Molecular Imprinting -- A Way to Make "Smart Polymers".
In order to protect our health and the environment we need to be able to detect very small quantities of drugs or pollutants. This has stimulated the search for sensitive and highly specific methods to isolate these compounds from complex matrices. One technique that has been introduced lately is the molecular imprinting of polymers, leading to materials that contain receptor sites, designed to recognize and preferentially hind a particular molecule . The concept is simple: a polymer is constructed around the target molecule, which serves as a template and is extracted after the polymerization is completed. This leaves a polymer with cavities, or imprints, that are complementary in shape and electronic environment to the compound of interest.
Although still in the experimental stage, molecular imprints (MIPs), have been tested for practical application in several areas of analytical chemistry, for instance in chromatography. Structurally similar molecules, such as enantiomers, were successfully separated by HPLC using an imprint of one of the enantiomers as the stationary phase. The imprinted species was preferentially retained while the other was eluted. This is particularly important in view of the fact that many drugs are chiral and are synthesized as racemates, while only one of the enantiomers is of pharmaceutical interest.
An elegant use of MIPs, pioneered by Mosbach et al., is as artificial antibodies in immunoassays.  These assays provide a sensitive method for the quantification of very small, sometimes picogram, amounts of drugs. Replacing the natural antibodies with MIPs, however, offers some distinct advantages. First, there is a large saving in time. It takes at least several weeks to raise natural antibodies in animals at considerable cost, while MIPs can be prepared in the laboratory in days using inexpensive ingredients. Moreover, the MIPs are inert, can conveniently be stored at room temperature, and are reusable.
Potentially, molecular imprints are useful wherever molecular recognition is part of, or can facilitate analyses. It is often necessary to extract the analyte from a biological sample prior to detection and quantification and this can be labourintensive and tedious. The advantages of using a molecularly imprinted solid phase for extractions are clear.
Finally, the use of molecularly imprinted membranes as the recognition element in chemical sensors should be mentioned. These devices can be small and portable and are particularly practical for the analyses of pollutants in the environment since they can easily be taken out in the field. Recently, it was reported that it was possible to detect traces of nerve gases in fieldwater in concentrations as low as seven parts per trillion with a MIP membrane chemical sensor.
Mechanical and chemical properties
The performance of a MIP is, of course, dependent on the initial choice of polymerizable ingredients and the reaction conditions that are used to synthesize the polymer. In order to make a good MIP there are several factors to consider, for instance the physical quality of the matrix. The polymer must have sufficient mechanical stability to withstand manipulation, not break up when shaken vigorously and, if it is to be used for HPLC, not collapse under pressure. Heat resistance is also desirable and extends the purposes for which the MIP can be used. It should be rigid enough to retain a permanent memory of the imprint, yet it needs to he macro-porous to allow the template to easily diffuse in and out. It was found experimentally that these criteria are satisfied by using a high ratio, typically 80 per cent, of cross-linking monomer in the polymerization mixture. A cross- linker, as the name implies, is a molecule that contains two or more polymerizable end groups and is responsible for forming a polymer netw ork. At the moment, the cross- linkers of choice for making MIPs are ethyleneglycol dimethacrylate and trimethylolpropyl trimethacrylate, both of which form polymers that have the required properties. The rest of the polymer is made up of a functional monomer. This is a polymerizable molecule which bears one or more functional groups and is selected for its ability to attractively interact with the chosen template. The role of the functional monomer is to provide points of electronic recognition for rebinding of the template. The choice of functional monomer is often based on its ability to form hydrogen bonds or an ion pair with the template. There is a wide selection of acidic, basic or neutral compounds commercially available for the chemist to choose from. Methacrylic acid is the monomer most often used to interact with templates bearing electron donating functionalities, while vinylpyridine makes a good hydrogen acceptor. Interestingly, a combination of the two has also been used with good success. Other forces of attraction, such as metal ion chelation can also be instrumental in producing strong binding sites, in analogy to nature's binding of porphyrin rings to various electron deficient metal ions. Nature, of course, continually makes use of all these forces for receptor site mediated biological processes. However, what nature accomplishes seemingly without effort still requires experimental trial and error in the laboratory.
Figure 1 shows a schematic representation of the process by which we recently imprinted a polymer with ethynylestradiol to be used as an artificial antibody . The template has four fused rings, making it a stiff molecule with few degrees of freedom, which is conducive to good rebinding. On the negative side, the molecule only has two functional groups that can participate in hydrogen bonding. After trying out several different polymer recipes and optimizing the conditions, it was found that the polymer made with ethyleneglycol dimethacrylate as cross-linker and vinylpyridine as functional monomer performed best when tested by immunoassay. In fact, the MIP exhibited strong rebinding characteristics and was highly selective for ethynylestradiol.
The actual synthesis of the MIPs in the laboratory does not take much time and is decidedly not a high-tech operation. The MIP ingredients are mixed in a test tube with a suitable solvent and irradiated with a UV lamp. The resulting hard polymer monolith is taken out of the test tube, ground to small particles and sieved to obtain a uniformly sized fraction. The polymer is then washed to remove the template and after drying is ready for use.
Presently, extensive research is being done to perfect the molecular imprinting techniques. It is to be expected that in the near future these versatile materials will be available as additional tools for chemists.
Irene Idziak is a principal scientist at MDS Pharma Services, Montreal, QC.
(1.) Owens, P.K., L. Karlsson, E.S.M. Lutz and L.I. Anderson, "Tailor-made materials for tailor-made applications: application of molecular imprints in chemical analysis', Trends Anal. Chem., 18: 146-154, 1999.
(2.) Vlatakis, G., L.I. Anderson, R. Muller and K. Mosbach, 'Drug assay using antibody mimics made by molecular imprinting', Nature, 361: 645-647, 1993.
(3.) Idziak, I. and A. Benrebouh, 'A molecularly imprinted polymer for 17[alpha]-ethynylestradiol evaluated by immunoassay', The Analyst, in press.