Trapping and Imaging of Micron-sized

Short Communication

Trapping and imaging of micron-sizedembryos using dielectrophoresis

Development of dielectrophoretic (DEP) arrays for real-time imaging of embryonic

organisms is described. Microelectrode arrays were used for trapping both embryonated

eggs and larval stages of Antarctic nematode Panagrolaimus davidi. Ellipsoid single-shellmodel was also applied to study the interactions between DEP fields and developing

multicellular organisms. This work provides proof-of-concept application of chip-based

technologies for the analysis of individual embryos trapped under DEP force.

Keywords:

Dielecrophoresis / Embryo / Lab-on-a-chip / Microfluidics / Real-time imagingDOI 10.1002/elps.201100160

Small multicellular organisms such as nematodes are

emerging models for an increasing number of biomedical

studies [13]. They offer substantial advantages over cell

lines and isolated tissues, providing analysis of cells in the

context of cellcell and cellextracellular matrix interactions

and under normal physiological milieu of the whole

organism. This provides analytical capabilities that cannot

be easily replicated in vitro, such as organogenesis and

tissue regeneration [4]. Small size, optical transparency of

organs and ease of husbandry make them ideal models for

large-scale genetic and pharmacological studies. Moreover, a

large number of environmental and ecotoxicological tests

can only be performed using small model organisms [2].

Automated manipulation and immobilisation of

micron-sized small-model organisms such as the nematodes

is, however, a challenging task [5, 6]. Several innovative

microfabricated technologies for manipulation, immobili-

sation and sorting of a popular laboratory roundworm

(Caenorhabditis elegans) have recently been reported. Theseamong others include microfluidic chambers, clamps,

pneumatic valves, microsuction manifolds, thermal and

chemical immobilisation and segmented flow [7, 8].

Interestingly, despite increasing numbers of reports on

the immobilisation and on chip culture of adult and larval

stages of C. elegans, no attempts have been so far made totrap developing emryonated eggs. Moreover, virtually no

work has been performed on non-invasive, dielectrophoretic

(DEP) immobilisation of nematodes. DEP refers to the

motion of polarisable particles induced by a spatially non-

uniform electric field. DEP has already found noteworthy

applications in immobilisation, trapping and sorting of

human cells, yeast, bacteria, and cell organelles [913]. Here,

for the first time, we describe the development of a mini-

mally invasive chip that can be used for rapid immobilisa-

tion and imaging of developing multicellular organisms

such as the nematode Panagrolaimus davidi under positiveDEP (pDEP).

Thin films of chromium/gold were deposited to a

thickness of 500 A/1500 A on a glass substrate using elec-

tron beam evaporation, and then patterned using standard

photolithography techniques to form an array of curved

Khashayar Khoshmanesh1,2

Nimrod Kiss3

Saeid Nahavandi2

Clive W. Evans3

Jonathan M. Cooper4

David E. Williams5

Donald Wlodkowic1

1The BioMEMS Research Group,Department of Chemistry,University of Auckland,Auckland, New Zealand2Centre for Intelligent SystemsResearch, Deakin University,Waurn Ponds, Australia3School of Biological Sciences,University of Auckland,Auckland, New Zealand4The Bioelectronics ResearchCentre, Department ofElectronics and ElectricalEngineering, University ofGlasgow, Glasgow, UK5Department of Chemistry andMacDiarmid Institute forAdvanced Materials andNanotechnology, University ofAuckland, Auckland, NewZealand

Received March 13, 2011Revised May 4, 2011Accepted May 5, 2011

Abbreviations: DEP, dielectrophoretic/dielectrophoresis;pDEP, positive DEP

Additional corresponding author: Dr. Khashayar KhoshmaneshE-mail: [email protected]

Colour Online: See the article online to view Figs. 1 and 3 in colour.

Correspondence: Dr. Donald Wlodkowic, The BioMEMSResearch Group, Department of Chemistry, University of Auck-land, Auckland 1142, New ZealandE-mail: [email protected]: 164-9-3737422

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2011, 32, 31293132 3129

microelectrodes (Fig. 1A and Supporting Information) [14].

A detachable poly(dimethylsiloxane) (PDMS; Sylgard 184,

Dow Corning) microculture chamber with a height of

approximately 4 mm was then assembled onto the micro-

electrode arrays (Fig. 1A).

The nematode P. davidi was originally collected from theMcMurdo Sound region in Antarctica and was maintained

in vitro as a parthenogenetic strain [15]. Nematodes were

grown at 15201C on standard nematode growth medium(NGM) agar plates seeded with E. coli as a food source [16].Embryonated eggs, larval stages and adults were rinsed

from culture plates with distilled water (DI) and filtered

through a 40-mm cell strainer to separate the embryonatedeggs from hatched stages.

Assuming the eggs and worms as ellipsoid structures,

the time-averaged DEP force applied on them is defined as

below [17, 18]:

FDEP pr2L

3 emed RefCM HE2rms 1

where r and L are the radius and the length of ellipsoid (seeSupporting Information), emed is the permittivity of themedium, Erms is the root-mean-square value of the appliedelectric field and Re[fCM] is the real part of the Clausius-

Mossotti factor, which reflects the polarisation of the

bioparticles relative to the surrounding medium. The

Re[fCM] was calculated using the ellipsoid single-shell modelfor both eggs and larval stages at different aspect ratios

(length [L]/diameter [D]), (Fig. 1B and C) [19]. The details ofcalculations, including equations, structural and dielectric

properties of eggs/worms, as well as the dielectric properties

of the medium are given in the Supporting Information.

Interestingly, the Re[fCM] was stronger along the major axisof eggs/worms and strongly depended on the aspect ratio of

the specimens. For example, at L/D5 1.5, the eggs exhibiteda crossover frequency of 31.5 kHz above which they

demonstrated pDEP response. The Re[fCM] reached a peakvalue of 0.6870.035 within the frequency range of0.210 MHz, and decreased thereafter until reaching 0.27 at

40 MHz (Fig. 1B). In contrast, the larval stages exhibited a

pDEP response across the whole frequency range. For

example, at L/D5 15, the Re[fCM] reached a peak value of20.171.1 within the frequency range of 65450 kHz,and decreased thereafter until reaching 1.04 at 40 MHz

(Fig. 1C).

Numerical analysis was further performed using the

finite-volume-based Fluent 6.3 software (Fluent, Lebanon,

NH, USA) to evaluate the performance of the DEP system.

Figure 1. The principles of theDEP system: (A) assembledDEP manifold with an arrayof microelectrodes and amicroculture PDMS chamber:glass substrate, microfabri-cated chrome/gold electrodearray, interconnecting padsproviding AC signal actuation,and microculture chamber;(B, C) application of ellipsoidsingle-shell model to charac-terise the DEP responsesof multicellular organisms.Re[fCM] factor was calculatedalong the major and minoraxes of eggs/worms at differ-ent aspect ratios (length/diameter), as detailed inSupporting Information; and(D) distribution of DEP force,at z5 10 mm with respect tothe bottom surface of the chipfor eggs (L-axis, L/D5 1.5 @20MHz) and worms (L-axis,L/D5 15 @ 0.11MHz and20MHz).

Electrophoresis 2011, 32, 312931323130 K. Khoshmanesh et al.


In doing so, the Laplace equation was solved to obtain the

electric potential H2j 0, and consequently the electricfield was obtained by taking the gradient of electric potential

E Hj [14]. Figure 1D shows the distribution of DEPforces, at z5 10 mmwith respect to the bottom surface of thechip. Due to the curved geometry of the microelectrodes, the

attractive force increased along the microelectrodes, reach-

ing a peak value at the tips, where the field strength was

highest. As the eggs/worms exhibit pDEP responses at the

applied frequencies of 0.120 MHz, the curved microelec-

trodes will provide large delta-shaped immobilisation areas

close to the tips (Fig. 1D). The eggs and worms experienced

different forces due to their different dimensions and

Re[fCM] values (see Supporting Information). For example,at z5 10 mm, the eggs experienced a maximum pDEP forceof 2.07 108 N at 20 MHz, the worms experienced amaximum force of 1.12 108 N at the same frequency butexperienced an intensified force of 9.61 108N at0.11 MHz.

To immobilise the specimens, embryonated eggs and L1

larval stages were resuspended in DI water of 5 104 S/mand 200 mL of the sample was injected to the PDMS cham-ber. Microelectrode arrays were energised at 10 Vp-p and

20MHz. Joule heating and electrolysis were minimised due

to the low conductivity of DI water and high frequency of

the field. Eggs were trapped under the pDEP force with

major axis oriented along the electric field (Fig. 2A). They

become polarised and thus subsequently attracted remain-

ing eggs to their free ends, creating characteristic pearl

chains [20] (Fig. 2A). Interestingly, the larval stages could

also be trapped under the pDEP force and remain viable for

over 15 h, as evidenced by their continous locomotion. The

trapping DEP force was, however, not strong enough to

immobilise them but locomotion was restricted to the

electrodes surface area where they were retained (Fig. 2B).

Interestingly, decreasing the frequency to 0.11 MHz

induced an immediate immobilisation and stretching of

larval worms along the electric field (Fig. 2C and Supporting

Information). The DEP force at such low frequencies was

strong enough to overcome the locomotory functions of

P. davidi, which is in line with our numerical models(Fig. 1C). Viability of the worms was, however, greatly

reduced down to only 2 h, most likely due to a number of

events occurring at low frequencies, including heating of the

medium due to Joule effect, imposing intensive trans-

membrane voltage on the cells, and unwanted chemical

reactions at the surface of microelectrodes [21]. Never-

theless, decreasing the frequency to 0.11 MHz did not

considerably affect the immobilisation of eggs (see

Supporting Information).

To demonstrate the feasibility of DEP-based arrays for

dynamic analysis of developing embryos, a non-synchro-

nised population of embryonated eggs was trapped

approximately 10001400 mm away from the tips at 10 Vp-pand 1MHz frequency (Fig. 3). Specimens immobilised at

this location experienced a weaker electric field and

temperature rise. They could survive over long periods of

1015 h. As presented in Fig. 3, the pDEP trapping did notaffect the embryo development that progressed normally till

hatching, as compared with control chips without energised

electrodes (not shown). The eggs remained stably immobi-

lised except for some minor displacements caused by the

movements of the hatched P. davidi larvae.In conclusion, the DEP microelectrode array greatly

reduces the complexity of conventional protocols and

permitted for rapid immobilisation of both nematode eggs

and larval stages. Due to their suspending nature and result-

ing dislodgment, positioning and imaging of microscale-sized

eggs represents a particular challenge. Our work provides

proof-of-concept data that DEP immobilisation neither

adversely affects egg development nor viability of early larval

stages while at the same time allows for real-time analysis at a

single egg level. The major advantages of DEP-based immo-

bilisation are that specimens can be (i) trapped for extended

periods of time, (ii) stimulated with chemical compounds of

interest and analysed and (iii) subsequently released for

further processing [10, 22], which should be further investi-

gated in the future research. Integration with microfluidic

devices featuring on chip valving systems is currnetly ongoing

that will fully suport on chip small model organisms studies,

specimen recovery post-analysis and potential for automated

Figure 2. The response of P. davidi embryonated eggs and L1larval stages to the DEP field: (A) the eggs trapped along thesurface of microelectrodes under the pDEP force at 20MHz;(B) juvenile worms trapped along the surface of microelectrodesunder the pDEP force at 20MHz are able to move only at theclose proximity of the trapped location but not able to leave themicroelectrodes; and (C) immobilisation and stretching ofjuvenile worms that overcomes their locomotory functionsunder reduced frequency (0.11MHz).

Electrophoresis 2011, 32, 31293132 Microfluidics and Miniaturization 3131


hight-througput pharmacological screening. We postulate that

manipulating micron-sized embryos and larval stages of

nematodes using DEP principles avoids extensive preparative

procedures and is therefore likely amenable for automated

handling of model organisms on a chip.

Grant sponsor: Supported by the Endeavour ResearchFellowships, Department of Education, Employment andWorkplace Relations, Australia and the Centre for IntelligentSystems Research, Deakin University, Australia (K. K.); Foun-dation for Research Science and Technology (FRST), NewZealand (D. W., D. E. W.); Faculty Research and DevelopmentFund, University of Auckland. New Zealand (D. W.); andBBSRC, EPSRC and Scottish Funding Council, United King-dom, funded under the RASOR Program (J. M. C.).

The authors have declared no conflict of interest.

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Figure 3. Dynamic analysis of developing embryonated P. davidieggs patterned along the energised microelectrodes. pDEPtrapping does not affect the egg development (red arrow) orthe hatching of larval stages (yellow arrow) for up to 15 h.

Electrophoresis 2011, 32, 312931323132 K. Khoshmanesh et al.


Trapping and Imaging of Micron-sized

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