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Development of Electrochemical Immunosensor forProgesterone Analysis in MilkMark P. Kreuzer a b , Rafael McCarthy a , Miloslav Pravda a & George G. Guilbault aa Department of Chemistry, Analytical and Biological Chemistry Research Facility,University College Cork, Irelandb Applied Molecular Receptors Group (AMRg), Department of Biological OrganicChemistry, IIQAB‐CSIC, Jordi Girona, 18–26, Barcelona, 08034, SpainPublished online: 22 Aug 2007.

To cite this article: Mark P. Kreuzer , Rafael McCarthy , Miloslav Pravda & George G. Guilbault (2005) Developmentof Electrochemical Immunosensor for Progesterone Analysis in Milk, Analytical Letters, 37:5, 943-956, DOI: 10.1081/AL-120030289

To link to this article: http://dx.doi.org/10.1081/AL-120030289

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CHEMICAL AND BIOSENSORS

Development of ElectrochemicalImmunosensor for Progesterone

Analysis in Milk

Mark P. Kreuzer,* Rafael McCarthy, Miloslav Pravda,

and George G. Guilbault

Department of Chemistry, Analytical and Biological Chemistry Research

Facility, University College Cork, Ireland

ABSTRACT

A disposable electrochemical biosensor, based on a screen-printed carbon

electrode (SPE) coated with a progesterone (prog)–BSA conjugate, has

been prepared and evaluated for measuring progesterone in cows’ milk.

The immunosensor was employed in an indirect competitive assay format

involving anti-progesterone monoclonal antibody and anti-species anti-

body labeled with the enzyme alkaline phosphatase (AP). Differential

pulse voltammetry (DPV) and amperometry were used as electrochemical

means to detect the product of the enzymatic reaction [p-aminophenol

(p-AP)]. Amperometric detection was carried out at þ350 mV vs. Ag/AgCl reference electrode. The DPV detection was in the potential range

of þ100 to þ500 mV vs. Ag/AgCl reference electrode. Progesterone was

943

DOI: 10.1081/AL-120030289 0003-2719 (Print); 1532-236X (Online)

Copyright # 2004 by Marcel Dekker, Inc. www.dekker.com

*Correspondence: Mark P. Kreuzer, Applied Molecular Receptors Group (AMRg),

Department of Biological Organic Chemistry, IIQAB-CSIC, Jordi Girona, 18–26,

Barcelona 08034, Spain; E-mail: mkrqob@iiqab.csic.es.

ANALYTICAL LETTERS

Vol. 37, No. 5, pp. 943–956, 2004

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detectable in milk matrix by sigmoidal curve (variable slope) method

from 13 to 189 ng/mL and from a linear calibration between 16 and

256 ng/mL with an associated limit of detection (LOD) of 3+2 ng/mL

progesterone. The use of DPV improved the accuracy of our measure-

ments over conventional amperometric detection by electrode back-

ground correction. Errors were significantly lower by this method and

conditions. Assays can be performed directly in full fat milk, with a C.V.

of 4% and total analysis time of less than 30 minutes.

Key Words: Progesterone; Milk; Immunosensor; Electrochemistry;

Differential pulse voltammetry; Screen-printed electrode; Estrous.

INTRODUCTION

Milk sample is a complex matrix containing protein and dispersed fat.

Since progesterone is fat-soluble, strong matrix effects may be expected. Thus,

proper sample handling is required through the whole analytical procedure in

order to avoid problems.[1] Progesterone is present in the fat so liquid–liquid

extraction (LLE) has been used with diethylether and evaporated to

dryness.[1,2] HPLC on C18 columns fitted with a UV detector operating at

245 nm is the dominant technique for progesterone.[2,3]

Over the past 10 years various immunoassay formats have been proposed

for progesterone detection in bovine milk such as the BIAcoree biosensor[4]

and Heap et al.[5] developed a radioimmunoassay using H3 labeled progesterone

resulting in a large volume of publications in this area. A rapid enzyme

immunoassay based on HRP with TMB/H2O2 substrate was developed by

Claycomb et al. for progesterone in bovine milk during the milking process.[6]

The assay was linear from 0.2 to 20 ng/mL. A competitive immunochromato-

graphic assay of milk progesterone (Prog) was developed by Laitinen and

Vuento.[7] Their limit of detection (LOD) was 50 ng/mL in a milk sample.

Killard et al.[8] measured biotin in a competitive assay using a SPCE based

immunosensor in an amperometric flow cell. Abad-Villar et al.[9] have shown

the use of flow immunoassay employing planar working electrodes for IgG

labeled alkaline phosphatase systems, with naphthol phosphate as substrate.

Hart et al.[10] and Pemberton et al.[11,12] have shown that an

immunosensor for progesterone could be developed using monoclonal anti-

progesterone antibody in a batch method. Later Pemberton et al.[13]

incorporated a SPCE-based immunosensor for progesterone into a thin-layer

flow cell, to develop a more rapid analysis time with better precision.

4-Aminophenyl phosphate was used as substrate, and a C.V. of 12.5%[13]

observed was better than the 25 to 50% deviation observed in earlier work.[10]

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In this work, we have developed a progesterone immunosensor based on an

indirect competitive format. A secondary antibody labeled with alkaline

phosphatase was used in conjunction with p-aminophenyl phosphate substrate.

The product of the enzyme reaction, p-aminophenol, was detected by

differential pulse voltammetry in a cell consisting of a single drop (100 mL)

placed onto horizontal screen-printed three-electrode strip. The DPV technique

was discovered to give vastly improved results over square wave voltammetry,

or any of the techniques offered on the BAS Instrument. A full report on this

will appear in Ref.[14]. With this technology we envision the possibility of

decentralization from the laboratory environment allowing on-site (farm)

determinations of oestrous. This is under current investigation and will soon be

published. Vast improvement in the precision and accuracy is obtained, C.V. of

4%, with total analysis time (after pre-production of the immunosensors) of less

than 30 minutes. Assays can be performed directly in full fat milk.

EXPERIMENTAL

Reagents

All reagents were of analytical grade or better. Chemicals purchased from

Sigma (Dublin, Ireland) included anti-rat IgG (whole molecule) AP conjugate

(developed from rabbit), bovine serum albumin, butylamine, diethyl ether,

N-ethyl-N0-(3-dimethylaminopropyl) carbodiimide (EDC), monoclonal anti-

progesterone, clone 2H4, potassium chloride, prog (4-pregene-3,20-dione),

prog water-soluble (cyclodextrin-encapsulated progesterone), anhydrous

sodium carbonate, anhydrous magnesium chloride, sodium chloride,

Trizmaw Base (tris[hydroxymethyl]aminoethane), Trizmaw hydrochloride

(tris[hydroxymethyl]aminomethane hydrochloride), Tween-20 (polyoxy

ethylene-sorbitan monolaurate), and zinc chloride. HPLC grade acetonitrile

and methanol were obtained from Lab Scan Analytical Sciences (Dublin,

Ireland). Other chemicals included butan-1-ol Analar grade (BDH chemicals,

Poole, UK), SMA White (SMA Nutrition, Dublin, Ireland), sodium

dichromate dehydrate (Merck, Darmstadt, Germany), and anhydrous sodium

hydrogen phosphate (Fluka, Dublin, Ireland). Milk bought from retailers was

from CMP Dairies (Ireland).

Buffers and Solutions

Carbonate buffer (100 mmol Na2CO3, pH 9.6) was used for primary

protein coating. Phosphate-buffered saline (PBS) contained 50 mmol

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Na2HPO4 þ 100 mmol NaCl. Tris-HCl washing buffer 50 mmol, pH 7.4

included 0.05% (v/v) Tween-20. Freshly prepared blocking buffer consisted

of 3% (w/v) skim milk powder in 50 mmol Tris-HCl (Tris-SMA). Substrate

solution for AP was prepared with 50 mmol Tris-Base, 100 mmol NaCl,

1.0 mmol MgCl2, and 0.1 mmol ZnCl2, adjusted to pH 9.

Incubations at elevated temperatures were carried out in a thermostated

oven supplied by Heraeus Instruments. Centrifugation vials (30,000 MWCO)

were obtained from Millipore (Cork, Ireland). Slide-a-lyzer dialysis membranes

were purchased from Pierce (IL, USA). DEK 247 automatic screen-printer

(Dorset, England), together with a stainless steel mesh screen (400 counts per

inch, mesh orientation of 458) was used for printing the three electrode strips.

The inks used in screen-printing (Electrodagw B-0851, 423-SS and Ag/AgCl,

5% AgCl, Electrodagw 477 SS) were purchased from Acheson (Plymouth, UK).

Electrochemical experiments were performed using the BAS 100B/W electro-

chemical workstation (IN, USA). p-Aminophenyl phosphate was synthesized

in-house with slight variation from the original synthetic route[15] and is now

available from Universal Sensors, Inc. (Kinsale, Ireland).

METHODS

Conjugation of Progesterone to Bovine Serum Albumin

The Prog–BSA conjugate has been prepared as follows. To a 500mL

solution of BSA (7.5 � 1028 mol, 5.1 mg) made in phosphate buffer (pH 5)

were added a 100mL solution of EDC (3.4 � 1025 mol, 11.5 mg) also made in

phosphate buffer (pH 5) and then a 50mL solution of hemisuccinate Prog

(2.25 � 1026 mol, 2.1 mg) made in DMSO. The mixture was allowed to react

at room temperature for 3 hr while being vortexed at low speed. The protein

solution was then dialyzed by syringing into a slide-a-lyzer cassette (MWCO:

10,000), against three exchanges of PBS pH 7.4 for 1 hr each. The conjugates

were retained in the cassette due to their large molecular size.

Immobilization Procedures

The electrochemical immunoassay configuration employed in this paper

involved an indirect method beginning with the immobilization of the Prog–

BSA on the carbon working area of the SPE followed by sequential additions

of antibody. The SPEs were coated individually with 5mL of Prog–BSA

conjugate solution (carbonate buffer, 0.1 M, pH 9.6) using a micro-syringe and

incubated at 378C for 45 min. The SPEs were then washed and allowed to dry

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in air to remove excess moisture. Washing of the electrodes was carried

out in this manner between each step. During initial stages, SPEs were

blocked using 50mL of SMA buffer for 30 min at 378C. This blocking step

was used to ensure that all available sites on the carbon working area

after immobilization of conjugate were “blocked,” thus reducing non-specific

binding of antibody. Rat anti-progesterone monoclonal antibody (rat a-prog

mAb) dilutions were made up in SMA buffer or milk and pipetted onto

coated electrodes (10mL) and incubated at 378C for 45 min. Anti-rat

immunoglobulin (raised in rabbit) labeled with AP (a-rat-IgG-AP) solution

in SMA-buffer (10mL) was subsequently pipetted onto each electrode

and incubated for a further 45 min at 378C. Electrochemical studies were

realized by addition of substrate and measurement of the signal produced

by oxidation of the enzymatic product at the electrode surface poised at

þ350 mV vs. Ag/AgCl reference electrode.

Indirect Competitive Immunoassay for Progesterone in Milk

The immunoassay was optimized in competitive mode by mixing

progesterone in solution with the primary antibody (rat a-prog mAb) followed

by the addition of secondary labeled antibody for signal production. Figure 1

demonstrates the competitive format. Binding of the secondary antibody and

thus AP is indirectly proportional to the amount of free prog in solution. The

biosensor can quantitatively measure the amount of prog that was in the

sample through correlation with enzyme activity monitored electrochemi-

cally. Progesterone standards solutions were prepared as either organic

Figure 1. An indirect competitive immunoassay format for progesterone, whereby

surface bound prog and that free in solution compete for primary antibody. Secondary

labeled species subsequently follows. Theoretical sigmoidal trend expected with this

type of assay set-up.

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(THF) or aqueous solutions (cyclodextrin-encapsulated prog) allowing us to

monitor any effect due to the presence of organic solvent. The advantage of

using cyclodextrin-encapsulated prog is that complete solubility of the prog

is assured. When using organic solvents doubt may be associated with the

partitioning of prog between the aqueous and organic phases. Various

dilutions of the 3 mg/mL stock solutions were made before dilution into buffer

or cow’s milk.

Electrochemical Protocols

Amperometric studies were conducted in a 5 mL batch cell with

constant stirring. Measurements were taken at a potential of þ350 mV,

first in the buffer alone for background reading followed by addition of

50mL of pAPP solution (5 mg/mL). For DPV measurements a single drop

of the sample solution (100mL) was placed to horizontally positioned

SPE ensuring all three electrodes were coated. The electrode was scanned

from þ100 to þ500 mV using conditions optimized by Pravda et al.[14]

Substrate buffer was first measured to establish the background signal.

Substrate concentration used for DPV was 0.5 mg/mL. Readings were

taken at the peak maximum. A comparison of this new single drop

differential pulse voltammetric system will soon be published.[14] Results

with DPV are better than square-wave voltammetry or any other technique

available on the BAS Instrument.

Liquid–Liquid Extraction of Progesterone from Milk

The extraction of prog from milk is a two phase procedure, which firstly

involves the isolation of the milk-fat/lipid fraction that contains the prog

from the aqueous fraction; followed by LLE to isolate the steroids. To

2.5 mL of milk, 0.5 mL of the following mixture was added: 132 mL butan-

1-ol, 420 mL n-butylamine, and 310 mL water. The mixture was vortexed

for a few seconds and then heated to 858C for 1.5 min in a water bath.

After cooling for 1 min at room temperature the mixture was centrifuged

at 6400 rpm. The milk-fat appears as a yellow, oily layer at the top of the

solution that solidifies if let stand at room temperature. The aqueous layer at

the bottom was discarded. Milk-fat extract (0.5 mL) was extracted with

2 mL diethylether twice. Samples were vortexed for 1 min and flash frozen

with acetone/dry ice mixture. The aqueous layer was discarded and ether

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extracts vacuo evaporated. Residue reconstituted with suitable solvent, i.e.,

acetonitrile–water (70 : 30, v/v).

RESULTS AND DISCUSSION

Optimization of Protein Concentrations

The Prog–BSA conjugate was initially optimized using amperometry and

finally by DPV. All results (trends) were similar using the two methods but

higher reproducibility was noted for all cases when DPV was used. Hence

the measurements taken using DPV only are presented. All electrodes were

prepared in triplicate and the immunoassay was carried out according to

the conditions described in section Immobilization Procedures. Two blank

electrodes were prepared in order to ascertain the degree of non-specific

binding of the antibodies to surfaces other than their antigens. Dilutions of

Prog–BSA were in the range 1 : 50–1 : 6250. A blocking step was employed

to ensure specificity in the immunoassay. A high dilution (1 : 50) of the

primary and secondary antibodies was used. An optimum dilution for the

coating conjugate of 1 : 50 was chosen as it gave the maximum electro-

chemical response. At this point we are close to saturation as can be seen from

the Fig. 2(a). The error associated with the assay is quite low (RSDave 3%;

n ¼ 3) and the closeness of fit was quite acceptable (0.9903). Non-specific

binding of primary and secondary antibody signals were low (1.5% and 2.6%,

respectively, of the maximum signal) highlighting strong affinity of antibody

for its antigen and not the blocker.

Rat a-Prog Ab (Primary Antibody)

When the primary antibody dilution was determined using optimized

Prog–BSA, excellent reproducibility was once again observed with DPV

(RSDave 3% over 7 points, n ¼ 3) when compared to amperometry (RSDave

9% over 5 points, n ¼ 3; 0.9191). The graph on this occasion [Fig. 2(b)] did

not reach saturation in the dilution range investigated but the goodness of fit

(0.9909) mirrored that earlier enhancing the use of DPV as method. A dilution

of 1 : 40 of the primary antibody was decided upon yielding a signal of

adequate height (ca. 1.5mA). A higher dilution of 1 : 20 was decided against

for reagent conserving reasons.

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a-Rat IgG-AP (Secondary Antibody)

On this occasion, saturation was again not achieved [Fig. 2(c)] but this is

normal for commercial secondary antibody sources. A dilution of 1 : 40 of

the secondary antibody was decided upon, again giving a quantitative and

adequate signal at this dilution (ca. 2mA).

Figure 2. Optimization studies on screen-printed electrodes with DPV, scanning from

þ100 to þ500 mV vs. Ag/AgCl for (a) prog-BSA coating conjugate, (b) primary

monoclonal antibody (a-prog mAb), and (c) secondary labeled anti-species (IgG-AP).

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Michaelis–Menten Substrate Optimization by Differential

Pulse Voltammetry and Amperometry

The optimization of substrate concentration (Michaelis–Menten) using

DPV took the form of a hyperbola and can be described as a hyperbolic-

dependence on concentration. With enzyme-catalyzed reactions the rate

usually increases linearly with the substrate concentration at low concen-

trations, but then levels off at high concentrations where the enzyme cannot

turnover substrate any quicker. In order for the enzyme to affect the activation

free energy (DG‡), the substrate must bind to the active site. At very low

concentrations of substrate, the active sites of most of the enzyme molecules in

the solution will be unoccupied. Under these conditions, increasing the

substrate concentration will bring more enzyme molecules into play, and the

reaction will speed up. At high concentrations, on the other hand, most of

the enzyme molecules will have their active sites occupied, and the observed

rate will depend only on the rate at which the bound reactants are converted

into products; further increases in the substrate will then have little effect.

Km of 0.5 mg/mL was chosen for DPV measurements using the prog

immunoassay. Similarly a concentration of 5 mg/mL was established as the

optimum substrate concentration for amperometric analysis (data not shown).

Determination of Working Range and Limit of Detection

In order to determine the working range and thus, a calibration curve,

solutions of milk and SMA buffer containing known concentrations of prog

covering the range 500–0.0 ng/mL were prepared. The prog immunoassay

was carried out in an indirect competitive mode. Blank control containing

no free prog was included in each assay. The response to this blank was

labeled “max” as it should show maximum signal height. Calibration plots

were constructed using GraphPad Prismw using the sigmoidal dose-response

(variable slope) parameters. A typical competitive response is shown in

Fig. 3.

The results from four such calibration immunoassays in full fat milk

matrix, including parameters such as EC50, Hill slope, LOD, and working

range, are depicted in Table 1. It can be seen from this table that all parameters

were determined with good precision. The EC50 value of 55+7 ng/mL (RSD

4% when n ¼ 3) and goodness of fit of 0.987+0.003 (RSD 0.3%, n ¼ 4) show

highly reproducible results. The overall C.V. (Fig. 4) is 4% and the LOD is

3+2 ng/mL.

In order to accurately determine the linear portion of the range and thus a

calibration curve based on the indirect relationship between signal and prog

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concentration, assays were set-up. A typical result can be seen in Fig. 4. The

line is represented by the equation [y ¼ 20.233ln(x)þ2.094] with an

associated R2 value of 0.9951 (N ¼ 5). The linear portion ranges from 16 and

256 ng/mL with an average RSD of all 5 points (n ¼ 3) determined as 5% (P

value 0.0001). Repetition of such an assay within the same range returned a R2

value of 0.9944 (N ¼ 5).

Figure 3. A typical sigmoidal curve for prog in milk matrix on screen-printed

electrodes using DPV as detection mode (n ¼ 3). Range: þ100 to þ500 mV vs. Ag/AgCl. Trend is similar to theoretical highlighted in Fig. 1. Max control (zero) is that

value returned when no prog competitor is present.

Table 1. Comparison of four indirect competitive assays for prog in milk matrix on

screen-printed electrodes with DPV with essential parameters listed (sigmoidal curve

data).

Assay EC50a Hill Slope R2 LOD� LR1� LR2�

1 44.3 21.007 0.984 2.0 8.5 167.0

2 58.5 21.068 0.984 5.6 17.0 199.9

3 56.4 20.626 0.991 0.6 6.8 192.7

4 60.7 21.122 0.988 5.1 18.7 195.9

Average 55 20.9556 0.987 3 13 189

SD+ 7 0.2250 0.003 2 6 15

aProgestersone concentration (ng/mL).

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For the development of the prog immunosensor certain criteria were set

out at the beginning. Initially the sensor had to work in a concentration range

from 5 to 25 ng/mL in milk matrix. This was an issue that we feel has largely

been achieved with the optimized sensor operating in ranges from 13 to

189 ng/mL for the sigmoidal curves (variable slope) and 16–256 ng/mL for

the linear dependence with excellent goodness of fit for both methods.

Although sensitivity of the assay does not reach 5 ng/mL required for estrous

detection, the immunosensor developed is not far off that target and still could

operate as an estrous detection sensor. The sensor has an associated LOD of

3+2 ng/mL prog. A few changes in the assay configuration could measure

lower concentrations of analyte. Reduction of the amount of antibody bound

to the surface is one such option without compromising the absolute signal, of

course.

CONCLUSIONS

Table 2 shows a comparison of our method over the work of Pemberton

et al.[11,12] In essence our sensor works with a larger range than either

work,[11,13] but both cases our DPSV results show a much more concise and

lower error (4% compared to 12.5–50%, Pemberton et al.[11,13] considering

we are also measuring in whole milk). Vast improvement in the precision and

Figure 4. Linear calibration curve in milk matrix using DPV and screen-printed

electrodes for prog (n ¼ 3, R2 ¼ 0.9951). Range: þ100 to þ500 mV vs. Ag/AgCl.

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accuracy is obtained using DPV. The DPV technique was discovered to give

vastly improved results over square-wave voltammetry, or any of the

techniques offered on the BAS Instrument. The use of DPV has increased the

reproducibility of our method whereby it allows us to baseline correct our

overall signal and thus eliminate problems that would in other ways contribute

to increased error by individual electrodes. A full report on this will appear in

Ref.[14]. With this technology from the laboratory environment allowing on-

site (farm and veterinary) determinations of oestrous. This is under current

investigation and will soon be published.

The use of disposible screen-printed electrodes means that the system is

much less expensive than other immunoassays currently used incorporating

techniques like SPR.[4] The immunosensor works in full fat milk, with no

pre-treatment other than pH adjustment. Thus the method is fast (less than

30 minutes).

ACKNOWLEDGMENTS

The authors would like to thank the Higher Education Authority of

Ireland (HEA) for generously funding the project under the ABCRF program

and to MK for his swift and thorough drafting of the manuscript.

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Accepted February 5, 2004

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