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CHAPTER 1
Nano-sized Molecularly Imprinted Polymers as Artificial Antibodies
F. CANFAROTTAa, A. CECCHINI,*b,c AND S. PILETSKYd
a MIP Diagnostics Ltd, University Road, Leicester LE1 7RH, UK;
b IMSaT, University of Dundee, Wilson House, 1 Wurzburg Loan, Dundee DD2 1FD, UK;
c Bond Life Sciences Center, University of Missouri – Columbia, 1201 E. Rollins St., Columbia, MO 65211-7310, USA;
d Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, UK
1.1 Molecularly Imprinted Polymers: Different Formats for Different Applications
Molecularly imprinted polymers (MIPs) emerged about 40 years ago, when Wulff and Sahan proposed the strategy of polymerisation performed in the presence of a target-template.1 Since then, MIPs have been exploited for multiple applications thanks to their remarkable properties, such as high affinity and selectivity, and resistance to extremes of temperature, pressure and pH variations. In molecular imprinting, functional and cross-linking monomers are polymerised in an appropriate porogenic solvent in the presence of the compounds to be imprinted (called “templates”). After removal of the template, the polymer matrix retains recognition cavities that are complementary to this template in terms of size, shape and functionality. More precisely, the three-dimensional arrangement of cavity recognizing sites is driven by the structure of the template molecule. Therefore, after template removal the synthesized polymer is capable of binding the target, which can be the analyte itself. Compared with synthesis of monoclonal antibodies (mAbs), the synthesis of MIPs is simpler and cheaper, and can be performed without any preclinical development involving animals. In addition, MIPs show high stability and excellent mechanical properties. Moreover, they can be prepared for a wide variety of targets.2,3 Template removal, however, is often difficult and incomplete, with the possible disadvantage of subsequent analyte leaching from the matrix, thus resulting in inaccurate performance in analytical applications.4 Moreover, it is quite laborious to integrate them with signal transducers in sensors (unless electropolymerisation is involved, see Section 1.3.1.2, below) or to convert the template binding into an electric signal.5 In general, MIPs can be manufactured in different ways (Table 1.1) and in several formats, for example as films or membranes, microparticles or nanoparticles. Compared to other formats, the nanoparticles present several advantages. In particular, this format allows the system to exhibit a much higher surface-to-volume ratio and larger total surface area per weight unit of polymer. The imprinted cavities are more easily accessible by the analytes, thus improving binding kinetics and template removal and, hence, enhancing their recognition capabilities.6 Several authors have started developing nanoMIPs for diagnostic and therapeutic applications, for instance as drug delivery systems7,8 and sensing elements in assays or sensors.9,10 In virtue of their features, nanoMIPs represent an attractive option for a wide range of applications. One interesting characteristic of nanoMIPs is their property of remaining in solution, rendering them suitable for in vitro studies.11 However, it is crucial to obtain particle batches with a very narrow size distribution and with high yield, especially for biomedical studies. Two other great advantages of MIPs, compared to natural ligands, are their relatively straightforward preparation and, particularly, their inexpensive fabrication. Indeed, the availability of cheap reagents for MIP syntheses has led researchers to explore novel polymerisation approaches for devising smaller and monodispersed MIPs, from the most intuitive techniques (i.e. precipitation polymerisation) to more sophisticated ones (i.e. the use of solid-phase polymerisation and automated reactors).
Table 1.1 List of synthetic procedures available for the synthesis of MIPsa
| Approach | Procedure | Advantages | Drawbacks | Ref. |
| Bulk | Performed using organic solvents. A block is obtained, and then crushed and sieved | Simple method | Wide particle size distribution and heterogeneity of active sites | 37,38 |
| Precipitation polymerisation | Polymer chains grow in solution, precipitating when their size makes them insoluble | Easy and fast with high yields. Low amount of reagents required | The low monomer concentration required might affect the interactions with the template | 19,39 |
| Emulsion polymerisation | Use of surfactants and high-shear homogenisation to emulsify the water phase with the organic one | Possible to obtain very small NPs (50 nm) | Surfactants might interfere with the imprinting process. Difficult removal of surfactants | 19,40 |
| Core-shell emulsion polymerisation | Deposition of an MIP layer on preformed nanoparticles (made of metals, silica, polymers) | Suitable for large-scale production. High yields | The presence of surfactants and the aqueous phase can decrease the imprinting effect | 41,42 |
| Core-shell grafting | Chemical linkage of MIP to preformed nanoparticles modified with double bonds or iniferter | Excellent control over shell thickness. Sequential shell polymerisation | Imprinted shell might be too thin for imprinting of bulky templates like proteins | 43,44 |
| Living radical polymerisation | Use of nitroxide species, metal-containing or dithiocarbonyl initiators. Polymer chains grow at similar rates | Excellent control over particle size and PD. Useful for thermolabile templates | Low yield. Removal of catalyst needed (in NMP and ATRP). Not suitable for photolabile templates | 45,46 |
| Solid-phase polymerisation | The template is immobilised on the surface of a solid support (typically micro-sized glass beads). High affinity nanoMIPs are collected by a temperature-based affinity separation step | High affinity and selectivity (nano- or picomolar). High purity with low template contamination. Fully automatable process | The template must have functional groups for immobilisation. Typically, one binding site per particle (low binding capacity) | 9,32,33,35 |
1.2 Advances in the Synthesis of NanoMIPs; Different Approaches to Preparation of MIPs as Nanoparticles
Molecular imprinting involves three main steps; i.e. (i) the formation of the monomer–template complexes, (ii) polymerisation, and (iii) removal of the template and collection of the MIPs. In general, MIPs can be fabricated by means of two main approaches: covalent and non-covalent imprinting. In the former, developed by Wulff,12 reversible chemical bonds are created between the monomer and the template during the polymerisation, and the same bonds are then re-formed in the analyte binding step. The advantage is that only the monomer’s functional groups interact with the template and cavities with more homogeneous recognizing sites are generated. However, not many compounds are suitable for this approach to be used and, therefore, they need preliminary derivatization with the monomer. Furthermore, the template removal is quite difficult and the analyte binding step is slower compared to that of other approaches.5 In the non-covalent approach pioneered by Mosbach and co-workers,13 hydrogen bonding as well as electrostatic and hydrophobic interactions are involved in the formation of the monomer–template pre-polymerisation complexes, as well as in the following analyte recognition. Since weak interactions are involved, an excess of monomer is usually employed to stabilise the monomer–template complex. This method is easier and more versatile than the covalent approach, although issues related to heterogeneity of the binding sites within cavities generated might arise.14 Considering the advantages of the aforementioned two approaches, some authors have combined them, thus using a template covalently linked to the monomer and the following analyte binding step designed in a non-covalent way, thus introducing the concept of semi-covalent imprinting.14 In this section, we will explore the main polymerisation modalities optimised so far for the synthesis of MIP nanoparticles (nanoMIPs), ...
