Investigation of the nature of MIP recognition: The development and characterisation of a MIP for Ibuprofen

Biosensors and Bioelectronics 22 (2007) 1138–1146

Investigation of the nature of MIP recognition: The development and characterisation of a MIP for Ibuprofen Keith Farrington, Fiona Regan ∗ School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland Received 20 January 2006; received in revised form 19 May 2006; accepted 20 June 2006 Available online 2 October 2006

Abstract This paper describes the rational design, generation and testing of a molecularly imprinted polymer specific for Ibuprofen. Ibuprofen is a member of the class of drugs termed non-steroidal anti-inflammatory drugs (NSAIDS). In the present study, Ibuprofen was used as a template molecule for the preparation of molecularly imprinted polymers. A MIP has been produced which is capable of recognising Ibuprofen in aqueous media. Furthermore, Ibuprofen can be selectively extracted from aqueous conditions by molecularly imprinted solid phase extraction (MISPE). Recoveries were typically high (>80%) and good selectivity for Ibuprofen over structurally related analogues was seen. Moreover, the nature of the recognition between MIP and template has been investigated by NMR and molecular modelling to analyse whether or not it is possible to predict how well a given MIP will perform under set conditions. In addition, the physical characteristics of the MIP have been investigated including the particle size distribution on exposure of the MIP to different solvents. This has been related to the ability of the MIP to rebind Ibuprofen under the same conditions. The data from the characterisation of the MIP has been used to further enhance the understanding of the nature of MIP recognition. © 2006 Elsevier B.V. All rights reserved. Keywords: Molecularly imprinted polymers; NMR; Ibuprofen; Solid phase extraction; Particle size; Swelling

1. Introduction Molecular imprinting technology involves the generation of cavities containing functional sites within a highly crosslinked polymer matrix. The construction of these “chemical imprints” is based initially on the formation of a templatefunctional monomer pre-polymerisation complex. The polymerisation of this complex then generates a defined spatial configuration. This highly cross-linked polymer matrix is a rigid inflexible network. The template molecule is subsequently removed from the network leaving behind the cavities which are complementary in chemical functionality, size and spatial arrangement. Recently, molecularly imprinted synthetic receptors, capable of recognising analytes under aqueous conditions has achieved significant attention (Dirion et al., 2003; Cheng et al., 2004; Carabias-Martinez et al., 2000) This aspect of molecular imprinting is particularly relevant to molecularly imprinted

∗

Corresponding author. Tel.: +353 1 7005765; fax: +353 1 7005503. E-mail address: [email protected] (F. Regan).

0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.06.025

solid phase extraction (MISPE) techniques as applied to environmental samples (Caro et al., 2003; Watabe et al., 2005), samples containing biomolecules (as reviewed by Molinelli et al. (2005)) and pharmaceutical preparations (Martin et al., 2004). Caro et al. (2005) have developed a molecularly imprinted solid phase extraction method for determination of non-steroidal antiinflammatory drugs in river water for which Ibuprofen was used as the template molecule. The mechanism of template recognition by the MIP under aqueous conditions has been shown to be essentially dependent on factors that affect the polymer formation and arrangement as shown by Turner et al. (2004). Three important factors that affect the formation of the prepolymerisation complex are: the type of functional monomer employed for complexation with the template; the relative ratio of functional monomer to template will determine the degree of complexation of the template and finally, the solvent (or porogen). The latter will regulate important features in the prepolymerisation complex such as hydrogen bond formation and the formation of pores of differing sizes and volumes. The functions of the porogen are to dissolve all of the components of the polymer and to allow the arrangement of the template-functional monomer pre-polymerisation complex. Moreover, the nature

K. Farrington, F. Regan / Biosensors and Bioelectronics 22 (2007) 1138–1146

of the porogen employed for the rebinding step will influence events such as the relative swelling of the polymer(s). An important aspect related to the polymer swelling which has achieved relatively little attention to date, is the particle size distribution of the MIP. The actual particle sizes of the polymer itself are not uniform. This is related to the fact that in traditional bulk polymerisation, following crushing and grinding the polymer is sieved to particle sizes within the range 25 and 75 ␮m. Numerous reports have dealt with alternative MIP formats such as sol–gel MIPs (Olwill et al., 2004) or suspension polymerisation (Fairhurst et al., 2004). However when bulk polymerisation is the method used the MIP particles will be subject to increases in size (diameter) depending on the type of solvent used in rebinding. Studies investigating swelling (Turner et al., 2004) have centered on the fact that swelling of the polymer will cause changes in the binding site cavities. The latter leads to a loss of recognition although this is not likely to affect the individual functionalities but will affect interactions and will change the arrangement of the functional groups, i.e. will alter the proximity of the interacting groups. It is probable that shape complementarity is at least as important a factor in affecting analyte rebinding as the functional sites themselves. In order to successfully compete with antibodies as viable tools for use in analytical assays, it is imperative that MIPs are capable of functioning under aqueous conditions. Typically, MIPs have performed well when used in the same solvent of polymerisation i.e. a higher level of rebinding has been observed in contrast to when a different rebinding solvent is used. Of vital importance in generating a MIP competent in an aqueous environment such as water or urine is the choice of porogen (Chassaing et al., 2004). MIPs manufactured using porogens typically employed in molecular imprinting such as chloroform, dichloromethane or toluene are expected to be incompatible with template-MIP rebinding under aqueous conditions. To produce a MIP capable of recognising a given template in an aqueous environment a relatively polar aprotic solvent is required. Furthermore, in order to strengthen the pre-polymerisation complex certain solvents with a high capacity to form hydrogen bonds with either the template or the functional monomer should not be employed. Typical polar aprotic solvents include acetonitrile, dimethylsulfoxide (DMSO) and dimethylformamide (DMF). DMSO has a high propensity to form hydrogen bonds however. Turner et al. (2004) have used DMF in the manufacture of a MIP capable of recognising ochratoxin A under aqueous conditions. The solvent was chosen due to its ability to dissolve all of the components of the polymerisation mixture and its expected compatibility with aqueous conditions. Lubke et al. (2000), employed both DMSO and THF as porogen in the development of a MIP for ampicillin. The MIP prepared in DMSO was shown to be capable of efficient binding of ampicillin from aqueous buffers. When investigating the nature of the pre-polymerisation complex NMR is one of the most useful tools available. NMR has been employed to study the nature of MIP recognition in a MIP produced for anti-17␣-ethnylstradiol (Idziak et al., 2001). NMR has been employed by O’Mahony et al. (2005) to investigate hydrophobicity and ion pair interactions as forces promoting

1139

selectivity by imprinted polymers. Further, Lubke et al. (2000) have studied complexation between a synthetic monomer 2-(4vinylphenoxy)-3,5,6-trichlorobenzoquinone and ampicillin in d6 -DMSO showing a chemical shift of the proton associated with the amino group. This is indicative of hydrogen bond formation. This paper describes the development of a molecularly imprinted polymer for Ibuprofen by using the tools of rational design—namely NMR and molecular modelling is described. The effectiveness of the MIP is assessed by both batch rebinding studies and solid phase extraction. An investigation into nature of the rebinding under aqueous and organic conditions and by altering the pH is described. Studies have been performed on the physical characteristics of the polymer itself. This includes the degree of swelling on exposure to different solvents. The measurement of average pore diameters and pore volumes of successful and unsuccessful imprints are compared. The latter includes the degree of swelling on exposure to different solvents are discussed and, average pore diameters and pore volumes of successful and unsuccessful imprints are compared. In contrast to the production of a range of polymers based on the trial and error approach, we have designed the imprinted polymer based on data obtained from NMR spectroscopy and molecular modelling studies using the HyperchemTM molecular modelling software. The authors propose that certain physical characteristics of the polymer are important in understanding the nature of recognition in MIPs. 2. Experimental 2.1. Materials Acetonitrile, methanol, dimethylformamide (DMF), water (HPLC grade), Ibuprofen, methacrylic acid, (MAA), ethylene glycol dimethacrylate, 2-vinylpyridine, sodium phosphate were purchased from Sigma–Aldrich (Dublin, Ireland). Azoisobutyronitrile was purchased from Fisher Scientific (UK). Deuretated acetonitrile, methanol and dimethylsulfoxide (DMSO) were purchased from Apollo Scientific (Bredbury, UK) 2.2. Instrumentation High performance liquid chromatography (HPLC) was performed on a Hewlett-Packard 1050 (HP 1050) LC system (pump, injector, detector) employing Chemstation software. The variable wavelength detector was operated at 220 nm for Ibuprofen determinations. A 10 ␮l injection volume was used. Separations were performed on a 25 cm × 4.6 mm, 5 ␮m Alltech Bravda BDS C18 column. 2.3. HPLC measurements The mobile phase was a mixture of acetonitrile and 0.05 M sodium phosphate (80:20 (v/v) and the flow was maintained at 1.0 ml min−1 . For quantitation of Ibuprofen, a standard curve was constructed. Standard solutions of Ibuprofen, at the following concentrations: 100, 50, 25, 12.5, 5, 1, 0.5 and 0.25 ␮g ml−1

1140

K. Farrington, F. Regan / Biosensors and Bioelectronics 22 (2007) 1138–1146

were determined five consecutive times and the average peak areas were plotted against the concentration 2.4. Synthesis of polymers A mole ratio of 1:4 template:functional monomer was used during there polymerisation processes. A 206.28 mg weight of Ibuprofen was dissolved in 4 ml of DMF in a borosilicate test tube. To this was added either 4 mmol of MAA (344.36 mg) or 2Vpy (420.56 mg) or allylamine (228.40 mg). The mixture was stirred for 5 min. To this 20 mmol of EGDMA (4167 ␮l) was added and the mixture stirred for 5 min at room temperature. A 50 mg quantity of AIBN was added and the mixture was stirred for a further 10 min to ensure complete dissolution of all components. The mixture was sonicated for 10 min and sparged with N2 for 5 min to degas it. The tubes were plugged under vacuum and gradually heated to 60 ◦ C in a water bath and held for 16 h. A gel type matrix was observed after 2–3 h before solidification to form the polymer monolithic matrix. Following polymerisation, the tubes were smashed and the resultant hard, polymer was removed. The polymer was broken up before being ground with a mortar and pestle and finally ground by hand. The ground particles were sieved to between 75 and 45 ␮m in size. In order to verify that retention of template was due to molecular recognition and not to non-specific binding, a control (nonimprinted polymer) was prepared following the same procedure, including washing, but with the omission of the target molecule, i.e. Ibuprofen. To remove the template bound within the polymer matrix, the polymer was repeatedly washed with a mixture of hot methanol and acetic acid (10%) with vigorous mixing. The procedure was repeated until no trace of Ibuprofen could be detected in the washing solution. The extracted particles were then washed with methanol alone numerous times to remove residual acetic acid and then oven dried at 70 ◦ C for 6 h. 2.5. NMR studies All NMR spectra were recorded on a Brucker 500 MHz instrument at 25 ◦ C. The volume used for analysis was 750 ␮l and the concentrations of both Ibuprofen (template) and functional monomer were 0.04 M. Typically, the Ibuprofen was dissolved in the appropriate deuterated solvent and the monomer was added. For the purpose of spectral clarity, 2-vinylpyridine was replaced with deuterated pyridine. Data analysis was performed using the Mestrec software program. (http://www.mestrec.com). 2.6. Molecular modelling studies The molecular modelling was performed using the Hyperchem software (Hypercube inc. Gainsville, Florida, US). The structure of Ibuprofen and the functional monomers (methacrylic acid and 2-vinylpyridine) were drawn in the Hyperchem program and minimised to the lowest energy conformation allowed by the molecular mechanics (MM+) method and then refined using the semi-empirical mechanic (PM3) method. The

conformation of lowest energy was refined with an ab initio (3-21 G) quantum mechanic basis set. To analyse possible interactions between template and functional monomer and for calculation of binding energies, the Amber MM method was used. The force–field was set up with constant dielectric, van der Waals and electrostatic scale factors at 0.5. The calculation of the binding energies (between template and monomer) was performed using Eq. (1) (below): binding energy = E = [Ecomplex − Etemplate − Emonomer ] (1) The methods employed for the modelling studies were based on those of Chapuis et al. (2004) and Dong et al. (2005). For calculation of the molecular volumes the Accerlys DS Viewer program was used. (http://www.accerlys.com) 2.7. Batch rebinding studies For batch rebinding studies, a 30 mg aliquot of the dry polymer was placed in a 1.5 ml volume eppendorf tube. A 1 ml volume of a solution of Ibuprofen at 20 ␮g/ml was added. Uptake was assessed in the following in water with increasing volumes of acetonitrile using a 4 h incubation period. Following this, the sample was centrifuged at high speed for 5 min and the Ibuprofen present in the supernatant was quantified. The value obtained was subtracted from the initial concentration of Ibuprofen added and this was taken as the amount of Ibuprofen rebound. This was expressed as a percentage. All uptake studies were carried out at room temperature. 2.8. Solid phase extraction (SPE) studies Empty solid phase extraction cartridges were washed with methanol before use. The cartridges were then dried and 200 mg of the dry polymer (or corresponding control polymer) was placed in between two frits. The cartridge was washed with 10% acetic acid in methanol and then with methanol four times until no trace of Ibuprofen could be detected. All SPE experiments were performed on a VacMaster SPE processing station manifold. Before analyte loading, the polymer was conditioned with 1 ml methanol, 1 ml acetonitrile and 1 ml water. In the loading step, 20 ␮g/ml Ibuprofen in 10% acetonitrile in water was passed through. For the washing step, 1 ml of 1% triethylamine (TEA) in acetonitrile was used and Ibuprofen was eluted with 1 ml of methanol. The experiments were repeated in triplicate. After each experiment the cartridge was regenerated by washing with 3 ml of water and 3 ml of methanol. 2.9. Sample preparation NurofenTM liquid capsules were used for the study. Each capsule contained 200 mg of Ibuprofen. The contents of each capsule were dissolved in 10 ml acetonitrile and made up to 2 l with water to give a final concentration of 100 ␮g/ml. This was further diluted to a concentration of 20 ␮g/ml. This was

K. Farrington, F. Regan / Biosensors and Bioelectronics 22 (2007) 1138–1146

1141

passed through the SPE cartridge packed with appropriate polymer, washed and eluted as described above.

Table 1 Binding scores calculated for various functional monomers with Ibuprofen in vacuo by the Hyperchem molecular modelling program

2.10. Pore size distribution studies

Polymer

Functional monomer

Binding energy (kJ mol−1 )

IBU 1 IBU 2 – – – IBU 3 –

Allylamine 2-Vinylpyridine 4-Vinylpyridine Acrylamide 1-Vinylimidazole Methacrylic acid HEMA

−87.94 −55.12 −54.62 −43.50 −12.01 −11.80 −3.74

Pore size distribution and surface areas of the washed polymers were analysed by the Brunauer–Emmett–Teller (BET) method. The analysis performed on an ASAP 2010 from RMIT Applied Chemistry (Micromeritic). A 300 mg quantity of the dry polymer was used for analysis. The sample was degassed for 4 h at 150 ◦ C before analysis. Relevant information obtained from this method included surface area, total pore volume and average pore diameter of the polymer. 2.11. Particle size studies Particle size analysis was carried out on a Malvern Mastersizer 2000 instrument equipped with light scattering technology. The IbuprofenIbuprofen MIP (100 mg) was exposed to 20 ml of the solvent (methanol, acetonitrile or water) for 24 h before analysis. The particle size of the MIP was also acquired using the dry mode for comparison. 2.12. Swelling studies Analysis of polymer swelling was conducted based on the method described by Lu et al. (2004). Typically, 200 mg (the same mass used for SPE studies) was weighed into an empty SPE cartridge. A 1 ml aliquot of the appropriate solvent was added and left for 4 h at room temperature. Excess solvent was then removed and weights recorded. The swelling ratio was worked out using the following equation: Mw − M d Md

Ibuprofen would undergo electrostatic interaction and potentially hydrogen bonding with the nitrogen of allylamine and this is shown in Fig. 2. The dashed lines are representative of hydrogen bond formation. On washing the allylamine MIP (MIP 1), it was found that template bleeding was observed consistently. This continued despite vigorous washing in hot methanol with 10% acetic acid and also washing in acetonitrile. The template bleeding may be explained by the very high value obtained for the binding energy of allylamine. This is indicative of a strong complex formation between allylamine and Ibuprofen. When DMF was switched for acetonitrile as the porogen, Ibuprofen precipitated from solution during the nitrogen sparging process. Due to these physico-chemical factors, the development of a MIP using allylamine as functional monomer was not pursued.

(2)

where Mw is the mass of the wet polymer and Md is the mass of the dry polymer. 3. Results and discussion 3.1. Analysis of the pre-polymerisation complex Two polymers were produced using the methodology described. The porogen chosen for analysis was dimethylformamide (DMF). This porogen was chosen due to its ability to dissolve Ibuprofen (and all of the components of the polymerisation mixture). As a polar aprotic solvent, it was expected that DMF would be more compatible with aqueous environments than less polar solvents such as toluene or dichloromethane. DMF is classified as having a medium strength ability to form hydrogen bonds (Sellergren, 2001). Based on the molecular modelling data obtained (Table 1), allylamine was deemed to be the most suitable monomer for complexation with Ibuprofen due to its relatively high binding energy value. Given the presence of a carboxylic acid functionality on the Ibuprofen molecule (Fig. 1a) it was expected that

Fig. 1. Structures of Ibuprofen (a) and its structural analogues ketoprofen (b) and naproxen (c).

1142

K. Farrington, F. Regan / Biosensors and Bioelectronics 22 (2007) 1138–1146

Fig. 2. The hyperchem derived energy minimized structure of Ibuprofen and allylamine. The presence of hydrogen bonds is indicated by the dashed lines.

The monomer(s) tested, that demonstrated the next highest binding score, were both 2- and 4-vinylpyridine. Both of these molecules were expected to demonstrate hydrogen bonding with the –COOH group of Ibuprofen. Furthermore, recent studies have attested to the shape complementarity of the prepolymerisation complex being an important aspect for the generation of high affinity binding sites in the resultant polymer (Kim and Spivak, 2003). Moreover, given the presence of aromatic rings in both the template and functional monomer, it is possible that there is an electrostatic interaction between the two rings of the molecules. The HyperchemTM molecular modelling package was employed to model potential interactions of the two functional monomers (2-vinylpyridine and methacrylic acid—another commonly used monomer in molecular imprinting). Methacrylic acid was shown to have a significantly lower binding score than 2-vinylpyridine and thus was not expected to form as strong a complex with Ibuprofen. Table 1 shows that a stronger complex was attained when using 2-vinylpyridine. This result is in agreement with that of Haginaka et al. (1999) and Caro et al. (2004) who developed an imprinted polymer for Ibuprofen and naproxen respectively using 4-vinylpyridine as functional monomer finding high selectivity. Moreover, given the position of the complex structure with the lowest energy minimum (Fig. 3) the earlier theory of electrostatic interaction between the aromatic rings of monomer and template looked increasingly likely from modelling investigations. To investigate this more fully 1 H NMR experiments were carried out. From this study, aromatic protons are seen further downfield due to the deshielding effect of the ring current on the aromatic ring. The latter contributes strongly to the strength of the magnetic field experienced by the protons located in the plane of the ring. At either side of the ring however, the extra magnetic field produced by the second ring opposes the applied magnetic field. Consequentially, protons that interact with this field will be deshielded and will resonate further upfield (O’Mahony et al., 2005) As a result of this phenomenon, if the aromatic protons of Ibuprofen undergo electrostatic interaction (or ␲–␲ delocalisation) with the aromatic protons of the 2-vinylpyridine monomer then an upfield shift will be observed. Fig. 4 illustrates this point. An upfield shift was observed for all aromatic protons on addition of deuterated pyridine to a solution of Ibuprofen in deuterated dimethylformamide. Interestingly, this

Fig. 3. The hyperchem derived probable ␲–␲ stacking conformations for Ibuprofen and vinylpyridine showing: (a) the face to face stacking and (b) the edge on conformation.

Fig. 4. NMR (in deuterated DMF) showing the chemical shift of the aromatic protons of Ibuprofen on formation of ␲–␲ stacking conformation of Ibuprofen with vinylpyridine functional monomer. Above shows Ibuprofen alone. Below shows Ibuprofen with deuterated pyridine 1:10 ratio. The peak at 8.03 is deuterated DMF.

K. Farrington, F. Regan / Biosensors and Bioelectronics 22 (2007) 1138–1146

1143

effect was not observed when deuterated dimethylformamide was replaced with either deuterated methanol or dimethylsulfoxide. This would suggest that Ibuprofen undergoes a stronger interaction with the latter two solvents than with the vinylpyridine, i.e. the Ibuprofen is completely solvated in these solvents which precludes the access of vinylpyridine. Given the nature of the pre-polymerisation complex, this provides two points of interaction. Firstly, there is the chemical functionality of the ␲–␲ stacking effect. Secondly, there is the shape complementarity brought about by the proximity of the functional monomer and the incorporation of the complex into the cross-linked matrix of the resultant nano-cavities of the MIP. As pointed out by Turner et al. (2004), the shape of the binding cavity is likely to be as important in terms of rebinding as the distribution of the functional groups themselves. Joshi et al. (1998) demonstrated that the shape and size of the cavity in addition to hydrogen bonding determines the selectivity of separation of phenols from anisole. Spivak et al. (2004) have studied in detail structure-binding relationships between α-methylbenzylamine derivatives and have provided substantiation of shape selectivity in non-covalent MIPs. Shape complementarity then qualifies the prediction of molecular recognition in terms of rebinding based on the data obtained from the study of the pre-polymerisation complex. 3.2. Rebinding studies For the purpose of this work, rebinding is defined as the percentage uptake of Ibuprofen by the MIP when a defined concentration of Ibuprofen is added to a known weight of MIP. The porogen used for the generation of the MIPs was DMF. Due to the polar aprotic nature of this solvent, it was expected that DMF would be more compatible with rebinding in an aqueous system. Initially, the rebinding was assessed in the solvent in which the MIP was prepared, i.e. DMF. Fig. 5a shows that the % rebinding was high (<85%) for Ibuprofen in DMF at the three concentrations tested, 10, 20 and 50 ␮g/ml. The uptake of Ibuprofen on the control polymer was low and below 10% for the same concentrations of template. The rebinding of Ibuprofen to the MIPs was assessed in an aqueous environment with an increasing ratio of acetonitrile. Given the relative insolubility of Ibuprofen in water, it was necessary to include acetonitrile in each rebinding solution. Hence, the ratio of the rebinding solution varied from 100% acetonitrile to 10:90 acetonitrile:water. The percentage uptake, for these experiments is defined as he quantity of Ibuprofen present in the supernatant subtracted from the original added concentration. The % uptake of Ibuprofen on MIP IBU 2 as compared to the corresponding control polymer is shown in Fig. 5b. As is evident from the results obtained rebinding was found to be highly efficient when lower ratios of acetonitrile were included in the rebinding solution. At a ratio of greater than 30% acetonitrile, a drop in specific rebinding of approximately 40% was observed. At higher ratios of acetonitrile, the percentage rebinding was only approximately 20%. This study indicates that the rebinding is solvent specific and that the conditions for rebinding are driven by the aqueous rather than the organic solvent. It was necessary to include a

Fig. 5. Bar graph showing the uptake of Ibuprofen at different concentrations in dimethylformamide (DMF) alone (a) and showing the effect of increasing ratios of acetonitrile in the rebinding solution on the % uptake of Ibuprofen by the MIP IBU 2 and its corresponding control polymer (b).

quantity of acetonitrile to in all the binding solutions due to the poor solubility of Ibuprofen in water. As rebinding had already been shown to be efficient in DMF, acetonitrile was chosen as it is also a polar aprotic solvent and also due to its expected compatibility with the ␲–␲ interaction environment. As can be seen though, a significant decrease in rebinding was observed on addition of acetonitrile to the binding solution (see below morphological characterisation of the imprinted polymers and Table 4). As the % rebinding was higher when a greater volume of water was present the effect of pH on rebinding was investigated. pH values from 5 to 8 were tested with the same uptake solution ratios as discussed above. The pH dependent uptake is presented graphically in Fig. 6. From this study, it can be seen that the % rebinding decreases by approximately 20% at pH 5, whereas there is no significant variation between pH 6 and 8. This illustrates that the nature of the rebinding of Ibuprofen is pH independent above the pKa value. The pKa of Ibuprofen is 4.8 (Janjikhel and Adeyeye, 1999). At one pH unit above the pKa value, a certain percentage of the molecule will be deprotonated (negatively charged), i.e. at values below pH 5.8. As the pH nears the pKa value the amount of molecules negatively charged increases (it will be 50% at the pKa value).

1144

K. Farrington, F. Regan / Biosensors and Bioelectronics 22 (2007) 1138–1146

Fig. 6. The effect of pH on the specific uptake of Ibuprofen by the MIP IBU 2.

This may indicate a small contribution towards complex formation from hydrogen bonding. As an acid–base equilibrium or hydrogen bond interaction is subject to changes in pH however, it can be argued that contribution to the selectivity of the MIP from these interactions is minimal or at best augments the dominant ␲–␲ stacking effect. Furthermore no evidence for hydrogen bond formation in the pre-polymerisation complex was observed by NMR studies. It should be noted however, that the degree of contribution of hydrogen bonding to the selectivity of the MIP cannot be determined using Hyperchem. NMR studies did not show shifts in the –OH proton of Ibuprofen. If hydrogen bonding was present to a significant degree, it would be expected to observe upfield shifts associated with this in the –OH proton.

monomer was different in each case. When 2-vinylpyridine was replaced with methacrylic acid significant reduction in specific elution was observed. Furthermore it was also observed that with the MAA MIP, a higher level of non-specific binding was seen. This validates the initial modelling studies (Fig. 3) which had predicted that a MIP for Ibuprofen prepared with vinylpyridine would give a stronger interaction than one prepared with methacrylic acid as monomer. In addition, as described previously, the rationale supporting the significantly higher binding score of vinylpyridine over methacrylic acid is the nature of the specific interaction and orientation of the pre-polymerisation complex. Several other approaches to the SPE studies were undertaken. It was found that loading in greater ratios of acetonitrile:water led to a reduction in the % recovery on elution. Loading in methanol was found to result in non-retention of the Ibuprofen (data not shown). This was attributed to the high degree of solubility of Ibuprofen in methanol leading to a large degree of self-elution during the loading phase. To investigate the ability of the SPE method to extract Ibuprofen from a pharmaceutical preparation, a NurofenTM liquid caplet containing 200 mg Ibuprofen in gel form) was used. The liquid Ibuprofen was dissolved in a minimum volume of acetonitrile and made up to a concentration of 20 ␮g/ml. SPE was conducted as before. Fig. 8 shows the recovery of Ibuprofen from the liquid NurofenTM capsule. As can be seen the % recovery on elution was high in comparison to the control. The MIP has thus shown to bind Ibuprofen very strongly. A highly efficient clean up system for extraction of Ibuprofen from pharmaceutical preparations has been demonstrated using MIP technology.

3.3. Solid phase extraction studies

3.4. Selectivity in solid phase extraction

Given the results obtained for the preliminary rebinding studies, the solid phase extraction procedure was designed as follows. The analyte was loaded in 10:90 ACN:water, washed with 1% TEA in ACN and eluted with methanol. Fig. 7 shows the recovery on elution for two MIPs studied. IBU 2 and IBU 3. Both MIPS were made using DMF as porogen however, the functional

The selectivity of the MIP for Ibuprofen over the Ibuprofen analogues naproxen and ketoprofen was investigated using solid phase extraction. Table 2 shows the recovery on elution of the three compounds. Given the structure of the molecules presented in Fig. 1, both ketoprofen (Fig. 1b) and naproxen (Fig. 1c) contain two aromatic rings. However, the molecular volume (as

Fig. 7. Comparison of the recovery of Ibuprofen by MIPs IBU 2 and IBU 3 by solid phase extraction along with the corresponding controls.

Fig. 8. The specific recovery of Ibuprofen from a NurofenTM caplet by MIP IBU 2 alongside the corresponding control polymer.

K. Farrington, F. Regan / Biosensors and Bioelectronics 22 (2007) 1138–1146 Table 2 Recoveries of Ibuprofen and analogues on MIPs IBU 2 and IBU 3 along with the corresponding controls (CON 2 and CON 3) Compound

IBU 2 % recovery

CON 2 % recovery

IBU 3 % recovery

CON 3 % recovery

Ibuprofen Naproxen Ketoprofen

83.34 30.96 17.94

2.81 3.25 1.44

21.20 14.32 4.52

2.51 2.57 1.08

1145

Table 4 Data obtained from particle size and swelling studies Solvent of exposure

d50 (␮m)a

Swelling ratiob

Water Acetonitrile Methanol Dimethylformamide

63.260 ± 2.4 69.525 ± 6.1 71.859 ± 8.2

1.325 1.815 2.090 1.414

c

Defined as the size (in ␮m) below which 50% of the particles (sample) lies. Defined as (Mw − Md )/Md , where Mw is the mass of the wet polymer and Md is the mass of the dry polymer. c Particle size data not available. a

b

calculated by Accerlys) for ketoprofen is significantly larger than that of naproxen (ketoprofen having a molecular volume of ˚ and naproxen being 151 A). ˚ The molecular volume of 178.4 A ˚ Overall the strucIbuprofen has been calculated to be 149.8 A. ture of naproxen is more similar to Ibuprofen than ketoprofen. The study by Haginaka et al. (1999) found a similar trend in terms of specific binding in HPLC studies. Given the probability of Ibuprofen forming ␲–␲ interactions with both ketoprofen and with naproxen, there are two possible explanations for the selectivity of the MIP for Ibuprofen over the analogues. In the first case, the incorporation of the pre-polymerisation complex into the cross-linked matrix leads to pores with structural memory as well as specific functional groups. Secondly, the nature and strength of the ␲–␲ interaction(s) with the analogues will be different. Ketoprofen contains a carbonyl group in between the two aromatic rings. This group is electron withdrawing and will disrupt the interaction of vinylpyridine with either of the aromatic rings. A similar situation applies to naproxen. Two adjoined aromatic rings will have different resonance to a single ring and will lead to changes in the nature of the interaction. The possibility of structural memory of the MIP warranted further investigation, as described below. 3.5. Morphological characterisation of the imprinted polymers To further probe the nature of the structural selectivity, morphological studies were conducted on the Ibuprofen MIPs. The results of these studies are presented in Tables 3 and 4. The pore structure and surface area were studied using the BET adsorption method (Table 3) and the particle size distribution was studied using Malvern instruments light scattering technology (Table 4). As reported by Br¨uggemann (2001) MIPs tend to have higher surface areas than the corresponding controls. A result of this phenomenon is that the MIP will have more binding sites which are better distributed throughout the cavity. A greater total pore volume is indicative of the MIP having a superior sample load Table 3 Data obtained from BET analysis for both MIPs and the corresponding control polymers Polymer

Surface area (m2 /g)

Total pore volume (cm3 /g)

Average pore diameter (nm)

IBU 2 CON 2 IBU 3 CON 3

316.67 213.39 270.77 174.36

0.638 0.144 0.179 0.152

7.60 2.62 2.56 3.32

capacity. As can be seen from Table 3 the MIP IBU 2 expresses over 4 times the total pore volume of its corresponding control polymer (CON 2). A point of note from Table 3 is the larger average pore diameter of the MIP compared with the pore size of the control. The IBU 2 has an average pore size of almost three times that of CON 2. Given that the relative ratio of template: functional monomer may be 1:2 or greater, the molecular volume of the complex will be quite large. Preliminary studies ˚ (data not shown). indicate a value in the region of 500–600 A This will invariably lead to polymers with larger sized cavities allowing easier access of template during rebinding. Particle size studies were carried out to investigate the effect of solvents on MIP particles. Turner et al. (2004) have demonstrated that swelling of MIPs has a detrimental effect on their performance in terms of rebinding finding that a MIP with a higher average particle size performed poorly in terms of rebinding relative to a MIP with a smaller average particle size. Here it is shown that exposure of the IBU 2 MIP to different solvents significantly affects the particle size distribution (Table 4, left column). The MIP (IBU 2) showed that exposure to water caused the least increase in particle size, whereas exposure to methanol caused the greatest. The particle size study compares well with the simple swelling analysis study (Table 4, right column). From this study it can be seen that exposure to water causes the smallest increase in weight ratio whereas exposure to acetonitrile and methanol significantly enhance the swelling. Referring to Fig. 5b, it can be seen that rebinding was significantly better in water (with 10% acetonitrile) than in acetonitrile alone. In addition, exposure of the MIP to DMF causes only marginally more swelling than exposure to water. In general MIPs perform better in the solvent in which they are prepared. The aim of this study was to design a MIP for Ibuprofen capable of rebinding under aqueous conditions and to rationally explain the physico-chemical nature of this. From these studies, it is confirmed that, the particle size analysis and swelling study along with the rebinding study (Fig. 5) show the importance of conformational changes of the polymer on recognition. It is proposed that there are two mechanisms of selectivity affecting the rebinding of Ibuprofen to IBU 2. Firstly there is the chemical functionality of the ␲–␲ interaction (stacking effect) imparting functionality memory to the resultant binding sites. Secondly, there is structural or conformational memory associated with the solvent of rebinding. It is shown that both of these criteria are necessary for efficient and selec-

1146

K. Farrington, F. Regan / Biosensors and Bioelectronics 22 (2007) 1138–1146

tive rebinding and if either of these mechanisms of memory is abrogated, selective rebinding is adversely affected.

ogy (IRCSET) and the Materials and Surface Science Institute, University of Limerick for use of equipment.

4. Conclusion

References

The aim of this study was rationally design a molecularly imprinted polymer for Ibuprofen capable of use in an aqueous environment. This was successfully synthesised. This MIP is capable of recognising Ibuprofen in aqueous samples with acceptable selectivity over the structurally related analogues naproxen and ketoprofen. A non-imprinted polymer (NIP or control), constructed in parallel with the MIP did not possess recognition ability. A further aim was to investigate the chemical and physical nature of the recognition. For this, studies were carried out into the nature of the pre-polymerisation complex formation. NMR and molecular modelling software was used. Data attained by these methods were used to construct the MIPs in a rational design approach. The nature of the complex formation between Ibuprofen and 2-vinylpyridine is understood to be a ␲–␲ stacking arrangement between the aromatic rings of monomer and template. Following construction of the MIP, the percentage rebinding was evaluated and selectivity assessed by solid phase extraction. The MIP, IBU 2–made using 2-vinylpyridine as a monomer–showed high selectivity for Ibuprofen over structurally related analogues. It was furthermore capable of quantitatively extracting Ibuprofen from a pharmaceutical preparation. Physical characterisation of the polymer was performed by analysis of the porous environment and by particle size analysis. The surface area and pore volume were significantly higher for the MIPs over the controls and for IBU 2 in comparison to IBU 3. This is indicative of better arrangement of functional sites within the pores. The higher total pore volume is suggestive of the MIP having a higher load capacity. Swelling was investigated using particle size studies on exposure of the MIP to different solvents. A higher degree of swelling corresponded to poorer rebinding studies in the same solvents.

Br¨uggemann, O., 2001. Biomol. Eng. 18, 1–7. Carabias-Martinez, R., Rodriguez-Gonzaol, E., Herrero-HJernandez, E., 2000. J. Chromatogr. A. 1085, 199–206. Caro, E., Marce, R.M., Cormack, P.A., Sherrington, D.C., Borrell, F., 2003. J. Chromatogr. A 995, 233–238. Caro, E., Marce, R.M., Cormack, P.A., Sherrington, D.C., Borrell, F., 2004. J. Chromatogr. B: Analyt. Technol. Biomed. Life. Sci. 813, 137–143. Caro, E., Marce, R.M., Cormack, P.A., Sherrington, D.C., Borrull, F., 2005. J. Sep. Sci. 28, 2080–2085. Chapuis, F., Pichon, V., Lanza, F., Sellergren, B., Hennion, M.-C., 2004. J. Chromatogr. B 804, 93–101. Chassaing, C., Stokes, J., Venn, R.F., Lanza, F., Sellergren, B., Holmberg, A., Berggren, C., 2004. J. Chromatogr. B: Analyt. Technol. Biomed. Life. Sci. 804, 71–81. Cheng, Z., Zhang, L., Li, Y., 2004. Chemistry 10, 3555–3561. Dirion, B., Cobb, Z., Schillinger, E., Andersson, L., Sellergeren, B., 2003. J. Am. Chem. Soc. 125, 15101–15109. Dong, W., Yan, M., Zhang, M., Liu, Z., Li, Y., 2005. Anal. Chim. Acta 542, 186–192. Fairhurst, R.E., Chassaing, C., Venn, R.F., Mayes, A.G., 2004. Biosens. Bioelectron. 20, 1098–1105. Haginaka, J., Sanbe, H., Takehira, H., 1999. J. Chromatogr. A 857, 117–125. Idziak, I., Benrebouh, A., Deschamps, F., 2001. Anal. Chim. Acta 435, 137–140. Janjikhel, R.K., Adeyeye, C.M., 1999. Pharm. Dev. Technol. 4, 9–17. Joshi, V.P., Karode, S.K., Kulkarni, M.G., Mashelkar, R.A., 1998. Chem. Eng. Sci. 53, 2271–2284. Kim, H., Spivak, D.H., 2003. Org. Lett. 18 (5), 3415–3418. Lu, Y., Li, C., Wang, X., Sun, P., Xing, X., 2004. J. Chromatogr. B 804, 53–59. Lubke, C., Lubke, M., Whitcombe, M.J., Vulfson, E.V., 2000. Macromolecules 33, 5098–5105. Martin, P.D., Jones, G.R., Stringer, F., Wilson, I.D., 2004. J. Pharm. Biomed. Anal. 35, 1231–1239. Molinelli, A., Janotta, M., Mizaikoff, B., 2005. Meth. Mol. Biol. 300, 243–254. Olwill, A., Hughes, H., O’Riordain, M., McLoughlin, P., 2004. Biosens. Bioelectron. 20, 1045–1050. O’Mahony, J., Molinelli, A., Noplan, K., Smyth, M.R., Mizaikoff, B., 2005. Biosens. Bioelectron. 20, 1884–1893. Sellergren, B. (Ed.), 2001. Molecularly Imprinted Polymers. Man Made Mimics of Antibodies and Their Application in Analytical Chemistry. Elsevier, Amsterdam. Spivak, D., Simon, R., Campbell, J., 2004. Anal. Chim. Acta 504, 23–30. Turner, N.W., Piletska, E.V., Karim, K., Whiccombe, M., Malecha, M., Magan, N., Baggiani, C., Piletsky, S.A., 2004. Biosens. Bioelectron. 20, 1060–1067. Watabe, Y., Hosoya, K., Tanaka, N., Kubo, T., Kondo, T., Morita, M., 2005. J. Chromatogr. A 1073, 363–370.

Acknowledgements The authors wish to acknowledge funding provided by the Irish Research Council for Science, engineering and Technol-