Biomedical subjects

Francesco Damin

Publications and source records attributed to Francesco Damin.

7 recordsLinked to original sources

PubMed2006

Surface behavior and molecular recognition in DNA microarrays from N,N-dimethylacrylamide terpolymers with activated esters as linking groups.

A series of terpolymers made of DMA, NAS and MAPS were synthesized by free radical copolymerization and used as functional coatings for the fabrication of glass slide DNA microarrays. The surface properties of coated glass slides were investigated through contact angle measurements, ellipsometry and atomic force microscopy. The terpolymer molecular weight showed a moderate effect on surface tension (gamma(s) = 56-62 mN x m(-1)), but no clear effect on polymeric layer thickness (5-8 nm) and roughness. Hybridization experiments with amine-functionalized oligonucleotides gave the best fluorescence intensity results for microarrays coated with intermediate-molecular-weight terpolymers. Finally, an accelerated ageing test of the microarray in a humidity chamber showed a nice relationship between decay curves of contact angle against water and fluorescence intensity.

Acrylamides↗

PubMed2006

Microarray glass slides coated with block copolymer brushes obtained by reversible addition chain-transfer polymerization.

The reversible addition-fragmentation chain-transfer polymerization was used to prepare microarray slides grafted with polymer brushes for DNA-based applications. Block copolymer brushes of N,N-dimethylacrylamide (DMA) and glycidyl methacrylate (GMA), poly(DMA-b-GMA) were prepared by extending living poly(dimethylacrylamide) chains. The functional surface was used as a substrate for oligonucleotide hybridization experiments. The results were compared to those provided by glass slides coated by a self-assembled monolayer made of (3-glycidyloxypropyl)trimethoxysilane. Surfaces coated with block polymer brushes bearing oxirane groups are more efficient as substrates for oligonucleotide hybridization than surfaces coated with nonpolymeric self-assembled monolayers containing the same functional group. The high probe grafting density and hybridization efficiency achieved with this polymeric coating reveal the importance of the block architecture to ensure good accessibility of the immobilized probe. The new surface was characterized by static angle measurements and diffuse reflectance FT-IR spectroscopy on a silica model system.

Acrylic Resins↗

PubMed2006

Protein and peptide arrays: recent trends and new directions.

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.

Combinatorial Chemistry Techniques↗

PubMed2006

Different approaches for noninvasive prenatal diagnosis of genetic diseases based on PNA-mediated enriched PCR.

The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer-directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority-mutated allele in the presence of an excess of wild-type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild-type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild-type DNA in the beta-globin gene region, where four beta-thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild-type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.

DNA↗

PubMed2005

Peptide microarrays for the characterization of antigenic regions of human chromogranin A.

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.

Amino Acid Sequence↗

PubMed2004

A new polymeric coating for protein microarrays.

Despite the increasing interest in arraying proteins in a high-density format, several technical issues still impede the development of protein microarray technology. One of the major problems is the availability of substrates that are able to bind native proteins with high density. In this study, we investigated the suitability of a novel surface as a support for protein microarrays. A polymeric glass coating is obtained by physical adsorption of a N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS), and [3-(methacryloyl-oxy)propyl]trimethoxysilyl (MAPS) copolymer. The coating procedure provides a fast and inexpensive method of producing hydrophilic functional surfaces. The slide performance was investigated in a protein-protein interaction experiment and in the assessment of rheumatoid factor (RF) in human serum samples. The results demonstrate that the ligands immobilized on the polymeric surface maintain an active conformation and are easily accessible, providing a detection limit of 54amol/spot. Moreover, in the RF assay, after hybridization with the sera, the slides have a low background, leading to a detection limit of 900amol/spot.

Acrylamides↗

PubMed2004

Characterization of a polymeric adsorbed coating for DNA microarray glass slides.

A new method was developed to covalently attach target molecules onto the surface of glass substrates such as microwell plates, beads, tubes, and microscope slides, for hybridization assays with fluorescent targets. The innovative concept introduced by this work is to physically adsorb onto underivatized glass surfaces a functional copolymer, able to graft amino-modified DNA molecules. The polymer, obtained by radical copolymerization of N,N-dimethylacrylamide, N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate, copoly(DMA-NAS-MAPS), self-adsorbs onto the glass surface very quickly, typically in 5-30 min. The film, formed on the surface, bears active esters, which react with amino-modified DNA targets. The surface layer is stable in an aqueous buffer containing various additives (SDS, urea, salt), even at boiling temperature. It should be emphasized that the coating is formed by the immersion of glass slides in a diluted aqueous solution of the polymer. Therefore, the procedure is fast, inexpensive, robust, and reliable, and it does not require time-consuming glass pretreatments. Slides, coated with copoly(DMA-NAS-MAPS), were profitably used as substrates for the preparation of low-density DNA microarrays. The density and the thickness of the films were evaluated by X-ray reflectivity measurements whereas the extent of reaction of functional groups with DNA molecules was determined by a functional test. The experiments indicate that half of the active groups present on the surface reacts with oligonucleotide probes.

Acrylamides↗