University of Houston • University of Houston-Clear Lake • ISSO Annual Report Y2002—pp. 91-96

Dielectric Responses of Living Organisms

John H. Miller, Jr. (UH) and James R. Claycomb (UH)

Abstract
We have developed and tested several highly sensitive approaches for measuring the linear and nonlinear dielectric responses of live cell suspensions at low frequencies. We observe enormous linear dielectric constants at very low frequencies (with relative dielectric constants of over a hundred million at 10 Hz) attributed to the high polarizability of live cells resulting from their finite membrane potential. In addition, we find that nonlinear dielectric responses are affected by the metabolic states of the cells, such as their response to glucose. The tools reported here are powerful noninvasive methods with numerous potential applications in pharmacology, medicine, and biology.

THE ELECTRIC CHARGES IN LIVE CELLS INTERACT WITH OR produce electric fields, which results in enormous dielectric responses, flexoelectricity, and related phenomena. The electromagnetic properties of live organisms are of considerable fundamental interest and also hold the potential for numerous practical applications. For example, dielectric spectroscopy can potentially be used to detect biological warfare agents without requiring direct contact with the organisms. Additional applications include monitoring human cells grown in NASA bioreactors for microgravity studies, noninvasive glucose sensors, and detection of cancer cells.

Conventional methods of performing dielectric spectroscopy at very low frequencies require large electric field amplitudes, which can induce electrorotation, electroflexion, and other strongly perturbative influences. The reason is that previous techniques lack sufficient sensitivity to attain the nonperturbative weak field limit at low frequencies. Thus, there is a need for developing methods of measuring low-frequency dielectric response with greatly improved sensitivity.

Technical Plan and Equipment
We have developed several approaches for measuring both the linear and the nonlinear dielectric responses of live cell suspensions at low frequencies. One method is to use sensitive superconducting quantum interference devices (SQUIDs) to measure the tiny magnetic fields produced by the small displacement currents flowing through the cell suspension. The SQUID sensor is coupled to flux locked loop electronics, the output of which is fed into a lock-in amplifier. The suspension is placed between the two electrodes of a parallel plate capacitor, across which an ac voltage is applied at the frequency of interest.

Another approach is to employ a similar parallel plate capacitor and to use a bridge circuit to measure the alternating current (both magnitude and phase relative to the applied ac voltage); this information is utilized to compute the complex dielectric response as a function of frequency. The full complex dielectric spectrum from 10 Hz to 100 kHz is thereby acquired with an FFT spectrum analyzer. In order to measure nonlinear dielectric response, a signal at a fixed fundamental frequency is applied and the induced harmonics are measured with the FFT spectrum analyzer. We have also developed a SQUID-based approach to improve the sensitivity of nonlinear dielectric response measurements.

Experimental Activity
Experiments carried out in this project are designed to probe the dielectric properties of live cells. Interiors of living cells are electrically negative with respect to their environment,¹ and this charge separation is utilized for a variety of cellular properties, from energy production to cell-to-cell communication. The charges that create the membrane potential can be polarized by an electric field, leading to a net dipole moment, as illustrated in Fig. 1(a). The linear dielectric response, which measures the ability of the charges to respond to an alternating field, decreases with frequency. Most importantly, its frequency-dependence depends on the type of organism and also helps differentiate between living organisms and nonliving materials.

One of the set-ups we use to measure linear dielectric response^2,3 is illustrated in Fig. 1(b). An ac voltage applied to the electrodes produces both a conduction current and a displacement current through the specimen. The real and imaginary parts of the transfer function V₂(w)/V₁(w) are proportional to the conductivity and the dielectric constant , respectively. This method demonstrates the lowest noise levels thus far at low amplitudes and frequencies^4-8 and, unlike electrorotation methods,⁹ is weakly perturbative.

Discussion
Figure 2 shows dielectric spectra of suspensions of Schizosaccharomyces pombe (yeast-10⁸ cells/ml), Escherichia coli (10⁹ cells/ml), and particles of the inert compound Y₂O₃ in aqueous solutions. The dielectric responses of the living cell suspensions are seen to be enormous at low frequencies and to decrease with frequency. We find that these data are consistent with theoretical predictions¹⁰ and that the low-frequency dielectric constant decreases if the cells die. One can see that strong frequency dependence is a unique property of living organisms, by comparing it to the flat responses of water, ethylene glycol, and inert powder suspensions (Fig. 2).

Results
More recently, we have developed a powerful tool to probe the nonlinear dielectric responses of live cells to a singe frequency excitation. We have measured nonlinear dielectric properties of live cell suspensions using both a conventional electrode technique and a novel set-up employing a high-T_c superconducting quantum interference device (SQUID), as illustrated in Fig. 3. For example, we have measured the nonlinear dielectric properties of Saccharomyces cerevisiae (bakers’ yeast) cells,¹¹ as shown in Figs. 4 and 5. Woodward and Kell^12-14 have demonstrated that nonlinear dielectric spectroscopy is a powerful tool for characterizing biological cell suspensions and their metabolic states. Enzymes embedded in the cell membrane are not free to rotate in the presence of an ac electric field but, instead, change shape or conformation.¹⁵ These conformational changes, which depend on the metabolic state, result in a distortion of the excitation waveform that is detected as a superposition of harmonics of the fundamental excitation frequency.

To measure higher harmonics with the electrode technique, the four-electrode probe is suspended vertically in the cell suspension. An ac voltage is supplied across the outer electrodes, and the cell response across the inner pair is measured with an FFT spectrum analyzer. Nonlinear dielectric spectroscopy is an extremely sensitive probe in which the spectra depend on the type of organism and its metabolic state. For example, enzyme conformation changes result in harmonics that can be monitored with a fast-Fourier transform (FFT) spectrum analyzer. Figure 4 shows the voltage power spectra recorded on the FFT spectrum analyzer using a four-electrode probe immersed in (a) water, (b) a yeast (S. cerevisiae) cell suspension, and (c) a yeast suspension with glucose. Alarge peak is observed at the driving frequency (1 kHz) in each medium. A small third harmonic is observed in the water medium, probably due to residual polarization effects. More pronounced second and third harmonics are observed in the cell suspension, with less pronounced fourth and fifth harmonics. An enhanced second harmonic is observed in the cell suspension after the addition of glucose. This type of behavior has been attributed to changes in the metabolic state of the yeast cells.¹⁵

SQUIDs are extremely sensitive magnetic sensors that can measure the magnetic fields generated by displacement currents flowing in the cell suspension. The SQUID-based configuration, shown in Fig. 3 (right), provides greatly enhanced sensitivity at low frequencies and eliminates spurious harmonics resulting from polarization effects at the electrode surfaces. Figure 5(a) shows the harmonic response to a 17 Hz excitation frequency at a fixed amplitude, while Figs. 5(b) and 5(c) show the field amplitude-dependence and frequency-dependence, respectively, of the third harmonic response.

The sensitive linear and nonlinear dielectric spectroscopy methods reported here are powerful noninvasive tools with numerous potential applications in pharmacology, medicine, and biology.

Acknowledgments
The investigators gratefully acknowledge support by the Institute for Space Systems Operations (ISSO), the Texas Center for Superconductivity and Advanced Materials (TcSAM), and the Robert A. Welch Foundation (E-1221).

References
¹H. P. Schwan. "Electrical Properties of Tissue and Cell Suspensions," in Advances in Biological and Medical Physics, Vol. 5. J. H. Lawrence and C. A. Tobias, ed. New York: Academic Press, 1957. 147-209.
²C. Prodan, J. R. Claycomb, E. Prodan, and J. H. Miller, Jr. "High-Tc SQUID-Based Impedance Spectroscopy of Living Cell Suspensions," Physica C 341-348 (2000): 2693-94.
³C. Prodan, F. Mayo, J. R. Claycomb, J. H. Miller, Jr., and M. J. Benedik. "Low-Frequency, Low-Field Dielectric Spectroscopy of Living Cell Suspensions," J. Appl. Phys. (Submitted.)
⁴J. Gimsa and D. Wachne r. "A Unified Resistor-Capacitor Model for Impedance, Dielectrophoresis, Electrorotation, and Induced Transmembrane Potential," Biophys. J. 75 (1998): 1107-16.
⁵K. Asami, E. Gheorghiu, and T. Yonezawa. "Real-Ti me Monitoring of Yeast Cell Division by Dielectric Spectroscopy," Biophys. J. 76 (1998): 3345-48.
⁶Y. Polevaya, I. Ermolina, M. Schlesinger, B.-Z. Ginzburg, and Y. Feldman. "Time Domain Dielectric Spectroscopy Study of Human Cells. II. Normal and Malignant White Blood Cells," Biochimica et Biophysica Acta 1419 (1999): 257-71.
⁷V. Raicu, C. Gusbeth, D. F. Anghel, and G. Turcu. "Effects of Cetyltrimethylammonium Bromide (CTAB) Surfactant Upon the Dielectric Properties of Yeast Cells," Biochimica et Biophysica Acta 1379 (1998): 7-15.
⁸K. Asami, E. Gheorghiu, and T. Yonezawa. "Dielectric Behavior of Budding Yeast in Cell Separation," Biochimica et Biophysica Acta 1381 (1998): 234-40.
⁹R. Georgieva, B. Neu, V. M. Shilov, E. Knippel, A. Budde, R. Latza, E. Donath, H. Kiesewetter, and H. Bäumle. "Low Frequency Electrorotation of Fixed Red Blood Cells," Biophys. J. 74 (1998): 2114-20.
¹⁰C. Prodan and E. Prodan. "The Dielectric Behavior of Living Cell Suspensions," J. Phys. D: Appl. Phys. 32 (1999): 335-43.
¹¹J. R. Claycomb, C. Prodan, D. Nawarathna, and J. H. Miller, J r. "Nonlinear Dielectric Spectroscopy of Living Cell Suspensions," 2nd Joint Meeting of the IEEE Engineering in Medicine & Biology Society and the Biomedical Engineering Society, Houston, TX, Oct. 23-26, 2002. (In press.)
¹²A. M. Woodward and D. B. Kell. "On the Nonlinear Dielectric Properties of Biological Systems: Saccharomyces cerevisiae," Bioelectrochem. & Bioenergetics 24 (1990): 83-100.
¹³A. M. Woodward and D. B. Kell. "Dual-Frequency Excitation: A Novel Method for Probing the Nonlinear Dielectric Properties of Biological Systems, and its Application to Suspensions of S. cerevisiae," Bioelectrochem. & Bioenergetics 25 (1991): 395-413.
¹⁴A. M. Woodward and D. B. Kell. "On the Relationship between the Nonlinear Dielectric Properties and Respiratory Activity of the Obligately Aerobic Bacterium Micrococcus luteus," Bioelectrochem. & Bioenergetics 26 (1991): 423-39.
¹⁵A. Jones. "Computational Aspects of Nonlinear Biological Dielectric Spectroscopy," Ph.D. Thesis, Dept. of Computer Science, University of Whales, Aberystwyth, 2001.

Publications
Claycomb, J. R. and J. H. Miller, Jr. "Superconducting Shields for SQUID Applications," Review of Scientific Instruments 70 (1999): 4562-68.
Claycomb, J. R., N. Tralshawala, and J. H. Miller, Jr. "Theoretical Investigation of Eddy-Current Induction for Nondestructive Evaluation by Superconducting Quantum Interference Devices," IEEE Trans. Magn. 36 (2000): 292-98.
Claycomb, J. R., M. Nersesyan, D. Luss, and J. H. Miller, Jr. "SQUID Detection of Magnetic Fields Produced by Chemical Reactions," IEEE Trans. on Applied Superconductivity 11.1 (2001): 863-66.
Claycomb, J. R., C. Prodan, D. Nawarathna, and J. H. Miller, Jr. "Nonlinear Dielectric Spectroscopy of Living Cell Suspensions," IEEE Trans. Engineering in Medicine and Biology Transactions, 2003. (In press.)
Claycomb, J. R., K. E. Bassler, J. H. Miller, Jr., M. Nersesyan, and D. Luss. "Avalanche Behavior in the Dynamics of Chemical Reactions," Physical Review Letters 87 (2001): 178303-1-4.
Claycomb, J. R., A. Brazdeikis, R. A. Yarbrough, G. Gogoshin, and J. H. Miller, Jr. "Nondestructive Testing of PEM Fuel Cells," IEEE Trans. Appl. Supercond., 2003. (In press.)
Claycomb, J. R., M. Nersesyan, W. LeGrand, J. T. Ritchie, D. Luss, and J. H. Miller, Jr. "Chemomagnetic Characterization of Chemical Reactions using High-T_c SQUIDs," Physica C 341-348 (2000): 2641-44.
Fang, H., J. R. Claycomb, Y. X. Zhou, P. T. Putman, S. Padmanabhan, J. H. Miller, Jr., K. Ravi-Chandar, and K. Salam. "Melt-Textured YBCO Superconducting Tube for Magnetic Shielding," IEEE Trans. Appl. Supercond., 2003. (In press.)
Lobera-Serrano, J. A., J. R. Claycomb, J. H. Miller, Jr. and K. Salama. "Hybrid Double-D Sheet-Inducer for SQUID-Based NDT," IEEE Trans. on Applied Superconductivity 11.1 (2001): 1283-86.
Martirosyan, K. S., J. R. Claycomb, G. Gogoshin, R. A. Yarbrough, J. H. Miller, Jr., and D. Luss. "Spontaneous Magnetization Generated by Spin, Pulsating and Planar Combustion Synthesis," J. of Appl. Phys. (Submitted.)
McCarten, J. P., T. C. Jones, X. Wu, J. H. Miller, Jr., I. Pirtle, X. Xu, J. R. Claycomb, J.-R. Liu, and W.-K. Chu. "Phase Slip Scaling Relationship for the 59 K and 143 K Charge-Density Waves in NbSe3," Journal de Physique IV (France) 9 (1999): 129-32.
Nersesyan, M. D., J. R. Claycomb, J. T. Ritchie, J. H. Miller, Jr., and D. Luss. "Magnetic Fields Produced by the Combustion of Metals in Oxygen," Combustion Science and Technology 169 (2001): 89-106.
Nersesyan, M. D., J. R. Claycomb, Q. Ming, J. H. Miller, Jr., J. T. Richardson, and D. Luss. "Chemomagnetic Fields Produced by Solid Combustion Reactions," Applied Physics Letters 75 (1999): 1170-72.
Nersesyan, M. D., J. R. Claycomb, J. T. Ritchie, J. H. Miller, Jr., J. T. Richardson, and Dan Luss. "Electric and Magnetic Fields Generated by SHS," J. Materials Synthesis and Processing 9 (2001): 63-72.
Prodan, C., J. R. Claycomb, E. Prodan, and J. H. Miller, Jr. "High-T_c SQUID-Based Impedance Spectroscopy of Living Cell Suspensions," Physica C 341-348 (2000): 2693-94.
Prodan, C., F. Mayo, J. R. Claycomb, J. H. Miller, Jr., and M. J. Benedik. "Low-Frequency, Low-Field Dielectric Spectroscopy of Living Cell Suspensions," J. Appl. Phys. (Submitted.)

Presentations
Claycomb, J. R., C. Prodan, D. Nawarathna, and J. H. Miller, Jr. "Nonlinear Dielectric Spectroscopy of Living Cell Suspensions," 2nd Joint Meeting of the IEEE Engineering in Medicine & Biology Society and the Biomedical Engineering Society, Houston, TX, Oct. 23-26, 2002.
Claycomb, J. R., D. Nawarathna, K. E. Bassler, C. Prodan, and J. H. Miller Jr. "Is Electro-Chemical Noise a SOC Phenomenon," March Meeting of the American Physical Society, Austin, TX, March 3-7, 2003.
Claycomb, J. R., R. A. Yarbrough, G. Gogoshin, K. E. Bassler, and J. H. Miller, Jr. "Self Organized Criticality in Electrochemical Reactions," March Meeting of the American Physical Society, Indianapolis, IN, March 18-22, 2002.
Claycomb, J. R, A. Brazdeikis, R. A. Yarbrough, G. Gogoshin, J. H. Miller, Jr., and M. Le. "Nondestructive Testing of PEM Fuel Cells," Applied Superconductivity Conference, Houston, TX, Aug. 4-9, 2002.
Fang, H., J. R. Claycomb, Y. X. Zhou, P. T. Putman, S. Padmanabhan, J. H. Miller, Jr., K. Ravi-Chandar, and K. Salama. "Melt-Textured YBCO Superconducting Tube for Magnetic Shielding," Applied Superconductivity Conference, Houston, TX, Aug. 4-9, 2002.
Nawarathna, D. "Nonlinear Dielectric Spectroscopy of Living Cell Suspensions," 24th Semiannual TcSAM Student Symposium, Houston, TX, Dec. 17, 2002.
Nawarathna, D., J. R. Claycomb, C. Prodan, and J. H. Miller, Jr. "Non-Linear Dielectric Spectroscopy of Living Cell Suspensions using SQUIDs," March Meeting of the American Physical Society, Austin, TX, March 3-7, 2003.
Prodan, C., F. Mayo, J. R. Claycomb, and J. H. Miller, Jr. "Dielectric Spectroscopy of Living Cell Suspensions," March Meeting of the American Physical Society, Indianapolis, IN, March 18-22, 2002.
Prodan, C., F. Mayo, J.R. Claycomb, D. Nawarathna, and J. H. Miller, Jr. "Techniques of Measuring the Membrane Potential of Biological Cells: Measurements and Modeling," March Meeting of the American Physical Society, Austin, TX, March 3-7, 2003.
Sanabria, H., G. Cardenas, X. Yang, J. H. Miller, Jr., L. T. Wood, and M. J. Benedik. "Optical Projection Microscopy of Live Cells," March Meeting of the American Physical Society, Austin, TX, March 3-7, 2003.
Vajrala, V., D. Nawarathna, J. R. Claycomb, C. Prodan, and J. H. Miller, Jr. "Impedance Magnetocardiography using High-T_c SQUIDs," March Meeting of the American Physical Society, Austin, TX, March 3-7, 2003.

Funding and proposals
"Dielectric Spectroscopy of Biological Agents," Naval Surface Warfare Center, requested for two years, $757,726 (submitted).
"Novel Applications of High-T_c Superconducting Sensors." Robert A. Welch Foundation, June 1, 2001-May 31, 2004, $150,000.
"Novel Live Cell Imaging and Monitoring Technologies." NASA, Office of Biological and Physical Research, requested for four years, $813,468 (submitted).

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Institute for Space Systems Operations - Y2002 Annual Report
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