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Sensors and Actuators B 160 (2011) 1322– 1327

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

j o ur nal homep a ge: www.elsev ier .com/ locate /snb

evelopment of an amperometric, screen-printed, single-enzyme phosphate ioniosensor and its application to the analysis of biomedical and environmentalamples

. Gilberta, A.T.A. Jenkinsb, S. Browningc, J.P. Harta,∗

Faculty of Health and Life Sciences, University of the West of England, Bristol, Coldharbour Lane, Bristol BS16 1QY, United KingdomDepartment of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, United KingdomThe Environment Agency, Rivers House, Lower Bristol Road, Bath BA2 9ES, United Kingdom

r t i c l e i n f o

rticle history:eceived 4 August 2011eceived in revised form6 September 2011ccepted 21 September 2011vailable online 29 September 2011

eywords:

a b s t r a c t

An amperometric phosphate biosensor, based on a cobalt phthalocyanine screen-printed carbon elec-trode (CoPC-SPCE) is described. The immobilisation of the enzyme pyruvate oxidase (PyOd) wasinvestigated using pre-formed cellulose acetate/cellulose nitrate membranes, of different pore sizes, andthe cross-linking agent, glutaraldehyde (GLA). The latter method was found to be superior in terms of per-formance characteristics and also ease of fabrication. A linear range of 2.5–130 �M and limit of detectionof 2 �M was obtained under optimal conditions. The biosensor also exhibited an excellent response timeof just 13 s ± 1, n = 3. The biosensor was successfully applied to the measurement of phosphate in pond

hosphateiosensormperometriccreen-printedater analysis

water samples; the mean recovery of spiked water samples was 103.2%, n = 3. The original concentrationof phosphate calculated in the water sample (48 �M) was found to be in good agreement with that foundusing a standard colourimetric method. In addition to the analysis of water samples the biosensor wasalso applied to the analysis of human urine, with only a simple dilution of the sample, directly into theelectrochemical cell, required for analysis. The precision of the biosensors, obtained during the urine

rine analysis analysis was 6.4%, n = 6.

. Introduction

There has been continued interest in the development of ahosphate biosensor, for application in both biological and environ-ental samples for a number of years [1–3]. From an environmental

erspective, the concentration of phosphate in water is crucial dueo its role in eutrophication. Eutrophic water is described as havingn increased concentration of nutrients resulting in proliferation oflgae, a reduced oxygen concentration and can be potentially dam-ging to aquatic life [4]. As a result, water authorities in the UK andround the world monitor phosphate levels in order to comply withegislation brought into force, which aims to protect water bodies.or example, the Environment Agency must reduce the concentra-ion of phosphate in UK waters as laid out in the Water Frameworkirective (2000/60/EC) [5]. In addition, the analysis of bodily fluids

uch as urine can aid in the diagnosis of diseases such as vitamin D

eficiency and hyperparathyroidism [6].

Methods used for the determination of phosphate such asolourimetry or spectrophotometry have been used in the past

∗ Corresponding author. Tel.: +44 117 328 2469.E-mail address: john.hart@uwe.ac.uk (J.P. Hart).

925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2011.09.069

© 2011 Elsevier B.V. All rights reserved.

[7,8]. However, such methods do not lend themselves particularlywell to in situ analysis due to the use of potentially toxic agents andlengthy preparation and analysis times. An alternative approach isto develop electrochemical biosensors as they have the potentialto be used in situ, and when combined with hand-held instrumen-tation [9], offer a high degree of selectivity and specificity and maybe operated by lay personnel. We have previously reported both anamperometric assay [10] and biosensor [11] for the measurementof phosphate. The method of physical entrapment using an in situformed membrane, which consisted of a solution of 1.5% celluloseacetate in acetone, was investigated and applied to the analysisof urine and tap water samples [11]. While the measurement ofphosphate in urine samples was successful, the limit of detection(100 �M) was not sufficient to quantify phosphate in environmen-tal samples. In this paper we describe investigations into alternativemethods of enzyme immobilisation in order to amplify the sensitiv-ity with the aim of quantifying phosphate in water samples. Variousmethods of immobilising PyOd have been reported in the literatureand include: entrapment within a hydrogel layer [12–14], cova-lent attachment to a nano-conducting polymer [15], cross-linking

with glutaraldehyde [16] and photo cross-linking onto a celluloseacetate membrane using PVA-SbQ [17]. Here, we have investigatedthe cross-linking agent, glutaraldehyde (GLA) as our group has pre-viously employed this in a very successful biosensor system [18,19].

ctuators B 160 (2011) 1322– 1327 1323

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L. Gilbert et al. / Sensors and A

. Materials and methods

.1. Chemicals and reagents

All chemicals were of analytical-reagent grade and obtainedrom the following sources: pyruvate oxidase (PyOd; E.C. 1.2.3.3)rom Aerococcus viridans was obtained as lyophilised powderith 50.8 U mg−1 enzyme activity from Calbiochem/Merck Chem-

cals Ltd., thiamine pyrophosphate (TPP) and flavin adenineinucleotide (FAD) were purchased from Sigma–Aldrich, pyru-ic acid (PA) sodium salt, sodium dihydrogen orthophosphate,odium hydroxide, sodium chloride, magnesium sulphate, (3-(N-orpholino)propanesulphonic acid) (MOPS) and GLA 50% solutionere purchased from Fisher Scientific. Membrane filter discs,A/CN mix pore sizes 0.22 and 0.45 �m, 20 �m thickness and Mil-

ipore Millex HA 0.45 �m syringe filter units were purchased fromillipore, UK. Pond water was sampled from a local park, in glass

ottles, on the morning of analysis.The supporting electrolyte used was 0.05 M MOPS buffer, pre-

ared by dissolving the appropriate mass of (3-(N-morpholino)ropanesulphonic acid) in deionised water and adjusted to pH.3 using sodium hydroxide. Stock solutions of TPP, FAD, PA,agnesium sulphate, sodium chloride and sodium dihydrogen

rthophosphate were prepared in 0.05 M MOPS, pH 7.3 and storedt +4 ◦C. PyOd was also prepared in 0.05 M MOPS, pH 7.3 contain-ng 200 �M TPP and 20 �M FAD and stored at −20 ◦C until required.odium chloride was added to the electrochemical cell solution inll studies at a concentration of 0.1 M to maintain a constant halfell potential for the Ag/AgCl reference/counter electrode [20].

.2. Apparatus

All electrochemical measurements were carried out using acreen-printed two-electrode system comprising a CoPC workinglectrode and a screen-printed Ag/AgCl pseudo-reference/counterlectrode deposited onto a poly(vinyl chloride) (PVC) substrate,went Electronic Materials (Pontypool, UK). The working area of

he CoPC-SPCE electrode was defined using insulating tape to anrea of 3 mm × 3 mm; both this and the Ag/AgCl electrode wereonnected to the potentiostat by two gold clips. The cell contentsere stirred at a constant rate using a magnetic stirring disc and

tirrer, Stuart Scientific Ltd. (Staffs, UK). Experiments were tem-erature controlled at 25 ◦C using a water jacket and circulatingater bath, Haake P5 (Germany). A Amel model 466 Polarographicnalyzer or LC-4B amperometric detector, BAS (USA) connected to

ABB SE120 chart recorder was employed in all electrochemicaltudies.

.3. Procedures

.3.1. Fabrication of biosensors using pre-formed membranesA 12 �L aliquot of PyOd solution containing 0.6 U PyOd, 200 �M

PP and 20 �M FAD was deposited onto the CoPC and driedvernight in a desiccator. Squares of the appropriate pre-formedA/NC membrane filter, 0.22 or 0.45 �m pore size, both 20 �mhickness, were cut to the approximate size of the WE area of theoPC. The squares of membrane were then fixed to the PVC usinghin strips of insulating tape, firmly pressed down, along each edgef the membrane square. A diagram illustrating these is shown inig. 1; these biosensors are referred to as CA/CN-PyOd-CoPC-SPCEs.

.3.2. Fabrication of biosensors using PyOd cross-linked with GLA

A 12 �L aliquot of PyOd solution containing 0.6 U PyOd, 200 �M

PP and 20 �M FAD was deposited onto the CoPC-SPCE. Theiosensors were stored in a desiccator, shielded from light at 4 ◦Cvernight to allow the enzyme layer to dry. A freshly prepared 3 �L

Fig. 1. Diagram of CA/CN-PyOd-CoPC-SPCE. CA/CN is cellulose acetate/cellulosenitrate, CoPC-SPCE is cobalt phthalocyanine screen-printed carbon electrode, WEis working electrode, RE is reference electrode.

aliquot of 0.01% (v/v) GLA was drop-coated onto the enzyme layer toinitiate cross-linking and was allowed to dry at room temperaturefor 1 h before use. These biosensors are referred to as GLA-PyOd-CoPC-SPCEs.

2.3.3. Calibration of the biosensors using amperometry in stirredsolution

For each of the biosensors (CA/CN-PyOd-CoPC-SPCE andGLA-PyOd-CoPC-SPCE) calibration studies were performed usingamperometry in stirred solution by adding phosphate standardsolutions to an electrochemical cell, maintained at a constant tem-perature of 25 ◦C, containing 0.05 M MOPS pH 7.3, 2 mM PA, 10 mMMgSO4 and 0.1 M NaCl to give a total cell volume of 5 ml. Whenusing the GLA-PyOd-CoPC-SPCEs 200 �M TPP and 20 �M FAD werealso added to the electrochemical cell. Calibration studies were car-ried out at an applied potential of +0.4 V as this was found to beoptimal when using the previously reported phosphate ion biosen-sor [11].

2.3.4. Application of optimised amperometric biosensor(GLA-PyOd-CoPC-SPCEs) to the determination of phosphate inpond water

The concentration of phosphate in pond water was determinedusing the optimised design of the amperometric biosensor desig-nated as a GLA-PyOd-CoPC-SPCE; this was used in conjunction withamperometry in stirred solution. Initially, a 5 ml aliquot of 0.05 MMOPS pH 7.3 containing 2 mM PA, 10 mM MgSO4, 0.2 mM TPP,20 �M FAD and 0.1 M NaCl was introduced to the voltammetric cell.The amperometric biosensor was inserted into the stirred solution,then a potential of +0.4 V vs Ag/AgCl was applied. The backgroundcurrent was allowed to reach steady state then a 5 ml aliquot ofpond water was added; the resulting increase in steady state cur-rent was measured. Quantification was performed by the methodof standard addition by making 40 �L additions of standard phos-phate solution (10 �M) to the cell contents; in all, a total of eightadditions were made. The reproducibility of the biosensor assayfor pond water was determined by repeating the whole procedurethree times with three individual biosensors.

2.3.5. Analysis of pond water samples by colourimetryIn order to determine the accuracy of the biosensor method,

pond water samples were simultaneously analysed using thevanadomolybdophosphoric acid colorimetric method [21]. Briefly,

5 ml of a solution of vanadate–molybdate reagent was added to20 ml of either phosphate standard solution or a sample of fil-tered pond water (filtered using 0.45 �m syringe filter units). Theabsorbance of the solutions was then measured at a wavelength

1 Actuators B 160 (2011) 1322– 1327

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324 L. Gilbert et al. / Sensors and

f 400 nm using a Sanyo SP50 spectrophotometer (Watford, UK);0 min was allowed to elapse before analysis to allow sufficientolour development. A calibration curve was constructed using thehosphate standard solution absorbance and the concentration ofhosphate in the samples was calculated.

.3.6. Application of optimum amperometric biosensorGLA-PyOd-CoPC-SPCEs) to the determination of phosphate inuman urine

The endogenous concentration of phosphate in human urine,rom a healthy female subject without dietary control, was deter-

ined using the optimised design of the amperometric biosensorn a similar way to that described above for pond water analysis.owever, additions of only 25 �L of urine, to the 5 ml buffered mix-

ure in the voltammetric cell were required, owing to the relativelyigh levels of phosphate present. The reproducibility of the biosen-or assay for urine analysis was deduced by repeating the wholessay six times with six individual biosensors.

. Results and discussion

.1. Principle of operation of the amperometric biosensor

As seen in Fig. 1, the biosensor is based on a screen-printed car-on electrode (SPCE), which has been modified by the addition ofobalt phthalocyanine (CoPC). In order to produce the biosensor(s)he enzyme pyruvate oxidase (PyOd) is immobilised on the surfacef this CoPC-SPCE. The following sections describe three differentnzyme immobilisation methods; however the principle of opera-ion for the measurement of phosphate ions is essentially the same.he sequence of reactions may be summarised in the followingquations:

yruvate + phosphate + O2PyOd−→acetylphosphate + H2O2 + CO2

(1)

Co2+ + H2O2 → 2Co+ + 2H+ + O2 (2)

Co+ → 2Co2+ + 2e− (3)

q. (1) shows the enzymatic decarboxylation of pyruvate inhe presence of inorganic phosphate and oxygen to producecetylphosphate, CO2 and H2O2. The cofactors thiamine pyrophos-hate (TPP) and flavin adenine dinucleotide (FAD) are involved inhis reaction. Briefly, deprotonation of the C2 atom in TPP is fol-owed by binding of pyruvate (at the active site), the newly formedactyl-TPP (LTPP) is then decarboxylated forming hydroxyethyl-TPPHETPP), which is oxidised by FAD to form acetyl-TPP (AcTPP) andADH2. FADH2 is re-oxidised by O2 to form H2O2 and AcTPP is phos-horetically cleaved to produce acetyl phosphate and the TPP C2arbanion which may participate in further enzyme reactions [22].

The analytical response of the biosensor is based on the electro-atalytic oxidation of H2O2; this proceeds by the interaction of H2O2ith Co2+ to produce Co+ (Eq. (2)) this species is then re-oxidised

o Co2+ to generate the analytical response (Eq. (3)). It should beentioned that we carried out preliminary studies with solutions

ontaining hydrogen peroxide and pyruvic acid and showed thatnly the former produced an electrocatalytic oxidation peak at0.4 V.

.2. Characterisation of the three different amperometriciosensors

We investigated three different methods of immobilising thenzyme on the surface of the CoPC-SPCE in order to deduce theest approach for high sensitivity, together with the appropriate

follows: +0.4 V, pH 7.3 MOPS 0.05 M, 2 mM PA, 10 mM MgSO4 and 0.1 M NaCl. Plotsshown are mean values, n = 3; (a) 58% CV and (b) 47% CV.

selectivity for the measurement of phosphate in pond water andurine samples. As we intended using amperometry in stirred solu-tion for these applications, this technique was used in conjunctionwith each of the biosensors for their characterisation.

3.3. Calibration studies using CA/CN-PyOd-CoPC-SPCE biosensors

The first approach investigated involved depositing a pre-formed membrane over the enzyme layer, which was previouslydrop-coated onto the CoPC-SPCE (Fig. 1). This approach eliminatesthe need to fabricate a permselective membrane by drop-coatingthe polymer contained within organic solvents such as acetone.Additionally, variations in membrane thickness and pore size aresignificantly reduced when pre-formed materials are used. CA/CN-PyOd-CoPC-SPCE biosensors were prepared using two membranepore sizes, 0.22 and 0.45 �m. Calibration studies were performedusing the biosensors with phosphate standard solutions usingamperometry in stirred solution. Fig. 2(a) and (b) shows that the useof the CA/CN membrane allows the measurement of phosphate overthe range 250–1250 �M; it is also apparent that PyOd is retained inthe layer between the membrane and CoPC-SPCE. The precision ofthe biosensors fabricated using pre-formed membranes with poresizes of 0.22 and 0.45 �m was found to be 58% and 47% (n = 3),respectively. Clearly this precision would not be acceptable for theproposed environmental and biomedical applications. The reasonfor poor precision may be due to some loss of the biosensor com-ponents during the measurement; the slopes of the plots in Fig. 2

suggests greater leakage through the 0.45 �m membrane. In pre-vious studies by our group, we have fabricated biosensors using anenzyme cross-linking approach [18]. The precision obtained previ-ously with these amperometric biosensors was 4.8%; consequently

L. Gilbert et al. / Sensors and Actuators B 160 (2011) 1322– 1327 1325

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ual samples and found to be 6.4%. In addition, the mean phosphateconcentration was found to be 26.8 mM,which is well within therange of concentrations determined by Classen et al. in 1990[28] (between approximately 8–45 mM), using a method based on

ig. 3. Calibration of phosphate using GLA-PyOd-CoPC-SPCE biosensors obtained bymperometry in stirred solution, conditions as Fig. 2. Error bars represent standardeviations (n = 4).

e decided to investigate this approach for the development of theurrent phosphate biosensor.

.4. Characterisation of GLA-PyOd-CoPC-SPCE biosensors

.4.1. Immobilisation of PyOd using GLAThe cross-linking agent GLA was investigated as a method of

mmobilising PyOd onto the CoPC-SPCE. It has been reported thatross-linking agents can be detrimental to enzyme activity at highoncentrations [23] and in a separate study the use of GLA cross-inking was found not to be suitable for PyOd immobilisation [24].owever, it has been shown by other groups [17,25] that careful

election of the concentration of the cross-linking agent resulted inood enzyme activity in the resulting device. The following resultsre in agreement with the latter findings.

A calibration study was performed by amperometry in stirredolution using phosphate standard solutions over the range0–350 �M. Fig. 3 shows that the biosensors obtained usinghis cross-linking system are clearly able to measure the phos-hate concentrations studied. The sensitivity of the biosensor0.032 nA �M−1) using this immobilisation method represents avefold increase above the previously reported biosensor [11] andhe CA/CN-PyOd-CoPC-SPCEs discussed in the previous section.urthermore, the time to reach 95% of the steady-state currentti95%) using the GLA-PyOd-CoPC-SPCEs was found to be 13 s (±1 s,

= 3); this is a significant improvement over the previous immobil-sation method, which had a response time of close to 5 min [11].he faster response time obtained with the new design is proba-ly due to a significantly enhanced diffusion of phosphate to thenderlying enzyme layer.

.4.2. Effect of increasing concentration of co-factors onLA-PyOd-CoPC-SPCE biosensor response

At this stage in our studies we were able to achieve a detec-ion limit of 50 �M phosphate, which was not considered suitableor our proposed analysis of pond water samples. An explana-ion for the lack of sensitivity could be that a portion of themmobilised co-factors had leached from the reaction layer on theiosensor into the bulk solution during analysis. Consequently, we

nvestigated the possibility of enhancing the sensitivity by includ-ng TPP and FAD in the electrochemical cell at an appropriateoncentration.

Amperometric studies using the GLA-PyOd-CoPC-SPCE together

ith 200 �M TPP and 20 �M FAD led to a significant increase in ana-

ytical signal. Fig. 4 shows sharp increases in anodic current usinghe biosensor, which correspond to the addition of these cofactors.t is known from previous studies (data not shown) that neither

Fig. 4. The effect of additional cofactors during calibration of phosphate using GLA-PyOd-CoPC-SPCE biosensors.

of these compounds gives an anodic response at the CoPC-SPCEwith the operating potential (+0.4 V vs Ag/AgCl) of the biosen-sor. The mean sensitivity (n = 5) was calculated as 0.406 nA �M−1

phosphate and showed a 14-fold increase in analytical signal overthe biosensors without cofactors present in the electrochemicalcell.

In order to deduce the sensitivity of the new biosensor approacha calibration study was performed at low phosphate concentra-tions. Fig. 5 shows the resulting calibration plot for phosphate inthe range 2.5–50 �M; the limit of detection (LOD), calculated astwice the noise divided by the sensitivity is 2.28 �M phosphate. TheLOD is equal to 0.2 mg L−1 phosphate and is therefore in the regionof interest for monitoring by the Environment Agency (0.1 mg L−1)[26]. Clearly the sensitivity of the biosensor is enhanced signifi-cantly using this procedure; therefore this new approach was usedin all further studies.

3.4.3. Application of optimum amperometric biosensor(GLA-PyOd-CoPC-SPCEs) to the determination of phosphate inhuman urine

The amperometric biosensors were evaluated for the measure-ment of endogenous phosphate using six replicate samples from ahealthy female subject; six individual biosensors were used for theassay. The results obtained are shown in Table 1.

The coefficient of variation was calculated for the six individ-

Fig. 5. Calibration of phosphate using GLA-PyOd-CoPC-SPCE biosensors. Conditionsas follows: +0.4 V, pH 7.3 MOPS 0.05 M, 2 mM PA, 10 mM MgSO4, 200 �M TPP, 20 �MFAD and 0.1 M NaCl. Error bars represent standard deviations (n = 4).

1326 L. Gilbert et al. / Sensors and Actuat

Table 1Endogenous phosphate concentrations in human urine obtained with an ampero-metric biosensor.

Sample number Concentration (mM)

1 24.82 28.93 25.94 28.75 25.56 26.9

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on chromatography for 80 different urine samples. These results

emonstrate that the phosphate biosensor shows promise withhis complex biological fluid and may have application in a clinicalaboratory.

ig. 6. (a) Typical amperogram obtained with unspiked pond water using a GLA-yOd-CoPC-SPCE and (b) typical standard addition calibration plot of unspiked pondater using a GLA-PyOd-CoPC.

ors B 160 (2011) 1322– 1327

3.4.4. Analysis of pond water samples usingGLA-PyOd-CoPC-SPCEs

The GLA-PyOd-CoPC-SPCEs biosensors were used to determinephosphate in three replicate pond water samples using the methodof standard addition. Fig. 6(a) shows a typical amperogram and (b)shows a typical standard addition calibration plot obtained usingthe biosensor. From this data, the original mean phosphate con-centration was calculated and found to be 48 �M (n = 3). The meanrecovery was 103.2% for samples fortified with 10 �M phosphate,n = 3 with a coefficient of variation of 19.7%. It should be noted thatrecoveries were examined for deionised water fortified with 25 �Musing an identical method to that described for the pond water;the mean recovery was calculated to be 95.6% (n = 3) with coeffi-cient of variation of 1.5%. The increase in coefficient of variationin real samples is probably due to the presence of naturally occur-ring substances such humic acids; it should be feasible to improvethe precision by using a pre-treatment step such as ion exchange.However, the present method offers a very simple and economicapproach for the rapid screening of eutrophication. In order todeduce the accuracy of the current method using the biosensorwe compared it to a standard colourimetric procedure. The linearregression equation (Eq. (4)) obtained by the later method is shownbelow (r2 = 0.999). The original phosphate concentration in thepond water was calculated as 53.9 �M ± 0.71 (5.1 mg L−1 ± 0.07),n = 5.

y(Ab) = 0.0231(�M) + 0.0025(Ab) (4)

Statistical analysis of the phosphate concentration obtained byeach method (mean values of 53.9 �M for colourimetry vs 48.0 �Mfor biosensor) was performed using a two-tailed, unpaired Stu-dent’s t-test; the calculated p-value was found to be 0.39. Thisindicates that the difference between the two mean values is notsignificant (at 99% probability level) and therefore there is goodagreement between the biosensor and the standard colourimetricmethod.

The concentration of phosphate found in the pond water sam-ples by both methods indicates that the pond is eutrophic. Aconcentration of phosphate above 0.1 mg L−1 is considered to bean excess of this limiting nutrient [26,27].

4. Conclusion

This paper has demonstrated that the use of GLA to immo-bilise PyOd onto the surface of a CoPC-SPCE for the fabrication of aphosphate biosensor is the superior method in comparison to pre-formed CA/NC membranes and the cellulose acetate method [11]previously described. The biosensor with GLA immobilised PyOdwas successfully applied to the determination of phosphate in pondwater samples. The concentration of phosphate measured by thebiosensor and the standard colorimetric method were found notto be statistically different. The biosensor was successfully used toquantify phosphate in the complex water sample matrix withoutany sample pre-treatment, other than dilution. The determinationof phosphate in urine was also possible using only a simple dilutionstep directly into the electrochemical cell. It should be mentionedthat the method of fabrication of our biosensors is considerablysimpler than many of those reported previously [3]. Our devicerequires only one enzyme, namely PyOd, and this can be readilyimmobilised using a simple covalent cross-linking step with GLA.

The resulting device is stable for up to two days in a stored vesselcontaining silica gel, at 4 ◦C. For commercialisation of such devices,it should be feasible to add enzyme stabilisers to increase the shelflife [29].

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cknowledgements

The authors wish to thank the Environment Agency, Universityf the West of England, Bristol, and Great Western Research forunding. Gwent Electronic Materials are thanked for the provisionf the CoPC-SPCEs.

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Biographies

Lucy Gilbert received her BSc in Forensic Science in 2006, and PhD in Electroanalyt-ical (Bio)sensor Science in 2011, from the University of the West of England, BristolUK. She currently holds a position within the Royal Society of Chemistry, Cambridge,UK.

Toby Jenkins received his BSc in Chemistry in 1992, and PhD in Electrochemistryand Materials in 1995, from the University of Newcastle Upon Tyne. He is Lecturer inBiophysical Chemistry in the Department of Chemistry, University of Bath, UK. Cur-rent research interests include electrochemical gene sensing and electrochemicaldetection for clinical diagnosis.

Simon Browning is Senior Scientist in the Environment Agency, Bristol, UK. Oneof his current interests is in the development and application of (bio)sensors forremote analysis of anions and cations in river water.

John P. Hart received his PhD in Electroanalytical Chemistry in 1978 from ChelseaCollege, University of London, UK. He is Professor in Biosensor and Electroana-

lytical Sciences, and Head of the Electrochemical (Bio)sensors Research Group, inthe Faculty of Health and Life Sciences, University of the West of England, Bristol,UK. Current research interests include the development of disposable amperomet-ric/voltammetric (bio)sensors, based on screen printing technology, for biomedical,agri-food and environmental applications and gas detection.