De-aluminated zeolite Y as a tool to study endocytosis, a delivery system revealing differences between human peripheral dendritic cells.

We report the use of nanometre-sized zeolite particles as a novel approach to follow the endosomal acidification and proteolysis inside a viable cell. The method was verified by using human peripheral monocytes, a well known endocytosing cell population. Zeolite particles were subsequently used to investigate the endocytosing mechanisms of human peripheral dendritic cells (DC). Probes detecting pH neutral and acidic endosomes were adsorbed to de-aluminated zeolite Y, and used to detect endocytosis in immature human peripheral blood DC. Both the myeloid (mDC) and the plasmacytoid (pDC) dendritic cell subsets had an endocytosing capacity comparable with peripheral blood monocytes. However, the majority of both subsets of DC retained their endosomes at a neutral pH during the first hours after endocytosis and only a small number of the mDC showed any formation of acidic endosomes. Proteolytic degradation of endocytosed proteins was detected using self-quenched DQ-ovalbumin adsorbed to zeolite particles. Interestingly, a clear difference in proteolytic degradation of endocytosed ovalbumin was observed between the two subsets of DC. The mDC showed an efficient degradation of ovalbumin, while the pDC population displayed no or only minor proteolytic degradation. In conclusion, zeolite particles provide a useful tool to study the endocytosing mechanisms, and an efficient carrier of bio-molecules into the endosomal pathways of viable cells.

Selective solid-phase extraction of bio- and environmental samples using molecularly imprinted polymers.

Of the many applications of molecular imprinting in analytical separation science, the one with highest potential of soon being used in routine analysis is that of solid-phase extraction. Already several examples of selective pre-concentration of biological and environmental samples have been reported. The interest in imprinted extraction sorbents originates from the high selectivities and affinities obtainable, properties which can be qualitatively and quantitatively pre-determined for a particular analyte and separation by the imprinting process. This review summarises work published on molecular imprinted solid-phase extraction and discusses some imprinted-sorbent specific method development issues.

Efficient sample pre-concentration of bupivacaine from human plasma by solid-phase extraction on molecularly imprinted polymers.

The ability to use imprinted polymers for solid-phase extraction is demonstrated in a model pre-concentration of bupivacaine from human plasma samples prior to gas chromatography. Imprinting of the structural analogue pentycaine yielded a sorbent which efficiently extracted analyte and internal standard, while possible interference on analyte quantification from leakage of remaining template molecules was eliminated. Human plasma samples were diluted with citrate buffer pH 5, and applied onto solid phase extraction columns containing 15 mg of imprinted sorbent. Wash steps with 20% methanol in water followed by acetonitrile preceded elution with 2% triethylamine in acetonitrile. A direct comparison with conventional sample pre-treatment methods showed the high selectivity of the imprinted sorbent resulted in distinctly cleaner chromatographic traces than were obtained both after liquid-liquid extraction and C18-based solid-phase extraction.

Application of imprinted synthetic polymers in binding assay development.

The first part of the review describes a method for the synthesis of molecularly imprinted polymers for use in binding assays. The method considers the many factors involved that affect the recognition properties of the materials and describes an approach to screening and optimization of these factors. The second part describes the development of binding assays using such polymers. This includes the use of different labels, the effect of solvent and buffer, the scale of the assay (amount of solid polymer), and how these influence the quality of the assay in terms of sensitivity, selectivity, and speed of analysis.

Molecular imprinting: developments and applications in the analytical chemistry field.

In analytical separation science, molecularly imprinted polymers have been applied in several analytical techniques, such as liquid chromatography, capillary electrochromatography and capillary electrophoresis, solid phase extraction, immunoassay, and as a selective sorbent in chemical sensors. A benefit of imprinted polymers is the possibility to prepare sorbents with selectivity pre-determined for a particular substance, or group of structural analogues. The application most close to a wider acceptance is probably that of solid phase extraction for clean-up of environmental and biological samples. The improved selectivity of imprinted polymers compared with conventional sorbents may lead to cleaner chromatographic traces in the subsequent analytical separation. Furthermore, the solid phase extraction application does not suffer from drawbacks generally associated with imprinted polymers in chromatography, such as peak broadening and tailing. Most liquid chromatographic studies have focused on using imprinted polymers as chiral stationary phases for enantiomer separations. Also, the use of imprinted polymers as selective sorbents in capillary electrochromatography has been presented. For this purpose, a protocol to prepare superporous, monolithic imprinted polymer-based capillary columns has been developed. Due to the high affinities and selectivities often achievable, imprinted polymers have been considered as alternative binding entities in biosensors and in immunoassay type protocols. Here, high stability, easy preparation and ability to be used for assay of both aqueous and organic solvent based samples are advantages of the polymers.

Molecular imprinting for drug bioanalysis. A review on the application of imprinted polymers to solid-phase extraction and binding assay.

Molecularly imprinted polymers have been applied as selective sorbents in several analytical techniques, including liquid chromatography, capillary electrophoresis and capillary electrochromatography, solid-phase extraction, and 'immunoassay'. An advantage of this type of sorbent is the possibility to synthesize polymers with selectivity pre-determined for a particular analyte. This review critically discusses the use of imprinted polymers for analysis of drugs and other compounds in biological samples, with emphasis on their use as highly selective solid-phase extraction sorbents for sample pre-concentration and alternative binding entities in immunoassay type protocols.

Application of molecular imprinting to the development of aqueous buffer and organic solvent based radioligand binding assays for (s)-propranolol.

Antibody mimics have been prepared by molecular imprinting of (S)-propranolol and applied in the development of radioligand binding assays for the imprint species. In the assays, polymer particles, radioligand, and analyte were incubated either in an organic solvent or an aqueous buffer, with similarly high sensitivity under both set of conditions. Optimization of the assay conditions led to 100-1000-fold improvements in the limit of determination and 100-1000-fold reductions of the amount of imprinted polymer used, compared with previous studies of this novel type of assay. The present assay uses only 10-50 µg of polymer, and the limit of determination is about 6 nM. The toluene-based assay showed excellent enantioselectivity, the cross-reactivity of the R enantiomer being only 1%, which is better than that demonstrated by biological antibodies. The aqueous buffer based assay showed high substrate selectivity for propranolol in the presence of structurally similar ß-blockers. The different selectivity profiles obtained are due to a different balance between hydrophobic and polar interactions in toluene and water, since polar interactions, such as hydrogen bonds, are strong in apolar solvents and hydrophobic interactions are strong in water.

Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine.

Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed.

Sugar binding polymers showing high anomeric and epimeric discrimination obtained by noncovalent molecular imprinting.

Noncovalent molecular imprinting of sugar compounds in ethylene glycol dimethacrylate-methacrylic acid copolymers yielded materials containing highly selective sugar binding sites. Investigation of a range of polymers demonstrated that the resulting polymer imprints have a high degree of both anomeric and epimeric selectivity favoring the original print molecule. In HPLC assays, a polymer imprinted using p-nitrophenyl-alpha-D-galactoside could separate the alpha and beta anomers of the same compound with near baseline resolution. A polymer imprinted using p-nitrophenyl-beta-D-galactoside showed similar results but with the beta anomers retained longer as expected. Anomeric discrimination of closely related sugars was also possible, with the degree of separation depending on the structural resemblance to the print molecule. Similarly, a polymer imprinted with p-nitrophenyl-alpha-L-fucoside could separate alpha/beta mixtures of p-nitrophenyl-L-fucoside with baseline resolution. Using radioligand displacement assays a polymer imprinted using octyl-alpha-D-glucoside was shown to bind methyl-alpha-D-glucoside with a 40-fold higher affinity than that for the beta-anomer. The epimeric selectivity was even more impressive: methyl-alpha-D-mannoside and methyl-alpha-D-galactoside had 130- and 240-fold lower affinity, respectively, than methyl-alpha-D-glucoside. The results are discussed in relation to possible uses of such polymers in separation and analysis.

Exposure of workers in the electric power industry to electric and magnetic fields.

A survey of 50 Hz electric and magnetic fields in the electric power industry has distinguished differences in exposure among the employees making it possible to compare health effects as a function of exposure. The workers that were most exposed to electric and magnetic fields were linemen and substation personnel who worked on facilities at operating voltages above 20 kV. On average, they were exposed to an electric field strength above 30 V/m for approximately 1.5 to 3 hours per day and to a magnetic flux density of around 1 to 2 microT. The least exposed group were linemen working in the 0.4 to 20 kV part of the network with exposure to a magnetic field of 0.10 microT. The electric field seldom exceeded 30 V/m.

Drug assay using antibody mimics made by molecular imprinting.

Ligand-binding assays are used for determination of minute amounts of substances in the bloodstream. Such assays require a receptor that specifically binds the substance of interest. The receptor used is often an antibody, but antibodies require special handling and a costly production procedure. We have used molecular imprinting, a method for creating selective recognition sites in synthetic polymers, to prepare polymers that mimic antibody combining sites. Molecular imprints made against theophylline and diazepam showed strong binding and cross-reactivity profiles similar to those of antibodies. Here we describe a new radiolabelled ligand-binding assay, the molecularly imprinted sorbent assay, which uses antibody mimics. This assay accurately measures drug levels in human serum, with results comparable to those obtained using a well established immunoassay technique. Antibody mimics, which are stable and readily prepared by molecular imprinting, may provide a useful general alternative to antibodies.

Enantiomeric resolution on molecularly imprinted polymers prepared with only non-covalent and non-ionic interactions.

Molecular imprints were prepared utilizing only weak bonds between the print molecule and functional monomers; the bonding forces used in the imprinting process were only those weaker than covalent and ionic bonds. Methacrylate-based molecular imprints were prepared using a number of chiral compounds, including N-protected amino acid derivatives, as print molecules. Methacrylic acid was used as the functional monomer because the acid function of the monomer forms hydrogen bonds with a variety of polar functionalities, such as carboxylic acids, carbamates, heteroatoms and carboxylic esters, of the print molecule. Bulk polymers were prepared, ground and sieved to particles of size less than 25 microns, packed into high-performance liquid chromatographic (HPLC) columns and used for enantiomeric separations in the HPLC mode. The polymers were shown to effect efficient enantiomeric resolution of a racemate of the print molecule in addition to substrate selectivity for the print molecule in a mixture of substrates with very similar structures. For example, the enantiomers of Cbz-aspartic acid and Cbz-glutamic acid (Cbz = carbobenzoxy) were resolved with separation factors of 1.9 and 2.5, respectively, on polymers with molecular imprints of the L-form of the respective compounds. In addition, these polymers, prepared against Cbz-L-aspartic acid and Cbz-L-glutamic acid, respectively, had the ability to bind selectively the print molecule from a mixture of both racemates, although the two compounds differ only by one methylene group. The results presented represent a substantial widening of the scope of molecular imprinting in that it may now be possible to prepare molecular imprints against a very large number of compounds.

Enantiomeric resolution of amino acid derivatives on molecularly imprinted polymers as monitored by potentiometric measurements.

Potentiometric measurements have been applied to the detection of enantiomeric separations on molecularly imprinted polymers. A flow-through column electrode, based on the use of polymers imprinted against L-phenylalanine anilide, is described. The electrode consisted of a glass column in which the polymer was packed and where the end frits constituted the electrodes. The flow stream potential across the column can be continuously recorded as solvent is pumped through the system. The column resolved the enantiomers of phenylalanine anilide as detected by both UV absorption and potentiometric measurements and the recorded signals could be correlated with the concentration of phenylalanine anilide. The calibration graphs obtained for the UV absorption of phenylalanine anilide were linear over the concentration range investigated, whereas the potentiometric signal was shown to be exponentially linear with concentration. The application of molecular imprints to the preparation of supports suitable for chromatographic separations of enantiomers and for the preparation of specific electrodes is discussed.

The synthesis of an active derivative of cyclomaltoheptaose for the hydrolysis of esters and the formation of amide bonds.

The synthesis is described of a derivative of cyclomaltoheptaose (beta-cyclodextrin) to which the tripeptide Ser-His-Asp, the catalytic triad found in chymotrypsin, has been coupled. The derivative enhanced the rates of hydrolysis of activated esters, as measured by the release of p-nitrophenol, and the formation of amine bonds.

Molecular recognition in synthetic polymers. Enantiomeric resolution of amide derivatives of amino acids on molecularly imprinted polymers.

Molecular imprints were prepared using L-phenylalanine anilide as the print molecule and methacrylic acid as the functional monomer. Methacrylic acid interacts ionically with the primary amine of the print molecule and via hydrogen bonding with the amide function. In the HPLC mode such polymers were shown to exhibit efficient enantiomeric resolution of a racemic mixture of the original print molecule. Enantiomeric resolution was shown to be dependent on the ratio of methacrylic acid to print molecule in the pre-polymerization mixture and specific for the presence of both print molecule and functional monomer. Further analyses showed the importance of both the primary amino and amide functions in the correct stereochemistry for recognition and enantiomeric resolution of compounds on such polymers. Other amide derivatives of amino acids including p-nitroanilides, beta-naphthylamides and amides were recognized by such polymers, and enantiomeric resolution was obtained for amide derivatives of amino acid ranging from alanine to tryptophan on a single polymer. The implications of these findings with respect to the mechanism of recognition and the ability to predict enantiomeric resolution of molecules on molecularly imprinted polymers will be discussed.

Comparison between two power-frequency electric-field dosimeters.

Two instruments, an EFEM meter and a BE-log meter, have been tested on people working in switchyards for voltages between 70 kV and 400 kV. The results from the BE-log meter correlate well with the short circuit currents through people connected to the ground. The EFEM meter showed lower measured doses than the BE-log meter, and the relation between the two instruments was around 0.6.