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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, USA
b
 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 2006Available online 18 September 2006
Abstract
Disposable microsensors on polymer substrates consisting of fully integrated on-chip planar cobalt (Co) microelectrodes, Ag/AgCl referenceelectrodes, and microfluidic channels have been designed, fabricated, and characterized for phosphate concentration measurement in aqueoussolution. The planar Co microelectrode shows phosphate-selective potential response over the range from 10
−
5
to 10
−
2
M in acidic medium (pH5.0) for both inorganic (KH
2
PO
4
) 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%)onasinglechipandasmallchip-to-chipdeviation(i.e.R.S.D.<2.5%).Specifically,whilekeepingthehighselectivity,sensitivity,andstability of a conventional bulk Co-wire electrode, the proposed phosphate sensor yields advantages such as ease of use, cost effectiveness, reducedanalyte consumption, and ease of integrating into disposable polymer lab-on-a-chip devices. The capability to sense both inorganic and organicphosphatecompoundsmakesthissensorapplicableindiverseareassuchasenvironmentalmonitoring,soilextractanalysis,andclinicaldiagnostics.© 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 continuedresearch for over three decades (Engblom, 1998a)because of its ubiquitous significance. Determination of its concentrations inaqueous samples is important in applied analytical chemistryand clinical, horticultural, or environmental sample analysis(Engblom, 1998a; Antonisse and Reinhoudt, 1999; Moorcroftet 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 diagnosticsis 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 nutrientfor all plants, monitoring its concentration in soil extract is
∗
Corresponding author. Tel.: +1 513 556 0852; fax: +1 513 556 7326.
 E-mail address:
zouz@ececs.uc.edu(Z. Zou).
anotherhighlydesiredapplicationforsensingphosphatelevelinfertilizer 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 methodfor phosphate detection and has demonstrated a lot of promise.Considerable efforts have been directed to develop ISE formonitoringphosphateconcentrationssensitivelyandselectively.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 beenexploredandutilizedtoprovidephosphateselectivesensingandexhibited good sensitivity and selectivity. Although this typeof phosphate sensor experiences limitations such as relativelycomplicated membrane structure and complicated preparationsteps,itstillholdsgreatpotentialinsomeapplications.Enzyme-basedamperimetricorpotentiometricbiosensingisanotherverycommonly used method for phosphate ion detection. Pyru-vate oxidase (POD) is one of the most widely used enzymesfor phosphate-selective biosensors. Phosphate biosensors usingPOD 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
 
 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 theuse of enzyme-based phosphate sensors. Both low cost and highstability are necessary in disposable biochips for point-of-caretesting(POCT)andmassenvironmentaldatacollection.Inaddi-tiontotheuseofbiologicalcomponentsforphosphatedetection,other non-biological approaches are also under investigation.Xiao et al. (1995)introduced cobalt (Co) metal as aphosphate-sensitive electrode material. They showed that themetallic Co-wire has a selective electromotive force (EMF)response to dihydrogen phosphate (H
2
PO
4
−
) in acidic medium.Meruva and Meyerhoff (1996)reported that the Co-wire alsoresponded to hydrogen phosphate (HPO
42
−
) and phosphate(PO
43
−
) 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 tomake,longlifetime,andproduceslownoiseorinterferencefromother common anions (De Marco and Phan, 2003).MostreportsonCo-basedphosphatesensorshaveusedabulk Co-wire as the working electrode and used another isolated cellas the reference electrode. More recently, miniaturized on-chipelectrochemical sensors with planar microelectrodes draw greatattention for their numerous benefits (Bakker, 2004).Work has been done in Ahn’s research group to develop various microelectrochemical biosensors with fully integrated on-chip work-ing electrodes, reference electrodes, and microfluidic channelsfor monitoring pH,
p
O
2
, 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 partsof the micro total analysis system (microTAS) and lab-on-a-chip device, which provides a platform to conduct chemical andbiological analysis in a miniaturized format and is a rapidlygrowing 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) havebeenextensivelyutilizedforlab-on-a-chipsinsteadoftraditionalsiliconandglasssubstratesduetotheiruniquepropertiesofbio-compatibility,highopticaltransparency,andverylowcost(Ahnet al., 2004).The main goal of this work was to develop a miniatur-ized phosphate sensor with on-chip planar Co microelectrodeand integrated microfluidic channels (Fig. 1)using standard BioMEMS fabrication technology. The proposed sensor hasbeen realized very cheaply and is suited for large-scale massproduction 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 sensitivityto traditional Co-wire electrodes. Eventually, this sensor can beused 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 sensorwith planar Co electrodes on polymer substrates.
it can be easily integrated into lab-on-a-chip devices, coupledwith sample preparation and additional analyses. In addition,while most of previously reported Co-based phosphate sensorshave only been tested for the inorganic phosphate salt (e.g.KH
2
PO
4
and NaH
2
PO
4
), another aim of this work is to investi-gate the potential of the proposed sensor for organic phosphatecompoundsmeasurement.Adenosine5
-triphosphate(ATP)andadenosine5
-diphosphates(ADP)havebeenselectedasanalytesfor organic phosphate measurement in this work. ATP and ADPare good indicators of cellular viability due to their critical rolesas 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 tomeasure the intracellular free ATP and ADP concentration onisolated 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 thenon-stoichiometricCoOlayerprovidesspecificcavitiesthatcanaccommodate H
2
PO
4
−
and where the specific equilibrium of H
2
PO
4
−
within these cavities is responsible for the phosphate-selective EMF response. A more broadly accepted explanationwasgivenbyMeruvaandMeyerhoff(1996),inwhichthepoten- tiometric response originates from a mixed potential resultingfrom the slow oxidation of Co and the simultaneous reductionofoxygen(Eqs.(1a),(1b)and(1)).Inthepresenceofphosphate ions in the solution, Co
3
(PO
4
)
2
is formed at the electrode sur-face(Eq.(2)).Thiscoupledreactionshiftstheequilibriumofthe net electrochemical reaction (Eq.(1)),hence alters the steady- state mixed potential due to the combined anodic and cathodiccomponents of these reactions.
 
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Z. Zou et al. / Biosensors and Bioelectronics 22 (2007) 1902–1907 
Under acidic conditions,2Co
+
2H
2
O
⇔
2CoO
+
4H
+
+
4e
−
(1a)O
2
+
4H
+
+
4e
−
⇔
2H
2
O (1b)2Co
+
O
2
⇔
2CoO (1)3CoO
+
2H
2
PO
4
−
+
2H
+
⇔
Co
3
(PO
4
)
2
+
3H
2
O (2)Briefly, when phosphate contacts the Co electrode, theCo
2+
 /Co
0
redox couple will be influenced at the electrode sur-face due to the formation of Co
3
(PO
4
)
2
. The electrode potentialis determined by the Co
2+
concentration at the electrode surfaceand is therefore dependent on the mass transport of phosphateions to the electrode surface as shown inFig. 1.The electrode potentialresponsecanbederivedintermsoftheNernstequationwiththisassumptionthattheelectrodepotentialisdeterminedbybulk concentrations of phosphate ions, and thus a linear poten-tial response to the logarithmic phosphate concentration can beexpected (Chen et al., 1998).
3. Experimental
3.1. Materials and apparatus
KH
2
PO
4
(FisherScientificInternationalInc.,NH,USA)wasusedasthereferenceinorganicphosphatesourceandwasdilutedto several different concentrations using buffer solution. Thebuffer solution was made by 25mM potassium hydrogen phtha-late (KHP, Sigma–Aldrich Corp., MO, USA) and 1mM KCl(Fisher Scientific International Inc.) in de-ionized (DI) waterat pH 5.0. Disodium adenosine 5
-triphosphate and disodiumadenosine 5
-diphosphate were obtained from Sigma–Aldrichas organic phosphate sample. The buffer solution for ATP andADP was prepared by 15mM KHP and 1mM KCl in DI waterat pH 5.0. Co rods (99.95%) were purchased from Alfa Aesar(MA, USA) and used as metal source for the e-beam evaporatorto 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 SL
TM
Software (Labtronics Inc., Ontario,Canada).Thesamplesolutionwasinjectedintothesensingchan-
Fig. 2. Fabrication processes of the on-chip phosphate sensor with polymer microfluidic channels.
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