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Amino-terminated surfaces can be effectively obtained by means of silanizing agents, realizing surfaces suitable for the purification of biomarkers of several pathologies. Since the level of biomarkers, such as microRNAs and cell-free DNA, into circulation may be extremely low, new and ameliorated capturing molecules and protocols are highly required. In this work, a new silane, acetone-imine propyl trimethoxysilane (AIPTMS), is synthesized with a simple and elegant reaction, via the nucleophilic addition of the primary amino group to the carbonyl group of acetone. AIPTMS and APTMS were used to silanize silicon oxide surfaces, which were characterized chemically (XPS) and morphologically (AFM). The two types of surfaces were chemically similar, but behaved very differently both for surface morphology and functional properties. The AIPTMS-modified surface was indeed very smooth and homogeneous with respect to the APTMS-modified surface. Moreover, the AIPTMS surface captured larger amounts of nucleic acids almost immediately after preparation, while APTMS-based functional surfaces needed longer time to reach comparable efficiency. AIPTMS shows several advantages over standard aminosilanes, as it realizes a more homogeneous surface coverage that, in turn, produces an improved response towards the capture of nucleic acids. AIPTMS is a very promising reagent for the reliable and reproducible preparation of active biofunctional surfaces for the purification and analysis of circulating biomarkers.

Polydimethylsiloxane (PDMS) is a well-known biocompatible polymer employed for many applications in the biomedical field. In this study, the biocompatibility and versatility of PDMS was tested setting up a microdevice devoted to the purification and analysis of nucleic acids. The PDMS microdevice was demonstrated to successfully fulfill all requirements of genetic analyses such as genotyping and pathogen DNA identification both in multiplex and real-time PCR, suggesting the possibility to carry out a molecular test directly on-chip. Moreover, the PDMS microdevice was successfully applied to the purification and detection of disease biomarkers, such as microRNAs related to cancer or heart disease. On-chip microRNA purification was demonstrated starting from clinically relevant samples, i.e. plasma, serum, tissue biopsies. Significantly, the purification and the transcription of microRNA into cDNA occur in the same PDMS chamber, saving time and labor for the overall analysis. Again, the PDMS microdevice was confirmed as a notable candidate for compact, rapid, easy-to-use molecular tests.

The neuroscience field has increased enormously over the last decades, achieving the possible real application of neuronal cultures not only for reproducing neural architectures resembling in vivo tissues, but also for the development of functional devices. In this context, surface patterning for cell confinement is crucial, and new active materials together with new protocols for preparing substrates suitable for confining cells, guiding their processes in the desired configuration are extremely appreciated. Here, TiO2 sol-gel derived films were selected as proof-of-concept materials to grow neurons in suitable confined configurations, taking advantage of the biocompatible properties of modified TiO2 substrates. TiO2 sol-gel derived films were made compatible with the growth of neurons thanks to a stable and controlled poly-lysine coating, obtained by silanization chemistry and streptavidin-biotin interactions. Moreover, a spotting protocol, here described and optimized, allowed the simple preparation of arrays of neurons, where cell adhesion was guided in specific areas and the neurites development driven in the desired arrangement. The resulting arrays were successfully tested for the growth and differentiation of neurons, demonstrating the possible adhesion of cells in specific areas of the film, therefore paving the way to applications such as the direct growth of excitable cells nearby electrodes of devices, with an evident enhancement of cell-electrodes communication.

BackgroundAmyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease’s complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell’s physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification.MethodsWe analyzed plasma-derived EVs of ALS patients (n = 106) and controls (n = 96), and SOD1G93Aand TDP-43Q331Kmouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis.ResultsOur procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. EVs in the plasma of ALS patients and the two mouse models of ALS had a distinctive size distribution and lower HSP90 levels compared to the controls. In terms of disease progression, the levels of cyclophilin A with the EV size distribution distinguished fast and slow disease progressors, a possibly new means for patient stratification. Immuno-electron microscopy also suggested that phosphorylated TDP-43 is not an intravesicular cargo of plasma-derived EVs.ConclusionsOur analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis.

BackgroundAmyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease’s complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell’s physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification.MethodsWe analyzed plasma-derived EVs of ALS patients (n= 106) and controls (n=96), and SOD1G93A and TDP-43Q331K mouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis.ResultsOur procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. There were more particles in the plasma of ALS patients and the two mouse models of ALS while their average diameter was smaller. HSP90 was differentially represented in ALS patients and mice compared to the controls. In terms of disease progression, the levels of cyclophilin A, with the EV size distribution, distinguished fast and slow disease progressors, suggesting a new means for patient stratification. We also measured the levels of phosphorylated TDP-43 and showed that is not an intravesicular cargo of plasma-derived EVs.ConclusionsOur analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis.

Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers. We recently demonstrated that nickel-based isolation (NBI) is a valuable method for fast, efficient, and easy recovery of heterogeneous EVs from biological fluids. NBI exploits nickel cations to capture negatively charged vesicles. Then, a mix of balanced chelating agents elutes EVs while preserving their integrity and stability in solution. Here, we describe steps and quality controls to functionalize a matrix of agarose beads, obtain an efficient elution of EVs, and extract nucleic acids carried by them. We demonstrate the versatility of NBI method in isolating EVs from media of primary mouse astrocytes, from human blood, urine, and saliva processed in parallel, as well as outer membrane vesicles (OMVs) from cultured Gram-negative bacteria.

We report a comprehensive study of the biocompatibility and neurocompatibility of titanium dioxide films (TiO2) prepared by Pulsed Microplasma Cluster Source (PMCS). This technique uses supersonic pulsed beams seeded by clusters of the metal oxide synthesized in a plasma discharge. The final stoichiometry of the TiO2 thin films is tuned changing the gas mixture, achieving stoichiometric or oxygen overstoichiometric films. All the films showed consistent biocompatibility and a spontaneous absorption of poly-d-lysine (PDL) that favors the adhesion and growth of murine cortical neurons. Moreover, the bioelectrical activity of the neuronal culture grown on the TiO2 film can be modulated by changing the chemistry of the surface. This work paves the way to develop a bio-hybrid neuromorphic device, where viable nerve cells are grown directly over a titanium dioxide film showing a network of memristors.

Background Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice. Methods We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility. Findings From plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 1010/ml) and large EV (~560 nm of mean diameter; ~5 × 108/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies. Interpretation We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. Fund Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Universita e Ricerca (Miur).

This study explores the feasibility to use electroconductive Ti-doped polycrystalline diamond layers as scaffolds for tissue engineering. The synthesis of the diamond-based materials is accomplished in a HFCVD reactor where Ti(IV) acetyl acetonate powders are delivered by N2 fluxes to the growing diamond phase. In-depth investigations (Raman spectroscopy, SEM, AFM, XRD, XPS) allowed the characterization of the morphological/structural/compositional features and the properties of charge transport (KPFM, I-V) induced in the diamond layers by the incorporation of Ti-species. The bioactivity of the Ti-doped diamond surface was verified investigating the growth of MG-63 osteoblast-like cells by using MTT assays and confocal microscopy. The study evidenced a net increase of cell replication rate on diamond scaffolds after 4 days of incubation. After 6-days incubation, the cell growth on the Ti-doped diamond scaffolds increased up to 150% compared with the reference polystyrene tissue culture vessel, with a dominant presence of cells in active division. The cell behavior is discussed and related to the structural and functional surface properties of the Ti-diamond systems, acting as bioactive platforms able to offer an extremely beneficial environment for cell proliferation and viability. © 2020 Elsevier B.V.

The detection of low abundant biomarkers, such as circulating microRNAs, demands innovative detection methods with increased resolution, sensitivity and specificity. Here, a biofunctional surface was implemented for the selective capture of microRNAs, which were detected through fluorescence enhancement directly on a photonic crystal. To set up the optimal biofunctional surface, epoxy-coated commercially available microscope slides were spotted with specific anti-microRNA probes. The optimal concentration of probe as well as of passivating agent were selected and employed for titrating the microRNA hybridization. Cross-hybridization of different microRNAs was also tested, resulting negligible. Once optimized, the protocol was adapted to the photonic crystal surface, where fluorescent synthetic miR-16 was hybridized and imaged with a dedicated equipment. The photonic crystal consists of a dielectric multilayer patterned with a grating structure. In this way, it is possible to take advantage from both a resonant excitation of fluorophores and an angularly redirection of the emitted radiation. As a result, a significant fluorescence enhancement due to the resonant structure is collected from the patterned photonic crystal with respect to the outer non-structured surface. The dedicated read-out system is compact and based on a wide-field imaging detection, with little or no optical alignment issues, which makes this approach particularly interesting for further development such as for example in microarray-type bioassays. (C) 2016 Elsevier B.V. All rights reserved.

Aspergillus is known as one of the most frequent toxigenic fungi in Europe. It produces aflatoxin B1 transformed into aflatoxin M1 (AFM1) present in milk. The International Agency for Research on Cancer (IARC) has included AFM1 in the group I human carcinogens. Acceptable maximum level of AFM1 in milk according to EU regulation is 50 ppt equivalent to 15.2×10-2 nM (the molecular weight of AFM1 is 328.27 g/mol). Up to now, the most used techniques for AFM1 detection in milk are time consuming Enzyme-Linked Immuno-Sorbent Assay (ELISA) and high-performance liquid chromatography associated to fluorescence (HPLC). Silicon photonics based biosensors such as Mach-Zehnder Interferometers and Microring Resonators thanks to their ability to be miniaturize and integrated with electronics and microfluidics in lab-on-a-chip devices, are new candidates to become faster, cheaper and more accurate tools for AFM1 detection in milk. Here, we validate photonic AFM1 biosensors based on an array of four Si3N4 asymmetric Mach-Zehnder Interferometers (aMZI) functionalized by F(ab’)2 fragments and passivated with casein. Analyses of AFM1 binding by Fab’ in MES buffer and in real milk samples, using different concentrations of AFM1, are performed. The dependence of the phase shifts to the AFM1 concentration allows to calculate the association and dissociation rate constants. The proposed biosensor is capable to detect AFM1 concentration down to 50 ppt. For the average dissociation constant (KD) of AFM1-Fab’ interaction, values of (1.8÷5)×10-8 M in MES buffer and 0.8×10-9 M in milk samples are measured. These are in the same order of magnitude as published results. The difference of one order of magnitude between KD in MES buffer and in milk might be caused by the fact that during the preparation of milk samples, an additional concentration of salt is added to the solution which yields a stronger ionic interaction to occur. Finally, the specificity of the interaction is confirmed by using blank solutions that are free from AFM1.

Additive manufacturing techniques using three dimensional (3D) printing have been shown to be suitable for a wide range of applications. In this study, stereolithography (SLA) is applied to the field of microfluidic fabrication of lab-on-a-chip (LOC) devices. LOCs deal with different milli/microsized channels and chambers, which are the key features of the devices, so an appropriate manufacturing process should provide high precision as well as high versatility. In this work, the goal was to overcome the common drawbacks of 3D printing and multistep processes, by implementing multiple polymeric materials in the same printing process. Using a customized SLA machine, a novel process was developed to print microfluidic channels enclosed between two poly(methyl methacrylate) layers in a sandwichlike structure. For microfluidic walls, two distinct commercial resins with different properties were used. Once thermal and pressure resistance of the obtained LOCs were assessed, deoxyribose nucleic acid was amplified by polymerase chain reaction inside the microfluidic chambers. Test results indicated favorable mechanical and thermal resistance, as well as chemical compatibility with the assay reagents. Such observations suggest that this novel approach can be applied to 3D printing of customized microfluidics with embedded features.

Traditional implants made of bulk titanium are much stiffer than human bone and this mismatch can induce stress shielding. Although more complex to produce and with less predictable properties compared to bulk implants, implants with a highly porous structure can be produced to match the bone stiffness and at the same time favor bone ingrowth and regeneration. This paper presents the results of the mechanical and dimensional characterization of different regular cubic open-cell cellular structures produced by Selective Laser Melting (SLM) of Ti6Al4V alloy, all with the same nominal elastic modulus of 3 GPa that matches that of human trabecular bone. The main objective of this research was to determine which structure has the best fatigue resistance through fully reversed fatigue tests on cellular specimens. The quality of the manufacturing process and the discrepancy between the actual measured cell parameters and the nominal CAD values were assessed through an extensive metrological analysis. The results of the metrological assessment allowed us to discuss the effect of manufacturing defects (porosity, surface roughness and geometrical inaccuracies) on the mechanical properties. Half of the specimens was subjected to a stress relief thermal treatment while the other half to Hot Isostatic Pressing (HIP), and we compared the effect of the treatments on porosity and on the mechanical properties. Fatigue strength seems to be highly dependent on the surface irregularities and notches introduced during the manufacturing process. In fully reversed fatigue tests, the high performances of stretching dominated structures compared to bending dominated structures are not found. In fact, with thicker struts, such structures proved to be more resistant, even if bending actions were present.

The demand of miniaturized, accurate and robust micro-tools for minimally invasive surgery or in general for micro-manipulation, has grown tremendously in recent years. To meet this need, a new-concept comb-driven microgripper was designed and fabricated. Two microgripper prototypes differing for both the number of links and the number of conjugate surface flexure hinges are presented. Their design takes advantage of an innovative concept based on the pseudo-rigid body model, while the study of microgripper mechanical potentialities in different configurations is supported by finite elements' simulations. These microgrippers, realized by the deep reactive-ion etching technology, are intended as micro-tools for tissue or cell manipulation and for minimally invasive surgery; therefore, their biocompatibility in terms of protein fouling was assessed. Serum albumin dissolved in phosphate buffer was selected to mimic the physiological environment and its adsorption on microgrippers was measured. The presented microgrippers demonstrated having great potential as biomedical tools, showing a modest propensity to adsorb proteins, independently from the protein concentration and time of incubation.

We report a novel sensing method for fluorescence-labelled microRNAs (miRNAs) spotted on an all-dielectric photonic structure. Such a photonic structure provides an enhanced excitation and a directional beaming of the emitted fluorescence, resulting in a significant improvement of the overall signal collected. As a result, the Limit of Detection (LoD) is demonstrated to decrease by a factor of about 50. A compact read-out system allows a wide-field imaging-based detection, with little or no optical alignment issues, which makes this approach particularly interesting for further development for example in microarray-type bioassays.

We report a comprehensive study of the biocompatibility and neurocompatibility of titanium dioxide films (TiO

A reliable clinical assay based on circulating microRNAs (miRNAs) as biomarkers is highly required. Microdevices offer an attractive solution as a fast and inexpensive way of concentrating these biomarkers from a low sample volume. A previously developed polydimethylsiloxane (PDMS) microdevice able to purify and detect circulating miRNAs was here optimized. The optimization of the morphological and chemical surface properties by nanopatterning and functionalization is presented. Surfaces were firstly characterized by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), fluorescamine assay and s-SDTB (sulphosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate) assay and subsequently tested for their capacity to adsorb a fluorescent miRNA. From our analysis, modification of surface charge with 0.1% APTMS ((3-Aminopropyl)trimethoxysilane) and 0.9% PEG-s (2-[Methoxy-(polyethyleneoxy)propyl]trimethoxysilane) performed at 60 °C for 10 min was identified as the best purification condition. Our optimized microdevice integrated with real-time PCR detection, was demonstrated to selectively purify both synthetic and natural circulating miRNAs with a sensitivity of 0.01 pM.

MicroRNAs (miRNAs) modulate gene expression at post-transcriptional level, while their aberrant presence in circulation correlates with the most common human disorders such as cancer, neuro-degenerative and immune-related diseases. Currently, the pre-concentration of such important bio-markers present at low concentrations in biological fluids, which would make their identification and quantification easier, remains a challenging issue for biosensor-based non-invasive analyses. This paper describes a new nanostructure-based polymeric platform for enhancing adsorption capability of microRNAs, such as the cancer-associated miRNA-21. In this purification strategy, a nano-hole pattern was manufactured by Replica Molding (REM) and fabricated on Polydimethylsiloxane (PDMS) large-areas. Interestingly, the microRNAs adsorption is resulted favoured by this proper topography. In planning to concentrate the patient’s miRNAs used as biomarkers, PDMS surfaces with nanostructures were not further coated with any adhesive molecules to prevent forced surface-biomolecule adhesive regions and evaluate the mere influence of the surface topography and the ionic microenvironment. Both the nanopattern and the solution ionic strength promote adsorption of miRNAs by a sensitive and efficient method, which do not need probe immobilization, enzymatic reaction or further treatments. These studies revealed a two-fold higher fluorescent signal after solid-phase purification protocol compared to standard PDMS obtained by spin-coating. While this paper focuses on nanostructure-miRNA adsorption, the general strategy of trapping miRNAs on nano-hole patterns should be broadly applicable for purification of other miRNAs through microfluidic biosensors and should have basic as well as clinical research applications.

A new communication route among cells was reported in recent years, via extracellular vesicles and their cargo. Exosomes in particular are attracting increasing interest as privileged mediators of this cell communication route. The exosome-mediated transfer of nucleic acids, especially of microRNAs, is particularly promising for their use both as biomarkers of pathologies and as a therapeutic tool. Here, a simplified model of interaction among cells, microRNAs and vesicles is studied using a biophysical approach. A synthetic and fluorescent microRNA (i.e. miR-1246 conjugated with TAMRA) was selected to model cell communication, monitoring its internalization in cells. The fluorescent miR-1246, either naked or included in synthetic or natural vesicles, was incubated with human breast adenocarcinoma cells (MCF7) for different times. A comparison between this human microRNA and its DNA copy or an exogenous microRNA (from Caenorhabditis elegans) allowed assessment of the specificity of the information transfer through microRNAs, and especially associated with exosomes. The uptake of naked miR-1246 was indeed higher both in terms of number of targeted cells and intensity of fluorescence signal with respect to the other nucleic acids tested. The same occurred with miR-1246 loaded exosomes, evidencing a specific uptake only partially due to the lipidic components and present only when the human microRNA was loaded in exosomes, which were themselves derived from the same MCF7 cells.

Fatigue resistance and biocompatibility are key parameters for the successful implantation of hard-tissue prostheses, which nowadays are more and more frequently manufactured by selective laser melting (SLM). For this purpose, the present paper is aimed at investigating the effect of post-sintering treatments on the fatigue behavior and biological properties of Ti samples produced by SLM. After the building process, all samples are heat treated to achieve a complete stress relief. The remaining ones are tribofinished with the aim of reducing the surface roughness of the as-sintered condition. Part of the tribofinished samples are then subjected to one of the following post-sintering treatments: (i) shot peening, (ii) hot isostatic pressing (HIP), and (iii) electropolishing. It is found that shot peening and HIP are the most effective treatments to improve the high and the very-high cycle fatigue resistance, respectively. At the same time, they preserve the good biocompatibility ensured by the biomedical Titanium Grade 23.

Aflatoxins (AF) are naturally occurring mycotoxins, produced by many species of Aspergillus. Among aflatoxins, Aflatoxin M1 (AFM1) is one of the most frequent and dangerous for human health. The acceptable maximum level of AFM1 in milk according to EU regulation is 50 ppt, equivalent to 152 pM, and 25 ppt, equivalent to 76 pM, for adults and infants, respectively. Here, we study a photonic biosensor based on Si 3 N 4 asymmetric Mach&ndash, Zehnder Interferometers (aMZI) functionalized with Fab&rsquo, for AFM1 detection in milk samples (eluates). The minimum concentration of AFM1 detected by our aMZI sensors is 48 pM (16.8 pg/mL) in purified and concentrated milk samples. Moreover, the real-time detection of the ligand-analyte binding enables the study of the kinetics of the reaction. We measured the kinetic rate constants of the Fab&rsquo, AFM1 interaction.

International audience; PVL (Panton-Valentine leukocidin) and other Staphylococcus aureus β-stranded pore-forming toxins are important virulence factors involved in various pathologies that are often necrotizing. The present study characterized leukotoxin inhibition by selected SCns (p-sulfonato-calix[n]arenes): SC4, SC6 and SC8. These chemicals have no toxic effects on human erythrocytes or neutrophils, and some are able to inhibit both the activity of and the cell lysis by leukotoxins in a dose-dependent manner. Depending on the type of leukotoxins and SCns, flow cytometry revealed IC50 values of 6-22 μM for Ca2+ activation and of 2-50 μM for cell lysis. SCns were observed to affect membrane binding of class S proteins responsible for cell specificity. Electrospray MS and surface plasmon resonance established supramolecular interactions (1:1 stoichiometry) between SCns and class S proteins in solution, but not class F proteins. The membrane-binding affinity of S proteins was Kd=0.07-6.2 nM. The binding ability was completely abolished by SCns at different concentrations according to the number of benzenes (30-300 μM; SC8

Economical, religious, and health reasons demand an accurate control of food in order to protect consumers from falsely labeled products. Meat in particular is easily susceptible to fraudulent labeling, mainly through contamination with species of lower value. New methods and protocols for rapid, sensitive and reliable identification of extraneous species in food are therefore required. The miniaturization and optimization of analytical methodologies are powerful tools in this direction, especially when connected to Lab-on-a-chip (LOC) microdevices. LOCs possess many advantages, such as the reduction of the analysis cost, the possibility to save time and labor, the easiness of use and not last, the possibility to bring a complex technique out of the laboratory. Here we present a new concept for the food quality control, i.e. the use of LOC for the detection of exogenous DNA in meat via on-chip PCR in real-time. LOC surfaces were treated with different coatings in order to optimize the DNA extraction directly from meat homogenates (bovine, pork, horse). On the same LOC used for DNA purification, we set up the on-chip PCR with real-time detection. Over 1,000 beef genomes, up to 0.01 horse or pork genomes were successfully detected in binary mixtures of pre-purified DNA and similarly, up to 0.01 % parts of exogenous meat were detected in binary mixtures of meat homogenates. The successful on-chip detection of exogenous DNA is a promising step toward the production of an effective microdevice for rapid, sensitive, and reliable identification of meat adulteration.

MicroRNAs (miRNAs) are endogenous, small (18–24 nt), non-coding RNAs that regulate gene expression. Among miRNAs, those bound to the AGO2 protein are the functionally active fraction which mediates the cell regulatory processes and regulate messages exchanged by cells. Several methods have been developed to purify this fraction of microRNAs, such as immunoprecipitation and immunoprecipitation-derived techniques. However, all these techniques are generally recognized as technically complicated and time consuming. Here, a new bio-functional surface for the specific capture of AGO2-bound microRNAs is proposed. Starting from a silicon oxide surface, a protein A layer was covalently bound via epoxy chemistry to orient specific anti-AGO2 antibodies on the surface. The anti-AGO2 antibodies captured the AGO2 protein present in cell lysate and in human plasma. The AGO2-bound microRNAs were then released by enzymatic digestion and detected via RT-qPCR. Control surfaces were also prepared and tested. Every step in the preparation of the bio-functional surfaces was fully characterized from the chemical, morphological and functional point of view. The resulting bio-functional surface is able to specifically capture the AGO2-bound miRNAs from biologically-relevant samples, such as cell lysate and human plasma. These samples contain different proportions of AGO2-bound microRNAs, as reliably detected with the immunocapture method here proposed. This work opens new perspectives for a simple and faster method to isolate not only AGO2-bound microRNAs, but also the multiprotein complex containing AGO2 and miRNAs.

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease worldwide. The diagnosis and monitoring of HCV infection is a crucial need in the clinical management. The conventional diagnostic technologies are challenged when trying to address molecular diagnostics, especially because they require a complex and time-consuming sample preparation phase. Here, a new concept based on surface functionalization was applied to viral RNA purification: first of all polydimethylsiloxane (PDMS) flat surfaces were modified to hold RNA adsorption. After a careful chemical and morphological analysis of the modified surfaces, the functionalization protocols giving the best RNA adsorbing surfaces were applied to PDMS microdevices. The functionalized microdevices were then used for RNA purification from HCV infected human plasma samples. RNA purification and RT were successfully performed in the same microdevice chamber, saving time of analysis, reagents, and labor. The PCR protocol for HCV cDNA amplification was also implemented in the microdevice, demonstrating that the entire process of HCV analysis, from plasma to molecular readout, could be performed on-chip. Not only HCV but also other microdevice-based viral RNA detection could therefore result in a successful Point-of-Care (POC) diagnostics for resource-limited settings.

Modern Lab-on-a-chip (LOC) platforms for genomic applications integrate several biological tasks in a single device. Combination of these processes into a single device minimizes sample loss and contamination problems as well as reducing analysis time and costs. Here we present a study of a microchip platform aimed at analyzing issues arising from the combination of different functions, such as DNA purification from blood, target amplification by PCR and DNA detection in a single silicon-based device. DNA purification is realized through two different strategies: 1) amine groups coating microchannel surfaces and 2) magnetic nanoparticles coated by chitosan. In the first strategy silicon/Pyrex microdevices coated with 3-aminopropyltriethoxysilane (APTES) or 3-2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) were examined and their efficiency in human genomic DNA adsorption/desorption was evaluated. APTES treatment was the most suitable for the purification of a reasonable amount of DNA in a state suitable for the following PCR step. The second strategy has instead the main advantage of avoiding an elution step, since the DNA adsorbed on the magnetic nanoparticles can be used as PCR template. On-chip PCR was performed in a custom thermocycler, while the detection of PCR products was carried out by fluorescence reading. A complete genetic analysis was demonstrated on the monolithic silicon/Pyrex microchip, starting from less than 1 [Formula: see text]L of human whole blood and arriving at SNPs identification. The successful integration of DNA purification, amplification and detection on a single microdevice was proven without the need for biological passivation steps and possibly simplifying the realization of genomic detection devices.

The present generation of mechanical heart-valves prostheses offers significantly improved blood compatibility, combined with appropriate physical and mechanical properties and durability; however, thromboembolism persists as a crucial clinical complication because of the key role played by adsorbed plasma proteins and adherent platelets in determining hemocompatibility. In the present work human plasma protein adsorption and platelet adhesion and activation were evaluated on four materials, selected according to their different physico-chemical and morphological properties: pyrolytic carbon (PyC), titanium alloy Ti–6Al–4V (Ti), tissue culture polystyrene (TCPS) and polystyrene sterilized by γ-irradiation (StPS). Morphological and chemical properties of surfaces were assessed by Time of Flight Secondary Ion Mass Spectrometry (Tof-SIMS), X-ray Photoemission Spectroscopy (XPS), Atomic Force Microscopy (AFM) and Contact Angle (CA) analysis, while protein adsorption and platelet adhesion and activation were evaluated by colorimetric and immunofluorescence tests and Scanning Electron Microscopy (SEM), respectively. Among the analyzed materials, PyC induced the lowest level of protein adsorption and platelet adhesion and activation. In particular, immunofluorescence analysis of fibronectin (FN), fibrinogen (FG), von Willebrand factor (vWF), Hageman factor (FXII) and albumin (ALB) showed that PyC surface was characterized by high levels of FG adsorption, low levels of FN, ALB and vWF and the absence of FXII. Finally, when analyzing the biological response as a function of surface properties, it was found that protein adhesiveness increased with increasing contact angle values. To the contrary, platelet activation related to the total amount of adherent proteins was poor while cell activation was possibly dependent on the detailed composition of the underlying protein layer. Furthermore, differences in surface roughness of the examined materials did not seem to influence the biological response in terms of platelet activation and protein adsorption.

This work deals with the design, fabrication, and thermal characterization of a disposable miniaturized Polymerase Chain Reaction (PCR) module that will be integrated in a portable and fast DNA analysis system. It is composed of two independent parts: a silicon substrate with embedded heater and thermometers and a PDMS (PolyDiMethylSiloxane) chamber reactor as disposable element; the contact between the two parts is assured by a mechanical clamping obtained using a Plastic Leaded Chip Carrier (PLCC). This PLCC is also useful, avoid the PCR mix evaporation during the thermal cycles. Finite Element Analysis was used to evaluate the thermal requirements of the device. The thermal behaviour of the device was characterized revealing that the temperature can be controlled with a precision of ±0.5°C. Different concentrations of carbon nanopowder were mixed to the PDMS curing agent in order to increase the PDMS thermal conductivity and so the temperature control accuracy.

This work presents the design, fabrication and characterization of a Si-based microseparation module for liquid chromatography (LC), targeted for agrofood applications and in particular for wine quality monitoring. The microsystem chip is a Silicon/Pyrex structure realized by anodic bonding. The basic Si-chip modules are an open tubular microcolumn with inlet/outlet for fluidic connections, a platinum heater and a Pt three-electrode amperometric sensor. Analytical calculations and finite element analysis have been performed to optimize the microcolumn design. Microchannels and inlet/outlet have been fabricated by deep reactive ion etching (DRIE) and tetra methyl ammonium hydroxide (TMAH) wet etching, respectively. The column has been functionalized with n-octyltriethoxysilane (C8-TEOS). Preliminary characterization tests of the microcolumn have been performed by evaluating the capability of the system to retain test solutions of acetic acid.

The need for decentralized clinical tests together with the concept of time and cost saving are pushing the development of portable, miniaturized, compact biosensors with diagnostic and prognostic purpose. Here, we propose an innovative detection system based on a Single Photon Avalanche Diode (SPAD) with high sensitivity and low noise, crucial features for an efficient chemiluminescence biosensor. The SPAD detector, having 60. ?m diameter, has a Photon Detection Efficiency higher than 55% at 425. nm and a Dark Count Rate lower than 100. Hz at room temperature. Our design allows a good optical coupling efficiency between sample and detector. A specific biofunctional surface was implemented taking advantage of aptamers, short DNA sequences having high selectivity and affinity toward their targets. We successfully detected physiological levels of Vascular Endothelial Growth Factor (VEGF), a circulating protein biomarker highly correlated with cancer. The SPAD aptasensor showed a Limit of Detection (LoD) in the pM range, stability (up to 42 days) and re-usability (up to seven cycles). This compact biosensor is therefore a promising step toward the actual use of portable microdevices in diagnostics.

A growing number of receptors, often associated with the innate immune response, are being identified as targets for bacterial toxins of the beta-stranded pore-forming family. These findings raise the new question of whether the receptors are activated or merely used as docking points facilitating the formation of a pore. To elucidate whether the Staphylococcus aureus Panton-Valentine leukocidin and the leukotoxin HlgC/HlgB act through the C5a receptor (C5aR) as agonists, antagonists or differ from the C5a complement-derived peptide, their activity is explored on C5aR-expressing cells. Both leukotoxins equally bound C5aR in neutrophils and in stable transfected U937 cells and initiated mobilization of intracellular Ca(2+) . HlgC/HlgB requires the presence of robust intracellular acidic Ca(2+) stores in order to evoke a rise in free [Ca(2+) ]i , while the LukS-PV/LukF-PV directly altered reticular Ca(2+) stores. Intracellular target specificity is conferred by the F-subunit associated to the S-subunit binding the receptor. Furthermore, internalization of the two leukotoxin components (S- and F-subunits) associated to C5aR is required for the initiation of [Ca(2+) ]i mobilization. Electrophysiological recordings on living cells demonstrated that LukS-PV/LukF-PV does not alter the membrane resistance of C5aR-expressing cells. The present observations suggest that part of the pore-forming process occurs in distinct intracellular compartments rather than at the plasma membrane.