Simultaneous evaluation of multiple microarray surface chemistries through real-time interferometric imaging.

Surface chemistry is a crucial aspect for microarray modality biosensor development. The immobilization capability of the functionalized surface is indeed a limiting factor for the final yield of the binding reaction. In this work, we were able to simultaneously compare the functionality of protein ligands that were locally immobilized on different polymers, while on the same solid support, therefore demonstrating a new way of multiplexing. Our goal was to investigate, in a single experiment, both the immobilization efficiency of a group of reactive polymers and the resulting affinity of the tethered molecules. This idea was demonstrated by spotting many reactive polymers on a Si/SiO2 chip and depositing the molecular probes on the spots immediately after. As a proof of concept, we focused on which polymers would better immobilize a model protein (α-Lactalbumin) and a peptide (LAC-1). We successfully showed that this protocol is applicable to proteins and peptides with a good efficiency. By means of real-time binding measurements performed with the interferometric reflectance imaging sensor (IRIS), local functionalization proved to be comparable to the classical flat coating solution. The final outcome highlights the multiplexing power of this method: first, it allows to characterize dozens of polymers at once. Secondly, it removes the limitation, related to coated surfaces, that only molecules with the same functional groups can be tethered to the same solid support. By applying this protocol, many types of molecules can be studied simultaneously and immobilization for each probe can be individually optimized.766466 (INDEX) - Horizon 2020 Framework Programmehttps://s3-eu-west-1.amazonaws.com/itempdf74155353254prod/8976347/Simultaneous_Evaluation_of_Multiple_Microarray_Surface_Chemistries_Through_Real-Time_Interferometric_Imaging_v1.pdfFirst author draf

Optical sensing in microchip capillary electrophoresis by femtosecond laser written waveguides

Capillary electrophoresis separation in an on-chip integrated microfluidic channel is typically monitored with bulky, bench-top optical excitation/detection instrumentation. Optical waveguides allow confinement and transport of light in the chip directing it to a small volume of the microfluidic channel and collecting the emitted/transmitted radiation. However, the fabrication of optical waveguides or more complex photonic components integrated with the microfluidic channels is not a straightforward process, since it requires a localized increase of the refractive index of the substrate.\ud Recently, a novel technique has emerged for the direct writing of waveguides and photonic circuits in transparent glass substrates by focused femtosecond laser pulses.\ud In this work we demonstrate the integration of femtosecond laser written optical waveguides into a commercial microfluidic chip. We fabricate high quality waveguides intersecting the microchannels at arbitrary positions and use them to optically address with high spatial selectivity their content. In particular, we apply our technique to integrate optical detection in microchip capillary electrophoresis. Waveguides are inscribed at the end of the separation channel in order to optically excite the different plugs reaching that point; fluorescence from the labelled biomolecules crossing the waveguide output is efficiently collected at a 90° angle by a high numerical aperture optical fiber. The sensitivity of the integrated optical detection system was first evaluated filling the chip with a dye solution, obtaining a minimum detectable concentration of 40 pM. \ud After dynamic coating of the microchannels with an EPDMA polymer we demonstrate electrophoresis of an oligonucleotide plug with concentration down to 1 nM and wavelength-selective monitoring of on-chip separation of three fluorescent dyes. Work is in progress on separation and detection of fluorescent-labeled DNA fragments, targeting specific, diagnostically relevant regions of a template DNA, for application to the detection of chromosome aberrations

Синдром эмоционального выгорания в профессиональной деятельности женщин-провизоров

выгорания синдромПРОФЕССИОНАЛЬНАЯ ОПУСТОШЕННОСТЬ, СТРЕССОВЫЕ РЕАКЦИИФАРМАЦЕВТ

Parameterized Complexity of Asynchronous Border Minimization

Microarrays are research tools used in gene discovery as well as disease and cancer diagnostics. Two prominent but challenging problems related to microarrays are the Border Minimization Problem (BMP) and the Border Minimization Problem with given placement (P-BMP). In this paper we investigate the parameterized complexity of natural variants of BMP and P-BMP under several natural parameters. We show that BMP and P-BMP are in FPT under the following two combinations of parameters: 1) the size of the alphabet (c), the maximum length of a sequence (string) in the input (l) and the number of rows of the microarray (r); and, 2) the size of the alphabet and the size of the border length (o). Furthermore, P-BMP is in FPT when parameterized by c and l. We complement our tractability results with corresponding hardness results

Digital detection of exosomes by interferometric imaging

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.This work was supported by Regione Lombardia and Fondazione Cariplo through POR-FESR, project MINER (ID 46875467); Italian Ministry of Health, Ricerca Corrente. This work was partially supported by The Scientific and Technological Research Council of Turkey (grant #113E643). (Regione Lombardia; 46875467 - Fondazione Cariplo through POR-FESR, project MINER; Italian Ministry of Health, Ricerca Corrente; 113E643 - Scientific and Technological Research Council of Turkey)Published versio

Clickable cellulosic surfaces for peptide-based bioassays

The use of peptides in paper-based analytics is a highly appealing field, yet it suffers from severe limitations. This is mostly due to the loss of effective target recognition properties of this relatively small probes upon nonspecific adsorption onto cellulose substrates. Here we address this issue by introducing a simple polymer-based strategy to obtain clickable cellulose surfaces, that we exploited for the chemoselective bioconjugation of peptide bioprobes. Our method largely outperformed standard adsorption-based immobilization strategy in a challenging, real case immunoassay, namely the diagnostic discrimination of Zika + individuals from healthy controls. Of note, the clickable polymeric coating not only allows efficient peptides bioconjugation, but it provides favorable anti-fouling properties to the cellulosic support. We envisage our strategy to broaden the repertoire of cellulosic materials manipulation and promote a renewed interest in peptide-based paper bioassays

Membrane-binding peptides for extracellular vesicles on-chip analysis

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a "universal" marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation

BPSL1626 : Reverse and Structural Vaccinology Reveal a Novel Candidate for Vaccine Design Against Burkholderia pseudomallei

Due to significant advances in computational biology, protein prediction, together with antigen and epitope design, have rapidly moved from conventional methods, based on experimental approaches, to in silico-based bioinformatics methods. In this context, we report a reverse vaccinology study that identified a panel of 104 candidate antigens from the Gram-negative bacterial pathogen Burkholderia pseudomallei, which is responsible for the disease melioidosis. B. pseudomallei can cause fatal sepsis in endemic populations in the tropical regions of the world and treatment with antibiotics is mostly ineffective. With the aim of identifying potential vaccine candidates, we report the experimental validation of predicted antigen and type I fimbrial subunit, BPSL1626, which we show is able to recognize and bind human antibodies from the sera of Burkholderia infected patients and to stimulate T-lymphocytes in vitro. The prerequisite for a melioidosis vaccine, in fact, is that both antibody- and cell-mediated immune responses must be triggered. In order to reveal potential antigenic regions of the protein that may aid immunogen re-design, we also report the crystal structure of BPSL1626 at 1.9 angstrom resolution on which structure-based epitope predictions were based. Overall, our data suggest that BPSL1626 and three epitope regions here-identified can represent viable candidates as potential antigenic molecules