development of a sensitive, selective electrochemical immunoassay for progesterone in cow's...

Development of a sensitive, selectiveelectrochemical immunoassay for progesterone incow's milk based on a disposable screen-printed

amperometric biosensor

R. M. Pemberton,a J. P. Harta* and J. A. Foulkesb

aFaculty of Applied Sciences, University of the West of England, Frenchay Campus, Coldharbour Lane,Bristol, BS16 1QY, U.K.

bRidgeway Science Ltd., Rodmore Mill Farm, Alvington, Gloucestershire, GL15 6AH, U.K.

(Received 18 September 1997; in revised form 6 November 1997)

AbstractÐA disposable amperometric biosensor, based on a screen-printed carbon electrode (SPCE) coatedwith antibodies, has been prepared and evaluated for measuring progesterone in cow's milk. This immuno-sensor was employed in a competitive assay involving progesterone labelled with alkaline phosphatase. In-itially, phenylphosphate was used as the enzyme substrate and the product of the reaction, phenol, wasdetected by chronoamperometry at +0.70 V vs saturated calomel (SCE). However, this approach provedunsatisfactory, since an unidenti®ed component of cow's milk adsorbed to the electrode and underwent avariable oxidation at this potential. This resulted in a calibration curve which did not have the character-istic sigmoidal shape. An alternative enzyme substrate, 4-aminophenolphosphate, was therefore examined,the product of the reaction, 4-aminophenol, being detectable at +0.20 V vs SCE; at this lower potential,the interferent in milk did not undergo oxidation. Consistently low background currents were obtained,enabling construction of a sigmoidal-shaped calibration curve for progesterone concentration in the milksamples investigated. Progesterone levels determined by the proposed electrochemical immunoassay com-pared favourably with a colorimetric immunoassay method. # 1998 Elsevier Science Ltd. All rightsreserved

Key words: screen-printed, antibodies, milk progesterone, competitive immunoassay, chronoamperometry.

INTRODUCTION

In the cattle breeding industry, where arti®cial inse-

mination techniques are employed, the successful

prediction of oestrus onset leads to considerablecost saving in herd management [1]. One way of

detecting oestrus onset is to monitor the progester-one levels in milk, optimum fertility rates being

achieved when insemination is performed three days

after the progesterone level has fallen to below5 ng mlÿ1 of whole milk [2, 3]. On-site monitoring of

cow's milk for progesterone levels is highly desir-

able, both for convenience and for economicreasons and such measurements have been con-

ducted to date using immunoassay methods invol-

ving colorimetry [4]. The present study describes an

alternative approach involving electrochemical

measurement with a view to developing a disposa-

ble amperometric biosensor.

Electrochemical immunoassays have been investi-

gated for analytes other than progesterone. For

example, electrochemical detection, following liquid

chromatography or ¯ow injection, has been per-

formed on solutions which have undergone a com-

petitive assay [5±7], and similar approaches have

been employed with voltammetric detection [8].

Recently, workers have reported immobilisation

onto membranes connected over a carbon transdu-

cer, then removed after each analysis [9]. In all of

these cases, the biological element was fabricated

separately from the transducer. In contrast, a ®nal

biosensor for use on the farm should ideally be

Electrochimica Acta, Vol. 43, No. 23, pp. 3567±3574, 1998# 1998 Published by Elsevier Science Ltd. All rights reserved

Printed in Great Britain0013±4686/98 $19.00+0.00PII: S0013-4686(98)00104-2

*Author to whom correspondence should be addressed.

3567

robust, reliable and low cost, in which case a moreintegrated fabrication would be desirable. Hence,

with a minimum of instrumentation and manipu-lation steps, the whole biosensor, consisting of thetransducer with the immobilised biorecognition el-

ement could be disposed of after a single use.A previous preliminary study reported the fabri-

cation of a biosensor based on a monoclonal anti-

progesterone antibody immobilised directly onto ascreen-printed carbon electrode [10] and demon-strated that it might be feasible to monitor pro-

gesterone levels over the range required in cattlebreeding applications. The device employed alkalinephosphatase-labelled progesterone in a competitiveenzyme immunoassay and was e�ective in aqueous

bu�ered solutions but had not been applied to de-terminations in cow's milk.In the present study, the possibility was explored

of determining progesterone in milk samples with adetection limit of 5 ng mlÿ1 or lower. As milk is acomplex mixture of biomolecules, many of which

are electroactive, it is perhaps not surprising thatproblems can arise when trace determinations arerequired. Our approaches towards overcoming such

di�culties and achieving the desired selectivity andsensitivity for a progesterone biosensor aredescribed in this paper.

EXPERIMENTAL

Chemicals and reagents

Unless stated otherwise, chemicals were AR-

grade, purchased from Merck, Poole, Dorset. Allsolutions were prepared in water drawn from aMilli-Q Water Puri®cation System (Millipore U.K.,Watford, Herts.). Bu�er solutions: carbonate coat-

ing bu�er (CB), pH 9.6 containing 0.015 mol dmÿ3

Na2CO3 and 0.035 mol dmÿ3 NaHCO3;100 mmol dmÿ3 diethanolamine±HCl bu�er (DB),

pH 7.2, or pH 9.8 containing 10ÿ2 mol dmÿ3

MgCl2. Phenol (Fisons Scienti®c Equipment,Loughborough) and 4-aminophenol (Sigma-Aldrich

Company, Poole, Dorset) were dissolved, respect-ively, in water or 0.1 mol dmÿ3 NaOH before ®naldilution in bu�er.The puri®ed IgG fraction of a polyclonal rabbit

antibody against sheep IgG was purchased fromSigma-Aldrich (rIg). Crude culture ¯uid containinga sheep monoclonal antibody (mAb) against

progesterone [11], and a solution containing pro-gesterone labelled with alkaline phosphatase (AP-prog) were kindly provided by Ridgeway Science.

Unlabelled progesterone (formula wt = 314.5), p-nitrophenylphosphate and phenylphosphate (diso-dium salt) were purchased from Sigma-Aldrich; 4-

aminophenylphosphate (monosodium salt) was pur-chased from ICN Biomedicals, Thame, Oxon.Samples of cow's milk without progesterone and

with progesterone were obtained by sampling at

various times throughout the oestrus cycle. Thesewere kindly provided by Ridgeway Science.

Preparation of sensors

Screen-printed carbon electrodes (SPCEs), havinga 3 mm� 3 mm working area were printed fromD14 carbon ink onto PVC substrate at Gwent

Electronic Materials (Mamhilad, Gwent) asdescribed previously [12]. SPCEs were coated indivi-dually with antibodies according to the optimum

conditions established previously [10]. Brie¯y, mAb,in DB, was immobilised onto the working area viaa layer of rIg adsorbed previously in CB. These

antibody-coated SPCEs (mAb-SPCEs) were used onthe day of preparation.

Cyclic voltammetry and chronoamperometry

All electrochemical procedures were carried out

using an E612 VA Scanner and an E611 VADetector (Metrohm, Herisau), combined with a JJInstuments chart recorder (Fareham, Hampshire).In order to allow operation within small volumes of

enzyme substrate solution, a dual electrode setupwas adopted throughout, using a screen-printedworking electrode in conjunction with a saturated

calomel counter/reference electrode [10]. Thisallowed cyclic voltammetric and chronoampero-metric analyses to be performed using 0.5 ml

volumes of analyte solution contained within coni-cal polypropylene cells.

Calibration studies on cow's milk

Progesterone standards were prepared initially as

a 1 mg mlÿ1 solution in methanol. Various aliquotsof this were transferred to glass tubes and evapor-ated under N2, before subsequent dilution into buf-fer solution or cow's milk.

Colorimetric and chronoamperometric calibrationstudies were carried out using freshly-preparedmAb-SPCEs. These were ®rst rinsed in DB, pH 7.2,

before the addition of 8 ml volumes of a mixturecomprising 3 ml unlabelled progesterone (standardsolution or milk sample) plus 5 ml AP-prog sol-

ution.For colorimetric determinations, mAb-SPCEs

were then rinsed twice in DB, pH 9.8, dried andlocated individually into 96-well immunoassay plate

microwells (Dynatech Immulon, Farenheit LabSupplies, Milton Keynes). Each well then received120 ml pH 9.8 DB containing 1 mg mlÿ1 p-nitrophe-

nylphosphate, prior to 60 min incubation at RT.The presence of p-nitrophenol was determined, fol-lowing removal of mAb-SPCEs from the wells, by

measuring absorbance at 405 nm with an AnthosHTII plate reader (Labtech International, Uck®eld,East Sussex).

For calibration by chronoamperometry, the bio-sensors were washed in DB, pH 9.8, then placedinto 0.5 ml DB, pH 9.8, or into the same bu�ercontaining 1 mg mlÿ1 phenylphosphate or 4-amino-

R. M. Pemberton et al.3568

phenylphosphate, as speci®ed. Following a 15 minincubation period, chronoamperometry was per-

formed by stepping from open circuit to +700 mV(substrate, phenyl phosphate) or +200 mV (sub-strate, 4-aminophenylphosphate). The current±time

response was recorded and peak current (ip)measured; calibration graphs were constructed byplotting ip against progesterone concentration.

RESULTS AND DISCUSSION

Electrochemical immunoassay for progesterone in

cow's milk using phenylphosphate as substrate

In a previous preliminary study [10], an electro-chemical immunoassay for the measurement of pro-gesterone was described and optimised usingprogesterone standards in aqueous bu�er solutions;

the scheme for this competitive assay is shown inFig. 1. Our present studies began by performingthis immunoassay, involving our antibody based

screen-printed carbon electrodes (mAb-SPCEs), onmilk samples containing known concentrations ofprogesterone and reading these against a calibration

curve constructed from standard progesterone sol-utions in bu�er. Figure 2(a) shows the typical sig-moidal shape expected for a competitive

immunoassay. The current responses obtained forthe milk samples were referred to this plot and thecalculated progesterone concentrations are shown inTable 1, together with the actual concentrations.

Clearly, there is some discrepancy at all but the

lowest concentrations.

As the calibration graph in Fig. 2(a) had been

constructed from data obtained on bu�ered pro-

gesterone standards, the possibility was explored

that, despite the inclusion of a wash step (Fig. 1),

some matrix e�ects from the milk were responsible

for the discrepancy shown in Table 1. Therefore, a

calibration plot was constructed from a single pro-

gesterone-free milk sample, divided into aliquots,

and spiked with increasing known concentrations of

the analyte [Fig. 2(b)]. There are two main features

to note: ®rst all the currents are of greater magni-

tude than those obtained for corresponding concen-

trations shown in Fig. 2(a); secondly, an increase in

current appears for concentrations between 10 and

50 ng mlÿ1.A possible explanation for this ``V''-shaped cali-

bration curve was that at concentrations above

10 ng mlÿ1 (unlabelled progesterone) some seques-

tration had occurred during step 1 (Fig. 1), result-

ing in more enzyme-labelled progesterone being

bound to mAb and a corresponding increase in

phenol production. In order to test this hypothesis,

the assay was repeated using a colorimetric rather

than an electrochemical readout, in order to focus

on the antigen±antibody interaction. Figure 3

shows that a characteristic dose±response curve was

obtained for progesterone standards prepared in

milk, indicating the absence of sequestration.

Fig. 1. Competitive electrochemical enzymeimmunoassay for progesterone. Step 1 = competitive binding; step 2 = wash;

step 3 = product formation; step 4 = chronoamperometry. [Prog.AP = alkaline phosphatase-labelled progesterone;

Prog = unlabelled progesterone; ArOH= dephosphorylated phenol or 4-aminophenol].

Development of an electrochemical immunoassay 3569

An alternative possibility was that the ``V'' shape

observed in Fig. 2(b) related to the electrochemicalmeasurement and that since the currents were

obtained by chronoamperometry at an applied po-

tential of +0.7 V vs SCE, some electroactive com-

ponent of milk was being oxidised at the same

potential as the enzymatically produced phenol

(Fig. 1, step 4). In order to test this hypothesis, cyc-lic voltammetry was performed in the absence of

phenylphosphate (and therefore phenol) on mAb-

SPCEs following the incubation (with milk and

bu�er containing the same progesterone concen-

trations) and wash steps. Figure 4(a) shows that thevoltammogram for an mAb-SPCE exposed to milk

exhibits a large unresolved anodic peak around

+0.6 to +0.7 V vs SCE. This contrasts with vol-

tammograms for a mAb-SPCE [Fig. 4(b); a smallpeak occurs in this potential region, due to the

mAb/antigen coating] and a mAb-free SPCE

[Fig. 4(c)] exposed to bu�er, demonstrating that the®rst peak must have resulted from an adsorbed

electroactive compound present in milk. Adsorption

of the interfering substance in milk might beexpected to occur mainly at the SPCE itself and not

on the Ab-paratope (antigen binding site) which is

borne out by the results for milk shown in Fig. 3.This e�ect on its own would explain the observed

increases in the magnitude of current in milk over

bu�er, but not the ``V''-shaped plot shown inFig. 2(b).

The next consideration was that the nature of the

problem related to the molecular size of the electro-

active interference in milk, i.e., the ``V'' e�ect mayarise because displacement of a large antigen-

enzyme conjugate by a smaller unlabelled antigen

would allow more of the interferent to di�usethrough to the SPCE surface. In order to test this

possibility, the immunoassay was performed on

milk samples as described above, but phenylpho-sphate was eliminated from step 3 so that no phe-

nol was present during step 4 (Fig. 1). Figure 5

indeed shows that currents increased with increasingunlabelled progesterone (closed circles) and that

Fig. 2. Chronoamperometric calibration plots for standard

progesterone solutions prepared in (a) DB, pH 7.2 and (b)

cow's milk. ip values are means (2s.e.m.) for n= 3 elec-

trodes.

Fig. 3. Colorimetric calibration plot for standard pro-

gesterone solutions prepared in cow's milk. Absorbance

values are means (2s.e.m.) for n= 3 microwells/mAb-

SPCEs.

Table 1.

Comparison of actual values for progesterone concen-

tration in milk samples with values obtained by chron-

oamperometric calibration in bu�er

Progesterone concentration (ng/ml)

Sample Actual Obtained from calibration curve

A 1.2 1.4

B 1.0 1.4

C 27.0 7.9

D 31.6 6.3

E 9.7 35.5

F 6.3 28.2


this phenomenon did not occur following incu-

bation in bu�er (open circles).

At this stage, an attempt was made to eliminate

the electroactive interferent from the SPCE surface

prior to the measurement step. Although the inter-ferent was unidenti®ed it was probably hydrophobic

in nature as it seemed to be retained during thewash step with aqueous bu�ers. Therefore, a varietyof solvents were introduced into the wash step

(Fig. 1, step 2) including methanol, dimethylforma-mide and 1.4-dioxan, and also a solution of Tween;none of these eliminated the chronoamperometric

background current.

Electrochemical immunoassay for progesterone in

cow's milk using 4-aminophenylphosphate as enzyme

substrate

Since the electroactive interferent was oxidised at

around +0.6 V vs SCE [Fig. 4(a)], the possibilitywas considered that operating the immunosensor atpotentials well below this value could achieve thedesired selectivity. This approach would require an

alternative enzyme substrate.4-aminophenylphosphate, which is converted to

4-aminophenol by the antigen-label, alkaline phos-

phatase, was a good candidate. Figure 6(a) showsthe cyclic voltammogram of 4-aminophenol andFig. 6(b) its precursor 4-aminophenylphosphate;

Fig. 6(c) and (d) show the voltammograms of phe-nol and phenylphosphate for comparison. It is clearthat the oxidation peak for 4-aminophenol occurs

at a much lower potential than phenol and a valueof +0.2 V vs SCE was selected since it would pro-vide maximum sensitivity but minimum interferencefrom the milk [see Fig. 4(a)]. This potential was

Fig. 4. Cyclic voltammograms performed in DB, pH 9.8 containing 10ÿ2 mol dmÿ3 MgCl2 for mAb-SPCEs incubated in

(a) AP-prog in milk or (b) AP-prog in bu�er and (c) untreated SPCEs (mAb-free). Scan rate = 20 mV sÿ1.

Fig. 5. Chronoamperometric background response (mean

ip2s.e.m.) for mAb-SPCEs (n= 3) incubated with AP-

prog plus standard progesterone solutions prepared in

milk (*) or in bu�er (w).


also su�ciently low that the 4-aminophenylpho-

sphate precursor would not undergo oxidation at

the SPCE [Fig. 6(b)].

In order to establish that 4-aminophenol could be

detected by chronoamperometry over a wide con-

centration range, without passivating the SPCEs, a

calibration study was performed using individual

SPCEs for each measurement. A linear calibration

plot was indeed obtained over the concentration

range 10ÿ2 to 10ÿ4 mol dmÿ3 when an applied po-

tential of +0.2 V was employed. Initial evaluation

of mAb-SPCEs for susceptibility to milk interfer-

ents at +0.2 V was performed by examining back-

ground currents following incubation of the

immunosensor in milk, with increasing unlabelled

progesterone concentrations (enzyme substrate was

absent from the bu�er during the chronoampero-

metric measurement step). Background currents

Fig. 6. Cyclic voltammograms, at SPCEs, of 10ÿ3 mol dmÿ3 solutions of (a) 4-aminophenol, (b) 4-aminophenylphosphate,

(c) phenol and (d) phenylphosphate in DB, pH 9.8 containing 10ÿ2 mol dmÿ3 MgCl2. Scan rate = 20 mV sÿ1.


were generally less than 300 nA and, most impor-

tantly, did not vary over the concentration range 0±500 ng mlÿ1 unlabelled progesterone.

Since these results satis®ed the criteria for pro-

posed biosensor operation, a full calibration studywas performed with milk containing known concen-

trations of unlabelled progesterone. A new immu-

nosensor was used for each measurement and

triplicate biosensors were taken for each milk

sample. The resulting calibration plot is shown in

Fig. 7 (closed circles). It should be noted that thisplot no longer exhibits the ``V'' shape seen in

Fig. 2(b) and is typical for that obtained by immu-

noassay.

In order to evaluate the competitive assay as a

means of predicting oestrus onset, milk sampleswere taken from the same cow at various times inits oestrus cycle. Figure 8 and Table 2 compare theresults obtained by the electrochemical immunoas-

say and by a currently-used method employingmicrotitre plates with colorimetry (RidgewayScience) (correlation coe�cient, r= 0.937). These

data suggest that the electrochemical method showspromise for the sensitive determination of low pro-gesterone concentrations in milk.

CONCLUSIONS

An amperometric immunosensor for progesteronehas been constructed and is based on a SPCEcoated with a monoclonal antibody. The latter is

immobilised onto the SPCE by interaction with asurface-bound polyclonal antibody. This device hasbeen evaluated for the measurement of progesteronein milk using a competitive assay (Fig. 1). Initial

studies involved phenylphosphate as the substratefor the progesterone enzyme label, alkaline phos-phatase. In this approach, it was found that an

interferent present in milk adsorbed onto the SPCEand was not removed during the wash step. Thisresulted in a large background current in the chron-

oamperometric measurement step which varied inmagnitude with the concentration of unlabelled pro-gesterone. As chronoamperometry was performedat a potential of +0.7 V vs SCE, where phenol

undergoes oxidation, it was decided to investigatean alternative enzyme substrate.4-aminophenol gave maximum sensitivity at

+0.2 V vs SCE and the background current wasreduced considerably at this potential, whilstremaining una�ected by di�erent unlabelled pro-

gesterone levels. Therefore, the selectivity of theelectrochemical immunoassay for progesterone incow's milk has been greatly improved by the incor-

poration of an alternative enzyme substrate (4-ami-nophenylphosphate).It should be added that other workers have also

found 4-aminophenol to be advantageous in immu-

Fig. 7. Calibration plot of mean ip (2s.e.m.) vs progester-

one concentration (*), including background currents

without 4-aminophenylphosphate (w).

Fig. 8. Correlation plot of electrochemical vs colorimetric

assay results for progesterone (prog) in cow's milk samples

taken at various times during the oestrus cycle.

Table 2.

Comparison of actual values for progesterone concen-

tration in milk samples with those obtained by chronoam-

perometric calibration using 4-aminophenolphosphate as

substrate

Progesterone concentration (ng/ml)

Cow sample date actual

Obtained by

chronoamperometry

28/02/97 2.0 5.0

04/03/97 <1.0 4.4

07/03/97 <1.0 4.0

11/03/97 10.0 8.7

14/03/97 26.0 14.4

19/03/97 23.0 19.9


noassays where electrode materials other thanscreen-printed carbon have been used as part of the

biosensor. These include a carbon electrode whichwas used for Apolipoprotein (apo) E [9] and amicroporous gold electrode [13] to determine

human chorionic gonadotrophin (hCG). In our ear-lier investigation we had also examined 4-amino-phenol together with phenol and phenolphthalein at

our SPCEs [10]. This was done by amperometry instirred solution, using one electrode for each sub-stance, and it appeared that the sensitivity for 4-

aminophenol was somewhat less than for phenol,with apparent passivation at high concentrations.However, bearing in mind that in the present study,chronoamperometry was performed with a fresh

electrode for each measurement, it is unlikely thatany signi®cant passivation could occur. Indeed, thecalibration graph for 4-aminophenol gave a linear

response over the range studied. Therefore, our dis-posable mAb-SPCEs now have the required selec-tivity and sensitivity to determine down to 5 ng/ml

progesterone in milk.In future work, this electrochemical immunoassay

will be further evaluated on milk taken from a

number of cows at various stages in the oestruscycle. It may be possible to use this approach todetermine when arti®cial insemination can be car-ried out most e�ectively.

ACKNOWLEDGEMENTS

The authors would like to thank Mr. R. Pittsonand his sta� at Gwent Electronic Materials Ltd.(GEM), Mamhilad, Gwent for kindly providing the

SPCEs. They are also grateful to Dr. D. Cowell,Dr. R. Luxton and R. Wedge for their interest in

the work and Mr. M. Norman for artwork (all atUWE). Drs D. J. Groves and B. A. Morris fromthe Heterohybridoma Antibody Group,

Department of Biochemistry, University of Surreyare gratefully acknowledged as the originators ofthe anti-progesterone mAb. Financial support for

this project was provided by the HEFCE.

REFERENCES

1. P. J. Ball, Br. Vet. J. 138, 546 (1982).2. J. A. Foulkes, A. D. Cookson and M. J. Sauer, Vet.

Rec. 111, 302 (1982).3. J. A. Foulkes and R. G. Goodey, Vet. Rec. 122,

135 (1988).4. J. A. Foulkes, A. D. Cookson and M. J. Sauer, Br.

Vet. J. 138, 515 (1982).5. M. J. Doyle, H. B. Halsall and W. R. Heineman,

Anal. Chem. 56, 2355 (1984).6. H. T. Tang, C. E. Lunte, H. B. Halsall and W. R.

Heineman, Anal. Chim. Acta 214, 187 (1988).7. K. R. Wehmeyer, H. B. Halsall, W. R. Heineman, C.

P. Volle and I.-W. Chen, Anal. Chem. 58, 135 (1986).8. W. R. Heineman and H. B. Halsall, Anal. Chem. 57,

1321A (1985).9. M. Meusel, R. Renneberg, F. Spener and G. Schmitz,

Biosensors Bioelectron. 10, 577 (1995).10. J. P. Hart, R. M. Pemberton, R. Luxton and R.

Wedge, Biosensors Bioelectron., 12, 1113 (1997).11. D. J. Groves, M. J. Sauer, P. Rayment, J. A. Foulkes

and B. A. Morris, J. Endocrinol. 126, 217 (1990).12. S. D. Sprules, J. P. Hart, R. Pittson and S. A. Wring,

Electroanalysis 8, 539 (1996).13. C. Duan and M. E. Meyerho�, Anal. Chem. 66,

1369 (1994).


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