Dynamics of nanoparticles in complex fluids.

Soft matter is a subfield of condensed matter including polymers, colloidal dispersions, surfactants, and liquid crystals. These materials are familiar from our everyday life- glues, paints, soaps, and plastics are examples of soft materials. Many phenomena in these systems have the same underlying physical mechanics. Moreover, it has been recognized that combinations of these systems, like for example polymers and colloids, exhibit new properties which are found in each system separately. These mixed systems have a higher degree of complexity than the separate systems. In order to understand their behavior, knowledge from each subfields of soft matter has to be put together. One of these complex systems is the mixture of nanoparticles with macromolecules such as polymers, proteins, etc. Understanding the interactions in these systems is essential for solving various problems in technological and medical fields, such as developing high performance polymeric materials, chromatography, and drug delivery vehicles. The author of this dissertation investigates fundemental soft matter systems, including colloid dispersions in polymer solutions and binary mixture. The diffusion of gold nanoparticles in semidilute and entangled solutions of polystyrene (PS) in toluene were studied using fluctuation correlation spectroscopy (FCS). In our experiments, the particle radius (R ≈ 2.5 nm) was much smaller compared to the radius of gyration of the chain but comparable to the average mesh size of the fluctuating polymer network. The diffusion coefficient (D) of the particles decreased monotonically with polymer concentration and it can be fitted with a stretched exponential function. At high concentration of the polymer, a clear subdiffusive motion of the particles was observed. The results were compared with the diffusion of free dyes, which showed normal diffusive behavior for all concentrations. In another polymer solution, Poly ethylene glycol (PEG) in water, the diffusion of the gold nanoparticles depends on the dimentionlesss length scale R/ξ, where R is the radius of the nanoparticle and ξ is the average mesh size of the fluctuating polymer network. FCS were used to study the critical adsorption on curved surfaces by utilizing spherical nanoparticles immersed in a critical binary liquid mixture of 2,6 lutidine + water. The temperature dependence of the adsorbed film thickness and excess adsorption was determined from FCS measurements of the enlarged effective hydrodynamic radius of the particles. Our results indicated that the adsorbed film thickness is of the order of correlation length associated with concentration fluctuations. The excess adsorption per unit area increases following a power law in reduced temperature with an exponent of -1, which is the mean-field value for the bulk susceptibility exponent. The kinetics of adsorption of gold nanoparticles in polymer solutions on silicon substrate was studied using ellipsometry by measuring the thickness of the adsorbed layer versus time. The data was fitted using exponential growth with relaxation time constants proportional to the diffusion of the gold nanoparticles in polymer solution

Development of Optical Biosensors Based on Metal Nanostructures for Pollution (Mycotoxins) Detection

This work aims to develop optical biosensors for detection of the bio-toxins, particularly mycotoxins. The main detection technology chosen is a combination of a localized surface plasmon resonance (LSPR) transducer with direct immunoassay with specific bio-receptors antibodies or aptamers immobilized on the gold surface. The LSPR platform is based on gold nano-islands produced by thermal annealing of thin gold films deposited on glass. Thermal annealing was substituted with much quicker and more efficient microwave annealing in later stages of the project. The gold nano-structures produced were analyzed with scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD), optical absorption spectroscopy, and spectroscopic ellipsometry. The method of total internal reflection ellipsometry (TIRE) was chosen for LSPR bio-sensing because of its superior sensitivity as compared to conventional UV-vis absorption spectroscopy. The TIRE spectroscopic measurements were performed using the experimental set-up developed on the basis of J.A. Woollam M2000 spectroscopic instrument. The sensitivity of LSPR measurements is however limited by a finite evanescent field decay length in gold nano-islands which is typically in the range of tens of nanometres. In order to achieve the best results, the bio-receptors must be small and placed as close to the gold surface as possible. In our case the problem was solved using either halved antibodies or aptamers immobilized covalently on the surface of gold. A series of bio-sensing experiments to detect mycotoxins, i. e. aflatoxin B1, M1, zearalenone, and ochratoxin A, went successfully and resulted in the detection of the above mycotoxins in concentrations down to 0.01ng/mL. In this work we attempted, for the first time, the TIRE detection of Aflatoxin B1, M1 and ochratoxin A (OTA) in an assay with a specific aptamer. We compared the results obtained for the detection of mycotoxins using bio-receptors of different dimensions: large-size whole antibodies electrostatically immobilized on the surface of gold nano-islands, and small-size split antibodies or specific aptamers immobilised via thiol (SH) groups. For small-size receptors, the low detection limit (LDL) was 0.01ng/ml which is one order of magnitude lower than for whole antibodies. The results obtained demonstrate the advantages of using small bio-receptors in LSPR bio-sensing. The minimal detected concentration OTA was 0.01ng/ml, which is a remarkable result for direct aptamer assay format. The mycotoxin/aptamer binding kinetics were analysed using dynamic TIRE measurements and yeilded an association constant KA in the range of 107 Mol-1 which confirmed the high specificity of aptamer. An attempt to make gold nanostructures for SERS biosensing using nanosphere lithography was successful

FABRICATION AND CHARACTERIZATION OF MESOSCALE PROTEIN PATTERNS USING ATOMIC FORCE MICROSCOPY (AFM)

A versatile AFM local oxidation lithography was developed for fabricating clean protein patterns ranging from nanometer to sub-millimeter scale on octadecyltrichlorosilane (OTS) layer of Si (100) wafer. This protein patterning method can generate bio-active protein pattern with a clean background without the need of the anti-fouling the surface or repetitive rinsing. As a model system, lysozyme protein patterns were investigated through their binding reactions with antibodies and aptamers by AFM. Polyclonal anti-lysozyme antibodies and anti-lysozyme aptamer are found to preferentially bind to the lysozyme molecules on the edge of a protein pattern before their binding to the interior ones. It was also demonstrated that the topography of the immobilized protein pattern affects the antibody binding direction. We found that the anti-lysozyme antibodies binding to the edge lysozyme molecules on the half-buried pattern started from the top but the binding on the extruded pattern started from the side because of their different spatial accessibility. In addition, after incubating lysozyme pattern with anti-lysozyme aptamer in buffer solution for enough long time, some fractal-shaped aptamer fibers with 1-6nm high and up to tens of micrometers long were formed by the self-assembling of aptamer molecules on the surface. The aptamer fibers anchor specifically on the edge of protein patterns, which originates from the biospecific recognition between the aptamer and its target protein. Once these edge-bound fibers have formed, they can serve as scaffolds for further assembly processes. We used these aptamer fibers as templates to fabricate palladium and streptavidin nanowires, which anchored on the pattern edges and never cross over or collapse over each other. The aptamer fiber scaffold potentially can lead to an effective means to fabricate and interface nanowires to existing surface patterns

Physical properties of the plasma membrane studied by local probe techniques

Im Rahmen dieser Arbeit wurden physikalische Meßmethoden entwickelt, um die Struktur und Dynamik der Plasmamembran lebender Säugetierzellen zu untersuchen. Dabei lag der Schwerpunkt zum einem auf der mechanischen Kopplung der Membranlipidschicht zu dem darunter liegenden, unterstützend wirkenden Zytoskelett, und zum anderen auf der Beweglichkeit einzelner Membrankomponenten innnerhalb der Membranlipidschicht. Ausgangspunkte für die Entwicklung neuer Methoden waren die Rasterkraftmikroskopie (RKM) und ´single-particle tracking’ (SPT). Die RKMermöglicht die Abbildung von Ober‡ächen mit hoher Au‡ösung in physiologischer Umgebung, so lange die Wechselwirkungskräfte die Probenober‡äche nicht zu sehr deformieren. SPT ist eine Technik, um die Bewegung von einzelnen Molekülen zu verfolgen. Dafür werden diese mit Polysteren-kügelchen oder kolloidem Gold markiert und mit Videomikroskopie beobachtet. Auf diesen Techniken aufbauend wurden folgende Verfahren und Geräte für diese Arbeit entwickelt: Das Scanning-Photonic Force Microscope (SPFM) als Analogon zum RKM: In einer Laserfalle (einem mit einem Objektiv hoher Apperatur (100x, NA 1.3) fokussierten infraroten (IR) Laser) wird ein Fluorophor gefülltes Polysterenkügelchen (bead, r=0.1¹m) gefangen. Die Fluorophore des als Sonde verwendeten Kügelchens werden über einen Zwei-Photonen Prozeß durch den IR-Laser zur Fluoreszenz angeregt. Eine Auslenkung aus der Halteposition durch eine äußere Kraft führt zum Abfall der Fluoreszenz. Im SPFM wird die Sonde mit Hilfe des Lasers analog zum mechanischen Hebelarm im RKM über die Probe bewegt. Dabei wird wie beim RKM die Auslenkung und somit Kraft gemessen. Die wesentlichen Unterschiede sind die fehlende mechanische Verbindung zur Umwelt, die ein Abrastern beliebiger transparenter 3D-Strukturen ermöglicht, die extrem kleine Federkonstante des Sensors (0.1- 1¹N/m), welche daher besser an die Elastiztät von biologischen Objekten angepaßt ist, sowie die stark reduzierte viskose Dämpfung der kleineren Sonde, was schnelleres Messen ermöglicht. Das Photonic Force Microscope (PFM) ist die Weiterentwicklung des SPFM. Bei der verwendeten kleinen Federkonstante werden die thermischen Positions‡ uktuationen sehr groß. Daher wurde ein Detektor entwickelt, der Messung der Position der Sonde in der Falle in drei Dimensionen mit Nanometer Ortsau‡ösung und 50kHz-Bandbreite ermöglicht. Der Detektor basiert auf der Interferenz des an der Sonde vorwärts gestreuten Laserlichts mit dem ungestreuten Licht. Dieses Prinzip wurde zuvor nur für seitliche Auslenkungen entlang einer Achse verwendet und erstmals in dieser Arbeit drei dimensional angewandt. Die vollständige theoretische Beschriebung des Detektorsignals ist ebenfalls ein Novum. Im PFM werden mit diesem Detektor die thermischen 3D-Positions‡uktuationen einer durch den Laser in einem Volumen von 0.1*0.1*0.6¹m3 gehaltenen Sonde analysiert. Zusätzlich wirkende Potentiale und die Viskosität des umgebenden Mediums können gemessen werden