planar 555

8/14/2019 Planar 555

http://slidepdf.com/reader/full/planar-555 1/6

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

mailto:[email protected]

http://dx.doi.org/10.1016/j.bios.2006.08.004

http://dx.doi.org/10.1016/j.bios.2006.08.004

mailto:[email protected]

8/14/2019 Planar 555


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.

8/14/2019 Planar 555


1904 Z. Zou et al. / Biosensors and Bioelectronics 22 (2007) 1902–1907

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.

8/14/2019 Planar 555



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

8/14/2019 Planar 555


1906 Z. Zou et al. / Biosensors and Bioelectronics 22 (2007) 1902–1907

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.

8/14/2019 Planar 555



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.

References

Ahn, C.H., Choi, J.W., Beaucage, G., Nevin, J.H., Lee, J.B., Puntambekar, A.,Lee, J.Y., 2004. Proc. IEEE 92 (1), 154–173.

Antonisse, M.M.G., Reinhoudt, D.N., 1999. Electroanalysis 11 (14), 1035–

1048.

Aoki, H., Hasegawa, K., Tohda, K., Umezawa, Y., 2003. Biosens. Bioelectron.

18 (2), 261–267.

Bakker, E., 2004. Anal. Chem. 76 (12), 3285–3298.

Bernengo, J.C., Brau, F., Steghens, J.P., 1996. Proceedings of 18th Interna-

tional Annual Conference of the IEEE Engineering in Medicineand Biology

Society, Amsterdam, the Netherlands, October 31–November 3, pp. 1907–

1908.Carey, C.M., Riggan Jr., W.B., 1994. Anal. Chem. 66 (21), 3587–3591.

Chen, Z., De Marco, R., Alexander, P.W., 1997. Anal. Commun. 34 (3), 93–95.

Chen, Z., Grierson, P., Adams, M.A., 1998. Anal. Chim. Acta 363 (2), 191–

197.

Choi, J.W., Kim,S., Trichur, R., Cho, H.J.,Puntambekar,A., Cole,R.L.,Simkins,

J.R., Murugesan, S., Kim, K.S., Lee, J.B., Beaucage, G., Nevin, J.H., Ahn,

C.H., 2001. Proceedings of Fifth International Conference on Micro Total

Analysis Systems (micro-TAS 2001), Monterey, CA, USA, October 21–25,

pp. 411–412.

De Marco, R., Phan, C., 2003. Talanta 60 (6), 1215–1221.

Engblom, S.O., 1998a. Biosens. Bioelectron. 13 (9), 981–994.

Engblom, S.O., 1998b. Plant Soil 206 (2), 173–179.

Ganjali, M.R., Mizani, F., Niasari, M.S., 2003a. Anal. Chim. Acta 481 (1),

85–90.

Ganjali, M.R., Mizani, F., Emami, M., Niasari, M.S., Shamsipur, M., Yousefi,M., Javanbakhtd, M., 2003b. Electroanalysis 15 (2), 139–144.

Ganjali, M.R., Norouzi, P., Ghomi, M., Niasari, M.S., 2006. Anal. Chim. Acta

567 (2), 196–201.

Gao, C., 2005. Ph.D. Dissertation. University of Cincinnati.

Han, J., Lee, S.H., Puntambekar, A., Murugesan, S., Choi, J.W., Beaucage,

G., Ahn, C.H., 2003. Proceedings of Seventh International Conference on

Micro Total Analysis Systems (micro-TAS 2003), Squaw Valley, CA, USA,

October 5–9, pp. 1113–1116.

Janasek, D., Franzke, J., Manz, A., 2006. Nature 442 (7101), 374–380.

Kueng, A., Kranz, C., Mizaikoff, B., 2004. Biosens. Bioelectron. 19 (10),

1301–1307.

Liu, D., Chen, W.C., Yang, R.H., Shen, G.L., Yu, R.Q., 1997. Anal. Chim. Acta

338 (3), 209–214.

Mak, W.C., Chan, C., Barford, J., Renneberg, R., 2003. Bioelectronics 19 (3),

233–237.

Manz, A., Graber, N., Widmer, H.M., 1990. Sens. Actuators B 1 (5), 244–248.

Meruva, R.K., Meyerhoff, M.E., 1996. Anal. Chem. 68 (13), 2022–2026.

Moorcroft, M.J., Davis, J., Compton, R.G., 2001. Talanta 54 (5), 785–803.

Nakamura, H., Ikebukuro, K., McNiven, S., Karube, I., Yamamoto, H., Hayashi,

K., Suzuki, M., Kubo, I., 1997. Biosens. Bioelectron. 12 (9–10), 959–966.

Nishizawa, S., Yokobori, T., Kato, R., Yoshimoto, K., Kamaishi, T., Teramae,

N., 2003. Analyst 128 (6), 663–669.

Parra, A., Ramon, M.,Alonso,J.N., Lemos, S.G.,Vieira, E.C.,Nogueira, A.R.A.,

2005. J. Agric. Food Chem. 53 (20), 7644–7648.

Rahman, M.A., Park, D.S., Chang, S.C., McNeil, C.J., Shim, Y.B., 2006.

Biosens. Bioelectron. 21 (7), 1116–1124.

Salins, L.L.E., Deo, S.K., Daunert, S., 2004. Sens. Actuators B 97 (1), 81–89.

Xiao, D., Yuan, H.Y., Li, J., Yu, R.Q., 1995. Anal. Chem. 67 (2), 288–291.

Zhu, X., Gao, C., Choi, J.W., Bishop, P.L., Ahn, C.H., 2005. Lab Chip 5 (2),

212–217.

planar 555

Documents