| Abstract |
In order to gain a better insight into the metallome of a biological organism and investigate the impact of changes in the concentration levels of elements on biochemical processes taking place in a given biological organism, it is important to quantitate metals in individual cells rather than at the level of a tissue, consisting of different types of cells. The conventional method for quantitating the metal content of a biological sample by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is by digesting an ensemble of cells and analyzing the resulting digest in order to determine the average metal mass per cell. However, the given average value gives us only half the picture, as the average metal content may not reflect a possible metal content variation.
The development of the method for characterizing metallic nanoparticles through the use of the ICP-MS (SP ICP-MS) set the foundation for the development of a method for determining metals at the single cell level through the use of the ICP-MS (SC ICP-MS). The operation of SC ICP-MS involves the introduction of dilute cell suspensions, that is 104 -105 cells/mL, via pneumatic aspiration into a high-temperature Ar plasma. Upon entering the plasma, each aerosol droplet that contains a cell is evaporated and the remaining single cell is atomized into its constituent elements. The resulting atoms and their isotopes are then ionized, separated by the mass analyzer and detected.
The number of cells entering the plasma per unit of time, depending on the transport efficiency of the introduction system and the cell number density in the suspension, and the fast (μs scale) data acquisition are fundamental goals for the successive application of SC ICP-MS. The fast data acquisition is needed because a cell signal lasts between 150-500 μs. Furthermore, cells should keep their integrity throughout their transport to the plasma. This work demonstrates the development and application of SC ICP-MS to the study of heavy metal, such as lead (Pb), cadmium (Cd) and arsenic (As) in the form of arsenous (AsO33-) and arsenic (AsO43-) anions, uptake by Chlamydomonas reinhardtii cells upon incubation in concentrations of the given metals, as well as to the determination of endogenous metals such as calcium (Ca). A High Efficiency Introduction System (HEN Introduction System) is assembled for the purpose of SC ICP-MS, and the SC ICP-MS results taken with the two Introduction Systems are compared. Histograms depicting the metal amount per cell show that the mean Cd and As uptake is determined at 1-3 fg while the mean Pb uptake is determined at 12-17 fg for cells incubated in the highest metal concentrations. Results emerging from the SC ICP-MS analysis of the cells following their treatment with ethylenediaminotetraacetic acid (EDTA) suggest that Ca is located on the cell wall and is affected by the concentration of the existing dissolved Ca outside the cell. The SC ICP-MS technique demonstrates great sensitivity, as it is in the position to measure ag amounts of metals in single cells. SC ICP-MS is compared with Conventional ICP-MS through mass balance calculations, and the agreement of the two techniques demonstrates the reliability of SC ICP-MS.
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Ανάπτυξη μεθόδου φασματομετρίας μάζας επαγωγικά συζευγμένου πλάσματος για τον προσδιορισμό μετάλλων σε μεμονωμένα κύτταρα με σκοπό τη μελέτη της πρόσληψης βαρέων μετάλλων (Pb, Cd, As) από τα Chlamydomonas reinhardtii κύτταρα
