Nano/biosensors Based On Large-Area Graphene

Two dimensional materials have properties that make them ideal for applications in chemical and biomolecular sensing. Their high surface/volume ratio implies that all atoms are exposed to the environment, in contrast to three dimensional materials with most atoms shielded from interactions inside the bulk. Graphene additionally has an extremely high carrier mobility, even at ambient temperature and pressure, which makes it ideal as a transduction device. The work presented in this thesis describes large-scale fabrication of Graphene Field Effect Transistors (GFETs), their physical and chemical characterization, and their application as biomolecular sensors. Initially, work was focused on developing an easily scalable fabrication process. A large-area graphene growth, transfer and photolithography process was developed that allowed the scaling of production of devices from a few devices per single transfer in a chip, to over a thousand devices per transfer in a full wafer of fabrication. Two approaches to biomolecules sensing were then investigated, through nanoparticles and through chemical linkers. Gold and platinum Nanoparticles were used as intermediary agents to immobilize a biomolecule. First, gold nanoparticles were monodispersed and functionalized with thiolated probe DNA to yield DNA biosensors with a detection limit of 1 nM and high specificity against noncomplementary DNA. Second, devices are modified with platinum nanoparticles and functionalized with thiolated genetically engineered scFv HER3 antibodies to realize a HER3 biosensor. Sensors retain the high affinity from the scFv fragment and show a detection limit of 300 pM. We then show covalent and non-covalent chemical linkers between graphene and antibodies. The chemical linker 1-pyrenebutanoic acid succinimidyl ester (pyrene) stacks to the graphene by Van der Waals interaction, being a completely non-covalent interaction. The linker 4-Azide-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester (azide) is a photoactivated perfluorophenyl azide that covalently binds to graphene. A comparison is shown for genetically engineered scFv HER3 antibodies and show a low detection limit of 10 nM and 100 pM for the pyrene and azide, respectively. Finally, we use the azide linker to demonstrate a large-scale fabrication of a multiplexed array for Lyme disease. Simultaneous detection of a mixture of two target proteins of the Lyme disease bacterium (Borrelia burgdorferi), this is done by separating the antibodies corresponding to each target in the mixture to different regions of the chip. We show we can differentiate concentrations of the two targets

Recent Achievements in Electrochemical and Surface Plasmon Resonance Aptasensors for Mycotoxins Detection

Mycotoxins are secondary metabolites of fungi that contaminate agriculture products. Their release in the environment can cause severe damage to human health. Aptasensors are compact analytical devices that are intended for the fast and reliable detection of various species able to specifically interact with aptamers attached to the transducer surface. In this review, assembly of electrochemical and surface plasmon resonance (SPR) aptasensors are considered with emphasis on the mechanism of signal generation. Moreover, the properties of mycotoxins and the aptamers selected for their recognition are briefly considered. The analytical performance of bio-sensors developed within last three years makes it possible to determine mycotoxin residues in water and agriculture/food products on the levels below their maximal admissible concentrations. Requirements for the development of sample treatment and future trends in aptasensors are also discussed. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Funding: T.K. acknowledges funding by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities (grant No 0671–2020−0063). T.H. acknowledges funding from the Science Grant Agency VEGA, project No.: 1/0419/20

Molecular Imaging

The present book gives an exceptional overview of molecular imaging. Practical approach represents the red thread through the whole book, covering at the same time detailed background information that goes very deep into molecular as well as cellular level. Ideas how molecular imaging will develop in the near future present a special delicacy. This should be of special interest as the contributors are members of leading research groups from all over the world

Delivery of Molecules Using Nanoscale Systems for Cancer Treatment and/or Diagnosis

This book focuses on how nanoscale systems can be used to deliver molecules to help with cancer management. It provides a broad overview of some of the key strategies for nanocarrier design. These strategies are brought together by the wide compositional variety of these systems and the diversity of molecules that may be carried. Additionally, functionalization strategies, codelivery, and combination with other treatment modalities highlight a very active research field

Study of novel nanoparticle transport and drug release for cancer treatment.

Nano-scale particles sized 10—400 nm administered systemically preferentially extravasate from tumor vasculature due to the enhanced permeability and retention effect. Therapeutic success remains elusive, however, because of inhomogeneous particle distribution within tumor tissue. Insufficient tumor vascularization limits particle transport and also results in avascular hypoxic regions with non-proliferating cells, which can regenerate tissue after nanoparticle-delivered cytotoxicity or thermal ablation. In this study, gold nanoparticles were functionalized with phosphatidylcholine (two-layer) or phosphatidylcholine and HDL (three-layer) in the formation of “layered” nanoparticles. The diffusivity of both two- and three layered colloidal gold nanoparticles and silica gold nanoshells were examined in 3D cell cultures. Both two- and three layered nanoparticles showed enhanced diffusivity in comparison to previously developed PEGylated nanoparticles. As the two layer nanoparticles displayed enhanced diffusivity in comparison to three layer nanoparticles, the two layered nanoparticles were further examined in vivo using mice implanted with orthotopic pancreatic adenocarcinomas. The two layer colloidal gold nanoparticles showed enhanced diffusivity in comparison to silica gold nanoshells in vivo, suggesting that smaller nanoparticles were able to localize and diffuse from vasculature better than larger nanoparticles. Overall accumulation of solid gold nanoparticle accumulated in the tumor and filtering organs (liver and spleen) was 2X higher than silica gold nanoshells. Thus, two layer colloidal gold nanoparticles were loaded with cisplatin or paclitaxel to determine optimal drug release kinetics. Drug release from paclitaxel-loaded nanoparticles displayed a slower release while cisplatin-loaded nanoparticles experienced an initial burst of drug release followed by a slower release of remaining drug. Lastly, drug-loaded colloidal gold nanoparticles were tested in 3D cell cultures to determine their cytotoxicity. Both two and three layer nanoparticles loaded with cisplatin orpaclitaxel showed similar efficacy to drug alone, suggesting their viable use in vivo for cancer treatment. This study has demonstrated the potential use of layered nanoparticles for increasing the delivery of chemotherapeutics deeper into tumor tissue

Aptamères ADN : du Cell-SELEX à l'imagerie

Les aptamères ADN sont des séquences simple-brins capables de se lier spécifiquement à une cible. Ils constituent de nouveaux outils pour la détection et/ou l'inhibition de molécules biologiques. Ce travail s'inscrit dans le cadre du diagnostic et de la thérapie du cancer ovarien. Les cibles sélectionnées dans ce contexte sont les interleukines humaines IL-6 et IL-8 dans le but d'inhiber leurs effets angiogéniques, et le biomarqueur du cancer épithélial ovarien Mucine16 sous sa forme sécrétée (CA 125) et membranaire (Mucine16) dans le but de faire du diagnostic sanguin et cellulaire. Sélections IL-6 et IL-8 : Des séquences ont été sélectionnées par Filter-Binding SELEX. Sélection Mucine16 : Les cellules cancéreuses ovariennes présentent pour la plupart la particularité de surexprimer les deux formes de cette protéine. Ce qui en fait le candidat pour le ciblage des tumeurs. Des séquences ont été identifiées après 20 cycles de Cell-SELEX. D'autre part, nous avons comparé le marquage fluorescent de sphéroïdes MCF-7 par un anticorps anti-Mucine1 et un aptamère anti-Mucine1. L'aptamère montre une pénétration plus profonde dans le sphéroïde ainsi qu'une internalisation.DNA Aptamers are single-stranded sequences able to specifically bind a target molecule. They constitute new tools for the detection and/or the inhibition of biological molecules. This work is in the field of ovarian cancer diagnostic and therapy. In this concept, selected targets are the human interleukins IL-6 and IL-8 for inhibiting their angiogenic effect and the epithelial ovarian cancer biomarker Mucine16 on its secreted (CA 125) and membranal (Mucine16) forms in the aim of doing blood and cellular diagnostic. IL-6 and IL-8 selections: Sequences have been selected by Filter-Binding SELEX. Mucine16 selection: Most of ovarian cancer cells overexpress the two forms of this protein. That is why Mucine16 is a candidate for tumor targeting. Sequences have been identified after 20 cycles of Cell-SELEX. In addition, we compared the fluorescent labeling of MCF-7 spheroids by an anti-Mucine1 antibody and its DNA aptamer analog. The aptamer exhibits a deeper spheroid penetration and is internalized

Extracellular vesicles from induced neurons trigger epigenetic silencing of a brain neurotransmitter.

Introduction: Antithrombin (AT) is a glycoprotein involved in the regulation of blood coagulation. It belongs to the family of serine-protease inhibitors and acts as the most important antagonist of different clot- ting factors. Two types of inherited AT deficiency can be distinguished: Type I (quantitative deficit), and Type II (qualitative deficit). The latter is characterized by an impaired inhibitory activity related to dysfunc- tional domains of the protein. Three Type II subtypes can be defined: Type IIa (reactive site defect), Type IIb (heparin binding site defect) and Type IIc (pleiotropic defect). This classification has clinical importance since these subtypes have a different thrombotic risk. No functional routine diagnostic assay, however, can be assumed to detect all forms of Type II deficiencies since false-negative results may hamper the diagnosis. Methods: We analysed the biochemical/biophysical association of ATT to EVs. We separated EVs from plasma of healthy or Type II affected patients or from cultured hepatocytes through differential ultracentrifu- gation followed by sucrose density gradient and/or immunoprecipitation. We next combined dot blot ana- lysis, WB, 2D electrophoresis and enzymatic assays to reveal the nature of ATT association to EVs. Results: We evidenced that ATT is associated to the external leaflet of EVs. We also found that specific ATT isoforms are enriched in EV preparations in respect to total plasma and that those isoforms are selectively associated to EVs when comparing healthy or ATT type II deficient patients. Summary/Conclusion: ATT selective association pat- tern to EVs might be related either to mutations in the primary sequence of the protein or alterations in the glycosylation process, hence experiments are ongoing to reveal the nature of this phenomenon. Our findings suggest that analysis of ATT enriched in EV prepara- tions might be useful to gain insights into the patho- genesis and be of support in the diagnostic algorithm of ATT deficiency. Funding: This work acknowledges FFABR (Fondo finanziamento attività Base di ricerca from MIUR, Ministry of Education, Universities and Research, Italy) for financial support

Phosphonium-functionalised gold nanoparticles for mitochondria targeted therapeutics.

The work presented in this thesis demonstrates that triarylphosphoniopropyl-thiosulfate zwitterions and w-thioacetylpropyl(triphenyl)phosphonium salts can be used to prepare cationic, water-soluble gold nanoparticles with mean core sizes in the range of 2.5-5 nm. Phosphonium-functionalised gold nanoparticles have been characterised by a number of techniques including NMR, LDI-MS, SIMS, XPS, TGA, ICP-MS, MALDI-MS and TEM.Cytotoxicity studies illustrated that phosphonium ligands are relatively non-toxic to human prostate cancer cells and therefore can be used as a delivery vector to delivery gold nanoparticles specifically to the site of the mitochondria for other therapeutic applications such as photothermal therapy. Cellular uptake studies of phosphonium ligands by MALDI-MS showed that they are rapidly taken-up by cells within ten minutes.Phosphonium-functionalised gold nanoparticles are soluble in biological media which is of great importance for cell biology studies. Initial photothermal therapy studies demonstrated that the gold nanoparticles responds specifically to a green light excitation source (510-550 nm) which overlaps the surface plasmon resonance band of the phosphonium-functionalised gold nanoparticles at 525 nm. Preliminary data also showed that phosphonium-functionalised gold nanoparticles can selectively induce apoptosis in cells followed by irradiation, this was confirmed by Hoechst and caspase-3 staining. Quantification studies of phosphonium-functionalised gold nanoparticles by ICP-MS illustrated that these nanoparticle have good uptake in cells (above 75%). TEM data confirmed that phosphonium-functionalised gold nanoparticles are taken-up by human prostate cancer cells and are localised in the mitochondria