Gabriele Di Carlo

High sensitivity protein assays on microarray silicon slides.

In this work, we report on the improvement of microarray sensitivity provided by a crystalline silicon substrate coated with thermal silicon oxide functionalized by a polymeric coating. The improvement is intended for experimental procedures and instrumentations typically involved in microarray technology, such as fluorescence labeling and a confocal laser scanning apparatus. The optimized layer of thermally grown silicon oxide (SiO(2)) of a highly reproducible thickness, low roughness, and fluorescence background provides fluorescence intensification due to the constructive interference between the incident and reflected waves of the fluorescence radiation. The oxide surface is coated by a copolymer of N,N-dimethylacrylamide, N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate, copoly(DMA-NAS-MAPS), which forms, by a simple and robust procedure, a functional nanometric film. The polymeric coating with a thickness that does not appreciably alter the optical properties of the silicon oxide confers to the slides optimal binding specificity leading to a high signal-to-noise ratio. The present work aims to demonstrate the great potential that exists by combining an optimized reflective substrate with a high performance surface chemistry. Moreover, the techniques chosen for both the substrate and surface chemistry are simple, inexpensive, and amenable to mass production. The present application highlights their potential use for diagnostic applications of real clinical relevance. The coated silicon slides, tested in protein and peptide microarrays for detection of specific antibodies, lead to a 5-10-fold enhancement of the fluorescence signals in comparison to glass slides.

Direct observation of conformation of a polymeric coating with implications in microarray applications.

The conformation of a three-dimensional polymeric coating (copoly(DMA-NAS-MAPS)) and immobilization and hybridization of DNA strands on the polymer coated surface are investigated. A conformational change, specifically the swelling of the surface adsorbed polymer upon hydration, is quantified in conjunction with the application of this polymer coating for DNA microarray applications. Fluorescently labeled short DNA strands (23mers) covalently linked to the functional groups on the adsorbed polymer are used as probes to measure the swelling of the polymer. A fluorescence microscopy technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), is utilized to directly measure the change in axial position of fluorophores due to swelling with subnanometer accuracy. Additionally, immobilization characteristics of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) probes, as well as hybridization of ssDNA with target strands have been studied. The results show that ssDNA further away from the surface is hybridized more efficiently, which strengthens the earlier analysis of this polymeric coating as a simple but highly efficient and robust DNA microarray surface chemistry.

Tetraaryl ZnIIPorphyrinates Substituted at β-Pyrrolic Positions as Sensitizers in Dye-Sensitized Solar Cells: A Comparison withmeso-Disubstituted Push-Pull ZnIIPorphyrinates

A facile and fast approach, based on microwave-enhanced Sonogashira coupling, has been employed to obtain in good yields both mono- and, for the first time, disubstituted push-pull Zn(II) porphyrinates bearing a variety of ethynylphenyl moieties at the β-pyrrolic position(s). Furthermore, a comparative experimental, electrochemical, and theoretical investigation has been carried out on these β-mono- or disubstituted Zn(II) porphyrinates and meso-disubstituted push-pull Zn(II) porphyrinates. We have obtained evidence that, although the HOMO-LUMO energy gap of the meso-substituted push-pull dyes is lower, so that charge transfer along the push-pull system therein is easier, the β-mono- or disubstituted push-pull porphyrinic dyes show comparable or better efficiencies when acting as sensitizers in DSSCs. This behavior is apparently not attributable to more intense B and Q bands, but rather to more facile charge injection. This is suggested by the DFT electron distribution in a model of a β-monosubstituted porphyrinic dye interacting with a TiO2 surface and by the positive effect of the β substitution on the incident photon-to-current conversion efficiency (IPCE) spectra, which show a significant intensity over a broad wavelength range (350-650 nm). In contrast, meso-substitution produces IPCE spectra with two less intense and well-separated peaks. The positive effect exerted by a cyanoacrylic acid group attached to the ethynylphenyl substituent has been analyzed by a photophysical and theoretical approach. This provided supporting evidence of a contribution from charge-transfer transitions to both the B and Q bands, thus producing, through conjugation, excited electrons close to the carboxylic anchoring group. Finally, the straightforward and effective synthetic procedures developed, as well as the efficiencies observed by photoelectrochemical measurements, make the described β-monosubstituted Zn(II) porphyrinates extremely promising sensitizers for use in DSSCs.

Detection of allergen specific immunoglobulins by microarrays coupled to microfluidics.

Allergen microarrays are under development for a component‐resolved diagnosis of Type I (IgE‐mediated) allergies. Here we report an improved microarray coupled to microfluidics for the detection of allergen specific immunoglobulin E (IgE). The signal intensity for IgE detection in serum has been improved by using glass slides coated with a novel poly[DMA‐co‐NAS] brush copolymer which is able to immobilize allergens in their native conformation and by carrying out the incubation step in dynamic conditions. The assay, fully automated, was performed in a microcell, using a software‐controlled fluidic processor, to bring assay reagents on the surface of the array. Microfluidics turns the binding between serum immunoglobulins and immobilized allergens from a diffusion‐limited to a kinetic‐limited process by ensuring an efficient mixing of serum samples on the surface of the microarray. As a result of this, the binding of high affinity IgE antibodies is enhanced whereas that of low affinity IgG antibodies, which are present at higher concentration, is impaired paving the way to more accurate and sensitive results.

Advanced polymers for molecular recognition and sensing at the interface

In the few last years, the need of reliable, fast and inexpensive methods for selective analysis of specific substances in complex mixtures has grown exponentially. In particular, the detection of biomolecules, such as oligonucleotides, proteins, peptides and carbohydrates is of outstanding importance in gene expression, drug design and medicine studies. To these purposes, molecular recognition on microarray-configured devices is one of the most promising tools. This technology uses a number of different substrates such as glass, silicon, alumina or gold-coated slides. The use of polymers is a very effective way to tailor surface properties introducing functional groups able to bind biomolecules and prevent denaturation and non-specific binding. Furthermore, advanced polymers, thanks to their particular physico-chemical properties, can be used to improve selectivity and sensitivity during assays. This review will provide very recent examples of polymer-mediated molecular recognition between guest molecules in solution and host molecules located at the solid phase.

Influence of Porphyrinic Structure on Electron Transfer Processes at the Electrolyte/Dye/TiO2 Interface in PSSCs: a Comparison between meso Push–Pull and β-Pyrrolic Architectures

Time-resolved photophysical and photoelectrochemical investigations have been carried out to compare the electron transfer dynamics of a 2-β-substituted tetraarylporphyrinic dye (ZnB) and a 5,15-meso-disubstituted diarylporphyrinic one (ZnM) at the electrolyte/dye/TiO2 interface in PSSCs. Although the meso push-pull structural arrangement has shown, up to now, to have the best performing architecture for solar cell applications, we have obtained superior energy conversion efficiencies for ZnB (6.1%) rather than for ZnM (3.9%), by using the I(-)/I3(-)-based electrolyte. To gain deeper insights about these unexpected results, we have investigated whether the intrinsic structural features of the two different porphyrinic dyes can play a key role on electron transfer processes occurring at the dye-sensitized TiO2 interface. We have found that charge injection yields into TiO2 are quite similar for both dyes and that the regeneration efficiencies by I(-), are also comparable and in the range of 75-85%. Moreover, besides injection quantum yields above 80%, identical dye loading, for both ZnB and ZnM, has been evidenced by spectrophotometric measurements on transparent thin TiO2 layers after the same adsorption period. Conversely, major differences have emerged by DC and AC (electrochemical impedance spectroscopy) photoelectrochemical investigations, pointing out a slower charge recombination rate when ZnB is adsorbed on TiO2. This may result from its more sterically hindered macrocyclic core which, besides guaranteeing a decrease of π-staking aggregation of the dye, promotes a superior shielding of the TiO2 surface against charge recombination involving oxidized species of the electrolyte.

Advances in Parallel Screening of Drug Candidates

In the hit to lead process, a drug candidate is selected from a set of potential leads by screening its binding with potential targets. This review focuses on the lead identification assays that employ a bio-chemical or bio-physical test to detect molecular recognition events between proteins and small molecules in a parallel format. These tests require either the lead or the target immobilization followed by incubation with the set of potential interaction partners and detection of a signal related to the target-ligand binding. In the first part of the review the different detection strategies amenable for drug screening are discussed. In the second part, a review of immobilization approaches for leads or targets, allowing the parallel screening of arrays of molecules, is presented.

Epitope Mapping of Human Chromogranin A by Peptide Microarrays

In this chapter we report on the characterization of linear antigenic sites of human chromogranin A (CgA), a useful tissue and serum marker for neuroendocrine tumours and a precursor of many biologically active peptides. The epitope mapping of CgA has been carried out by peptide microarrays on glass slides coated by a copolymer of N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS) and [3-(methacryloyl-oxy) propyl] trimethoxysilyl (MAPS). 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, despite peptide random orientation, linear epitopes are freely exposed. The results reported are in accordance with those obtained in conventional ELISA assays using biotinylated and non-biotinylated peptides.

Light-Induced Regiospecific Bromination of meso-Tetra(3,5-di-tert-butylphenyl)Porphyrin on 2,12 β-Pyrrolic Positions

The antipodal introduction of two bromine atoms on the 2,12 β-pyrrolic position of 5,10,15,20-tetra(3,5-di-tert-butylphenyl)porphyrin was successfully achieved by a light-induced reaction of the substrate with excess NBS. Complexation with Ni(II) of the major regioisomer led to good quality crystals, suitable for X-ray structure determination with unprecedented probability levels. The regiospecific character of the synthetic procedure and the exactness of the bromine atom position assignment were thus confirmed, suggesting an unexpected electrophilic aromatic substitution pathway rather than a free-radical halogenation process. A QTAIM topological analysis on the DFT-optimized wave function of the monosubstituted free-base porphyrin intermediate carrying a bromine atom in C2 β-pyrrolic position confirmed the largest negative charge for the C12 carbon atom in antipodal position, in agreement with the proposed electrophilic aromatic substitution mechanism.

Development of new substrates for high-sensitive genotyping of minority mutated alleles

An unsurpassed level of sensitivity was reached in the detection of minority mutated alleles. A low‐density microarray was printed on a substrate specifically designed to provide an interference effect which amplifies the collection of the light emitted on the support and reinforces the intensity of excitation light. Optimal performance of the array was obtained by maximizing the probe density and the binding efficiency to the target through a polymeric coating made by the adsorption of a copolymer of N,N‐dimethylacrylamide (97% of moles), N,N‐acryloyloxysuccinimide (2%) and 3‐(trimethoxysilyl)propyl methacrylate (1%) synthesized by free radical copolymerization. The new substrate was used in the identification of fetal mutations in the maternal plasma DNA. Amino‐modified amplicons from genomic DNA corresponding to the locus of eight β‐thalassemia mutations were immobilized and interrogated with dual‐color oligonucleotide targets. Compared with the conventional glass substrates, the new substrate showed a great enhancement of fluorescence signals thanks to the combination of the optics with the highly efficient polymeric coating, allowing specific detection of all mutations. The high sensitivity and selectivity obtained made it possible to develop assays for the identification of paternally inherited mutations on fetal DNA in the maternal plasma in couples at risk for β‐thalassemia.