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Κωδικός Πόρου 000455773
Τίτλος Development of novel atomic mass spectrometric techniques for the analysis of single particles and single cells
Άλλος τίτλος Ανάπτυξη νέων τεχνικών ατομικής φασματομετρίας μάζας για την ανάλυση διακριτών σωματιδίων και κυττάρων
Συγγραφέας Μαυράκης, Εμμανουήλ Α
Μέλος κριτικής επιτροπής Λυδάκης – Σημαντήρης Νικόλαος
Άγγλος, Δημήτριος
Τσιώτης, Γεώργιος
Bettmer, Jorg
Σπύρος, Απόστολος
Χατζηιωάννου, Χρυσοβαλάντου-Αναστασία
Σύμβουλος διατριβής Περγαντής, Σπυρίδων
Περίληψη In response to the need for performing metal determination at the level of single particles and single cells, this dissertation reports on the development of advanced methods based on inductively-coupled plasma mass spectrometry (ICP-MS). Due to the evident advantages of microfluidics in manipulating volume-limited biological samples, including single cells/particles, emphasis is given on pairing microfluidic chips to ICP-MS. Structurally, this dissertation is comprised of 5 studies. The first study concerns the development and application of single-cell (SC)-ICP-MS to determine the uptake of arsenate by individual C. reinhardtii cells, exposed to 12.5, 22.5 and 30 μg As mL-1. Compared to conventional analytical schemes, SC-ICP-MS revealed the distribution of As among single cells. C. reinhardtii demonstrated heterogeneity with respect to As uptake, as evidenced by the broad As mass histograms that were fitted with a lognormal probability function. All exposure concentrations exhibited a similar most frequent As mass of 1.5-1.8 fg cell-1, indicating a saturation mass reached by the majority of the cell population, while the lognormal mean As mass of 2.7-4.1 fg cell-1 indicated that a significant portion of the cell population internalized higher amounts of arsenate. A significant cellular As amount was wall-bound, as evidenced by a 30% drop in the cellular As of washed cells. The second study reports on the development of a data processing method (DP) that can be employed to analyze single nanoparticles (NPs) using single-particle (sp)-ICP-MS by means of μs dwell times. Herein, the developed (A) DP-75μs-sp-ICP-MS is validated through comparisons with the benchmark (B) DP-5σ-10ms-Syngistix™ Nano method, as well as by conducting silver (Ag) mass recovery experiments in seawater samples. Analysis of a reference 60 nm AgNP suspension showed excellent agreement between A and B in terms of NP peak area (38.8 and 37 counts for A and B, respectively) and transport efficiency (2.3 and 2.1 % for A and B, respectively). Analysis of seawater mesocosm tanks, spiked with 60 nm AgNPs, across a concentration range of 50-500 ng L-1, yielded Ag mass recoveries of 83 ± 22% and 86 ± 21% for A and B, respectively. The applicability of DP-μs-sp-ICP-MS was demonstrated in a seawater mesocosm experiment, where Ag-containing NPs could be tracked at the early stages of formation. In a third study, coupling of a microfluidic chip to ICP-MS was accomplished using a combination of commercially available components, including a pneumatic high-efficiency nebulizer and a spray chamber designed to allow for the addition of a laminar flow makeup gas. To demonstrate the efficiency of this coupling, standard dilution analysis (SDA) was employed for the first time for chip-based ICP-MS. High average recoveries (97.4-100.1%) and low average relative standard deviations (2.9-4.8%) were achieved for the determined elements (Cd, Co, Pb, Cr) across several spiked matrices and certified reference materials, whereas only 140 μL of sample is required for SDA in triplicate or 40 μL for a single analysis. The fourth study demonstrates, for the first time, the direct coupling of a chip-based supersonic microfluidic nebulizer (chip-μf-Neb) to ICP-MS. The system exhibited efficient operation at liquid flow rates as low as 0.5 μL min-1, with minimum dead volumes, while sensitive metal isotope detection was evidenced by an attained indium (In) sensitivity of 40000 cps (μg L-1)-1 at 10 μL min-1. The system featured a transport efficiency of 46% for Ag nanoparticles. Finally, the capabilities for conducting single-cell analysis were demonstrated with the detection of 80Se, 75As and 31P16O in single Chlamydomas reinhardtii cells. The fifth study reports on the use of inertial microfluidics for cell focusing and size-based sorting of cell populations prior to SC-ICP-MS detection. Microfluidic chips with spiral channels were operated at a range of Reynolds and Dean numbers, while multiple outlet channels allowed for collection of cell-enriched and cell-free samples. A high degree of cell focusing was achieved for C. reinhardtii, as 80% of the infused cells were collected from a single outlet. Individual sorting of BMDMS cells (diameter of 12 μm) and C. reinhardtii cells (diameter of 7 μm) at a De of 69.9 indicated that the 2 cell lines could be collected with 100% and 70% purity, respectively, when present in the same cell suspension; thus, equipping SC-ICP-MS with flow cytometry capabilities.
Φυσική περιγραφή 229 σ. : πίν., σχήμ., εικ. (μερ. εγχρ.) ; 30 εκ.
Γλώσσα Αγγλικά
Θέμα Cell sorting
Chip-based ICP-MS
Inductively-coupled plasma mass spectrometry (ICP-MS)
Inertial microfluidics
Microfluidics
SC-ICP-MS
Single-cell analysis
Single-particle analysis
Spiral channels
Standard dilution analysis
Supersonic microfluidic nebulizer
sp-ICP-MS
μs-sp-ICP-MS
Ημερομηνία έκδοσης 2023-06-30
Συλλογή   Σχολή/Τμήμα--Σχολή Θετικών και Τεχνολογικών Επιστημών--Τμήμα Χημείας--Διδακτορικές διατριβές
  Τύπος Εργασίας--Διδακτορικές διατριβές
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