MOLECULARLY IMPRINTED POLYMERS REVIEW SEMINAR 13

REVIEW SEMINAR ON
MOLECULARLY IMPRINTED POLYMERS

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1. HISTORY
In 1931 Polyakov and co workers, from Kiev, reported some unusual adsorption properties in silica particles prepared using a novel synthesis procedure .1 Sodium silicate had been polymerized in water using (NH4)2CO3 as the gelating agent. After two weeks, additives (benzene, toluene or xylene) had been added. The silica was subsequently allowed to dry for 2030 days, after which the additive was removed by extensive washing in hot water. Subsequent adsorption studies revealed a higher capacity for uptake of the additive by the silica than for structurally related ligands, i.e. some kind of memory for the additive was apparent, at least in the cases of benzene and toluene Linus Pauling,2 in 1940 proposed that antibody formation took place in the presence of an antigen, captured by the cell, which served as a template for antibody formation. Pauling suggested that the primary structure of any antibody was identical and that the template-induced conformational effect gave rise to the remarkable selectivities exhibited by antibodies. In addition, he proposed that this ability could be investigated by precipitating globulins under denaturing conditions in the presence of an antigen and by slow removal and redissolution of the globulins. The globulins would then exhibit specificity for the antigen. A similar methodology to that of Polyakov, was carried out by Dickey 3 in 1949 in his laboratory ,but in this case methyl orange (and other alkyl orange dyes) was used as the template. The results of Dickey were striking demonstrating pronounced selectivity for the pattern dye which had been present during polymerization as related to the other dyes After two decades of rather intense research in the area, a decline of molecular imprinting in silica appears to have coincided with the introduction of molecular imprinting in organic polymers, made independently by Wulff and Klotzin 19724-5, Owens et al6 in 1999 reviewed the potential of MIP-based analytical techniques in bio- and pharmaceutical analysis They mentioned that earlier work of MIP-based chiral stationary phases (CSPs) was almost exclusively carried out with LC and that the number of MIP applications for CE and CEC was relatively small despite their usefulness. Ansell 7 in 2005 reviewed the literature on chiral separation of drug enantiomers using MIPs via HPLC, TLC,csupercritical fluid chromatography (SFC), and CEC. . Wei and Mizaikoff 8 in 2007 gave a review on recent advances of noncovalent MIPs for affinity separations. They reviewed MIP applications in affinity separations as well as bio-mimetic assays emphasizing the preparation of shape- and size-uniform particles Vasapollo et al.9 in 2011 presented a very comprehensive review on present and future prospective of MIPs aiming to summarize the molecularly imprinting processes and principal application fields of MIPs, focusing on separation science (mostly HPLC), chemical sensing, drug delivery, and catalysis.

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2.INTRODUCTION
In molecular imprinting, the target molecule (or a derivative thereof) acts as the template around which interacting and cross-linking monomers are arranged and co-polymerized to form a cast-like shell (Figure 1). Initially, the monomers form a complex with the template through covalent or noncovalent interactions. After polymerization, the template is removed, and binding sites are exposed that are complementary to the template in size, shape, and position of the functional groups. In essence, a molecular memory is imprinted on the polymer, which is now capable of selectively rebinding the template. Thus, molecularly imprinted polymers (MIPs) possess two of the most important features of biological receptorsthe ability to recognize and bind specific target molecules. However, MIPs differ from biological receptors in that they are large, rigid, and insoluble, whereas their natural counterparts are smaller, flexible, and, in most instances, soluble. Depending on their size, MIPs can have thousands or millions of binding sites, whereas biological receptors have a few or even just one. Moreover, the population of binding sites in MIPs, especially those imprinted using noncovalent manner monomertemplate interactions, is heterogeneous because of the influence of the equilibria that govern the monomertemplate complex formation and the dynamic of the growing polymer chains prior to copolymerization. In addition, the chaotic structure of many of the polymers used for imprinting, the heterogeneous pore size distribution, and the fact that the binding sites are contained within the bulk material often make mass transfer slow. Although not always problematic, these characteristics can prevent MIPs from being substituted for natural receptors in certain applications, and part of the current MIP research is focused on finding solutions or workarounds to these shortcomings.

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FIGURE 1. Creating a molecular imprint in a synthetic polymer.

(a) Functional monomers, (b) a cross-linker, and (c) a template molecule are mixed together. (1) The functional monomers form a complex with the template molecule. (2) The functional monomers copolymerize with the cross-linker. (3) As polymerization proceeds, an insoluble, highly cross-linked polymeric network is formed around the template. (4) Removing the template liberates complementary binding sites that can reaccommodate the template in a highly selective

3. APPROACHES IN MOLECULAR IMPRINTING

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Figure :2 Schematic representation of covalent and non-covalent molecular imprinting procedures MIPs can be synthesised following three different imprinting approaches [1], as follows: 1. The non-covalent procedure is the most widely used because it is relatively simple experimentally and the complexation step during the synthesis is achieved by mixing the template with an appropriate functional monomer, or monomers, in a suitable porogen (solvent). After synthesis, the template is removed from the resultant polymer simply by

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washing it with a solvent or a mixture of solvents. Then the step rebinding the template to the MIP exploits non-covalent interactions. 2. All these features offer several advantages over the covalent protocol, in which formation of covalent bonds between the template and the functional monomer is necessary prior to polymerisation. Furthermore, to remove the template from the polymer matrix after synthesis via the covalent protocol, it is necessary to cleave the covalent bonds. To this end, the polymer is then refluxed in a Solvent extraction or treated with reagents in solution 3. The semi-covalent approach is a hybrid of the two previous methods. Thus, covalent bonds are established between the template and the functional monomer before polymerisation, while, once the template has been removed from the polymer matrix, the subsequent re-binding of the analyte to the MIP exploits non-covalent interactions, as per the non-covalent imprinting protocol.

4. TEMPLATE REMOVAL

Most of the developments in MIP production during the last decade have come in the form of new polymerization techniques in an attempt to control the arrangement of monomers and therefore the polymers structure. However, there have been very few advances in the efficient removal of the template from the MIP once it has been
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polymerized. Due to this neglect, the process of template removal is now the least cost efficient and most time consuming process in MIP production. [22] Furthermore, in order of MIPs to reach their full potential in analytical and biotechnological applications, an efficient removal process must be demonstrated. There are several different methods of extraction which are currently being used for template removal. These have been grouped into 3 main categories: Solvent extraction, physically assisted extraction, and subcritical or supercritical solvent extraction.

9.1

Soxhlet Extraction

Solvent Extraction technique consists of placing the MIP particles into a cartridge inside the extraction chamber, and the extraction solvent in poured into a flask connected to the extractor chamber. The solvent is then heated and condenses inside the cartridge thereby contacting the MIP particles and extracting the template. The main advantages to this technique are the repeated washing of MIP particles with fresh extracting solvent, favors solubilization because it uses hot solvent, no filtration is required upon completion to collect the MIP particles, the equipment is affordable, and it is very versatile and can be applied to nearly any polymer matrix. The main disadvantages are the long extraction time, the large amount of organic solvent used, the possibility or degradation for temperature sensitive polymers, the static nature of the technique does not facilitate solvent flow through MIP, and the automation is difficult Incubation This involves the immersion of the MIPs into solvents that can induce swelling of the polymer network and simultaneously favor the dissociation of the template from the polymer. Generally this method is carried out under mild conditions and the stability of the

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polymer is not affected. However, much like the Solvant extraction technique, this method also is very time consuming.

9.2 Physically-Assisted Extraction

Ultrasound-assisted extraction (UAE) This method uses Ultrasound which is a cyclic sound pressure with a frequency greater than 20 kHz. This method works through the process known as cavitation which forms small bubbles in liquids and the mechanical erosion of solid particles. This causes a local increase in temperature and pressure which favor solubility, diffusivity, penetration and transport of solvent and template molecules. Microwave-Assisted Extraction (MAE) This method uses microwaves which directly interact with the molecules causing Ionic conduction and dipole rotation. The use of microwaves for extraction make the extraction of the template occur rapidly, however, one must be careful to avoid excessively high temperatures if the polymers are heat sensitive. This has the best results when the technique is used in concert with strong organic acids, however, this poses another problem because it may cause partial MIP degradation as well. This method does have some benefits in that it significantly reduces the time required to extract the template, decreases the solvent costs, and is considered to be a clean technique.

9.3

Subcritical or Supercritical Solvent Extraction

This method employs the use of water, which is the cheapest and greenest solvent, under high temperatures (100374 C) and pressures ( 1060 bar). This method is based upon the high reduction in polarity that liquid water undergoes when heated to high temperatures. This allows water to solubilize a wide variety of polar, ionic and non-polar compounds. The decreased surface tension and viscosity under these conditions also favor diffusivity. Furthermore, the high thermal energy helps break intermolecular forces such as dipole-dipole interactions, vander Waals forces, and hydrogen bonding between the template and the matrix.

5.OPTIMIZATION OF MIPs SYNTHESIS

There are several variables, such as kind and amount of monomer or nature of cross-linker and solvent that affect the final characteristics of the obtained materials in terms of capacity, affinity and selectivity for the target analytes. Thus, the obtainment of the optimum MIP to be used in several applications might take several weeks of trial-anderror experiments using different formulations. This fact has provoked an overuse of certain standard formulations (i.e. the typical 1:4:20 template:monomer:cross-linker molar ratio) [14]. Therefore, some attempts dealing with the optimisation of MIP formulations in
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a simple, fast and rational way for the obtainment of MIPs with improved molecular recognition capabilities have been proposed.

1. Combinatorial imprinting
As mentioned above, due to the variety of parameters influencing the molecular recognition properties of MIPs, the combinatorial approach can be an ideal tool for making easier and faster the screening and optimisation of MIP formulations. This strategy was proposed independently by the groups of Sellergren and co-worker [20] and Takeuchi et al. [21] and consisted of the preparation of a quite large number of polymers directly in HPLC vials as small monoliths (mini-MIPs). Then, the obtained mini-MIPs were screened in rebinding experiments by measuring the template release after incubation in the presence of a suitable solvent. This methodology has been successfully employed in the rapid evaluation and selection of the best MIP for different analytes (triazines [20,21] and sulphonylurea herbicides [22]). The selection of the optimum formulation can be eased by the use of experimental design and multivariate analysis methods since such methods allow identifying the main factors affecting the properties of MIPs [23,24]. However, in spite of the mentioned advantages, one of the main drawbacks is the limited range of methodologies for the screening of mini-MIPs, being restricted to rebinding experiments in equilibrium. In addition, the extrapolation of the obtained results has to be made with caution since the fate of a mini-MIP (monolith) might differ from that obtained after crushing and sieving the polymer.

2. Computational approach
This approach uses molecular modelling software to design and screen a virtual library of monomers against the desired template. Through this approach it is possible to calculate binding energies and predict templatemonomer interaction positions, making easier to select the best functional monomer to be used [1518]. Following this methodology, polymers with high binding capacity and selectivity have been obtained for different analytes. In this sense, it is remarkable the results presented recently by Chianella et al. [19] where the use of a computational protocol allowed the preparation of a MIP for the drug abacavir with a surprisingly high binding capacity of up to 157 mg of drug per gram of polymer. It is necessary to point out that this approach is relatively new and thus, before being routinely used, it is still necessary to prepare and evaluate the best polymers (also for the worst ones it would be desirable) to confirm the trueness of the computational prediction.

5 EFFECTING OF SPECIAL MOLECULAR RECOGNITION

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Optimization of the Polymer Structure

The synthesis of molecularly imprinted polymers is a chemically complex pursuit and demands a good understanding of chemical equilibrium, molecular recognition theory, thermodynamics and polymer chemistry in order to ensure a high level of molecular recognition . The polymers should be rather rigid to preserve the structure of the cavity after splitting off the template. On the other hand, a high flexibility of the polymers should be present to facilitate a fast equilibrium between release and reuptake of the template in the cavity. These two properties are contradictory to each other, and a careful optimization became necessary.

2 Template
. Optically active templates have been used in most cases during optimization. In these cases the accuracy of the structure of the imprint (the cavity with binding sites) could be measured by its ability for racemic resolution, which was tested either in a batch procedure or by using the polymeric materials as chromatographic supports. One of the many attractive features of the molecular imprinting method is that it can be applied to a diverse range of analytes, however, not all templates are directly amenable to molecular imprinting processes..

3 Monomers
The careful choice of functional monomer is one of the utmost importance to provide complementary interactions with the template and substrates. For covalent molecular imprinting, the effects of changing the template to functional monomer ratio is not necessary because the template dictates the number of functional monomers that can be covalently attached; furthermore, the functional monomers are attached in a stoichiometric manner. For non-covalent imprinting, the optimal template /monomer ratio is achieved empirically by evaluating several polymers made with different formulations with increasing template.. It is very important to match the functionality of the template with the functionality of the functional monomer in a complementary fashion (e.g. H-bond donor with H-bond acceptor) in order to maximise complex formation and thus the imprinting effect.

4 Crosslinkers
The selectivity is greatly influenced by the kind and amount of crosslinking agent used in the synthesis of the imprinted polymer. The careful choice of functional monomer is another importance choice to provide complementary interactions
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with the template and substrates (Figure 5). In an imprinted polymer, the cross-linker fulfils three major functions: First of all, the cross-linker is important in controlling the morphology of the polymer matrix, whether it is gel-type, macroporous or a microgel powder. Secondly, it serves to stabilize the imprinted binding site. Finally, it imparts mechanical stability to the polymer matrix

5 Porogenic solvents
Porogenic solvents play an important role in formation of the porous structure of MIP, which known as macroporous polymers. It is known that the nature and level of porogenic solvents determines the strength of non-covalent interactions and influences polymer morphology which, obviously, directly affects the performance of MIP. Firstly, template molecule, initiator, monomer and cross-linker have to be soluble in the porogenic solvents. Secondly, the porogenic solvents should produce large pores, in order to assure good flow-through properties of the resulting polymer. Thirdly, the porogenic solvents should be relatively low polarity, in order to reduce the interferences during complex formation between the imprint molecule and the monomer, as the latter is very important to obtain high selectivity MIP.

6 Initiators
Many chemical initiators with different chemical properties can be used as the radical source in free radical polymerization Normally they are used at low levels compared to the monomer, e.g. 1 wt. %, or 1 mol. % with respect to the total number of moles of polymerisable double bonds. The rate and mode of decomposition of an initiator to radicals can be triggered and controlled in a number of ways, including heat, light and by chemical/electrochemical means, depending upon its chemical nature. For example, the azoinitiator azobisisobutyronitrile (AIBN) can be conveniently decomposed by photolysis (UV) or thermolysis to give stabilised, carbon-centred radicals capable of initiating the growth of a number of vinyl monomers.

7 Polymerization condition
Several studies have shown that polymerization of MIP at lower temperatures forms polymers with greater selectivity versus polymers made at elevated temperatures. Usually, most people using 60 as the polymerization temperature. However, the initiation of the polymerization reaction was very fast and therefore hard to control at this temperature and
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resulted in low reproducibility of molecular imprinted polymer. Furthermore, the relatively high temperatures have a negative impact on the complex stability, which reduced the reproducibility of the monolithic stationary phases and produced high column pressure drops. Thus, the relatively low temperatures of with a prolonged reaction time were selected in order to yield a more reproducible polymerization.

8 PREPERATION METHODS OF MIP

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Figure 3. Polymerization approaches forMIP-based HPLC stationary phases: (A) bulk polymerization, (B) in situ polymerization, (C) one-step suspension or precipitation polymerization, (D) MIP composite beads.

Figure 4. Synthetic schemes for MIP composite beads. (A) Shell-imprinted coreshell beads, (B) MIP-silica composite beads made from iniferter-modified support, (C) multistep swelling polymerization for the production of MIP beads. Reprinted with permission from reference [5].

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Fig. 2. Scanning electron micrographs of spherical MIP particles prepared by: (A) suspension polymerization (500 magnification), from Ref. [25]; (B) multistep swelling polymerization (2000 magnification), from Ref. [27]; (C) precipitation polymerization (the scale is 2 m), from Ref. [31]; (D) precipitation polymerization (the bar is 22 m), from Ref. [33].

New strategies in the synthesis of MIPs

6.1. Microwave-assisted synthesis.

A microwave-heating technique for preparing magnetically- imprinted polymer beads was first proposed by Li and co-workers [7]. The preparation was performed by dispersing the polymerization solution, which comprised the self-assembled mixture of template, functional monomer, copolymer monomer, cross-liner and initiator. Then, polymerization was initiated by microwave heating. The reaction time was dramatically shortened compared to conventional heating. In addition, the resultant polymer beads exhibited good characteristics (e.g., narrow size distribution, uniform morphology, and superior selectivity) and showed rather higher imprinting efficiency. We conclude that microwave heating is a powerful technique to prepare magnetic MIP beads in this simple, efficient manner. So far, the microwave-assisted synthetic method has been applied to bulk
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polymerization, precipitation polymerization, surface-graft polymerization and sol-gel synthesis, and shortened the polymerization time significantly. It has great potential to be used for the efficient preparation of MIPs of different forms.

6.2. Reversible addition-fragmentation chain-transfer (RAFT) polymerization.

So far, free-radical polymerization is the major technique employed in preparing MIPs. However, the MIPs obtained generally have heterogeneous structures because of the uncontrollable chaintransfer and termination reactions. Controlled or living free-radical polymerization methods have great potential to overcomthese intrinsic limitations. Due to its versatility and simplicity, RAFT is an ideal candidate for the controlled or living freeradical polymerization method, where almost all the conventional radical-polymerization monomers can be employed. Recently, MIPs with different formats have been successfully synthesized via RAFT polymerization. Most research focuses on preparing surfaceimprinted polymer. Control of the grafting can be achieved using immobilized azoinitiators [8], iniferters or RAFT agents [9] on the surface of supports. The immobilization of RAFT agent is the commonly used method. MIPs could also be directly grafted onto silica NPs modified with vinyl groups by RAFT polymerization [10]. With the RAFT process, thin imprinted films have been successfully coated on a variety of supports (e.g., silica gel, silica NPs, Fe3O4 microspheres, graphene oxide and polystyrene). RAFT-precipitation polymerization (RAFTPP) was first proposed to prepare regular MIP beads by Zhang and coworkers [11]. The presence of surface-immobilized reactive functional groups on the microspheres obtained allows their further surface modification. Functional polymer brushes or hydrogel layers could be used to modify these MIPs via RAFT polymerization to prepare water-compatible MIPs. Recently, a novel efficient one-pot approach obtained pure-water-compatible and narrowly dispersed MIPs with surface-grafted hydrophilic polymer brushes by RAFTPP [12]. Other formats (e.g., powders and soluble MIPs) have been reported. In general, uniform imprinted shells with adjustable thickness, higher selectivity, improved masstransfer properties, much higher binding capacity and better column efficiency were observed with RAFT polymerization compared with conventional polymerization.

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10 CONCLUSION AND FUTURE OUTLOOK 9 REFERENCE JOURNALS

1. Polyakov MV. 1931. Adsorption properties and structure of silica gel. Zhur. Fiz. Khim. 2: 799805. 2. Pauling L. 1940. A theory of the structure and process of formation of antibodies. J. Am. Chem. Soc. 62: 2643 2657. 3 .Dickey FH. 1949. The preparation of specific adsorbents. Proc. Natl. Acad. Sci. USA 35: 227229. 4. Wulff G, Sarhan A. 1972. The use of polymers with enzyme-analogous structures for the resolution of racemates. Angew. Chem., Intl. Ed. Engl. 11: 341. DOI: 10.1002/anie.197203341 5. Takagishi T, Klotz IM. 1972. Macromolecule-small molecule interactions; introduction of additional binding sites in polyethyleneimine by disulfide cross-linkages. Biopolymers 11: 483491. DOI: 10.1002/bip.1972. 360110213 6 Owens, P. K., Karlsson, L., Lutz, E. S. M., Andersson, L. I., Trends Anal. Chem. 1999, 18, 146154. 7 Ansell, D. J., Adv. Drug Delivery Rev. 2005, 57, 18091835. 8 Wei, S., Mizaikoff, B., J. Sep. Sci. 2007, 30, 17941805. 9 Vasapollo, G., Sole, R. D., Mergola, L., Lazzoi, M. R., Scardino, A., Scorrano, S.,Mele, G., Int. J. Mol. Sci. 2011, 12, 59085945.

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