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.
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