DEVELOPMENT OF A MICROFLUIDIC ION SENSOR ARRAY (MISA) TO MONITOR GRAVITYDEPENDENT CALCIUM FLUXES IN CERATOPTERIS SPORES
A.R. De Carlo1, M. Rokkam2, A. ul Haque3, S.T. Wereley3, P.P. Irazoqui4, H.W. Wells5, W.T. McLamb6, S.J.
Roux7, D.M. Porterfield1,8
1
Dept. of Ag. & Bio. Eng., 2Dept. of Elec. & Comp. Eng., 3Dept. of Mech. Eng., 4Weldon School of Biomed. Eng.,
Purdue University, West Lafayette, IN, 5Bionetics Corporation, Kennedy Space Center, FL, 6Dynamac
Corporation, Kennedy Space Center, FL., 7Molecular, Cellular & Devel. Bio., Univ. of Texas, Austin, TX, 8Dept.
of Hort. & Landscape Arch., Purdue Univ., West Lafayette, IN.
Early development of the germinating Ceratopteris fern
spore involves the establishment of cellular polarity based
on gravity sensing in the single cell system (Edwards and
Roux; 1994). This process of cell polarization culminates
in directional nuclear migration which determines the
plane of the first cell division. The product of this first
mitotic division is a prothalial cell (top) and a rhizoid
(bottom).
Previous experiments have shown that polar Ca2+
currents
are
involved
with
gravimorphogenic
development in Ceratopteris fern spores (Chatterjee et al.,
2000). In a series of experiments that covered the time
period of polarity development that leads up to spore
germination, it was possible to measure calcium flux at
the top, bottom, and sides of the spore using a selfreferencing Ca2+ electrode (Porterfield, 2002). Using the
single sensor we were able to determine that there is a
transcellular Ca2+ current (into the bottom and out of the
top) that correlates with responsiveness of the cell to
gravity. More recently we have attempted to identify the
differential role of ion channels and pumps in this process
of cell polarity development using both molecular and
electrophysiological methods. We used a microsystem to
rotate individual cells 180° and measure changes in Ca2+
ion currents after reorientation using a single Ca2+selective microelectrode operated as a self-referencing
sensor. This approach was limited as we could only
effectively measure before and after signals from a single
position, and was only able to do these experiments in a
40-second time frame (Stout et al., 2003). These
experiments were not able to measure how fast the
transcellular calcium current reorients, because of the
basic limitations in the sensor technology used.
In order to overcome the technical limitations to
studying this system associated with the current sensor
technology we have been working towards developing
new sensor systems based on adaptation of MEMS
fabrication techniques. The goal is to fabricate and test
dynamic sensing systems for advanced-throughput
physiological measurements of calcium signaling events
at the cellular level. Using silicon micromachining we
have constructed and tested prototype versions of the
Microfluidic Ionic Sensor Array (MISA) lab-on-a-chip
device for conducting real-time multidimensional calcium
flux measurements around 16 fern spores arranged in a
vertically positioned 4x4 matrix. A microfluidic pore
sustains each fern spore in basic growth media while four
solid-state calcium-selective electrodes measure the
calcium concentration differential pairwise between four
positions around the cell (1 top, 1 bottom, and 2 sides).
The microfluidic system and sensor array are fabricated
on a silicon substrate. The pyramidal pores which hold the
fern spores in place are etched at an angle of 54° using
KOH etching, and the electrodes and bonding pads are
defined by depositing silver and gold on top of photoresist
and lifting the photoresist off. Chloride plating of the
electrodes is accomplished through immersion in 6%
(w/w) NaClO. SU-8 photoresist (MicroChem, USA)is
applied to the chip as a structural and insulating layer, and
Ca++-selective PVC membrane made with ETH-5234
ionophore (Sigma-Aldrich, St. Louis MO) is spin coated
onto the electrode surfaces. The dimensions of the whole
chip are 9.7mm x 11mm, with 250µm x 1000µm
silver/gold composite bonding pads on all sides and 20µm
x 20µm electrodes. For a single wafer there are 41 MISA
chips (Figure 1), of which the current process yields
approximately 80-90%.
Figure 1. Picture of the 9x11 mm silicon fabricated MISA chip.
The MISA chips are mounted and wire bonded
in the center of a printed circuit board (PCB) that contains
an array of 64 non-inverting amplifiers, using low-noise
(~3nV/√Hz),
low-drift
(~0.8µV/°C)
operational
amplifiers, corresponding to the 64 electrodes. Each of the
amplifiers provides a signal gain of 30 and filters out
noise above 50Hz. The PCB is connected to four data
cables through 16-pin connectors with 2mm spacing. The
total size of the board is 30.25in2. The output from the
PCB is then multiplexed by a 512-point crosspoint matrix,
then processed and stored using a 32-input 18-bit data
acquisition (DAQ) card in differential mode. MISAPlot
2.0 software was written in LabVIEW (National
Instruments, Austin TX) for the DAQ. There are four
main modules in the software to facilitae experimental
functionality. The software also provides accesss
tomoothing and compression functions are written in, as
well as functions for logging acquired data, switching
data and notes from the user. Data extraction and rapid
calibration (Figure 2) are also possible.
Gravitational and Space Biology Bulletin 19(2) August 2006
123
AR DeCarlo et al. – Microfluidic Ion Sensor Array
We have also developed a novel coupling
method, dual-electrode differential coupling (DEDC), so
that the differentials between two working electrodes
could be amplified and digitized directly without the use
of reference electrode. This method was validated in tests
(Figure 3) that involved placing the two ion-selective
probes in separate Ca++ solutions, connected by a 3M
KCl salt bridge. One probe was calibrated in 100µM,
1mM and 10mM solution while the second electrode
remained in a standard solution. Each calibration was
done three times in each of the standard solutions.
Raw Electrode Output (mV)
-60
-80
-100
-120
REFERENCES
-140
-160
0.1
1
10
Calcium Concentration (mM)
Figure 2. Calibration (Average +/- SD) of a planar array of 64
separate calcium sensors using the ETH-5234 calcium-selective
membrane (Anker et al. 1981, Bühlmann et al. 1998). After spin
coating to form the membrane, the sensor array was left to sit
overnight in a dust-free container, then soaked in a solution of
10µM CaCl2 + 10mM NaNO3 (Konopka et al., 2004). Potentials
for the electrodes were measured in calibration solutions of
100µM, 1mM and 10mM CaCl2. The potentials were normally
distributed around a slope of 27mV which is in good agreement
with the theoretical slope predicted by the Nernst equation.
60
Raw Electrode output (mV)
40
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20
0
-20
10.0mM
1.0mM
0.1mM
-40
-60
0.01
0.1
1
10
100
Calcium Concentration (mM)
Figure 3. The DEDC (dual-electrode differential coupling)
method was developed so that transcellular ions concentration
differentials could be measured without need for system offset
settings. Two glass-bodied probes were placed in separate
Ca++ solutions, connected by a salt bridge. Each series was
replicated using each of three standard solutions as a base
solution (figure legend). For each series the Nernst slope was
approximately 27mV, with the potential equal to zero when both
electrodes are in the same solution.
These basic tests have validated the functionality
of the technology. The MISA chip will be able to measure
the calcium concentration differentials associated with
transcellular currents from individual fern spores in real
time. The array of 64 electrodes is organized in quads
within 16 fluidic pores, enabling simultaneous
124
measurement of calcium concentrations differentials
around sixteen spores. The MISA system will be used in
parabolic
flight/microgravity based
physiological
experimentation, to study the role of polar calcium
currents in gravity-dependent cellular development.
Future uses of this in silico cell physiology lab-on-a-chip
technology include biomedical applications such as
advanced through-put cell viability and cancer detection.
Possible pharmacological applications include testing the
effectiveness and dose-response curves of drugs. We
anticipate adapting this base system by development and
adaptation of ionophores for other ions. We are also
developing different versions of this system for
amperometric electroanalytical chemistry, which will be
used for analytes like oxygen, nitric oxide, and various
neurotransmitters to name a few. This will also provide
the technical foundation for adapting enzyme based
biosensors for use on the system.
Gravitational and Space Biology Bulletin 19(2) August 2006
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potentiometric response of all-solid-state solvent
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