Richard J. Ansell

MOLECULAR IMPRINTING TECHNOLOGY : CHALLENGES AND PROSPECTS FOR THE FUTURE

Molecular imprinting is a technique for the fabrication of biomimetic polymeric recognition sites or “plastic antibodies/receptors” which is attracting rapidly increasing interest. By this technology, recognition matrices can be prepared which possess high substrate selectivity and specificity. In the development of this technology, several applications have been foreseen in which imprinted materials may be exchanged for natural recognition elements. Thus, molecularly imprinted polymers have been used as antibody/receptor binding mimics in immunoassay-type analyses, as enzyme mimics in catalytic applications and as recognition matrices in biosensors. The best developed application area for imprinted materials, though, has been as stationary phases for chromatography, in general, and chiral chromatography, in particular. This review seeks to highlight some of the more intriguing advantages of the technique as well as pointing out some of the difficulties encountered. The prospects for future development will also be considered. Chirality 10:195–209, 1998.

Magnetic molecularly imprinted polymer beads for drug radioligand binding assay

Molecularly imprinted polymer-magnetic iron oxide composite materials which exhibit recognition properties and can be withdrawn from solution by application of a magnetic field were prepared for the first time. Magnetic iron oxide was incorporated using a suspension polymerisation methodology with a perfluorocarbon liquid as the dispersing phase for the preparation of methacrylic acid-1,1,1-trimethylolpropane trimethacrylate copolymer beads molecularly imprinted with the beta-blocker (S)-propranolol. The resulting superparamagnetic imprinted polymer beads were capable of binding [3H]-(S)-propranolol more strongly than a non-imprinted, otherwise identical, polymer. In a competitive radioligand binding assay using a magnet to separate polymer from solution, (R)-propranolol and (R,S)-metoprolol exhibited cross-reactivities of 19 and 0.7%, respectively, compared with (S)-propranolol.

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.

Molecularly imprinted polymers by suspension polymerisation in perfluorocarbon liquids, with emphasis on the influence of the porogenic solvent

Methacrylic acid (MAA)-trimethylolpropane trimethacryl ate (TRIM) copolymer beads imprinted with tert.-butoxy-carbonyl-l-phenylalanine (Boc-l-Phe) have been prepared by suspension polymerisation in a perfluorocarbon liquid. The suspension polymerisation procedure has been adapted to work with a number of different porogenic solvents, and with UV initiated or thermal initiated polymerisation. The resulting polymers have been evaluated as stationary phases for the chromatographic separation of Boc-d,l-Phe. Polymer beads of average diameter 8–25 μm packed into 100×4.6 mm chromatography columns were used with a mobile phase of dichloromethane-acetic acid (99:1, v/v) at flow-rates up to 5 ml min−1. A polymer prepared with 1,2-dichloroethane as porogenic solvent (average bead diameter 15.1 μm) was capable of the baseline separation of 100 μg Boc-d,l-Phe at a flow-rate of 0.5 ml min−1.

Use of molecularly imprinted polymers in a biotransformation process

Molecularly imprinted polymers are highly stable and can be sterilised, making them ideal for use in biotransformation process. In this communication, we describe a novel application of molecularly imprinted polymers in an enzymatic reaction. The enzymatic condensation of Z-L-aspartic acid with L-phenylalanine methyl ester to give Z-L-Asp-L-Phe-OMe (Z-aspartame) was chosen as a model system to evaluate the applicability of using molecularly imprinted polymers to facilitate product formation. When the product-imprinted polymer is present, a considerable increase (40%) in product yield is obtained. Besides their use to enhance product yields, as demonstrated here, we suggest that imprinted polymers may also find use in the continuous removal of toxic compounds during biochemical reactions.

A fluorescence polarisation molecular imprint sorbent assay for 2,4-D: a non-separation pseudo-immunoassay.

The first pseudo-immunoassay which employs a molecularly imprinted receptor and a fluorescent probe, and quantifies the bound analyte directly using the fluorescence anisotropy of the polymer-probe-analyte suspension, is described.

Imprinted polymers for chiral resolution of (±)-ephedrine, 4: Packed column supercritical fluid chromatography using molecularly imprinted chiral stationary phases

(-)-Ephedrine-molecularly imprinted polymers (MIPs) have been successfully used as stationary phases in supercritical fluid chromatography for the separation of (±)-ephedrine enantiomers. This approach combines the simple preparation and predictable elution order of MIP stationary phases with the superior mobile phase diffusivity and low viscosity of supercritical fluid mobile phases. The optimised mobile phase comprised supercritical carbon dioxide with a modifier consisting of MeOH/isopropylamine/H(2)O 93:5:2 (v/v/v). In many cases, better resolution separations were observed compared to when liquid mobile phases were used, and better separations achieved at high sample loads, although interestingly the MIPs which work best in SFC are different from the MIPs that work best in HPLC with an amine modifier. The MIP stationary phases were stable under the conditions employed and the chromatography was reproducible. This work opens the door to exploiting MIP stationary phases in preparative SFC.

Molecularly imprinted polymer combinatorial libraries for multiple simultaneous chiral separations

We demonstrate that the ligand cross-reactivities of molecularly imprinted polymers can be beneficially employed for the simultaneous separation of different stereoisomeric structures. In this study, a limited molecularly imprinted polymer combinatorial library has been prepared and used for the simultaneous separation of a mixture of N-tert-butyloxycarbonyl-L-phenylalanine, N-tert-butyloxycarbonyl-D-phenylalanine, N-acetyl-L-phenylalaninyl-L-tryptophanyl methyl ester, N-acetyl-D-phenylalaninyl-D- tryptophanyl methyl ester, yohimbine and corynanthine.

Use of fluorescence shift and fluorescence anisotropy to evaluate the re-binding of template to (S)-propranolol imprinted polymers

The binding of (R)- and (S)-propranolol to an (S)-propranolol imprinted polymer in organic and aqueous solutions has been studied using fluorescence. The amount of propranolol that binds can be measured by separating non-bound propranolol from the polymer by centrifugation, and measuring the fluorescence intensity. However, this work demonstrates that other measurements can indicate how much propranolol has bound without the need to separate bound and non-bound analyte. In toluene + 0.5% AcOH, and in aqueous buffer (25 mM citrate pH 6 + 0.5% Triton X100) the fluorescence anisotropy increases as the fraction of analyte bound to the polymer increases. In aqueous buffer, binding to the polymer is also accompanied by a change in the relative intensities of fluorescence at 322 nm and at 352 nm. These non-separation techniques have been used to show that the imprinted polymer binds more (S)-propranolol than a non-imprinted polymer, and at least in organic solvent, shows selectivity for (S)- over (R)-propranolol.

MIP-ligand binding assays (pseudo-immunoassays).

Molecular imprint sorbent assays (MIAs) have been applied to an increasing number of analytes of medical and environmental interest: the sensitivities and selectivities of these assays are comparable to immunoassays employing biological antibodies. In a number of cases complete analytical procedures starting from raw samples (blood, plasma and urine) have been demonstrated. There have been significant advances in applying MIPs in new formats and in the use of non-radioisotope labels. Progress in the field is reviewed, with particular emphasis on the technical aspects and new innovations. It is demonstrated that many of the perceived drawbacks of molecular imprinted polymers (MIPs) do not hinder their application in competitive binding assays: Many MIAs have been applied in aqueous systems and a heterogenous distribution of binding sites is not problematic, provided the recognition sites which bind the probe most strongly are selective.