detection of progesterone in bovine milk using an electrochemical immunosensor

ORIGINALRESEARCH Detection of progesterone in bovine milk using an

electrochemical immunosensor

HONG YOU ZHANG,1 XIA YAN DU,2 QIAN LIU,1 CHENG XIA1* andLING WEI SUN1

1College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, 163319,China, and 2School of Public Health, Harbin Medical University, Harbin, 150081, China

To improve the detection of ovulation and early diagnosis of pregnancy on dairy farms, a dispos-able electrochemical immunosensor was developed using indirect competitive immunoassays andscreen-printed carbon electrodes. After optimisation of the assay conditions, the linear range wasfrom 0.16 to 50 ng/mL. The limit of detection was 0.16 ng/mL. There was no cross-reactivity withtestosterone, 17-b-estradiol and estriol. The intra-assay coefficient of variation was less than 13%and interassay coefficient of variation was < 9%. The percentage recovery was from 95.8% to115.7%. Stability lasts 4 days at 4 °C. The results of preliminary application trials indicate that themethod could have practical potential.

Keywords Cows, Milk progesterone, Immunosensor, ELISA, Screen-printed electrode.

INTRODUCTION

The main tasks performed in the reproductivemanagement of dairy cattle are detection of ovu-lation and early diagnosis of pregnancy, whichreflect to a great extent the progesterone status.The concentration of progesterone, in bothplasma and milk, varies with the oestrous cycle.Progesterone secretion is very low on the day ofoestrous (<5 ng/mL of milk), increases rapidlyfrom days 4 to 9 of the cycle (10–40 ng/mL ofmilk), and remains stable until approximately3 day before the next oestrous on day 21 (Frig-gens and Chagunda 2005; Pache 2007). Thepresent work on the determination of the proges-terone concentration in milk resulted from thedrive to develop a rapid means of determiningthe onset of oestrous in dairy cattle. This is theperiod of 12–24 h before ovulation when cattledisplay standing to be mounted behaviour. Ide-ally artificial insemination should be performedapproximately 12 h after the onset of standingoestrous (Brandt et al. 2010; Foulkes andGoodey 1988). The onset of oestrous is indicatedby a rapid fall in the concentration of milk pro-gesterone, to below 2–5 ng/mL (Bulman 1979;Sauer et al. 1986). Monitoring progesterone inmilk is a quite effective method for predicting

ovulation, the detection of pregnancy and fertil-ity problems. Enzyme-linked immunosorbentassay (ELISA) test kits (BIOTANG Inc., Wal-tham, MA, USA) allow the oestrous detectionwith 98% specificity and have a 0.12 ng/mLlower detection limit (sensitivity) and a 0.12–40.0 ng/mL detection range for serum sample,but they require time and skills that are notcommonly found on dairy farms. Developing areal-time milk progesterone biosensor wouldprovide a very useful tool for monitoring fertil-ity (Maria and Toby 2003; Pemberton et al.2001).Several approaches have been developed to

determine progesterone concentrations directlyin milk. A disposable screen-printed amperomet-ric progesterone biosensor is used in an existingimmunoassay (Pemberton et al. 1998). The bio-sensor relies upon a reduction in the binding ofalkaline phosphatase-labelled progesterone to thesensor surface in the presence of milk progester-one. The enzyme substrate is naphthyl phos-phate. The 1-naphthol generated in the reactionis oxidised electrochemically, which produces asignal that is inversely proportional to the con-centration of unlabelled progesterone in milk. Itis possible to produce the transducer elementand fabricate sensors using screen-printing at

*Author forcorrespondence. E-mail:[email protected]

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doi: 10.1111/1471-0307.12076

low cost, which enables the screen-printed electrodes to beused as disposable devices. Other researchers have devel-oped an automated ovulation detection system that measuresprogesterone directly in whole fresh milk (Maria and Toby2003; Pemberton et al. 2001).The ideal method for the screening of anabolic steroids

should be fast, simple, easy to perform, and enable measure-ment in a small volume of biological fluid (Popii and Bau-mann 2004). An immunoassay is based on the recognitionof antigens by antibodies during the formation of a stablecomplex. They are among the more sensitive assays avail-able due to the high affinity of antibodies. An immunosen-sor combines the sensitivity of the antibody–antigeninteraction with other advantages, including low cost, fastanalysis and portable screening, which allows decentralisa-tion from the laboratory environment. They are appliedwidely in human and animal health care, the food and fer-mentation industries, environmental monitoring, agricultureand defence (Hock1997; D’Orazio 2003). Disposablescreen-printed electrodes (SPEs), used in conjunction withelectrochemical immunosensors, provide an approach todevelop a cost-effective device and to miniaturise the ana-lytical system (Conneely et al. 2007; Lu et al. 2006). TheSPEs were modified with progesterone-ovalbumin (P4-OVA). In this work, the electrochemical sensor consisted ofimmobilised protein hapten conjugate, an antibody and ahorseradish peroxidase (HRP)-labelled antispecies. The elec-trochemical technique uses 3,3′,5,5′-tetramethylbenzidine(TMB) hydrogen peroxide (H2O2) substrate mixture. Theprocess of screen-printing carbon inks provides an efficientmeans to mass-produce electrodes, which can then be tai-lored for specific applications (Hart et al. 2004). Samplepretreatment is kept to a minimum, with only a simple dilu-tion step required. This paper presents the results of optimi-sation studies, but the ultimate goal of this work is todevelop a sensor for progesterone that, in the future, couldbe incorporated into a milking parlour to provide in-linemonitoring of the cow’s reproductive status.

MATERIALS AND METHODS

Apparatus and reagentsThe 96-well plates were purchased from Costar (Shanghai,China). An ELISA plate reader (ELX800), used for spectro-photometric detection, was supplied by Bio-Tek Instruments(Winooski, VT, USA). Electrochemical measurements wereperformed using a CHI660A electrochemical workstation(Shanghai Jiao Tong University, China) with a three-elec-trode setup. The electrodes were screen-printed using ascreen printer (Nanjing Mckesson Electronics Co., Ltd.,Xixia District, Nanjing, China).Antiprogesterone monoclonal antibody (MAb) and pro-

gesterone complete antigen (11a-OH-P4-HS-OVA, P4-OVA) were prepared by our laboratory (Gao et al. 2009).

Progesterone (P4) and anti-mouse-IgG-HRP were purchasedfrom Sigma-Aldrich (Trading Co., Ltd., Shanghai, China).The 3,3′,5,5′-tetramethylbenzidine 2HCl salt (TMB-2HCl)was purchased from AMRESCO LLC (Solon, OH, USA).Bovine serum albumin (BSA), H2O2 and polyoxyethylene-sorbitan monolaurate (Tween 20�) were purchased fromBeijing Biosynthesis Biotechnology Co., Ltd. (Peiking,China). Bovine ovalbumin (OVA) was purchased from Beij-ing Dingguo Biotechnology Co., Ltd. (Peiking, China). Allother solvents and reagents were of analytical grade.Phosphate-buffered saline (PBS) consisted of NaCl

(137 mM), Na2HPO4 (8 mM), KCl (2.6 mM) andKH2HPO4 (1.5 mM), pH 7.4. Bicarbonate buffer consistedof Na2CO3 (15 mM) and Na2HCO3 (45 mM), pH 9.6.Phosphate citrate buffer contained anhydrous citric acid(0.2 M), Na2HPO4 (0.1 M), KCl (0.1 M), pH 5.0. Washingbuffer consisted of PBS containing 0.05% Tween� 20.Blocking buffer contained BSA (1%, w/v) in PBS buffer,and phosphate buffer contained Na2HPO4 (8 mM) andKH2HPO4 (1.5 mM). The pH of each solution was adjustedusing either HCl or NaOH.

Preparation of antiprogesterone monoclonal antibodyProgesterone is a hapten. To prepare the antigen, 11a-hy-droxyprogesterone hemisuccinate was conjugated, respec-tively, to the carrier proteins BSA and OVA bydicyclohexylcarbodiimide. P4-BSA was used for the immu-nisation of mice, and P4-OVA was used as the coating anti-gen for the ELISA. Both antigens have been verified bysodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE).Male mice, 6–8 weeks of age, were immunised with P4-

BSA four times at 1-month intervals. The spleen wasremoved on the third day after the fourth immunisation. Thespleen cells were fused with SP2/0 myeloma cells usingPEG, and 96-cell culture boards were incubated at 37 °C,5%CO2 (at 2–3 days half of the medium was changed) andselected with hypoxanthine–aminopterin–thymidine (HAT)and hypoxanthine–thymidine (HT) medium. Two hybridomacell lines, 8G6 and 11A11, were obtained by indirectELISA using P4-OVA as the coating antigen and cloned bythe limiting dilution method three times. The titres of theculture medium for subtype IgG1 and IgG2a were1:160009 and 1:5120009, of ascetic fluid were 1:80009and 1:2160009. When analysed by indirect competitiveELISA, the antiprogesterone monoclonal antibodies showedgood sensitivity that has a 0.10 ng/mL detection limit ofprogesterone. The affinity of 8G6 was higher than that of11A11, and therefore, 8G6 was used in the following assay.

Enzyme-linked immunosorbent assay (ELISA)The ELISA procedures were carried out in a 96-well microt-itre plate. The wells were coated with 100 lL of a 1:800dilution of P4-OVA in bicarbonate buffer and incubated at

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37 °C for 2 h. The plate was washed three times with wash-ing buffer (PBS buffer containing 0.05%, v/v, Tween 20)and blocked using 200 lL blocking buffer (PBS buffercontaining 0.5%, w/v, BSA) for 1 h at 37 °C. The competi-tive assay was performed by the addition of 50 lL of seri-ally diluted progesterone (taken from a stock solution of1 mg/mL) and 50 lL of antiprogesterone monoclonal anti-body solution (0.94 lg/mL), both prepared in blocking buf-fer, to a final volume of 100 lL in each well. The plate wasagain incubated at 37 °C for 1 h, followed by washing. Analiquot of 100 lL of enzyme-labelled anti-mouse-IgG-HRPprepared in blocking buffer was added to each well andincubated at 37 °C for 1 h.Colour development was facilitated by the addition of

100 lL of substrate solution to each well. The substrateconsisted of TMB (0.1 mg/mL) in phosphate citrate buffercontaining H2O2 (1 mM). Colour was allowed to develop at37 °C for 25 min; the reaction was stopped by the additionof 50 lL of 2M H2SO4. Colour development was measuredspectrophotometrically at 450 nm.

Immunosensor protocolImmunosensors were prepared by adsorbing 5 lL P4-OVA(1:100 dilution in PBS buffer) passively onto the carbonarea of the SPE. The electrodes were blocked by the appli-cation of 50 lL blocking solution to the electrode. Duringthe competitive step, electrodes were exposed simulta-neously to 2.5 lL of antiprogesterone monoclonal antibodysolution (final concentration 2.5 lg/mL) and 5 lL proges-terone. Anti-mouse-IgG-HRP was diluted to 1:1000, and10 lL was placed on the electrode. The antibody, analyteand label solutions were prepared in blocking buffer. Theelectrodes were washed for a final time with distilled water,and the concentration was measured using chronoamperom-etry by adding TMB substrate solution (0.4 mM TMB inphosphate citrate buffer, pH 5, with 1 mM H2O2). Eachelectrode was assessed chronoamperometrically and themeasured current was recorded. The parameters for detec-tion were as follows: Pretreatment – first conditioningpotential 0.2 V (or open circuit potential), duration 20 s,equilibrium time 5 s; measurement: interval time 0.1 s,standby potential 0.2 V (or open circuit potential), numberof potential steps 1; potential 0.1 V; duration 10 s.The study was approved by the Veterinary Medical Ethi-

cal Committee of Local Agricultural Department (Mishan,Heilongjiang, China). All mice and cows were treatedaccording to International Guiding Principles for BiomedicalResearch Involving Animals (CIOMS, 2002).

RESULTS

Characterisation of ELISA methodOptimum working concentrations of both P4-OVA conjugateand antibodies were obtained by performing chessboard

titrations, in which serial dilutions of both proteins were car-ried out in duplicate. The optimum concentration of P4-OVA conjugate was found to be a 1:800 dilution with a1:8000 dilution of the antiprogesterone MAb. A volume of100 lL of HRP-labelled anti-mouse antibody (1:4000 dilu-tion) was added to each well; this was followed by the addi-tion of substrate and colour development ensued.Competitive assays were developed using concentrations

obtained from the checkerboard assays, and calibrationcurves were fitted by nonlinear regression. The logarithmicconcentration of P4 was used on the abscissa, and Logit(B/B0) was used on the ordinate, where B0 is the optical den-sity (OD) value with no P4 and B is the OD value of serialdilutions of P4. Our previous results (Gao et al. 2009)showed that the standard curve equation of an indirect com-petitive ELISA was y = �1.9271x �0.1125 (R2 = 0.9909),the limitation of optical density (LOD) of this assay was0.21 ng/mL, and the linear range of the curve was quitebroad, from 0.21 to 40 ng/mL.

Standard curve of progesterone immunosensorThe immunoassay system was transferred subsequently toan immunosensor format using disposable SPEs. Prelimin-ary concentrations of the assay components were againdetermined using a checkerboard assay. The dilution of P4-OVA (1:200) was immobilised to the carbon working areaof a screen-printed electrode. Following blocking proce-dures, 10 lL of antiprogesterone MAb was applied to theelectrodes. Labelled anti-mouse-IgG-HRP was used at adilution of 1:1000. The optimum concentrations were cho-sen by evaluation of the signal:noise ratio. For developmentof the competitive assay, the P4-OVA was diluted to 1:200and 5 lL of this solution was adsorbed passively onto thecarbon working area. Following addition of the blockingsolution, 25 lL volumes of the analyte and antiprogesteroneMAb solution were mixed in a 1:1 ratio (to yield a finalconcentration of 2.5 lg/mL of antibody). A 10 lL volumeof this mixture was placed immediately on the working areaand incubated. Anti-mouse-IgG-HRP was diluted to 1:1000,and 5 lL was placed on the electrode. Following incuba-tion, the electrode was assessed chronoamperometrically.The logarithm concentration of P4 was used on the abscissa,and Log i was used as ordinates, where i is current. Resultsshowed that chronoamperometrical curve (Figure 1a) andcalibration curve (Figure 1b) were obtained from the result-ing currents, the limitation of optical density (LOD) was0.16 ng/mL, the linear range of the assay was 0.16–50 ng/mL, with a regression value of 0.9915.

Optimisation of incubation timeTo obtain the optimal incubation time for the competitiveassay, the incubation periods of 10, 20, 30, 40, 50 and60 min at 37 °C, respectively, were used. Five sets (n = 3)of identical electrodes were prepared simultaneously, and a

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competitive assay was carried out. P4-OVA (1:100 dilution)was immobilised; the electrodes were blocked, and acompetitive assay was performed using 0.5 ng/mL ofprogesterone and 2.5 lg/mL of antiprogesterone MAb. Theelectrodes were placed in an incubator in a humid environ-ment for the specified period of time; each set was washed,followed by the application of the enzyme-labelled anti-mouse antibody (1:1000 dilution), and chronoamperometricmeasurements were made. A large, almost twofold, increasein signal was observed from 20 to 30 min and a more grad-ual increase to 45 min. The recorded current increasedslightly from 45 to 60 min; however, this increase was notconsidered significant enough to warrant increasing theassay time further. An incubation time of 45 min was there-fore chosen for all further electrochemical assays (Figure 2).

Optimisation of pHHorseradish peroxidase has maximum activity between pH5 and 7. To establish the maximum activity of HRP usingthe TMB/H2O2 substrate system, a pH study was carriedout with the substrate prepared in buffers of different pH.Phosphate citrate buffer was used at pH 5 and pH 6, andphosphate buffer adjusted to pH 7 and pH 8 to determine

the effects of higher pH. A capture assay was performedusing a 1:100 dilution of P4-OVA immobilised on the work-ing area and 2.5 lg/mL of antiprogesterone MAb. Thelabelled anti-mouse-IgG-HRP was used at 1:1000 dilution.The results indicated that pH 5 and pH 6 appeared to be themost suitable, with good specific:blank ratios. Although ahigher specific signal was obtained using pH 7, the blankvalues were unacceptably high, with very high error attrib-uted to both specific and blank values. It was thereforedecided to prepare the substrate at pH 5.5 (Figure 3).

Stability of substrateThe stability of the substrate was investigated following theaddition of H2O2. It was necessary to establish a time periodwithin which the measurements should be completed.A noncompetitive assay, which used a 1:100 dilution of im-mobilised P4-OVA, followed by the addition of 2.5 lg/mLof antiprogesterone and a 1:1000 dilution of the labelledanti-mouse-IgG-HRP, was performed on 10 sets of elec-trodes (n = 3) simultaneously, so that each set of electrodeswas measured at a different time. The substrate was pre-

y = –4.9569x + 8.8305R2

= 0.9915

–1

–0.5

0

0.5

1

1.5

2

1.2 1.4 1.6 1.8 2

Log C (ng/mL)

Log

i (uA

)

(b)(a)

Figure 1 The chronoamperometrical curves for increasing concentration of progesterone (P4) on the modified screen-printed electrodes (SPE) (a) andcalibration graphs for progesterone (b). (a) Chronoamperometry measurement. A standard curve of indirect competitive ELISA can be establishedusing the current value. The time was used as abscissa, and current was used as ordinates. (b) The logarithm of P4 concentration was used asabscissa, and Log i was used as ordinate, where i is current. The linear range of the assay was 0.16–50 ng/mL, with a regression value of 0.9915.

00.5

11.5

22.5

33.5

44.5

10 20 30 40 50 60Incubation time (min)

IP/u

A

Figure 2 Optimisation of incubation time. The incubation time for acompetitive assay was used as abscissa, and signal strength (IP/uA) wasused as ordinate.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5 5 5.5 6 6.5 7 7.5 8 8.5pH

IP/u

A

Figure 3 Optimisation of pH. The pH for horseradish peroxidase (HRP)was used as abscissa and signal strength (IP/uA) was used as ordinate.



pared and allowed to sit at room temperature under lightexclusion until measurement was performed. The substrateappeared to be sufficiently stable for approximately 20 min;after this time, the current decreased by almost 20%. There-fore, the measurements should be completed within 15 minif possible (Figure 4).

Specificity of the immunosensorSpecificity was evaluated by assessment of cross-reactivity.The chronoamperometric currents of progesterone, 17-b-estradiol, estriol and testosterone in serial concentrationswere determined using the indirect competitive assay. Atypical calibration curve is shown in Figure 5. The curvefor progesterone declined gradually with increased concen-

tration of the inhibitor. The curves for the other three hor-mones were almost flat. This indicated that the specificity ofthe immunosensor was excellent.

Precision of the immunosensorIn the study of precision, the reproducibility (intra-assay)and repeatability (interassay) of the competitive assays werecompared. Three different concentrations of standard pro-gesterone, 0.5, 10 and 40 ng/mL, representing low, mediumand high concentrations based on the calibration curve, werestudied. Reproducibility was assessed by preparing fourelectrodes for each concentration. These were preparedsimultaneously from the same solutions of coating conju-gate, antibody and labels (intraday assay). Repeatability wasdetermined by preparing four sets of electrodes on differentdays and comparing the resulting currents. These electrodeswere prepared from different solutions, unlike the reproduc-ibility study. The results of these two studies are summar-ised in Table 1.The intra-assay study showed good reproducibility for the

higher concentrations of analyte, with only the lowest con-centration showing 13% variability. Interestingly, the interas-say performance was significantly better, at 8.75% coefficientof variation (CV). It would be expected that the intradayassay variance would be lower than that of procedures carriedout on different days. At higher concentrations of the analyte,the intra- and interday assay variance was satisfactory.

Stability of the immunosensorThe SPEs were dried and stored in a sealed container at4 °C, in the presence of a desiccant, until the day of mea-surement, at which point they were prepared and measuredas described previously. The electrodes showed good stabil-ity for the first 4 days of the study. The signals dropped byapproximately 20% by day 5 and on subsequent days. Thedifference in the signal, measured as part of the stabilitystudy on day 5 and later, was considerably greater than thisvalue. Therefore, the lack of stability must have been aresult of the storage of the electrodes. The P4-OVA solutioncan be stored as a stock solution for 2 months at 4 °C and

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 10 20 30 40 50Detection time (min)

IP/u

A

Figure 4 Stability of substrate. The current detection time was used asabscissa, and signal strength (IP/uA) was used as ordinate.

0

0.2

0.4

0.6

0.8

1

1.2

0 0.625 1.25 2.5 5 10 20 40ng/mL

A/A

o

Progesterone Testosterone

17-β-oestradiol Oestriol

Figure 5 Inhibition curve for the immunosensor. A0 is the value of thecurrent with no hormone, and A is the value following inhibition ofhormones. The progesterone concentration was used as abscissa, andA/A0 was used as ordinate, where A0 is no progesterone inhibited thecurrent value; A suppressed when progesterone concentrations for eachcurrent value.

Table 1 Precision and accuracy of progesterone immunosensor

Progesteronea

(ng/mL)

Precision Accuracy

Intra-assayCVb (%)

InterassayCV (%)

Recovered(ng/mL) % Recoveryc

40 4.6 5.8 38.3 95.810 6.8 8.72 11 109.50.5 13.2 8.75 0.58 115.7aStandard progesterone was used to detect precision and accuracy of

immunosensor.bCV, coefficient of variation.crecovery = (actual concentration/recovery concentraion) 9 100.



for at least 2 years at �20 °C without any noticeable lossof performance. When the P4-OVA is diluted and coated onan electrode, the stability drops significantly to approxi-mately 4 days.

RecoveryThe accuracy of the immunosensor was studied by measur-ing the recovery of progesterone samples. The concentra-tions chosen were 0.5, 10 and 40 ng/mL, which representedlow, medium and high concentrations, respectively. A com-petitive assay was performed simultaneously so that the con-centrations of the samples could be calculated using thisstandard curve. The results are shown in Table 1. Therecovery for all samples was satisfactory. The highest recov-ery was obtained for the highest concentration (40 ng/mL),which deviated by <2% from the spiked concentration. Theconcentration was overestimated for both the medium(10 ng/mL) and low (0.5 ng/mL) concentrations. The over-estimation of the medium value was 10%, which is accept-able. The overestimation of the lower concentration washigh, at 17%, but this was due primarily to a small degreeof error in the standard curve.

Analysis of field samplesElectrodes were coated and blocked as described previously.Fresh milk samples were taken from three oestrous cows thathad marked oestrous behaviour at ovulatory phase by rectumexamination during 60–90 day postpartum (Williams andEsslemont 1993) from the same dairy farm and followingremoval of 98% of hormones using a method of active carbonand dextran (Gao et al. 2009) and were used as test samplesto investigate the performance of the immunosensor. Threeother milk samples without hormone were spiked with pro-gesterone at 0.5, 10 and 40 ng/mL, respectively, to create astandard curve with a wide linear range of progesterone. Sofar, a gold standard progesterone ELISA assay has not beenavailable. In this study, 0.5, 10, and 40 ng/mL of progester-one in milk typically represent low, median and high concen-trations of progesterone, which should meet the requirementof detecting progesterone for oestrous and pregnancy accord-ing to the previous research methods (Engeland et al. 1997;Comin et al. 2005). All samples were assayed according tothe immunosensor protocol. A competitive assay was per-formed simultaneously, and the concentrations of the sampleswere determined using this assay. The results of the analysisshow that the progesterone concentration of three test samples(ng/mL) was 2.53, 5.13 and 2.53 and suggested that theimmunosensor has a good sensitivity to the low milk proges-terone concentrations for oestrous. Levels in the other threemilk samples were 0.6, 10.31 and 40.19 which indicated thatthe immunosensor was accurate at the different milk proges-terone concentrations found during pregnancy. The resultsobtained show that the method should be helpful for assessingreproductive performances in the future.

DISCUSSION

The results of this study demonstrate that an electrochemicalimmunosensor can be used for the analysis of progesteronein fresh bovine milk. A simple and rapid assay was devel-oped, which required minimal hands-on time. The assayswere developed initially using an ELISA with an LOD of0.21 ng/mL. The linear range of the calibration curveextended from 0.21 to 40 ng/mL, with a regression coeffi-cient of 0.99. The assay system was transferred to screen-printed electrodes. Competitive assays were developed inbuffer and used to determine the concentration of progester-one in cow’s milk. The linear range obtained for the assayin buffer extended from 0.16 to 50 ng/mL.Other biosensor assays for milk progesterone that use dif-

ferent detection systems were reported over a decade ago.An amperometric biosensor was described, in which single-use, disposable screen-printed carbon electrodes were coatedwith antibody (Pemberton et al.1998, 1999). The assayinvolved a 30-min competitive binding step, during whichsample or standard progesterone competed with enzyme-labelled progesterone for binding to antibody, followed bycontinuous flow electrochemical detection of bound enzyme.Although the sensitivity of this assay appeared to be good,the authors refer to the need for a significant reduction inassay time and improvement in precision. Others report thedevelopment of a biosensor for progesterone that is basedon a rapid competitive enzyme immunoassay using cova-lently immobilised antibody, horseradish peroxidase as theenzyme label and tetramethylbenzidine as the substrate. Thedynamic range of the assay was from 0.10 to 5.00 ng/mL,and the assay time was 8 min (Claycomb and Delwiche1998; Claycomb et al. 1998). However, the ability to reusethe test wells was very poor (approximately 15–20 cycles)owing to noise from residual enzyme activity.The biosensor assay developed in this study is based on

immobilised progesterone rather than immobilised anti-body, which accounts for the much greater stability of thesensor surface when compared with the above methods.An antiprogesterone MAb was also used in this assay. TheMAb did not appear to cross-react to any significant extentwith the other steroids studied. The precision of the singleanalyte sensor was satisfactory, as measured by its repro-ducibility (CV <13%) and repeatability (CV <9%). Accept-able recovery was measured as part of the assessment ofaccuracy. Coated and blocked electrodes were stable for4 days at 4 °C, with some loss of performance after thistime.

CONCLUSIONS

A sensitive and specific single analyte immunosensor wasdeveloped for the detection of progesterone in bovinemlk. Precision studies for the single analyte sensor were



satisfactory: assessment of reproducibility gave a CV of<13% and repeatability CV <9%. Acceptable recovery wasrecorded during the assessment of accuracy. Coated andblocked electrodes were stable for 4 days at 4 °C, withsome loss of performance after this time.An in-line assay of milk progesterone could contribute

significantly to the improved reproductive management ofdairy herds. It would allow daily monitoring of milk proges-terone concentrations to advance accuracy of heat identifica-tion, early pregnancy diagnosis and detection of ovarianfailure. In addition, the introduction of a small portable bio-sensor technology into the milking parlour would facilitatecontinuous monitoring of milk progesterone and other ana-lytes, which are very important to improve productive andreproductive performance and even milk quality in thefuture.

ACKNOWLEDGEMENTS

The authors are very grateful to the support of HeilongjiangProvince Project (No. GB07B205) and English revision fromInternational Science Editing Compuscript Ltd.

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detection of progesterone in bovine milk using an electrochemical immunosensor

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