Plasmonic and Magnetic Nanostructures for Multiplexing Detection and Multiple Imaging Techniques in Bionanomed

In the last years nanotechnology is strongly influencing the progresses of medicine, in particular against cancer, both for diagnostic and therapeutic purposes. Nanostructures can be planned with properties useful for a specific application. However, the same nanostructure can be synthesized also for more diagnostic techniques. In addiction, one can also plan these nanostructures for what is called multiplexing, namely the presence in the same diagnostic signal of information related to many analytes at the same time. The nanostructures can be synthesized for obtaining targeting, which increases the ability of discriminating different type of tumour associated antigens or, in the case of therapeutic purposes, for the ability of being directed to a specific site. In the present thesis, nanostructures have been synthesized for their applications in particular with two techniques like surface enhanced Raman spectroscopy (SERS) and magnetic resonance imaging (MRI), which are complementary techniques for imaging. The nanostructures have been functionalized also with antibodies for targeting antigens. SERS exploits the huge enhancement of electromagnetic fields of plamonic nanostructures. It is shown that this technique can be used for quantitative measurements of clinically interesting anticancer drugs, with a linear range response in the order of nanomole per millimetre square of SERS active area, which is appropriate also for clinical purposes. It is also demonstrated how molecules with a poor Raman cross section could be revealed with the help of a protocol, called Reactive-SERS, which considers a simple photoactivated reaction. Nanoparticles are obtained with laser ablation and they are found easy to functionalize due to their unique naked surface. It is shown that their properties are useful in laser desorption ionization mass spectrometry. Used as a matrix, they are compared with chemically produced particles and with a widely used organic matrix and they always show the lowest background especially in the low mass region under 500 Da, which is important for the identification of small molecules. Multiplexing analysis are carried out with gold nanoparticles (AuNP) functionalized with different dyes, as SERS reporters, associated to different antibodies. The library of optimized thiolated reporters is increased and makes possible to investigate more complex biological samples. This application is also translated to the cultural heritage research field for the characterization of paint stratigraphic samples. Multimodal contrast agents are developed, coupling the SERS activity, typical of dye functionalized gold nanoparticles, with magnetic sensitive moieties, like iron atoms in alloy nanoparticles or with Gd3+ ions linked to gold nanoparticles. A new polymer, called SuperDOTA, is synthetized for achieving high Gd3+ loadings on the surface of nanoparticles. Preliminary results show that AuNPs, functionalized with both a SERS reporter dye and SuperDOTA-Gd, have useful properties for in vivo MRI tumour analysis and ex-vivo SERS imaging

Use of nano gold obtained by laser ablation for SEIRA analyses of colorants

The analysis of dyes in cultural heritage samples is a well-known challenging task, due to their inherent high tinting strength and consequent low concentration in the carrying matrix a fact that severely limits the number of analytical techniques that can be efficiently and micro-destructively employed for their detection and unambiguous identification. In the present study, an advanced and alternative SEIRA based analytical protocol for the analysis of small quantities of synthetic colorants has been proposed. The method has been set up for the identification of Acid Orange 7 (AO7) using Au nanoparticles obtained by laser ablation in solution (LASiS). Analyses have been performed applying a drop containing a mixture between the colorant and the Au colloidal solution in its unaggregated state on a gold coated glass slide for RAS (Reflection Absorption Spectroscopy) analysis. The first results showed that, thanks to the enhancement produced by the nanoparticles, it is possible to analyze small amount of diluted solutions containing the colorant. Thus, the method has been successfully applied for the analysis of few pieces of dyed wool, after the development of a suitable micro extraction procedure

Single File Flow of Biomimetic Beads for Continuous SERS Recording in a Microfluidic Device

A major challenge in cancer treatment is the quantification of biomarkers associated with a specific cancer type. Important biomarkers are the circulating tumor cells (CTCs) detached from the main cancer and circulating in the blood. CTCs are very rare and their identification is still an issue. Although CTCs quantification can be estimated by using fluorescent markers, all the fluorescence techniques are strongly limited by the number of emissions (therefore markers) that can be discriminated with one exciting line, by their bleaching characteristics, and by the intrinsic autofluorescence of biological samples. An emerging technique that can overcome these limitations is Surface Enhanced Raman Scattering (SERS). Signals of vibrational origin with intensity similar to those of fluorescence, but narrower bandwidths, can be easily discriminated even by exciting with a single laser line. We recently showed the benefit of this method with cells fixed on a surface. However, this approach is too demanding to be applied in clinical routine. To effectively increase the throughput of the SERS analysis, microfluidics represents a promising tool. We report two different hydrodynamic strategies, based on device geometry and liquids viscosity, to successfully combine a microfluidic design with SERS

Manipulating chemistry through nanoparticle morphology

We demonstrate that the protonation chemistry of molecules adsorbed at nanometer distances from the surface of anisotropic gold nanoparticles can be manipulated through the effect of surface morphology on the local proton density of an organic coating. Direct evidence of this remarkable effect was obtained by monitoring surface-enhanced Raman scattering (SERS) from mercaptobenzoic acid and 4-aminobenzenethiol molecules adsorbed on gold nanostars. By smoothing the initially sharp nanostar tips through a mild thermal treatment, changes were induced on protonation of the molecules, which can be observed through changes in the measured SERS spectra. These results shed light on the local chemical environment near anisotropic colloidal nanoparticles and open an alternative avenue to actively control chemistry through surface morphology.LL and LML-M acknowledge funding from European Commission Grant (EUSMI 731019). Funding is also acknowledged from the Spanish MINECO (MAT2017-86659-R and MDM-2017-0720 to LML-M; MAT2017-88492-R and SEV2015-0522 to JGA) and the European Research Council (Advanced Grant 787510 4DBIOSERS to LML-M; Advanced Grant 789104-eNANO to JGA)

Energy rationale for the Use of the Thermophilic Mode of Anaerobic Bioconversion of Liquid Organic Waste in the Climatic Conditions of the Russian Federation

The transition of livestock production to industrial processes and the concentration of animals associated with this process on large farms and complexes has caused a sharp increase in the volume of manure that must be disposed of without pollution. One of the ways of processing organic waste (biomass) is its anaerobic digestion in biogas plants through the vital activity of microorganisms (methanogenesis).Biogas obtained using microbiological processing of biomass can be used as a raw material for heat and electric energy. Annually, 0.17% of the total livestock manure produced at Russian agricultural enterprisesis used for biogas production.The main component of a biogas plant is a manure fermentation reactor, the required volume of which is determined by the daily output of manure from the livestock farm, the temperature and the hydraulic retention time of treatment. This research explored thermal energy consumption of biogas plants, using the example of a biogas plant of a modular design that depended on the average annual outdoor temperature. Based on the calculations, the thermophilic mode was found to be more energy-efficient than the mesophilic one; thus, with the thermophilic mode, the specific energy consumption needed for the plant was lower at the average annual outdoor temperatures of all the constituent entities of the Russian Federation. At the same time, the specific biogas yield in the thermophilic regime was 20-50%higher than in the mesophilic regime. Keywords: anaerobic processing, agricultural waste, thermophilicmode, mesophilicmode, energy costs, energy rational

SERS nanostructures with engineered active peptides against an immune checkpoint protein

The immune checkpoint programmed death ligand 1 (PD-L1) protein is expressed by tumor cells and it suppresses the killer activity of CD8+ T-lymphocyte cells binding to the programmed death 1 (PD-1) protein of these immune cells. Binding to either PD-L1 or PD1 is used for avoiding the inactivation of CD8+ T-lymphocyte cells. We report, for the first time, Au plasmonic nanostructures with surface-enhanced Raman scattering (SERS) properties (SERS nanostructures) and functionalized with an engineered peptide (CLP002: Trp-His-Arg-Ser-Tyr-Tyr-Thr-Trp-Asn-Leu-Asn-Thr), which targets PD-L1. Molecular dynamics calculations are used to describe the interaction of the targeting peptide with PD-L1 in the region where the interaction with PD-1 occurs, showing also the poor targeting activity of a peptide with the same amino acids, but a scrambled sequence. The results are confirmed experimentally since a very good targeting activity is observed against the MDA-MB-231 breast adenocarcinoma cancer cell line, which overexpresses PD-L1. A good activity is observed, in particular, for SERS nanostructures where the CLP002-engineered peptide is linked to the nanostructure surface with a short charged amino acid sequence and a long PEG chain. The results show that the functionalized SERS nanostructures show very good targeting of the immune checkpoint PD-L1.Engineered peptides on plasmonic nanostructures show a good targeting activity against the immune checkpoint programmed death ligand 1 protein (PD-L1) expressed by tumor cells. SERS signals allow quantitative evaluation of the targeting activity

Manipulating chemistry through nanoparticle morphology

We demonstrate that the protonation chemistry of molecules adsorbed at nanometer distances from the surface of anisotropic gold nanoparticles can be manipulated through the effect of surface morphology on the local proton density of an organic coating. Direct evidence of this remarkable effect was obtained by monitoring surface-enhanced Raman scattering (SERS) from mercaptobenzoic acid and 4-aminobenzenethiol molecules adsorbed on gold nanostars. By smoothing the initially sharp nanostar tips through a mild thermal treatment, changes were induced on protonation of the molecules, which can be observed through changes in the measured SERS spectra. These results shed light on the local chemical environment near anisotropic colloidal nanoparticles and open an alternative avenue to actively control chemistry through surface morphology.LL and LML-M acknowledge funding from European Commission Grant (EUSMI 731019). Funding is also acknowledged from the Spanish MINECO (MAT2017-86659-R and MDM-2017-0720 to LML-M; MAT2017-88492-R and SEV2015-0522 to JGA) and the European Research Council (Advanced Grant 787510 4DBIOSERS to LML-M; Advanced Grant 789104-eNANO to JGA)

Research of interferential factors of accounting and evaluation of cryptocurrency in the practical business model of a company

Cryptocurrency is a modern phenomenon of the digital economy, which is gradually becoming part of the business processes of companies of various profiles and economic sectors. The presence of unsettled issues at the legislative level of jurisdictions, as well as the ambiguity of approaches to the classification and assessment of cryptocurrency in financial reporting, gives rise to discussions of both practical and scientific nature. For trade organizations, the need to resolve this issue is evident, since already now businesses are considering the possibility of accepting payment for goods with some types of cryptocurrencies, as well as using cryptocurrencies to pay for the resources the company needs

SERRS Multiplexing with Multivalent Nanostructures for the Identification and Enumeration of Epithelial and Mesenchymal Cells

Liquid biopsy represents a new frontier of cancer diagnosis and prognosis, which allows the isolation of tumor cells released in the blood stream. The extremely low abundance of these cells needs appropriate methodologies for their identification and enumeration. Herein we present a new protocol based on surface enhanced resonance Raman scattering (SERRS) gold multivalent nanostructures to identify and enumerate tumor cells with epithelial and mesenchimal markers. The validation of the protocol is obtained with spiked samples of peripheral blood mononuclear cells (PBMC). Gold nanostructures are functionalized with SERRS labels and with antibodies to link the tumor cells. Three types of such nanosystems were simultaneously used and the protocol allows obtaining the identification of all individual tumor cells with the help of a Random Forest ensemble learning method