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Characterization and quality assessment of binding properties of the monocrotophos molecularly imprinted microspheres prepared by precipitation polymerization in toluene.

INTRODUCTION

Polymers have played an important role as superabsorbents and water treatment chemicals in recent years, but lack of selectivity to the target substrates greatly limits the application of the polymers in many analytical fields [1]. During the last years, molecularly imprinted polymers (MIP), which often utilize in solid-phase extraction, sensor, and chromatography for their potential absorptive selectivity to target molecule and the related compounds [2, 3], have also been used as synthetic materials to rebind the target analyte since 1972 when Paling's production of antibodies in vitro and Fischer's lock and key principle was described by Wuff et al., 1972.

The general principle of molecular imprinting is as follows: (1) Specific complex formed based on (non)-covalent bonding interactions between template molecule and polymerizable functional monomer in an apolar and aprotic solvent before polymerization by assembling the functional monomers around the template molecule; (2) A rigid and porous copolymer yields in the presence of crosslinker and initiator; (3) The distinct cavities remains in the copolymer with the removal of the target template, which is tailor-made complementary to the template molecule in size, shape and functionality.

At present, many methods have been developed for the preparation of MIP, such as bulk polymerization [4], suspend polymerization [5], emulsion polymerization, two-step polymerization, precipitation polymerization [6, 7], and so on. Among them precipitation polymerization method is the easiest one, and it can overcome the drawbacks of the other methods with obtaining size-uniformed and conglobated microspheres. Consequently, a kind of desired copolymer particles with a narrow particle size and more homogeneous binding sites can be obtained by this method.

In a precipitation polymerization process, the polymerization takes place in a medium, which is a solvent for the functional monomer, template molecule, crosslinker, and initiator but nonsolvent for the polymer. The polymer particles are not stable and tend to agglomerate and precipitate from the solvent to form a narrow size particle. In the process there is not any emulsifier or suspending reagent added, so the copolymer is at a high purity.

Growing public concerns on the contamination of the food, drinking water supplies, and the environment with the organic pollutant, especially the organophosphate pesticides residues, have stimulated vigorous research on the development of various determination technologies [8]. Submicrogram detection limits for most pesticides are now in routine in many analyses by gas chromatography (GC), high performance liquid chromatography (HPLC), capillary electrophoresis, immunoassay bioassays [9], and so on. However, the trace pesticides residues in real samples must be cleaned up and enriched prior to the detection by GC or HPLC, and this process is time-consuming and laborious. In the immunoassay bioassays, the antibody is easily wrecked in extreme environment, and the yield of antibody is affected by the bio-factors. Thus, the development of adsorbents with good-selectivity and excellent stability to certain organophosphate pesticides is necessary.

In this work, precipitation polymerization was employed to prepare organophosphorous pesticide molecularly imprinted microspheres using monocrotophos (MCP) as template, methacylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as crosslinker, azobisisobutyronitrile (AIBN) as initiator and toluene as porogen, respectively. The characterization of the obtained particles were performed through environmental scan electronic microscope (ESEM), the rebinding properties were demonstrated by computer simulation, equilibrium rebinding experiments, and Scatchard analysis. The selectivity of the obtained particles was elucidated by the different rebinding capability of the MCP and the related compounds. Desired organophosphorous pesticide imprinted polymer microspheres were obtained for the separation, enrichment, and analysis of trace pesticides residue in soils and foods.

EXPERIMENTAL

Chemicals

All chemicals and reagents were of analytical grade without further purification. MCP, methamidophos, omethoate, dimethoate, phosdrin, dichlorvos, and phosphamidon were purchased from ChemService (USA). MAA, EGDMA, and AIBN were obtained from Sigma-Aldrich (USA). Toluene, methanol, and acetic acid were purchased from Tianjin No.1 Chemical Reagent Factory (Tianjin, China).

Preparation of Molecularly Imprinted Microspheres and Nonmolecularly Imprinted Microspheres

In a 50 mL glass flask, functional monomer MAA, crosslinker EGDMA, and reaction initiator AIBN were dissolved in toluene. The ratio of the template, monomer, crosslinker, initiator, and porogen are shown in Table 1. The mixture solution was put into an ultrasonic bath for 10 min for complete dissolution. Under this condition the complex of the template and the functional monomer formed by hydrogen bond, ion bond or other interactions. Then the solution was sparged with nitrogen gas for about 5 min to remove oxygen, which inhibits the polymerization, and the flask was sealed under nitrogen gas. Polymerization was performed in a water bath at 60[degrees]C for 24 h. After polymerization, the microspheres were collected by centrifugation at 5000 rpm for 5 min, and then the template molecules were removed by washing with a mixture of methanol and acetic acid (9:1, V/V) until no template molecules were detected. Finally, the microspheres were put into an oven at 40[degrees]C under vacuum to dry. Blank microspheres were made with the similar procedure in the absence of the template molecule; the obtained polymer was nonimprinted polymer microspheres (Non-MIP).

Characterization of the Size and Shape of the Obtained Microspheres

The surface, the size, and the shape of the microspheres prepared by precipitation polymerization were studied using an environmental scanning electron microscope (Philips XL30 ESEM).

Computer Simulation

The server used to simulate monomer-template interactions was a Silicon Graphics Origin 350 running IRIX 6.5 operating system. The server was configured with four 4700 MHz reduced instruction set processors; 2 GB memory and a 72 GB fixed drive. This system used to execute the software packages SYBYL 6.9 (Tripos, St. Louis, MO). The computational design was performed in three steps [10, 11]. In the first steps, the molecular model of MCP (template) and that of the most commonly used functional monomer were designed. These structures were charged using the molecular mechanics method applying an energy minimization with the MINIMIZE command. In the second step, the Leapfrog algorithm was applied to investigate the possible interactions between the template and functional monomer. The program was activated for 600,000 steps. The results from the runs were examined evaluating the empirical binding scores.

Equilibrium Rebinding Experiments and Scatchard Analysis

About 50 mg imprinted and blank microspheres were put into 2 mL Eppendorf tubes, respectively. Then, 1.5 mL MCP toluene solution with the concentration varying from 0.0125 to 0.8 mmol/L was added. The mixtures were incubated at room temperature for 24 h and then were centrifuged for 10 min at 5000 rpm; the free MCP in toluene was determined by GC detector. Bound MCP to the microspheres was obtained by the difference between the initial concentration and the free concentration of MCP.

The selectivity of MIP was investigated using MCP and structurally related organophosphorous pesticides adsorption on the imprinted microspheres and nonimprinted microspheres.

GC-Analysis

The GC system of a Agilent 6890 gas chromatograph with a FPD detector at 250[degrees]C, a Hewlett Packard 7673 auto-sampler, and an j & w DB-17capillary column. The oven temperature program was as follows: the initial temperature of 150[degrees]C was kept for 2 min, and then increased to 250[degrees]C at a rate of 8[degrees]C/min and the final temperature of 250[degrees]C was kept for 1.5 min. The carrier gas was nitrogen at a flow velocity of 10.0 mL/min. Injection in the split-less mode was carried out at 220[degrees]C. Data collection was performed using HP ChemStation Software.

RESULTS AND DISCUSSION

Preparation of Imprinted Microspheres and Nonimprinted Microspheres

Functional monomers are responsible for the bonding interactions in the imprinted binding sites, and for noncovalent molecular imprinting protocols, functional monomers are normally used in excess relative to the number of moles of template to favor the formation of template and functional monomer assemblies. The molar ratio of template, monomer, crosslinker, and initiator was applied from a reported literature [12]. At present the most commonly used monomer is MAA, which can form hydrogen bond with template in porogen prior to the polymerization. In this study, -COOH in MAA can form hydrogen bond with -NH- or =C=O in MCP, the specific and positioned hydrogen bond would contribute the MIP's selective affinity. EGDMA and AIBN were used as cross-linker and reaction initiator, respectively. Crosslinker fulfills three functions in an imprinted polymer. First of all, it is important in controlling the morphology of the polymer matrix; secondly, it serves to stabilize the imprinted binding sites; finally, it imparts mechanical stability to the polymer matrix. High crosslinker ratio is generously preferred in order to access permanently porous materials and in order to be able to generate materials with adequate mechanical stability. Polymers with crosslinker ration in excess of 80% are often the norm.

The choice of the solvent of polymerization is crucial for the resulting selectivity of the imprinted polymers. The solvent serves to bring all the components in the polymerization into one phase, and it serves a second important function for creating the pores in macroporous polymers, for this reason it is quite common to refer to the solvent as the "porogen", when macroporous polymers are being prepared, the nature and the level of the porogen can be used to control the morphology and the total pore volume. Besides its dual roles mentioned above, the appropriate solvent of polymerization is generally an aprotic and a non- or weakly polar solvent, especially in noncovalent imprinting polymerization, the solvent must be judiciously chosen such that it simultaneously maximizes the likelihood of template and monomer complex formation by hydrogen bond or other interactions. So in this work the toluene, aprotic, and weakly polar was chosen as the porogen [13]. The volume of the toluene in the polymerization experiment was found out by a series of small-scale format polymerization experiments.

Under the conditions above-mentioned (Table 1), the desired and uniformly sized MCP imprinted microspheres and nonimprinted microspheres were produced with the yield of 90.3 and 87.4%, respectively (see Fig. 1), which was calculated from the ratio between the yield of the obtained dry particles after removing the template molecules and other unpolymerized molecules and that of the dry particles after polymerization. The diameter of the microspheres is about 2 [micro]m. In this work, the size of the microspheres was larger than that prepared in acetonitrile (the work has not published), the possible reason was that the density of toluene used as porogen was larger than that of acetonitrile. In this work precipitation polymerization was used to produce microspheres, and the mechanism of precipitation polymerization suggested that the particles would not precipitate from the polymerization system until it agglomerated too large. So the microspheres obtained in this work were 2 [micro]m in size and uniform. It was reported that the size of imprinted microspheres prepared by precipitation polymerization was smaller than that of nonimprinted ones [14], but in this work there was almost no difference in size between the obtained imprinted and nonimprinted microspheres.

Computer Simulation

There are many methods to illustrate the recognition mechanism between the template and the functional monomer, Zhu et al. had reported that the existence of multi-molecular complexes formed by hydrogen-bonding interactions between MCP and MAA by [.sup.1.H] NMR study [15]. With the development of chemometrics and computer science the computer simulation on the interactions between atoms is available, so in this work the interactions between MCP and MAA was calculated by this technology. The computational design results were showed in Fig. 2, there were two binding sites on MCP where hydrogen bond can form between MCP and MAA, the distances between the atoms which can form hydrogen bonds between them were 1.895. 2.008, and 2.238 [Angstrom], respectively, which confirmed the interactions between MCP and MAA. The results of the computer simulation also more completely demonstrated the spatial structure of the MCP-MAA complex than the previous methods did.

[FIGURE 1 OMITTED]

Adsorption Analysis

In an attempt to investigate the affinity of the imprinted microspheres for MCP, binding experiments and subsequence Scatchard analysis were carried out. The binding isotherms of MCP to the imprinted microspheres and nonimprinted ones were determined in the range of 0.0125 to 0.8 mmol/L (initial concentration) (Fig. 3). The amount of MCP bound to per gram imprinted microspheres at equilibrium experiment increased along with increasing the initial concentration of MCP and reached saturation at high MCP concentration. But the amount of MCP bound to per gram nonimprinted polymer microspheres at equilibrium experiment only increased along with increasing the initial concentration of MCP and did not reach saturation at high MCP concentration obviously in the studied range, and the amount of MCP bound to imprinted microspheres was dramatically higher than that bound to nonimprinted microspheres at the same initial concentration. This suggested that MCP binding to imprinted microspheres might be caused by the specific binding to a certain number of binding sites populated in the imprinted microspheres.

[FIGURE 2 OMITTED]

The adsorption of MCP on the imprinted and nonimprinted microspheres was compared in Fig. 3, in which Langmuir isotherm model was used to fit the obtained adsorption data [16]:

B = [[B.sub.max] x C]/[[K.sub.D] + C]. (1)

[FIGURE 3 OMITTED]

Where C is the free equilibrium concentration of MCP, B is the amount of MCP bound to per gram microspheres, [B.sub.max] is the maximum amount bound to per gram microspheres, and [K.sub.D] is the equilibrium dissociation constant. The apparent constants fitted by Langmuir isotherm model could be seen in Table 2. The results indicated that the imprinted polymer microspheres had higher affinity to the template molecule than nonimprinted ones did.

The saturation binding data were further processed with Scatchard equation to estimate the binding properties of MCP on the imprinted and nonimprinted microspheres.

[FIGURE 4 OMITTED]

The Scatchard equation was as follows:

B/C - ([B.sub.max] - B)/[K.sub.D]. (2)

Figure 4b shows that the Scatchard plots were only a single straight line, which illustrated that there was only a class of binding sites populated in the nonimprinted microspheres. Figure 4a is the Scatchard plots according to the Scatchard equation. The Scatchard plots were not a single straight line, but consisted of two distinct linear sections with different slopes, which indicated that there exit two kinds of binding sites populated in the imprinted microspheres. The adsorption data could be fitted by two sites Langmuir isotherm model as follows:

B = [[[B.sub.max1] x C]/[[K.sub.D1] + C]] + [[[B.sub.max2] x C]/[[K.sub.D2] + C]]. (3)

The fitted equation in Fig. 5 B = [[4.86 x C]/[0.0019 + C]] + [[43.24 x C]/[0.12 + C]] ([R.sup.2] = 0.9975) suggested that the binding sites in the MIPs were heterogeneous in respect to the affinity for MCP. Most MIPs prepared with noncovalent imprinted approach suffered from a heterogeneous distribution of binding sites for the amorphous nature of MIP; the binding sites were not identical, somewhat similar to a polyclonal preparation of antibodies and the incompleteness or inaccuracy of the monomer-template association. Under high concentration of monomer and template the template cannot accurately associate with the functional monomer to produce selective binding sites due to the major part of the functional monomer existing in a dimerized or polymerized form; however, under diluted concentration, the template could accurately associate with the functional monomer because of the functional monomer existing in a free form. The accurate form of noncovalence between template and monomer produced desired selective binding sites for MCP. In this method of precipitation polymerization, the concentration of template and monomer was very low, the inaccurate association between template and functional monomer could be omitted, and the microspheres obtained by precipitation polymerization were in good and uniform shape. So the adsorption properties of imprinted microspheres prepared by precipitation polymerization were better than those by the tradition method mentioned above.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

It was reported that the MIPs showed high affinity and selectivity for the template in the same solvent, which was used as the porogen in the polymerization [17]. As the selectivity of the template to imprinted polymer microspheres for noncovalence imprinting approach strongly depended on the solvent used. The imprinted polymers often showed different swelling properties in solvent due to the different solution properties of solvents for a given type of polymer. The various degree of swelling in different solvents might considerably change the morphology of the polymer network, size, shape, and relative position of the functional groups, and this would inevitably affect the specific binding of the template molecule to the imprinted polymer microspheres because of the essential complementary interaction between them. So in the adsorption analysis experiments, the MCP imprinted microspheres rebinding the template was carried out only in toluene, which was used as porogen prior to polymerization.

Binding Specificity of the Imprinted Microspheres

In order to verify whether the imprinted polymer microspheres are selective to MCP, the binding experiments of MCP and some structurally related organophosphorous pesticides to the microspheres were conducted. The different compounds and their structures are listed in Fig. 6. The imprinted polymer microspheres obviously exhibited high binding affinity for MCP, methamidophos and omethoate, while other organophosphorous pesticides showed less binding capacity. As for the nonimprinted polymer microspheres, they showed considerably less binding for most of the analytes although some of them such as methamidophos and omethoate seemed to show high binding to the nonimprinted polymer microspheres. The obtained MCP imprinted microspheres can used to simultaneously enrich the MCP related organophosphorous pesticides from real samples before detection.

The selectivity of the imprinted polymer microspheres might also give some insights into the molecular recognition mechanism in MIP. Table 3 shows that besides the template MCP, methamidophos and omethoate also had relatively high affinity for the imprinted polymer microspheres compared to other organophosphorous pesticides. This could be easily explained by their close homologous to MCP and having the same functional groups in the specific position. It could be seen in Fig. 6 that there is a slight difference between the structure of MCP and that of omethoate. For omethoate, the structural difference is -SC[H.sub.2]- instead of -OC(C[H.sub.3])CH- as in MCP. The sizes of omethoate are smaller than that of MCP, and thus, for methamidophos, the structural difference is obvious compared with MCP, and the size of methamidophos is much smaller than that of MCP. It could be reasonable to assume that omethoate and methamidophos are able to fit into the specific binding sites in the imprinted polymer microspheres. Table 3 also lists that other organophosphorous pesticides had relatively low affinity for the imprinted microspheres compared with MCP. From Fig. 6, the slight structural difference between them and MCP is the functional groups such as O=P[equivalent to], O=C=, and =NH. Figure 6 also showed that the structure of MCP is very similar to that of phosdrin, the only difference between the two compounds is that -NHC[H.sub.3] in MCP is substituted by -OC[H.sub.3] in phosdrin. However, the phosdrin have low affinity for the MCP imprinted microspheres. It indicated that the group of -NH- in the organophosphorous pesticides used played an important role in molecular imprinting and molecular recognition, which could be involved in hydrogen bonding with -COOH of MAA. Figure 6 shows that omethoate and dimethoate almost have the same structure, the only structural difference is the group of S=P[equivalent to] in Dimethoate instead of O=P[equivalent to]. What is more, the affinity of dimethoate for MCP imprinted microspheres was low; however, affinity of omethoate for MCP imprinted microspheres is higher than that of MCP. It indicated that the functional group O=P[equivalent to] also played an important role in the molecular imprinting and molecular recognition. To sum up, we postulate that the imprinting creates binding sites with positioned -NH- group, O=P[equivalent to] group and shape selectivity that recognize the template molecules and the structurally related compounds. From the results of the computer simulation and the Binding specificity of the imprinted microspheres, the possible polymerization procedure of MCP MIP can be calculated as follows (see Fig. 7).

[FIGURE 7 OMITTED]

CONCLUSIONS

Desired uniform MCP imprinted polymer microspheres were synthesized with satisfactory yield by precipitation polymerization, the regular form and narrow size distribution of the microspheres made them easy to handle in various application. The microspheres exhibited highly selective affinity for MCP in toluene, which was used as the porogen in polymerization. Nonspecific binding of different organophosphorous pesticides on the microspheres was low. In a word, it can be concluded that the imprinted polymer microspheres could be used as a good material for analytical purposes, such as for enrichment, purification, and analysis of trace organophosphorous pesticides in foods or soil matrixes.

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Shoulei Yan, (1) Yanjun Fang, (2) Wei Yao, (2) Zhixian Gao (1,2)

(1) College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China

(2) Institute of Hygienic and Environmental Medicine, Tianjin 300050, People's Republic of China

Correspondence to: Zhixian Gao; e-mail: gaozhx@163.com

Contract grant sponsor: National Natural Science Foundation of China; contract grant number: 30371218; Contract grant sponsor: Tianjin Natural Science Foundation; contract grant number: 06YFJMJC07700.
TABLE 1. Composition of the polymerization mixture for MIP and non-MIP.

 Composition of the polymer (mass or volume)
 Imprinted Nonimprinted
Function Compound microspheres microspheres

Template MCP 0.25 mmol 0 mmol
Monomer MAA 1 mmol 1 mmol
Crosslinker EGDMA 5 mmol 5 mmol
Initiator AIBN 10 mg 10 mg
Porogen Toluene 50 mL 50 mL

TABLE 2. Apparent constants of the adsorption data fitted by Langmuir
isotherm model.

 [B.sub.max]
Polymer [K.sub.D] (mol/L) ([micro]mol/g)

Imprinted microspheres 6.62 x [10.sup.-5] 45.52
Nonimprinted microspheres 4.33 x [10.sup.-4] 41.26

TABLE 3. Binding selectivity of the imprinted polymer microspheres and
non-imprinted ones (a).

Analytes MIPs ([micro]mol/g) Non-MIPs ([micro]mol/g)

Monocrotophos 32.6 [+ or -] 2.5 26.2 [+ or -] 2.4
Phosdrin 14.8 [+ or -] 0.6 12.1 [+ or -] 0.8
Methamidophos 34.2 [+ or -] 1.1 31.6 [+ or -] 0.3
Dichlorvos 22.5 [+ or -] 0.2 14.6 [+ or -] 0.9
Phosphamidon 14.6 [+ or -] 0.4 14.4 [+ or -] 1.2
Dimethoate 17.9 [+ or -] 0.7 15.4 [+ or -] 0.6
Omethoate 29.9 [+ or -] 0.9 11.1 [+ or -] 0.3

(a) Nonimprinted polymer microspheres and imprinted polymer
microspheres: 10 mg, respectively; initial concentration of analyte:
0.45 mmol/L; solvent: toluene; volume: 1 mL; incubation time: 24 h, at
room temperature; n = 3.
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Author:Yan, Shoulei; Fang, Yanjun; Yao, Wei; Gao, Zhixian
Publication:Polymer Engineering and Science
Geographic Code:1USA
Date:Sep 1, 2007
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