Biomedical subjects

Giovanna Pirri

Publications and source records attributed to Giovanna Pirri.

11 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↗

PubMed2005

Electroosmotic flow suppression in capillary electrophoresis: chemisorption of trimethoxy silane-modified polydimethylacrylamide.

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.

Acrylamides↗

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↗

PubMed2003

Nonconventional synthesis and characterization of ultrahigh-molar-mass polyacrylamides.

In spite of the significant progresses in the field of replaceable sieving matrices for separating DNA in capillary electrophoresis (CE), an intense research activity is still going on to improve the separation of large size DNA sequencing fragments. There are evidences, both from experimental and theoretical sides that the resolution of these fragments, at the single base, requires the use of sieving matrices comprised of long chain linear polymers. In the separation of DNA fragments by CE are of upmost importance: (i) the complete solubility of the polymer, (ii) the linearity of the chain, (iii) the achievement of ultrahigh viscosity in dilute solutions. The aim of this work is the synthesis of ultrahigh-molecular-weight polymers which possess the three requirements mentioned above by employing a nonconventional method. We demonstrate that the sieving performance of polyacrylamide is directly correlated to its intrinsic viscosity.

Acrylic Resins↗

PubMed2003

Separation of proteins in a multicompartment electrolyzer with chambers defined by a bed of gel beads.

Multicompartment electrolyzers (MEs) with isoelectric membranes were introduced in 1989 for purifying proteins in an electric field. At the basis of ME technology there are membranes consisting of cross-linked copolymers of acrylamide and acrylamido monomers bearing protolytic groups. The technology employed for casting the membranes is an extension of the isoelectric focusing in immobilized pH gradient technique for which specific acrylamido monomers, known with the trade name of Immobiline, have been developed. However, the use of continuous membranes presents several disadvantages. Due to the mechanical characteristics of polyacrylamide, the gel must physically adhere onto a rigid support, which prevents it from collapsing. The support must have a highly porous structure in order to be permeable to proteins. The mechanical fragility of the membranes is one of the main problems that hinders the industrial scale application of ME separators. In order to overcome this problem, we propose to substitute the continuous membranes with a bed of gel beads of identical comonomer composition, obtained by an inverse emulsion polymerization process.

Electrophoresis↗

PubMed2002

Use of peptide nucleic acid probes for detecting DNA single-base mutations by capillary electrophoresis.

Peptide nucleic acid (PNA) oligomers can be used as probes in pre-gel hybridization experiments, as an alternative to Southern hybridization. In this technique, the PNA probe is hybridized to a cyanine-5 labeled DNA sample denatured at low ionic strength, and the mixture is directly injected for size separation into a capillary electrophoresis (CE) system equipped with laser-induced fluorescence (LIF) detector. The neutral backbone of PNA allows hybridization to occur at low ionic strength and assures an efficient CE separation of the PNA/DNA hybrids from both double-stranded and single-stranded DNA. We have used as a model system the cystic fibrosis R553X and R1162X single-base mutations and we have assessed the influence of various factors, such as temperature and denaturants concentration on DNA/PNA hybrid stability in order to achieve the high specificity required for a single base pair discrimination.

Base Pairing↗

PubMed2002

Multimodal open-tubular capillary electrochromatographic analysis of amines and peptides.

This study describes a comparison of different modes of open-tubular electrochromatography (OTCEC) in bare and etched capillaries. To carry out the investigation, the separation of impurities of two synthetic peptides and the separation of a mixture of five heterocyclic aromatic amines were studied. Three different types of stationary phase were evaluated: (i) fluorosurfactants (anionic and zwitterionic) adsorbed in the inner wall of the capillary (electrochromatography with dynamically modified stationary phases (DMS)CEC); (ii) physically adsorbed polymers (DMA-SO(3-) and DMA-N(+)(CH(3))(3)) and (iii) chemically modified capillaries (C(18), cholesteryl 10-undecanoate and diol). The results confirm that electrochromatography can be a viable alternative to capillary electrophoresis (CE) and liquid chromatography, more established separation techniques. It is possible to differentiate some minor species for the synthetic peptides that cannot be resolved by CE or high-performance liquid chromatography (HPLC). Moreover the separation of the amine mixture depends strongly on the stationary phase used.

Amines↗