Electrokinetic system to determine differences of electrorotation and traveling-wave electrophoresis...

Colloids and Surfaces A: Physicochem. Eng. Aspects 262 (2005) 57–64

Electrokinetic system to determine differences ofelectrorotation and traveling-wave electrophoresis between

autotrophic and heterotrophic algal cells

Yifan Wua,1, Chengjun Huangb,c,1, Lei Wangb,c, Xiaoling Miaob,Wanli Xingb,c, Jing Chengb,c,∗

a Department of Electrical and Computer Engineering, McMaster University, 1280 Main St West, Hamilton, ON, Canada L8S 4K1b Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China

c National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, PR China

Received 2 February 2005; received in revised form 2 April 2005; accepted 2 April 2005Available online 31 May 2005

Abstract

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Electrorotation and traveling-wave dielectrophoresis are non-invasive methods and can be used to determine the dielectric particles. In the present study, we have developed an electrokinetic system using microfabricated electrorotation and traveling-w

rophoresis electrodes to determine the dielectric differences of algal cells.Chlorella protothecoidesis a microalgae that can be photoautotrocally or heterotrophically grown under different culture conditions. Different culture conditions resulted in biochemical differencesutotrophic and heterotrophic cells ofC. protothecoides, and therefore, distinctions of dielectric properties. We presented differenceslectrorotational responses, which were also confirmed by traveling-wave dielectrophoresis. Based on these electrical and biochem

t was suggested that electrorotation and traveling-wave dielectrophoresis responses could be used to reflect the biochemical diological particles including algal cells.2005 Elsevier B.V. All rights reserved.

eywords:Electrorotation; Traveling-wave dielectrophoresis; Auotrophic and heterotrophic; Biochemical differences; Algal cells

. Introduction

Use of non-uniform electric field causing particles toove (dielectrophoresis) or spin (electrorotation, ROT)[1]

s a promising method in many areas to determine the di-lectric properties of particles. This method is non-invasive,oes not require the use of antibodies or other labeling andan be incorporated into ‘laboratory-on-a-chip’ devices. Di-lectrophoresis (DEP) includes conventional dielectrophore-is (cDEP)[2], the move of the particle to strong or weaklectric regions and traveling-wave dielectrophoresis (TWD)

3], the move of particle induced by traveling-wave electric

∗ Corresponding author. Tel.: +86 10 62772239; fax: +86 10 62773059.E-mail address:[email protected] (J. Cheng).

1 These authors contributed equally to this work.

field. It is usually used to perform selective separatioparticles.

Applications of electrokinetic effects are extensivinvestigated in recent years including the study ofdielectric properties as a function of cell physiological stthe investigation of the physical–chemical properties ofloidal particles, the detection of the presence of viabilitmicroorganisms in water, the manipulation and positioof individual cells in aqueous suspensions and cell soof clinical significance[4]. Biological particles investigateby this approach include white and red blood cells, viand non-viable yeast, Gram-positive and Gram-negbacteria, cancer cells, CD34+ stem cells, and DNA[5].Samples of successful applications are isolation of viand non-viable yeast, detection of the apoptosis of cacell using electrorotation (ROT) and the establishment

927-7757/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2005.04.008

58 Y. Wu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 262 (2005) 57–64

theoretical multi-shell model to fully describe the motion ofparticles in ac electric field. However, there have been fewreports on the relationship between the dielectric behaviorsof bioparticles and corresponding biochemical properties. Inaddition, no reports showed that micro-algal cells had everbeen investigated. In order to investigate the electrodynamicsof micro-algal in different biochemical components, a strat-egy of autotrophic and heterotrophic growth of algal cellswas taken to result in distinctive differences of biochemicalcomponents in one strain ofChlorella protothecoides(greenalgal). In this report, we developed an electrokinetic systemto study the behaviors of algal cells using ROT and TWD.It would be of great significance to relate their biologicaland chemical components with dielectric properties. At lastthe potential uses of ROT and TWD for determination ofcell biochemical components and isolation of those cells arediscussed.

2. Experimental methods

2.1. Cell cultures

The strain ofC. protothecoideswas provided by the cul-ture collection of algae at the University of Texas (Austin, TX,U heyc ellsw resu eso b-b 0 mlfKg -l andsp sc ROTa

si-t urefl thatf cedt -t erej origi-n at or-g wayo hic.Ta WDm fp tionsm ellsb

2.2. ROT measurement

Both of the ROT and TWD chips were designed and fabri-cated by�TAS (micrototal analysis system) group, NationalEngineering Research Center for Biochip Technology, Bei-jing, China.

AC and HC cells were measured separately in the ROTchip. Green AC cells and yellow HC cells were harvestedseparately by centrifuge from their culture medium for 3 m(speed was 3000 rpm). Then the sample was rinsed two timeswith electrolyte solution pre-configured (medium conduc-tivity was 300�S m−1, tested by TDSTester 20, OAKTONInstruments, Vernon Hills, IL). It was made by combiningsucrose (85 mg/ml) solution with phosphate-buffered saline(PBS, pH 7.0). The low conductivity of suspending solu-tion reduced electrolysis and heat production at the electrodeedges.

After AC or HC cells entirely dissolved into electrolytemedium, 25�l suspension with about 104 cells per ml waspipetted into the electrode chamber and covered with a coverslip. Cells in the chamber would rotate against or with thedirection of the rotating electric field depending upon therelative dielectric properties of them and frequency applied.Under the temperature at 25◦C, ROT effects were monitoredfor 3 m for each type of cells by microscope and recordedusing attached color CCD camera (Panasonic WV-GP410)a wella ratew turec Aftere ashedp riedu

ntralr top-w eatedf , ine anda nallyn e thei inedb s.

2

heirc be-f ep-a glasss t, twof uen-c eld,c

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SA). The color of the algal cells was originally green as tontained chlorophylls for photosynthesis. The algal cas grown autotrophically and axenically in batch cultunder 26 (±1)◦C with continuous illumination at intensitif 40�mol m−2 s−1. Aeration was provided by continue buling air at regular pressure. The culture medium per 100

or autotrophic growth of cells contained 0.7 g KH2PO4, 0.3 g2HPO4, 0.3 g MgSO4·7H2O, 3.0 mg FeSO4·7H2O, 5.0 glycine, 10�g Vitamin B1, and 1 ml Arnon’s A5 trace so

ution [6]. It took about 5 days for each batch culturepecific growth rate (µ) was about 0.032 h−1. After 5 daysropagation, the normal green autotrophicC. protothecoideells (here after refereed as AC cells) were harvested fornd TWD measurements.

To obtain the cells with different biochemical compoion, 5 ml of AC cells were transferred into another cultask which contained the medium (1000 ml) as same asor AC cell culture except the amount of glycine was reduo 0.1 and another 10 g/l glucose was added[6]. The condiions including temperature, illumination, and aeration wust the same as before. During 5 days cell propagation,al green cells turned to yellow cells. It was suggested thanic carbon nutrition in later medium changed the pathf nutrition metabolism from autotrophic to heterotrophen the yellow heterotrophicC. protothecoidescells (herefter refereed as HC cells) were harvested for ROT and Teasurements. Autotrophic and heterotrophic growth oC.rotothecoidesat a same propagation rate at above condiay lead to biochemical variations in those two kinds of celonged to one strain.

nd a Panasonic NV-HD500 video recorder. The ROT ass TWD speed of each cell was obtained by timing theith a stopwatch from recorded videotape. No temperahange was observed during the short record time (3 m).ach sample had been investigated, the chamber was wrior to the next experiment with ultra pure water and dnder a stream of nitrogen.

In each period of frequency, 5–10 cells around the ceegion of the chamber were individually timed by the satch. The same procedure for each cell sample was rep

or 4–5 times to eliminate experimental error. So totallyach frequency, there were around 80–100 cells timedveraged to give the angular velocity. The speed was fiormalized against the square of the voltage to remov

nfluence of the electric field. Rotating spectra was obtay plotting spin rates against corresponding frequencie

.3. TWD measurement

Procedure for re-suspending AC and HC cells from tulture medium to buffer was the same as mentionedore. After that, AC or HC cell samples were pipetted srately into the electrode channel and covered with aubstrate to build an enclosed fluid. In the measuremenrequencies (20 and 40 kHz) were applied. At those freqies dipole moment of cell was opposite to the applied fiausing negative DEP (repulsion from the electrodes)[7].

AC and HC cells experienced a linear force and tendeove in between electrodes from the top of the chambottom or from bottom to top when the direction of elec

Y. Wu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 262 (2005) 57–64 59

field was reversed. TWD effects were quantified by deter-mining the linear velocity of the cell traveling in betweenelectrodes. Same set of experiments was repeated 3–4 times.Motions were captured by the identical equipments and con-ditions in ROT measurements. After each sample had beeninvestigated, the chamber was washed prior to the next ex-periment with ultra pure water and dried under a stream ofnitrogen.

2.4. Quantification of the main chemical components

To understand the differences in biochemical compositionbetween AC and HC cells, the main biochemical componentsincluding chlorophyll, crude protein, crude lipids, and totalcarbohydrate were quantified.

Chlorophyll was determined from the visible absorptionspectrum of the acetone extracts of AC and HC cells[8]. Thecrude protein was determined by dye binding method[9]. Ex-traction of crude lipids from biomass powder was performedaccording to the procedure of Zhu et al.[10]. Solvent phasewas recovered by centrifugation, and evaporated on circula-tion baths at 70◦C and dried under infrared light at 30◦C.The content of carbohydrate was determined by the methodof 3,5-dinitrosalicylic acid colorimetry[11]. Powder (0.5 g)was acidified with HCl to a final concentration of 2.5N andh ◦ l-u ltings

tiali

3

3

dedtv e ong

where a rotating electric filed was established by address-ing four electrodes with sinusoidal ac voltages at phases of0◦, 90◦, 180◦, and 270◦, respectively (Fig. 1b). A layer oftitanium (50 nm) was first deposited onto the glass substrateusing standard photolithography processes[12] followed bya 150 nm layer of platinum. Each of the four polynomial mi-croelectrodes on the glass substrate was connected to onepair of bonding pad on the PCB to provide voltage supply.An “O” shaped ring with height 2 mm and radius 4 mm wasplaced on the glass chip to build the chamber. Small scales ofthe electrodes were used to lower the operating voltages for agiven desired ROT torque, resulting in the reduction in elec-trical heating and electrochemical effects. The “hyperbola”electrode design was used to maintain the uniformity of theelectric field in the chamber.

The design and fabrication procedure for TWD chips(Fig. 2a) were ultimately the same except for the use of amulti-electrode structure (Fig. 2b) [13] instead of four poly-nomial electrodes of the central chamber. Each single elec-trode was 20�m wide and was spaced at 20�m. The TWDchip consisted of 100 individual electrodes and every four ofthem were addressed by ac signals with the same magnitudeand frequency but phase differences.

A purpose-built generator controlled by computer-produced such ac signals to both ROT and TWD boards. Theuse of a computer in this experiment allowed better controlo lue)a

3

wedt lls at4d id ath ellsr xtrac-t and4 llsc flect-

F the RO oarT h and e beo erated h phasess

ydrolyzed at 100C for 30 min. After neutralization, the vome was adjusted to 100 ml and then filtered. The resuolution was used for assay of carbohydrate.

AC and HC cells were further observed with a differennterference microscope and electron microscope.

. Results and discussion

.1. Electrodes

The essential part of the electrokinetic system incluhe ROT (8 cm× 4 cm) and TWD chips (12 cm× 6 cm) de-eloped by us. The ROT chip had an essential part madlass substrate using microelectrodes technology (Fig. 1a)

ig. 1. Figures associated with ROT measurement. (a) Photograph ofhe white-insulating tape surrounded was used to protect from scratcpposite polynomial electrodes was 400�m. A rotating field can be genpaced 90◦ apart.

f the magnitude (ranging from 0 to 22 V, peak to peak vand frequency (ranging from 0 to 40 MHz) of the signal.

.2. General characteristics of AC and HC cells

Determination by the visible absorption spectrum shohe absorption peaks of the acetone extraction of AC ce32.3, 451.5, 462.5, and 665.4 nm (Fig. 3, curve a), whichemonstrated the presence of chlorophyll and carotenoigh concentrations. The chlorophyll content in AC ceached as high as 1.5% (w/v). However, the acetone eion of HC cells only showed absorption peaks at 451.576.7 nm (Fig. 3, curve b). Compared with curve a, HC ceompletely lost the absorption at 432.3 and 665.4 nm, re

T experimental device. It was basically a double layer-printed circuit bd (PCB).rosion. (b) The schematic outline of the glass substrate chip. Distancetween twobetween four electrodes by applying sinusoidal voltages to them wit


Fig. 2. Figures associated with TWD measurement. (a) Photograph of the TWD experimental device. It was a double layer PCB. (b) Schematic outline ofthe traveling-wave dielectrophoresis arrays made on glass substrate for operation with a four-phase signal. On the rectangular chamber, 100 electrodes wereconstrued with 40�m apart and each connected to a bonding pad.

Fig. 3. The visible absorbance spectra of acetone extracts from autotrophic(a) and heterotrophic (b)C. protothecoidescells.

ing the disappearance of chlorophyll in HC cells. Almost nochlorophyll content was detected in HC cells. The absorp-tion at 451.5 and 476.7 nm reflected a very low concentrationof carotenoid. The HC cells contained many lipid vesicles,which can be easily observed under differential interference

microscope (Fig. 4b). Whereas, almost no lipid vesicles wereobserved in AC cells (Fig. 4a).

3.3. ROT and TWD differences

Fig. 5shows the ROT spectra of AC and HC cells by usingthe approach described above. During each time of experi-ment, the voltage applied was set to 8.2 V (peak to peak) andthe frequency was sequentially changed from 100 to 15 MHz.Simultaneously, an oscilloscope (Tektronix TDS210) wasconnected from the voltage supply in order to confirm the con-tinued operation of the stimulus signals. Three types of sus-pending buffers were considered and tested with conductivi-ties of 300, 580 and 1000�S m−1. A buffer with 300�S m−1

was finally preferred because it provided the largest differ-ence in spinning rate.

Rotation speed of cell was related to the torque created bythe ac electric field and the equations were as follows givenby a particle with radiusR exposed to an electric field ofmagnitudeE and angular frequencyω

�(ω) = −Im{m(ω)}E (1)

Fig. 4. Cells of autotrophic (a) and heterotrophic (b)C. protothecoidesunder diffe ved inAC cells. HC cells were full of lipid vesicles.

rential interference microscope. Almost no lipid vesicles were obser


Fig. 5. ROT spectra of AC and HC cells. (�) ROT spectrum of AC; (�)ROT spectrum of HC. See text for details.

m(ω) = 4πεm f (ε∗m, ε∗

p)R3E (2)

(Following the theories of Maxwell[14] and Wagner[15].)where m(ω) was the effective dipole moment,�(ω) the rota-tion torque.f (ε∗

m, ε∗p) the well known Clausius–Mossotti fac-

tor defined asf (ε∗m, ε∗

p) = (ε∗p − ε∗

m)/(ε∗p + 2ε∗

m) in whichε∗p

andε∗m were the particle and surrounding medium complex

permittivities, respectively, defined byε∗ = ε − j(σ/ω) with ε

the permittivity,σ the conductivity andj =√−1. Im in Eq.

(1) referred to the imaginary part of the Clausius–Mossottifactor.

Velocity of those cells that were rotating with the electricfield was preset to be positive in our plotting, and vice versa.As shown inFig. 5, there were three rotating peaks for eachspectrum, two positive and one negative. Great differencesoccurred in the frequency window 10 kHz to 10 MHz wherethe spinning rates of AC cells were faster than HC. In partic-ular at the frequency of 1 MHz, the most evident differenceof rotating rate was observed, where speed of AC cells wasalmost two times that of HC cells. Additionally, it was ob-served that AC and HC cells rotated in opposite directions at7 kHz (positive rate for HC and negative for AC), allowingfor easy discrimination of the two kinds of cells.

Linear speed of AC and HC were obtained by using theTWD measurement approach described above. In both twofrequencies (20 and 40 kHz), velocities of AC cells wereg Tr WDf aryct dingm arer si , soi mentw

dt s oft ctricp ico-c tiono tric

Fig. 6. Linear speed of AC and HC in TWD electric field. Unit is m s−1 V−2,normalized against the square of voltage. In both two frequencies, speed ofAC cells were faster than HC, which proved our ROT results.

properties, namely in this experiment, the conductivity andpermittivity of the cell walls and membranes[17].

3.4. Biochemical components in AC and HC cells

In order to understand why there were such obvious differ-ences of AC and HC cells in ROT and TWD (Figs. 5 and 6),main biochemical components of AC and HC cells were ob-tained (Table 1) using the qualification method describedabove. As shown inFig. 4, heterotrophic growth ofC. pro-tothecoidesresulted in not only the disappearance of chloro-phyll, which turned the color of the cell into yellow, but alsoaccumulation of high lipid content in cells. Lipid content inHC cells reached was as high as 55.20% (Table 1), which wasabout four times that of AC cells (14.57%,Table 1). Proteincontent in HC cells was 10.28%, only 1/5 of that of AC cells(52.64%). The difference of carbohydrate content (10.6% inAC and 15.43% in HC) was not great as compared to that ofproteins and lipids (Table 1).

The above biochemical results were confirmed by elec-tron micrographs of AC and HC cells. As clearly shown inFig. 7, AC cells were full of cup-like chloroplasts and lamel-lae. However, many electron-transparent granules were ob-served in HC cells, which were inferred to be lipid granules.This confirmed the accumulation of large amounts of lipidi tivei sedl

TC

C

PLCO

reater than HC (Fig. 6). This is in consistent with our ROesults. It is well established that the time-averaged Torce acting on a particle is proportional to the imaginomponent of the induced dipole moment[16]. According tohat and Eq.(1), given the same type of cells and suspenedium with equal conductivity, ROT and TWD speed

elated only to the imaginary part of m(ω) [13], which meanf the ROT speed of AC cells is faster than that of HC cellss its TWD speed. Those TWD results are in good agreeith our ROT results.The field-induced dipole moment (m(ω)) is closely relate

hrough the relative dielectric and conductive propertiehe particles and their surrounding medium. The dieleroperties of the particle are in turn related to the physhemical properties of the particle. Thus the induced rotaf the particle is a sensitive function of the particle’s dielec

n HC cells (Table 1). Besides, the graph gives quantitanformation about AC and HC cell walls, which were uater in the three-shell model to simulate ROT spectra.

able 1ontents of the main chemical components of AC and HC cells (%)

omponent AC HC

rotein 52.64± 0.26 10.28± 0.10ipid 14.57± 0.16 55.20± 0.28arbohydrate 10.62± 0.14 15.43± 0.17thers 22.17± 0.74 19.09± 0.67


Fig. 7. Cells of AC and HC under electron microscope. AC cell had nucleus, cup-like chloroplast and lamellae. However, many electron-transparent granuleswere observed in HC cell, which were inferred to be lipid granules.

According to the measurements, there are huge differencesin the biochemical compositions of AC and HC cells. As wementioned before, these two cells were from the same algalstrain, the only change we made was the culture conditions.No other cells can exhibit such great biochemical distinctionsso far regardless of what kind of methods applied. Therefore,AC and HC cells are the best suitable example to study. Basedon the biochemical differences, we suggested AC and HCcells would behave differently under ROT and TWD electricfields because biochemical components are closely related toelectrical properties. For example, the cell membrane consistsof a lipid bilayer containing many proteins, which makes themembrane highly insulating. Thus, the conductivity of thecell membrane tends to be around 10−7S m−1. The results ofROT and TWD rates (Figs. 5 and 6) are in agreement withthe above suggestion.

In addition to the variation of the biochemical components,was there a difference in ROT and TWD effects between ACand HC cells due to the size variation? To address this ques-tion, we monitored the AC cells of different sizes, and oncemore, monitored the HC cells of different sizes in other time.The results showed that there was little effect of size varia-tion within the scale we observed on the spin or line speedin ROT and TWD experiments. Therefore, we suggested thatthe size variation was not the key factor to affect the be-

haviors of algal cells in the ROT and TWD electric field.Since the size difference between AC and HC cells was soslight, its effect on the difference in ROT and TWD could benegligible.

3.5. Three-shell model

Theoretical ROT (solid line) and cDEP (dotted line) spec-tra exhibited by AC and HC cells are shown inFig. 8a andb, respectively. Lines are best fit according to the three-shell model, which is simulated from a two-shell model[18,19]. The following dielectric properties (literature fromyeast) of the suspending medium and the cell compartmentswere taken into account: measured medium conductivity(300�S m−1); cell diameter (7�m) and thickness of cellmembrane (4.5 nm). The thickness of the cell wall for HCcells was 0.22�m and that for AC was two times that of HC(Fig. 7).

The three-shell model gives the relative conductivitiesand capacitances of AC and HC cell walls and membranes(Table 2). Cell wall capacitance for AC is 0.045�F/cm2 whilecapacitance for HC is 0.341�F/cm2. This result is confirmedby previous research[20], which demonstrated that the ACcell wall was smooth and the HC cell wall was wrinkled. Asa result, the surface area of the HC cell wall is greater than

and HC
Fig. 8. Ideal cDEP (· · ·) and ROT (—) spectra of AC (a) (b) cells according to three-shell model. See text for details.


Table 2Dielectric properties of each cell compartments of AC and HC cells

AC HC

Membrane Wall Membrane Wall

Capacitances (�F/cm2) 0.121 0.045 0.367 0.341Conductivites (S m−1) 5.68E−7 0.002 3.834E−7 0.008

AC and thus, cell wall capacitance of HC is larger than ACcells.

As shown inFig. 8, our theoretical model did not give apositive peak in the frequency window 100 Hz to 10 kHz. Thatis because our three-shell model was based on Eqs.(1)and(2)and most importantly on Clausius–Mossotti factor. Accord-ing to the Clausius–Mossotti factor, the limit off (ε∗

m, ε∗p)

goes to zero when the frequency approaches zero. Therefore,the Clausius–Mossotti factor must be modified to justify ourthree-shell model in the further work. However, the rest of thetheoretical curve matches well with our experimental curveand explains the different behaviors of those two cells in ROTmeasurements.

3.6. Potential applications of the system

The first application is cell isolation. TWD measurementwas taken in our experiment to support ROT results becauseROT and TWD behaviors were related to each other as de-scribed above. However, the TWD device developed by uswas mainly used to perform cell manipulation and separa-tion before. Mixtures of different kinds of particles (usuallytwo) can be pipetted onto the channel region of the elec-trode structure. Two forces can be applied to them, caus-ing the movement of cells in two perpendicular directions.O vet ove-m thatd fer-e ppliest lec-t WDa ointsd andf iumc dif-f y ofR ACs dif-f OTs ctiv-i ont canp HCc

onset undt peed

in ROT and TWD measurement compared to those that hadfewer proteins. An explanation for this phenomenon is thatproteins can carry more negative electrical charges than docarbohydrates and lipids. With the applied electric field suchas ROT or TWD, particles with more proteins will becomemore polarized and consequently, exhibiting larger rotatingtorque or linear force. Although the protein content is themost important factor to determine the dielectric property inalgal cells, we do not think the differences of ROT and TWDeffects are only resulted from the content of differences ofprotein. The variations in conductivities and capacitancesresulted from the changes of cell wall, cell membrane ordifferences in lipid content in those two kinds of cells maybe the other effective factors. This proposal needs to beverified in the future research. If found to be true, it willbe possible to estimate the amount of proteins, lipids, andother biochemical components contained in particles usingROT spectra. That would provide another way to determinethe biochemical compositions of particles rather thanthe conventional chemical and biological method usuallyapplied.

Acknowledgements

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ne is the fluid flow, cells undergoing the flow will hahe same speed following the fluid. The other is the ment generated by the applied TWD electric field. Inirection, there will be displacement differences of difnt particles and cause them to be separated. This a

o the case of AC and HC cells as well. Changes in dieric properties of AC and HC cells can be detected by Tnd used for selective separation among them. Key petermining cell separation experiment are the voltage

requency supplied and conductivity of suspending medhosen[13]. Through repeated ROT experiments usingerent kinds of suspending mediums and detailed studOT spectrum, conductivity of buffer, and frequency ofignal can be found to give the most evident velocityerences of AC and HC cells. In our case, from the Rpectra, the great difference occurred when the conduty was 300�S m−1 and frequency was 1 MHz. Basedhe dielectric properties studied by the experiment, weerform selective separation and purification of AC andells.

The other application we propose is using ROT respo determine biochemical components of cells. It was fohat cells with high percentages of proteins had faster s

We are grateful for Dr. M. Guo at National Engineeresearch Center for Beijing Biochip Technology, China,r. J.P. Xu at McMaster University, Canada, for their valuauggestions in manuscript preparing.

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