Molecularly Imprinted Polymers and Their Synthesis by Different Methods

Molecularly Imprinted Polymers and Their Synthesis by Different Methods Molecularly Imprinted Polymers and Their Synthesis by Different Methods Fadim Yemiş 1*, Pinar Alkan 2, Berin Yenigül 2, and Mesut Yenigül 3 1 Celal Bayar University, Faculty of Science and Art, Department of Chemistry, Division of Analytical Chemistry, 45040, Muradiye-Manisa, Turkey 2 Ege University, Faculty of Science, Department of Chemistry, Division of Analytical Chemistry, 35100, Bornova- Izmir, Turkey 3 Ege University, Faculty of Engineering, Department of Chemical Engineering, 35100, Bornova-Izmir, Turkey Summary Over the past thirty years, molecular imprinting has aroused great research interest for scientists. This attention can be explained by the serious potential advantages of using molecularly imprinted polymers in place of natural receptors and enzymes such as their superior durability, low-cost and easy preparation. Furthermore molecular imprinting is a promising technique for the preparation of polymer composites with predetermined selectivity and high affinity. This mini review examines and evaluates the preparation technique and utility application of molecularly imprinted polymers (MIPs) in several areas. The review begins with a brief introduction to MIPs with a focus on their preparations and continues with application areas. Keywords: Molecularly imprinted polymers, Selective recognition sites, Molecular imprinting, Imprinted polymers 1. Molecular Imprinting Natural receptors, enzymes and antibodies are used as the recognition element within a wide variety of assays and sensor systems. However, the biorecognition elements have some disadvantages, such as low stability, high price, poor performance in nonaqueous media, etc. The search for alternative ways has led researchers to the development of stable synthetic analogues of biorecognition elements. One of the most promising methods is molecular imprinting. The polymers prepared by this method have numerous advantages such as robust, inexpensive, high performance in organic solvents at low and high phs, etc. in comparison with biorecognition elements 1,2. 1* Corresponding author. Tel.: +90-236-2013179; fax: +90-236-2412158; e-mail: fadimyemis@hotmail.com (F. Yemiş) 2 fepinar@hotmail.com, berrin.yenigul@ege.edu.tr 3 mesut.yenigul@ege.edu.tr Smithers Rapra Technology, 2013 Molecular imprinting is a technique for producing chemically selective binding sites in a three-dimensional, which recognize a particular molecule, in a macroporous crosslinked polymer matrix 3. This technique included formation of the complex between functional monomer and template molecule in appropriate solution and freezing of this complex by polymerization in the presence of high concentration of crosslinker. Following template removal by washing, polymer binding sites (imprints) were left in the structure, which were specific or complementary to size, shape and functional group of the template molecule 4. Since their beginnings in the 1970s, MIPs have provided the focus for scientists and engineers involved with the development of chromatographic adsorbents, membranes, sensors and enzyme and receptor mimics. Initially, MIP research was aimed to the design of specific adsorbents and understanding the nature of their specificity. Comparatively new areas in which MIPs provide opportunities for advancement in the last twenty years include the development of binding assays and sensors, catalysts, membranes, capillary electrophoresis (CE), and the production of polymers with special functions such as drugrelease matrices 2. Generally, the fabrication of MIPs consists of three main steps: 1. Pre-arrangement of the functional monomers around the template molecule, 2. Co-polymerization in the presence of excess crosslinker, 3. Removal of the template molecule from the polymer by extraction produce 3,5,6. Polymers & Polymer Composites, Vol. 21, No. 3, 2013 145

Fadim Yemiş, Pınar Alkan, Berin Yenigül, and Mesut Yenigül Figure 1. Schematic representation of the molecular imprinting procedure In pre-arrangement step, depending on the nature of binding interaction between the functional monomer and the template molecule during polymerization, MIPs can be classified according to whether they are obtained using the covalent, non-covalent or semicovalent approach. In the non-covalent imprinting-the most extensively used, a template is mixed with an appropriate functional monomer, a suitable porogenic solvent, a crosslinking agent and a polymerization initiator. Specific binding sites are formed by selfassembling of template and the functional monomer, which should be capable to form a fairly stable complex with the template via dipole interaction, hydrophobic interaction, Van der Waals forces, hydrogen bonding, ion pair, etc. After the polymerization, the template is removed by extracting the polymer with appropriate solvents. The guest binding by the polymer occurs through the corresponding non-covalent interactions. On the covalent approach, the template and the functional monomer are covalently bound prior to polymerization. After the polymerization, the covalent linkage is cleaved and the template is removed from the polymer. Upon the guest binding by the imprinted polymers, the same covalent linkage is formed. Sorbents prepared using covalent imprinting tend to have well defined and more homogeneous binding sites than those resulting from non-covalent approach: the interaction between template and functional monomers are much more stable and stoichiometric during the polymerization. Compared to non-covalent MIP (where analyte binding to imprinted sites take place by weaker interactions), this leads to higher selectivity, with less nonspecific retention, and better extraction efficiencies. Finally, on non-covalent MIP preparation the synthesis of the adsorbent is carried out through a covalent procedure but the extraction mechanism is more related to a noncovalently prepared MIP 6-9. Another synthetic protocol for obtaining MIPs is known as the semi-covalent approach. Semi-covalent imprinting combines the advantages of both covalent and non-covalent approaches. In this case, the interaction between the functional monomer and the template molecule before the polymerization process is covalent, whereas the interaction of the target analyte and the MIP once the polymer is in use is through non-covalent interactions 1,7,8. Due to the different interactions established between the template and the functional monomer before the polymerization process in the non-covalent or semicovalent approach, a different ratio of template molecule to functional monomer is needed to ensure the best imprint on the final polymer. The ratio normally used in the semicovalent approach is rather low and is generally 1:1 or 1:2, whereas, for the non-covalent approach, ratios typically range from 1:4 to 1:8, depending upon the complexity of the template and the affinity of the functional monomer to the template 9. 2. MIP Synthesis Several synthetic protocols have been developed to obtain MIPs. Since the first appearance of MIPs, the most widely-used synthetic protocol has been traditional polymerization (TP). Traditionally, molecular imprints have mostly been made by bulk polymerization. In this process, polymerizable functional monomer (most frequently-methacrylic acid) is prearranged around a template molecule in organic solvent (e.g. chloroform, acetonitrile). The resulting prepolymer complexes are copolymerized with an excess of crosslinker (e.g. ethylene glycol dimethacrylate) in the presence of a free radical initiator under thermal or photochemical conditions. The block of resulting polymer is then 146 Polymers & Polymer Composites, Vol. 21, No. 3, 2013

Molecularly Imprinted Polymers and Their Synthesis by Different Methods ground and sieved by hand in order to produce particles of an appropriate size for experiments. After the removal of the template by extraction, binding sites complementary to the template molecule both in shape and chemical functionality are left within the polymer matrices that allow rebinding of the template with quite specificity 1,3,10-13. However, the molecular imprinted polymers prepared by traditional method have some disadvantages, such as time-consuming and complicated preparation process, less recognition sites inside matrices particles thereby poor binding capacity and lower binding kinetic of MIPs for the template molecules 9,14. Moreover, the non-regular shape of the particles obtained by bulk polymerization, although not a real limitation for off-line SPE applications, can cause broad asymmetric peaks when using it as stationary phase in LC, CEC or in on-line coupling with LC. Therefore, other methods of polymerization are used in order to prepare spherical and monodispersed particles of MIPs. They include suspension polymerization, seed polymerization and dispersion/ precipitation polymerization 14. In order to overcome these drawbacks effectively, the surface imprinting technique has been developed in recent years 9,13,14. The material with binding sites situated at the surface present many advantages including high selectivity, more accessible sites, fast mass transfer and binding kinetics 9. washing and centrifugation operations. This approach offers an efficient methodology for preparing MIP beads in high yield that can be used for a wide range of applications. However, as the monomer needs to be soluble in the solvent while the resulting MIP gel beads should not be, careful optimization of the system in terms of the monomer type and concentration is required. In addition, aggregation of the MIP beads could occur, resulting in poor particle yields. This technique can also be employed to prepare MIP nanospheres. These nanobeads can either be used directly, as in capillary electrochromatography, or they can be loaded onto a membrane for template extraction. Besides the method mentioned above, other less common approaches for spherical MIP preparation include twostep swelling and polymerization, miniemulsion polymerization and novel micelle formation based on diblock copolymers 9,15,16. Another approach for obtaining spherical particles is by multi-step swelling polymerization (MSS). In this technique, preformed uniformlysized seed particles are suspended in water and, after several additions of suitable organic solvents, the initial particles swell to a final size in the range 5-10 µm. Once the particles have swollen to the desired size, all the components involved in the production of the MIP are added to the solution and incorporated into the particles in this swelling state and afterwards polymerization is induced 9. A different approach to obtaining spherical particles is to use suspension polymerization (SP). Templates such as metal ions 17, drugs 18 and proteins 19 are suitable for this system. The suspension polymerization consisted in a heterogeneous polymerization based on the suspension of droplets of prepolymerization mixture in a continuous phase (water, mineral oil or Figure 2. (a) Examples of functional monomers that can be used in non-covalent molecular imprinting, (b) Examples of functional monomers that can be used in covalent molecular imprinting, (c) Examples of crosslinking monomers that can be used for the synthesis of MIPs Amongst the techniques developed to overcome the drawbacks of TP, precipitation polymerization (PP) is the most widely-used technique for obtaining spherical particles. The basic principle of this approach is that, when the polymeric chains growing in solution reach a certain critical mass, they precipitate from the solution. Starting with a very dilute monomer solution highly crosslinked polymeric microgels precipitate out after polymerization due to their low solubility in the solvent. Then the polymer beads are easily recovered by Polymers & Polymer Composites, Vol. 21, No. 3, 2013 147

Fadim Yemiş, Pınar Alkan, Berin Yenigül, and Mesut Yenigül perfluorocarbon), each droplet acting like a mini bulk reactor that allows the production of spherical beads in a broad size range starting at a few micrometers and reaching up to millimeter size. In this case, all the components involved in the polymerization process are dissolved together in an appropriate organic solvent and this solution is further added to a larger volume of an immiscible solvent. Afterwards, this system is vigorously stirred in order to form droplets and then the polymerization reaction is induced. The biggest advantage of this method is its excellent heat dispersion, which makes it suitable for industrial scaleup without heat transfer limitations 9,16. Grafting is another polymerization technique aimed at delivering spherical particles, but it is also used to produce composites materials. Surface grafting of MIP layers onto preformed beads has also been proposed as a technique to obtain chromatography-grade imprinted polymers. In this method, thin imprinted layers have been used as coatings on chromatography-grade porous silica or spherical polymers using several techniques to retain the radical polymerization at the surface of the beads 20. In this case, the starting material comprises silica particles, and all the components involved in the polymerization process are adsorbed within these particles before the polymerization process starts. Once the polymer is formed, the silica is etched away to reveal a final product of spherical particles, which are, in turn, the specular image of the original silica particle 9,15. In recent years molecularly imprinted polymer membranes have attracted significant interest. The phase inversion technique was used for the synthesis of a number of MIP membranes. Despite their good recognition properties in aqueous media, these membranes were not stable in organic environment. Simultaneous formation of the pore structure and imprinting sites in the phase inversion membranes becomes a significant limitation, since different optimal conditions for that are required. Another approach for MIP membranes synthesis used the method of in situ crosslinking polymerization. The main problem of this approach is high levels of crosslinking traditionally used in molecular imprinting for achieving a desirable selectivity. Poor mechanical stability as well as extremely low as consequences of high degrees of MIP crosslinking were the special features of the membranes obtained in the early attempts. Porous free-standing molecularly imprinted polymer membranes were synthesized by the method of in situ polymerization using the principle of synthesis of interpenetrating polymer Networks. Addition of linear flexible oligomer (oligourethane acrylate) provided formation of the highly crosslinked MIP in the form of a free-standing 60 μm thick flexible membrane. However, for separation purposes low membrane permeability was still the main obstacle for their application in separation processing 20,21. Monolithic MIPs have also been prepared by a simple, one-step, in situ free-radical polymerization process directly within the confines of a chromatographic column without the need of grinding, sieving and column packing 20. In PhD thesis of Yemiş, the novel method has been suggested for MIP preparation in environmental-friendly and practical ways. In this novel imprinting method, sulphonated styrene-divinylbenzene resin was swelled in dimethyl formamide (DMF) in the presence of Nitrofurantoin (NF) as a template. The maximum amount of DMF for swelling of 1.00 g of resin was established. This amount of resin was added to the saturated solution of NF in suitable amount of DMF. The mixture was allowed to react overnight at room temperature. The swelled resin was dried at 50 C for 3 hours in an oven 22. 3. Applications of Imprinted Polymers Molecularly imprinted polymers (MIPs) have been used as materials of molecular recognition in many scientific and technical fields, such as solid-phase extraction, chromatograph separation, membrane separations, sensors, drug releases, catalysts, etc. The larger part of applications of MIPs is their use as solid-phase adsorbents for HPLC 23. 3.1 Separation The field of separations is continuing to expand, driven by the promise of increased market opportunities in the chemical and pharmaceutical industries, water purification and waste-material treatment. MIP materials are expected to part of this market (1-3%) in the niche areas of affinity separation, solidphase extraction (SPE) and separation under extreme conditions (for example, organic and toxic environments, low and high phs, and high temperatures and pressures) 2. MIP adsorbents are used for protein separation, the solid-phase extraction of drugs, the separation and purification of amino acids, DNA and RNA, peptides, hormones and carbohydrates, and for the recovery of flavour compounds. Another area for application of MIPs is in the separation of chiral compounds, which are important for basic research, drug design, optics and polymer chemistry. The ability of MIPs to operate in extreme conditions, in particular in organic solvents, is attractive to the chemical industry because it might allow multistep enrichment and purification processes to be substituted with a one-step separation on a MIP 2. Chromatography, capillary electrochromatography, and solidphase extraction are the main fields of MIPs used in separation. 148 Polymers & Polymer Composites, Vol. 21, No. 3, 2013

Molecularly Imprinted Polymers and Their Synthesis by Different Methods 3.2 Sensors In chemical sensors and biosensors, a chemical or physical signal is generated upon the binding of the analyte to the recognition element, a transducer then translates this signal into a quantifiable output signal 24. Typical problems that remain unsolved in connection with biological materials used in biosensors are: (1) low stability; (2) high cost; and (3) an absence of enzymes or receptors that are able to recognize certain target analytes. MIPs are considered as appropriate alternatives to the biological receptors for use in sensors principally because of their high stability. MIPs can be synthesized for analytes for which no receptor or enzyme is available or when they are too expensive. Additionally, the polymerization step is fully compatible with microfabrication used in sensor technology. MIP sensors have been developed for herbicides, sugars, nucleic acid and amino acid derivatives, drugs, toxins, solvents and vapors 2. Field effect devices, diffusion through selective membranes, Electrochemical and Fluorescence are few sensor types of MIPs 25. 3.3 Other Applications The other some application areas of MIPs are binding assays-antibody/ receptor mimics 24, catalysis 26 and drug development and screening 27. Some commercial interest was also shown recently. 4. Conclusions Because of the excellent properties, such as low cost, high stability, easy preparation, applicability for many scientific fields, molecular imprinting has attracted much attention in recent years. Molecular imprinting is a technique for imparting molecular recognition properties to a synthetic polymeric matrix, which is called the lock and key model. Conventionally, the technique is easily carried out using bulk imprinting, where molecularly imprinted polymers (MIPs) are prepared in large chunks and then processes like grinding and sieving are required. The tedious grinding and sieving process commonly yields sharpedged and nonuniform particles in shape and size, which causes numerous problems such as limitations in direct application and chromatographic efficiency. Furthermore, the act of sieving may result in material loss as particles too small are discarded. In addition, due to the creation of binding sites within the polymeric bulk, the issue of the hindrance of adsorbate diffusion (especially in the case of macromolecules) during template rebinding makes the MIPs prepared through this approach unsuitable for practical applications. New imprinting techniques instead of bulk polymerization which can improve the properties of MIPs particles, should promote future development of MIPs in different application fields. MIPs have been widely studied as HPLC stationary phases, MISPE cartridge material and selective material for sensors and biosensors catalysts. There are still problems or advantages need to be evaluated for any specific MIP application. Thus over the years, many efforts to address the limitations of conventional molecular imprinting techniques have resulted in new imprinting methodologies. Systems like suspension and precipitation polymerization, where MIPs with tunable morphologies can be prepared, have been developed. Additionally, strategies like surface imprinting have also been employed. MIPs prepared in this manner show significant promise due to their desirable physical morphologies and favorable adsorption kinetics in industrial applications. One particular goal is to synthesize suitable templates are expected to be helpful and new imprinted polymers with more simplicity and higher selectivity. 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