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Biosensors and Bioelectronics 22 (2007) 1902–1907
A disposable on-chip phosphate sensor with planar cobaltmicroelectrodes on polymer substrate
Zhiwei Zou a,∗, Jungyoup Han a, Am Jang b, Paul L. Bishop b, Chong H. Ahn a
a Microsystems and BioMEMS Laboratory, Department of Electrical and Computer Engineering,
University of Cincinnati, Cincinnati, OH 45221-0030, USAb Department of Civil and Environment Engineering, University of Cincinnati, Cincinnati, OH 45221-0030, USA
Received 23 April 2006; received in revised form 2 August 2006; accepted 9 August 2006
Available online 18 September 2006
Abstract
Disposable microsensors on polymer substrates consisting of fully integrated on-chip planar cobalt (Co) microelectrodes, Ag/AgCl reference
electrodes, and microfluidic channels have been designed, fabricated, and characterized for phosphate concentration measurement in aqueous
solution. The planar Co microelectrode shows phosphate-selective potential response over the range from 10−5 to 10−2 M in acidic medium (pH
5.0) for both inorganic (KH2PO4) and organic (adenosine 5-triphosphate (ATP) and adenosine 5-diphosphates (ADP)) phosphate compounds.
This microfabricated sensor also demonstrates significant reproducibility with a small repeated sensing deviation (i.e. relative standard deviation
(R.S.D.) < 1%) on a single chipand a small chip-to-chip deviation (i.e.R.S.D. < 2.5%).Specifically, whilekeepingthe highselectivity, sensitivity, and
stability of a conventional bulk Co-wire electrode, the proposed phosphate sensor yields advantages such as ease of use, cost effectiveness, reduced
analyte consumption, and ease of integrating into disposable polymer lab-on-a-chip devices. The capability to sense both inorganic and organic
phosphate compounds makes this sensor applicable in diverse areas such as environmental monitoring, soil extract analysis,and clinical diagnostics.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Phosphate sensor; Cobalt electrode; Polymer biosensor; Lab-on-a-chip
1. Introduction
Aqueous phosphate ion has been the subject of continued
research for over three decades (Engblom, 1998a) because of its
ubiquitous significance. Determination of its concentrations in
aqueous samples is important in applied analytical chemistry
and clinical, horticultural, or environmental sample analysis
(Engblom, 1998a; Antonisse and Reinhoudt, 1999; Moorcroft
et al., 2001). For example, phosphate is the major source of
eutrophication of rivers and lakes. Therefore, sensitive, cheap,
and portable phosphate sensors are in high demand for moni-
toring the effective eutrophication process. Clinical diagnostics
is another field where phosphate measurement is in demand.
Hyperparathyroidism, Vitamin D deficiency, and Fanconi syn-
drome can be diagnosed based on specific phosphate concentra-
tions in body fluids. Because phosphate is an essential nutrient
for all plants, monitoring its concentration in soil extract is
∗ Corresponding author. Tel.: +1 513 556 0852; fax: +1 513 556 7326.
E-mail address: [email protected] (Z. Zou).
another highly desired application for sensing phosphate level in
fertilizer to serve the agricultural science (Engblom, 1998a,b).
The diversity of applications and examples represents a signifi-
cant need for sensitive and affordable phosphate sensors.
The ion selective electrode (ISE) is a normally used method
for phosphate detection and has demonstrated a lot of promise.
Considerable efforts have been directed to develop ISE for
monitoringphosphate concentrations sensitively and selectively.
For instance, liquid-membrane electrode with different mem-
brane materials (Carey and Riggan, 1994; Liu et al., 1997;
Nishizawa et al., 2003; Ganjali et al., 2003a,b, 2006) have been
explored and utilized to provide phosphate selective sensing and
exhibited good sensitivity and selectivity. Although this type
of phosphate sensor experiences limitations such as relatively
complicated membrane structure and complicated preparation
steps, it still holds great potential in some applications. Enzyme-
based amperimetric or potentiometric biosensing is another very
commonly used method for phosphate ion detection. Pyru-
vate oxidase (POD) is one of the most widely used enzymes
for phosphate-selective biosensors. Phosphate biosensors using
POD have been realized with different sensing mechanisms
0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2006.08.004
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Z. Zou et al. / Biosensors and Bioelectronics 22 (2007) 1902–1907 1903
(Nakamura et al., 1997; Mak et al., 2003; Rahman et al., 2006).
Biosensors based on phosphate binding protein (Salins et al.,
2004) and ion-selective-channels (Aoki et al., 2003) have also
been reported for phosphate sensing. However, the compara-
tively high cost and instability of enzyme materials limit the
use of enzyme-based phosphate sensors. Both low cost and high
stability are necessary in disposable biochips for point-of-care
testing (POCT) and mass environmental data collection. In addi-
tion to the useof biological components for phosphate detection,
other non-biological approaches are also under investigation.
Xiao et al. (1995) introduced cobalt (Co) metal as a
phosphate-sensitive electrode material. They showed that the
metallic Co-wire has a selective electromotive force (EMF)
response to dihydrogen phosphate (H2PO4−) in acidic medium.
Meruva and Meyerhoff (1996) reported that the Co-wire also
responded to hydrogen phosphate (HPO42−) and phosphate
(PO43−) ions in different pH solutions. Several other groups
(Chen et al., 1997; Engblom, 1998b; Parra et al., 2005) showed
that this Co-wire based phosphate sensor had excellent sensi-
tivity and low detection limit in a broad detection range. TheCo-wire sensor is particularly attractive because of its ease to
make, long lifetime,and produces low noise or interferencefrom
other common anions (De Marco and Phan, 2003).
Most reports on Co-based phosphatesensors have used a bulk
Co-wire as the working electrode and used another isolated cell
as the reference electrode. More recently, miniaturized on-chip
electrochemical sensors with planar microelectrodes draw great
attention for their numerous benefits (Bakker, 2004). Work has
been done in Ahn’s research group to develop various micro
electrochemical biosensors with fully integrated on-chip work-
ing electrodes, reference electrodes, and microfluidic channels
for monitoring pH, pO2 , glucose, lactate (Ahn et al., 2004),insulin (Gao, 2005), and heavy metal ions (Zhu et al., 2005).
These on-chip microsensors have also been integrated as parts
of the micro total analysis system (microTAS) and lab-on-a-
chip device, which provides a platform to conduct chemical and
biological analysis in a miniaturized format and is a rapidly
growing field for biochemical analysis and clinical diagnos-
tics (Manz et al., 1990; Ahn et al., 2004; Janasek et al., 2006).
Polymer substrates such as cyclic olefin copolymer (COC) have
beenextensivelyutilized for lab-on-a-chips instead of traditional
silicon and glass substrates due to their unique properties of bio-
compatibility, high optical transparency, and very low cost (Ahn
et al., 2004).
The main goal of this work was to develop a miniatur-ized phosphate sensor with on-chip planar Co microelectrode
and integrated microfluidic channels (Fig. 1) using standard
BioMEMS fabrication technology. The proposed sensor has
been realized very cheaply and is suited for large-scale mass
production and disposable usage without cross contamination.
Further benefits of the proposed sensor include low volume of
analyte consumption and waste generation, rapid sensing time,
and elimination of the extensive polishing step used for bulk
Co-wire, while maintaining comparable stability and sensitivity
to traditional Co-wire electrodes. Eventually, this sensor can be
used for large-scale field deployment for environment applica-
tions and disposable POCT in clinical diagnostics. Moreover,
Fig. 1. Schematic view and working principle of the on-chip phosphate sensor
with planar Co electrodes on polymer substrates.
it can be easily integrated into lab-on-a-chip devices, coupled
with sample preparation and additional analyses. In addition,
while most of previously reported Co-based phosphate sensors
have only been tested for the inorganic phosphate salt (e.g.
KH2PO4 and NaH2PO4), another aim of this work is to investi-
gate the potential of the proposed sensor for organic phosphate
compounds measurement. Adenosine 5-triphosphate (ATP) and
adenosine 5-diphosphates (ADP) have been selected as analytes
for organic phosphate measurement in this work. ATP and ADP
are good indicators of cellular viability due to their critical roles
as the energy source for many biochemical reactions. Cellu-
lar contractile phenomena are directly related to ATP and ADPconcentration locally. For these reasons, a reliable technique to
measure the intracellular free ATP and ADP concentration on
isolated or cultured single cells addresses worthy actual physi-
ological and pharmacological interests (Bernengo et al., 1996;
Kueng et al., 2004).
2. Theoretical background
Several theories have been introduced to explain the sens-
ing mechanism of Co towards phosphate ions. Xiao et al.
(1995) first proposed a host–guest mechanism in which the
non-stoichiometric CoO layer provides specific cavities that canaccommodate H2PO4
− and where the specific equilibrium of
H2PO4− within these cavities is responsible for the phosphate-
selective EMF response. A more broadly accepted explanation
was given by Meruva and Meyerhoff (1996), in which the poten-
tiometric response originates from a mixed potential resulting
from the slow oxidation of Co and the simultaneous reduction
of oxygen (Eqs. (1a), (1b) and (1)). In the presence of phosphate
ions in the solution, Co3(PO4)2 is formed at the electrode sur-
face (Eq. (2)). This coupled reaction shifts the equilibrium of the
net electrochemical reaction (Eq. (1)), hence alters the steady-
state mixed potential due to the combined anodic and cathodic
components of these reactions.
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Under acidic conditions,
2Co + 2H2O ⇔ 2CoO + 4H++ 4e− (1a)
O2+ 4H++ 4e−⇔ 2H2O (1b)
2Co + O2 ⇔ 2CoO (1)
3CoO + 2H2PO4−+ 2H+⇔ Co3(PO4)2+ 3H2O (2)
Briefly, when phosphate contacts the Co electrode, the
Co2+ /Co0 redox couple will be influenced at the electrode sur-
face due to the formation of Co3(PO4)2. The electrode potential
is determined by the Co2+ concentration at the electrode surface
and is therefore dependent on the mass transport of phosphate
ions to the electrode surface as shown in Fig. 1. The electrode
potential response can be derived in terms of the Nernst equation
with this assumption that theelectrode potentialis determined bybulk concentrations of phosphate ions, and thus a linear poten-
tial response to the logarithmic phosphate concentration can be
expected (Chen et al., 1998).
3. Experimental
3.1. Materials and apparatus
KH2PO4 (Fisher Scientific International Inc., NH, USA) was
used as thereferenceinorganic phosphatesource andwas diluted
to several different concentrations using buffer solution. The
buffer solution was made by 25 mM potassium hydrogen phtha-
late (KHP, Sigma–Aldrich Corp., MO, USA) and 1 mM KCl
(Fisher Scientific International Inc.) in de-ionized (DI) water
at pH 5.0. Disodium adenosine 5-triphosphate and disodium
adenosine 5-diphosphate were obtained from Sigma–Aldrich
as organic phosphate sample. The buffer solution for ATP and
ADP was prepared by 15 mM KHP and 1 mM KCl in DI water
at pH 5.0. Co rods (99.95%) were purchased from Alfa Aesar
(MA, USA) and used as metal source for the e-beam evaporator
to fabricate the planar Co microelectrodes.
The fabricated phosphate microsensor was electrically con-
nected to the model 215 benchtop research-grade pH/mV meter
(Denver Instrument Corp., CO, USA). The potential was mea-sured at room temperature and the data was collected and ana-
lyzed by BalanceTalk SLTM Software (Labtronics Inc., Ontario,
Canada). The samplesolutionwas injected into thesensingchan-
Fig. 2. Fabrication processes of the on-chip phosphate sensor with polymer microfluidic channels.
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Fig. 3. Photographsof thefabricateddeviceand themicroscope imageof theon-
chip phosphate sensor composed of Co working electrodes (WE) and Ag/AgCl
reference electrodes (RE).
nel through the inlet using the pump 33 dual syringes pump(Harvard Apparatus, MA,USA) and kept in the sensing chamber
for test until washed out through the outlet. For each measure-
ment, after obtained signals became stable for about 2 min, a
washing step was performed using DI water, and then the next
sample solution was applied.
3.2. Fabrication of phosphate sensor and microfluidic chip
on polymer substrate
Standard microfabrication processes were used and summa-
rized in Fig. 2. Briefly, an Au layer of 100 nm and Co layer of
300 nm were deposited on the 3-inch blank COC wafer usingthe e-beam metal evaporator. Au and Co electrodes were pat-
terned by photolithography technique and etched by Co (0.5%
HNO3) and Au (TFA) etchant. The Ag/AgCl (∼1m) layer was
depositedon the reference electrode using electroplating method
on the Au seed layer (Gao, 2005).
The analyte consumption and sensing time of the proposed
sensor can be significantly reduced by using the integrated poly-
mer microfluidic chip. The plastic injection molding and UV
adhesive bonding technique have been developed in our group
for high throughput polymer biochip fabrication. The fabrica-
tion detail has been reported previously (Choi et al., 2001) and
summarized in Fig. 2. After drilling holes for fluidic intercon-
nection at inlet and outlet, the microfluidic chip was bondedwiththe sensor chip using UV adhesive bonding technique at room
temperature (Han et al., 2003) to achieve the final device.
Photographs of the fabricated device have been shown
in Fig. 3, which illustrate the microelectrode array, electri-
cal connections, and microchannels. The entire chip size is
1.5 cm× 2 cm, and the inlet and outlet channels have width of
200m and depth of 100m. The reaction chamber has width
of 2 mm, length of 10 mm, depth of 100m, and volume of
2l. The detail of the Co working electrode and the Ag/AgCl
reference electrode has been clearly shown in Fig. 3 inset. Both
electrodes have length of 1.5 mm, width of 200m, and a spac-
ing of 200m.
Fig. 4. Potentiometric response of the phosphate sensor in different concen-
trations of KH2PO4 at pH 5.0: (a) dynamic measurement and (b) calibration
curve.
4. Results and discussion
Bulk Co-wire based phosphate sensors have been charac-
terized for inorganic phosphate (Meruva and Meyerhoff, 1996;
Chen et al., 1997; Engblom, 1998b; Parra et al., 2005). These
Co-wire electrodes show a very good response to inorganicphos-
phate (KH2PO4 and NaH2PO4) in a very wide dynamic range
from 5× 10−5 to 5× 10−2 M with a detection limit less than
10−5 M. The Co-wire electrode has high selectivity for phos-
phate ions with respect to many other common anions (Chen et
al., 1997). In this research, an on-chip Co microelectrode phos-phate sensor was evaluated for its performance in comparison
to traditional bulk Co-wire based phosphate sensors.
Measurements were performed using this sensor as the con-
centration of KH2PO4 was dynamically varied from 10−5 to
10−2 M. A stepwise response to different KH2PO4 concentra-
tions and the steady-state potential for each sample concentra-
tion has be observed and shown in Fig. 4a. It is also evident
that due to the miniaturized sensing size and reaction volume,
the on-chip sensor reaches an equilibrium response rapidly, in
approximately 1 min for 10−5 M and in less than 30 s for higher
concentrations above 10−5 M. Calibration curve derived from
Fig. 4a is shown in Fig. 4b as well. The dynamic range is from
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Fig. 5. Potentiometric response of the phosphate sensor in different concentrations of ATP and ADP at pH 5.0.
10−5 to 10−2 M by using the proposed sensor and is the same as
most bulk Co-wire phosphate sensors (Xiao et al., 1995; Chen
et al., 1998; De Marco and Phan, 2003). A higher base line
potential can be observed in this sensing system compared to
bulk Co-wire sensors but can likely be due to the different Cl−
concentrations used for the Ag/AgCl reference electrode.
The sensor response to the organic phosphate was performed
using standard ATP and ADP samples. The KHP concentration
in buffer solution was adjusted to 15 mM according to the opti-
mized value for ATP and ADP (Xiao et al., 1995). As shown in
Fig. 5, the sensor exhibits a potentiometric response to ATP and
ADP in the range between 10−5 and 10−2 M. It is also noticed
that ADP displays a larger potentiometric slope than ATP does.
A similar phenomenon was observed and explained by Xiao et
al. (1995). This is in agreement with the fact that the number of “additional” units of phosphate binding to the electrode is two
for ATP and only one for ADP as illustrated in Fig. 5.
The on-chip sensor presents a steady-state response for more
than 30 min in 10−5 M KH2PO4 solution (Fig. 6), which is
sufficient for disposable sensor applications. In addition, this
Fig. 6. Long-term potentiometric response of the phosphate sensor in 10−5 M
KH2PO4 at pH 5.0.
Fig. 7. Reproducibility of the fabricated sensor: (a) potential responses to 10-
time repeated injections of 10−3 M ADP to the same phosphate sensor and (b)
chip-to-chip deviation of four different phosphate sensors in measuring 10−3 M
KH2PO4 and 10
−3
M ATP.
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sensor has high reproducibility which is another vital require-
ment for mass-produced microsensors. For example, injections
of 10−3 M ADP into the same phosphate sensor for 10 times
reveal good measurement reproducibility (i.e. 526± 4mV or
relative standard deviation (R.S.D.) of 0.6%) as shown in Fig.7a.
This result is comparable with reported data using bulk Co-
wire (3.0% R.S.D., Chen et al., 1997; 3.8% R.S.D., Chen et al.,
1998; 2–4% R.S.D., De Marco andPhan, 2003). Reasonably low
chip-to-chip deviation hasbeen obtained by measuring KH2PO4
and ATP at 10−3 M on four different sensors with variances of
2.5% R.S.D. for KH2PO4 and 2.1% R.S.D. for ATP (Fig. 7b).
The proposed on-chip sensor also exhibited high selectivity for
H2PO4− (e.g. K i, j(Cl−)=4.1× 10−3, K i, j(NO3
−) = 8× 10−4,
K i, j(SO42−)=8.2× 10−4, K i, j(I−)=1.1× 10−2) which is com-
parable to bulk Co-wire based phosphate sensors.
5. Conclusions
The new on-chip phosphate sensor using planar Co micro-
electrodes has been developed and fully characterized in thiswork. The feasibility of this electrochemical sensor to moni-
tor both inorganic and organic phosphate compounds has been
fully demonstrated. By incorporating the mass-produced micro-
fabrication technique and high throughput plastic micromachin-
ing, the proposed on-chip phosphate sensor with the integrated
microfluidic chip can be batch fabricated with very low cost and
high yield compared to the conventional bulk Co-wire based
sensor,while maintaining the excellent performance. The minia-
turized sensing system is especially suitable for large-scale field
deployment for mass environmental data collections and dis-
posable POCT in clinical diagnostics. Moreover, the proposed
on-chip microsensor is fully integrated with polymer microflu-idic system and can be easy developed as multi-analyte polymer
lab-on-a-chips for a wide range of applications.
Acknowledgements
The authors gratefully thank Mr. Ron Flenniken in the Insti-
tute for Nanoscale Science and Technology at the University of
Cincinnati, for his technical support, and also thank Mr. Andrew
Browne for discussion.
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