# Iridium Oxide pH Sensor for Nanoscale Electrochemical Cell Monitoring

Erik Douglas, E298A-EE290B, 6 May 2004

# Abstract

A device was developed to record a spatial and temporal map of
cell-surface pH on the sub-cellular size scale. The pH can be correlated
with cell metabolism, thereby yielding valuable information about the
status and activity of the cell. An array of 100 nm wide gold electrodes
was patterned with the LBL CXRO electron beam writer, and a pH sensitive
layer of iridium oxide was electrodeposited on the surface. The pH
response of the electrodeposited IrO~2~ film sensor was characterized,
and found to be linear, with a slightly super-Nernstian response of
-65.3 mV/pH unit.

![](media/image1.jpeg){width="2.375in" height="2.5208333333333335in"}

# I. Introduction

Single cells are the basic unit of biology, and nanofabrication holds
great promise for electrochemical interaction with these micron-scale
components. Communication with single cells could lead to biomimetic
neuroprosthetics and produce personalized drug screening. Since many
cells are on the size scale of a few microns in diameter, nanoscale
devices produced by electron beam lithography could allow for high
resolution spatial and temporal interrogation of the surface of a cell.

A device is envisioned which would isolate a target cell on an array of
nanoscale electrodes, with surfaces tailored to detect biologically
relevant signals and provide electrical stimulation. For example,
because cells acidify their environment in the course of normal
metabolism, the extra-cellular pH can be used to measure cell metabolic
rate. This concept has lead to the development of the cytosensor
microphysiometer[^1] which has been a valuable tool in cell surface
receptor pharmacology. The directed isolation of a single cell also
provides a considerable challenge, but the needs of this project could
be met by elevating the surface around the array, thereby placing the
electrodes in a sunken well where a cell will settle during flow.[^2] It
would also be possible to capture the target cell on an electrochemical
device by modifying either the device or cell surface. Figure 1 displays
an example of a single cell captured on a microfabricated platinum dot
using protein adhesion facilitated by this type of cell-surface
modification.

The present work describes the fabrication of an array of 100 nm wide pH
sensitive electrodes for use in cell monitoring. To measure pH using
this array, the potential was measured between an electrode and a
common, distant Ag/AgCl reference electrode. The voltage and pH are
linearly related by the Nernst equation, which describes the expected
equilibrium potential of an electrode: E=E~0~ + (2.3RT/nF)logA~H+~.
Calibrating the sensor at two points gives the slope of the line, which
allows the conversion from voltage to pH. The pH response of the device
was characterized using standardized pH buffer solutions over the
physiologically relevant range of 4 to 10.

![](media/image2.png){width="1.9984251968503937in"
height="2.6251968503937007in"}**II. Experiment**

# Fabrication

A two-by-sixteen rectangular array of 100 nm wide gold electrodes with a
chromium adhesion layer was patterned on a glass substrate by metal
evaporation and the lift-off technique (20 and 80 nm thick,
respectively). Glass was chosen as the substrate because it will
facilitate visualization of the cells in subsequent experiments.

To allow for the use of glass, the KRS resist was coated with a layer of
the water-soluble conductive polymer AquaSave to prevent charge buildup.
While the results were generally good with this approach, Figure 4b
illustrates the fact that the AquaSave was sometimes not effective in
preventing distortion of the pattern due to charging. The AquaSave was
readily removed in the development process by rinsing with deioized
water after exposure.

The electron beam part of the array pattern is roughly 500 μm on a side,
and the larger fan-out leads were be patterned by optical lithography in
the UC Berkeley Microlab. The layout was designed to ensure that the
leads are all of equal area to prevent differences in resistance. In
order to fabricate the array with one electron beam exposure, a simple
array pattern was chosen. The pattern, shown schematically in Figure 2,
is two columns of 100 nm wide lines separated within a column by 400 nm,
and between columns by 1 μm.

![](media/image3.jpeg){width="2.760236220472441in"
height="2.072834645669291in"}The larger, optically patterned, leads
terminated in 100-by-400 μm pads which were to be addressed by gold
wires with a wire bonder. This fan-out contact layer was aligned to the
features in the first layer, and completed with metal evaporation and
lift-off. The gold layer was poorly adhered, however, and pulled off of
the surface when the wire bonder was used. This resulted in tests of the
device being limited to the collective response of each column, with a
wire connected across the contact pads.

The active area was isolated by masking the rest with a 15 μm thick
layer of the rugged negative photoresist SU-8, which will insulate the
gold wires, and also form a well into which a single cell will settle
during flow in upcoming experiments. The remaining exposed area of the
gold electrodes was electroplated with iridium oxide (IrO~2~) following
the protocol of Yamanaka.[^3] Anodic electrodeposition at 10mA/cm^2^ for
150 sec using a gold counter electrode produced a 120 nm thick layer of
IrO~2~. A second "dummy" gold electrode, with an area of 0.15 cm^2^ was
also plated in order to improve control over the total current density.
A bluish film was observed on this electrode after deposition which
confirmed the presence of the iridium oxide.

**Characterization**

The electrode device was characterized using calibrated pH buffer
solutions at 4, 7 and 10, and an Ag/AgCl reference electrode. Initial
tests recorded an overload resistance between the nanoelectrodes and the
reference, which prevented measurements of voltage. Instead, the larger
electrode which was simultaneously plated with IrO~2~ was used to
characterize the sensitivity of the device. This may be due to blockage
by, or misalignment of, the SU-8 layer, or disconnects in the electrode
array. The results which were obtained for the larger electrode should
be very similar to those with a functional nanoelectrode device.

![](media/image4.jpeg){width="2.1811023622047245in"
height="1.6362204724409448in"}![](media/image5.jpeg){width="1.6362204724409448in"
height="1.7271653543307086in"}Though it has not yet been accomplished, a
single-cell experiment would make use of a PDMS microchannel formed with
a mold made from SU-8. A syringe pump would drive the liquid flow, and
as the cells passed over the well, one would eventually fall in and fill
it, so the rest would pass by. The cell surface pH would be monitored,
and then the cell would be killed with heat. If the device was
successful, the cell death would register as an increased pH, since it
is no longer acidifying its local environment by pumping out H^+^ ions.
The characterization of the device has so far been limited to its native
pH sensing properties, due to the complex measurement compensation which
is required when the interrogation volume is made very small by the
presence of a cell, as well as the aforementioned complications with
fabrication. As discussed by Slowinska and Majda,[^4] the very small
volume causes the measurement to use up the ions being sensed and
substantially alter the pH being measured. Single cell experiments are
planned in the near future which will take this compensation into
account and allow the device to serve its intended purpose of
single-cell monitoring.

# III. Results and Discussion

Following fabrication and electrodeposition, the pH response was
characterized by measuring calibrated buffer solutions over the
physiological range of pH 4 to 10. Because the array device seemed to be
inoperable, the characterization data in Figure 5 were measured with the
larger (0.15 cm^2^) iridium oxide coated gold electrode. This electrode
was seen to have a linear pH response of -65.3 mV/pH units over the
range of 4-10, with an accuracy of ±0.25 pH units. This result is in
close agreement with the literature, and presents an exciting
opportunity for further development of similar nanoscale sensors.

Iridium oxide appears to be a good choice for a pH sensitive layer
because of its strong response and straightforward patterning. Iridium
oxide films have the attractive qualities of being stable over a wide pH
and temperature range, fast response, and low disturbance by redox
interference and stirring.[^5] Marzouk *et al.* have demonstrated the
use of anodic electrodeposited iridium oxide film (AEIROF) for use in
physiological measurements.[^6] They reported a super-Nernstian
(exceeding the Nernst equation which describes the expected equilibrium
potential of an electrode) response of --63.5 mV/pH unit after
conditioning, and an accuracy of ±0.02 pH units, which is quite close to
the sensitivity described in the current work. They also found that the
pH selectivity of the film was not compromised by the presence of other
physiological cations such as Na^+^, K^+^, Ca^2+^, and Mg^2+^. This
property is very encouraging for use in future integrated
multi-ion-selective devices, and allowed that group to conduct the
simultaneous measurement of pH, K^+^ and lactate using other selective
electrodes. The probes they used were on the order of 1 mm in diameter,
but they state that greater spatial and temporal resolution would allow
them to better understand the mechanism and interactions of the
biological system in question.

![](media/image6.wmf){width="3.875in" height="2.7180555555555554in"} One
immediate application of this technology could be a feedback-control
system for electroporation of single cells. Electroporation is often
used to introduce foreign material into cells, such as DNA for
transfection, but the instrumentation provides only a crude interface
with cells. Huang and Rubinsky described a system which detects the
formation of pores in a single cell membrane by measuring current
passing through them under applied potential.[^7] Such a system
indicates the onset of membrane permeability, but does not provide
information about the location or the number of pores. A nanoscale array
of electrodes with some having a pH sensitive layer and others of bare
metal could do just that, by measuring the amount and location of the
change in pH levels.

# IV. Conclusions

Nanoscale single-cell interface devices promise to elucidate the
fundamental workings of the cell, and hold potential for applications
such as drug screening and neural prosthetics. Electron beam lithography
allows for the generation of probes which are well below the size scale
of a single cell. The pH sensitive electrode demonstrated herein could
be an important step toward this goal.

# V. Acknowledgements

The author would like to thank Alex Liddle, Erik Anderson, and Josei Lee
for their enormous help in fabrication, and Nick Toriello for his help
with electrodeposition. The work took place in the CXRO Nanofabrication
Laboratory and the UC Berkeley Microlab.

[^1]: McConnell, H. et al. SCIENCE 257 (5078): 1906-1912 SEP 25 1992.

[^2]: Huang et al. ANAL CHEM 76 (2): 483-488 JAN 15 2004.

[^3]: Yamanaka, K. JAPANESE J APP PHYS 28 (4): 632-637 APR 1998.

[^4]: Slowinska, K. and Majda, M. J. SOLID STATE ELEC CHEM In Press.

[^5]: Fog, A. and Buck, R.P. SENS ACTUAT 5: 137-146 1984.

[^6]: Marzouk, S.A.M. et al. ANAL CHEM 70 (23): 5054-5061 DEC 1 1998.

[^7]: Huang, Y. and Rubinsky, B. SENS ACTUAT A 89: 242-249, 2001.