| Abstract |
Nanoparticles are the materials with at least two dimensions between 1 and 100
nm. These particles classified in two main categories: the natural products (e.g.
Macromolecules, Bio-colloids) and the artificially synthesized (engineered nanoparticles).
Nanoparticles, have elicited much interest since can be used in numerous applications
because of their unique physical, chemical and biological properties. These materials can be
characterized using various techniques including Electron Microscopy, Size Exclusion
Chromatography, X-ray crystallography and Nuclear Magnetic Resonance spectroscopy.
Contrary to the wide variety of available techniques, the need for fast, label free and
statistical strong analysis remains. Over the past decade considerable progress has been
made in the development of such analytical techniques. One of these, involves the use of
nanoelectrospray (nES) coupled with differential ion mobility spectrometry (IMS) coupled
on-line with condensation particle counting (CPC). In several of the relevant publications the
term GEMMA (gas-phase electrophoretic mobility molecular analyzer) is used instead of
IMS. GEMMA has been successfully used to determine the size and Relative Molecular Mass
of macromolecules of biological origin and a plurality of inorganic engineered nanoparticles.
The focus of this work was the evaluation and the development of GEMMA for the
determination of the size and elemental composition of Nanoparticles. To achieve this wide
number of biological samples (protein, protein complexes) prepared in-house using routine
biochemical isolation and purification techniques has been analyzed. Results were
compared with two hydrodynamic approaches quasi elastic (QELS) and multi-angle (MALLS)
laser light scattering and an established MS technique (LTQ-Orbitrap), in terms of sensitivity,
accuracy, reproducibility, speed, maintaining intact oligomeric assemblies and other
measurement characteristics. Furthermore, GEMMA has been used for the determination of
the oligomeric state of protein with different point mutations. DNA samples are also
analyzed. The most challenging project of this work was the development of theoretical
model for the determination of the conformational changes of the covalent protein complex
GFP-GBP-YFP (Fluo) in addition of substrate.
Inorganic nanoparticles of known size, shape and agglomeration are used to
evaluate the techniques accuracy for characterizing particles. The study extended to
laboratory synthesized nanoparticles (Carbogenic Quantum Dots) and the results compared
with TEM measures.
Improving of GEMMA was one of the main objectives of this project. The major
disadvantage of this technique is the lack of chemical information that is provided by the
CPC detector. This extremely sensitive detector does not provide information regarding the
elemental composition of the Nanoparticles detected. To overcome this shortcoming
coupling with other detectors was tried. The off-line combination of GEMMA with ICP-MS
(operate in the single particle mode) for the characterization of engineered nanoparticles in
aqueous solution was achieved. The resulting off-line analytical technique exhibits potential
for determining nanoparticle concentration, size and metal content. The efforts were
focused, also, in the optimization of GEMMA operating characteristics by adjusting of various capillaries in the electrospray unit. The main axes of this thesis can be summarized as: 1) evaluation of GEMMA for
determining the properties (size, relative molecular mass, oligomerization, structure) of
biomolecules and inorganic nanoparticles, 2) technique improvement (coupling with other
detectors, changes in instrumentation) in order to eliminate the drawbacks and expand the
analytical capabilities.
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Ανάπτυξη αναλυτικών μεθόδων φασματομετρίας κινητικότητας ιόντων για τον προσδιορισμό μεγέθους και σύστασης νανοσωματιδίων
