Chemische Charakterisierung und Spurenanalytik
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An overview about different analytical approaches will be presented of how to detect metals in individual biological cells by use of ICP-MS. For this purpose, we are using different sample introduction systems for ICP-MS for detection, imaging and quantification of metals at cellular levels.
By use of laser ablation, we have studied the up-take by and distribution of nanoparticles in single cells. Recently we have developed staining techniques to measure protein and DNA content of cells and identifying the cell status by immunoassays using metal-tagging of antibodies. New research based on cell arrays will be shortly discussed.
Using pneumatic nebulization and microdroplet generation, we have also studied the up-take of nanoparticles and toxic metals as well as essential elements in single cells using different ICP-MS mass spectrometric concepts (sector field instrument, triple-quad instrument, time of flight (CyTOF) instrument).
The different ICP-MS based methods will be compared concerning their analytical figures of merit and their strengths and weaknesses will be evaluated.
An overview about different analytical approaches will be presented of how to detect metals in individual biological cells by use of ICP-MS. For this purpose, we are using different sample introduction systems for ICP-MS for detection, imaging and quantification of metals at cellular levels.
By use of laser ablation, we have studied the up-take by and distribution of nanoparticles in single cells. Recently we have developed staining techniques to measure protein and DNA content of cells and identifying the cell status by immunoassays using metal-tagging of antibodies. New research based on cell arrays will be shortly discussed.
Using pneumatic nebulization and microdroplet generation, we have also studied the up-take of nanoparticles and toxic metals as well as essential elements in single cells using different ICP-MS mass spectrometric concepts (sector field instrument, triple-quad instrument, time of flight (CyTOF) instrument).
The different ICP-MS based methods will be compared concerning their analytical figures of merit and their strengths and weaknesses will be evaluated.
We are using LA-ICP-MS to quantify metals in biological cells and thin cuts of tissues from varies organs. Different applications will be presented to demonstrate the state of the art of bioimaging to visualize the elemental distribution pattern in soft bio-materials (tissue, single cells) of metals, metal containing stains and metal-tagged antibodies. For this purpose, different strategies for metal tagging will be presented and will be compared in terms of analytical figures of merit. First applications for detection of biomarkers in animal and human tissue samples will be presented.
In a first example, we have applied LA-ICP-MS to visualize the local distribution of proteins, which are used as bio-markers for neurodegenerative diseases. For this purpose, brain tissues from mice experiments have been stained by metal-tagged antibodies. House-keeping proteins have been investigated as internal cellular standards. Additionally, ink-jet printing of metal doped inks onto the surface of tissue samples has been applied for drift corrections and quantification. Validation of our results are achieved in comparison to immune-histochemical staining and optical microscopy.
In a second example, we used specific metal-tagged antibodies for detection of biomarker specific for prostate cancer. For this purpose, micro tissue arrays are incubated with metal-tagged antibodies for bioimaging of samples from many patients using simultaneous detection of all relevant biomarkers and their tags.
For improvement of sensitivity in the next example application nanoparticle tagged antibodies for detection of metallothionines in eye lens tissue samples will be discussed.
Recently we have used our tagging and staining strategies to determine the cell cycle of single cells, which is of future interest for toxicological studies.
Finally, future trends in elemental microscopy and mass cytometry imaging will be discussed.
An overview about different analytical approaches will be presented of how to detect metals in individual biological cells by use of ICP-MS. For this purpose, we are using different sample introduction systems for ICP-MS for detection, imaging and quantification of metals at cellular levels.
By use of laser ablation, we have studied the up-take by and distribution of nanoparticles in single cells. Recently we have developed staining techniques to measure protein and DNA content of cells and identifying the cell status by immunoassays using metal-tagging of antibodies. New research based on cell arrays will be shortly discussed.
Using pneumatic nebulization and microdroplet generation, we have also studied the up-take of nanoparticles and toxic metals as well as essential elements in single cells using different ICP-MS mass spectrometric concepts (sector field instrument, triple-quad instrument, time of flight (CyTOF) instrument).
The different ICP-MS based methods will be compared concerning their analytical figures of merit and their strengths and weaknesses will be evaluated.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been revealed as a convenient technique for trace elemental imaging in tissue sections, providing elemental 2D distribution at a quantitative level. For quantification purposes, in the last years several approaches have been proposed in the literature such as the use of CRMs or matrix matched standards. The use of Isotope Dilution (ID) for quantification by LA-ICP-MS has been also described, being mainly useful for bulk analysis but not feasible for spatial measurements so far. In this work, a quantification method based on ID analysis was developed by printing isotope-enriched inks onto kidney slices from rats treated with antitumoral Pt-based drugs using a commercial ink-jet device, in order to perform an elemental quantification in different areas from bio-images. For the ID experiments ¹⁹⁴Pt enriched platinum was used. The methodology was validated by deposition of natural Pt standard droplets with a known amount of Pt onto the surface of a control tissue, where could be quantified even 50 pg of Pt, with recoveries higher than 90%. The amount of Pt present in the whole kidney slices was quantified for cisplatin, carboplatin and oxaliplatin-treated rats. The results obtained were in accordance with those previously reported. The amount of Pt distributed between the medullar and cortical areas was also quantified, observing different behavior for the three drugs.
Quantification of metals in single cells by LA-ICP-MS comparison of single spot analysis and imaging
(2018)
LA-ICP-MS is increasingly used for single cell analysis in two different detection modes using either the imaging mode with subcellular resolution or alternatively single spot analysis of cells with a larger laser spot size. This study compares the analytical figures of merit of both detection modes (signal to noise, precision, accuracy, throughput), as well as ease of operation and data evaluation. Adherent 3T3 fibroblast cells were stained with two metal dyes (mDOTA-Ho, Ir-DNA-intercalator) and several dozen cells were measured using both modes. We found a ten times higher throughput for single spot analysis, which has as well a straightforward data analysis, shortening the total analysis time further. The signal to noise ratio for single spot analysis was found to be slightly better compared to the signal to noise of pixels in imaging. The mean metal intensity per single cell differed by only 10% between both modes and obtained distributions were found to show no statistically significant differences. Using matrix matched calibration based on standards spotted onto nitrocellulose membrane, we achieved detection limits (10 σ) of 12 fg for Ir and 30 fg for Ho and quantified 57 ± 35 fg Ir and 1,192 ± 707 fg Ho per single cell. Compared to a conventional ICP-MS measurement of a digest of ~60,000 cells, 54 % of Ir content and 358 % Ho content was found using quantitative LA-ICP-MS. The difference might be a consequence of the two metal dyes binding to different structures of the cell and therefore might behave differently in sample preparation for conventional and LA-ICP-MS.
LA-ICP-MS is increasingly used for single cell analysis in two different detection modes using either the imaging mode with subcellular resolution or alternatively single spot analysis of cells with a larger laser spot size. This study compares the analytical figures of merit of both detection modes (signal to noise, precision, accuracy, throughput), as well as ease of operation and data evaluation. Adherent 3T3 fibroblast cells were stained with two metal dyes (mDOTA-Ho, Ir-DNA-intercalator) and several dozen cells were measured using both modes. We found a ten times higher throughput for single spot analysis, which has as well a straightforward data analysis, shortening the total analysis time further. The signal to noise ratio for single spot analysis was found to be slightly better compared to the signal to noise of pixels in imaging. The mean metal intensity per single cell differed by only 10% between both modes and obtained distributions were found to show no statistically significant differences. Using matrix matched calibration based on standards spotted onto nitrocellulose membrane, we achieved detection limits (10s) of 12 fg for Ir and 30 fg for Ho and quantified 57 +/-35 fg Ir and 1192 +/- 707 fg Ho per single cell.
Compared to a conventional ICP-MS measurement of a digest of about 60000 cells, 54% of Ir content and 358% Ho content was found using quantitative LA-ICP-MS. The difference might be a consequence of the two metal dyes binding to different structures of the cell and therefore might behave differently in sample preparation for conventional and LA-ICP-MS.
Iron nanoparticles (NPs) metabolism is directly associated to human health due to their use as anemia treatment and should be studied in detail in cells. Here we present a speciation strategy for the determination of the metabolic products of iron oxide nanoparticles coated by tartaric and adipic acids in enterocytes-like cell models (Caco-2 and HT-29). Such methodology is based on the use of SDS-modified reversed phase high performance liquid chromatography (HPLC) separation using inductively coupled plasma-mass spectrometry (ICP-MS) as Fe selective detector. Post-column isotope dilution analysis is used as quantification tool by adding Fe-57 as isotopically enriched standard. To assess the separation capability of the method, two different iron nanostructures: iron sucrose nanoparticles -Venofer®- used as model suspension and iron tartrate/adipate-modified nanoparticles, both of about 4 nm (core size) were evaluated. The two nanostructures were injected into the system showing good peak profiles and quantitative elution recoveries (>80%) in both cases. In addition, both nanoparticulate fractions could be based-line separated from ionic iron species, which needed to be complexed with 1mM citrate to elute from the column. Exposed cells up to 0.5mM of iron tartrate/adipate-modified nanoparticles were specifically treated to extract the internalized NPs and the extracts examined using the proposed strategy. The obtained results revealed the presence of three different fractions corresponding to nanoparticle aggregates, dispersed nanoparticles and soluble iron respectively in a single chromatographic run. Quantitative experiments (column recoveries ranging from 60 to 80%) revealed the presence of the majority of the Fe in the nanoparticulated form (>75%) by summing up the dispersed and aggregate particles. Such experiments point out the high uptake and low solubilization rate of the tartrate/adipate NPs making these structures highly suitable as Fe supplements in oral anemia treatments.
In this work, we describe a simple solvothermal route for the synthesis of Eu3+-doped gadolinium orthovanadate nanocrystals (Eu:GdVO4−PAA) functionalized with poly(acrylic)acid (PAA), that are applicable as cell labeling probes for multimodal cellular imaging. The Eu3+ doping of the vanadate matrix provides optical functionality, due to red photoluminescence after illumination with UV light. The Gd3+ ions of the nanocrystals reduce the T1 relaxation time of surrounding water protons, allowing these nanocrystals to act as a positive MRI contrast agent with a r1 relaxivity of 1.97 mM−1 s−1. Low background levels of Eu3+, Gd3+, and V5+ in biological systems make them an excellent label for elemental microscopy by Laser Ablation (LA)-ICP-MS. Synthesis resulted in polycrystalline nanocrystals with a hydrodynamic diameter of 55 nm and a crystal size of 36.7 nm, which were further characterized by X-ray diffraction (XRD), photoluminescence spectroscopy (PL) and transmission electron microscopy (TEM). The multifunctional nanocrystals were subsequently used for intracellular labeling of both human adipose-derived stem cells (MSCs) and A549 (adenocarcinomic human alveolar basal epithelial) cells.
The mechanism of action of zirconium permanent modifiers on graphite surfaces was investigated in order to understand its influence on the analytical signal in atomic and molecular absorption spectrometry (AAS/MAS). For this, the molecule formation of CaF was studied, which is used for the indirect analytical determination of fluorine in high-resolution continuum source graphite furnace molecular absorption spectrometry (HR-CS-GFMAS). The kinetics of this reaction was established by monitoring its molecular spectrum at different atomisation temperatures. An Arrhenius plot showed a pseudo-first order reaction with respect to fluorine (n = 1). An intermediate state was isolated, and its structure was elucidated by spectroscopic methods: scanning electron microscopy with energy dispersive X-ray spectroscopy (SEMEDX), X-ray photoelectron spectroscopy (XPS), X-ray absorption spectroscopy (XANES and EXAFS), and Raman microspectroscopy. We propose here a mechanism, where ZrO2 acts as a heterogeneous catalyst: after a pyrolytic step, an intermediate state of ZrO(OCaF) is activated, and at higher temperatures, CaF(g) is released from the zirconium-coated graphite surface. No evidence of the formation of zirconium carbide was found. Consequently, as the CaF formation is catalysed by a heterogeneous catalyst, surface modifications with ZrO2 nanoparticles and ZrO xerogels were investigated in order to increase the surface area. Their influence was evaluated in the molecule formation of CaF, CaCl, CaBr, and CaI. Graphite furnace modification with zirconium oxide nanoparticles proves to be the best choice for fluorine analysis with a signal enhancement of more than eleven times with respect a non-coated graphite furnace. However, the influence of zirconium modifications in the analytical signals of Cl, and I is lower than the F signals or even negative in case of the Br. Understanding zirconium modifiers as heterogeneous catalysts offers a new perspective to AAS and MAS, and reveals the potential of surface analytical methods for development of improved permanent modifiers and graphite furnace coatings.