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Curcumin is a natural polyphenol that exhibits a variety of beneficial effects on health, including anti-inflammatory, antioxidant, and hepato-protective properties. Due to its poor water solubility and membrane permeability, in the present study, we prepared and characterized a water-stable, freely dispersible nanoformulation of curcumin. Although the potential of curcumin nanoformulations in the hepatic field has been studied, there are no investigations on their effect in fibrotic pathological conditions involving cholangiocytes. Exploiting an in vitro model of transforming growth factor-β (TGF-β)-stimulated cholangiocytes, we applied the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomic approaches to study the proteome modulation induced by curcumin nanoformulation. Our results confirmed the well-documented anti-inflammatory properties of this nutraceutic, highlighting the induction of programmed cell death as a mechanism to counteract the cellular damages induced by TGF-β. Moreover, curcumin nanoformulation positively influenced the expression of several proteins involved in TGF-β-mediated fibrosis. Given the crucial importance of deregulated cholangiocyte functions during cholangiopathies, our results provide the basis for a better understanding of the mechanisms associated with this pathology and could represent a rationale for the development of more targeted therapies.

BRAF represents one of the most frequently mutated protein kinase genes and BRAFV600E mutation may be found in many types of cancer, including hairy cell leukemia (HCL), anaplastic thyroid cancer (ATC), colorectal cancer and melanoma. Herein, a fluorescent probe, based on the structure of the highly specific BRAFV600E inhibitor Vemurafenib (Vem, 1) and featuring the NIR fluorophore cyanine-5 (Cy5), was straightforwardly synthesized and characterized (Vem-L-Cy5, 3), showing promising spectroscopic properties. Biological validation in BRAFV600E-mutated cancer cells evidenced the ability of 3 to penetrate inside the cells, specifically binding to its elective target BRAFV600E with high affinity, and inhibiting MEK phosphorylation and cell growth with a potency comparable to that of native Vem 1. Taken together, these data highlight Vem-L-Cy5 3 as a useful tool to probe BRAFV600E mutation in cancer cells, and suitable to acquire precious insights for future developments of more informed BRAF inhibitors-centered therapeutic strategies.

Matrix metalloproteinases (MMPs) have been proposed as markers of many pathological conditions for their ability to degrade extracellular matrix components and remodel tissues. This review presents an overview of biosensors for the measurement of MMPs in serum, sputum and cell cultures. Major benefits and limitations of these technologies are discussed with respect to zymography and immunoassays, which are the reference methods to measure MMP activity and concentration. The paper also compares the analytical performances of sensors to the specific requirements for the application in clinical research, and comments on future trends in this field.

Ubiquitous in nature, polyamines (PAs) are a class of low-molecular aliphatic amines critically involved in cell growth, survival and differentiation. The polycation behavior is validated as a successful strategy in delivery systems to enhance oligonucleotide loading and cellular uptake. In this study, the chemical features and the functional roles of the PA spermidine are synergistically exploited in the synthesis and bioactive functionalization of SiO2-based structures. Inspired by biosilicification, the role of spermidine is assessed both as catalyst and template in a biomimetic one-pot synthesis of dense silica-based particles (SPs) and as a competitive agent in an interfacial reassembly strategy, to empty out SPs and generate spermidine-decorated hollow silica nanoporous pods (spd-SNPs). Spermidine bioactivity is then employed for targeting tumor cell over-expressed polyamine transport system (PTS) and for effective delivery of functional miRNA into melanoma cells. Spermidine decoration promotes spd-SNP cell internalization mediated by PTS and along with hollow structure enhances oligonucleotide loading. Accordingly, the functional delivery of the tumor suppressor miR-34a 3p resulted in intracellular accumulation of histone-complexed DNA fragments associated with apoptosis. Overall, the results highlight the potential of spd-SNP as a multi-agent anticancer therapy.

This paper presents the development of a lab-on-chip, based on thin-film sensors, suitable for DNA treatments. In particular, the system performs on-chip DNA amplification and separation of double-strand DNA into single-strand DNA, combining a polydimethylsiloxane microfluidic network, thin-film electronic devices, and surface chemistry. Both the analytical procedures rely on the integration on the same glass substrate of thin-film metal heaters and amorphous silicon temperature sensors to achieve a uniform temperature distribution (within ±1 °C) in the heated area and a precise temperature control (within ±0.5 °C). The DNA separation also counts on the binding between biotinylated dsDNA and a layer of streptavidin immobilized into a microfluidic channel through polymer-brushes-based layer. This approach results in a fast and low reagents consumption system. The tested DNA treatments can be applied for carrying out the on-chip systematic evolution of ligands by exponential enrichment process, a chemistry technique for the selection of aptamers.

Here we report on a high-sensitivity interferometric technique, namely Interferogram Averaged over Wavelength - IAW-reflectance spectroscopy, enabling sub-nanomolar detection (both nonspecific and specific) of proteins to be accomplished in direct (without any amplification) label-free mode using porous silicon (PSi) interferometers. The IAW reflectance spectroscopy is based on both calculation and elaboration of spectral interferograms. Interferograms are obtained by subtracting (intensity, wavelength by wavelength) reflectance spectra acquired on PSi interferometers in buffer solution after protein injection (at different concentrations) from a reference reflectance spectrum acquired in buffer solution before protein injection. The output signal, namely IAW value, is eventually obtained as the average value, calculated over a given wavelength interval, of each interferogram. Using the IAW technique nonspecific adsorption of Bovine Serum Albumin (BSA) down to 0.15 nM (DL=20 pM) and specific detection of Tumor Necrosis Factor (TNF?) down to 3 nM (DL=0.2 nM) was reliably achieved in direct label-free mode using PSi interferometers. Both the results represent a 10000-fold reduction in DL with respect to the current literature on the use of PSi for direct label-free measurements.

Porous silicon (PSi) is a nanostructured and interferometric optical transducer for label-free (bio)sensing really appealing for ease of fabrication and low costs, though currently suffering of poor sensitivity (with micromolar DLs in biosensing) commonly ascribed to hindered diffusion of biomolecules (tens of KDa) inside the columnar nanometric pores. In this study the development of a novel interferometric technique (i.e. Interferogram Average over Wavelength - IAW - reflectance spectroscopy) is reported for ultrasensitive and label-free (bio)sensing using nanostructured PSi interferometers. The IAW reflectance spectroscopy is based on the calculation of spectral interferograms by subtraction (intensity, wavelength by wavelength) of the reflection spectrum acquired after diffusion of the (bio)molecules of interest inside the PSi nanopores from a reference reflection spectrum recorded in buffer. The IAW signal, related to the analyte concentration, is calculated as average value from the obtained interferograms. As case of study of refractometric applications, a minimum refractive index variation of 10-7 RIU was measured through IAW reflectance spectroscopy using NaCl aqueous solutions, exploiting Electrical Double Layer-Induced Ion Surface Accumulationof Na+ ions onto the oxidized PSi surface. As proof of concept of biosensing applications, an assay making use of an anti-TNF? aptamer to specifically target TNF?, a peptidic biomarker of sepsis, was optimized onto the PSi surface. The IAW reflectance spectroscopy allowed a minimum concentration of 3.0 nM to be selectively measured, with a theoretical DL of 200 pM. Both the presented results, obtained with IAW reflectance spectroscopy, represent at least a 1000-fold improvement compared to the Fast Fourier Transform (FFT) reflectance spectroscopy commonly used for PSi interferometers and with which only ~ 10-4 RIU variations (in bulk refractive index sensing) and micromolar concentration (in biosensing) can be detected.

Porous silicon (PSi) is a promising nanostructured material for optical/interferometric (bio)sensing characterized by a huge specific surface (hundreds of m2/g) and low-cost preparation (~ 0.01$ per 8''silicon wafer) but lacking in sensitivity for refractive index (r.i.) sensing (DLs ~ 10-5 RIU) and biosensing (DLs ~ µM) . In this study we report on the development of a novel interferometric technique (i.e. Interferogram Average over Wavelength reflectance spectroscopy, IAW-RS) for ultrasensitive and label-free (bio)sensing with PSi interferometers. The IAW-RS is based on the calculation of spectral interferograms by subtraction (intensity, wavelength by wavelength) of the reflection spectrum acquired after accumulation/binding of (bio)molecules inside the PSi nanopores from a reference reflection spectrum recorded in buffer. The IAW signal is calculated as average value from the obtained interferograms . Using the IAW-RS in r.i.variation measurements with NaCl/KCl aqueous solutions, a r.i. variation of 10-7 RIU was detected exploiting also the electrical double layer-induced ion surface accumulation of Na+/K+ ions inside the oxidized PSi nanopores. Moreover employing the IAW-RS for the monitoring of BSA nonspecific adsorption inside the oxidized PSi nanopores (model for biosensing) a DL=20 pM was reached. For biosensing applications, an anti-TNF? aptamer was covalently immobilized inside the oxidized PSi nanopores. The IAW-RS pushed the selective TNF? detection down to a DL of 200 pM . The reported results prove that the IAW-RS significantly reduces the DLs (~2-4 orders of magnitude) in r.i. sensing and label-free biosensing with PSi interferometric transducers close to the most sensitive optical and label-free transduction techniques, e.g., Surface Plasmon Resonance.

Performing cell biology experiments in space imposes the use of hardware that essentially allows fluid exchange in a contained environment. Given the technical and logistical peculiarities, the limited opportunities and the high cost of access to space, a great effort during mission preparation of scientific studies is devoted to preventing loss of the experiment. The European Space Agency (ESA) requires, at the end of the preparation phase, the execution of an Experiment Sequence Test (EST), a dry-run version of the space experiment to check all procedures. At conclusion of the EST of our experiment 'ENDO' (ESA ILSRA-2009-1026), we found pitting corrosion of metal parts and biofilm formation within the cell-culture devices. The subsequent chemical (spectral assays), instrumental (OGP SmartScope) and microbiological (MALDI-TOF, 16S rRNA gene sequencing) investigations allowed the identification in contaminated material of Paenibacillus glucanolyticus, a ubiquitous, aerobic, facultative anaerobic, endospore forming, acid-producing, Gram-positive microorganism. A concurrence of P. glucanolyticus contamination and galvanic corrosion determined massive fouling, rust precipitation and damage to cells and cell-culture devices being, to our knowledge, the association between this microbe and corrosion never reported before in literature. As a consequence of the episode a critical procedure of experiment set up, i.e. hardware sterilization, was modified. The ENDO experiment was successfully launched to the International Space Station on September 2nd 2015 and returned to the PI laboratory on September 13th, with all cell culture samples in optimal condition.

In this research we report on a novel ultrasensitive interferometric reflectance spectroscopy technique, namely Interferogram Average over Wavelength (IAW), enabling the sub-nanomolar and label-free detection of proteins with porous silicon (PSi) interferometers. The IAW reflectance spectroscopy improves of 4 orders of magnitude the detection limit (DL) of PSi interferometers in biosensing compared to the FFT reflectance spectroscopy technique conventionally employed.

As the endothelium still represents the ideal surface for cardiovascular devices, different endothelialization strategies have been attempted for biocompatibility and nonthrombogenicity enhancement. Since endothelial progenitor cells (EPCs) could accelerate endothelialization, preventing thrombosis and restenosis, the aim of this study was to use oligonucleotides (ONs) to biofunctionalize stents for EPC binding. In order to optimize the functionalization procedure before its application to cobalt-chromium (Co/Cr) stents, discs of the same material were preliminarily used. Surface aminosilanization was assessed by infrared spectroscopy and scanning electron microscopy. A fluorescent endothelial-specific ON was immobilized on aminosilanized surfaces and its presence was visualized by confocal microscopy. Fluorescent ON binding to porcine blood EPCs was assessed by flow cytometry. Viability assay was performed on EPCs cultured on unmodified, nontargeting ON or specific ON-coated discs; fluorescent staining of nuclei and F-actin was then performed on EPCs cultured on unmodified or specific ON-coated discs and stents. Disc biofunctionalization significantly increased EPC viability as compared to both unmodified and nontargeting ON-coated surfaces; cell adhesion was also significantly increased. Stents were successfully functionalized with the specific ON, and EPC binding was confirmed by confocal microscopy. In conclusion, stent biofunctionalization for EPC binding was successfully achieved in vitro, suggesting its use to obtain in vivo endothelialization, exploiting the natural regenerative potential of the human body.

Porous silicon (PSi) is a promising nanostructured material for label-free interferometric biosensing, though it currently suffers of restricted “real” applications in medical diagnostics. This is usually ascribed to PSi interferometer limited sensitivity due to mass-limited diffusion of (bio)molecules inside its nanopores. Here we report on a high-sensitivity label-free interferometric technique (i.e. Interferogram Average over Wavelength, IAW, reflectance spectroscopy) for, though not limited to, PSi interferometers. The IAW reflectance spectroscopy is based on the calculation of spectral interferograms obtained by subtraction of reflectance spectra acquired after interaction of target biomolecules with the PSi interferometer inner surface, from a reference reflection spectrum acquired in buffer before injection of target molecules. The IAW reflectance spectroscopy was set up studying nonpecific adsorption of Bovine Serum Albumin (BSA) onto oxidized PSi interferometers and then validated through the development of an aptasensor using PSi interferometers for the specific detection of Tumor Necrosis Factor a (TNFa, a sepsis biomarker). In both the cases we demonstrated that sub-nanomolar detection limits (DLs) can be achieved, which are 4 orders of magnitude lower than DLs achieved using Fast Fourier Transform (FFT) reflectance spectroscopy (usually in the micromolar range), in direct label-free operation. These results open exciting perspectives for point of care (POC) analysis based on PSi interferometers, for which increasingly lower detection limits are required.

In-field analysis (e.g., clinical and diagnostics) using nanostructured porous silicon (PSi) for label-free optical biosensing has been hindered so far by insufficient sensitivity of PSi biosensors. Here we report on a label-free PSi interferometric aptasensor able to specifically detect tumor necrosis factor alpha (TNFα, a protein biomarker of inflammation and sepsis) at concentration down to 3.0 nM with signal-to-noise ratio (S/N) of 10.6 and detection limit (DL) of 200 pM. This represents a 10 000-fold improvement with respect to direct (i.e., nonamplified) label-free PSi biosensors and pushes PSi biosensors close to the most sensitive optical and label-free transduction techniques, e.g., surface plasmon resonance (SPR) for which a lowest DL of 100 pM in aptasensing has been reported. A factor 1000 in improvement is achieved by introducing a novel signal-processing technique for the optical readout of PSi interferometers, namely, interferogram average over wavelength (IAW) reflectance spectroscopy. The...

Polycrystalline silicon–germanium (poly-SiGe) is a promising structural material for the post-processing of micro electro-mechanical systems (MEMS) on top of complementary metal-oxide-semiconductor (CMOS) substrates. Combining MEMS and CMOS allows for the development of high-performance devices. We present for the first time selective protein immobilization on top of poly-SiGe surfaces, an enabling technique for the development of novel poly-SiGe based MEMS biosensors. Active regions made of 3-aminopropyl-triethoxysilane (APTES) were defined using silane deposition onto photoresist patterns followed by lift-off in organic solvents. Subsequently, proteins were covalently bound on the created APTES patterns. Fluorescein-labeled human serum albumin (HSA) was used to verify the immobilization procedure while the binding capability of the protein layer was tested by an antigen-labeled antibody pair. Inspection by fluorescence microscopy showed protein immobilization inside the desired bioactive areas and low non-specific adsorption outside the APTES pattern. Furthermore, the quality of the silane patches was investigated by treatment with 30 nm-diameter gold nanoparticles and scanning electron microscope observation. The developed technique is therefore a promising first step towards the realization of poly-SiGe based biosensors.

Gene function assessment is a main task in biological networking investigations and system biology. High throughput technologies provide an impressive body of data that enables the design of hypotheses linking genes to phenotypes. When a putative scenario is depicted, gene knockdown technologies and RNA-dependent gene silencing are the most frequent approaches to assess the role of key effectors. In this paper, we discuss the relevance of hammerhead ribozymes in target discovery and validation, describing their properties and applications and highlighting their selectivity. In particular, similarities with siRNAs are presented and advantages and drawbacks are discussed. A description of the perspectives of ribozyme application in wide range studies is also provided, strengthening the value of these inhibitors for target validation purposes.

The Nuclear Factor kappa B (NF-kB), a transcription factor regulating a battery of inflammatory genes, has been implicated as playing a fundamental role in the development of numerous pathological states. The NF-kB dimer is located in the cytoplasm of cells in an inactive form that can be activated by many stimuli (e.g. cytokines, bacterial or viral products, UV-radiation, etc.). It has been demonstrated that anti-inflammatory agents, both steroid and non steroid, besides their principal mechanism of action, have a secondary mechanism with anti-NF-kB effects. Therefore, when anti-inflammatory therapy is applied to an inflammatory disease, for which detection of the efficacy of the therapeutic agent is particularly important, monitoring of NF-kB-active form concentration (for example in cellular lysate) is an important target. The critical need for a simple and direct method to evaluate the presence of active NF-kB in a biological sample can be addressed using a suitable and reusable biosensor. For this purpose, a novel method, using fluorescence resonance energy transfer (FRET), to detect the active form of NF-kB binding a specific DNA sequence has been developed. In order to evaluate FRET due to the DNA/protein binding interaction taking place between dsDNA immobilized in a capillary wall and p50 proteins, a highly sensitive FRET-based biosensor system developed in our laboratory was used.

A study of antibody immobilisation techniques on quartz and fibre optic surfaces for immunosensors has been carried out. Methods of covalent antibody immobilisation which have not previously been applied to optical fibres were investigated, and compared with classical methods found in the literature. Preliminary experiments on covalent immobilisation methods on planar quartz surfaces were conducted to enable us to choose the most suitable protein immobilisation technique for sensor applications. The immobilisation studies were directed in particular towards obtaining a high density of binding sites for the analyte of interest. Two of the most promising methods, antibody immobilisation on surfaces coated with dextran based hydrogel and F(ab′)-SH fragments bound to silanised glass, which resulted in surface densities of active sites of above 0.45 pmol/cm2, were selected for further experiments on a fibre optic total internal reflection fluorescence immunosensor and gave satisfactory responses to changes in analyte concentrations of the order of 10−8 M. The efficiency of polar organic solvents, such as dimethylsulfoxide, in dissociating the antigen–antibody complex and hence to regenerate the immunosensor surface was also evaluated.

Smart, MEMS-based biosensors are a promising new platform for the delivery of diagnostic services, but inclusion of on-chip CMOS electronics requires the definition a CMOS compatible techniques for the bio-activation of the sensor surface. In this work, a comparison between a sensor functionalization procedure by complete immersion of the sensor chip or by exposure to a single drop of the reagents (the latter approach being more compatible with the presence of on-chip CMOS components) is presented.

With the disclosure of the human genome a new era for bio-medicine has arisen, characterized by the challenge to investigate pathogenic mechanisms, studying simultaneously metabolites, DNA, RNA, and proteins. As a result, the "omics" revolution boomed, giving birth to a new medicine named "omics-based medicine". Among the other "omics", proteomics has been widely used in medicine, since it can produce a more "holistic" overview of a disease and provide a "constellation" of possible specific markers, a molecular fingerprinting that defines the clinical condition of an individual. Endpoint of this comprehensive and detailed analysis is the "diagnostic-omics", i.e. the achievement of personalized diagnoses with obvious benefits for prevention and therapy and this goal can be reached only with a perfect integration between clinicians and proteomists. To impact on the possible key factors involved in the pathological processes, oligonucleotide-based knock-down strategies can be helpful. They exploit omics-derived molecular tools (antisense, siRNA, ribozymes, decoys, and aptamers) that can be used to inhibit, at transcriptional or post-transcriptional levels, the events leading to protein synthesis, thus decreasing its expression. The identification of the pivotal mechanisms involved in diseases using global, "scenic" approaches such as the "omics" ones, and the subsequent validation and detailed description of the processes by specific molecular tools, can result in a more preventive, predictive and personalized medicine.

Vascular restenosis is affecting 30-40% of patients treated by percutaneous coronary angioplasty (PTCA). The advent of stenting reduced but not abolished restenosis. The introduction of drug eluting stent (DES) further reduced restenosis, but impaired endothelization exposed to intracoronary thrombosis as late adverse event. It is widely accepted that the endothelial denudation and coronary wall damages expose Vascular Smooth Muscle Cells (VSMC) to multiple growth factors and plasma circulating agents thus activating migration and proliferative pathways leading to restenosis. Among the major players of this processes, phosphorylated Elk-1, forming the Elk-1/SRF transcription complex, controls the expression of a different set of genes responsible for cell proliferation. Therefore, it is feasible that gene-specific oligonucleotide therapy targeting VSMC migration and proliferation genes can be a promising therapeutic approach. While a plethora of vehicles is suitably working in static in vitro cultures, methods for in vivo delivery of oligonucleotides are still under investigation. Recently, we have patented a novel erythrocyte-based drug delivery system with high capability to fuse with targeted cells thus improving drug bioavailability at the site of action. Here, the potential of these engineered porcine erythrocytes to deliver a synthetic DNA Elk-1 decoy inside syngenic porcine VSMC was tested. The results of this study indicate that Elk-1 decoy is actually able to inhibit cell proliferation and migration of VSMC. Our data also suggest that erythrocyte-based carriers are more efficient in delivering these oligonucleotides in comparison to conventional vehicles. As a consequence, a lower dose of Elk-1 decoy, delivered by engineered erythrocytes, was sufficient to inhibit cell growth and migration. This approach represents the translational step to reach in vivo experiments in pigs after PTCA and/or stent implantation where oligonucleotide drugs will be site-specific administered by using erythrocyte-based carriers to prevent restenosis.

A simple and fast methodology for protein patterning on silicon substrates is presented, providing an insight into possible issues related to the interaction between biological and microelectronic technologies. The method makes use of standard photoresist lithography and is oriented towards the implementation of biosensors containing Complementary Metal-Oxide-Semiconductor (CMOS) conditioning circuitry. Silicon surfaces with photoresist patterns were prepared and hydroxylated by means of resist- and CMOS backend-compatible solutions. Subsequent aminosilane deposition and resist lift-off in organic solvents resulted into well-controlled amino-terminated geometries. The discussion is focused on resist- and CMOS-compatibility problems related to the used chemicals. Some samples underwent gold nanoparticle (Au NP) labeling and Scanning Electron Microscopy (SEM) observation, in order to investigate the quality of the silane layer. Antibodies were immobilized on other samples, which were subsequently exposed to a fluorescently labeled antigen. Fluorescence microscopy observation showed that this method provides spatially selective immobilization of protein layers onto APTES-patterned silicon samples, while preserving protein reactivity inside the desired areas and low non-specific adsorption elsewhere. Strong covalent biomolecule binding was achieved, giving stable protein layers, which allows stringent binding conditions and a good binding specificity, really useful for biosensing.

We selectively immobilized organofunctional silanes on top of polycrystalline silicon-germanium (poly-SiGe) layers, as a first step towards the fabrication of poly-SiGe-based bioMEMS (biomedical MicroElectroMechanicalSystems) by means of standard UV photolithography. 3-aminopropyl-dimethyl-ethoxysilane (APDMES) and 3-aminopropyl-triethoxysilane (APTES) molecules were immobilized onto resist-patterned poly-SiGe surfaces. The protocols for surface hydroxylation and silane immobilization were designed to be CMOS-compatible and to avoid damage to photoresist. Silanized surfaces were investigated both by means of fluorescence microscopy, and by FEG-SEM observation after labeling with 30 nm-diameter gold nanoparticles (NPs). We report the silanization protocols, together with the results indicating successful organic functionalization of the samples.

A novel optical platform for point of care testing application was developed. The core of the platform is a miniaturised polymethylmetacrylate (PMMA) chip through which the analysed sample flows. Thanks to the fluorescence anisotropy exhibited by any dipole emitting at a distance from a medium interface of the order of the emitted wavelength, a large fraction of the fluorescence emitted by the sensing layer immobilized onto the internal flow-channel travels along the thickness of the PMMA up to its end-face where it is collected by a plastic optical fibre connected to a spectrum analyser. The potentiality of the optical chip was investigated with a sandwich assay for the C-reactive protein, one of the markers for inflammatory diseases.

A new optical platform for the interrogation of a polymethylmethacrylate (PMMA) multichannel array for chemical and biochemical parameters is described. It consists of a plastic chip formed by two pieces of PMMA properly shaped in order to obtain different microchannels, 500 ¼m in width and 400 ¼m in height. A fluorescent sensing layer is immobilized on the internal wall of the microchannel. The emitted light travels along the thickness of the upper PMMA piece and it is detected with an optical fiber connected with a spectrum analyzer. The anisotropy of the fluorescence emitted by the fluorophore immobilized at the liquid/plastic interface gives rise to preferential directions of the emitted fluorescence. The collection of the fluorescence at an angle of 50 degrees implies an amplification of roughly one order of magnitude in the ratio between the fluorescence signal and the scattered light. The optical platform was further characterized as a pH sensor and as potential chip for immunoassay. In the first case the fluorescein dye is directly immobilized onto the internal wall of the channel and the fluorescence changes are measured as a function of the pH of the flowing sample. In the second case a direct antigen-antibody interaction is carried out. The mouse-IgG is covalently immobilized onto the internal wall of the channel and the Cy5-labeled anti-mouse IgG is used for the specific interaction.

The present paper is concerned with the development of an optical system capable of interrogating an array of silicon microcantilevers. The use of a laser line-generator and of a CCD camera allows the simultaneous interrogation of the eight cantilevers of the chip without movable parts. The resolution achieved in terms of displacement of each cantilever end is better than 7.5 nm. As an example of the operation, the system has been characterized as a refractometer by filling the cell with liquids having different index of refraction.

Background Vascular Smooth Muscle Cells (VSMCs), due to their plasticity and ability to shift from a physiological contractile-quiescent phenotype to a pathological proliferating-activated status, play a central role in the onset and progression of atherosclerosis and cardiovascular diseases. PDGF-BB, among a series of cytokines and growth factors, has been identified as the critical factor in this phenotypic switch. In order to obtain new insights on the molecular effects triggered by PDGF-BB, a hammerhead ribozyme targeting the membrane receptor PDGFR-β was applied to inhibit PDGF pathway in porcine VSMCs. Findings Ribozymes, loaded on a cationic polymer-based vehicle, were delivered into cultured VSMCs. A significant impairment of the activation mechanisms triggered by PDGF-BB was demonstrated since cell migration decreased after treatments. In order to functionally validate the effects of PDGFR-β partial knock down we focused on the phosphorylation status of two proteins, protein disulfide isomerase-A3 (PDI-A3) and heat shock protein-60 (HSP-60), previously identified as indicative of VSMC phenotypic switch after PDGF-BB stimulation. Interestingly, while PDI-A3 phosphorylation was counteracted by the ribozyme administration indicating that PDI-A3 is a factor downstream the receptor signalling cascade, the HSP-60 phosphorylation status was greatly increased by the ribozyme administration. Conclusion These contradictory observations suggested that PDGF-BB might trigger different parallel pathways that could be modulated by alternative isoforms of the receptors for the growth factor. In conclusion the knock down strategy here described enables to discriminate between two tightly intermingled pathways. Moreover it opens new attractive perspectives in functional investigations where combined gene knock down and proteomic technologies would allow the identification of key factors and pathways involved in VSMC-linked pathological disorders.

Background The use of chromatography coupled with mass spectrometry (MS) analysis is a powerful approach to identify proteins, owing to its capacity to fractionate molecules according to different chemical features. The first protein expression map of vascular smooth muscle cells (VSMC) was published in 2001 and since then other papers have been produced. The most detailed two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map was presented by Mayr et al who identified 235 proteins, corresponding to the 154 most abundant unique proteins in mouse aortic VSMC. A chromatographic approach aimed at fractionating the VSMC proteome has never been used before. Results This paper describes a strategy for the study of the VSMC proteome. Our approach was based on pre-fractionation with ion exchange chromatography coupled with matrix assisted laser desorption-time of flight mass spectrometry analysis assisted by a liquid chromatography (LC-MALDI-TOF/TOF). Ion exchange chromatography resulted in a good strategy designed to simplify the complexity of the cellular extract and to identify a large number of proteins. Selectivity based on the ion-exchange chemical features was adequate if evaluated on the basis of protein pI. The LC-MALDI approach proved to be highly reproducible and sensitive since we were able to identify up to 815 proteins with a concentration dynamic range of 7 orders of magnitude. Conclusions In our opinion, the large number of identified proteins and the promising quantitative reproducibility made this approach a powerful method to analyze complex protein mixtures in a high throughput way and to obtain statistical data for the discovery of key factors involved in VSMC activation and to analyze a label-free differential protein expression.