Karsten Haupt

Assay System for the Herbicide 2,4-Dichlorophenoxyacetic Acid Using a Molecularly Imprinted Polymer as an Artificial Recognition Element

Noncovalent molecular imprinting of a synthetic polymer with the herbicide 2,4-dichlorophenoxyacetic acid has been achieved in the presence of the polar solvents methanol and water. Formation of the prearranged complex relied on hydrophobic and ionic interactions between the template and the functional monomer 4-vinylpyridine. The polymer obtained binds the original template with an appreciable selectivity over structurally related compounds. The potential use of micrometer-sized imprinted polymer particles as the recognition element in a radioligand binding assay for 2,4-dichlorophenoxyacetic acid is demonstrated.

Plastic antibodies: developments and applications

Antibodies are natural receptor molecules produced after contact with an antigen. In an attempt to mimic nature, the technique of molecular imprinting has been developed, which allows specific recognition sites to be formed in synthetic polymers through the use of templates. These recognition sites mimic the binding sites of antibodies and may be substituted for them in applications such as affinity separation, assay systems and biosensors. The stability and low cost of these polymers make them particularly attractive to industry.

Molecularly Imprinted Microgels as Enzyme Inhibitors

We demonstrate, on the example of trypsin, the use of water-soluble molecularly imprinted polymer microgels as specific enzyme inhibitors. Using a strong anchoring monomer, methacryloylaminobenzamidine, the growing polymer chains are confined to close proximity of the substrate recognition site of our model enzyme. The microgels bind selectively trypsin over other proteins of similar size and molecular weight, and show competitive inhibition of trypsin with an inhibition constant K(i) of 79 nM, making them more potent inhibitors than the low molecular-weight competitive inhibitor benzamidine by almost 3 orders of magnitude. We believe that these tailor-made materials with biological activity have potential for future drug development that extends beyond enzyme inhibition.

Molecularly Imprinted Polymers

Molecular imprinting is a process that allows for the synthesis of artificial receptors for a given target molecule based on synthetic polymers. The target molecule acts as a template around which interacting and cross-linking monomers are arranged and co-polymerized to form a cast-like shell. In essence, a molecular memory is imprinted in the polymer, which is now capable of selectively binding the target. Molecularly imprinted polymers (MIPs) thus possess the most important feature of biological antibodies - specific molecular recognition. They can thus be used in applications where selective binding events are of importance, such as immunoassays, affinity separation, biosensors, and directed synthesis and catalysis. Since its beginnings in the 1970s, the technique of molecular imprinting has greatly diversified during the last decade both from a materials point of view and from an application point of view. Still, there is much room for further improvement. The key challenges, in particular the binding site homogeneity and water compatibility of MIPs, and the possibility of synthesizing MIPs specific for proteins, are actively addressed by research groups over the World. Other important points are the conception of composite materials based on MIPs, in order to include additional interesting properties into the material, and the synthesis of very small and quasi-soluble MIPs, close in size to proteins.

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.

Imprinted polymers—Tailor-made mimics of antibodies and receptors

The technique of molecular imprinting allows the formation of specific recognition sites in synthetic polymers through the use of templates or imprint molecules. These recognition sites mimic the binding sites of antibodies and other biological receptor molecules. Molecularly imprinted polymers can therefore be used in applications relying on specific molecular binding events. The stability, ease of preparation and low cost of these materials make them particularly attractive. This review focuses on recent developments and advances in the field of molecularly imprinted materials, with special emphasis on applications in immunoassays and sensors recently developed by our group and by others.

Photopolymerization and photostructuring of molecularly imprinted polymers for sensor applications--a review.

Molecularly Imprinted Polymers (MIP) were prepared by photochemical route. Photoinduced polymerization was used to achieve the preparation of the MIP and at the same time spatially controlled irradiation allowed shaping the material to confer it an optical function useful for interrogation. Such route significantly simplifies the integration of MIP for sensor applications. Specific photopolymerizable MIP were designed for photopolymerization at different wavelengths and advanced methods of photopatterning were used including optical near-field, interference or self-guiding lithography. Photopatterning appears thereby as one of the most suitable methods for patterning MIP at the micro and nano scale, directly on the transducer surface.

Molecularly imprinted polymer for metsulfuron-methyl and its binding characteristics for sulfonylurea herbicides

A molecularly imprinted polymer (MIP) was prepared using metsulfuron-methyl (MSM) as the template molecule. A combinatorial protocol has been employed to optimize the polymer in terms of the kind and relative amounts of functional and cross-linking monomers. A copolymer of 2-(trifluoromethyl)acrylic acid (TFMAA) and divinylbenzene (DVB) showed the highest binding capacity for MSM. The binding characteristics of the imprinted polymers and MSM were evaluated in various solvents using equilibrium binding experiments. The results showed that the MIP binds MSM only in dichloromethane, which was used as the porogen during polymerization. Scatchard plot analysis revealed that two classes of binding sites were formed in the imprinted polymer with dissociation constants of 32.3 mumol 1(-1) and 1.7 mmol 1(-1), respectively. The specificity of the imprinted polymer was investigated by binding assays using MSM and other structurally related sulforylurea herbicides. The results indicated that the imprinted polymer showed a marked selectivity for MSM.

Imprinted polymer-based enantioselective acoustic sensor using a quartz crystal microbalance

An enantioselective chemical sensor has been designed and fabricated. The sensor is based on a molecularly imprinted polymer, serving as the recognition element, and a quartz crystal microbalance (QCM), used as the transducer. The polymer, imprinted with the chiral β-blocking drug S-propranolol, was cast as a thin permeable film onto a gold electrode deposited on the quartz crystal vibrator. The mass increase of the polymer due to analyte binding was quantified by piezoelectric microgravimetry with the QCM. The sensor was able to discriminate between the R- and S-propranolol enantiomers in acidified acetonitrile solutions owing to the enantioselectivity of the imprinted sites. Detectability of S-propranolol was 50 µmol dm–3. The general procedure developed here for preparation of the sensor can be adapted for fabrication of a range of different stable analytical sensing devices for numerous analytes by using conventional molecular imprinting protocols.

Separation of immunoglobulin G from human serum by pseudobioaffinity chromatography using immobilized l-histidine in hollow fibre membranes

L-Histidine, intended as a pseudobiospecific ligand, was immobilized on poly(ethylenevinyl alcohol) hollow fibre membranes after their activation with epichlorohydrin or butanediol diglycidyl ether. The affinity membranes obtained allowed the one-step separation of immunoglobulin G (IgG) from untreated human serum. Elution was possible under mild conditions with discontinuous pH or salt gradients. IgM was also adsorbed to a certain extent and partially separated from IgG by pH gradient elution. The bound IgG fractions showed pI values between 8 and 9.5 and contained IgG1 and IgG3. The dissociation constants for IgG on the bisoxirane- and epichlorohydrin-activated membranes coupled with histidine were determined by equilibrium binding analysis to be 2.5 x 10(-5) and 2.0 x 10(-5) M, respectively. The maximum binding capacity of the affinity hollow fibre membranes was 80 and 70 mg of IgG per gram of support, respectively. With a cartridge of surface area 1 m2 (about 19 g of fibres), during a 60-min run, theoretically up to 1.5 g of IgG can be removed from human serum. The histidine affinity membranes are very stable owing to the simple nature of the ligand and the coupling via an ether linkage. Reproducible results were obtained over more than 1 year even with untreated human serum being used regularly.