CELL CLINICS FOR BLOELECTRONIC INTERFACE
WITH SINGLE CELLS
P. Abshiret, J.-M Lauensteid Y. L i d E. Smeld
Electrical and Computer Engineering’ and Mechanical Engineering’
University of Maryland
College Park,Maryland 20742, USA
ABSTRACT
We
describe
integrated
microstructures
and
instrumentation for capture and characterization of large
numbers of individual cells. Each of these “cell clinics”
consists of a cell-sized cavity and a lid that can be opened
and closed by hinges constructed from polypyrrole
microactuators linking rigid plates to the substrate. These
clinics are fabricated on integrated circuit substrates to
bring sensors, actuation, computation, and control together
at the scale of a single cell. Sensing modalities are
tailored for specific applications; we have designed and
begun to fabricate and test prototypes of cell clinics for
impedance measurements, extracellular recording and
stimulation of electrically active cells, and optical
measurements.
The first generation of cell clinics is constructed on a
silicon substrate with electrodes incorporated into the
bottom of the vials. Dissociated adult Xenopus laevis
brainstem neurons have been cultured to test these single
cell biosensors. Custom integrated complementary metal
oxide semiconductor (CMOS) electronics including
amplifiers, filters, and optical detection circuitry will be
incorporated as the substrate for the next generation of cell
clinics. Large arrays of cell clinics can be produced at
high volume and low cost using simple low-temperature
post-processing steps on commercially fabricated
integrated circuits.
The potential applications are
numerous, spanning a spectrum from detailed
physiological studies of specific mechanisms to whole cell
studies of ecology or developmental biology to collecting
concentrated cell secretions to statistical studies of assayed
cell properties.
A significant challenge in modem microbiology is to
determine how genomic information is translated into
protein expression (proteomics) and cell metabolism
(metabolomics). These studies require novel tools.
Microelectromechanical systems (MEMS) technology can
contribute these tools since devices can be made with
dimensions of the same order of magnitude as individual
cells. Microfabrication can be used to produce devices
with completely new functions unavailable with classical
methods. Classical cell-biology studies carried out on
petri-disb cell cultures provide averages of large
populations. The possibility of studying individual
responses, such as metabolic activity or metabolism or
secretion, would be of great value because individuals
could be studied over time, providing insight into
variations among cells as well as statistical distributions.
Microbiologists are therefore gaining interest in the study
of single cells or single-cell organisms. While
conventional methods exist for separating cells based on
various characteristics, only recently have appropriate
techniques begun to be developed for examining large
arrays of single cells, obtaining data on each one rather
than on ensembles of cells. Micromachining technology
enables the fabrication of such systems.
Folch and Toner [I] recently reviewed research on the
engineering of cell-substrate, cell-cell, and cell-medium
interactions on the micrometer scale. Some single cell
1. INTRODUCTION
Biological agents as transducers between stimuli and
electronic sensors have applications in many fields
including healthcare, military defense, environmental
monitoring, and scientific research. Interfacing electronics
to biological systems leads to the possibility of creating a
range of devices capable of being used as biosensors,
monitoring units for individual biological cells, and hybrid
bioelectronic computational engines.
0-7803-7761-31031S17.00 0 2 0 0 3 IEEE
Figure I : Sketch of an individual cell clinic, showing the
vial with a controllable lid, surface electrodes within the
vial, and a captured cell.
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devices have been presented recently. For example, Lin et
al. [2] developed a force transducer for measuring tension
in single heart cells. Merz and Fromherz [3] positioned
the cell bodies of neurons using polyester SU-8
topographical structures.
Cells were successfully
immobilized using walls with heights of 30 pm. Le
Pioufle et al. [4] described a microsystem for living cell
manipulation and DNA injection, They used an array of
wells without lids, using antibodies for attaching and
immobilizing the cells to the bottoms of the wells.
Andreou and Blain.have developed a micromachined well
array for patch clamp and PCR studies [5]. They use an
array of wells etched into a bulk silicon substrate, with
small perforations for whole cell patch clamping,
integrated heaters, temperature sensors, and amplifiers.
However, studying single cells can he difficult because
some cells are very mobile. A method to keep the cells
contained in a vial would reduce the necessity of using
immobilization agents. The containment of single cells is
the first step towards single cell studies. The next step is
to perform characterization with, for example,
fluorescence probing, optical microscopy, chemical
analysis of metabolites, and impedance measurements. An
actuated microsystem designed for electrical stimulation
and recording of electrically active cells is shown in
Figure 1; this “cell clinic” consists of a sample cavity and
closable lid with electrodes on the bottom surface of the
cavity.
2. CELL CLINICS DESIGN
The prototypical cell clinic shown in Figure 1 incorporates
electrodes into the bottom of a vial together with an
actuated hinge connected to a closeable lid. An array of
test structures has been designed to investigate and
optimize design parameters; the layout is shown in Figure
2. Sixteen groups of cell clinics are shown, including
eight groups of eighteen instrumented clinics with
electrodes in the vials connected to pads for bonding (total
of 144 instrumented cell clinics) and eight groups of
uninstrumented cell clinics lacking electrodes (total of
1584 uninstrumented cell clinics). The uninstrumented
structures will he used to investigate mechanical
characteristics of the design such as varying the hinge
length and design, the lid size, and the thickness of
deposited polypyrrole and gold bilayers.
The
instrumented clinics will be used to test the interface to
electrically active cells as well as to investigate design
parameters such as lid size, hinge length, and electrode
configuration.
In the following sections we will describe the components
of the cell clinic briefly, give more detailed layout
examples from each layer, and describe the range and
nature of design parameters for these prototypes. Initial
prototypes have been fabricated; testing and refinement of
the prototypes is currently underway.
2.1 Actuated Hinge
Conjugated polymers are organic semiconductors with
many interesting properties and potential applications,
including static dissipation, light emitting diodes,
photovoltaic devices, batteries, electrochromic windows,
smart membranes, chemical sensors, and molecular
electronics. They are characterized by alternating single
and double bonds along the polymer backbone
(conjugation), a structure that results in delocalization of
the positive charge if an electron is removed 60m the
polymer. Conjugated polymer actuators have a number of
features that makes them attractive for use as artificial
muscles. They work at room or body temperature, require
low voltages (typically 1 V or less), have a large strain that
can he exploited in either linear or bending actuators, can
be positioned continuously between minimum and
maximum values, hold a fairly constant strain under DC
voltage, have high strength, are light weight, and can
operate in liquid electrolytes, including body fluids.
Figure 2: Layout for cell clinics prototypes
For electrochemically doped polymers the main
mechanism for volume change is mass transport: when
ions and solvent enter the polymer, it expands, and when
they exit, it contracts. This volume change can be cycled
many times by oxidizing and reducing the polymer.
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The key reason for choosing polypyrrole (PPy) actuators
is that they will work in virtually any aqueous salt
solutions; we have operated them in blood plasma, urine,
and cell culture medium [6]. Therefore, the actuators are
perfectly suited for operation in cell culture medium.
Microfabricated bilayer microactuators have been used to
rotate rigid plates (see Figure 3) to form self-folding boxes
[7] and rotating Si plates [XI with electrochromic "pixels"
[9]. They have also been used to grab micron-sized
objects [6, 10-121 and to cover and uncover microcavities
[ I I , 131.
inputs to signal processing stages or a5 outputs for direct
measurement and control.
For the prototypes described above we are investigating
three configurations of electrodes as shown in Figure 4
below, with electrode sizes of 20pmx30pm,
20pmx100pm, and 4 0 p m x 4 0 p from left to right. The
present designs are constrained by a minimum linewidth
of 20pm, determined by the mask quality and cost. Future
designs need not be limited to a 20pm feature size.
2Opmx3Opm
114
$0"
1m)r
Figure 3: PPy/Au bilayer of 30 pm length can rotate a rigid
plate 180" [8].
A
gold
layer
defines
the
electrode
for
electropolymerization of the polypyrrole hinges and
control of the fabricated hinges and serves as the
attachment site for the hinge. The gold bends upon PPy
volume change and permits use of the differential
adhesion method of actuator fabrication.
The prototype cell clinics include three different hinge
designs: single hinges consisting of a single PPy/Au
bilayer and double hinges constructed from two parallel
bilayers, with and without a cross-linking segment. Each
of these hinges is fabricated in lengths of 120, 150, 200
250, 300, and 350 pm. The eight groups of cell clinics
will be fabricated with different combinations of PPy and
Au layer thicknesses. The gold will he fabricated in
2000A and 4000A thicknesses, and the PPy will be
deposited in I, 2, 4, and 8pm thicknesses. These
combinations will support an empirical investigation of the
bending and force obtained for different absolute and
relative bilayer thickness. This will enable us to optimize
the hinge design for subsequent generations of cell clinics.
2.2 Electrodes
Electrodes are the key to interface with electrically active
cells. The electrodes within the wells of the cell clinics
permit field stimulation to induce action potentials and
recording of spontaneous and induced potentials.
Additional electrodes are included inside the well to
provide a reference potential for recording and a return
current path for stimulation.
Electrodes can be defined by metal layers in an integrated
circuit layout and fabricated with the remainder of the
integrated circuit [ 5 ] . These electrodes can be designed as
20pmxlOOpm
4Opmx4Op1
Figure 4: Three electrode configurations used in cell clinic
prototypes.
The electrodes will be plated with silver or platinum using
standard electrochemical methods to provide functional
electrode-electrolyte interfaces for accurate measurements
of extracellular electrical potentials.
Although in future designs the electrodes may be
aluminum layers defined by an integrated circuit layout,
the electrodes for the prototype cell clinics are constructed
from gold on top of a goldchromium layer for adhesion to
the silicon substrate.
2.3 Vial a n d Lid
SU-8 negative photoresist provides a thick plastic film to
define the well and lid for cell containment. The
photoresist can be patterned with U V light, aligner, and
developer. Three different lid sizes are included in the
prototypes: 140xI40pm, 160xI60pm, and 18Ox180pm.
2.4 Silicon S u b s t r a t e
While the first prototypes of cell clinics will be fabricated
on blank silicon wafers, the silicon substrate permits
future integration of CMOS electronics. Amplifiers,
filters, and optical detectors are currently under
development.
2.5 Fabrication Procedure (Masks)
A small section of the mask is shown in Figure 5 for two
cell clinic prototypes. The MEMS fabrication sequence is
as follows: start with a clean silicon wafer; (a) thermally
evaporate chrome/gold layer and perform wet etching for
differential adhesion of gold structural layer; (b) thermally
evaporate gold structural layer and perform wet etching to
define electrodes, polypyrrole-gold bilayer, and wires; (c)
deposit and pattern SU-X negative photoresist for lids,
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vials, and insulation of wires; (d) electropolymerize to
deposit polypyrrole hinges; (e) final etching of adhered
goldchromium layer.
sophisticated sensor-actuator structures which are well
suited for many biomedical applications.
4. ACKNOWLEDGEMENTS
Funding provided by NSF 0225489. Thanks to Dr. B. Emmanuel
Akinshols Elizabeth Fryer, Dr. Martha Davila-Garcia. and Shiny
Matthews, Dept. of Pharmacology at Howard University College
of Medicine, and to Dr. Roger Davenport, Dept. of Biology,
University of Maryland, College Park.
5. REFERENCES
[I] Folch, A., and M. Toner, Microengineering ofcellular
interactions. Annual Revicw of Riomedical Engineering, 2000.
2: p. 227-256.
121
. _Lin., G... K.S.J.Pister. and K.P. Roos. Surface
micromochinedpolysilicon heart cell force transducer. J.
Microelectromech. Syst., 2000. 9(1): p. 9-17.
Figure 5: Mask design showing MEMS layer sequence for
two instrumented cell clinics.
[3] Men, M. and P. Fromherz, Polyester microstructuresfor
topogrophcial control of outgrowth and synapse.fn,.mation of
snailneurons Adv. Mat., 2002. 14(2): p. 141-44.
[4] Le Pioufle, B., P. Surbleda, H. Nagaib, Y. Murakamib,K.
S. Chuna, E. Tamiyaio, and H. Fujitaa, Living cells captured on
a bio-microsystem devoted to DNA injection. Mat. Sci. Eng. C.
2000. 12(1-2): p. 77-81
[5] Blain, I., C. E. Davis, M. Li. and A. G. Andreou. Robust
polymeric techniquesfor design and post-processing and
packaging arrays of bio-MEMS devices. in International
Symposium on Circuits and Systems. 2002. Phoenix, AZ.
[6] Jager. E.W.H.,E. Smela. and 0. Inganas, Microfabricating
conjugatedpolymer actuators. Science, 2000. 290: p. 1540-1545.
171 Smela, E., 0.Inganas, and 1. LundstrBm, Conlroiledfolding
ofmicrometer-size structures. Science, 1995. 268(23 June): p.
1735-1738.
2.6 Electrically Active Cells
For the:purposes of testing the cell clinics, a protocol has
been .developed to culture electrically active cells.
Xenopus luevis brain stem neurons are electrically active,
of appropriate size (20-3Opm cell body afier attachment),
and viable at 18-23'C.
The protocol consists of:
dissection of Xenopus Iuevis brain tissue followed by
mechanical dissaggregation, enzymatic dissociation by
incubation in trypsinhalanced salt solution, tituration and
centrifugation, resuspension in culture medium, and
plating on poly-L-lysine pretreated silicon. This protocol
was tested and shown to produce viable cells suitable for
testinghe hio-electronic interface.
3. SUMMARY
An array of test structures has been designed to investigate
and \optimize design parameters. Microfabrication of
initial prototypes has been completed; testing and
refmement of the prototypes is currently underway.
[8] Smela, E., M. Kallenbach, and J . Holdenried,
Electrochemically driven polypyrrole hilayersfor moving and
positioning bulk micromachined silicon plates. J.
Microelectromech. Syst., 1999. 8(4): p. 373-383.
[9] Smela, E., A microfabricatedmovable electrochromic
'i?pixel"basedonpolypyrrole. Adv. Mat., 1999. lI(16): p. 1343-
45.
[IO] Smela, E., 0. Inganas, and I. Lundstrom. New devices made
from combining silicon microfabricafion and conducting
polymers, in Molecular Manufacturing, C. Nicolini, Editor.
1996, Plenum Press: New York. p. 189-213.
[I I] Jager, E.W.H., et al. Applications ofpolypyrrole
microactuators. in Proc. SPIE.Int. Soc. Opt. Eng., Smart
Structures andMaterials, EAPAD'99. 1999. Newuort Beach,
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[I21 lager, E.W.H., 0. Inganas, and 1. Lundstrom, Microrobots
for micromerer-size objects in aqueous media: potential lools foor
single-cell manipulation. Science, 2000. 288(5475): p. 2335-
2338.
[I31 Jager, E.W.H., et al., Polypyrrole microactuators. Synth.
Met., 1999. 102(1-3):p. 1309-IO.
The ;integration of polymer micro-actuation with low
power CMOS electronics enables new classes of
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