Development of a competitive format sorbent assay for the determination of parathion in water using molecular imprinted polymer as specific sorbent carrier

Article abstract of DOI:10.1016/j.cclet.2010.05.014

A rapid, simple, and reliable competitive molecular imprinted polymer sorbent assay (MIPSA) was developed and validated for measurement of parathion in water samples. This assay employed a molecular imprinted polymer (MIP) that was synthesized with non-covalent imprinting method as capture reagent and p-aminoparathion conjugate of horseradish peroxidase (para-HRP) as an enzyme label. The assay depended on a competitive binding reaction between the enzyme conjugate and analyte for the binding sites of the MIP. The optimized analysis conditions of 10ngmL^-1 para-HRP and 10mgmL^-1 polymer were found. The assay was acceptable to detect parathion in water samples under the optimized conditions, with a limit of detection of 50ngmL^-1. Mean analytical recoveries of added parathion in water samples ranged from 101.2% to 105%. The precision of the assay was satisfactory; relative standard deviation ranged from 4.3% to 6%.

Full text of DOI:10.1016/j.cclet.2010.05.014

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Chinese Chemical Letters 21 (2010) 1361–1365
www.elsevier.com/locate/cclet
Development of a competitive format sorbent assay
for the determination of parathion in water using
molecular imprinted polymer as specific sorbent carrier
*
Jian She Tang , Li Xiang
National Experimental Teaching Demonstration Laboratory of Water Pollution Control and Recovery Utilization from Wastewater,
Anhui University of Architecture, Hefei 230022, China
Received 4 March 2010
Abstract
A rapid, simple, and reliable competitive molecular imprinted polymer sorbent assay (MIPSA) was developed and validated for
measurement of parathion in water samples. This assay employed a molecular imprinted polymer (MIP) that was synthesized with
non-covalent imprinting method as capture reagent and p-aminoparathion conjugate of horseradish peroxidase ( para-HRP) as an
enzyme label. The assay depended on a competitive binding reaction between the enzyme conjugate and analyte for the binding
À1
À1
sites of the MIP. The optimized analysis conditions of 10 ng mL para-HRP and 10 mg mL polymer were found. The assay was
À1
acceptable to detect parathion in water samples under the optimized conditions, with a limit of detection of 50 ng mL . Mean
analytical recoveries of added parathion in water samples ranged from 101.2% to 105%. The precision of the assay was satisfactory;
relative standard deviation ranged from 4.3% to 6%.
#
2010 Jian She Tang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Molecular imprinting; Conjugated enzyme; Sorbent assay; Parathion; Water sample
Enzyme-linked immunosorbent assay (ELISA) is a widely used method for measuring the concentration of a
particular molecule [1]. The ELISA based on the antibody suffers from the high expense of producing antibodies and
the reagents stability. The molecular imprinted polymers (MIPs) have been identified as excellent mimics, which have
been used in the place of antibodies in ELISA. Using an enzyme-labelled conjugate as a competitive probe to the
determination of 2,4-dichlorophenoxyacetic (2,4-D) was demonstrated by Surugiu firstly [2]. Sergey et al. [1]
developed subsequently a new technique for coating microtitre plates with MIPs through oxidation of 3-
aminophenylboronic acid (APBA), 3-thiopheneboronic acid (TBA) and aniline. It was proved in their report that the
high stability of the polymers and good reproducibility of the measurements make MIP coating an attractive
alternative to conventional antibodies or receptors used in ELISA. Adapting the ionic liquid of 1-butyl-3-
methylimidazoliumhexafluorophosphate to an oxidative polymerization, Wang et al. [3] developed a direct
competitive biomimetic enzyme-linked immunosorbent assay for the determination of estrone in environmental water.
In the present paper, we describe the design of a competitive molecular imprinted polymer sorbent assay (MIPSA) for
*
Corresponding author.
E-mail address: tjs28@126.com (J.S. Tang).
1001-8417/$ – see front matter # 2010 Jian She Tang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2010.05.014

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J.S. Tang, L. Xiang / Chinese Chemical Letters 21 (2010) 1361–1365
the pesticide parathion in water samples. The results of analysis were satisfactory in terms of recovery and relative
standard deviation (RSD).
1
. Experimental
Parathion was purchased from the Institute of Environmental Protection, Ministry of Agriculture (Tianjin, China).
Horseradish peroxidase (HRP), tetramethylbenzidine (TMB) and Tween 20 were purchased from Sigma Chemical Co.
Polypropylene test tubes were purchased from Shanghai Dobio Biotech Co., Ltd. Double distilled water and MilliQ
water were prepared in our laboratory. All other chemicals were of analytical grade. The UV-2401 was from Shimadzu
Corporation. The MIP for parathion was synthesized using non-covalent imprinting method as described previously
[4]. The ratio of template (parathion) to monomer is 1:4. The para-HRP conjugate was prepared by conjugating the p-
aminoparathion with HRP using diazotization method in our laboratory. The p-aminoparathion was obtained from
parathion by reduction reaction in the condition of acid and zinc powder [5].
1
.1. MIPSA procedure
The appropriate concentration of para-HRP and 50 mL parathion standards or 50 mL water sample was mixed.
The mixture was added into the tube containing 10 mg MIP microspheres. The phosphate-buffered saline
0.2 mol/L pH 7.4) with 0.05% Tween-20 was added to give a total volume of 1 mL. The tube was incubated on an
oscillator for 2 h, after which the particles were removed by centrifugation for 10 min at 5000 rpm or filtering.
(
À1
À1
TMB solution (0.4 mg mL TMB-50 mg L hydrogen peroxide in citric acid buffer, pH 5.5) was used as
substrate for colorimetric measurements in the experiments. 500 mL of supernatant was withdrawn and added to
1
0
mL of TMB solution in a tube. Then the solution was read immediately by a spectrophotometer at 652 nm within
.5 cm glass cuvette.
1
.2. Preparation and analysis of samples
Water samples were collected from a tap in the laboratory. The samples were filtered by 0.45 mm filters without any
other pretreatment prior to MIPSA analysis. For recovery studies, a standard dilution series of parathion was spiked
À1
À1
À1
into tap water. Aliquots were fortified with parathion to 100 ng mL , 500 ng mL , and 1000 ng mL . The spiked
samples were then analyzed with MIPSA.
2
. Results and discussion
2
.1. Optimum concentration of enzymes
Binding of the enzyme ( para-HRP, HRP) with the polymer in the glass tube was checked by confirming the binding
À1
reactivity of enzyme at different concentrations from 5000-fold dilution (approx. 40 ng mL ) to 80,000-fold dilution
À1
(
approx. 2.5 ng mL ). A significantly different binding between para-HRP and HRP to polymer is shown in Fig. 1
clearly. This different binding indicated that the conjugated HRP characterized an essential role in the specific
recognition of MIP. As shown in Fig. 1, the absorbance decreased with the decreasing enzyme concentration in the
supernatant, which was due to dilution of enzyme and adsorption by polymer. It is worth noting that the absorbance
decreased drastically in some extent of the range of enzyme concentration. The ranges were from 5000-fold dilution
À1 À1
À1 À1
0 ng mL ) to 80,000-fold dilution (approx. 2.5 ng mL ) when the HRP and para-HRP were adopted, respectively.
(
approx. 40 ng mL ) to 20,000-fold dilution (approx. 10 ng mL ) and from 20,000-fold dilution (approx.
1
This phenomenon should be explained by specific binding between polymer and para-HRP and non-specific binding
between polymer and HRP.
The absorbance veritably decreased to the end point (0.093) when the para-HRP was adopted, while the absorbance
was 0.795 when the HRP was present instead of para-HRP in the assay at the same concentration, thus establishing the
sensitivity and workability of new developed MIP sorbent assay format for the pesticide determination. The non-
specific binding between NIP and para-HRP was also observed, because the typical downtrend of optical density (OD)

[(Fig._1)TD$FIG]
J.S. Tang, L. Xiang / Chinese Chemical Letters 21 (2010) 1361–1365
1363
Fig. 1. Primary data for colorimetric measurement at the different dilution times of enzyme.
value at the present of para-HRP within low concentration existed when the non-imprinted polymers (NIPs) were
adopted as sorbent material.
2
.2. Optimum concentration of polymer
In order to obtain higher sensitivity for the MIPSA, the optimum concentration of polymer microsphere for
competitive binding assay was established in terms of experiments under the condition of different microsphere
concentration. The obviously decreasing signal intensity with increasing MIP microsphere concentration could
indicate that binding to the control polymer was lower than that to the imprinted polymer (Fig. 2).
Due to hydrophobic interactions of the polymer with the biomacromolecule, namely enzyme, non-specific binding
of the polymer with HRP cannot be avoided and observed always, though the various concentration of non-ionic
surfactant was used in the assays. When the polymer concentration was 10 mg/mL and higher, the OD value was no
longer decreasing to MIP, while that was decreasing slightly to NIP for non-specific binding.
[(Fig._2)TD$FIG]
Fig. 2. OD value of substrate solution relative to MIP concentration (&) and NIP concentration (*). Each point represented the mean of triplicate
determinations Æ SD.

[(Fig._3)TD$FIG]
1
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J.S. Tang, L. Xiang / Chinese Chemical Letters 21 (2010) 1361–1365
Fig. 3. MIPSA standard curve for the determination of parathion. The MIPSA standard curve obtained under the optimized conditions of 20,000
À1
times dilution to para-HRP and 10 mg mL polymer. The error bar represents the SD calculated from triplicate determinations.
2
.3. Standard competitive curve
The representative MIPSA standard competitive curve for parathion is shown in Fig. 3. Under the optimized
À1
À1
experimental conditions of the polymer concentration (10 mg mL ) and the para-HRP concentration (10 ng mL ),
the OD value of control tube without analyte was only 0.092, whereas 0.783 of OD value was obtained from utmost of
À1
analyte (4000 ng mL ). It was shown that the parathion molecule could effectively compete with the para-HRP for
the binding sites in the MIP surface in the competitive assay format.
The sensitivity (50% inhibiting concentration, IC ) and limit of detection (LOD), calculated as the concentration
5
0
À1
À1
of standard solution cause 90% inhibition of color development, of MIPSA were 350 ng mL and 50 ng mL
,
respectively. In our preliminary experiments, it was established that the indirect competitive enzyme-linked
immunosorbent assay (IC-ELISA) could be used for the determination of the parathion sophisticatedly. The sensitivity
À1
À1
(
respectively [6]. The LOD of MIPSA for the analysis of parathion was only 3 times higher than that obtained from
IC50 value) and LOD (10% inhibiting concentration, IC ) of ELISA were 428 ng mL
and 12 ng mL
,
10
À1
traditional ELISA using polyclonal antibodies against parathion. The linear range of MIPSA was from 82.8 ng mL
À1
to 910.3 ng mL
.
2
.4. Recovery study in spiked water samples
In order to evaluate the applicability of the MIPSA method under the optimized conditions for measurement of the
À1
À1
real samples, the tap water samples were spiked with parathion at three levels (100 ng mL , 500 ng mL and
À1
1
000 ng mL ), and directly analyzed by the MIPSA with three replicates for each concentration. The analytical data
of each spiked water sample are shown in Table 1. The average recoveries for water samples at three concentrations
À1
À1
À1
were excellent as follows: 101.2%, 103.8%, and 105% for 100 ng mL , 500 ng mL
and 1000 ng mL ,
respectively. The good recovery indicated that the developed MIPSA was acceptable for the determination of
pesticides in the environmental water samples.
Table 1
Analysis results of parathion spiked into tap water samples by the developed MIPSA (n = 3).
À1
À1
)
Theoretical (ng mL
000
)
Detected (ng mL
Mean recovery (%)
RSD (%)
1
1049.8
518.8
101.2
105
6
5
1
00
00
103.8
101.2
6
4.3

J.S. Tang, L. Xiang / Chinese Chemical Letters 21 (2010) 1361–1365
1365
3
. Conclusion
In summary, we have developed a novel direct competitive mimetic enzyme-linked immunosorbent assay for the
determination of parathion in water samples using the molecular imprinted polymer of parathion as specific sorbent
À1
À1
carrier. It was found that the 10 ng mL para-HRP and 10 mg mL polymer were the optimum conditions for the
À1
assay with LOD of 50 ng mL . In the recovery study of spiked water samples, mean recoveries ranged from 101.21%
to 104.98%, with coefficients of variation ranging from 4.34% to 6.04%.
Acknowledgments
This work was supported by the Excellent Young Teacher Fund of Anhui Province, China (No. 2009SQRZ105) and
the Dr. Fund of Anhui University of Architecture (No. QD20090905).
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