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Boc-l -tryptophan imprinted polymeric microparticles for bioanalytical applications
Materials Science and Engineering C 29 (2009) 2141–2146
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Boc-L-tryptophan imprinted polymeric microparticles for bioanalytical applications Olympia Kotrotsiou, Sotiria Chaitidou, Costas Kiparissides ⁎ Department of Chemical Engineering, Aristotle University of Thessaloniki and Chemical Process Engineering Research Institute, P.O. Box 472, Thessaloniki, 54124, Greece
a r t i c l e
i n f o
Article history: Received 23 December 2008 Received in revised form 7 April 2009 Accepted 18 April 2009 Available online 24 April 2009 Keywords: Molecularly imprinted polymers Microparticles Protein separation Enantiomer selectivity
a b s t r a c t Molecularly imprinted polymer (MIP) microparticles with good chromatographic characteristics were synthesized via the suspension polymerization process in a single preparative step. Initially, the effects of process parameters (i.e., porogen concentration, polymerization temperature, types and concentrations of functional monomer and cross-linker) on the particle size distribution and particle morphology were experimentally investigated. Subsequently, various MIP microparticles were synthesized in the presence of an amino acid derivative (i.e., boc-Ltryptophan), acting as template molecule. Batch-wise guest binding experiments were then performed to determine the rebinding capacity of the synthesized MIP microparticles towards the template molecule. Competitive binding experiments were also carried out with boc-D-tryptophan (i.e., the enantiomer of boc-Ltryptophan) to assess the selectivity of the imprinted polymer microparticles towards the two enantiomers. Finally, a quantitative description of the experimentally measured rebinding isotherms was obtained using the well-known Freundlich–Langmuir models. The present results clearly demonstrate the potential application of the synthesized MIP microparticles for bioanalytical separation of peptides and proteins since the amino acid templates employed in this study are the building units of larger biomolecules. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Molecular imprinting is a process where a functional monomer and a cross-linker are co-polymerized (i.e., via a free-radical polymerization mechanism) in the presence of a target molecule (i.e., the so-called imprint molecule) that acts as a molecular template. Initially, the monomers are self-assembled around the template molecule through covalent or non-covalent molecular interactions of the functional groups attached to both the template and the functional monomer. This is followed by the copolymerization of the functional monomer with the bifunctional or trifunctional cross-linker. Subsequent removal of the imprint molecules reveals specific binding sites that are complementary in size and shape to the template molecule [1]. Thus, molecular imprinting is an efficient method for the synthesis of polymers with highly specific recognition sites [2–5]. In general, non-covalent imprinting is easy to achieve and applicable to a wide range of template molecules since many of practically important molecules (e.g., pharmaceuticals, herbicides, biologically active substances, and environmental contaminants) possess polar groups (e.g., hydroxyl, carboxyl, amino and amide) that can non-covalently interact with the functional groups of the monomer. The advantage of non-covalent imprinting is that the procedure is relatively simple and the synthesis of covalent conjugates is not required prior to polymerization. Furthermore, the template is easily removed from the polymer under very mild conditions since it is only weakly bound to the polymer
⁎ Corresponding author. Tel.: +302310 99 6211; fax: +2310 99 6198. E-mail address: [email protected] (C. Kiparissides). 0928-4931/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2009.04.014
matrix via non-covalent molecular interactions. Additionally, the guest binding-release, which takes advantage of the non-covalent interactions, is fast. Therefore, non-covalent molecular imprinting has been extensively studied due to its simplicity and versatility [1]. In principle, any kind of non-covalent molecular interactions, including ionic, hydrogen bonding, π–π interactions and hydrophobic interactions, can be effectively employed in molecular imprinting. However, hydrogen bonding is the most appropriate interaction for selective molecular recognition since this type of non-covalent force is highly dependent on both distance and direction between the functional monomer and the template molecule. Thus, monomers that bear the required functional groups (e.g., carboxyl, amino, pyridine, hydroxyl, and amide groups) complementary to the template molecule are commonly chosen for molecular imprinting [6,7]. Up to now, biomolecules of relatively low molecular weight (e.g., amino acid derivatives and oligopeptides) have been employed as templates in molecular imprinting. Commonly, the free-radical copolymerization of the functional monomer with the cross-linker is carried out in bulk. The cross-linked polymer is subsequently ground and sieved to obtain the final product in particulate form [8–12]. The free-radical bulk polymerization process is well established and is especially suitable for bioanalytical applications of MIPs. However, the process involves a number of steps (i.e., polymerization, grounding and sieving) that result in a low overall process efficiency due to the formation, during the polymer grounding step, and subsequent removal of a large amount of fine polymer particles. Commonly, the final polymer yield is less than 50%. Thus, there is a need for the development of alternative methods for the preparation of MIP microparticles with well-defined morphological characteristics [13–15].
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In principle, suspension polymerization offers an attractive alternative to the bulk polymerization process since it results in a high yield of spherical polymer particles. The polymer particles, produced by suspension polymerization, are suitable for different applications, including analytical chromatography, solid phase extraction, and other flow-through applications, since columns and cartridges packed with uniform spherical particles exhibit better flow characteristics than those packed with irregular particles [16]. Molecularly imprinted polymer microparticles were prepared by the inverse suspension polymerization method using a non-aqueous medium (i.e., liquid perfluorocarbon). However, the latter method suffers from a number of disadvantages, including the high cost of the perfluorocarbons and the need of a suitable fluorinated stabilizer that, usually, has to be synthesized. Alternatively, MIP microparticles can be prepared by the inverse suspension polymerization using mineral oil as the continuous phase. Key to the success of this method is that the dispersed liquid phase should not be soluble in the continuous oil phase. This sets some constraints regarding the selection of solvent employed as porogen. Thus, the relatively non-polar solvents (e.g., chloroform, dichloromethane and toluene) that generally favor the development of non-covalent interactions cannot be used with mineral oil. Additionally, MIP microparticles can be prepared by a multi-step process including the grafting of the imprinted polymer to preformed spherical particles (e.g., silica or acrylates) [16,17]. In the present study, the suspension polymerization method, in an aqueous continuous phase, was employed for the synthesis of molecularly imprinted polymer porous microparticles for bioanalytical applications. In particular, MIP microparticles with well-defined morphological characteristics and optimum rebinding properties were prepared using two types of functional monomers (i.e., methacrylic acid (MAA) and methacrylamide (MAm)) and two types of cross-linkers (i.e., ethylene glycol dimethacrylate (EGDMA) and trimethylopropane trimethacrylate (TRIM)). The protected amino acid (boc-L-tryptophan) was used as template molecule. Finally, chloroform dissolved in the dispersed liquid droplets was used as porogen. The effects of process parameters (i.e., porogen concentration, polymerization temperature, types and concentrations of functional monomer and cross-linker) on the particle size distribution and particle morphology were experimentally investigated. Batch-wise guest binding experiments were carried to determine the rebinding capacity of the synthesized MIP microparticles towards the template molecule. Competitive binding experiments were also carried out with boc-D-tryptophan (i.e., the enantiomer of boc-Ltryptophan) to assess the selectivity of the imprinted polymer microparticles towards the two enantiomers. 2. Materials and experimental methods 2.1. Materials Methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), acetonitrile and methanol were purchased from Merck. Chloroform was purchased from Riedel and acetic acid from Carlo Erba. Trimethylopropane trimethacrylate (TRIM) and 2, 2′ azobis-(2-isobutyronitrile) (AIBN) were purchased from Aldrich. Boc-L-tryptophan (boc-L-trp), boc-D-tryptophan (boc-D-trp), methacrylamide (MAm), methanol and poly(vinyl acetate) (PVA) of 100,000 MW and degree of hydrolysis of 86–89% were purchased from Fluka.
Table 1 Selected experimental conditions for the preparation of MIP and NIP microparticles. Polymer
Template
Monomer
Cross-linker
Chloroform
Temperature
MIP 1 NIP 1 NIP 2 MIP 3 NIP 3 MIP 4 NIP 4 MIP 5 NIP 5
553 – – 332 – 497 – 555 –
MAA, 0.62 ml MAA, 0.62ml MAA, 0.62 ml MAA, 0.37 ml MAA, 0.37ml MAA, 0.56 ml MAA, 0.56ml MAm, 0.62 g MAm, 0.62g
EGDMA, 6.88 ml EGDMA, 6.88ml EGDMA, 6.88 ml EGDMA, 4.13 ml EGDMA, 4.13ml TRIM, 6.94 ml TRIM, 6.94ml EGDMA, 6.82 ml EGDMA, 6.82ml
7.5 ml 7.5ml 7.5 ml 10.5 ml 10.5ml 7.5 ml 7.5ml 7.5 ml 7.5ml
60 60 80 60 60 60 60 60 60
mg
mg mg mg
°C °C °C °C °C °C °C °C °C
(2% w/w on the total monomers mass) were dissolved in chloroform. In Table 1, the experimental conditions selected for the preparation of the different types of MIP and NIP microparticles are reported. In all the experimental runs, the molar ratio of the cross-linker to the functional monomer was equal to 5:1 for EGDMA and 3.3:1 for TRIM so that the total double bonds concentration, for both types of the cross-linker, was the same [12]. Accordingly, the organic phase (15 ml in volume, containing the functional monomer, the cross-linker, the porogen, the amino acid derivative and the initiator) was dispersed into an aqueous PVA solution (35 ml in volume, 1% w/w) under the action of a mechanical agitator and a nitrogen atmosphere. Subsequently, the polymerization was carried out, at the specified temperature (i.e., 60 °C or 80 °C), for 24 h. The template molecules were then removed from the polymer microparticles by means of successive washing cycles with a methanol–acetic acid solution (9:1 v/v). The template removal was monitored via UV spectroscopy. Finally, the washed polymer microparticles were conditioned in methanol. Non-imprinted polymeric (NIP) microparticles were also prepared under the same polymerization conditions. 2.3. Characterization of the molecularly imprinted microparticles The size distributions of NIP and MIP microparticles were measured with the aid of a Malvern mastersizer 2000 light scattering instrument. The surface morphology of the microparticles was determined by a JSM6300 scanning electron microscope. The pore size distribution and the specific surface area of the washed microparticles were analyzed by Brunauer–Emmett–Teller (BET) analysis, using a Quantachrome Autosorb Automated Gas Sorption apparatus. 2.4. Binding experiments Equilibrium batch-wise guest binding experiments were conducted to evaluate the rebinding isotherms of both NIP and MIP microparticles. For each rebinding isotherm, five polymer samples of the same weight (i.e., 0.15 g) were employed. The polymer samples were equilibrated for 24 h, under mild agitation conditions, in respective analyte solutions (5 ml each) of known concentrations, using either chloroform or acetonitrile as solvent. Subsequently, the solid polymer microparticles were separated from the solvent, using a membrane filter with a pore diameter of 0.45 µm, and the concentration of the free analyte (F) in the solution was measured by a Lamda 35 UV/VIS spectrometer from Perkin Elmer. Accordingly, the corresponding bound analyte concentration (B) was calculated from the difference of the initial minus the final free analyte concentration. The quantification of boc-L-trp and boc-D-trp was realized at 280 nm.
2.2. Preparation of the molecularly imprinted microparticles 3. Results and discussion The polymerization experiments were carried out in a laboratory scale, water-jacketed glass reactor of 100 ml working volume, equipped with a six-blade impeller, an overhead condenser and a nitrogen purge line. The reaction mixture was thermostated to within ±0.05 °C with the aid of a constant temperature bath. Initially, the template molecule (i.e., boc-L-Trp), the functional monomer, the cross-linker and the initiator
In the suspension polymerization process, the organic phase, containing the functional monomer, the cross-linker, the solvent, the template molecules and the chemical initiator, is initially dispersed, with the aid of an agitation system, in the continuous aqueous phase containing the surface-active agent. Subsequently, the temperature is
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raised to the desired one so that the free-radical copolymerization can be initiated. As the polymerization progresses, the dispersed liquid droplets are gradually transformed from sticky liquid–solid particles into rigid, spherical polymer particles in the size range of 5–500 µm [18]. One of the most important issues in suspension polymerization is the control of the droplet/particle size distribution (DSD/PSD). Note that the initial liquid droplet size distribution depends on the type and concentration of the surface-active agent, the quality of agitation and the physical properties (i.e., densities, viscosities and interfacial tension) of the continuous and dispersed phases. Poly(vinyl acetate) partially hydrolyzed to poly(vinyl alcohol) (PVA), is commonly used as stabilizer. The degree of hydrolysis of PVA strongly affects its surface activity at the organic/aqueous interface. In general, the solubility of PVA in the aqueous phase depends on its molecular weight, the sequence length distribution of vinyl alcohol and vinyl acetate monomers in the copolymer chains, the degree of hydrolysis and temperature [19]. In the present study, a PVA grade of high molecular weight (i.e., 100,000 MW) and medium degree of hydrolysis (i.e., 86–89%) was selected as stabilizer [20,21]. It was found that the composition of the organic phase did not significantly affect the final PSD, which was mainly controlled by the concentration of the surface-active agent and the degree of agitation. In Fig. 1, the measured PSDs of MIP and NIP microparticles, prepared under similar polymerization conditions, are depicted. As can be seen, the two PSDs are almost identical.
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Table 2 Mean particle diameter, specific surface area and total pore volume of the synthesized NIPs. Polymer
Solvent volume
Temperature
Mean particle diameter (µm)
Specific surface area (m2/g)
Total pore volume (cm3/g)
NIP NIP NIP NIP NIP
7.5 ml 7.5 ml 10.5 ml 7.5 ml 7.5 ml
60 80 60 60 60
133 105 139 114 141
360 296 420 45 460
0.2331 0.1783 0.4854 0.0226 0.2878
1 2 3 4 5
°C °C °C °C °C
A critical parameter related to the binding–rebinding properties of MIPs is the microparticles' morphology (i.e., PSD, specific surface area and pore size distribution). In particular, the particle size distribution as well as the specific surface area is key parameter controlling the binding efficiency of MIPs. Thus, the formation of highly porous polymer microparticles can significantly improve their binding efficiency. This can be achieved by the addition of a suitable porogen into the dispersed organic phase prior to its polymerization. It should be pointed out that the use of a thermodynamically good solvent results in highly porous polymer microparticles (i.e., of high specific surface area). On the other hand, the use of a thermodynamically poor solvent leads to the formation of polymer microparticles of low porosity. Moreover, the porogen's polarity must be judiciously chosen, especially in noncovalent imprinting polymerization, to maximize the efficiency of template-functional monomer complex formation. As a result, solvents of very low dielectric constant are commonly selected since they can improve the stability of the hydrogen bonds [22].
Based on the above arguments, chloroform was selected as porogen medium. It was found that the surface morphology and the porosity of the microparticles strongly depended on the solvent amount as well as the polymerization temperature. The effects of solvent concentration and temperature on the mean particle size and the specific surface area of the NIP microparticles are reported in Table 2. From the results of Table 2, it is evident that the specific surface area of the microparticles as well as the total pore volume decrease as the polymerization temperature increases (i.e., NIP 1 and NIP 2). This can be explained by the fact that as the polymerization temperature increases the solvent evaporation rate increases. As a result, the solvent concentration in the polymerized monomer droplets is quickly depleted with concomitant decrease of total pore volume. Note that the presence of porogen in the dispersed polymerizable droplets affects the microstructure (i.e., the pore size distribution and pore volume) of the polymer microparticles. Thus, when the porogen concentration decreases the polymer phase becomes denser (i.e., absence of open pores) resulting in a lower specific surface area [23]. On the other hand, as the solvent concentration increases (i.e., NIP 1 and NIP 3) the polymer phase becomes more porous due to the presence of the porogen for longer polymerization time [24,25]. These results clearly show that both the solvent concentration and the polymerization temperature affect the morphological properties of the polymer microparticles. In Fig. 2, the surface morphology of typical microparticles (NIP 1) is shown. It was found that when the polymerization temperature increased the surface of the microparticles became smoother, indicating a less porous microstructure that was also confirmed by BET measurements. The effect of the cross-linker type on the polymer morphology was also studied. Thus, it was found that, in the presence of the trifunctional cross-linker TRIM, the polymer microparticles had a significantly lower specific surface area and a lower total pore volume (see Table 2, NIP 4: 45 m2/g and 0.02262 cm3/g) than those prepared in the presence of the bifunctional cross-linker EGDMA (see Table 2,
Fig. 1. Characteristic particle size distributions of MIP and NIP microparticles.
Fig. 2. Surface morphology of typical microparticles (NIP 1).
3.1. Effect of process parameters on particle morphology
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NIP 1, NIP 2 and NIP 3). This was attributed to the fact that in the presence of the trifunctional cross-linker a highly cross-linked polymer network, having a low solvent swelling capacity, is obtained [26]. This results in a lower porogen concentration during polymerization and, thus, to a lower pore volume formation. This observation is in agreement with the results of BET analysis that showed a much lower specific surface area in the case of the trifunctional cross-linker (see Table 2, NIP 4). Finally, it was found that the type of the functional monomer and the polymer solubility in the dispersed organic phase had a direct impact on the polymer morphology. More specifically, when MAm was used as functional monomer, porous polymer microparticles, having a large specific surface area (see Table 2, NIP 5: 460 m2/g), were produced due to the higher swelling capacity of the cross-linked polymer network. 3.2. Effect of process parameters on the selectivity and specificity of MIPs Fig. 4. Rebinding capacities of MIPs and NIPs in acetonitrile at 1 mM of initial analyte concentration.
To determine the rebinding capacity of MIPs, batch-wise guest binding experiments were carried out as described before. The binding capacities of the MIP microparticles were compared to those of the NIPs. It was found that the rebinding capacity (selectivity) of the MIP microparticles to the boc-L-tryptophan molecules was higher than that of the NIPs. Moreover, comparative rebinding experiments of boc-Ltryptophan and boc-D-tryptophan enantiomers showed that the binding of boc-L-tryptophan to the MIP microparticles was higher than that of boc-D-tryptophan, proving the specificity of synthesized MIPs towards the template molecule. It should be pointed out that the rebinding capacity of the MIP microparticles was further enhanced when the amount of porogen in the dispersed organic phase was increased (see Table 2 and Fig. 3). That was attributed to the increase of the total pore volume and specific surface area of the microparticles with concomitant increased accessibility of the specific binding sites [27]. The effects of the cross-linker and functional monomer type on the rebinding capacity of MIPs were also examined (see Table 2 and Fig. 3). More specifically, it was found that when the trifunctional cross-linker TRIM was used, the produced MIP microparticles exhibited a higher rebinding capacity and selectivity towards the template molecule despite the decrease in the overall specific surface area. This can be explained by the fact that in the presence of TRIM a more rigid polymer matrix, having a higher number of specific binding sites, is obtained as a result of the more favorable and efficient arrangement of the template molecules with the functional monomer and the trifunctional cross-linker [9,12]. With regard to the type of the functional monomer, it was found that the use of MAm, instead of MAA, resulted in a significant increase of the rebinding capacity of MIPs. Although no general agreement on the relative strength of amide and carboxylic acid hydrogen bonds has been reached, it has been suggested that the amide functional groups may be
more capable of forming stronger hydrogen bonds even in polar solvents than the carboxyl groups [28]. The latter is related to the significant differences in the values of the dielectric constants and dipole moments of the amide and carboxyl groups. This explanation is further supported by the fact that a number of investigations on the enantiomeric recognition of molecularly imprinted polymers, prepared via the bulk polymerization process and using the same functional monomers, reached the same conclusions [26]. Finally, the effect of the solvent polarity on the rebinding properties of the imprinted polymers was investigated (see Fig. 4). Thus, when a more polar solvent (i.e., acetonitrile) was employed in the batch-wise guest binding experiments, the rebinding capacity of both NIPs and MIPs was found to be lower than that obtained in the presence of chloroform. This is explained by the fact that the solvent polarity strongly affects the recognition capabilities of the imprinted polymers [26]. In principle, a solvent of high polarity competes with the template molecule for the functional groups of the recognition sites. As a result, the specific non-covalent interactions between the functional monomer and the template molecule are decreased [29]. Additionally, the degree of polymer swelling strongly depends on the solvent type that, in turn, affects the morphology of the polymer network (i.e., size and shape) and, thus, the relative positions of the functional groups at the template recognition sites [28]. Therefore, it is recommended that the solvent used in the rebinding experiments to be the same with that employed in the polymerization [30]. On the other hand, the use of a high polarity solvent (i.e., acetonitrile) in the rebinding experiments resulted in an increase of the selectivity (i.e., the ratio of the binding capacity of the template to that of its
Fig. 3. Rebinding capacities of MIPs and NIPs in chloroform at 1 mM of initial analyte concentration.
Fig. 5. Comparison of the rebinding isotherms of MIP 4 and NIP 4 microparticles.
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Table 3 Estimated values of the Langmuir model parameters for MIP 1, MIP 3, MIP 4 and MIP 5.
Fig. 6. Measured rebinding isotherms of MIP 1, MIP 3, MIP 4 and MIP 5 microparticles.
analogue) and specificity (i.e., the ratio of the binding capacity of MIPs to that of NIPs) of MIPs. Thus, when the solvent changed from chloroform to acetonitrile, the selectivity and specificity values of MIPs were increased from (1.1–1.5, see Fig. 3) to (1.5–3.1, see Fig. 4). This was especially true when MAA was used as functional monomer. The observed behavior was attributed to the fact that, in the presence of a more polar solvent (i.e., acetonitrile), the rebinding of the template molecules to the preformed binding sites became even more specific due to the less favorable solvent conditions [31]. This phenomenon was less intense when MAm was used as functional monomer. In the latter case, the values of selectivity and specificity did not change significantly, due to the stronger non-covalent interactions between the functional monomer and the template.
Polymer
K, mM− 1
Nt, µmol/g pol.
R2
MIP 1 MIP 3 MIP 4 MIP 5
0.1089 0.0741 0.1132 0.3644
73.7 180.6 109.6 157.6
0.965 0.987 0.924 0.997
have a significant impact on the rebinding capacity of MIPs. It is clear that the rebinding capacity of MIPs increases as the initial amount of chloroform (porogen) increases (see MIP 1 and MIP 3 curves). Similarly, when the cross-linker type changes from bifunctional (EGDMA, MIP 1) to trifunctional (TRIM, MIP 4), the rebinding capacity of MIPs increases. Finally, when MAm is used as functional monomer the microparticles exhibit the highest rebinding capacity. To obtain a quantitative description of the experimentally measured rebinding isotherms, in Fig. 6, different specific binding models can be applied [32]. It is well well-known that the Langmuir–Freundlich equation can describe the specific relationship between the equilibrium concentrations of bound (B) and free (F) analyte in a heterogeneous system of binding sites. B=
Nt aF m 1 + aF m
ð1Þ
where Nt is the total number of binding sites, α is a parameter related to the median binding affinity constant K (K = α1 / m) and m is the heterogeneity index that can vary from 1 (i.e., homogeneous sites) to 0 (i.e., heterogeneous sites). Eq. (1) is a composite of the Langmuir and Freundlich isotherms and, thus, can be reduced to either of the two. That is, for m = 1 Eq. (1) is reduced to the Langmuir isotherm (see Eq. (2)) and to the Freundlich isotherm (Eq. (3)) when either F or α approaches 0.
3.3. Estimation of Langmuir–Freundlich parameters In Fig. 5, the measured rebinding isotherms for MIP 4 and NIP 4 microparticles are depicted. These plots clearly verify the imprinting effect of MIPs over a wide range of variation of the analyte concentration. As can be seen, the MIP microparticles rebind the template molecule (boc-L-tryptophan) more than the NIP microparticles. In Fig. 6, the rebinding isotherms for the four types of the synthesized MIPs (see Table 1) are depicted. The results of Fig. 6, clearly show that the process parameters (i.e., porogen concentration, polymerization temperature, type and concentration of the functional monomer and cross-linker)
Fig. 7. Scatchard plots of MIP 1, MIP 3, MIP 4 and MIP 5 microparticles.
B=
Nt aF 1 + aF m
B = aF :
ð2Þ ð3Þ
The linear form of Eq. (2) (i.e., B /F versus B) was fitted to the experimentally measured rebinding isotherms of Fig. 6, via least-squares regression analysis (see Fig. 7), and the respective parameters K( =α) and Nt in Eq. (2) were accordingly estimated. In Table 3, the numerical
Fig. 8. Freundlich logarithmic rebinding isotherms of MIP 1, MIP 3, MIP 4 and MIP 5 microparticles.
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Table 4 Estimated values of the Freundlich model parameters for MIP 1, MIP 3, MIP 4 and MIP 5. Polymer
m
α
N(K1 − K2), µmol/g pol.
R2
MIP 1 MIP 3 MIP 4 MIP 5
0.739 0.828 0.741 0.607
7.51 12.53 11.47 38.52
11.6 16.7 17.7 58.4
0.996 0.995 0.999 0.978
values of the Langmuir model parameters (K and Nt) and the respective coefficients of determination are reported for the different polymer microparticles (i.e., MIP 1, MIP 3, MIP 4 and MIP 5). As can be seen, the total number of binding sites (Nt) increases when the trifunctional cross-linker TRIM is used instead of the bifunctional cross-linker EGDMA (MIP 1 and MIP 4). Moreover, the numerical value of Nt increases as the initial amount of chloroform (porogen) in the dispersed organic phase increases (MIP 1 and MIP 3) or MAm is used as functional monomer (MIP 1 and MIP 5). Notice that the value of the association constant (K) is mostly affected by the type of the functional monomer rather than the other process parameters (i.e., type of the cross-linker and initial amount of porogen in the dispersed organic phase). The basic assumption of the Langmuir model is that all the binding sites are homogeneous. However, this assumption might not hold true for all cases of MIPs (see numerical values of R2 for MIP 1 and MIP 4 in Table 3). Thus, the continuous distribution model of Freundlich was also applied to calculate the distribution of the heterogeneous binding sites [33]. Accordingly, the logarithmic form of Eq. (3) (i.e., logB versus logF) was fitted to the experimentally measured rebinding isotherms, via least-squares regression analysis (see Fig. 8), and the respective coefficients of determination (R2) were calculated. As can be seen in Table 4, the calculated values of R2 were equal or/and larger than 0.978, indicating a very good fit. Τhe estimated numerical values of the Freundlich model parameters (i.e., α and m) are also shown in Table 4 for the different types of MIP microparticles (i.e., MIP 1, MIP 3, MIP 4 and MIP 5). Notice that the numerical value of m is higher when the initial amount of porogen in the dispersed organic phase increases (see values of m for MIP 1 and MIP 3), indicating the presence of less heterogeneous binding sites. On the other hand, when MAm was employed as functional monomer (i.e., MIP 5), the numerical value of m decreased, indicating a higher number of heterogeneous binding sites. Subsequently, following the original developments of Umbledy et al. [33], the total number of binding sites, N(K1 − K2), over a specific range of variation of the analyte concentration (i.e., 0.86–7.2 mM), corresponding to a specific range of variation of the template-polymer binding affinity constant (i.e., K1 = 1.16 mM− 1 and K2 = 0.139 mM− 1), was calculated using Eq. (4). 2 −m −m K1 − K2 : NðK1 − K2 Þ = a 1 − m
ð4Þ
In Table 4, the calculated values of N(K1 − K2), are reported. As can be seen, these values are much lower than those calculated for Nt in Table 3, using the Langmuir model. The observed differences in the calculated values of Nt and N(K1 − K2) are not uncommon since it is generally accepted that the Freundlich model does not provide an accurate account on the total number of binding sites [33]. Nevertheless, it should be mentioned that the calculated values of N(K1 − K2) for the four types of MIPs follow a similar behavior with that observed for the values of Nt. That is, the value of N(K1 − K2) increases as the initial amount of porogen in the dispersed organic phase increases.
Similarly, the value of N(K1 − K2) increases in the presence of the trifunctional cross-linker TRIM. Finally, the type of the functional monomer has the highest impact on the calculated value of N(K1 − K2). 4. Conclusions The results of the present study show that molecularly imprinted polymeric microparticles (MIPs) with good chromatographic properties can be successfully prepared by the suspension polymerization process. More specifically, it was shown that the rebinding capacity (selectivity) of the MIPs to the boc-L-tryptophan was higher than that of the NIPs. Moreover, comparative rebinding experiments of boc-L-tryptophan and boc-D-tryptophan enantiomers showed that the binding of boc-Ltryptophan to the MIP microparticles was higher than that of boc-Dtryptophan, proving the specificity of the synthesized MIPs towards the template molecule. Consequently, the synthesized MIPs can be used as artificial receptors in analytical applications for the selective recognition and separation of biological molecules. Finally, it was shown that the process parameters (i.e., porogen concentration, types of functional monomer and cross-linker) can have a significant impact on the selectivity and specificity of the synthesized MIPs. Acknowledgement The authors gratefully acknowledge the European Commission for the financial support of the present work under the FP6 Project NMP4CT-2005-516981. References [1] M. Komiyama, T. Takeuchi, H. Mukawa, T. Mukawa, H. Asanuma, Molecular Imprinting: from Fundamentals to Applications, Wiley, Weinheim, 2003. [2] M. Kempe, K. Mosbach, J. Chromatogr. A 694 (1995) 3–13. [3] K. Nilsson, K. Sakaguchi, J. Chromatogr. A 707 (1995) 199–203. [4] G. Wulff, Angew. Chem. Int. Ed. Eng. 34 (1995) 1812–1832. [5] G. Wulff, S. Schanhaff, J. Org. Chem. 56 (1991) 395–400. [6] A. Rachkov, N. Minoura, Biochim. Biophys. Acta 1544 (2001) 255–266. [7] A. Rachkov, N. Minoura, J. Chromatogr. A 889 (2000) 111–118. [8] X. Huang, H. Zou, X. Chen, Q. Luo, L. Kong, J. of Chromatogr. A 984 (2003) 273–282. [9] M. Kempe, Anal. Chemistry 68 (1996) 1948–1953. [10] M. Kempe, K. Mosbach, J. Chromatogr. A 691 (1995) 317–323. [11] R. Sun, H. Yu, H. Luo, Z. Shen, J. Chromatogr. A 1055 (2004) 1–9. [12] C. Yu, K. Mosbach, J. Chromatogr. A 888 (2000) 63–72. [13] A. Cameron, H. Andersson, L. Andersson, R. Ansell, N. Kirsch, I. Nicholls, J. O'Mahony, M. Whitcombe, Journal of Molecular Recognition 19 (2006) 106–180. [14] N. Perez-Moral, A.G. Mayes, Anal. Chim. Acta 504 (2004) 15–21. [15] L. Ye, P. Cormack, K. Mosbach, Anal. Commun. 36 (1999) 35–38. [16] M. Kempe, H. Kempe, Macromol. Rapid Commun. 25 (2004) 315–320. [17] P. Spagel, L.I. Andersson, S. Nilsson, Methods Mol. Biol. 243 (2003) 217–229. [18] C. Kiparissides, Chemical Engin. Science 51 (1996) 1637–1659. [19] C. Kotoulas, C. Kiparissides, Chem. Eng. Sci. 61 (2006) 332–346. [20] E.G. Chatzi, C. Kiparissides, Chem. Eng. Sci. 47 (1992) 445–456. [21] E.G. Chatzi, C. Kiparissides, Chem. Eng. Sci. 49 (1994) 5039–5052. [22] P.A.G. Cormack, A.Z. Elorza, J. Chromatogr. B 804 (2004) 173–182. [23] M.J. Benes, D. Horak, F. Svec, J. Sep. Sci. 28 (2005) 1855–1875. [24] B. Sellergren, Molecularly Imprinted Polymers: Man-made Mimics of Antibodies and Their Applications in Analytical Chemistry, Elsevier Science B.V., Amsterdam, 2001. [25] G.P. Gonzalez, P.F. Hernando, J.S.D. Alegrva, Anal. Chim. Acta 557 (2006) 179–183. [26] J. Cederfur, Y. Pei, M. Zihui, M. Kempe, J. Com. Chem. 5 (2003) 67–72. [27] K. Farrington, E. Magner, F. Regan, Anal. Chim. Acta 566 (2006) 60–68. [28] C. Yu, K. Mosbach, J. Org. Chem. 62 (1997) 4057–4064. [29] D. Spivak, M. Gilmore, K. Shea, J. Am. Chem. Soc. 119 (1997) 4388–4393. [30] L. Ye, R. Weiss, K. Mosbach, Macromolecules 33 (2000) 8239–8245. [31] C. Allender, C. Heard, K. Brain, Chirality 9 (1997) 238–242. [32] M. Yan, O. Ramstrom, Molecularly Imprinted Materials: Science and Technology, Marcel Dekker, New York, 2005. [33] R.J. Umpleby, S.C. Baxter, A.M. Rampey, G.T. Rushton, Y. Chen, K.D. Shimizu, J. Anal. Commun. 804 (2004) 141–149.
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Boc-L-tryptophan imprinted polymeric microparticles for bioanalytical applications Olympia Kotrotsiou, Sotiria Chaitidou, Costas Kiparissides ⁎ Department of Chemical Engineering, Aristotle University of Thessaloniki and Chemical Process Engineering Research Institute, P.O. Box 472, Thessaloniki, 54124, Greece
a r t i c l e
i n f o
Article history: Received 23 December 2008 Received in revised form 7 April 2009 Accepted 18 April 2009 Available online 24 April 2009 Keywords: Molecularly imprinted polymers Microparticles Protein separation Enantiomer selectivity
a b s t r a c t Molecularly imprinted polymer (MIP) microparticles with good chromatographic characteristics were synthesized via the suspension polymerization process in a single preparative step. Initially, the effects of process parameters (i.e., porogen concentration, polymerization temperature, types and concentrations of functional monomer and cross-linker) on the particle size distribution and particle morphology were experimentally investigated. Subsequently, various MIP microparticles were synthesized in the presence of an amino acid derivative (i.e., boc-Ltryptophan), acting as template molecule. Batch-wise guest binding experiments were then performed to determine the rebinding capacity of the synthesized MIP microparticles towards the template molecule. Competitive binding experiments were also carried out with boc-D-tryptophan (i.e., the enantiomer of boc-Ltryptophan) to assess the selectivity of the imprinted polymer microparticles towards the two enantiomers. Finally, a quantitative description of the experimentally measured rebinding isotherms was obtained using the well-known Freundlich–Langmuir models. The present results clearly demonstrate the potential application of the synthesized MIP microparticles for bioanalytical separation of peptides and proteins since the amino acid templates employed in this study are the building units of larger biomolecules. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Molecular imprinting is a process where a functional monomer and a cross-linker are co-polymerized (i.e., via a free-radical polymerization mechanism) in the presence of a target molecule (i.e., the so-called imprint molecule) that acts as a molecular template. Initially, the monomers are self-assembled around the template molecule through covalent or non-covalent molecular interactions of the functional groups attached to both the template and the functional monomer. This is followed by the copolymerization of the functional monomer with the bifunctional or trifunctional cross-linker. Subsequent removal of the imprint molecules reveals specific binding sites that are complementary in size and shape to the template molecule [1]. Thus, molecular imprinting is an efficient method for the synthesis of polymers with highly specific recognition sites [2–5]. In general, non-covalent imprinting is easy to achieve and applicable to a wide range of template molecules since many of practically important molecules (e.g., pharmaceuticals, herbicides, biologically active substances, and environmental contaminants) possess polar groups (e.g., hydroxyl, carboxyl, amino and amide) that can non-covalently interact with the functional groups of the monomer. The advantage of non-covalent imprinting is that the procedure is relatively simple and the synthesis of covalent conjugates is not required prior to polymerization. Furthermore, the template is easily removed from the polymer under very mild conditions since it is only weakly bound to the polymer
⁎ Corresponding author. Tel.: +302310 99 6211; fax: +2310 99 6198. E-mail address: [email protected] (C. Kiparissides). 0928-4931/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2009.04.014
matrix via non-covalent molecular interactions. Additionally, the guest binding-release, which takes advantage of the non-covalent interactions, is fast. Therefore, non-covalent molecular imprinting has been extensively studied due to its simplicity and versatility [1]. In principle, any kind of non-covalent molecular interactions, including ionic, hydrogen bonding, π–π interactions and hydrophobic interactions, can be effectively employed in molecular imprinting. However, hydrogen bonding is the most appropriate interaction for selective molecular recognition since this type of non-covalent force is highly dependent on both distance and direction between the functional monomer and the template molecule. Thus, monomers that bear the required functional groups (e.g., carboxyl, amino, pyridine, hydroxyl, and amide groups) complementary to the template molecule are commonly chosen for molecular imprinting [6,7]. Up to now, biomolecules of relatively low molecular weight (e.g., amino acid derivatives and oligopeptides) have been employed as templates in molecular imprinting. Commonly, the free-radical copolymerization of the functional monomer with the cross-linker is carried out in bulk. The cross-linked polymer is subsequently ground and sieved to obtain the final product in particulate form [8–12]. The free-radical bulk polymerization process is well established and is especially suitable for bioanalytical applications of MIPs. However, the process involves a number of steps (i.e., polymerization, grounding and sieving) that result in a low overall process efficiency due to the formation, during the polymer grounding step, and subsequent removal of a large amount of fine polymer particles. Commonly, the final polymer yield is less than 50%. Thus, there is a need for the development of alternative methods for the preparation of MIP microparticles with well-defined morphological characteristics [13–15].
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In principle, suspension polymerization offers an attractive alternative to the bulk polymerization process since it results in a high yield of spherical polymer particles. The polymer particles, produced by suspension polymerization, are suitable for different applications, including analytical chromatography, solid phase extraction, and other flow-through applications, since columns and cartridges packed with uniform spherical particles exhibit better flow characteristics than those packed with irregular particles [16]. Molecularly imprinted polymer microparticles were prepared by the inverse suspension polymerization method using a non-aqueous medium (i.e., liquid perfluorocarbon). However, the latter method suffers from a number of disadvantages, including the high cost of the perfluorocarbons and the need of a suitable fluorinated stabilizer that, usually, has to be synthesized. Alternatively, MIP microparticles can be prepared by the inverse suspension polymerization using mineral oil as the continuous phase. Key to the success of this method is that the dispersed liquid phase should not be soluble in the continuous oil phase. This sets some constraints regarding the selection of solvent employed as porogen. Thus, the relatively non-polar solvents (e.g., chloroform, dichloromethane and toluene) that generally favor the development of non-covalent interactions cannot be used with mineral oil. Additionally, MIP microparticles can be prepared by a multi-step process including the grafting of the imprinted polymer to preformed spherical particles (e.g., silica or acrylates) [16,17]. In the present study, the suspension polymerization method, in an aqueous continuous phase, was employed for the synthesis of molecularly imprinted polymer porous microparticles for bioanalytical applications. In particular, MIP microparticles with well-defined morphological characteristics and optimum rebinding properties were prepared using two types of functional monomers (i.e., methacrylic acid (MAA) and methacrylamide (MAm)) and two types of cross-linkers (i.e., ethylene glycol dimethacrylate (EGDMA) and trimethylopropane trimethacrylate (TRIM)). The protected amino acid (boc-L-tryptophan) was used as template molecule. Finally, chloroform dissolved in the dispersed liquid droplets was used as porogen. The effects of process parameters (i.e., porogen concentration, polymerization temperature, types and concentrations of functional monomer and cross-linker) on the particle size distribution and particle morphology were experimentally investigated. Batch-wise guest binding experiments were carried to determine the rebinding capacity of the synthesized MIP microparticles towards the template molecule. Competitive binding experiments were also carried out with boc-D-tryptophan (i.e., the enantiomer of boc-Ltryptophan) to assess the selectivity of the imprinted polymer microparticles towards the two enantiomers. 2. Materials and experimental methods 2.1. Materials Methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), acetonitrile and methanol were purchased from Merck. Chloroform was purchased from Riedel and acetic acid from Carlo Erba. Trimethylopropane trimethacrylate (TRIM) and 2, 2′ azobis-(2-isobutyronitrile) (AIBN) were purchased from Aldrich. Boc-L-tryptophan (boc-L-trp), boc-D-tryptophan (boc-D-trp), methacrylamide (MAm), methanol and poly(vinyl acetate) (PVA) of 100,000 MW and degree of hydrolysis of 86–89% were purchased from Fluka.
Table 1 Selected experimental conditions for the preparation of MIP and NIP microparticles. Polymer
Template
Monomer
Cross-linker
Chloroform
Temperature
MIP 1 NIP 1 NIP 2 MIP 3 NIP 3 MIP 4 NIP 4 MIP 5 NIP 5
553 – – 332 – 497 – 555 –
MAA, 0.62 ml MAA, 0.62ml MAA, 0.62 ml MAA, 0.37 ml MAA, 0.37ml MAA, 0.56 ml MAA, 0.56ml MAm, 0.62 g MAm, 0.62g
EGDMA, 6.88 ml EGDMA, 6.88ml EGDMA, 6.88 ml EGDMA, 4.13 ml EGDMA, 4.13ml TRIM, 6.94 ml TRIM, 6.94ml EGDMA, 6.82 ml EGDMA, 6.82ml
7.5 ml 7.5ml 7.5 ml 10.5 ml 10.5ml 7.5 ml 7.5ml 7.5 ml 7.5ml
60 60 80 60 60 60 60 60 60
mg
mg mg mg
°C °C °C °C °C °C °C °C °C
(2% w/w on the total monomers mass) were dissolved in chloroform. In Table 1, the experimental conditions selected for the preparation of the different types of MIP and NIP microparticles are reported. In all the experimental runs, the molar ratio of the cross-linker to the functional monomer was equal to 5:1 for EGDMA and 3.3:1 for TRIM so that the total double bonds concentration, for both types of the cross-linker, was the same [12]. Accordingly, the organic phase (15 ml in volume, containing the functional monomer, the cross-linker, the porogen, the amino acid derivative and the initiator) was dispersed into an aqueous PVA solution (35 ml in volume, 1% w/w) under the action of a mechanical agitator and a nitrogen atmosphere. Subsequently, the polymerization was carried out, at the specified temperature (i.e., 60 °C or 80 °C), for 24 h. The template molecules were then removed from the polymer microparticles by means of successive washing cycles with a methanol–acetic acid solution (9:1 v/v). The template removal was monitored via UV spectroscopy. Finally, the washed polymer microparticles were conditioned in methanol. Non-imprinted polymeric (NIP) microparticles were also prepared under the same polymerization conditions. 2.3. Characterization of the molecularly imprinted microparticles The size distributions of NIP and MIP microparticles were measured with the aid of a Malvern mastersizer 2000 light scattering instrument. The surface morphology of the microparticles was determined by a JSM6300 scanning electron microscope. The pore size distribution and the specific surface area of the washed microparticles were analyzed by Brunauer–Emmett–Teller (BET) analysis, using a Quantachrome Autosorb Automated Gas Sorption apparatus. 2.4. Binding experiments Equilibrium batch-wise guest binding experiments were conducted to evaluate the rebinding isotherms of both NIP and MIP microparticles. For each rebinding isotherm, five polymer samples of the same weight (i.e., 0.15 g) were employed. The polymer samples were equilibrated for 24 h, under mild agitation conditions, in respective analyte solutions (5 ml each) of known concentrations, using either chloroform or acetonitrile as solvent. Subsequently, the solid polymer microparticles were separated from the solvent, using a membrane filter with a pore diameter of 0.45 µm, and the concentration of the free analyte (F) in the solution was measured by a Lamda 35 UV/VIS spectrometer from Perkin Elmer. Accordingly, the corresponding bound analyte concentration (B) was calculated from the difference of the initial minus the final free analyte concentration. The quantification of boc-L-trp and boc-D-trp was realized at 280 nm.
2.2. Preparation of the molecularly imprinted microparticles 3. Results and discussion The polymerization experiments were carried out in a laboratory scale, water-jacketed glass reactor of 100 ml working volume, equipped with a six-blade impeller, an overhead condenser and a nitrogen purge line. The reaction mixture was thermostated to within ±0.05 °C with the aid of a constant temperature bath. Initially, the template molecule (i.e., boc-L-Trp), the functional monomer, the cross-linker and the initiator
In the suspension polymerization process, the organic phase, containing the functional monomer, the cross-linker, the solvent, the template molecules and the chemical initiator, is initially dispersed, with the aid of an agitation system, in the continuous aqueous phase containing the surface-active agent. Subsequently, the temperature is
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raised to the desired one so that the free-radical copolymerization can be initiated. As the polymerization progresses, the dispersed liquid droplets are gradually transformed from sticky liquid–solid particles into rigid, spherical polymer particles in the size range of 5–500 µm [18]. One of the most important issues in suspension polymerization is the control of the droplet/particle size distribution (DSD/PSD). Note that the initial liquid droplet size distribution depends on the type and concentration of the surface-active agent, the quality of agitation and the physical properties (i.e., densities, viscosities and interfacial tension) of the continuous and dispersed phases. Poly(vinyl acetate) partially hydrolyzed to poly(vinyl alcohol) (PVA), is commonly used as stabilizer. The degree of hydrolysis of PVA strongly affects its surface activity at the organic/aqueous interface. In general, the solubility of PVA in the aqueous phase depends on its molecular weight, the sequence length distribution of vinyl alcohol and vinyl acetate monomers in the copolymer chains, the degree of hydrolysis and temperature [19]. In the present study, a PVA grade of high molecular weight (i.e., 100,000 MW) and medium degree of hydrolysis (i.e., 86–89%) was selected as stabilizer [20,21]. It was found that the composition of the organic phase did not significantly affect the final PSD, which was mainly controlled by the concentration of the surface-active agent and the degree of agitation. In Fig. 1, the measured PSDs of MIP and NIP microparticles, prepared under similar polymerization conditions, are depicted. As can be seen, the two PSDs are almost identical.
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Table 2 Mean particle diameter, specific surface area and total pore volume of the synthesized NIPs. Polymer
Solvent volume
Temperature
Mean particle diameter (µm)
Specific surface area (m2/g)
Total pore volume (cm3/g)
NIP NIP NIP NIP NIP
7.5 ml 7.5 ml 10.5 ml 7.5 ml 7.5 ml
60 80 60 60 60
133 105 139 114 141
360 296 420 45 460
0.2331 0.1783 0.4854 0.0226 0.2878
1 2 3 4 5
°C °C °C °C °C
A critical parameter related to the binding–rebinding properties of MIPs is the microparticles' morphology (i.e., PSD, specific surface area and pore size distribution). In particular, the particle size distribution as well as the specific surface area is key parameter controlling the binding efficiency of MIPs. Thus, the formation of highly porous polymer microparticles can significantly improve their binding efficiency. This can be achieved by the addition of a suitable porogen into the dispersed organic phase prior to its polymerization. It should be pointed out that the use of a thermodynamically good solvent results in highly porous polymer microparticles (i.e., of high specific surface area). On the other hand, the use of a thermodynamically poor solvent leads to the formation of polymer microparticles of low porosity. Moreover, the porogen's polarity must be judiciously chosen, especially in noncovalent imprinting polymerization, to maximize the efficiency of template-functional monomer complex formation. As a result, solvents of very low dielectric constant are commonly selected since they can improve the stability of the hydrogen bonds [22].
Based on the above arguments, chloroform was selected as porogen medium. It was found that the surface morphology and the porosity of the microparticles strongly depended on the solvent amount as well as the polymerization temperature. The effects of solvent concentration and temperature on the mean particle size and the specific surface area of the NIP microparticles are reported in Table 2. From the results of Table 2, it is evident that the specific surface area of the microparticles as well as the total pore volume decrease as the polymerization temperature increases (i.e., NIP 1 and NIP 2). This can be explained by the fact that as the polymerization temperature increases the solvent evaporation rate increases. As a result, the solvent concentration in the polymerized monomer droplets is quickly depleted with concomitant decrease of total pore volume. Note that the presence of porogen in the dispersed polymerizable droplets affects the microstructure (i.e., the pore size distribution and pore volume) of the polymer microparticles. Thus, when the porogen concentration decreases the polymer phase becomes denser (i.e., absence of open pores) resulting in a lower specific surface area [23]. On the other hand, as the solvent concentration increases (i.e., NIP 1 and NIP 3) the polymer phase becomes more porous due to the presence of the porogen for longer polymerization time [24,25]. These results clearly show that both the solvent concentration and the polymerization temperature affect the morphological properties of the polymer microparticles. In Fig. 2, the surface morphology of typical microparticles (NIP 1) is shown. It was found that when the polymerization temperature increased the surface of the microparticles became smoother, indicating a less porous microstructure that was also confirmed by BET measurements. The effect of the cross-linker type on the polymer morphology was also studied. Thus, it was found that, in the presence of the trifunctional cross-linker TRIM, the polymer microparticles had a significantly lower specific surface area and a lower total pore volume (see Table 2, NIP 4: 45 m2/g and 0.02262 cm3/g) than those prepared in the presence of the bifunctional cross-linker EGDMA (see Table 2,
Fig. 1. Characteristic particle size distributions of MIP and NIP microparticles.
Fig. 2. Surface morphology of typical microparticles (NIP 1).
3.1. Effect of process parameters on particle morphology
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NIP 1, NIP 2 and NIP 3). This was attributed to the fact that in the presence of the trifunctional cross-linker a highly cross-linked polymer network, having a low solvent swelling capacity, is obtained [26]. This results in a lower porogen concentration during polymerization and, thus, to a lower pore volume formation. This observation is in agreement with the results of BET analysis that showed a much lower specific surface area in the case of the trifunctional cross-linker (see Table 2, NIP 4). Finally, it was found that the type of the functional monomer and the polymer solubility in the dispersed organic phase had a direct impact on the polymer morphology. More specifically, when MAm was used as functional monomer, porous polymer microparticles, having a large specific surface area (see Table 2, NIP 5: 460 m2/g), were produced due to the higher swelling capacity of the cross-linked polymer network. 3.2. Effect of process parameters on the selectivity and specificity of MIPs Fig. 4. Rebinding capacities of MIPs and NIPs in acetonitrile at 1 mM of initial analyte concentration.
To determine the rebinding capacity of MIPs, batch-wise guest binding experiments were carried out as described before. The binding capacities of the MIP microparticles were compared to those of the NIPs. It was found that the rebinding capacity (selectivity) of the MIP microparticles to the boc-L-tryptophan molecules was higher than that of the NIPs. Moreover, comparative rebinding experiments of boc-Ltryptophan and boc-D-tryptophan enantiomers showed that the binding of boc-L-tryptophan to the MIP microparticles was higher than that of boc-D-tryptophan, proving the specificity of synthesized MIPs towards the template molecule. It should be pointed out that the rebinding capacity of the MIP microparticles was further enhanced when the amount of porogen in the dispersed organic phase was increased (see Table 2 and Fig. 3). That was attributed to the increase of the total pore volume and specific surface area of the microparticles with concomitant increased accessibility of the specific binding sites [27]. The effects of the cross-linker and functional monomer type on the rebinding capacity of MIPs were also examined (see Table 2 and Fig. 3). More specifically, it was found that when the trifunctional cross-linker TRIM was used, the produced MIP microparticles exhibited a higher rebinding capacity and selectivity towards the template molecule despite the decrease in the overall specific surface area. This can be explained by the fact that in the presence of TRIM a more rigid polymer matrix, having a higher number of specific binding sites, is obtained as a result of the more favorable and efficient arrangement of the template molecules with the functional monomer and the trifunctional cross-linker [9,12]. With regard to the type of the functional monomer, it was found that the use of MAm, instead of MAA, resulted in a significant increase of the rebinding capacity of MIPs. Although no general agreement on the relative strength of amide and carboxylic acid hydrogen bonds has been reached, it has been suggested that the amide functional groups may be
more capable of forming stronger hydrogen bonds even in polar solvents than the carboxyl groups [28]. The latter is related to the significant differences in the values of the dielectric constants and dipole moments of the amide and carboxyl groups. This explanation is further supported by the fact that a number of investigations on the enantiomeric recognition of molecularly imprinted polymers, prepared via the bulk polymerization process and using the same functional monomers, reached the same conclusions [26]. Finally, the effect of the solvent polarity on the rebinding properties of the imprinted polymers was investigated (see Fig. 4). Thus, when a more polar solvent (i.e., acetonitrile) was employed in the batch-wise guest binding experiments, the rebinding capacity of both NIPs and MIPs was found to be lower than that obtained in the presence of chloroform. This is explained by the fact that the solvent polarity strongly affects the recognition capabilities of the imprinted polymers [26]. In principle, a solvent of high polarity competes with the template molecule for the functional groups of the recognition sites. As a result, the specific non-covalent interactions between the functional monomer and the template molecule are decreased [29]. Additionally, the degree of polymer swelling strongly depends on the solvent type that, in turn, affects the morphology of the polymer network (i.e., size and shape) and, thus, the relative positions of the functional groups at the template recognition sites [28]. Therefore, it is recommended that the solvent used in the rebinding experiments to be the same with that employed in the polymerization [30]. On the other hand, the use of a high polarity solvent (i.e., acetonitrile) in the rebinding experiments resulted in an increase of the selectivity (i.e., the ratio of the binding capacity of the template to that of its
Fig. 3. Rebinding capacities of MIPs and NIPs in chloroform at 1 mM of initial analyte concentration.
Fig. 5. Comparison of the rebinding isotherms of MIP 4 and NIP 4 microparticles.
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Table 3 Estimated values of the Langmuir model parameters for MIP 1, MIP 3, MIP 4 and MIP 5.
Fig. 6. Measured rebinding isotherms of MIP 1, MIP 3, MIP 4 and MIP 5 microparticles.
analogue) and specificity (i.e., the ratio of the binding capacity of MIPs to that of NIPs) of MIPs. Thus, when the solvent changed from chloroform to acetonitrile, the selectivity and specificity values of MIPs were increased from (1.1–1.5, see Fig. 3) to (1.5–3.1, see Fig. 4). This was especially true when MAA was used as functional monomer. The observed behavior was attributed to the fact that, in the presence of a more polar solvent (i.e., acetonitrile), the rebinding of the template molecules to the preformed binding sites became even more specific due to the less favorable solvent conditions [31]. This phenomenon was less intense when MAm was used as functional monomer. In the latter case, the values of selectivity and specificity did not change significantly, due to the stronger non-covalent interactions between the functional monomer and the template.
Polymer
K, mM− 1
Nt, µmol/g pol.
R2
MIP 1 MIP 3 MIP 4 MIP 5
0.1089 0.0741 0.1132 0.3644
73.7 180.6 109.6 157.6
0.965 0.987 0.924 0.997
have a significant impact on the rebinding capacity of MIPs. It is clear that the rebinding capacity of MIPs increases as the initial amount of chloroform (porogen) increases (see MIP 1 and MIP 3 curves). Similarly, when the cross-linker type changes from bifunctional (EGDMA, MIP 1) to trifunctional (TRIM, MIP 4), the rebinding capacity of MIPs increases. Finally, when MAm is used as functional monomer the microparticles exhibit the highest rebinding capacity. To obtain a quantitative description of the experimentally measured rebinding isotherms, in Fig. 6, different specific binding models can be applied [32]. It is well well-known that the Langmuir–Freundlich equation can describe the specific relationship between the equilibrium concentrations of bound (B) and free (F) analyte in a heterogeneous system of binding sites. B=
Nt aF m 1 + aF m
ð1Þ
where Nt is the total number of binding sites, α is a parameter related to the median binding affinity constant K (K = α1 / m) and m is the heterogeneity index that can vary from 1 (i.e., homogeneous sites) to 0 (i.e., heterogeneous sites). Eq. (1) is a composite of the Langmuir and Freundlich isotherms and, thus, can be reduced to either of the two. That is, for m = 1 Eq. (1) is reduced to the Langmuir isotherm (see Eq. (2)) and to the Freundlich isotherm (Eq. (3)) when either F or α approaches 0.
3.3. Estimation of Langmuir–Freundlich parameters In Fig. 5, the measured rebinding isotherms for MIP 4 and NIP 4 microparticles are depicted. These plots clearly verify the imprinting effect of MIPs over a wide range of variation of the analyte concentration. As can be seen, the MIP microparticles rebind the template molecule (boc-L-tryptophan) more than the NIP microparticles. In Fig. 6, the rebinding isotherms for the four types of the synthesized MIPs (see Table 1) are depicted. The results of Fig. 6, clearly show that the process parameters (i.e., porogen concentration, polymerization temperature, type and concentration of the functional monomer and cross-linker)
Fig. 7. Scatchard plots of MIP 1, MIP 3, MIP 4 and MIP 5 microparticles.
B=
Nt aF 1 + aF m
B = aF :
ð2Þ ð3Þ
The linear form of Eq. (2) (i.e., B /F versus B) was fitted to the experimentally measured rebinding isotherms of Fig. 6, via least-squares regression analysis (see Fig. 7), and the respective parameters K( =α) and Nt in Eq. (2) were accordingly estimated. In Table 3, the numerical
Fig. 8. Freundlich logarithmic rebinding isotherms of MIP 1, MIP 3, MIP 4 and MIP 5 microparticles.
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Table 4 Estimated values of the Freundlich model parameters for MIP 1, MIP 3, MIP 4 and MIP 5. Polymer
m
α
N(K1 − K2), µmol/g pol.
R2
MIP 1 MIP 3 MIP 4 MIP 5
0.739 0.828 0.741 0.607
7.51 12.53 11.47 38.52
11.6 16.7 17.7 58.4
0.996 0.995 0.999 0.978
values of the Langmuir model parameters (K and Nt) and the respective coefficients of determination are reported for the different polymer microparticles (i.e., MIP 1, MIP 3, MIP 4 and MIP 5). As can be seen, the total number of binding sites (Nt) increases when the trifunctional cross-linker TRIM is used instead of the bifunctional cross-linker EGDMA (MIP 1 and MIP 4). Moreover, the numerical value of Nt increases as the initial amount of chloroform (porogen) in the dispersed organic phase increases (MIP 1 and MIP 3) or MAm is used as functional monomer (MIP 1 and MIP 5). Notice that the value of the association constant (K) is mostly affected by the type of the functional monomer rather than the other process parameters (i.e., type of the cross-linker and initial amount of porogen in the dispersed organic phase). The basic assumption of the Langmuir model is that all the binding sites are homogeneous. However, this assumption might not hold true for all cases of MIPs (see numerical values of R2 for MIP 1 and MIP 4 in Table 3). Thus, the continuous distribution model of Freundlich was also applied to calculate the distribution of the heterogeneous binding sites [33]. Accordingly, the logarithmic form of Eq. (3) (i.e., logB versus logF) was fitted to the experimentally measured rebinding isotherms, via least-squares regression analysis (see Fig. 8), and the respective coefficients of determination (R2) were calculated. As can be seen in Table 4, the calculated values of R2 were equal or/and larger than 0.978, indicating a very good fit. Τhe estimated numerical values of the Freundlich model parameters (i.e., α and m) are also shown in Table 4 for the different types of MIP microparticles (i.e., MIP 1, MIP 3, MIP 4 and MIP 5). Notice that the numerical value of m is higher when the initial amount of porogen in the dispersed organic phase increases (see values of m for MIP 1 and MIP 3), indicating the presence of less heterogeneous binding sites. On the other hand, when MAm was employed as functional monomer (i.e., MIP 5), the numerical value of m decreased, indicating a higher number of heterogeneous binding sites. Subsequently, following the original developments of Umbledy et al. [33], the total number of binding sites, N(K1 − K2), over a specific range of variation of the analyte concentration (i.e., 0.86–7.2 mM), corresponding to a specific range of variation of the template-polymer binding affinity constant (i.e., K1 = 1.16 mM− 1 and K2 = 0.139 mM− 1), was calculated using Eq. (4). 2 −m −m K1 − K2 : NðK1 − K2 Þ = a 1 − m
ð4Þ
In Table 4, the calculated values of N(K1 − K2), are reported. As can be seen, these values are much lower than those calculated for Nt in Table 3, using the Langmuir model. The observed differences in the calculated values of Nt and N(K1 − K2) are not uncommon since it is generally accepted that the Freundlich model does not provide an accurate account on the total number of binding sites [33]. Nevertheless, it should be mentioned that the calculated values of N(K1 − K2) for the four types of MIPs follow a similar behavior with that observed for the values of Nt. That is, the value of N(K1 − K2) increases as the initial amount of porogen in the dispersed organic phase increases.
Similarly, the value of N(K1 − K2) increases in the presence of the trifunctional cross-linker TRIM. Finally, the type of the functional monomer has the highest impact on the calculated value of N(K1 − K2). 4. Conclusions The results of the present study show that molecularly imprinted polymeric microparticles (MIPs) with good chromatographic properties can be successfully prepared by the suspension polymerization process. More specifically, it was shown that the rebinding capacity (selectivity) of the MIPs to the boc-L-tryptophan was higher than that of the NIPs. Moreover, comparative rebinding experiments of boc-L-tryptophan and boc-D-tryptophan enantiomers showed that the binding of boc-Ltryptophan to the MIP microparticles was higher than that of boc-Dtryptophan, proving the specificity of the synthesized MIPs towards the template molecule. Consequently, the synthesized MIPs can be used as artificial receptors in analytical applications for the selective recognition and separation of biological molecules. Finally, it was shown that the process parameters (i.e., porogen concentration, types of functional monomer and cross-linker) can have a significant impact on the selectivity and specificity of the synthesized MIPs. Acknowledgement The authors gratefully acknowledge the European Commission for the financial support of the present work under the FP6 Project NMP4CT-2005-516981. References [1] M. Komiyama, T. Takeuchi, H. Mukawa, T. Mukawa, H. Asanuma, Molecular Imprinting: from Fundamentals to Applications, Wiley, Weinheim, 2003. [2] M. Kempe, K. Mosbach, J. Chromatogr. A 694 (1995) 3–13. [3] K. Nilsson, K. Sakaguchi, J. Chromatogr. A 707 (1995) 199–203. [4] G. Wulff, Angew. Chem. Int. Ed. Eng. 34 (1995) 1812–1832. [5] G. Wulff, S. Schanhaff, J. Org. Chem. 56 (1991) 395–400. [6] A. Rachkov, N. Minoura, Biochim. Biophys. Acta 1544 (2001) 255–266. [7] A. Rachkov, N. Minoura, J. Chromatogr. A 889 (2000) 111–118. [8] X. Huang, H. Zou, X. Chen, Q. Luo, L. Kong, J. of Chromatogr. A 984 (2003) 273–282. [9] M. Kempe, Anal. Chemistry 68 (1996) 1948–1953. [10] M. Kempe, K. Mosbach, J. Chromatogr. A 691 (1995) 317–323. [11] R. Sun, H. Yu, H. Luo, Z. Shen, J. Chromatogr. A 1055 (2004) 1–9. [12] C. Yu, K. Mosbach, J. Chromatogr. A 888 (2000) 63–72. [13] A. Cameron, H. Andersson, L. Andersson, R. Ansell, N. Kirsch, I. Nicholls, J. O'Mahony, M. Whitcombe, Journal of Molecular Recognition 19 (2006) 106–180. [14] N. Perez-Moral, A.G. Mayes, Anal. Chim. Acta 504 (2004) 15–21. [15] L. Ye, P. Cormack, K. Mosbach, Anal. Commun. 36 (1999) 35–38. [16] M. Kempe, H. Kempe, Macromol. Rapid Commun. 25 (2004) 315–320. [17] P. Spagel, L.I. Andersson, S. Nilsson, Methods Mol. Biol. 243 (2003) 217–229. [18] C. Kiparissides, Chemical Engin. Science 51 (1996) 1637–1659. [19] C. Kotoulas, C. Kiparissides, Chem. Eng. Sci. 61 (2006) 332–346. [20] E.G. Chatzi, C. Kiparissides, Chem. Eng. Sci. 47 (1992) 445–456. [21] E.G. Chatzi, C. Kiparissides, Chem. Eng. Sci. 49 (1994) 5039–5052. [22] P.A.G. Cormack, A.Z. Elorza, J. Chromatogr. B 804 (2004) 173–182. [23] M.J. Benes, D. Horak, F. Svec, J. Sep. Sci. 28 (2005) 1855–1875. [24] B. Sellergren, Molecularly Imprinted Polymers: Man-made Mimics of Antibodies and Their Applications in Analytical Chemistry, Elsevier Science B.V., Amsterdam, 2001. [25] G.P. Gonzalez, P.F. Hernando, J.S.D. Alegrva, Anal. Chim. Acta 557 (2006) 179–183. [26] J. Cederfur, Y. Pei, M. Zihui, M. Kempe, J. Com. Chem. 5 (2003) 67–72. [27] K. Farrington, E. Magner, F. Regan, Anal. Chim. Acta 566 (2006) 60–68. [28] C. Yu, K. Mosbach, J. Org. Chem. 62 (1997) 4057–4064. [29] D. Spivak, M. Gilmore, K. Shea, J. Am. Chem. Soc. 119 (1997) 4388–4393. [30] L. Ye, R. Weiss, K. Mosbach, Macromolecules 33 (2000) 8239–8245. [31] C. Allender, C. Heard, K. Brain, Chirality 9 (1997) 238–242. [32] M. Yan, O. Ramstrom, Molecularly Imprinted Materials: Science and Technology, Marcel Dekker, New York, 2005. [33] R.J. Umpleby, S.C. Baxter, A.M. Rampey, G.T. Rushton, Y. Chen, K.D. Shimizu, J. Anal. Commun. 804 (2004) 141–149.