Hyperphosphorylation at tyrosine is commonly observed in tumor proteomes and, hence, specific phosphoproteins or phosphopeptides could serve as markers useful for cancer diagnostics and therapeutics. The analysis of such targets is, however, a challenging task, because of their commonly low abundance and the lack of robust and effective preconcentration techniques. As a robust alternative to the commonly used immunoaffinity techniques that rely on phosphotyrosine(pTyr)-specific antibodies, we have developed an epitope-imprinting strategy that leads to a synthetic pTyr-selective imprinted polymer receptor. The binding site incorporates two monourea ligands placed by preorganization around a pTyr dianion template. The tight binding site displayed good binding affinities for the pTyr template, in the range of that observed for corresponding antibodies, and a clear preference for pTyr over phosphoserine (pSer). In further analogy to the antibodies, the imprinted polymer was capable of capturing short tyrosine phosphorylated peptides in the presence of an excess of their non-phosphorylated counterparts or peptides phosphorylated at serine.

Based on a survey of the literature on pretreatment of fused silica capillaries, 3 etching procedures and 11 silanization protocols based on the vinylic silane 3-((trimethoxysilyl)propyl) methacrylate (gamma-MAPS) were found to be most representative as a means of ensuring attachment of in situ prepared vinylic polymers. These techniques were applied to fused silica capillaries and the success in establishing the intended surface modification was assessed. X-ray photoelectron spectroscopy (XPS) was used to characterize the chemical state of the surface, providing information regarding presence of the reagent bound to the capillary. Wetting angles were measured and correlated with the XPS results. An adherence test was done by photopolymerization of a 2 mm long plug of 1,6-butanediol dimethacrylate in the prepared capillaries and evaluation of its ability to withstand applied hydraulic pressure. SEM was also performed in cases where the plug was released or other irregularities were observed. Finally, the roughness of the etched surface, considered to be of importance, was assessed by atomic force microscopy. Alkaline etching at elevated temperature provided a surface roughness promoting adhesion. The commonly used silanization protocols involving water in the silanization or washing steps gave inadequate surface treatment. The best silanization procedure was based on toluene as a solvent.

A set of monolithic stationary phases representing a broad span of monomers and porogens have been characterized directly in their capillary chromatographic format by computational assessment of their pore structure from transmission electron micrographs obtained after in situ embedment of the monoliths in contrast resin, followed by dissolution of the fused-silica tubing, further encasement of the resin-embedded monolith, and microtomy. This technique has been compared to mercury intrusion, a more conventional technique for macroporosity estimation. Supplementing the embedding resin by lead methacrylate gave a negative staining, and the resulting micrographs showed a good contrast between the polymeric monoliths and the embedding resin that allowed studies on the pore formation and polymer development. The technique was also applied to a commercial monolithic silica column.

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Molecular imprinting has resulted in a range of robust polymer-based receptors that are being considered for use in a variety of applications based on molecular recognition. [1] The technique entails polymerization of mono-and polyfunctional monomers in the presence of a template, whose subsequent removal leaves sites that can be reoccupied by the template or a closely related compound. These synthetic receptors are distinguished by their robustness and ease of synthesis, but they also have drawbacks, notably their poor water compatibility and, with notable exceptions, [2][3][4][5] the lack of strategies for imprinting water-soluble target molecules. One approach towards overcoming the latter is to use a nonaqueous imprinting protocol, in which lipophilic templates representing a close structural analogue or substructure of the target are employed. [6,7] Here, a solvent/porogen is chosen such that the intrinsically weak monomer-template interactions based, for example, on hydrogen bonding, electrostatics, or charge transfer, are stabilized. Thus, the use of solvents with low polarity and hydrogen bonding strength is generally favored. Although polymers prepared by this route have displayed selective binding of their targets under aqueous conditions, the effects were typically too weak to be of practical value. Following this approach, we report here on molecularly imprinted polymers (MIPs) that recognize their targets (riboflavin) effectively under such conditions ( Figure 1). Careful fine-tuning of the synthesis conditions with respect to the choice of template and cross-linking monomer proved critical and resulted in a polymer which strongly and selectively bound riboflavin in water-rich media similar to those found in common alcoholic beverages. [8] For the design of the polymers, we first turned our attention to the functional monomer. The flavin ring system contains an imide functionality which contains an acceptordonor-acceptor (ADA) array of hydrogen-bonding sites capable of interacting with receptors containing a complimentary DAD array. Representative of such molecules are 2,6-bisamidopyridines, whose ability to bind to imide functionalities has been widely studied.[9] Accordingly, we chose 2,6-bis(acrylamido)pyridine (BAAPy) as our functional monomer (Scheme 1). [10,11] With regards the choice of template, we focused on flavin and riboflavin derivatives having high solubility in typical imprinting solvents, in this case chloroform (Scheme 1), initially attempting to use N(10)-alkylflavins.[12] However, the solubility of these compounds in chloroform was found to be too low (ca. 16 mm for 1 and only ca. 6 mm for 2) for a conventional template/monomer molar ratio to be used. As expected, polymers prepared using these analogues as templates exhibited low imprinting factors and capacities (see below). Riboflavin tetraesters performed better in this regard, their solubility in chloroform being far greater than that of the N(10)-alkylflavins described above (RfAc: 0.2 m, RfPr: 0.6 m, RfBu: 0.8 m). Furthermore...

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A trimethylolpropane trimethacrylate-based monolith of dimensions carefully chosen to fit exactly in a standard 4-mm solid-state CP/MAS NMR rotor was photopolymerized and subsequently molecularly imprinted with bupivacaine using a grafting protocol with methacrylic acid and ethylene dimethacrylate as monomers. As no crushing or grinding of the monolith was necessary, additional unspecific surface area was not created. This procedure ascertains that differences observed between imprinted and nonimprinted polymers are due only to graft imprinted surfaces and give therefore better results in NMR spectroscopy due to less unspecific interactions between analyte and monolith. This improves the comparability to chromatographic evaluations where uncrushed monolithic columns are also used. To track interactions between analyte and stationary phase, the saturation transfer difference (STD) technique was applied on the polymer in the suspended state using the same solvent as in the chromatographic evaluation. This relatively new NMR method has to our knowledge not been used on chromatographic materials before. By using STD NMR on pristine monoliths, it was possible to measure large differences between the imprinted or nonimprinted polymers and the analyte indicating significant differences in the interaction mechanisms. These could be directly correlated with retention differences observed in chromatographic evaluations.