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Determination of Estradiol by Biotin‐Avidin‐Amplified Electrochemical Enzyme

Immunoassay

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DOI: 10.1080/00032710701242089

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Determination of Estradiol by Biotin-Avidin-Amplified
Electrochemical Enzyme Immunoassay
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Analytical Letters, 40: 887–896, 2007

Copyright # Taylor & Francis Group, LLC
ISSN 0003-2719 print/1532-236X online
DOI: 10.1080/00032710701242089

CHEMICAL & BIOSENSORS

Determination of Estradiol by
Biotin-Avidin-Amplified Electrochemical
Enzyme Immunoassay

Shuhao Wang
College of Environmental Science and Engineering, Donghua University,
Shanghai, P. R. China and Institute of Chemistry and Chemical
Engineering, Liaocheng University, Liaocheng, P. R. China

Huisheng Zhuang
College of Environmental Science and Engineering, Donghua University,
Shanghai, P. R. China

Lingyun Du, Shilei Lin, and Chuantao Wang

Institute of Chemistry and Chemical Engineering, Liaocheng University,
Liaocheng, P. R. China

Abstract: A simple, sensitive biotin-avidin-amplified electrochemical enzyme-linked

immunosorbent assay (ELISA) for the determination of estradiol (E2) was proposed in
this paper. The complex of biotinylated anti-E2 antibody and horseradish peroxidase-
labeled avidin (HRP-avidin) were regarded as a probe in this system. The activity of
labeled enzyme was measured with electrochemical methods using o-phenylenedia-
mine as substrate. Coupled with the plate-coated antigen, indirect ELISA format
using E2-ovalbumin, the electrochemical detection was performed for E2 with the
detection limit of 21.0 pg/ml, and the linear range of determination of
50.0– 500.0 pg/ml. The proposed method has been used for the determination of E2

Received 26 July 2006; accepted 1 November 2006

We were grateful for the financial support of the National Natural Science Foun-
dation of China (No. 20277005) Doctor Discipline Scientific Research Foundation of
China University and Natural Science Foundation of Shandong Province, P. R.
China (No. 2005ZX12).
Address correspondence to Huisheng Zhuang, College of Environmental Science
and Engineering, Donghua University, Shanghai 201620, P. R. China. E-mail:
hszhuang@dhu.edu.cn

887
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888 S. Wang et al.

in river water with satisfactory results. Compared with the traditionally spectrophoto-
metric ELISA detection, this method shows greatly heightened sensitivity. The limit of
detection improved by about two orders of magnitude, which is very suitable for the
conditions with extremely low concentration of analyte or very small volumes of
sample present.

Keywords: Estradiol, horseradish peroxidase, o-phenylenediamine, ELISA,

voltammetry

INTRODUCTION

There has lately been a growing interest in chemicals that might be disrupting
the endocrine system of humans and wildlife. A variety of chemicals are
known to disrupt the endocrine systems of animals in laboratory studies.
The endocrine systems of certain wildlife have been affected by chemical con-
taminants (Colborn et al. 1993; McLachlan and Arnold 1996). The female
hormone (estrogen) such as 17b-estradiol, estrone, ethynyl estradiol, and
estriol is considered to be one of the endocrine-disrupting chemicals.
Estrogen in the environment is derived from the excreta of humans and
livestock, medicines, and so on. Since 17b-estradiol has the greatest estro-
genic activity and seems to have been discharged into the environment in
large quantities, this chemical has been monitored in an endocrine disruptor
survey. So it is important to establish a sensitive method for the detection
of E2 in the environment.
The number of analytical methods currently available for the determi-
nation of estrogens in water is limited because of low estrogens. Enzyme-
linked immunosorbent assay (ELISA) (Aherne et al. 1985; Shore et al.
1993; Valentini et al. 2002) and chromatographic techniques (Desbrow
et al. 1998; Belfroid et al. 1999; Ternes et al. 1999; Kuch et al. 2000; Ishii
et al. 2000) have been used for the analysis of estrogens in environmental
samples after sample preparation. But the chromatographic techniques for
the analysis of estrogens are time- consuming and require long preparation
steps as well as suitable volatile derivatives. The first official biological
technique employed for the assay of 17b-estradiol was radioimmunoassay
(RIA). This method allows rapid, sensitive, and inexpensive screening of a
large number of samples. However, the major disadvantage of this conven-
tional technique is that it requires radioisotopes and scintillation fluids. A
large number of different immunological non-isotopic screening methods,
based on colorimetric, fluorimetric, and chemiluminescent detection, have
been reported (El 1991; Bouve et al. 1992).
In recent years, electrochemical methods have also been successfully
combined with the immunoassay and the electrochemical immunoassay
(ECIA) has got great developments because of the simplicity and sensitivity
of electrochemical analysis (Jiao et al. 1995; Warsinke et al. 2000). In
principle, ECIA can be performed in direct and indirect formats. The direct
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Estradiol Determination by Enzyme Immunoassay 889

ECIAs use changes in charge densities or conductivities for transduction and

do not need any auxiliary reaction. Some electrochemical methods such as
potentiometry, capacitory or amperometry have been used to indicate the
antigen-antibody reaction directly. In indirect ECIAs, the binding reaction is
indicated via an auxiliary reaction with a labeling compound. Enzyme is
commonly used as the label substance, which is often labeled with antigen
(or antibody). It can catalyze the reaction of substrate and generate an
electro-active product, which can be detected by different electrochemical
methods. Because the signal can be amplified by the catalytic effect of
labeled-enzyme, the sensitivity can be greatly improved and the detection
limits of atto-mole level for some antigens can be achieved. Horseradish per-
oxidase (HRP) is the most commonly used labeling enzyme for immunoas-
says. It is also widely used in the electrochemical enzyme immunoassay
(Thompson et al. 1991; Niwa et al. 1993; Kaneki et al. 1994; Jiang et al.
1995; Zhang et al. 1999a –d; Jiao et al. 2000).
o-Phenylenediamine (OPD) is widely used as HRP substrate with spectro-
photometry (Porstmann and Porstmann 1988) and fluorometry (Mekler and
Bystryak 1992). Jiao et al. reported the electrochemical immunoassay for
determination of virus by using of OPD (Jiao et al. 1998; Sun et al. 2000;
2001).
In this work, the biotinylated anti-E2 antibody and horseradish peroxi-
dase-labelled avidin (HRP-avidin) were regarded as a probe, OPD was HRP
substrate in enzyme immunoassay (EIA) and voltammetric analyzer was
used as detector. A sensitive biotin-avidin-amplified electrochemical
enzyme immunoassay system was developed and applied to determining E2
in environmental water.

EXPERIMENTAL

Apparatus

All voltammetric determinations were performed on a model DS-2005

voltammetric analyzer (Jining Dongsheng Electric Apparatus Factory, P. R.
China), with a hanging mercury electrode as working electrode, a platinum-
wire auxiliary electrode, and an Ag/AgCl reference electrode. The commer-
cial 96-well polystyrene ELISA plates were purchased from Beijing
Dingguo Biotech Co.

Reagents

Estradiol, estrone, progesterone, bovine serum album (BSA), and ovalbumin

(OVA) were purchased from Sigma Chemicals Co. (St. Louis, MO). HRP
was purchased from Xueman Biochemical Technique Co., Ltd., Shanghai,
P. R. China (250 units per mg enzyme, RZ . 3.0), 1.0 mg/ml solution of
Downloaded By: [Ohio University] At: 13:42 27 April 2007

890 S. Wang et al.

HRP was prepared by dissolving 0.0500 g of HRP in 50 ml water, and

stored at 48C; 1.0
1022 mol/l o-phenylenediamine (Sigma Chemical Co.)
stock solution was prepared by dissolving 0.1081 g o-phenylenediamine
(OPD) in 100 ml methanol; 1.0
1023 mol/l H2O2 was prepared just
before use; Avidin-HRP was obtained from Beijing Biodee Biotech Co.,
Ltd. (P. R. China) and the working concentration of avidin-HRP provided
was 1:1000.

Buffers and Substrate

The buffer for ELISA was prepared as follows: coating buffer: 0.10 mol/l
sodium carbonate, pH 9.6; phosphate buffer (PBS, 0.2 mol/l phosphate
buffer, 137 mmol/l NaCl, 2.7 mmol/l KCl, pH 7.4); blocking and dilution
buffer: PBS containing 0.2% OVA; rinsing buffer (PBST): PBS containing
0.05% Tween-20. The substrate solution was prepared just before the measure-
ment, which is composed of 1.0
1023 mol/l OPD and 4.0
1024 mol/l
H2O2 in 0.01 mol/l HOAc-NaOAc buffer solution (pH 4.8). All chemicals
used were of analytical reagent grade and doubly deionized water was used
throughout.

Preparation of Antibody

The antibody was rabbit polyclonal antibody; it was prepared by immuniz-

ation with E2-3-CMO-BSA and purified by a modified caprylic acid-
saturated ammonium sulfate method (Tang et al. 1999).

Hapten Synthesis

The immune system does not recognize relatively small molecules, so we have to
use a trick to produce antibody with binding sites that are specific for estradiol. E2
is a small hapten with a molecular weight of 272.4 Da. To increase its immuno-
genicity, E2 was linked to BSA. Since E2 does not have any usual conjugation
groups such as carboxyl or amino groups, a two-step strategy was used to link
this molecule to BSA. The first step is to prepare estradiol-3-carboxymethyl
ether (E2-3-CME) (Dhar et al. 1988). The second step is a reaction during
which amino groups of BSA were linked to E2 through the carboxyl group.
The reaction procedure was showed in the following paragraph.

Preparation of Protein-Hapten Conjugate

Hapten used in this study was conjugated to protein via its carboxylic group by
the N-hydroxysuccinimide active ester method (Langone and Van 1975).
Downloaded By: [Ohio University] At: 13:42 27 April 2007

Estradiol Determination by Enzyme Immunoassay 891

The hapten was conjugated to BSA for immunogen and to OVA for
coating conjugate preparation. Briefly, 6.6 mg of E2-CME was dissolved
with 2.9 mg NHS and 5.2 mg DCC in a total volume of 1.0 ml of dry dimethyl-
sulfoxide (DMSO). This mixture was stirred gently about 2 h at room temp-
erature. Then the activated NHS ester was slowly added to 5 ml of
0.128 mol/l BSA or OVA in 130 mmol/l NaHCO3 under stirring. The
coupling reaction was allowed to continue for 2 h. The mixture was then
purified on a Sephadex G-25 column using 100 mmol/l phosphate buffer,
pH 7.4, and stored at 48C until use.

Preparation of Biotinylated Antibodies

Lyophilized antibodies were reconstituted in distilled water and then mixed

with 1.0 mg of biotin-N-hydroxysuccinimide ester in 0.5 ml of PBS, pH 7.2.
The reaction mixture was gently shaken at room temperature for 3 – 4 h and
then dialysed against PBS, pH 7.2 for 2 days.

Electrochemical EIA Procedure

The plate-coated antigen format, indirect ELISA, was performed as follows:

100 ml of the E2-OVA in coating buffer was applied to the microtiter plate
overnight at 48C. After washing three times with washing buffer, 150 ml
blocking buffer was added to each well, and incubated for 2 h at 378C in
order to saturate the attachment sites on the ELISA plate, after that washed
for further test. E2 was added to the limited biotinylated antibody (biotin-Ab)
solution and incubated for 1 h at 378C, then 100 ml of the mixed solution of
E2 and biotin-Ab was added to each well and incubated for 1 h at 378C.
Washing as before, the plate was incubated with avidin-HRP in dilution
buffer (100 ml) for 0.5 h at 378C. After washing three times, 150 ml of
substrate solution was added to each well and the plates were incubated in
the dark at 378C. After 30 min reaction 30 ml of 2.0 mol/l NaOH was added
to every well to stop the enzymatic reaction. The miniature three-electrode
system of DS-2005 voltammetric analyser was directly inserted into the well
of the ELISA plate and the second order derivative linear-sweep voltammetric
peak current of the product DAP at 0.93 V (vs. Ag/AgCl) was measured.

RESULTS AND DISCUSSION

Optimal Conditions for Enzyme-Catalysis and Voltammetric

Detection

The catalytic oxidization reaction of OPD and H2O2 by HRP has been
reported (Mekler et al. 1992; Jiao et al. 1998), the product of this reaction is
Downloaded By: [Ohio University] At: 13:42 27 April 2007

892 S. Wang et al.

2,3-diaminophenazine (DAP), and DAP has easily undergone the reductive

reaction. A stable voltammetric reduction peak of DAP was observed in
pH 4.8 of 0.10 mol/l HOAc-NaOAc buffer solution, so pH 4.8 was chosen for
this enzyme-catalyzed reaction. The reaction temperature is also studied and
at 378C HRP shows the greatest activity, which is in accordance to the nature
of enzyme. At this temperature, the enzymatic reaction equilibrium can
be achieved in 30 min. So the optimal reaction time was selected as 30 min.
The peak current was stable and maximum when pH of the supporting electrolyte
is in the range of 8.0–10.0 alkaline solution. So 2.0 mol/l NaOH was added to
the reaction solution both to stop the enzyme-catalyzed reaction and adjust the
pH of the supporting electrolyte for subsequent voltammetric detection.
The apparatus conditions of DS-2005 voltammetric analyser were chosen
as follows: the scanning rate, 300 mV/s; the dropping mercury standing time,
10 s; and the initial potential, 20.50 V.

Optimization of Reagents for Electrochemical EIA

The optimization of concentrations of biotinylated antibody and immobilized

antigen is very important to enhance the sensitivity and extend the working
range for immunoassay. Therefore, these concentrations were always
optimized firstly. When the dilution of avidin-HRP is 1:1000, dilution
curves of biotin-anti-E2 at different concentrations of coating conjugate
were shown in Fig. 1. The test indicated the optimal concentration of the

Figure 1. Dilution curves of Biotin-anti-E2 at different concentrations of coating conju-

gate C0 was the primary mass concentration of Biotin-anti-E2, which was 80 mg . ml21;
C was the concentration of serially diluted Biotin-anti-E2. The concentration of coated
E2-OVA: * 40 mg . ml21, O 20 mg . ml21, B 10 mg . ml21, V 5 mg . ml21.
Downloaded By: [Ohio University] At: 13:42 27 April 2007

Estradiol Determination by Enzyme Immunoassay 893

coated E2-OVA, and biotinylated antibody used in the biotin-avidin electro-

chemical ELISA were 40.0 mg/ml, and 20.0 mg/ml, respectively.

Determination of E2 by Electrochemical EIA

Plate-coated antigen-ELISA is used because it has the following advantages: it

is relatively simple, requires only a small amount of specific antiserum, allows
the use of commercially prepared enzyme-labeled antibody, and unfractio-
nated specific antisera can be used. The primary procedure of this indirect
ELISA format is shown in Fig. 2.
During this procedure, a high concentration of OVA was used in blocking
buffer to prevent non-specific binding. Otherwise, unoccupied sites of the plates
may adsorb other components such as biotinylated antibody and avidin-HRP
during the subsequent steps, which may cause unnecessary background.
Under the optimum conditions, the calibration curve of E2 with voltam-
metric detection is constructed and shown in Fig. 3. The linear range of the
purified E2 determination is 50.0– 500.0 pg/ml and the equation of linear

Figure 2. Procedures of electrochemical EIA with biotinylated antibody.

Figure 3. Calibration curves of the enzyme immunoassay for E2.

Downloaded By: [Ohio University] At: 13:42 27 April 2007

894 S. Wang et al.

regression equation is DI ¼ 3.384C þ 669.4, R2 ¼ 0.9975 (C is concentration

of E2). The detection limit is 21.0 pg/ml. The relative standard deviation for
11 parallel determinations with 200.0 pg/ml E2 is 7.3%.
Compared with the spectrophotometric ELISA method using OPD as
substrate was also performed with the following result: the detection limit is
2.0 ng/ml and the linear range is 6.0– 120.0 ng/ml. So this electrochemical
method gives about 100-fold increase in sensitivity than the traditional spec-
trophotometric method.

Specificity for Assay

Specificity of anti-E2 antibody is defined as the ratio of antigen concentration

to cross-reactant concentration at 50% inhibition of maximum binding. The
cross-reactivity date is as follows: 17b-estradiol 100%, 17a-estradiol 5%,
estrone 5%, estriol 2%, and progesterone 0.1%.

Sample Analysis

Samples were collected in pre-cleaned amber glass bottles, and the samples
were filtered through 0.45 mm HVLP filters (Millipore Bedford, MA) to
eliminate particulate matter and other suspended solid matter.
No significant matrix effect was observed when the ECIA was applied to
measure the spiked river water samples. The recovery results were shown in
Table 1. Compared with the results from CLIA (the routine CLIA kit),
these results confirmed that the ECIA with biotin-avidin amplification
system showed good performances in both the feasibility and recovery of
the assay.

CONCLUSION

With biotin-avidin amplification system, a simple, sensitive electrochemical

ELISA for the determination of E2 was proposed. The activity of labeled

Table 1. The results of determination of E2 in river water (n ¼ 6)

Added Determined Recovery CLIA

Sample (pg/ml) (pg/ml) (%) (pg/ml)

1 200.0 189.6 94.8 196.0

400.0 416.8 104.2 392.3
2 200.0 213.5 106.8 206.8
400.0 436.0 109.0 425.2
3 200.0 223.4 111.7 234.1
400.0 385.3 96.3 418.6
Downloaded By: [Ohio University] At: 13:42 27 April 2007

Estradiol Determination by Enzyme Immunoassay 895

enzyme was measured with electrochemical methods using o-phenylenedia-

mine as substrate. Coupled with the plate-coated antigen indirect ELISA
format using E2-OVA, the electrochemical detection was performed for E2
with the detection limit of 21.0 pg/ml, and the linear range of determination
of E2 was 50.0– 500.0 pg/ml. Compared with the traditionally spectrophoto-
metric detection, this system shows greatly heightened sensitivity. The limit of
detection improved by about two orders of magnitude, which is very suitable
for the conditions with extremely low concentration of analyte or very small
volumes of sample present.

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