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PAPERLiangti Qu and Liming DaiDirect growth of 3D multicomponentmicropatterns of vertically alignedsingle-walled carbon nanotubesinterposed with their multi-walledcounterparts

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COMMUNICATIONShaliniGuptaet al.On-chipelectricfielddrivenassemblyofbiocompositesfromlivecellsandfunctionalizedparticles

Soft Matter

COMMUNICATION www.rsc.org/softmatter | Soft Matter

On-chip electric field driven assembly of biocomposites from live cells andfunctionalized particles†

Shalini Gupta, Rossitza G. Alargova,‡ Peter K. Kilpatrick and Orlin D. Velev*

Received 19th November 2007, Accepted 21st January 2008

First published as an Advance Article on the web 12th February 2008

DOI: 10.1039/b717850f

Live cells and surface-functionalized synthetic microparticles are

co-assembled on electrically controlled chips to yield permanent

chains and one cell layer thick membranes that can be freely

manipulated in external magnetic fields.

The directed assembly of colloidal particles can be used to synthesize

microscopic functional structures. Conventional techniques for

assembly of particles in suspension by methods such as sedimenta-

tion,1 restricting free volume,2 capillary forces3 and convective evap-

oration,4 however, can be slow, inefficient, and difficult to control.

One way to achieve rapid, controllable and scalable colloidal

assembly is the use of alternating current (AC) electric fields. The

mobility and interactions of particles induced by AC electric fields

is called dielectrophoresis (DEP).5 The interactions between the

dipoles induced in the particles of vastly different systems (ferrofluids,

electrorheological fluids, anisotropic liquid-crystalline drops) lead to

formation of particle chains and crystalline phases.6–10 Examples of

colloidal assemblies obtained using DEP include microscopic

biosensor patches, gold nanoparticle microwires and switchable

particle crystals.11 These assemblies, however, represent only a small

fraction of the useful structures that can be fabricated by using

electrical fields on a chip. For example, the precise and controllable

fabrication of biomaterials from live cells still remains one of the

most promising, yet largely unrealized, applications of colloidal

assembly. We demonstrate here how directed assembly of cell–

particle composites can be achieved by transcribing the colloidal

on-chip assembly principles to the biomaterials domain.

Patterned biomaterials with live cells have previously been assem-

bled by cell adhesion to template surfaces that have been patterned

photolithographically or by microcontact printing with oligopepti-

des.12,13 Cells in microfluidic devices can also be patterned by laminar

flow14 and DEP over complex electrode patterns.15 A large number of

studies report preparation of biomaterials fabricated by cell infusion

of and adhesion to porous scaffolds16 and porous silica hosts modi-

fied with lipid layers.17 However, we are not aware of studies

describing the directional on-chip assembly of cells into freely

suspended and oriented structures, such as chains or membranes.

One of the problems in such a process is that live cells collected and

arranged on their own typically do not possess the mechanical

cohesiveness required to form materials and device components.

Department of Chemical and Biomolecular Engineering, North CarolinaState University, Raleigh, 27695-7905, NC, USA. E-mail: odvelev@unity.ncsu.edu; Tel: +1 919-513-4318

† Electronic supplementary information (ESI) available: Movies 1–6 plusother supplementary data. See DOI: 10.1039/b717850f

‡ Present address: Vertex Pharmaceuticals Inc., Cambridge, 02139-4242,MA, USA.

726 | Soft Matter, 2008, 4, 726–730

The challenge is thus to bind the live cells together in cohesive struc-

tures that are robust enough to be manipulated by external fields.

Here, we show the assembly of permanent materials by using

functionalized synthetic microparticles binding to the cells through

biospecific interactions.

Two types of electrode chambers were developed for electric field

manipulation of cell–particle suspensions. Chips with two coplanar

electrodes allowed applying the field in one direction (Fig. 1A). Chips

with four electrodes were used in experiments where the field could be

applied in two perpendicular directions by connecting the voltage

source to one of two opposing electrode pairs (Fig. 1B). The

electrodes were encapsulated in small fluidic chambers, where the

cell suspension was injected. The assembly process was continuously

monitored using optical microscopy.18 The surface of the chips and

the microfluidic chambers was treated with 0.5% F-127 Pluronic

surfactant for 45 min prior to the experiment to minimize non-

specific interactions of the cells and particles with the substrate. AC

electric fields of intensities between 5–20 V mm�1 and frequencies

in the range of 30 Hz–5 kHz were applied across the electrodes by a

square wave field generator connected to a voltage amplifier. A 1 mF

capacitor was connected in series to the chamber to filter any direct

component of the voltage.

Suspensions of brewer’s yeast (S. cerevisiae) cells were freshly

prepared in phosphate buffered saline at pH 6.3. The buffer was

diluted to an electrolyte concentration of 1.5 mM to avoid electrolysis

and to minimize electroosmotic flow of the solution. NIH/3T3 mouse

fibroblast cells were cultured in standard Dulbecco’s modified Eagle’s

medium (DMEM) for 2 days. The cells were trypsinized and resus-

pended in an isotonic 0.45 M dextrose solution with 1% v/v calf

serum at pH 7.2. 0.15 mM CaCl2 was added to the cell–particle

mixtures to facilitate the binding of the lectins to polysaccharides.19

Bovine serum albumin (� 0.1% w/v) and non-ionic surfactant

(� 0.1% w/v Tween 20) were added to prevent non-specific aggrega-

tion or adhesion of cells or particles.

In the beginning of the experiments the cells were randomly

dispersed throughout the chamber where they began slowly sedi-

menting towards the bottom. When the AC field was applied, the

cells began to align within 30–60 seconds into chains in the direction

of the field lines. This is a typical assembly pattern for particle suspen-

sions in electric fields resulting from the interaction between the

dipoles induced in the cells. Within 10–15 min most of the cells

were captured into chains. The chain formation was faster and the

resulting chains were longer at higher voltages and lower frequencies

of the electric field (for more details see the ESI†). The chains near the

electrodes were also pulled by positive DEP and AC electrohydrody-

namic currents into the areas of highest field intensities at the elec-

trode edges. A typical image of the yeast cell chains assembled in

less than 10 min using the two-electrode setup is shown in Fig. 1A.

This journal is ª The Royal Society of Chemistry 2008

Fig. 1 Schematics of the two electrode configurations used for on-chip assembly of cell composites and examples of structures obtained. (A) Chains

from live yeast cells assembled with a two-electrode cell; (B) 2D ordered arrays from live yeast cells made by using a four-electrode setup.

Fig. 2 Permanent composite structures from cells and Concanavalin-

A-coated microparticles assembled in AC electric field. (A–B) Linear

clusters of yeast cells and 1 mm fluorescent particles assembled at 15 V

mm�1 and 200 Hz. The specific organization of the particles in the junc-

tion between the cells (compare the regular and fluorescence microscope

images) is a result of induced dipolar interactions. (C) Live cell chain

assembled at 17 V mm�1 and 100 Hz from yeast cells and 0.95 mm

magnetic particles. (D) Rotation of this wire with an external magnet

after the electric field was switched off. (E–F) Small chains from NIH/

3T3 mouse fibroblast cells and 0.95 mm magnetic particles assembled at

10 V mm�1 and 100 Hz and manipulated with an external magnetic field.

Scale bars represent 20 mm.

The cell chains exhibited weak lateral interactions and did not

assemble into two-dimensional (2D) arrays. Isolated areas with local

ordering could be observed but the overall cell-packing efficiency was

low. These assembly dynamics are different from our previous results

for 2D crystal assembly of latex and silica particles using a two-

electrode cell.20 The process of DEP-driven latex crystallization of

microspheres involved the formation of chains (similar to the ones

observed with cells), as an intermediate structure, but the chains

also attracted laterally to form close-packed hexagonal 2D crystals.

The lack of attraction amongst the cell chains is probably a result

of the cell polydispersity and, weaker polarizability and dipolar inter-

actions in comparison to synthetic microspheres. We managed to

draw the cells into uniformly close-packed arrays by using the

four-electrode cell configuration in which the field was applied

consecutively in two perpendicular directions. We first applied the

field in one direction for approximately 15 min and then switched

between the two electrode pairs approximately every 5 min for a total

duration of 30–45 min. This drew the neighboring cells together into

closely packed single-layer cell arrays via axial dipolar interactions

(Fig. 1B, see also ESI† Movies 1 and 2).

The 1D and 2D cell arrays assembled by the field were not perma-

nent and started disassembling shortly after the voltage was turned

off. We used microparticles functionalized with lectins to perma-

nently bind the live cell structures. In one system, 1 mm FITC-labeled

fluorescent microparticles had chemically attached Concanavalin-A

on their surfaces, which could bind to the saccharide functional

groups on the surface of the cells.21 We found that AC field in the

low-frequency range of 10–200 Hz lead to sequestration of the parti-

cles into the junctions between the cells, resulting in the formation of

alternating cell–particle assemblies (Fig. 2A and B). These cell–

particle chains did not disassemble after the field was turned off

and remained as permanent chains of cells and particles.

The attraction and binding of the particles into the interstitial

space between the cells during the process of field-induced organiza-

tion thus proved crucial for the cell–particle chain assembly. A

numerical procedure was developed to simulate the polarization-

induced cell–particle interactions and dynamics of assembly under

complex electrostatic fields, and to predict the types of structures

that can be formed at different configurations and frequencies of

the field. The polarization, ~P, induced in the cells and particles

This journal is ª The Royal Society of Chemistry 2008

by the external field, ~E, was calculated as: ~P ¼ a~E ¼ 4p

R3 3m

�3p�3m

3pþ2 3m

�~E. Here, a is the polarizability, R is the radius, and

3m;p ¼ 3m;p � jusm;p are the complex permittivities of the medium

and cells or particles, respectively. The complex permittivity of the

Soft Matter, 2008, 4, 726–730 | 727

cells was calculated as a function of the frequency (u) by using multi-

shell models.22,23 The parameters of this model include the dielectric

constant of cytoplasm, e2, and its conductivity, s2, membrane capac-

itance, cm, and transconductance, gm, dielectric constant of the cell

wall, e1, conductivity of cell wall, s1, and, inner and outer radius of

the cell wall, R and R0, respectively. The effective membrane polariz-

ability and the surface conductance of the cells were calculated as

a function of membrane potentials and electrolyte ionic strength.

The calculated complex frequency-dependent Clausius–Mossotti

function for the cells was in good agreement with our experimental

data and literature.22,23 A similar model was used to calculate the effec-

tive polarizability of the charged particles in the electrolyte solution24,25

(Model details and numerical parameters are provided in the ESI†).

The 2D electrostatic field intensity distribution was determined by

a finite element calculation of the set of PDEs for the system

geometry using the FEMLAB multiphysics modeling package

(COMSOL, Burlington, MA) as shown in Fig. 3A. The electrostatic

force vector was calculated by the boundary integration of the stress

tensor on the exterior surface of each cell or particle:~FEF ¼ � #

S

ð�12~E~D þ ðn̂1

~EÞ ~DT Þ dS. Here, ~D ¼ 30~E þ ~P, is the

Fig. 3 Time-lapsed images of the co-assembly dynamics of a cell–

particle system at 100 Hz. (A–C) Three snapshots of the dynamic simu-

lation, where the field intensity distribution is color coded. The higher

polarizability of the cells leads to attraction and chaining. The simulation

illustrates how one of the particles is captured in the higher intensity area

between the cells in the ‘‘chain’’. This process is observed experimentally

(D–F) and is used to bind the structure. Scale bar represents 5 mm.

728 | Soft Matter, 2008, 4, 726–730

electric flux density and e0 is the permittivity of free space. This force

was used to calculate the cell or particle displacement at the next stage

of the simulation by calculating the object velocity from the hydrody-

namic resistance:~v ¼ ~FEF

6 p h r. The use of Stokes sphere dynamics does

not account for the largely increased fluid resistance at small cell–cell

separations, but we do not expect this to change much the macro-

scopic cell trajectory. After the new coordinates of each object

were obtained, the electrostatic field distribution and the force vectors

were recalculated and the loop was repeated iteratively until an

equilibrium cell–particle structure was reached.

An illustration of how this model captures the dynamics of

co-assembly in a system comprising two cells and two particles is

presented in a panel of snapshots of the electric field distribution

and particle re-arrangement at 100 Hz (Fig. 3A–C). The simulation

illustrates clearly a dynamic where synthetic particles are attracted

and bound between the larger cells. At this frequency both the cells

and the particles are more polarizable than the media due to the

high mobility of the counter-ionic layers and also due to the high

conductivity of the cell cytoplasm. The areas of higher field intensity

above and below the cells (collinear with the field direction) attract

and capture the small particles by positive DEP. This in-turn

makes the attraction of another cell easier, trapping the particle

in-between them. Thus, we confirm that the formation of chains

(and membranes) of alternating cells and particles is an intrinsic

characteristic of the on-chip field-driven process. The experimentally

observed dynamics of the cell–particle co-assembly shown in

Fig. 3D–F were in good agreement with this simulation (compare

Supplementary Movies 3 and 4, ESI†). Notably, the phenomenon

is not universal at any frequency as separation of the cells and

particles into cell-rich and particle-rich chains was observed at

frequencies above 10 kHz. The process is also suppressed by large

concentrations of electrolyte. The ‘‘automatic’’ co-assembly at low

frequencies and low to mid-electrolyte concentrations, however,

offers an intriguing range of possibilities for controlled fabrication

of soft biomaterials.

We demonstrated the potential of the DEP assembly of cells on

a chip by fabricating a variety of biocomposites. The particles

incorporated between the cells served not only as binding elements,

but also imparted an additional functionality such as making the

biomaterials responsive to magnetic fields. For example, by using

0.95 mm lectin-coated paramagnetic particles as binding units, we

assembled permanent responsive cell ‘‘wires’’. These chains could

be aligned, rotated, and transported throughout the microchambers

by magnetic fields (Fig. 2C–F). They remained intact during the

manipulation and for several days following these experiments.

The same approach could be used in the 4-electrode chip for the

assembly of large (� cm2) permanent magnetic biomembranes

from live cells. Single layer membranes made of organized yeast

and fibroblast cells bound by lectin-coated magnetic particles are

shown in Fig. 4A and C. The intimate structure of the membranes

consisting of cells and particles could be observed by field emission

SEM after fixation with 2% w/v glutaraldehye and sputtering with

gold (Fig. 4B). Due to their intrinsic magnetic functionality, the

biomembranes assembled on the chip could be stretched, translated,

folded and rotated in 2D and 3D by external magnets (Fig. 5A and

B, and Supplementary Movies 5 and 6, ESI†). The magnetic response

of the biomembranes could also be used to extract them from the

chamber where they are assembled. The ability to assemble, trans-

port, and manipulate live cell biomembranes has direct applications

This journal is ª The Royal Society of Chemistry 2008

Fig. 4 Micrographs of permanent cell membranes bound together by

Concanavalin-A functionalized microparticles. (A) Large magnetic yeast

cell membrane at low microscope magnification; note that the membrane

is only one cell layer thick. (B) SEM image of a closely-packed fixed yeast

membrane. (C) NIH/3T3 mouse fibroblast membrane.

Fig. 5 Magnetic manipulation of responsive cell membranes. (A)

Folding of a large magnetic yeast cell membrane by an external magnetic

field. (B) Extraction and transport of a yeast cell membrane through thin

Teflon tube using an external magnet. Inset: arrow points to the actual

position of the membrane in the tubing attached to the chip used in

the experiments.

in the creation of artificial tissues and cell assemblies for sensing and

microsurgery.

We performed fluorescence tests of cell viability by the FUN-1 dye

method (Molecular Probes, Eugene, OR) on some of the yeast chains

and membranes assembled to prove experimentally that the cells

retain their metabolic activity after treatment on the DEP chips.

The proportion of metabolically active cells in the biomagnetic arrays

after a few hours was found to be approximately the same as the one

in the original suspension (z 90%). The viability of the fibroblasts

assembled in such structures below 15 V mm�1 field intensities deter-

mined by Trypan Blue staining (Sigma, St. Louis, MO) was also

comparable to the viability of the cells in the original suspension

(z 90% in the beginning of the experiment). These data confirmed

that the electric field has little effect on the metabolic activity of the

cells (also see ESI†). In the preliminary experiments we encountered

the problem that the fibroblasts could only survive for about 45 min

This journal is ª The Royal Society of Chemistry 2008

(with or without the electric field) under the highly varied environ-

mental stresses. To compensate for the reduced osmotic pressure

outside the fibroblast cell membranes we added dextrose to the cell

suspensions, and this increased the cell survival time to 1.5 h. This

points out that the assembly of fibroblasts into chains and

membranes should be performed in a precisely controlled environ-

ment in order to prolong mammalian cell viability.

Biocomposite structures similar to the ones shown here could be

used in applications where the functionality of the synthetic particles

complements the one of the cells. The particles can impart magnetic,

electronic or optical properties to the assemblies. One of the potential

applications of cell–particle assemblies on a chip is in biosensors with

electrical detection of changes in cell impedance imparted by toxins

or changes in the environment. Such devices have been previously

fabricated by sedimenting single cells onto micropatterned elec-

trodes.26–28 The serial connection of cells in ‘‘wires’’ can increase the

sensor sensitivity to single events such as cell death, poisoning,

membrane potential shift and ion-channel activity. The parallel

connection of multiple chains in membranes could increase the preci-

sion of detection of low dose toxins that change the impedance of

each cell only slightly. The ability to manipulate the cell arrays

with magnetic fields might be used for rapid alignment and replace-

ment of the cell patches. Co-assemblies with metallic nanoparticles

could allow easier and more reliable electrical interfacing. The cell–

particle arrays can also find application as artificial tissues for micro-

surgery, advanced drugs and vaccines, or ‘‘smart’’ biomaterials or

reporters that respond to changes in the chemical composition of

the environment.

Soft Matter, 2008, 4, 726–730 | 729

Acknowledgements

This study was supported by CAREER and NER grants from the

National Science Foundation and in part by the STC Program of

the National Science Foundation under Agreement No. CHE-

9876674. We are grateful to Michael Weiger and Jason Haugh at

NCSU for providing the NIH/3T3 mouse fibroblast cells, and to

Valerie Knowlton and Dale Batchelor for assistance with the SEM

imaging.

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This journal is ª The Royal Society of Chemistry 2008