A sensitive immunoassay for parathion based on covalent linkage between small molecules hapten microtiter plates surface

Article abstract of DOI:10.1007/s13738-016-1009-5

A sensitive competitive inhibition enzyme-linked immunosorbent assay (CIELISA) for the detection of parathion has been developed. In this assay, a small molecule hapten II (O,O-diethyl O-4-aminophenyl phosphorothioate) was covalent linked to glutaric dialdehyde treated-microtiter plates. In addition, 4-(ethoxy(4-nitrophenoxy) phosphorothioylamino) butanoic acid-ovalbumin (hapten I–OVA) conjugate served as the coating antigen for comparison with directly hapten II covalent linked plates in the CIELISA format. The developed assay demonstrated highly sensitivity (IC₁₀ was 0.08?ng?mL^?1) selectivity and stability. In samples analysis, the results of parathion detected by this assay were in accordance with which obtained by high-performance liquid chromatography.

Full text of DOI:10.1007/s13738-016-1009-5

J IRAN CHEM SOC
DOI 10.1007/s13738-016-1009-5
A sensitive immunoassay for parathion based on covalent linkage
between small molecules hapten microtiter plates surface
1
1
1
1
1
Na Sai · Wenjing Sun · Yuntang Wu · Zhong Sun · Guowei Huang
Received: 3 June 2016 / Accepted: 5 November 2016
©
Iranian Chemical Society 2016
Abstract A sensitive competitive inhibition enzyme-linked
immunosorbent assay (CIELISA) for the detection of para-
thion has been developed. In this assay, a small molecule
hapten II (O,O-diethyl O-4-aminophenyl phosphorothioate)
was covalent linked to glutaric dialdehyde treated-microti-
ter plates. In addition, 4-(ethoxy(4-nitrophenoxy) phospho-
rothioylamino) butanoic acid-ovalbumin (hapten I–OVA)
conjugate served as the coating antigen for comparison
with directly hapten II covalent linked plates in the CIEL-
ISA format. The developed assay demonstrated highly sen-
HPLC
MWs
MS
NMR
NHS
OP
High-performance liquid chromatography
Molecular weights
Mass spectrum
Nuclear magnetic resonance
N-hydroxysuccinimide
Organophosphates
PBS
Phosphate-buffered saline
SM-hapten Small molecule hapten
LOD
TLC
OVA
EDC
The limit of detection
Thin-layer chromatography
6-Aminocaproic acid, ovalbumin
1-ethyl-3-(3-dimethylaminopropyl) carbodi-
imide hydrochloride
−1
sitivity (IC10 was 0.08 ng mL ) selectivity and stability. In
samples analysis, the results of parathion detected by this
assay were in accordance with which obtained by high-per-
formance liquid chromatography.
APTES
3-Aminopropyltriethoxysilane
Keywords Competive inhibition enzyme-linked
immunosorbent assay · Covalent linkage · Parathion ·
Small molecule hapten
Introduction
The pesticides contaminants in foods and environment are
great concern for the public and regulatory due to their
chemical hazards to human health [1]. In China, about 1
million tons of various pesticides were used per year, and a
quarter of them were organophosphates (OP) chemicals [2].
Parathion belongs to the OP pesticides and has the potential
to be involved in the activities of cholinesterase and disrupt
the nervous system of humans, resulting in convulsions,
poor vision, tremor, dyspnea, lung oedema as well as res-
piratory arrest [3, 4]. More critically, parathion is associ-
ated with an increasing risk of cancer in malignant transfor-
mation of some cells through altering genomic instability
[5–7]. Parathion was highly toxic (estimated median lethal
Abbreviations
CIELISA
Competitive inhibition enzyme-linked immu-
nosorbent assay
CR
GD
GM
GC
Cross-reactivity
Glutaric dialdehyde
Gas chromatography-mass
Gas chromatography
*
Guowei Huang
haungguowei@tmu.edu.cn
Na Sai
70979794@qq.com
−1
4
dose of 11.68 to 7.21 mg kg ), producing dose-depend-
ent inhibition of brain and plasma cholinesterase activity,
hyperglycemia and elevated plasma corticosterone con-
centration [8, 9]. Several organizations such as the United
1
Department of Nutrition and Food Hygiene, School of Public
Health, Tianjin Medical University, Tianjin 300070,
People’s Republic of China
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J IRAN CHEM SOC
States Environmental Protection Agency list parathion as a
possible human carcinogen [10, 11].
modified by a mixture of HNO –H SO and 3-aminopro-
3
2
4
pyltriethoxysilane (APTES) for directly linking bisphenol
A, atrazine and 2,4-dichlorophenoxyacetic acid [36, 38].
However, such method has potential risk on the operators
due to using strong acids. In addition, the small peptide
haptens were covalent attached to the plate surface treated
with ϒ-radiation activation [39]. The g-irradiated was
also used to modify the plate surfaces for directly binding
p-aminophenylalanine [40]. But, these methods are time-
consuming and difficult to control. A facile method was
later developed by treating the plate with a polymerized
glutaric dialdehyde (GD) to directly immobilize macromol-
ecules [42] and O-pinacolyl methylphosphonic acid [43].
Herein, a highly sensitive CIELISA was developed for
the detection of parathion based on a covalent linkage
between the SM-hapten II (O,O-diethyl O-4-aminophenyl
phosphorothioate) and microtiter plate surface. In this
assay, the plate surface was treated with GD network to
introduce aldehyde group that can cross-link with amino
group of hapten II. As a comparison, the other parathion
derivative (Hapten I) OVA conjugate (4-(ethoxy(4-nitro-
phenoxy) phosphorothioylamino) butanoic acid-ovalbu-
min (hapten I-OVA) conjugate) was prepared and coated
on the plate surface by hydrophobic interactions. After
optimization of reaction conditions, the developed assays
were further applied in food/water matrices including
corn, rice, cucumber and drinking water. Furthermore,
the detected amounts of parathion were also confirmed by
HPLC.
Due to its high chemical stability and persistence, para-
thion and their oxidation products can not only remain in
crops, but also disperse into the environment (water, soil
and so on), eventually transmitting into the food chain
through the bio-accumulation [12, 13]. The hazards of
parathion contaminants to human health are incontestable
[
14–16]. Therefore, it is still necessary to regular monitor
parathion residues in foods and environment. There have
been routine methods for monitoring parathion, including
gas chromatography-mass (GM) [17, 18], gas chromatog-
raphy (GC) [19–21], and high-performance liquid chroma-
tography (HPLC) [22, 23]. However, they usually involve
sophisticated equipment and a large number of separate
analytical procedures, resulting in more-complex, time-
consuming and laborious screening procedures. In addi-
tion, new detection assays for parathion have been devel-
oped recently, such as acetylcholinesterase biosensor [24],
molecule imprinted polymeric sensor [25], electrochemical
sensor [26], fluorescence probing strategy [27], nanoporous
gold/MWCNTs electrode sensor [28] and so on. Unfortu-
nately, they still required complicated fabrication processes
and special instruments. Alternatively, enzyme-linked
immunosorbent assays (ELISAs) is recognized as a valu-
able tool in pesticide residual analysis and a complements
conventional analytical method due to its cheap, rapid, sen-
sitive and selective characteristics [29–31].
Many competitive inhibition enzyme-linked immuno-
sorbent assays (CIELISAs) have been developed for moni-
toring OP pesticides to date [32–35]. In these CIELISAs,
the small molecule hapten (SM-hapten) should be conju-
gated with carrier protein when immobilized on the sur-
face of microtiter plates because SM-haptens do not have
multiepitopes. However, this approach has many disad-
vantages for some purposes. Firstly, the formation of SM-
hapten-protein conjugate is a time-consuming process and
has poor reproducibility, which is unfavourable for assay
standardization [36, 37]. Secondly, immobilization pro-
cess of SM-hapten-protein conjugates on the PS surface
depends on hydrophobic interactions between SM-hapten-
protein conjugate and the surface of plates. This interac-
tion often contains significant conformational changes
which lead carrier protein easily to screen SM-hapten, thus
resulting in unsuitable presentation and orientation of SM-
hapten [38].
Experimental
Materials
Parathion, methyl parathion, atrazine, monocrotophos,
chlorpyrifos, diazinon, n-hydroxysuccinimide (NHS),
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDC), 6-aminocaproic acid, ovalbumin (OVA)
and goat anti-rabbit IgG-HRP conjugate secondary anti-
body were purchased from Sigma-Aldrich (USA). The
rabbit polyclonal antibody against parathion was obtained
from Shanghai Biotechnology Institute (Shanghai, China).
Common solvents (including GD) and salts were obtained
from Tianjin Regent Corporation (Tianjin, China). The
reaction progress was monitored by thin-layer chromatog-
raphy (TLC) using GF254 silica gel on glass plates. The
water used was prepared by a Milli-Q system (Millipore,
Bedford, MA). Coating buffer was 0.05 M carbonate buffer
(pH 9.0). Washing buffer (PBST) was 0.01 M phosphate-
buffered saline (PBS) with 0.05% Tween 20 (pH 8.5). The
96 wells ELISA microtiter plates (medium binding) were
purchased from Wanger Biotech Co. (Beijing, China).
For avoiding these drawbacks, the direct attachment of
SM-hapten to the surface of microtiter plates would be an
interesting approach [39, 40]. The surface of plates can
be pre-activated and modified with different functional
groups, such as hydroxyl, amino, carbonyl and carboxyl;
these functional groups could link SM-hapten through
covalent linkage [41]. The polystyrene surface has been
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J IRAN CHEM SOC
1
The H nuclear magnetic resonance (NMR) spectrum of
steps. Ethyl dichlorothiophosphate (2.5 g), K CO (10 g),
2 3
hapten II was obtained with an AVANCE DMX 500 spec-
trometer (Bruke, Berlin, Germany). The hapten I–OVA
conjugates were determined by Reflex III MALDI-TOF-
Mass Spectrum (Bruker Inc., Germany). A Multiskan MK3
ELISA reader (Thermo, USA) was applied for the deter-
mination of absorbance. 6930 centrifuge (KUBOTA Inc.,
Japan) and MS3 digital orbital shaker (IKA Inc., Germany)
were used for the preparation of hapten I, hapten I–OVA
conjugate and hapten II. HPLC system with coupled with
UV–Vis UV-detector (Waters, USA) was employed for
detection of drinking water samples.
4-nitrophenol (1.5 g) were mixed thoroughly in 10 mL
of acetonitrile. After stirring for 1 h at 37 °C, the mixture
was filtered by Celite, and the solvent was removed under
reduced pressure. The obtained solid (250 mg) was dis-
solved in 2 mL of cooled MeOH and mixed with a mixture
of KOH (125 mg) and 4-aminobutyric acid in 1.5 mL of
MeOH. After stirring for 10 min, the reaction mixture was
filtered and extracted with 1 M HCl-chloroform. After dry-
ing over anhydrous sodium sulphate, hapten I was obtained.
1
H NMR (CDCl ): δ = 8.23 (d, J = 8.8, ArH, 2H), 7.37
3
(d, J = 8.0, ArH, 2H), 4.21 (m, CH OP, 2H), 3.40 (m, NH,
2
1
H), 3.18 (m,CH2NH, 2H), 2.47 (m,CH COOH, 2H), 1.87
2
Haptens syntheses
(m, CH CH CH , 2H), 1.38 (t, J = 6.8, CH3, 3H).
2
2
2
O,O-diethyl O-4-aminophenyl phosphorothioate (hap-
ten II) was synthesized by the following procedures. O,O-
diethyl-O(4-nitrophenyl) thiophosphate (4 g) was dis-
solved in ethyl ether (40 mL) and extracted with 1% cold
Na CO solution (4 × 10 mL). After removing the water
The synthetic routes for hapten I and hapten II are illus-
trated in Fig. 1. Two approaches were utilized in the hap-
tens syntheses: (1) use of a spacer attached to a carboxyl
group of parathion, (2) displacement of a nitro-group of
parathion by amino group. Their syntheses procedures
were described as follows.
2
3
layer, 20 mL of acetic acid/hydrochloric acid mixture (9:1,
V/V) and 4 g of zinc powder were added, then stirred and
reflowed for 45 min. After purification, an orange-red oily
hapten II was obtained.
4-(ethoxy (4-nitrophenoxy) phosphorothioylamino)
butanoic acid (hapten I) was prepared by the following
Fig. 1 Synthesis of hapten I, hapten I-OVA conjugate and hapten II.
a 4-(ethoxy (4-nitrophenoxy) phosphorothioylamino) butanoic acid
I was then conjugated with OVA to form hapten I-OVA conjugate. b
O,O-diethyl O-4-aminophenyl phosphorothioate (hapten II). A nitro-
group of parathion was displaced by amino group to form hapten II
(
hapten I) and hapten I-OVA conjugate. A spacer was attached to a
carboxyl group of parathion to form hapten I, and the obtained hapten
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J IRAN CHEM SOC
Hapten I–OVA conjugate preparation
Analysis of data
Hapten I–OVA conjugate was prepared using active ester
method [44, 45]. Hapten I–OVA conjugate was characterized
by mass spectrum (MS). Assuming that the molar absorptiv-
ity of hapten was the same for the conjugated and free forms
Experimental wells were blanked against wells in which
the rabbit polyclonal antibody against parathion was omit-
ted. Data analysis was performed as described elsewhere
[37–39]. The absorbance was recorded at 490 nm, and data
analysis was performed by normalizing the absorbance
[36, 41], the identification of hapten, i.e. coupling ratios (the
number of hapten molecules per molecule of protein carrier)
of the conjugate could be approximately estimated directly
by molecular weights (MWs) shown in Eq. (1):
using the absorbance based on %B/B value.
0
%
B/B = (A − A )/(A − A ) × 100
0 ex 0 ex
(2)
where A: absorbance of parathion at standard concentra-
Coupling ratio = (MWsConjugate − MWsOVA )/MWsHapten
tion, A : absorbance at zero parathion concentration, and
0
(1)
Aex: absorbance at excess parathion concentration.Curve
CIELISA procedures using the hapten I–OVA
conjugate coated plates
fitting was performed by use of Eq. (3).
Y = [100/(1 + (X/C))B]
(
3)
Microtiter plates were coated with hapten I–OVA conju-
gate by adding 100 μL of the conjugate. After incubating
at 4 °C overnight, the plates were treated with 50 μL the
rabbit polyclonal antibody against parathion and 50 μL dif-
ferent concentration of free parathion (100, 20, 4, 0.8, 0.16,
in which C is the test midpoint (Hapten value), B is the
slope of the curve at the IC -value, and X is the analyte
concentration.
5
0
Optimization of CIELISAs
−
1
0
.032, 0.0064, 0.00128, 0.000256 and 0 μg mL ) for 1.5 h
at 37 °C. The plates were thoroughly washed with PBST.
00 μL of goat anti-rabbit IgG-HRP conjugate secondary
To detect parathion in foods and environmental samples,
it is essential to develop CIELISA with optimum sensitiv-
ity. Therefore, effects of different concentration of coating
hapten and GD, blocking buffers and antibodies dilution
were studied. The main criteria used to evaluate immunoas-
say performance were competitive inhibition curve and the
absorbance values.
1
antibody at 1:3000 diluted in PBS was then added into each
well of the plates. Afterwards, 100 μL tetramethylbenzi-
dine substrate systems were added into each well and incu-
bated for 10 min at 37 °C in dark. Absorbance at 450 nm
was determined in an ELISA reader.
CIELISA procedures using the hapten II covalent
linked plates
Cross‑reactivity
In our study, cross-reactivity (CR) of CIELISAs was evalu-
ated by using its analogues: methyl parathion, atrazine,
monocrotophos, chlorpyrifos and diazinon. The CR values
were calculated according to Eq. (4) [37, 41].
Microtiter plates were covalent linked hapten II molecules
directly following the procedure of works with some modi-
fications [41]. Standard microtiter plates were pre-treated
with a dilution of GD in carbonate buffer (0.05 M, pH 9.0)
at 37 °C for 2 h. After washing 3 times with PBST buffer,
an optimized dilution of hapten II in PBS (0.05 M, pH
CR (%) = IC of parathion / IC of analogues × 100
50
50
(4)
Sample extraction and spiking method for CIELISAs
8
.5) was incubated at 37 °C for 2 h. The plates were then
washed with PBST buffer thoroughly. By incubating with
a selected blocking reagent in PBS (pH 8.5) at 37 °C for
Real samples including drinking water, cucumber, rice and
corn were spiked and analysed by CIELISAs. Cucumber,
rice and corn samples were supplied from a local supermar-
ket in Tianjin. Three kinds of drinking water were obtained
from the Haihe River, Tuanbo Lake and Dongli Lake,
respectively, in Tianjin.
1
h, the remaining binding sites on the modified surface of
microtiter plates were blocked. After washed thoroughly
with PBST, the plates were treated with 50 μL of the rab-
bit polyclonal antibody against parathion and 50 μL stand-
ard concentration of free parathion (100, 20, 4, 0.8, 0.16,
The cucumber sample preparation was carried out fol-
lowing the previous report [25]. Parathion was spiked in
−
1
0
.032, 0.0064, 0.00128, 0.000256 and 0 μg mL ) for 1.5 h
−
1
at 37 °C. Then, 1:3000 dilution of the goat anti-rabbit IgG-
HRP conjugate secondary antibody was added into each
well of the plates. The plates were incubated for 45 min at
cucumber at three final concentrations (5, 10, 20 ng mL ).
One-gram spiked cucumber was mixed with 2 mL methanol
and then shaken for 24 h. After centrifuging at 6000 rpm
for 15 min, the supernatant was evaporated to dryness
under vacuum. The obtained residue was then dissolved in
3
7 °C and washed with PBST for 6 times. The next steps
were followed as described above.
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J IRAN CHEM SOC
Fig. 2 Verification of hapten
I, hapten I-OVA conjugate and
hapten II. a TLC analysis of
hapten I. Compared with the
parathion original drug, there
was new product (hapten I) gen-
erated. b Mass spectra of hapten
I-OVA conjugate and OVA.
Their MWs were 47,296.88 and
1
4
4,656.13, respectively. c H
HMR spectrum of hapten II.
δ = 1.26 (t, J = 5.7, CH CH ,
2
3
6
4
6
H), 4.17–4.95 (m, CH CH ,
H), 5.02 (s, Ph–NH, 1H),
.503–6.867 (m, PhH, 4H)
2
3
1
mL PBS with 5% methanol and analysed by CIELISAs
with 2 mL of methanol/water (75:25, V/V) and then
shaken using a rotary shaker at 250 rpm for 15 min. The
mixture was kept for 24 h. The aliquot of the extract
was then diluted with 1 mL PBS with 5% methanol.
without any other purification procedure.
Rice and corn samples were prepared with the same
procedure. Concretely, the spiked samples were mixed
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J IRAN CHEM SOC
Fig. 3 Reaction mechanism of direct covalent linkage of hapten II
and the PS surface of microtiter plates. The GD was used to treat the
microtiter plate surface for 2 h to introduce aldehyde groups that can
covalent link hapten II. And, aminoethanoic acid was used to block
the excess aldehyde groups of the modified surface
Table 1 Effect of blocking
reagents on absorbance values
a
blocking
c
0
b
bg
d
Blocking reagents A
A
A
A
ex
(
n = 3)
BSA
-aminocaproic
acid
0.209 ± 0.04
0.335 ± 0.02
0.109 ± 0.03
0.234 ± 0.04
0.407 ± 0.03
1.127 ± 0.02
0.249 ± 0.02
0.375 ± 0.03
6
Aminoethanoic
acid
0.241 ± 0.05
0.102 ± 0.01
1.192 ± 0.03
0.234 ± 0.01
Ethanolamine
Guanidine
0.294 ± 0.03
0.275 ± 0.01
0.317 ± 0.04
0.339 ± 0.03
0.236 ± 0.02
0.375 ± 0.04
1.607 ± 0.05
0.829 ± 0.04
0.987 ± 0.02
0.768 ± 0.04
0.423 ± 0.03
0.846 ± 0.05
Acrylamide
Using aminoethanoic acid and BSA, there were higher A value and lower A value
0
bg
a
Surface only coated with 6-aminocaproic acid, aminoethanoic acid, BSA, ethanolamine, guanidine or
acrylamide, respectively
b
Absorbance at negative group
c
Absorbance at zero hapten concentration
d
Absorbance at excess hapten concentration
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J IRAN CHEM SOC
The following steps were just the same as the cucumber
preparation.
Column: C18 (250 mm × 4.6 mm, 5 μm)
Mobile phase: acetonitrile–water (50:50, v/v)
−1
The water sample was filtered by the Millipore filtration
Flow rate: 1.0 mL at room temperature
Detection wavelength: 270 nm.
(
5
0.45 μm) and then was spiked with parathion standard (1,
, 10 ng mL ) in methanol and diluted with 10% metha-
−1
nol-PBS for CIELISAs.
Results and discussion
Haptens syntheses
Lake water samples preparation for HPLC method
Two lake water samples (A and B) were collected from two
different lakes in Tianjin. HPLC method was carried out by
some reports [46, 47]. HPLC instrument was utilized for
the experiments, and the obtained data were collected and
processed by the empower software. The optimized chro-
matographic conditions were as follows:
The obtained hapten I was characterized by TLC analy-
sis. In Fig. 2a, it was learned that a new product was
generated and hapten I preparation was initially con-
firmed. Figure 2b shows the mass spectrograms of hap-
ten I–OVA conjugate and OVA. It was determined
that MWs of hapten I–OVA conjugate and OVA were
Fig. 4 Blocking effects of blocking reagents and BSA concentration
of diluting PBS. A value is the absorbance at zero hapten concentra-
0
tion; Aex value means the absorbance at excess hapten concentration;
Abg value is the absorbance at negative group. Each point represents
the mean ± SD (standard deviation, n = 3). a Effect of different
Fig. 5 Effect of hapten II concentration on genetic parameters of
CIELISA. A value is the absorbance at zero hapten concentration;
0
Aex value means the absorbance at excess hapten concentration.
Each point represents the mean ± SD (standard deviation, n = 3). a
Effect of the concentration of hapten II on A −A difference. From
blocking reagents on the absorbance values. A value is the absorb-
0
ance at zero hapten concentration. When using aminoethanoic acid,
0
ex
−1
there were higher A −A difference and lower value of Abg. b Effect
0.1 to 4 nmol mL of hapten II concentration, higher A −A dif-
0
ex
0
ex
of BSA with different concentration on the absorbance values. Higher
ference was achieved. b Effect of the concentration of hapten II on
A −A difference and lower Abg were achieved when BSA concen-
competitive inhibition. There was the obvious linear correlation using
2 nmol mL of hapten II
0
ex
−1
tration beyond 0.6% (M/V)
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J IRAN CHEM SOC
4
7,296.88 and 44,656.13, respectively, and the coupling
Selection of blocking effect
ratio was 7:1 according to Eq. (1). The synthesized hap-
1
1
ten II was characterized by H NMR in Fig. 2c.
H
Sensitivity of an assay depends on the solid phase con-
dition to a great extent. Ideal solid phase should be con-
sistent, stable and have low background signal. We chose
medium binding ELISA plates, because it can largely
eliminate non-specific absorption, as described in the
product manual.
In this assay, the non-specific binding of antibod-
ies on the surface was prevented by the following steps.
In the blocking step, several blocking reagents (BSA,
HMR (DMSO + D O): δ = 1.26 (t, J = 5.7, CH CH ,
2
2
3
6
6
H), 4.17–4.95 (m, CH CH , 4H), 5.02 (s, Ph–NH, 1H),
2
3
.503–6.867 (m, PhH, 4H).
As described in Fig. 3, microtiter plates were pre-treated
with GD at 37 °C to introduce aldehyde groups, which
were used to cross-link with amino groups of hapten II.
Aminoethanoic acid was then utilized for blocking excess
aldehyde groups of the surface.
Fig. 6 Effect of the concen-
tration of GD, antibodies on
genetic parameters of CIELISA
using the hapten II covalent
linked plates. Each point repre-
sents the mean ± SD (standard
deviation, n = 3). a Effect of the
concentration of GD solution
on competitive inhibition. When
using 6% a dilution of GD, the
linear correlation was achieved.
b Concentration of polyclonal
antibody against parathion (5,
−
1
1
0, 15, 20 ng mL ) and dilu-
tion level of goat anti-rabbit
IgG-HRP conjugate secondary
antibody (1:2000 and 1:3000)
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J IRAN CHEM SOC
6
-aminocaproic acid, acrylamide, ethanolamine, guani-
Optimization of GD concentration, the rabbit
polyclonal antibody against parathion and goat
anti‑rabbit IgG‑HRP conjugate secondary antibody
concentration
dine and aminoethanoic acid) were utilized for blocking
the excess aldehyde groups of the surface modified with
a GD layer. As shown in Table 1, aminoethanoic acid
and BSA prevented non-specific binding of assay com-
ponents to a large extent (A ) without influencing the
The competitive inhibition assay was performed to select
the optimized concentration of GD. According to the previ-
ous studies [41], a range of 1−8% GD was tested. Based
on Eq. (2), the linear correlation was manifest in the use of
a 6% dilution of GD in Fig. 6a. So 6% GD was chose in the
subsequent experiments. Figure 6b shows a chequerboard
titration of the rabbit polyclonal antibody against parathion
and goat anti-rabbit IgG-HRP conjugate secondary anti-
body concentration by using competitive inhibition assays.
From the result, it was learned that the optimal concentra-
tion of the rabbit polyclonal antibody against parathion was
bg
specific binding of the primary antibody (A ). In Fig. 4a,
0
there was higher A −A difference and lower value of
0
ex
A
while using aminoethanoic acid. It indicated that
bg
aminoethanoic acid could prevent non-specific binding
of antibodies to the surface and the high ratio A /A was
0
ex
desirable for assay purpose. On the other hand, the non-
specific binding could be further avoided when antibod-
ies were diluted in PBS with BSA. As shown in Fig. 4b,
A0, Aex and Abg values all trended to decrease with the
increase of BSA concentration. Aex and Abg almost run
−1
through the lowest values, and the positive value (A ) was
10 ng mL and the proper titer of goat anti-rabbit IgG-
HRP conjugate secondary antibody was 1:3000.
0
decreased greatly when BSA concentration beyond 0.6%
(
M/V). Thus, aminoethanoic acid was used for block-
ing the excess aldehyde groups of the modified surface
Fig. 4a), and 0.6% BSA in PBS was selected to dilute the
Sensitivity
(
antibodies (Fig. 4b).
The typical standard curves of CIELISAs are given in
Fig. 7. In the case of CIELISA using the hapten I–OVA
conjugate coated plates, the IC50 value and the limit of
detection (LOD, calculated as IC ) for parathion were
Optimization of hapten II concentration
1
0
−1
As shown in Fig. 5a, when the absorbance values A and
375 and 1.25 ng mL , respectively. However, in CIELISA
0
Aex were close to the background value (A ), it was indi-
cated that hapten II was not covalent linked on the surface
using the hapten II covalent linked plates, the IC value
bg
50
−1
and LOD were 15 and 0.08 ng mL , respectively. In view
of LOD, the sensitivity of the CIELISA using the hapten II
covalent linked plates was improved 15-fold compared with
that using the hapten I–OVA conjugate coated plates. The
of microplates. The A signals increased with increasing of
0
hapten II concentration, but run through a maximum value
−1
at approximately 1 nmol mL . This phenomenon may
be explained by steric effects, which arising from the fact
that each atom with a molecule occupies a certain amount
of space. If atoms are brought too close to each other, there
is an associated cost in energy due to overlapping electron
cloud, and this may influence the molecule preferred shape
(conformation) and reactivity. In other words, the steric
effects can affect the reaction between antibody and anti-
−1
gen. When the hapten II was beyond 1 nmol mL , the mass
of hapten II influenced each other to generate conforma-
tional changes, which resulted in unfavourable recognition
between the rabbit polyclonal antibody against parathion
and hapten II [36].
−
1
From 0.1 to 4 nmol mL of hapten II concentra-
tion, there was higher A −A difference (Fig. 5a). So we
0
ex
selected this range of concentration for further optimization
of hapten II concentration. According to Eq (2), the linear
correlation was obvious and sensitivity was highest when
−
1
Fig. 7 Standard curves of CIELISAs using the hapten I-OVA con-
hapten II concentration was 2 nmol mL in Fig. 5b. Thus,
jugate coated plates and the hapten II covalent linked plates. Their
−
1
we chose 2 nmol mL of hapten II as coating concentra-
tion in this study.
−1
−1
IC10 values were 1.25 ng mL and 0.08 ng mL , respectively. Each
point represents the mean ± SD (standard deviation, n = 3)
1
3

J IRAN CHEM SOC
−
1
great improvement of sensitivity would be resulted from
the following reasons. The reactive site of hapten I was eas-
ily shielded by carrier protein OVA in the preparation of
hapten I–OVA conjugate due to its low MWs and the sin-
gle reactive site of SM-hapten. In addition, the immobiliza-
tion of hapten I-OVA conjugate on the microtiter plate sur-
face mainly depended on hydrophobic interactions. It may
involve significant conformational changes which lead car-
rier protein easily to shield SM-hapten during this immobi-
lization. However, the directly covalent linkage of hapten
II on the PS plates may release more reactive sites of SM-
hapten since there was no interference of carrier protein,
thus improving the reaction efficiency of SM-hapten and
antibody reactions.
parathion (as low as 0.08 ng mL ), which was less than
or approximately close to the detection borderline of tra-
ditional chromatograph methods [18–21, 46–48] and the
reported CIELISAs [49, 50] and was below the legislated
maximum residue levels [51].
Specificity
In this study, CR data of CIELISA using the hapten II cova-
lent linked plates are summarized in Table 2. It was learned
that the assay had CR of 100% to parathion, 17% to methyl
parathion and less than 0.01% to other analogues. Except
for parathion, each IC50 value was greater than the high-
est concentration of compounds used (1500 ng mL ). So
the CIELISA using the hapten II covalent linked plates was
very specific against parathion.
−
1
As a result, our CIELISA using the hapten II cova-
lent linked plates achieved a highly sensitive detection of
Recovery and precision
Table 2 Cross-creativities of the inhibition assay with parathion,
methyl parathion, atrazine, monocrotophos, chlorpyrifos and diazinon
(
n = 3)
Four kinds of real samples (river water, cucumber, rice
and corn) were spiked with parathion and analysed by the
developed CIELISAs. As shown in Table 3, the recover-
ies for river water ranged from 83.0 to 101.0% with CVs
of 6.65–11.13% in CIELISA using the hapten II cova-
lent linked plates, which showed better stability than that
using the hapten I–OVA conjugate coated plates. The food
samples included cucumber, rice, corn were also analysed
by CIELISAs after being fortified with parathion at 5.0,
Compounds
CR (%)
Parathion
100
Methyl parathion
Atrazine
18
<0.01
<0.01
<0.01
<0.01
Monocrotophos
Chlorpyrifos
Diazinon
−
1
1
0, 20 ng mL . The recoveries of CIELISA using the
hapten II covalent linked plates were ranged from 86.3 to
10.2% with CVs of 5.95–13.59%. The fortifications of
CR for parathion was 100% and that was highest than the other ana-
logues
1
Although IC50 value could not be determined accurately due to the
limited solubility of the compounds at high concentrations, it was
food samples were carried out by incubating the chopped
ground rice in fortification solutions for 24 h. Therefore,
−
1
clear that inhibition was less than 50% at 1500 ng mL
Table 3 Accuracy and precision of parathion in spiked samples (River water, cucumber, rice and corn) by the CIELISAs using the hapten II
covalent linked plates and the hapten I-OVA conjugate (n = 3)
Sample
Spiked (ng/g)
Using the hapten II covalent linked plates
Using the hapten I–OVA conjugate coated plates
Measured (ng/g)
Recovery (%)
CV (%)
Measured (ng/g)
Recovery (%)
CV (%)
River water
1
5
0.8 ± 0.09
5.1 ± 0.37
9.5 ± 0.63
4.9 ± 0.66
10.2 ± 0.94
18.6 ± 1.17
5.4 ± 0.41
11.0 ± 0.67
20.8 ± 2.03
4.4 ± 0.47
9.1 ± 0.80
17.3 ± 1.41
83.0
101.0
94.7
11.13
7.46
6.65
13.59
9.24
6.32
7.48
5.95
9.75
10.71
8.69
8.21
0.8 ± 0.01
5.1 ± 0.37
9.3 ± 0.70
4.9 ± 0.62
9.6 ± 0.83
18.1 ± 1.09
5.7 ± 0.42
10.5 ± 0.63
21.7 ± 2.12
4.2 ± 0.42
8.9 ± 0.76
16.1 ± 1.30
80.3
102.0
93.3
12.28
7.44
7.42
12.61
8.66
6.02
7.44
6.03
9.77
9.98
8.54
8.06
1
0
5
0
0
5
0
0
5
0
0
Cucumber
Rice
97.6
98.2
1
2
102.3
92.9
95.6
90.5
108.8
110.2
104.1
88.2
112.9
105.2
108.7
84.3
1
2
Corn
1
2
91.3
88.5
86.3
80.6
1
3

J IRAN CHEM SOC
pesticides may have penetrated quite deeply into the inte-
rior of grains during the incubation period. However, the
CR data suggested that pesticides can be nearly com-
pletely recovered by the extraction procedure adopted in
this study.
Analysis of lake water samples
CIELISAs and HPLC were utilized to detect parathion in
lake water samples to assess the applicability and validity
of the developed methods. As shown in Table 4, determina-
tion of parathion measured by three methods was similar. It
was learned that the CIELISA using the hapten II covalent
linked plates could be used for the determination of para-
thion in real samples.
Compared with GC, HPLC and other methods [17–23],
our proposed approach eliminated sophisticated equip-
ment, and complicated operation processes. For low-pres-
sure gas chromatography–tandem mass spectrometry (LP-
GC/MS–MS) for parathion [52], the sample preparation
procedure consisted of ten steps, and for GC–MS analy-
sis [53], it generally needed eight steps to prepare real
samples, which were time-consuming and complicated
processes. For fluorescence probing sensors [22], acetyl-
cholinesterase biosensor [24], and molecule imprinted pol-
ymeric sensor [54, 55], complex fabrication was required
to prepare carbon spheres, CdTe quantum dots and molec-
ular imprinted polymer, and complex instruments (surface
plasmon resonance, electrochemical working station and
fluorescence spectrophotometer) were utilized to gener-
ate a readable optical or electronic signals. Therefore, our
constructed method was characterized by (a) low cost,
easy construction and simple operation; and (b) stable,
sensitive and selective detection.
Conclusion
In summary, we have developed a highly sensitive CIEL-
ISA in which a SM-hapten was directly covalent linked
the PS surface of microtiter plates for the detection of
parathion. The developed assay not only refrained from a
complex process of hapten-protein conjugation, but also
showed highly sensitivity, stability and selectivity. Further
research should be focused on exploiting more means to
directly link SM-hapten to microtiter plates and applying
some signal amplification systems simultaneously to fur-
ther improve the sensitivity and stability of immunoassays.
Acknowledgements The authors gratefully acknowledge the finan-
cial supports by National Natural Science Foundation of China
(
2
Grant 81302430), China Postdoctoral Science Foundation (Grant
014M560192) and the Tianjin Application of Basic and Cutting-
edge Technology Research Program (Grant 11JCZDJC 22400).
1
3

J IRAN CHEM SOC
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3
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3
3
3
3
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9. Y. Na, W. Sheng, M. Yuan, L.L. Li, B. Liu, Y. Zhang, S. Wang,
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