R. Pethig, J. P. H. Burt, and F. F. Becker, “Membrane changes accompanying the induced differentiation of friend murine erythroleukaemic cells studied by dielectrophoresis,” Biochim. Biophys. Acta., vol. 1149, pp. 119–126, 1993. [23] T. Muller, L. Kuchler, G. Fuhr, T. Schnelle, and A. Sokirko, “Dielektrische einzelzellspektroskopie an pollern verschiedener waldbaumartencharakterisierung der pollenvitalitat,” Silvia Genet., vol. 42, pp. 311–322, 1993. [24] V. L. Sukhorukov, W. M. Arnold, and U. Zimmerman, “Hypotonically induced changes in the plasma membrane of cultured mammalian cells,” J. Membr. Biol., vol. 132, pp. 27–40, 1993. [25] J. Gimsa, T. Muller, T. Schnelle, and G. Fuhr, “Dielectric spectroscopy of single human erythrocytes at physiological ionic strength: Dispersion of the cytoplasm,” Biophysics J., 1996. [26] J. Gimsa, P. Marszalek, U. Lowe, and T. Y. Tsong, “Dielectrophoresis and electrorotation of slime and murine myeloma cells,” Biophys. J., vol. 60, pp. 5–14, 1991. [27] H. E. Ayliffe, “Micromachined cellular characterization system for studying the biomechanics of individual cells,” presented at IEEE Transducers ’97, Chicago, IL. [28] H. U. Keller, “Crawling-like movements, adhesion to solid substrate and chemokinesis of neutrophil granuloctes,” J. Cell. Sci., vol. 64, pp. 89–106, 1983. [29] C. M. Lo, C. R. Keese, and I. Giaever, “Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing,” Biophys. J., 1995. [30] K. Takahashi, T. H. Tagaya, K. Higashitsuji, and S. Kittaka, Electrical Phenomena at Interfaces. New York: Marcel Dekker, vol. 15, 1984. [31] S. S. Dukhin and V. N. Shilov, Dielectric Phenomena and The Double Layer in Disperse Systems and Polyelectrolytes. New York: Wiley, 1974. [32] R. A. Normann, Principles of Bioinstrumentation. New York: Wiley, 1988. H. Edward Ayliffe received the B.S. degree with High Honors in mechanical engineering from Worcester Polytechnic Institute in 1989 and the Ph.D. degree in bioengineering from the University of Utah in 1998. Prior to his graduate studies at the University of Utah, he was employed as a Research Engineer and Product Development Engineer with M.A.N. Roland, Augsburg, Germany, and ALCOA, Denver, CO. He is currently a Postdoctorate Fellow with HP Labs, Palo Alto, CA, working on the miniaturization of product sensors. Concurrently, he is working with the University of Utah’s Bioengineering Department to develop systems for microelectric impedance spectroscopy of isolated living cells using MEMS technology. AYLIFFE et al.: ELECTRIC IMPEDANCE SPECTROSCOPY USING MICROCHANNELS A. Bruno Frazier (S’85–M’85) received the B.S. and M.S. degrees in electrical engineering from Auburn University in 1986 and 1987, respectively. In December 1993, he received the Ph.D. degree in electrical engineering from Georgia Institute of Technology. From 1987 to 1990 he worked for Intergraph Corporation in the development of computer-aided graphics systems. His experience at Intergraph Corporation ranged from the development of advanced printed circuitboard technology as a Process Engineer to system design at the component level to advanced packaging technologies for future CAD/CAM systems. From 1990 through 1993, he attended Georgia Institute of Technology and conducted research into micromachining processes for the fabrication of metallic microstructures, development and characterization of micromachining materials, as well as micromachined devices utilizing the previously developed processes and materials. He was awarded the IShGM Educational Fellowship two consecutive terms during his graduate studies. After graduating, he conducted research in micromachining technologies at the University of Michigan as a Visiting Scholar through June 1995. In August 195, he joined the bioengineering and electrical engineering faculty at the University of Utah. His current research interest is in the area of microinstrumentation including biomedical, optical, magnetic applications of micromachining technology. 57 R. D. Rabbitt received the Ph.D. degree in applied mechanics from Rensselaer Polytechnic Institute in 1986. He is currently an Associate Professor of Bioengineering at the University of Utah with primary research interests in auditory and vestibular end organ physiology, microscale electric impedance tomography, and analysis of nonlinear strain using medical image data. Dr. Rabbitt was awarded a Presidential Young Investigator for his research on the mechanics of the auditory system by the National Science Foundation shortly after graduating from Rensselaer Polytechnic Institute.