Single-Cell Measurements of Human Neutrophil Activation Using

Anal. Chem. 1998, 70, 2607-2612

Single-Cell Measurements of Human Neutrophil Activation Using Electrorotation Alun W. Griffith† and Jonathan M. Cooper

Bioelectronics Research Centre, Department of Electronics & Electrical Engineering, Glasgow University, Glasgow G12 8QQ, U.K.

We have used the technique of cell electrorotation within a microfabricated planar gold electrode array as a means of detecting changes in the physical properties of a single human neutrophil, when activated by the chemotactic factor, phorbol myristate acetate (PMA). The results, which have been analyzed using a double-shell model to represent the cell and its nucleus, provide an indication of the changes in biophysical parameters that occur during cell activation. The methods used in this study have potential applications in the development of single-cell assays for pharmaceutical screening, as a means of determining rapidly the action of drugs. Over the last 10 years, there has been an increased interest in the ability to perform assays on single cells,1-3 with the long-term aim of being able to establish well-characterized dose-response curves for highly characterized biological systems. More recently, the interest in this field has been further intensified by the activities of the pharmaceutical industry in exploring the applications of microtechnology in producing analytical arrays for the development of rapid, high-density biological screens.4 Most recently these trends have been exemplified by the adaptation of microfabrication methodologies, traditionally used in the semiconductor industry, to produce miniaturized sensing devices within which individual cells can be constrained or manipulated, either physically5 or by using electromagnetic cues.6-14 In this paper, we focus on the use of electromagnetic cues to manipulate single cells using a technique closely associated with electrokinetics,6-9 namely, electrorotation.10-14 The phenomenon, which involves the rotation of a particle in an electric field, occurs † Present address: Inverness Medical Ltd., Beechwood Business Park, Inverness, IV2 3ED, U.K. (1) Allue, I.; Gandelman, O.; Dementieva, E.; Ugarova, N.; Cobbold, P. H. Biochem. J. 1996, 319, 463-469. (2) Mailinski, T.; Taha, Z. Nature 1992, 358, 676-678. (3) Jankowski, J. A.; Tracht, S.; Sweedler, J. V. Trends Anal. Chem. 1995, 14, 170-176. (4) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-5026. (5) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1998, 70, 1164-1170. (6) Goater, A. D.; Burt, J. P. H.; Pethig, R. J. Phys. D: Appl. Phys. 1997, 30, L65-L69. (7) Talary, M. S.; Mills, K. I.; Hoy, T.; Burnett, A. K.; Pethig, R. Med. Biol. Eng. Comput. 1995, 33, 235-237. (8) Wang, X. B.; Huang, Y.; Hughes, M. P.; Pethig, R.; Gascoyne, P. R. C.; Becker, F. F. Biophys. J. 1994, 66, 281. (9) Fuhr, G.; Arnold, W. M.; Hagedorn, R.; Muller, T.; Benecke, W.; Wagner, B.; Zimmermann, U. Biochim. Biophys. Acta 1992, 1108, 215-223.

S0003-2700(98)00070-5 CCC: $15.00 Published on Web 05/15/1998

© 1998 American Chemical Society

as a result of an induced torque which is itself dependent upon the electrical properties of the particle and the medium in which it is rotating. As a methodology, electrorotation has recently gained popularity by providing a method for observing subtle biophysical changes in both external and internal cellular properties. For example, electrorotational spectra have been used to analyze the physiological changes which occur in oocytes upon fertilization12 and have provided estimated values for the membrane capacitance and the cytoplasm conductivity of lymphocytes13 and erythroleukemia cells.14 In this study, we have used electrorotation as a bioanalytical technique, providing a means of determining the changes in the electrical properties of human neutrophils upon activation by the chemotactic agent phorbol myristate acetate (PMA). The work focuses specifically on the use of PMA, since the durability of the cell’s activation response makes it particularly well suited to electrorotation studies. It is, however, also reasonable to consider the analysis of the interaction between PMA and the neutrophil as being a model cell-based screen, demonstrating the potential application of the methodology in the discovery of new medicines. The process of chemotactic activation of neutrophils is closely related to the rapid physiological and metabolic changes which occur during the “respiratory burst” 15 being characterized by free radical release.16,17 Symptomatic of the physical changes which occur during this change in metabolic activity is a massive production of superoxide anions with associated gating through the cell membrane,18 as well as an increase in cell volume by ca. 20%.19 Both of these events will produce changes in the dielectric properties of the cell and, hence, should cause a measurable shift in the electrorotational response. Other parameters which might also be expected to vary during neutrophil activation include changes in the cytoplasm conductivity and permittivity; the (10) Ziervogel, H.; Glaser, R.; Schadow, D.; Heymann, S. Biosci. Rep. 1986, 6, 973-982. (11) Foster, K. R.; Sauer, F. A.; Schwan, H. P. Biophys. J. 1992, 63, 180-190. (12) Arnold, W. M.; Schmutzler, R. K.; Alhasani, S.; Krebs, D.; Zimmermann, U. Biochim. Biophys. Acta 1989, 979, 142-146. (13) Hu, X.; Arnold, W. M.; Zimmermann, U. Biochim. Biophys. Acta 1990, 1021, 191-200. (14) Gascoyne, P. R. C.; Pethig, R.; Burt, J. P. H.; Becker, F. F. Biochim. Biophys. Acta 1993, 1149, 119-126. (15) Hill, H. A. O.; Tew, D. G.; Walton, N. J. FEBS Lett. 1985, 191, 257-263. (16) Maly, F. E.; Schurermaly, C. C. News Physiol. Sci. 1995, 10, 233-238. (17) Anderson, R. South Afr. Med. J. 1995, 85, 1024-1028. (18) Morel, F.; Doussiere, J.; Vignais, P. V. Eur. J. Biochem. 1991, 201, 523546. (19) Stoddard, J. S.; Steinbach, J. H.; Simchowitz, L. Am. J. Physiol. 1993, 265, C156-165.

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thickness and structure of the cell membrane; and alterations in the character of the cell nucleus. THEORY Dielectrophoresis. As stated earlier, electrorotation is an element of the broader phenomena of AC electrokinetics, which includes dielectrophoresis, and occurs specifically as a result of the induced torque exerted on a particle (or cell) in a rotating electric field. It has been shown that, for a spherical particle in an electric field of strength E, the induced dipole moment (m b (ω)) is given by 3 m b (ω) ) 4πmf(*p,* BRMS m)R E

(1)

where R is the radius of the particle and f(*p,*m) is the ClausiusMossotti factor,20 represented by

f(* p,* m) )

*p - *m *p + 2*m

(2)

The parameters *p, and *m represent the complex permittivities of the particle and the medium, respectively, and take the form

* i ) i - jσi/ω

(3)

where the subscript i refers to the particle (p), the medium (m), the cell (c, see later), the cytoplasm (cp, see later), or the nucleoplasm (np, see later), σ is the conductivity, and j is (-1)1/2. Electrorotation. The observed rotation of particles occurs as a result of the interaction of the induced dipole moment with a rotating electric field. The frequency-dependent torque (Γ(ω)) is related to effective dipole moment (see eq 1) as follows:21

Γ(ω) ) m b (ω)E B

(4)

The observed electrorotation is determined by the imaginary component of the Clausius-Mossotti factor and is dependent on the relative conductivities and permittivities of both the suspension medium and the particle as well as the strength of the applied electric field and its frequency. When the Im(f(*p,*m)) > 0 (see eqs 2 and 5), the induced dipole lags behind the applied field by less than half a period and an anti-field rotational torque is exerted on the particle. Conversely, when Im(f(*p,*m)) < 0, the induced dipole moment lags by greater than half a period and co-field torque is exerted (see also Figure 1). The nature of observed rotation rate (R(ω)) is the resultant force balance between the torque and the viscous forces acting on the particle in a particular medium, defined as14

R(ω) ) -

mE2 Im(f(*p,*m)) 2η

where η is the dynamic medium viscosity. Since the field is applied by a phase-shifted voltage, R(ω) can be also be expressed as

R(ω) ) -

mk2V2 Im(f(*p,* m)) 2η

(6)

where k is a scaling constant relating the applied RMS voltage (V), electrode geometry, and particle position. In practice, the electrorotation of viable cells follows a characteristic pattern. At low frequencies (1 MHz), the applied field permeates the membrane and the rotational response is dominated by the dielectric properties of the cellular interior, and co-field rotation is observed. Changes in cell physiology and the influence of cell organelles will be reflected in the speed and sense of the electrorotation.10 The Cell Model. In this study, the analysis of the electrorotational spectra involved the use of the double-shell model proposed by Asami et al.22 Although a single-shell model provides a simpler version of the theory, the double-shell model was preferred as it takes into consideration the potential influence of the large nucleus, characteristic of the human neutrophil. The analysis is based upon determining the effective complex permittivity of the whole cell (*c), using the double-shelled structure,22 illustrated in Figure 2b. Thus *c can be determined as

2(1 - r1) + (1 + 2r1)E1 * c ) * m (2 + r1) + (1 - r1)E1

(7)

(5)

(20) Arnold, W. M.; Schwan, H. P.; Zimmermann, U. J. Phys. Chem. 1987, 91, 5093-5098. (21) Pethig, R.; Huang, Y.; Wang, X. B.; Burt, J. P. J. Phys. D: Appl. Phys. 1992, 25, 881-888.

2608 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

Figure 1. In rotating electric fields, the induced dipole moment of a particle is out of phase with the direction of the electric field. The angle between the two (θ) reflects the time taken for the dipole to form. The phase difference will generate a variable torque (Γ) acting on the particle, causing it to rotate.

where r1 ) (1 - dm/R)3, *m is the complex membrane permittivity, (22) Asami, K.; Takahashi, Y.; Takashima, S. Biochim. Biophys. Acta 1989, 1010, 49-55.

Figure 2. Comparison of the components used in the single-shell (a) and double-shell (b) models of a cell. In the double-shell model, the inner, concentric circle represents the cell’s nucleus.

dm is the membrane thickness, R is the cell radius, and E1 is an intermediate parameter, given by

E1 )

*cp 2(1 - r2) + (1 + 2r2)E2 * m (2 + r2) + (1 - r2)E2

(8)

where r2 ) (rn/(R - dm)3, *cp is the complex permittivity of the cytoplasm, and rn is the radius of the nucleus. Similarly, the parameter E2 is given by

E2 )

*np 2(1 - r3) + (1 + 2r3)E3 * n (2 + r3) + (1 - r3)E3

(9)

where r3 ) (1 - dn/rn)3. *n and *np are the complex permittivities of the nuclear membrane and nucleoplasm, respectively; and dn is thickness of the nuclear membrane. Finally, the parameter E3 is given by

E3 ) *np/*n

(10)

Simulations of the electrorotational spectra were made using this model as MATLAB (Mathworks Inc.) subroutines, with the modeling output being the frequency-dependent variation of real and imaginary components of the Clausius-Mossotti factor. MATERIALS AND METHODS Suspension Media. All reagents were purchased from Sigma-Aldrich (Poole, U.K.). The solutions used in this study were prepared using reverse osmosis (RO) water, and their conductivities were measured at 20 °C on a Hewlett-Packard 4192A

impedance analyzer at 100 kHz, using a platinum black electrode. Additions of phosphate-buffered saline (PBS) were used to adjust solution conductivities to 400 µS cm-1. Cell suspension media were prepared as a stock solution, containing 270 mM sucrose. Electrodes and Instrumentation. Microelectrodes were designed using the Wavemaker circuit design package (Barnard Microsystems Ltd., U.K.) and fabricated using photolithographic techniques. “Polynomial” electrodes (where the tip curvature is defined by a polynomial function) of varying separations (200600 µm) were used, based upon the design of Huang et al.23 Glass was chosen as the substrate material, providing a flat rigid support for photolithographic processing and a transparent base, necessary for the inverted light microscopy. The electrodes were made by electron beam evaporation of Ti/Pd/Au (10/10/100 nm), which provided an inert multilayer metal electrode, with excellent adhesion to the glass substrate. Observations of the neutrophil’s electrorotational spectra, as well as the estimation of the magnitude of cell parameters (e.g., diameter), were made using a Nikon Microspot light microscope using both reflected and transmitted light, as appropriate. Phase contrast imaging helped to highlight cell profiles. The sinusoidal electric fields (1 Hz to 20 MHz, 0-12 V) were generated using a direct digital synthesis generator designed in-house and controlled by a software written in Borland Turbo Pascal (version 7.0) running on an IBM-compatible PC. Purification of Neutrophils. Whole human blood was obtained from the Glasgow Royal Infirmary Hospital, Glasgow, Scotland. The separation of neutrophils from blood was carried out using the methods of Chettibi et al.24 and involved the removal of the erythrocytes by dextran sedimentation to give a supernatant rich in neutrophils and lymphocytes. Neutrophils were subsequently purified by centrifugal separation using a Ficoll (Histopaque 1077, Sigma) density gradient, with the residual plasma and supernatant fractions being discarded. The neutrophil fraction was frequently contaminated with residual erythrocytes, which could be removed by a series of steps beginning with their rapid (20 s) hypotonic lysis in RO water. The cell lysis step was halted by excess (>10 times) dilution with PBS, and the residual neutrophils were concentrated by centrifugal washing. Cell viability (typically in the range 95-98%) was determined using the trypan blue exclusion test. Samples were then diluted in PBS to a concentration of ca. 5 × 104 cells mL-1. Collection of Electrorotational Spectra. Cell adhesion is associated with the neutrophil’s high motility and is particularly apparent after their activation.25 As a consequence, prior to the dispensing of cells onto the microelectrode structure (and thereafter, the collection of the spectra), the microelectrode structure was soaked in casein solution (1 mg mL-1 in 270 mM sucrose) for 15 min to provide a blocking layer that reduced the rate of cell attachment. Subsequently, a sample of cell suspension (ca. 800 µL) was pipetted into a measurement compartment (comprising a 2 mm tall and 20 mm diameter glass ring) attached to the microelectrode array using a thin layer of silicone grease. Increases in conductivity due to evaporation were reduced to a negligible level by covering the sample well with a cover slip. (23) Huang, Y.; Pethig, R. Meas. Sci. Technol. 1991, 2, 1142-1146. (24) Chettibi, S.; Lawrence, A. J.; Stevenson, R. D.; Young, J. D. FEMS Immunol. Med. Microbiol. 1994, 8, 271-281. (25) Tauber, A. I. Blood 1987, 69, 711-720.

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Electrorotational spectra were collected by observing both the rotation rate and sense (co-field or anti-field) of at least four points per frequency decade. The experimental observations were made by recording the rotation of a cell on videotape, which was later analyzed to determine the cell’s mean rotation speed (ω) in rad s-1. Each of the recorded spectral points represents the average of six measurements on cells of similar diameter. Activation of Neutrophils. Neutrophils were activated by the addition of the PMA chemotactic factor. The stimulant was first dissolved in dimethyl sulfoxide (DMSO) and then diluted to a concentration of 500 mg mL-1 in sucrose/PBS solution of the same conductivity and osmolarity as the cell suspension. Once the spectrum of the resting neutrophils had been collected, a 40 µL aliquot (i.e., to give a ca. 20 times dilution) of PMA was added to the cell suspension. The suspension was left to equilibrate for 60 s before collection of the spectrum of the activated neutrophil. Figure 3. Experimental electrorotation spectra of resting (O) and activated (0) human neutrophils. The spectra were collected using polynomial electrodes (360 µm tip-to-tip separation) in a field of ca. 56 V cm-1 (2 V p-p) in a sucrose/PBS medium (2.4% PBS, 400 µS cm-1). The neutrophils were activated using PMA at a final concentration of 25 µM. The solid line is a simulated spectrum of the resting human neutrophils generated with the double-shell cell model using the parameters given in Table 1.

RESULTS AND DISCUSSION An important consideration in the collection of the electrorotational spectra was to ensure that none of the chemicals used in the procedure (apart from PMA) contributed to the activation mechanism of the neutrophils. For this reason, cell suspension media such as glucose and mannitol solutions were avoided, and a salt solution free of divalent ions was chosen in preference to a simple culture medium (it has been shown that Ca2+ and Mg2+ ions can induce undesirable aggregation of neutrophils during the purification procedure).26 Likewise, control experiments involving, for example, different cell-handling protocols as well as the addition of appropriate amounts of DMSO were performed in order to ensure that the solvent did not contribute to the observed “activation” response. As expected, medium conductivity and permittivity were found to be crucial parameters in electrorotation experiments. In contrast to previous studies, which have been performed on human lymphocytes,13 a relatively high medium conductivity (400 mS cm-1) was used in this study to reduce the degree of neutrophil aggregation26 (this also had the effect of making the working solution less susceptible to fluctuations in conductivity due to cell cytoplasm leakage or ion release through membrane gating). Importantly, at the concentrations for salt (200 mM PBS) and sucrose (270 mM) used in this study, the solution permittivity remained constant (790)27 over the range of available frequencies (100 Hz to 20 MHz). In general, it has been found that induced dipoles in nearby rotating particles can effect the observed rotational response. To minimize this influence, rotational measurements were made only on cells which were separated by at least three cell diameters.28 In addition, spectra were collected only for particles positioned in an area defined by a circle located at the center of the electrode array (with a width of 0.3x, where x represents the separation between the electrode tips). For the polynomial electrode design used in these electrorotational experiments, Hughes et al.29 have shown that particles in this region experience a consistent (95%

confidence) rotational torque and minimal positive dielectrophoretic forces. Neutrophil Activation. It is already well established within the literature that neutrophils are extremely sensitive to a variety of chemotactic stimulants; in our hands, and those of others,30 PMA has proved to give a more consistent and durable chemotactic response for the activation of neutrophils, with concentrations as low as 10 nM generating the characteristic “respiratory burst”. Previously, a dose-dependent response for superoxide production by neutrophils has been demonstrated by established assay methods including chemiluminescence and cytochrome c reduction31 over a final PMA concentration range between 50 nM and 1 mM. In this study, a final PMA concentration of 25 µM was chosen specifically to give a rapid and kinetically “saturated” cellular response (i.e., one where the response was not PMAconcentration dependent). Electrorotational Spectra. For the polynomial electrode design chosen for this study, a broad range of tip-to-tip separations could be used to induce electrorotation in human neutrophils. The data presented in this study were obtained using an electrode separation of 360 µm, which provided a satisfactory compromise between the experimental demands of creating a suitable monitoring area while also generating an adequate range of rotation speeds with minimal dielectrophoretic force. The recorded electrorotational spectra for the neutrophils are shown in Figure 3 and are seen to have both co-field and anti-field rotational elements. Upon neutrophil activation, differences in the physical properties of the cell were reflected in the changing nature of

(26) O’Flaherty, J. T.; Dechatlet, L. R.; McCall, C. E.; Bass, D. A. Proc. Soc. Exp. Biol. Med. 1980, 165, 225-232. (27) Arnold, W. M.; Gessner, A. G.; Zimmermann, U. Biochim. Biophys. Acta 1993, 1157, 32-44. (28) Wang, X. B.; Huang, Y.; Gascoyne, P. R. C.; Becker, F. F.; Holzel, R.; Pethig, R. Biochim. Biophys. Acta 1994, 1193, 330-344.

(29) Hughes, M. P.; Wang, X. B.; Becker, F. F.; Gascoyne, P. R. C.; Pethig, R. J. Phys. D: Appl. Phys. 1994, 27, 1564-1570. (30) Lundqvist, H.; Kricka, L. J.; Stott, R. A.; Thorpe, G. H. G.; Dahlgren, C. J. Biolumin. Chemilumin. 1995, 10, 353-359. (31) Daval, J. L.; Ghersiegea, J. F.; Oillet, J.; Koziel, V. J. Cereb. Blood Flow Metab. 1995, 15, 71-77.

2610 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

Table 1. The Parameter Values and Literature Sources Used in the Double-Shell Model of a Human Neutrophil parameter

value

source

cell radius (R) cell membr thickness (dm) nucleus radius (rn) nuclear membr thickness (dn) medium conductivity (σmed) medium permittivity (med) cell membr conductivity (σm) cell membr permittivity (m) cytoplasm conductivity (σcp) cytoplasm permittivity (cp) nuclear membr conductivity (σn) nuclear membr permittivity (n) nucleoplasm conductivity (σnp) nucleoplasm permittivity (np) permittivity of free space (0)

6 µm 8 nm 3.5 µm 20 nm 0.4 mS cm-1 790 1 × 10-11 mS cm-1 6.20 3 mS cm-1 600 60 × 10-3 mS cm-1 280 13.5 mS cm-1 520 8.854 × 10-12 F m-1

measured Asami et al.22 Davis et al.36 Asami et al.22 measured Arnold et al.27 Ziervogel et al.10 Ziervogel et al.10 Ziervogel et al.10 Asami et al.22 Asami et al.22 Asami et al.22 Asami et al.22 Asami et al.22 CRC Handbook37

the electrorotational spectrum. To aid interpretation of these data, the electrorotational spectra of both the resting and the activated neutrophils were fitted using the double-shell model,22 as previously described. Key changes in the spectrum of the neutrophils upon activation are the shifts in crossover (fc) and the anti-field rotation peak (fp) frequencies. The differences between the spectra were attributed to physical changes in the character of the cellular membrane and cytoplasm and clearly demonstrated the use of the technique for identifying subtle physicochemical changes in single cells (with a potential for use in pharmaceutical screening and analysis). Notwithstanding effects due to variations in the size or biophysical character of the nuclei, and relying solely upon the spectral data collected in this study, it is difficult to precisely identify which physical change, or combination of changes, occurring upon neutrophil activation causes the observed shifts in the experimental data. As a consequence, a series of visual (microscopic) observations, together with literature estimates (summarized in Table 1), were used to supplement our knowledge. For example, previous work has found that, upon neutrophil activation, cell volumes increase on average by 21.7% (providing a 6.7% increase in the cell radius).32 Using this estimate (which was consistent with our own observations), together with mathematical simulations of the double-shell cell model,22 we explored the possible explanations for the observed experimental data. We limited ourselves to investigating four cellular parameters (R, dm, m, and σcp) which had the greatest influence on the electrorotational spectra. Figure 4 shows the results of these simulations, which illustrate how changes in the magnitudes of these parameters resulted in (theoretical) shifts in the rotational spectra, as predicted by the double-shell model.22 Given the size of our data set and the large parameter space within it, it was not, however, possible to deconvolute the observed changes and assign them to specific changes in the cellular structure and physiology. Nonetheless, from the simulated data (see Figure 4a-d) it is possible to identify those parameters which are of particular significance: for example, it is apparent from Figure 4a that increasing the radius (R) by up to 50% (nearly an order of magnitude greater than observed during activation)32 has a primary influence on the shift in crossover frequency (fc), as well (32) Grinstein, S.; Furuya, W.; Cragoe, E. J. J. Cell. Physiol. 1986, 128, 33-40.

Figure 4. Effect of the variation of four cellular parameters (R, dm, m, and σcp) on the nature of the modeled electrorotational response of human neutrophils. The modeled response of resting neutrophils (solid line) can be compared to the measured changes (see Figure 3). Conceivable physical changes in cellular properties upon cell activation are indicated by dotted lines.

as causing a downward shift of fp. The simulation also shows that the cell would require an unrealistic increase in volume of >300% to provide an acceptable fit to the activated neutrophil spectrum, assuming that the change in cell diameter alone was the only explanation for the observed results. In the same fashion, we can see from Figure 4d that a decrease in cytoplasm conductivity (σcp) is the dominant parameter change that explains the observed decrease in the peak rotation rate of the activated neutrophil. Analytical Chemistry, Vol. 70, No. 13, July 1, 1998

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Because of the dominance of these two parameters (R and σcp) in influencing the rotational spectra, it was possible to explain the experimental and the simulated results in terms of known physiological changes to the cell. Activation is known to induce blebs on the cell membrane surface resulting in variations in cell shape, associated with actin polymerization and depolymerization.33,34 In addition, membrane stretching (associated with the increased cell volume) and bleb formation on the cell membrane (leading to loss of material) will both lead to a reduction in the thickness of the cell membrane, see Figure 4b. Activation of the neutrophil is also known to result in the expulsion of ions, which might be expected to reduce the cytoplasm conductivity and increase the cell volume. Other associated physical factors, such as changes in membrane capacitance (which will include membrane double layer effects) measured during studies on cell differentiation,12 or membrane conductivity (σm), may also contribute to the nature of the observed electrorotational spectra. In this latter respect, it is known that the netrophil’s “respiratory burst” results in ion exchange,18 which requires membrane channel gating, and thus might be expected to change the value of σm. In order to investigate these effects, electrorotational spectra were simulated, as above, by changing values for σm over 4 orders of magnitude, that is, from that of its resting conductivity10 to values for σm that have previously been measured for other “leaky” cells.35 Interestingly, the double-shell model showed us that, for increases in σm, the anti-field rotational peak was found to increase, while there was no change in the crossover frequency (data not shown). Both of these results are in contrast to the experimental data that we observed and indicate that, while (33) Watts, R. G.; Crispens, M. A.; Howard, T. H. Cell Motil. 1991, 19, 159168. (34) Coates, T. D.; Watts, R. G.; Hartman, R.; Howard, T. H. J. Cell Biol. 1992, 117, 765-774. (35) Huang, Y.; Holzel, R.; Pethig, R.: Wang, X.-B. Phys. Med. Biol. 1992, 37, 1499-1517. (36) Davis, B. H.; Walter, R. J.; Pearson, C. B.; Becker, E. L.; Oliver, J. M. Am. J. Pathol. 1982, 108, 206-216. (37) CRC Handbook of Chemistry and Physics; Linde, D. R., Ed.; CRC Press: London, 1993.

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changes in σm may contribute to the overall dielectric properties of the cell, they do not contribute significantly to the observed electrorotational behavior. Finally, it should be noted that there are some particular experimental difficulties associated with the inevitable adhesion and motility mechanisms characteristic of activated neutrophils, which result in the cell attachment to the glass substrate in a nonspherical shape. At this stage of the investigation, the potential contribution of these effects should not be ruled out of the analysis. Although, for resting neutrophils, coating the substrate in milk protein (casein) was effective in limiting the effects of the activation mechanisms, the addition of PMA always accelerated cell adhesion and limited experiment time to 15-30 min before these effects became apparent, at which point a fresh sample was prepared. In conclusion, in order to allow a more precise interpretation of the data, in future studies we propose to record electrorotational spectra over a greater variety of experimental conditions. Notwithstanding this, there were clearly observable differences between the spectra of both the resting and the activated neutrophils attributable to neutrophil activation, which could, in the future, be used in the development of novel single-cell assays. Given the ease with which microfabricated electrodes can be produced, using photolithographic methods adapted from the semiconductor industry, there is clearly a potential to mass produce such devices and/or develop an array technology, for high-throughput screening. Acknowledgment is given to the Engineering and Physical Science Research Council, EPSRC (U.K.), for funding this research. The authors also thank Dr. Mike Hughes for his valuable opinions and Dr. Hywel Morgan for his help in the designing and building of instrumentation. Received for review January 26, 1998. Accepted March 23, 1998. AC980070C