Fabio Domenici

On-chip detection of multiple serum antibodies against epitopes of celiac disease by an array of amorphous silicon sensors

In this paper, we present the preliminary results of an ELISA-on-chip device, intended as a technological demonstrator of a novel analytical system suitable for the diagnosis and follow-up of celiac disease. The idea of the work is to combine an array of amorphous silicon photosensors with a pattern of a poly(2-hydroxyethyl methacrylate) polymer brush film, which acts as anchor for the immobilization of gliadin peptides containing the celiac disease epitopes. Recognition relies on a sandwich immunoassay between antibodies against the peptides and secondary antibodies marked with horseradish peroxidase to obtain a chemiluminescent signal. Detection is based on the measurement of photocurrent induced in the array of amorphous silicon photosensors by the chemiluminescent signal. An ad-hoc procedure has been developed in order to enable the fabrication of the photodiode array and the polymer brush pattern on the two sides of the same glass substrate ensuring the compatibility of the different technological steps. The sensitivity and the selectivity of the chip for multiplex immunoassays were demonstrated using two gliadin peptides (VEA and DEC). In particular, we found that the average amount of the bound HRP revealed by our analytical protocol is 3.5(±0.3) × 10−6 pg μm−2 and 0.85(±0.3) × 10−6 pg μm−2 for specific and non-specific interactions, respectively.

Ultrasound well below the intensity threshold of cavitation can promote efficient uptake of small drug model molecules in fibroblast cells

Abstract Ultrasound (US) induced enhancement of plasma membrane permeability is a hugely promising tool for delivering exogenous vectors at the specific biological site in a safe and efficient way. In this respect, here we report effects of membrane permeability alteration on fibroblast-like cells undergoing very low-intensity of US. The change in permeability was pointed out in terms of high uptake efficiency of the fluoroprobe calcein, thus resembling internalization of small cell-impermeable model drugs, as measured by fluorescence microscopy and flow cytometry. Fluorescence evidences moreover suggests that the higher the time of exposure, the larger will be the size of molecules can be internalized. The uptake events were related to the cell viability and also with structural changes occurring at membrane level as revealed by infrared spectroscopy and preliminary membrane fluidity and atomic force microscopy (AFM) investigation. Thus, the question of whether the uptake of cell-impermeable molecules is consistent with the presence of disruptions on the cell membrane (sonopore formation) has been addressed. In this framework, our findings may constitute experimental evidence in support of sub-cavitation sonoporation models recently proposed, and they may also provide some hints towards the actual working condition of medical US dealing with the optimum risk to benefit therapeutic ratio.

Graphene Meets Microbubbles: A Superior Contrast Agent for Photoacoustic Imaging

Coupling graphene with a soft polymer surface offers the possibility to build hybrid constructs with new electrical, optical, and mechanical properties. However, the low reactivity of graphene is a hurdle in the synthesis of such systems which is often bypassed by oxidizing its carbon planar structure. However, the defects introduced with this process jeopardize the properties of graphene. In this paper we present a different approach, applicable to many different polymer surfaces, which uses surfactant assisted ultrasonication to exfoliate, and simultaneously suspend, graphene in water in its intact form. Tethering pristine graphene sheets to the surfaces is accomplished by using suitable reactive functional groups of the surfactant scaffold. We focused on applying this approach to the fabrication of a hybrid system, made of pristine graphene tethered to poly(vinyl alcohol) based microbubbles (PVA MBs), designed for enhancing photoacoustic signals. Photoacoustic imaging (PAI) is a powerful preclinical diagnostic tool which provides real time images at a resolution of 40 μm. The leap toward clinical imaging has so far been hindered by the limited tissues penetration of near-infrared (NIR) pulsed laser radiation. Many academic and industrial research laboratories have met this challenge by designing devices, each with pros and cons, to enhance the photoacoustic (PA) signal. The major advantages of the hybrid graphene/PVA MBs construct, however, are (i) the preservation of graphene properties, (ii) biocompatibility, a consequence of the robust anchoring of pristine graphene to the bioinert surface of the PVA bubble, and (iii) a very good enhancement in a NIR spectral region of the PA signal, which does not overlap with the signals of PA active endogenous molecules such as hemoglobin.

Dimensional scale effects on surface enhanced Raman scattering efficiency of self-assembled silver nanoparticle clusters

A study of the Surface Enhanced Raman Scattering (SERS) from micrometric metallic nanoparticle aggregates is presented. The sample is obtained from the self-assembly on glass slides of micro-clusters of silver nanoparticles (60 and 100 nm diameter), functionalized with the organic molecule 4-aminothiophenol in water solution. For nanoparticle clusters at the micron scale, a maximum enhancement factor of 109 is estimated from the SERS over the Raman intensity ratio normalized to the single molecule contribution. Atomic force microscopy, correlated to spatially resolved Raman measurements, allows highlighting the connection between morphology and efficiency of the plasmonic system. The correlation between geometric features and SERS response of the metallic structures reveals a linear trend of the cluster maximum scattered intensity as a function of the surface area of the aggregate. On given clusters, the intensity turns out to be also influenced by the number of stacking planes of the aggregate, thus sugg...

Next generation ultrasound platforms for theranostics

Microbubbles are a well-established contrast agent which improves diagnostic ultrasound imaging. During the last decade research has focused on expanding their use to include molecular imaging, targeted therapy and imaging modalities other than ultrasound. However, bioadhesion of targeted microbubbles under physiological flow conditions is still difficult to achieve, the main challenge being connected to the poor stability of lipid microbubbles in the bodys circulation system. In this article, we investigate the use of polymeric microbubbles based on a poly (vinyl alcohol) shell as an alternative to lipid microbubbles. In particular, we report on the development of microbubble shell modification, using mild reaction conditions, with the aim of designing a multifunctional platform to enable diagnosis and therapy. Superparamagnetic iron oxide nanoparticles and a near infrared fluorescent probe, indocyanine green, are coupled to the bubbles surface in order to support magnetic resonance and fluorescence imaging. Furthermore, anchoring cyclic arginyl-glycyl-aspartic acid (RGD) peptide, and cyclodextrin molecules, allows targeting and drug loading, respectively. Last but not least, shell topography is provided by atomic force microscopy. These applications and features, together with the high echogenicity of poly (vinyl alcohol) microbubbles, may offer a more stable alternative to lipid microbubbles for the development of a multimodal theranostic platform.

Folate-based single cell screening using surface enhanced Raman microimaging

Recent progress in nanotechnology and its application to biomedical settings have generated great advantages in dealing with early cancer diagnosis. The identification of the specific properties of cancer cells, such as the expression of particular plasma membrane molecular receptors, has become crucial in revealing the presence and in assessing the stage of development of the disease. Here we report a single cell screening approach based on Surface Enhanced Raman Scattering (SERS) microimaging. We fabricated a SERS-labelled nanovector based on the biofunctionalization of gold nanoparticles with folic acid. After treating the cells with the nanovector, we were able to distinguish three different cell populations from different cell lines (cancer HeLa and PC-3, and normal HaCaT lines), suitably chosen for their different expressions of folate binding proteins. The nanovector, indeed, binds much more efficiently on cancer cell lines than on normal ones, resulting in a higher SERS signal measured on cancer cells. These results pave the way for applications in single cell diagnostics and, potentially, in theranostics.

Structural and permeability sensitivity of cells to low intensity ultrasound: Infrared and fluorescence evidence in vitro.

This work is focused on the in vitro study of the effects induced by medical ultrasound (US) in murine fibroblast cells (NIH-3T3) at a low-intensity of exposure (spatial peak temporal average intensity Ita<0.1Wcm(-2)). Conventional 1MHz and 3MHz US devices of therapeutic relevance were employed with varying intensity and exposure time parameters. In this framework, upon cells exposure to US, structural changes at the molecular level were evaluated by infrared spectroscopy; alterations in plasma membrane permeability were monitored in terms of uptake efficiency of small cell-impermeable model drug molecules, as measured by fluorescence microscopy and flow cytometry. The results were related to the cell viability and combined with the statistical PCA analysis, confirming that NIH-3T3 cells are sensitive to therapeutic US, mainly at 1MHz, with time-dependent increases in both efficiency of uptake, recovery of wild-type membrane permeability, and the size of molecules entering 3T3. On the contrary, the exposures from US equipment at 3MHz show uptakes comparable with untreated samples.

Potential genotoxic effects of low-intensity ultrasound on fibroblasts, evaluated with the cytokinesis-block micronucleus assay.

Although medical ultrasound offers compelling opportunities to improve therapy in principle, progress in the field has been limited because of an insufficient understanding of the potential genotoxic and cytotoxic effects of ultrasound on biological systems. This paper is mainly focused on an in vitro study of effects with respect to genotoxicity and viability induced by 1- and 3-MHz medical ultrasound in murine fibroblasts (NIH-3T3) at low-intensity exposure (spatial peak temporal average intensity Ita<0.1 W/cm(2)). The NIH-3T3 cells constitute a well-characterized in vitro cell model in which a genotoxic effect can be predicted by means of a reliable and precise murine cytokinesis-block micronucleus assay. A statistically significant increase in the incidence of micronuclei was observed in sonicated 3T3 cells. In particular, the effects were more evident at 1 MHz. Moreover, for each frequency investigated, the occurrence of micronuclei was comparatively more frequent with increasing time of exposure. The possible toxicological implications of the medical ultrasound employed herein deal with the existence of a window of exposure parameters (set well below the intensity of ultrasound cavitation) in which some genotoxic effects may occur without significant cytotoxicity. In this respect, they provide new insight toward the correct risk to benefit balancing of ultrasound-based treatments and for designing innovative therapeutic strategies.

Alamethicin–lipid interaction studied by energy dispersive X-ray diffraction

A detailed knowledge of the interaction between bacterial membranes and antibiotics provides important information to prevent high levels of antibiotic resistance exhibited by pathogenic strains. We investigated by energy dispersive X-ray diffraction (EDXD) the structure ordering of dioleoyl-phosphatidylcholine (DOPC) lipid interacting with antimicrobial peptide alamethicin, varying the lipid/peptide (L/P) molar ratio under two different hydration levels. In conditions of full hydration (100%) we found that the bilayer thickness is constant between L/P=20 and L/P=80 indicating that in this range, the system has reached the threshold value for the channel formation, while at the relative hydration of 45% a linear decrease of the bilayer thickness as function of L/P was revealed. The kinetic study of the complex alamethicin-DOPC at different L/P values, shows that the Bragg peak energy variation versus the hydration time has a biexponential behavior characterized by two different time constants.

Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies.