Klaus Mosbach

The Emerging Technique of Molecular Imprinting and Its Future Impact on Biotechnology

The technique of molecular imprinting allows the formation of specific recognition and catalytic sites in macromolecules by the use of templates. Molecularly imprinted polymers have been applied in an increasing number of applications where molecular binding events are of interest. These include (i) the use of molecularly imprinted polymers as tailor-made separation materials, (ii) antibody and receptor binding site mimics in recognition and assay systems, (iii) enzyme mimics for catalytic applications, and (iv) recognition elements in bio-sensors. The stability and low cost of molecularly imprinted polymers make them advantageous for use in analysis as well as in industrial scale production and application.

MOLECULARLY IMPRINTED POLYMERS : USEFUL MATERIALS FOR ANALYTICAL CHEMISTRY?

Abstract Molecular imprinting is a technique for producing chemically selective binding sites, which recognize a particular molecule, in a macroporous polymer matrix. This paper reviews recent advances in imprinting methodology, particularly emphasizing some recently introduced functional monomers and methods which directly yield polymeric material suitable for chromatography. Following this, attempts to incorporate imprinted polymers into a range of analytical methodologies are critically discussed, with some pointers to likely directions for future research.

Competitive amperometric morphine sensor based on an agarose immobilised molecularly imprinted polymer

Abstract A morphine-sensitive device was constructed based on a molecularly imprinted polymer. The imprinted polymer exhibited recognition properties previously. A method of detection based on competitive binding was used to measure morphine in the concentration range 0.1–10 μg/ml. A morphine concentration of 0.5 μg/ml gave a peak current (by oxidation) of 4 nA. The method of morphine detection involves two steps. In the first step, morphine binds selectively to the molecularly imprinted polymer in the sensor. In the second step, an electroinactive competitor (codeine) is added in excess, whence some of the bound morphine is released. The released morphine is detected by an amperometric method. The advantages of this type of sensor compared to biosensors based on antibodies, enzymes or cells are discussed. This sensor, based on an artificial recognition system, demonstrates autoclave compatibility, long-time stability and resistance to harsh chemical environments.

Synthesis and catalysis by molecularly imprinted materials.

Molecularly imprinted materials have been demonstrated to possess a very high degree of selectivity towards targeted substrates. In addition to such tailor-made molecular recognition, progress has been made in introducing reactive groups into the recognition sites. Putting teeth into imprinted matrices is one method of making true enzyme mimics or plastizymes, which are plastic polymer enzyme mimics.

Molecularly imprinted microspheres as antibody binding mimics

Molecular imprinting is an emerging technique for preparing artificial antibodies that have potential applications in affinity-based separations, biomimetic sensors and assays. The conventional imprinting methods either deliver a monolith that is mechanically ground to irregular particles - a time-consuming process with low yield, or involves complicated polymerization process by which spherical beads may be obtained, but with frequently compromised binding specificity. We have recently developed a novel molecular imprinting method that can deliver microspheres bearing imprinted binding sites, quickly, cleanly and in good yield. Imprinted uniform microspheres are synthesized by precipitation polymerization of a dilute solution of functional monomer and crosslinker in the presence of a print molecule. The method is generally applicable to a large variety of target molecules. For the first time, an enzyme probe is successfully used in a competitive herbicide assay using molecularly imprinted microspheres as the affinity adsorbents.

Imprinting of amino acid derivatives in macroporous polymers

Abstract Phenylalanine ethyl ester-selective polymers have been prepared using the ion-pair association of substrate and carboxyl-containing vinyl monomers in the polymerization step.

New configurations and applications of molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) are applicable in a variety of different configurations. For example, bulk polymers imprinted with beta-lactam antibiotics are presented to be used as stationary phases for the chromatographic separation of beta-lactam antibiotics with both aqueous and organic mobile phases. However, in some analytical applications, monosized spherical beads are preferred over the currently used ground bulk polymers. A precipitation polymerization technique allows preparation of monosized spherical imprinted beads with diameters down to 200 nm having excellent recognition properties for different target molecules. Nevertheless, with current imprinting protocols a substantial amount of template has to be used to prepare the polymer. This can be problematic if the template is poorly soluble, expensive or difficult to obtain. It is shown that for analytical applications, the functional monomer:template ratio can be drastically increased without jeopardizing the polymers recognition properties. Furthermore, a substantial reduction of the degree of crosslinking is demonstrated, resulting in much more flexible polymers that are useful for example the preparation of thin imprinted films and membranes for sensors. Apart from analysis, MIPs also are applicable in chemical or enzymatic synthesis. For example, MIPs using the product of an enzyme reaction as template are utilized for assisting the synthetic reaction by continuously removing the product from the bulk solution by complexation. This results in an equilibrium shift towards product formation.

Influence of mobile phase composition and cross-linking density on the enantiomeric recognition properties of molecularly imprinted polymers

A series of experiments were conducted to investigate elements which affect the enantiomeric recognition properties of molecularly imprinted polymers (MIPs) in the HPLC mode. Our results show that the recognition properties of MIPs are greatly influenced by the mobile phase used. For a polymer prepared in acetonitrile, a good enantiomeric separation was observed when acetonitrile-based mobile phase was used, when the mobile phase was changed to chloroform-based, no enantiomeric recognition was observed although the sample molecule was retarded. This indicates that the specific co-operative binding interactions between the functional groups at the imprinted polymers recognition sites and the sample molecule were considerably disrupted and only non-specific interactions remained. When the mobile phase was changed back to acetonitrile-based, the recognition was regained. In contrast, for polymers prepared in chloroform, chloroform-based mobile phase gave much better separation than acetonitrile-based mobile phase. When other solvents were tested, significant solvent effects were generally observed. Based on these observations, the recognition properties of the methacrylic acid (MAA)-co-ethylene glycol dimethacrylate (EGDMA) polymers were reinvestigated, and the results show that by simply using an optimised mobile phase system, significantly improved recognition over previously reported results was observed. For a polymer made against Cbz-L-Trp, 100 microg of Cbz-D,L-Trp was separated with a separation factor (alpha) of 4.23 and a resolution (Rs) of 3.87, whereas in the previous report, 10 microg of Cbz-D,L-Trp was only separated with alpha = 1.67 and Rs = 0.1. It is generally realised that the imprinted polymers recognition property is also very much influenced by the nature of the polymer network. It was shown that the recognition decreased with a decrease in the apparent degree of cross-linking (molar percentage of cross-linker in the polymerisation mixture). Nonetheless, our results show that in our optimised assay system a significant separation could still be obtained on a polymer which was only 22% cross-linked. We consider this to be of importance, since it may suggest a way of imprinting larger molecules because of the possibly improved mass transfer in low cross-linking density polymers. It was reported that when trifunctional cross-linkers [for example: trimethylolpropane trimethacrylate (TRIM)] were used as the cross-linker instead of EGDMA, considerably improved enantiomeric separation and resolving capability were observed. Our results show that the improved performance of the MAA-co-EGDMA MIPs is actually comparable to the performance of the MIPs prepared with those trifunctional cross-linkers. The combination of a hydrogen bonding functional monomer (acrylamide) with TRIM also did not give improved recognition. The results suggest that although the three-dimensional network of these two kinds of polymer may be quite different, the observed recognition improvements were probably largely due to solvent effect.

Molecularly imprinted polymers for bioanalysis: chromatography, binding assays and biomimetic sensors.

The field of molecular imprinting has undergone rapid expansion in recent years, and applications have been demonstrated for imprinted materials in separations, analysis and catalysis. The range of compounds that has been imprinted has also expanded. Imprinted polymers have been used for the chromatographic separation of amino acids, sugars, drugs and nucleotides. Drugs have been measured using imprinted polymers as antibody substitutes in radioligand binding assays. Applications are being found for imprinted polymers in biomimetic sensing devices and sensors. Particularly interesting developments from the past year include ligand binding assays for morphine in aqueous buffers with similar specificity to antibody-based assays and the imprinting of cholesterol using a novel procedure in which the template is covalently bonded to the monomer during polymerization, but after cleavage, rebinds the polymer only by non-covalent interactions.

Molecular imprinting of amino acid derivatives in macroporous polymers : Demonstration of substrate- and enantio-selectivity by chromatographic resolution of racemic mixtures of amino acid derivatives

Abstract Substrate-selective macroporous polymers were prepared by a non-covalent imprinting procedure. By this method, acrylic polymers selective for d or l -phenylalanine ethyl esters were prepared utilizing coulombic interactions, during polymerization, between the print molecule ( d or l -phenylalanine ethyl ester) and the carboxyl group carried by the vinyl monomers. These polymers, after removal of print molecules, were used in a chromatographic procedure to examine their enatio- and substrate-selectivity for some amino acid derivatives. The best separation factor, α, for d , l -phenylalanine ethyl ester was found to be 1.30. The selectivity persisted to several months after intermittent applications.