ΣΧΟΛΗ ΘΕΤΙΚΩΝ ΚΑΙ ΤΕΧΝΟΛΟΓΙΚΩΝ ΕΠΙΣΤΗΜΩΝ

TMHMA ΧΗΜΕΙΑΣ

Υποψήφιος Διδάκτωρ

Καπέλλιος Ευθύμιος

Τίτλος Διδακτορικής Διατριβής

Ανάπτυξη Αναλυτικών Μεθόδων Φασματομετρίας Κινητικότητας Ιόντων για τον Προσδιορισμό Μεγέθους και Σύστασης Νανοσωματιδίων

Επιβλέποντες

Επιβλέπων Καθηγητής : Περγαντής Σπυρίδων

Μέλη Επιτροπής : 

(1) Περγαντής Σπυρίδων

(2) Στεφάνου Ευριπίδης

(3) Αναστασιάδης Σπύρος

Στοιχεία Διατριβής

Έναρξη Διδακτορικής Έρευνας : 12/02/2009

Ημερομηνία Υποστήριξης Διδακτορικής Έρευνας : 30/04/2013

Εγκεκριμένος Π/Υ: 29.375

Περίληψη

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 characterization of the size, relative molecular weight, oligomeric state and structure of biomolecules. 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. Fluo operates as glucose biosensor. The model predicts the structure combining the experimentally measured spherically equivalent size (electrophoretic mobility diameter) and the calculated geometrical projected areas, Ai, of the protein complex. 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, also, focused in the reduction of “nES-induced” oligomers which formed as a result of the nES-GEMMA process. GEMMA analysis is based on measuring the electrophoretic mobility of singly-ionized particles in air. Thus, a bipolar ion source (Po-210) is used in order to neutralize the charge of the droplets produced by nES. The neutralizer stops the fission process at an early stage leading to larger final droplet sizes. Droplets containing more than one protein molecule will result in oligomer formation upon droplet drying according to the Charge Residue Model (CRM). The formation of nEs-induced oligomers has been described by statistical model and depends on the concentration and the initial droplet diameter. The purpose of this study was a thorough experimental investigation of the factors that contribute to the formation of non-specific “nES-induced” oligomers and subsequently the reduction of them. For this reason capillaries with different characteristics (e.g inner diameter, tip geometry: cone, plain) were adjusted in the electrospray unit. The primary droplet size distribution for various flow rates and for solutions with different conductivity was determined. A systematic analysis demonstrated, also, the effect of physicochemical properties of proteins to the formation of nES-induced oligomers. In addition, significant indications for the mechanisms involved in the nES of protein and protein complexes were observed. The main axes of 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.

Λέξεις κλειδιά

Φασματομετρία, Κινητικότητα Ιόντων, νανοσωματίδια, ionmobilityspectrometry, ΙΜS, νανο-ηλεκτροψεκασμός, nanoelectrospray, nES, Φασματομετρία Κινητικότητας Ιόντων (IMS), Φασματομετρία Μάζας Επαγωγικά Συζευγμένου Πλάσματος (ICP-MS), Βιοαναλυτικές Μέθοδοι, Χαρακτηρισμός Νανοσωματίδιων και Βιομορίων, ανάπτυξη τεχνικής ή Ion mobility spectrometry (IMS), Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Nanoparticles, Biomolecules, Bioanalytical methods, Nanoparticle and Biomolecule characterization, method development.

Παραδοτέα - Δημοσιεύσεις

1) Kapellios, E. A., Karamanou, S., Sardis, M. F., Aivaliotis, M., Economou, A. and Pergantis, S. A. (2011)  Using Nanoelectrospray Ion Mobility Spectrometry (GEMMA) to Determine the Size and Relative Molecular Mass of Proteins and Protein Assemblies:  A Comparison with MALLS and QELS.                                                                                                        Analytical and Bioanalytical Chemistry 399, 2421-33

2) Kapellios, E. A., Karamanou, S., Sardis, M. F., Aivaliotis, M., Economou A. and Pergantis S. A. (2010) Using Nanoelectrospray Ion Mobility Spectrometry (GEMMA) to Determine the Size and Relative Molecular Mass of Proteins and Protein Assemblies: A Comparison with MALLS and QELS.

1st International Proteomics Conference on Crete- IPCC01 (October 7-8, 2010)

3) Kapellios, E. A., Karamanou, S., Sardis, M. F., Papanastasiou, M., Aivaliotis, M., Pergantis, S. A. and Economou, A. (2011) A Multiplexed Approach for the Determination of Intact Protein Mass, Dimension and Oligomerization Using Nanoelectrospray Ion Mobility Spectrometry (GEMMA), LTQ-Orbitrap MS, MALLS and QELS.

59th ASMS conference on Mass Spectrometry (June 5-9, 2011), on Denver, Colorado

4) Ε. Α. Καπέλλιος, Σ. Α. Περγαντής, A. Οικονόμου (2011) Ανάπτυξη αναλυτικών μεθόδων φαμσατομετρίας κινητικότητας ιόντων για τον προσδιορισμό μεγέθους και σύστασης νανοσωματιδίων, 17ο Συνέδριο Μεταπτυχιακών Φοιτητών Χημείας, Ηράκλειο, Κρήτη, (15-18 Ιουλίου 2011).

5) Efthymios A. Kapellios and Spiros A. Pergantis: “Size and elemental composition of nanoparticles using ion mobility spectrometry with inductively coupled plasma mass spectrometry”. J. Anal. At. Spectrom., 2012, 27, 21-24 (Featured on Journal Inside Cover-2012's Top 25 most read JAAS articles) 

6) Τhe 60th American Society for Mass Spectrometry (ASMS) Conference on Mass Spectrometry, Vancouver, BC, Canada, May 20-24, 2012: “Evaluating Novel Nebulizer Configurations for Sonic Spray Ionization Mass Spectrometry” M. M. Antonakis, A. Tsirigotaki, E. Kapellios, S. A. Pergantis (Poster presentation).

7)  2012 Winter Conference on Plasma Spectrochemistry, Tucson, Arizona, January 9-14, 2012: “Hydrodynamic Liquid Chromatography and Ion Mobility Spectrometry with ICP-MS for Determining Nanoparticle Concentration, Size and Elemental Composition” S. A. Pergantis (Oral Presentation), E.A. Kapellios, T.L. Jones-Lepp, E. M.Heithmar

8) “Ανάπτυξη Αναλυτικών Μεθόδων Φασματομετρίας Κινητικότητας Ιόντων για τον Προσδιορισμό Μεγέθους και Σύστασης Νανοσωματιδίων” Παρουσίαση με πόστερ, Διημέρίδα Ηράκλειτος ΙΙ, Ηράκλειο, Κρήτη, 4-5 Δεκ. 2012.

9) Metal nanoparticles for advanced materials: From theory to practice, NanoTP Conference, 1-3 October 2012, Heraklion, Crete, Greece: “Current Analytical Strategies for Detecting Trace Amounts of Nanoparticles in the Environment” S.A. Pergantis (Oral Presentation), E.A. Kapellios