Börje Sellergren

NONCOVALENT MOLECULAR IMPRINTING : ANTIBODY-LIKE MOLECULAR RECOGNITION IN POLYMERIC NETWORK MATERIALS

Abstract Molecular imprinting techniques allow the preparation of polymeric receptors which bind small molecules with affinities and selectivities of the same order as those observed in the binding of antigens by antibodies. The molecular imprinting technology has now reached a stage where the commercial use of imprinted materials is being assessed, notably for separations requiring strong and selective binding of small molecules. This development is driven by the potential advantages of polymeric receptors over biological in terms of stability, capacity, cost and ease of preparation. In this short review the state of the art of noncovalent imprinting is summarised indicating a few areas in analytical chemistry where the technique may have future impact.

Use of silane monomers for molecular imprinting and enzyme entrapment in polysiloxane-coated porous silica

Abstract The use of organic silane monomers in the preparation of substrate-selective polymers by molecular imprinting is described. Silanes are allowed to polymerize on the surface of porous silica particles in aqueous solution. The resulting polysiloxane copolymer becomes covalently anchored to silanol groups of the original silica. such preparations retain the rigidity of the silica matrix and can therefore be used in high-performance liquid chromatography. Polysiloxane copolymers imprinted with the dyes rhodanile blue or safranine O showed preferential binding of the respective compound. The observed recognition is believed to occur because cavities containing specific binding groups for the dyes at defined positions are developed during the polymerization procedure. In this context the synthesis of a new silane, boronatesilane, was carried out. This compound was included in the monomer mixture used for the preparation of a polysiloxane-coated silica showing affinity for the glycoprotein transferrin. Organic silanes were also used for entrapment of enzymes, resulting in block polymers, which after fragmentation yielded relatively high recoveries of enzyme activity. Alternatively, the entrapment/polymerization was allowed to proceed on the surface of porous silica, in analogy with the imprinting procedure, resulting in entrapped enzyme preparations with high mechanical stability.

Polymer- and template-related factors influencing the efficiency in molecularly imprinted solid-phase extractions

Abstract This review identifies the factors of importance in the development of molecularly imprinted polymers for use in solid-phase extractions and summarizes the results obtained to date in the area of bioanalysis, pharmaceutical analysis and environmental analysis.

Method for synthesis and screening of large groups of molecularly imprinted polymers

A technique for the synthesis of molecularly imprinted polymers (MIPs) in small scale (∼55 mg) coupled with direct in situ processing and batch rebinding evaluation is reported. The primary assessment is based on quantification by HPLC or UV absorbance measurement of the amount of template released from the polymer in a given solvent. This method allows a rapid screening of the parameters of importance to reach a desired level of binding affinity capacity and selectivity for a given target molecule. This was demonstrated for the triazine herbicide terbutylazine, where an initial screening was performed for the type of functional monomer used in the MIP preparation. Thus among the six functional monomers tested, methyl methacrylate, 4-vinylpyridine, and N-vinyl-α-pyrrolidone led to rapid and quantitative extraction whereas methacrylic acid and (trifluoromethyl)acrylic acid led to polymers that retained the template the most. After having established useful functional monomers, a secondary screening for selectivity was performed. In this, nonimprinted blank polymers were prepared and a normal batch rebinding evaluation was performed. The polymer showing the highest selectivity was the one prepared using methacrylic acid as functional monomer. This polymer was shown to strongly retain chlorotriazines including atrazine when a normal-scale batch of the polymer was evaluated in chromatography.

Evaluation of methods aimed at complete removal of template from molecularly imprinted polymers

Polymers imprinted with clenbuterol were used to study the influence of various post-polymerization treatments [e.g., thermal annealing, microwave assisted extraction (MAE), Soxhlet extraction and supercritical fluid template desorption] on the bleeding of residual template. The aim of the study was to reduce the bleeding to levels that would allow the use of the materials as affinity phases for extraction of clenbuterol from bovine urine at concentrations below 1 ng ml-1. After treatment, the clenbuterol imprinted polymers were packed into solid-phase extraction columns and the bleeding was estimated by quantifying the amount of template released in 10 ml of methanol-acetic acid (9 + 1 v/v). This was followed by an assessment of selectivity and recovery in comparison with non-treated material. The lowest bleeding level was found after MAE using 100% trifluoroacetic acid for 3 x 20 min at 100 degrees C. The collected eluate contained in this case 3 ng ml-1 of clenbuterol. The same material was subsequently used for the extraction of clenbuterol from spiked bovine urine. The resulting selectivity and recovery were lower compared with those obtained using the untreated material. A milder but still efficient method to reduce the bleeding level was found to be MAE with formic acid. In this case a bleeding level of 14 ng ml-1 was found after only a 1 h extraction time. In a second model system, using a polymer imprinted with L-phenylalanine anilide, the bleeding was reduced to a similar level by extensive on-line washing in good swelling solvents containing acid or base additives and after thermal annealing of the polymers in the dry state.

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.

Imprinted dispersion polymers : a new class of easily accessible affinity stationary phases

Abstract Non-stabilizing dispersion polymerization in combination with molecular imprinting was used to prepare agglomerates of globular micron-sized particles exhibiting molecular recognition properties. These could be prepared either in situ in a chromatographic column or separately followed by wet or dry packing of the material. This allowed a rapid chromatographic evaluation of the molecular recognition properties of the materials. Depending on the monomer concentration and the solvency of the dispersion medium the particle dispersity, the degree of particle agglomeration and the average particle size varied. The choice of dispersion medium was mainly dictated by the template solubility and the nature of the interactions between the functionalized monomers (methacrylic acid) and the template used for producing the molecular recognition sites. Addition of water to the dispersion medium allowed imprinting of the poorly soluble template pentamidine (PAM), a drug used for the treatment of AIDS-related disorders. The PAM-imprinted materials prepared in situ in the chromatographic column strongly retained the drug in the chromatographic evaluation compared to the retention of PAM on a reference material prepared using benzamidine as template (separation factor α′ = 6.8). Meanwhile weakly or moderately basic templates from the group nucleotide bases (tri-O-acetyladenosine), herbicides (atrazine) and chiral amino acid derivatives ( l -phenylalanine anilide) required low temperature and exclusion of water during imprinting in order to produce the recognition effect.

Study of the thermodynamics and mass transfer kinetics of two enantiomers on a polymeric imprinted stationary phase

The adsorption isotherms of d- and l-phenylalanine anilide (PA) on an l-phenylalanine anilide imprinted stationary phase have been determined using staircase frontal analysis. An aqueous buffer–organic solvent mixture has been used as mobile phase. The measurements were done at temperatures of 40, 50, 60 and 70°C for sample concentrations ranging between 5·10−4 to 1 g/l. It was found that the adsorption data fit well to both the Freundlich and the Bi-Langmuir isotherm models. Examination of the best values of the numerical coefficients of the Bi-Langmuir model shows that the site class representing the binding sites with the highest binding energy exhibits a very low saturation capacity for the non-imprinted enantiomer, indicating a high selectivity for the imprinted l-enantiomer. The low energy site class also shows some selectivity for the l-enantiomer. Mass transfer rate coefficients were obtained for each single breakthrough curve by using the transport model of chromatography. It was found that the mass transfer coefficient of l-PA increases very rapidly with the sample concentration while there is only a slight increase for the other enantiomer.

Imprinted polymers as antibody mimetics and new affinity gels for selective separations in capillary electrophoresis

Abstract Methacrylate-based imprinted dispersion polymers could be prepared in situ in a fused-silica capillary as agglomerates ( ca . 10 μm) of micrometer-sized globular particles, exhibiting antibody mimetic, molecular recognition properties. Thus, in one example, imprinted polymer particles selective for pentamidine (PAM), a drug used for the treatment of AIDS-related pneumonia, could be prepared in situ in the capillary. The retention could be varied predictably by changing the electrolyte pH. Thus, whereas no observable caution of PAM was achieved at near neutral pH, the PAM-selective capillary gave a retention time of 18 min for PAM and 7.8 min for benzamidine at pH 3.5, whereas the retention times were 6.6 and 6.1 min, respectively, with a reference capillary. Importantly, the electrolyte could by pumped hydrodynamically through the capillaries, allowing rapid phase changes and micro-chromatographic possibilities with high plate numbers.

Extraction of clenbuterol from calf urine using a molecularly imprinted polymer followed by quantitation by high-performance liquid chromatography with UV detection

A method for the extraction of clenbuterol from calf urine samples using a molecularly imprinted polymer (MIP) has been developed. The aim was that the final extracts from the MIP should allow quantitation of clenbuterol down to 0.5 ng/mL urine using HPLC with UV detection. The MIP was produced using brombuterol as a template and the selectivity of the MIP, for clenbuterol, was tested against a non-imprinted polymer (produced without template) and was found to be high. After loading of 5 mL diluted centrifuged urine, selective binding was established in acetonitrile-acetic acid (98:2). For further elution of interferences, 0.5 M ammonium acetate buffer pH 5 and 70% acetonitrile in water was used. Clenbuterol was eluted using 1% trifluoroacetic acid in methanol, which was evaporated and reconstituted in buffer. Results from the HPLC analyses showed that the extraction of clenbuterol using MIP is linear in the range 0.5-100 ng/mL with good precision (4.3% for 0.6 ng/mL and 2.1% for 6.0 ng/mL) and accuracy (96.7% for 0.6 ng/mL and 96.7% for 6.0 ng/mL). The recoveries were 75%. The results show that the method offers a selectivity and sensitivity that make the quantitation of 0.5 ng clenbuterol/mL urine by HPLC-UV possible and a competitive alternative to state-of-the-art routine analytical methods.