Towards novel compact laser sources for non-invasive diagnostics and treatment

An important field of application of lasers is biomedical optics. Here, they offer great utility for diagnosis, therapy and surgery. For the development of novel methods of laser-based biomedical diagnostics careful study of light propagation in biological tissues is necessary to enhance our understanding of the optical measurements undertaken, increase research and development capacity and the diagnostic reliability of optical technologies. Ultimately, fulfilling these requirements will increase uptake in clinical applications of laser based diagnostics and therapeutics. To address these challenges informative biomarkers relevant to the biological and physiological function or disease state of the organism must be selected. These indicators are the results of the analysis of tissues and cells, such as blood. For non-invasive diagnostics peripheral blood, cells and tissue can potentially provide comprehensive information on the condition of the human organism. A detailed study of the light scattering and absorption characteristics can quickly detect physiological and morphological changes in the cells due to thermal, chemical, antibiotic treatments, etc [1-5]. The selection of a laser source to study the structure of biological particles also benefits from the fact that gross pathological changes are not induced and diagnostics make effective use of the monochromatic directional coherence properties of laser radiation.

Characterization of microvesicles released from human red blood cells

Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.

Obtenção e caracterização de complexos polieletrolíticos de quitosana e poli (metacrilato de sódio)

Polyelectrolyte complexes (PECs) nanoparticles were prepared using chitosan and sodium polymethacrylate. The complex formation was investigated using turbidimetry, conductometry, viscometry, and dynamic light scattering. The presence of excess positive charges was evidenced by zeta potential measurements. The particle diameter was characterized by dynamic light scattering and the morphology by atomic force microscopy. In all experiments an abrupt change in behavior was observed at a carboxyl:amino molar ratio around 0.7−0.8. Those changes in behavior were related to a proposed mechanism of complex formation based on the decrease of macromolecular dimensions of soluble polyelectrolyte complex clusters, followed by phase segregation

Síntese, caracterização e estudo da associação em meio aquoso de copolímeros responsivos de carboximetilcelulose-g-Jeffamine®

This thesis presents the synthesis, characterization and study of the associative behaviour in aqueous media of new responsive graft copolymers, based on carboxymethylcellulose as the water-soluble backbone and Jeffamine® M-2070 e Jeffamine® M-600 (commercial polyetheramines) as the thermoresponsive grafts with high cloud point temperatures in water. The synthesis was performed on aqueous medium, by using 1-ethyl-3- (3-(dimethylamino)-propyl)carbodiimide hydrochloride and N-hydroxysuccinimide as activators of the reaction between carboxylategroupsfrom carboxymethylcellulose and amino groups from polyetheramines. The grafting reaction was confirmed by infrared spectroscopy and the grafting percentage by 1H NMR. The molar mass of the polyetheramines was determined by 1H NMR, whereas the molar mass of CMC and graft copolymers was determined by static light scattering. The salt effect on the association behaviour of the copolymers was evaluated in different aqueous media (Milli-Q water, 0.5M NaCl, 0.5M K2CO3 and synthetic sea water), at different temperatures, through UV-vis, rheology and dynamic light scattering. None of the copolymers solutions, at 5 g/L, turned turbid in Milli-Q water when heated from 25 to 95 °C, probably because of the increase in hydrophibicity promoted by CMC backbone. However, they became turbid in the presence of salts, due to the salting out effect, where the lowest cloud point was observed in 0.5M K2CO3, which was attributed to the highest ionic strength in water, combined to the ability of CO3 2- to decrease polymer-solvents interactions. The hydrodynamic radius and apparent viscosity of the copolymers in aqueous medium changed as a function of salts dissolved in the medium, temperature and copolymer composition. Thermothickening behaviour was observed in 0.5M K2CO3 when the temperature was raised from 25 to 60°C. This performance can be attributed to intermolecular associations as a physical network, since the temperature is above the cloud point of the copolymers in this solvent.

Rapid quantification and validation of lipid concentrations within liposomes

Quantification of the lipid content in liposomal adjuvants for subunit vaccine formulation is of extreme importance, since this concentration impacts both efficacy and stability. In this paper, we outline a high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method that allows for the rapid and simultaneous quantification of lipid concentrations within liposomal systems prepared by three liposomal manufacturing techniques (lipid film hydration, high shear mixing, and microfluidics). The ELSD system was used to quantify four lipids: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, dimethyldioctadecylammonium (DDA) bromide, and D-(+)-trehalose 6,6′-dibehenate (TDB). The developed method offers rapidity, high sensitivity, direct linearity, and a good consistency on the responses (R2 > 0.993 for the four lipids tested). The corresponding limit of detection (LOD) and limit of quantification (LOQ) were 0.11 and 0.36 mg/mL (DMPC), 0.02 and 0.80 mg/mL (cholesterol), 0.06 and 0.20 mg/mL (DDA), and 0.05 and 0.16 mg/mL (TDB), respectively. HPLC-ELSD was shown to be a rapid and effective method for the quantification of lipids within liposome formulations without the need for lipid extraction processes.

Highly stable PEGylated gold nanoparticles in water: applications in biology and catalysis

Gold nanoparticles (Au NPs) with diameters ranging between 5-60 nm have been synthesised in water, and further stabilized with polyethylene glycol-based thiol polymers (mPEG-SH). Successful PEGylation of the Au NPs was confirmed by Dynamic Light scattering (DLS) and Zeta potential measurements. PEG coating of the Au NPs is the key of their colloidal stabilty, and its successful applications. Catalytic efficiency testing of the PEG-AuNPs were carried out on homocoupling of boronic acid. PEG-Au NPs with AuNps diameter < 30 nm were useful as catalyst in water. Finally, the PEG-Au NPs were also shown to be stable in biological fluid and not cytotoxic on B16.F10 cell line, making them attractive for further studies.

Synthesis & retention mechanisms of novel mixed mode stationary phase for detection of specific polar low molecular weight biological samples

The thesis primarily reports the synthesis, characterization and application of novel mixed mode stationary phases for Hydrophilic Interaction Liquid Chromatography (HILIC). HILIC is a rapidly emerging chromatographic mode that is finding great applicability in the analysis of polar organic molecules. In addition, there is a chapter on the analysis of Bisphenol A and related species using capillary electrophoresis (CE) coupled with boron-doped diamond electrodes for electrochemical detection. The synthesis and characterization of the novel mixed mode stationary phases prepared in this work is an important contribution to the field as the materials prepared exhibited better performance than similar materials obtained commercially. In addition a more thorough characterization of the materials (e.g.,thermogravimetric analysis, various NMR modes, elemental analysis, etc.) and resulting columns (e.g., H) than is typically encountered. The application of these new materials to the analysis of sugars using evaporative light scattering is also novel. In CE studies, electrochemical detection is sufficiently rare that the work is also novel.

Single particle characterization using the soot particle aerosol mass spectrometer (SP-AMS)

Understanding the impact of atmospheric black carbon (BC) containing particles on human health and radiative forcing requires knowledge of the mixing state of BC, including the characteristics of the materials with which it is internally mixed. In this study, we demonstrate for the first time the capabilities of the Aerodyne Soot-Particle Aerosol Mass Spectrometer equipped with a light scattering module (LS-SP-AMS) to examine the mixing state of refractory BC (rBC) and other aerosol components in an urban environment (downtown Toronto). K-means clustering analysis was used to classify single particle mass spectra into chemically distinct groups. One resultant cluster is dominated by rBC mass spectral signals (C+1 to C+5) while the organic signals fall into a few major clusters, identified as hydrocarbon-like organic aerosol (HOA), oxygenated organic aerosol (OOA), and cooking emission organic aerosol (COA). A nearly external mixing is observed with small BC particles only thinly coated by HOA ( 28% by mass on average), while over 90% of the HOA-rich particles did not contain detectable amounts of rBC. Most of the particles classified into other inorganic and organic clusters were not significantly associated with BC. The single particle results also suggest that HOA and COA emitted from anthropogenic sources were likely major contributors to organic-rich particles with low to mid-range aerodynamic diameter (dva). The similar temporal profiles and mass spectral features of the organic clusters and the factors from a positive matrix factorization (PMF) analysis of the ensemble aerosol dataset validate the conventional interpretation of the PMF results.

Hydroxylamine-O-sulfonic acid as a new reducing agent for the formation of nearly monodisperse gold nanoparticles in water: synthesis, characterisation and bioconjugation

Gold nanoparticles (Au NPs) with diameters ranging between 15 and 150 nm have been synthesised in water. 15 and 30 nm Au NPs were obtained by the Turkevich and Frens method using sodium citrate as both a reducing and stabilising agent at high temperature (Au NPs-citrate), while 60, 90 and 150 nm Au NPs were formed using hydroxylamine-o-sulfonic acid (HOS) as a reducing agent for HAuCl4 at room temperature. This new method using HOS is an extension of the approaches previously reported for producing Au NPs with mean diameters above 40 nm by direct reduction. Functionalised polyethylene glycol-based thiol polymers were used to stabilise the pre-synthesised Au NPs. The nanoparticles obtained were characterised using uv-visible spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM). Further bioconjugation on 15, 30 and 90 nm PEGylated Au NPs were performed by grafting Bovine Serum Albumin, Transferrin and Apolipoprotein E (ApoE).

Optical properties of chiral thin films and microparticles

In this work I study the optical properties of helical particles and chiral sculptured thin films, using computational modeling (discrete dipole approximation, Berreman calculus), and experimental techniques (glancing angle deposition, ellipsometry, scatterometry, and non-linear optical measurements). The first part of this work focuses on linear optics, namely light scattering from helical microparticles. I study the influence of structural parameters and orientation on the optical properties of particles: circular dichroism (CD) and optical rotation (OR), and show that as a consequence of random orientation, CD and OR can have the opposite sign, compared to that of the oriented particle, potentially resulting in ambiguity of measurement interpretation. Additionally, particles in random orientation scatter light with circular and elliptical polarization states, which implies that in order to study multiple scattering from randomly oriented chiral particles, the polarization state of light cannot be disregarded. To perform experiments and attempt to produce particles, a newly constructed multi stage thin film coating chamber is calibrated. It enables the simultaneous fabrication of multiple sculptured thin film coatings, each with different structure. With it I successfully produce helical thin film coatings with Ti and TiO_{2}. The second part of this work focuses on non-linear optics, with special emphasis on second-harmonic generation. The scientific literature shows extensive experimental and theoretical work on second harmonic generation from chiral thin films. Such films are expected to always show this non-linear effect, due to their lack of inversion symmetry. However no experimental studies report non-linear response of chiral sculptured thin films. In this work I grow films suitable for a second harmonic generation experiment, and report the first measurements of non-linear response.

Competing anisotropies in exchange biased nano-structured thin films

The magnetic anisotropies of a patterned, exchange biased Fe50Mn50/Ni80Fe20 system are studied using ferromagnetic resonance, supplemented by Brillouin light scattering experiments and Kerr magnetometry. The exchange biased bi-layer is partially etched into an antidot geometry so that the system approximates a Ni80 Fe20 layer in contact with antidot structured Fe50 Mn50 . Brillouin light scattering measurements of the spin wave frequency dependence on the wave vector reveal a magnonic band gap as expected for a periodic modulation of the magnetic properties. Analysis of the ferromagnetic resonance spectra reveals 8-fold and 4-fold contributions to the magnetic anisotropy. Additionally, the antidot patterning decreases the magnitude of the exchange bias and modifies strongly its angular dependence. Softening of all resonance modes is most pronounced for the applied magnetic field aligned within 10◦ of the antidot axis, in the direction of the bias. Given the degree to which one can tailor the ground state, the resulting asymmetry at low frequencies could make this an interesting candidate for applications such as selective/directional microwave filtering and multi-state magnetic logic.

Endonuclease Controlled Aggregation of Gold Nanoparticles for the Ultrasensitive Detection of Pathogenic Bacterial DNA

The development of an ultrasensitive biosensor for the low-cost and on-site detection of pathogenic DNA could transform detection capabilities within food safety, environmental monitoring and clinical diagnosis. Herein, we present an innovative approach exploiting endonuclease-controlled aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. The method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA. Once formed, the DNA-RNA heteroduplex is susceptible to RNAse H enzymatic cleavage of the RNA probe, allowing the target DNA to liberate and hybridize with another RNA probe. This continuously happens until all of the RNA probes are cleaved, leaving the nanoparticles unprotected and thus aggregated upon exposure to a high electrolytic medium. The assay is ultrasensitive, allowing the detection of target DNA at femtomolar level by simple spectroscopic analysis (40.7 fM and 2.45 fM as measured by UV-vis and dynamic light scattering (DLS), respectively). The target DNA spiked food matrix (chicken meat) is also successfully detected at a concentration of 1.2 pM (by UV-vis) or 18.0 fM (by DLS). In addition to the ultra-high sensitivity, the total analysis time of the assay is less than 3 hours, thus demonstrating its practicality for food analysis.

Storage stability of bevacizumab in polycarbonate and polypropylene syringes

Purpose To compare and examine the storage stability of compounded bevacizumab in polycarbonate (PC) and polypropylene (PP) syringes over a 6-month period. PC syringes have been used in a recent clinical study and bevacizumab stability has not been reported for this type of syringe. Methods Repackaged bevacizumab was obtained from Moorfields Pharmaceuticals in polycarbonate (PC) and polypropylene (PP) syringes. Bevacizumab from the stored syringes was analysed at monthly time points for a 6-month period and compared with bevacizumab from a freshly opened vial at each time point. SDS-PAGE electrophoresis and size-exclusion chromatography (SEC) was used to observe aggregation and degradation. Dynamic light scattering (DLS) provided information about the hydrodynamic size and particle size distribution of bevacizumab in solution. VEGF binding and the active concentration of bevacizumab was determined by surface plasmon resonance (SPR) using Biacore. Results SDS-PAGE and SEC analysis did not show any changes in the presence of higher molecular species (HMWS) or degradation products in PC and PP syringes from T0 to T6 compared to bevacizumab sampled from a freshly opened vial. The hydrodynamic diameter of bevacizumab in the PC syringe after six months of storage was not significantly different to bevacizumab taken from a freshly opened vial. Using SPR, the VEGF binding activity of bevacizumab in the PC syringe was comparable with bevacizumab taken from a freshly opened vial. Conclusion No significant difference over a 6-month period was observed in the quality of bevacizumab repackaged into prefilled PC polycarbonate and PP polypropylene syringes when compared to bevacizumab that is supplied from the vial.

Shapes and Dynamics of Blood Cells in Poiseuille and Shear Flows

The dynamics, shape, deformation, and orientation of red blood cells in microcirculation affect the rheology, flow resistance and transport properties of whole blood. This leads to important correlations of cellular and continuum scales. Furthermore, the dynamics of RBCs subject to different flow conditions and vessel geometries is relevant for both fundamental research and biomedical applications (e.g drug delivery). In this thesis, the behaviour of RBCs is investigated for different flow conditions via computer simulations. We use a combination of two mesoscopic particle-based simulation techniques, dissipative particle dynamics and smoothed dissipative particle dynamics. We focus on the microcapillary scale of several μm. At this scale, blood cannot be considered at the continuum but has to be studied at the cellular level. The connection between cellular motion and overall blood rheology will be investigated. Red blood cells are modelled as viscoelastic objects interacting hydrodynamically with a viscous fluid environment. The properties of the membrane, such as resistance against bending or shearing, are set to correspond to experimental values. Furthermore, thermal fluctuations are considered via random forces. Analyses corresponding to light scattering measurements are performed in order to compare to experiments and suggest for which situations this method is suitable. Static light scattering by red blood cells characterises their shape and allows comparison to objects such as spheres or cylinders, whose scattering signals have analytical solutions, in contrast to those of red blood cells. Dynamic light scattering by red blood cells is studied concerning its suitability to detect and analyse motion, deformation and membrane fluctuations. Dynamic light scattering analysis is performed for both diffusing and flowing cells. We find that scattering signals depend on various cell properties, thus allowing to distinguish different cells. The scattering of diffusing cells allows to draw conclusions on their bending rigidity via the effective diffusion coefficient. The scattering of flowing cells allows to draw conclusions on the shear rate via the scattering amplitude correlation. In flow, a RBC shows different shapes and dynamic states, depending on conditions such as confinement, physiological/pathological state and cell age. Here, two essential flow conditions are studied: simple shear flow and tube flow. Simple shear flow as a basic flow condition is part of any more complex flow. The velocity profile is linear and shear stress is homogeneous. In simple shear flow, we find a sequence of different cell shapes by increasing the shear rate. With increasing shear rate, we find rolling cells with cup shapes, trilobe shapes and quadrulobe shapes. This agrees with recent experiments. Furthermore, the impact of the initial orientation on the dynamics is studied. To study crowding and collective effects, systems with higher haematocrit are set up. Tube flow is an idealised model for the flow through cylindric microvessels. Without cell, a parabolic flow profile prevails. A single red blood cell is placed into the tube and subject to a Poiseuille profile. In tube flow, we find different cell shapes and dynamics depending on confinement, shear rate and cell properties. For strong confinements and high shear rates, we find parachute-like shapes. Although not perfectly symmetric, they are adjusted to the flow profile and maintain a stationary shape and orientation. For weak confinements and low shear rates, we find tumbling slippers that rotate and moderately change their shape. For weak confinements and high shear rates, we find tank-treading slippers that oscillate in a limited range of inclination angles and strongly change their shape. For the lowest shear rates, we find cells performing a snaking motion. Due to cell properties and resultant deformations, all shapes differ from hitherto descriptions, such as steady tank-treading or symmetric parachutes. We introduce phase diagrams to identify flow regimes for the different shapes and dynamics. Changing cell properties, the regime borders in the phase diagrams change. In both flow types, both the viscosity contrast and the choice of stress-free shape are important. For in vitro experiments, the solvent viscosity has often been higher than the cytosol viscosity, leading to a different pattern of dynamics, such as steady tank-treading. The stress-free state of a RBC, which is the state at zero shear stress, is still controversial, and computer simulations enable direct comparisons of possible candidates in equivalent flow conditions.