Lucio Renna

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.

Peptide Microarrays on Coated Silicon Slides for Highly Sensitive Antibody Detection

Peptides, with their well-established chemistry and fully automated synthesis, provide an invaluable tool for the screening of protein ligands, for epitope mapping, and for antibody diagnostics on the microarray format.The method described in this chapter shows that the sensitivity of a peptide-based microimmunoassay is greatly improved by using a new, specifically developed substrate made of silicon coated by an optimized layer of silicon oxide. A set of six peptides corresponding to the sequences of human and rat acetylcholine receptor subunits was immobilized on glass and silicon slides coated by a copolymer of N,N-dimethylacrylamide, N-acryloyloxysuccinimide, and 3-(trimethoxysilyl) propyl methacrylate, copoly(DMA-NAS-MAPS). The spotted probes were incubated with rabbit anti-sera and with purified antibodies raised against the corresponding peptides. The coated silicon slides, in comparison against the glass substrates, showed a five- to tenfold enhancement of the fluorescence signals, leading to the specific detection of the full set of antibodies down to a concentration of 0.5-1 ng/mL in serum. The sensitivity provided by the test allows its use for the diagnosis of antibodies in clinical samples.

UV-A Sensor Based on 6H-SiC Schottky Photodiode

In this paper, we propose a UV-A sensor based on a 6H-SiC Schottky photodiode with a thin Ni2Si front electrode and an integrated hydrogenated silicon nitride (SiN:H) dielectric filter. 6H-SiC prototypes were fabricated by using a manufacturing process to a large extent already implemented on 4H-SiC poly-type for the realization of a high signal-to-noise ratio ultraviolet (UV) sensor already commercialized. The results obtained on 6H-SiC prototypes show optimal electrical performance with dark current density lower than 0.2 nA/cm2 at room temperature and 10 V reverse bias. The use of 6H polytype allows shifting of the UV sensor response from 290 nm peak responsivity wavelength measured on standard 4H-SiC photodiodes to 360 nm wavelength in 6H-SiC prototypes with an integrated SiN:H filter exactly in the middle of the UV-A band (320–400 nm) with a simultaneous consistent reduction of the optical response in UV-C and UV-B regions below 320 nm wavelength.

Towards a high performing UV-A sensor based on Silicon Carbide and hydrogenated Silicon Nitride absorbing layers

Exposure to ultraviolet (UV) radiation is a major risk factor for most skin cancers. The sun is our primary natural source of UV radiation. The strength of the suns ultraviolet radiation is expressed as Solar UV Index (UVI). UV-A (320–400 nm) and UV-B (290–320 nm) rays mostly contribute to UVI. UV-B is typically the most destructive form of UV radiation because it has enough energy to cause photochemical damage to cellular DNA. Also overexposure to UV-A rays, although these are less energetic than UV-B photons, has been associated with toughening of the skin, suppression of the immune system, and cataract formation. The use of preventive measures to decrease sunlight UV radiation absorption is fundamental to reduce acute and irreversible health diseases to skin, eyes and immune system. In this perspective UV sensors able to monitor in a monolithic and compact chip the UV Index and relative UV-A and UV-B components of solar spectrum can play a relevant role for prevention, especially in view of the integration of these detectors in close at hand portable devices. Here we present the preliminary results obtained on our UV-A sensor technology based on the use of hydrogenated Silicon Nitride (SiN:H) thin passivating layers deposited on the surface of thin continuous metal film Ni2Si/4H-SiC Schottky detectors, already used for UV-Index monitoring. The first UV-A detector prototypes exhibit a very low leakage current density of about 0.2 pA/mm2 and a peak responsivity value of 0.027 A/W at 330 nm, both measured at 0V bias.

Strategy to discover full-length amyloid-beta peptide ligands using high-efficiency microarray technology

Although the formation of β-amyloid (Aβ) fibrils in neuronal tissues is a hallmark of Alzheimer disease (AD), small-sized Aβ oligomers rather than mature fibrils have been identified as the most neurotoxic species. Therefore, the design of new inhibitors, able to prevent the aggregation of Aβ, is believed to be a promising therapeutic approach to AD. Unfortunately, the short-lived intermediate structures that occur in a solution along the Aβ aggregation pathway escape conventional experimental investigations and there is urgent need of new tools aimed at the discovery of agents targeting monomeric Aβ and blocking the early steps of amyloid aggregation. Here, we show the combination of high-efficiency slides (HESs) with peptide microarrays as a promising tool for identifying small peptides that bind Aβ monomers. To this aim, HESs with two immobilized reference peptides, (i.e., KLVFF and Semax) with opposite behavior, were investigated for binding to fluorescently labeled Aβ peptide. Transmission electron microscopy was used to demonstrate Aβ fibrillar aggregates missing. The use of HESs was critical to ensure convenient output of the fluorescent microarrays. The resulting sensitivity, as well as the low sample consumption and the high potential for miniaturization, suggests that the proposed combination of peptide microarrays and highly efficient slides would be a very effective technology for molecule profiling in AD drug discovery.