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Background Microarray-based component-resolved diagnostics (CRD) has become an accepted tool to detect allergen specific IgE-sensitization towards hundreds of allergens in parallel from one drop of serum. Nevertheless specificity and sensitivity as well as a simultaneous detection of allergen specific IgG4, as a potential parameter for tolerance development, remain to be optimized. Objective We applied the recently introduced silicon chip coated with a functional polymer named copoly(DMA-NAS-MAPS) to the simultaneous detection of food allergen specific IgE and IgG4, and compared it with ImmunoCAP and ImmunoCAP ISAC. Inter- and intra- slide variation, linearity of signal and working range, sensitivity and application of internal calibrations for IgE and IgG4 were assessed. Methods Native and recombinant allergenic proteins from hen's egg and cow's milk were spotted on silicon chips coated with copoly(DMA-NAS-MAPS) along with known concentrations for human IgE and IgG4. A serum pool and 105 patient samples were assessed quantitatively and semi-quantitatively with the ImmunoCAP and ImmunoCAP ISAC and correlated with IgE- and IgG4-specific fluorescence on silicon microarrays. Results Allergen specific IgE and IgG4 were detected in parallel using two fluorescent dyes with no crosstalk. Results from the ImmunoCAP correlated better with microarray fluorescence than with ImmunoCAP ISAC except for the allergen ovomucoid. The working range of the silicon microarray for total hen's egg specific IgE was comparable to the range of 0.1 to >100 kUA/l of the ImmunoCAP system, whereas for total cow's milk the silicon microarray was less sensitive. Detectable allergen specific IgG4 could be determined only for low concentrations, but still correlated positively with ImmunoCAP results. Conclusions We confirmed the ability of the polymer coated silicon microarray to be comparably sensitive to the ImmunoCAP ISAC for various food allergens. This suggests that the copoly(DMA-NAS-MAPS) microarray is a low cost, self-producible alternative to the commercial ImmunoCAP ISAC in allergy research. This article is protected by copyright. All rights reserved.

A novel microchip electrophoresis instrument based on single-photon avalanche diodes was used for the molecular characterization of mutations in disease genes. The identification of the main mutation causing cystic fibrosis, named DeltaF508, by the Amplification Refractory Mutation System was used to validate the technology. In our implemented protocol the wild-type and mutant allele-specific primers are labeled with Cy5 and Cy5.5, respectively. The protocol enables the amplification of the DNA sample in a single PCR. The genotype was deduced from the fluorescence of the amplicons run in the CE microchip. Validation on 15 DNA samples from either homozygous wild-type or heterozygous and homozygous mutated control subjects proved the complete reliability of the system, thus confirming its high diagnostic potential.

In this work a high-throughput method based on the single-nucleotide extension (SNE) reaction and multicolour detection in a DNA sequencer was developed to screen for eight mutations in the human beta-globin gene: IVSI.110, cd39, IVSI.1, IVSI.6, IVSII.745, HbC, HbS and cd6. The method has been validated on a large number of samples for the two most common mutations causing beta-thalassemia in the Mediterranean area (IVSI.110 and cd39). The development of a high-throughput, fast and reliable method to assay beta-thalassemia mutations represents a significant improvement in molecular diagnosis of this disease. The multicolour detection and the use of multiple injections further enhances the throughput of mutational screening by the DNA sequencer and facilitates automated genotyping for routine molecular diagnostics.

Microarraying peptides is a powerful proteomics technique for studying molecular recognition events. Since peptides have small molecular mass, they are not easily accessible when adsorbed onto solid supports. Moreover, peptides can lack a well-defined three-dimensional structure, and therefore a correct orientation is essential to promote the interaction with their target. In this work, we investigated the suitability as a peptide array substrate of a glass slide coated with a copolymer of N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and [3-(methacryloyl-oxy)propyl]trimethoxysilyl. This polymeric surface was used as substrate for peptides in the characterization of linear antigenic sites of human chromogranin A, a useful tissue and serum marker for neuroendocrine tumors and a precursor of many biologically active peptides. The microarray support provided sufficient accessibility of the ligand, with no need for a spacer, as the polymer chains prevent interaction of immobilized peptides with substrate. In addition, the polymeric surface constitutes an aqueous micro-environment in which linear epitopes are freely exposed despite peptide random orientation. The results reported in this article are in accordance with those obtained in conventional ELISA assays using biotinylated and non-biotinylated peptides.

Adsorbed polymers are widely used to suppress electroosmotic flow (EOF) in capillary electrophoresis (CE). Polymeric coatings, physisorbed onto the surface of the capillary wall, are often unstable under harsh conditions. This can be attributed to the reversible nature of the coating which becomes apparent when the adsorbed layer competes with a second species in the electrophoresis buffer solution for attachment/interaction with the capillary surface. In an effort to overcome the problem of coating instability, trimethoxysilane-modified polydimethylacrylamide was synthesized. This copolymer rapidly adsorbs on the wall from ultradilute aqueous solutions. After incubation at a temperature of 60 degrees C silyl groups, which extend from the polymer backbone, form condensation bonds with the silanols on the capillary surface. This enables subsequent formation of strong covalent bonds between the copolymer and the capillary wall. In this research, we establish that physisorption of polymer chains to the surface is essential for close alignment of surface and polymer silane groups which facilitates the formation of covalent bonds.

A fast and simple method for the internal coating of capillaries in capillary zone electrophoresis (CZE) is that with epoxy-poly(dimethylacrylamide) (EPDMA). Duration of coating by that method is 30 min, compared with that of 24 h when using uncross-linked polyacrylamide (PA) under otherwise identical conditions. Under the conditions used for the CZE of proteins (pH 9.0, 2% polyethylene glycol), the capillary coating with EPDMA is stable for at least 50 consecutive runs as judged by the constancy of low electroosmotic flow, equalling the stability of coating achieved by PA. Protein mobilities and protein peak asymmetry (suggestive of reversible interaction with the capillary wall) are also found to be the same in EPDMA and PA coated capillaries. Differences between EPDMA and PA coating also exist: The former is unstable upon lowering the ionic strength of the buffer to 0.003, upon the addition of sodium dodecyl sulfate (SDS) to the buffer and in application to the hydrophobic analyte, polystyrene carboxylate.

We have synthesized two new sugar monomers, allylamine of gluconic and lactobionic acid, by opening the corresponding lactone ring with allylamine. These monomers were copolymerized with acrylamide leading to formation of copolymers with a relative molecular mass of 288000 and 180000 Da, respectively. Double-stranded DNA fragments were separated in entangled solutions of these linear polymers in capillary electrophoresis. Resolution, peak spacing and peak width were the parameters taken into account to evaluate the quality of the separation achieved with the new polymers. This work indicates that the copolymers of acrylamide and allyl gluconic acid have a high sieving capacity and provide a performance similar to that of hydroxyethylcellulose (HEC) of comparable viscosity. Unlike HEC, this copolymer selfcoats onto the capillary wall, allowing DNA fragments to be efficiently separated in an uncoated capillary.

A fully automated coating procedure was devised based on adsorption of a new polymer, poly(dimethylacrylamide-co-allyl glycidyl ether) onto the capillary surface. The whole procedure takes less than 30 min and does not require the use of organic solvents, viscous solutions, or elevated temperature. The coating is stable even under harsh conditions such as alkaline pH, elevated temperature, and denaturant conditions that destroy most other presently available adsorbed coatings. This new adsorbed coating is highly stable and easy to produce in quantity, making it quite unique, and further making it possible to operate with any DNA sieving matrix. Finally, the above-mentioned properties facilitate coating regeneration by a simple wash with a strongly alkaline solution, thus extending the lifetime of the capillary, a highly desirable property for any coating used in biopolymer separations.

The synthesis and characterization of a novel polymer consisting of an alkyl backbone and pendant β-cyclodextrin units, obtained by radical copolymerization of vinylpyrrolidone and methacryloyl-β-cyclodextrin (PVP-β-CD), was reported. The ability of this copolymer to act as a capillary electrophoresis (CE) chiral selector was investigated in the separation of a mixture of basic drugs. The influence of polymeric cyclodextrin concentration, temperature, and pH on the separation of the test analytes was assessed and the advantage of using the polymeric selector over native β-cyclodextrin was demonstrated.

Microarrays of proteins and peptides make it possible the screening of thousands of binding events in a parallel and high throughput fashion; therefore they are emerging as a powerful tool for proteomics and clinical assays. The complex nature of Proteome, the wide dynamic range of protein concentration in real samples and the critical role of immobilized protein orientation must be taken into account to maximize the utility of protein microarrays. Immobilization strategy and designing of an ideal local chemical environment on the solid surface are both essential for the success of a protein microarray experiment. This review article will focus on protein and peptide arrays highlighting their technical challenges and presenting new directions by means of a set of selected recent applications.

We have recently described [1] a fast and simple method for the "adsorbed static" coating of capillaries in capillary zone electrophoresis (CZE) with epoxy-poly(dimethylacrylamide) (EPDMA). Protein CZE peaks in the EPDMA-coated capillaries exhibited a peak asymmetry similar to that obtained in capillaries with "covalent static" coating of polyacrylamide, suggesting a similar degree of adsorption of the protein onto the coating [2]. Instability of such coating at very low ionic strength and its stripping from the capillary in the presence of sodium dodecyl sulfate (SDS) also indicated a hydrophilic bonding of EPDMA to the silanol surface of the capillary, while its stripping in the CZE of "carboxylate-modified" polystyrene suggested a competition between carboxylate and EPDMA for the hydrophilic bonds to silanol. To test those propositions, a number of EPDMA-derived coating agents with increased hydrophilicity were synthesized. Of a number of the hydrophilic coating agents tested (Table 1) only two, 2% hydrolyzed EPDMA (HPDMA) hydrolyzed in sulfuric acid to effect the conversion of the epoxy groups to diols (Table 1, No. 38), and 20% EPDMA (Table 1, No. 44) exhibited for representative proteins a decreased peak asymmetry and width while the stability of the suppression of electroosmotic flow (EOF), and the stability of mobility in consecutive CZE runs was reduced relative to EPDMA. Coating agents which were more highly hydrophilic than those two (Table 1, No. 49) or less hydrophilic than 2% EPDMA (Table 1, Nos. 57, 53, 46) provided no stable static coating.