Analytische Chemie
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- Laser ablation (15)
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- Nanoparticle (12)
- Bioimaging (10)
- Cell (5)
- HR-CS-MAS (5)
- Isotope dilution mass spectrometry (5)
- Lanthanide (5)
- Nanoparticles (5)
Organisationseinheit der BAM
- 1.1 Anorganische Spurenanalytik (68) (entfernen)
Eingeladener Vortrag
- nein (28)
Imaging of elemental distributions in single cell assays as well as tissue sections can be performed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). This powerful technique offers precise spatially resolved measurements at the trace and ultratrace level and has been established as an excellent tool to answer analytical, biological and biomedical questions. To date, imaging mass cytometry is already able to simultaneously detect up to 40 cellular targets due to conjugation of isotopically pure lanthanides to affinity binders, e.g. antibodies.
To further enhance the ability of multiparametric analysis to more than 100 analytes at once, we investigated lanthanide nanocrystals as new, highly sensitive metal tags for identification of targets in clinical cell assays and tissue samples. Multiparametric analysis will be possible by encoding the lanthanide composition of nanocrystals associated to the affinity binders. Nanocrystals showed remarkable potential for sensitive detection in MS due to high stability and signal amplification compared to e.g. polymer tags, carrying fewer metal atoms.
Synthesis of functionalized lanthanide nanocrystals for further bioconjugation was performed with high reproducibility and monodisperse size distribution. For proof of principle, the uptake and distribution of these nanocrystals within the monolayered cell line A549 were investigated by mapping the intensities at subcellular resolution using LA-ICP-MS. It could be shown, that the cells were efficiently labeled with the nanocrystals and mostly accumulate near the nucleus. Additionally, the bioconjugation of the nanocrystals to antibodies and particularly the preservation of the antibody specificity was investigated using Dot Blot experiments. All in all, the results imply high sensitivity and the possibility of multiparametric analysis by doting various lanthanides into the nanocrystals.
The impact of nanoparticles, NPs, at the single cell level has become a major field of toxicological research and different analytical methodologies are being investigated to obtain biological and toxicological information to better understand the mechanisms of cell–NP interactions. Here, inductively coupled plasma mass spectrometry in the single cell measurement mode (SC-ICP-MS) is proposed to study the uptake of silver NPs, AgNPs, with a diameter of 50 nm by human THP-1 monocytes in a proof-ofprinciple experiment. The main operating parameters of SC-ICP-MS have been optimized and applied for subsequent quantitative analysis of AgNPs to determine the number of particles in individual cells using AgNP suspensions for calibration. THP-1 cells were incubated with AgNP suspensions with concentrations of 0.1 and 1 µg/mL for 4 and 24 hours. The results reveal that the AgNP uptake by THP-1 monocytes is minimal at the lower dose of 0.1 µg/mL (roughly 1 AgNP per cell was determined), whereas a large cell-to-cell variance dependent on the exposure time is observed for a 10 times higher concentration (roughly 7 AgNPs per cell). The method was further applied to monitor the AgNP uptake by THP-1 cells differentiated macrophages incubated at the same AgNP concentration levels and exposure times demonstrating a much higher AgNP uptake (roughly from 9 to 45 AgNPs per cell) that was dependent on exposure concentration and remained constant over time. The results have been compared and validated by sample digestion followed by ICP-MS analysis as well as with other alternative promising techniques providing single cell analysis.
The interaction of nanoparticles (NPs) with cells has become a major field of interest, ranging from medical applications to nanotoxicology. Size, shape and surface modification of the NPs determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is an established quantitative multi-elemental analysis and mapping technique. In recent years, it was shown that LA-ICP-MS can provide quantitative as well as distribution information of NPs in cell samples.
Here LA-ICP-MS was applied for the imaging of individual cells to study the uptake and intracellular processing of metal-containing nanostructures. Additionally, the local distribution of naturally occurring elements in cells like P was measured to indicate cell morphology. The cells were incubated with different types of NPs under varying experimental conditions. For LA analysis, the cells were fixed and dried.
Our findings show, that LA-ICP-MS is suitable for the localisation of nanoparticle aggregates within cellular compartments. The studied NPs accumulate in the perinuclear region in the course of intracellular processing, but do not enter the cell nucleus. The uptake efficiency depends strongly on the physicochemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time.
The results demonstrate the potential of LA-ICP-MS providing insight into NP uptake, intracellular distribution and cell-to-cell variation dependent on experimental parameters.
An immunohistochemical method is described to visualize the distribution of metallothioneins 1/2 (MT 1/2) and metallothionein 3 (MT 3) in human ocular tissue. It is making use of (a) antibodies conjugated to gold nanoclusters (AuNCs) acting as labels, and (b) laser ablation (LA) coupled to inductively coupled plasma – mass spectrometry (ICP-MS).Water-soluble fluorescent AuNCs (with an average size of 2.7 nm) were synthesized and then conjugated to antibody by carbodiimide coupling. The surface of the modified AuNCs was then blocked with hydroxylamine to avoid nonspecific interactions with biological tissue. Immunoassays for MT 1/2 and MT 3 in ocular tissue sections (5 μm thick) from two post mortem human donors were performed. Imaging studies were then performed by fluorescence using confocal microscopy, and LA-ICP-MS was performed in the retina to measure the signal for gold. Signal amplification by the >500 gold atoms in each nanocluster allowed the antigens (MT 1/2 and MT 3) to be imaged by LA-ICP-MS using a laser spot size as small as 4 μm. The image patterns found in retina are in good agreement with those obtained by conventional fluorescence immunohistochemistry which was used as an established reference method.
Heritage Lecture
(2018)
After finishing my diploma thesis in plasma physics in 1981, I dreamt of a future in a research lab to develop novel fusion reactors for energy production or to study universal plasmas and their emission in the cosmos. This dream never became real, however I found my first job in a team to build up a new museum dedicated to “Energy”, and this first part of my career was already finished after a year, because the funding was not extended. So, I found immediately a new job as a young scientist in the institute for analytical sciences (originally ISAS: Institute for Plasmachemistry and Applied Spectroscopy) in 1982 to develop novel plasma ion sources for inorganic mass spectrometry. The first source of interest was based on a glow discharge for direct analysis of conducting solids (technically supported by Finnigan MAT, Bremen). Here I adopted the design of the Grimm type discharge for the first time, which was well known in optical emission spectroscopy, and coupled it to a quadrupole mass analyzer. The advantage of this design was that flat craters are produced by sputtering which made this source very powerful for in-depth analysis of technical layers. This then became the topic of my PhD, which was not originally planned, and I had to learn a lot about surface analysis. However, since the first project was too successful we established a small team (in cooperation with Jose Broekaert - an expert in ICP-OES) which started with the development of our own inductively coupled plasma ion source in 1986 coupled to a quadrupole and in 1989 to a sector field mass spectrometer (funded by the Minister of Science and Technology; again in cooperation with Finnigan MAT). The latter device was launched to the market in 1993 as the Element 1.
The second decade of my career was still related to instrumental development but mainly of glow discharge sources. In an EU funded project first an automated glow discharge sector field instrument was developed where the Grimm type geometry was combined with a fast flow concept (in cooperation with Volker Hoffmann at IFW in Dresden). This was done in cooperation with VG (which became later a part of Thermo Fisher Scientific together with Finnigan MAT), so that it is not surprising that this concept for the Element GD. This project was later continued in the third phase of my career, again funded by the EU and in cooperation with the group of Alfredo Sanz-Medel (Rosario Pereiro and Jorge Pisonero), to develop a fast flow, but now rf-powered GD ion source in combination with a time-of-flight mass spectrometer, which was later launched to the market commercially by Horiba Jobin Yvon (France) for in-depth profiling of thin layers even of non-conducting materials.
In the first decade of my career I started to study already “analytical chemistry” from the scratch because the instruments developed have been applied now for direct analysis of solid materials, technical layers and environmental samples. In case of environmental applications our ICP-MS (the quadrupole and the sector field instrument) was coupled with separation techniques, so that this period of instrumental development was dominated in the second decade by development of high efficiency sample introduction systems in combination with speciation studies of Pt group elements, arsenic, selenium and phosphorus (in DNA and phosphorylated proteins), Ni and Cr. Additionally, we continued with the analysis of solid ceramic materials (Al2O3, SiC, SiN) and ambient air-born particles. At the end of the second decade we complemented our instrumental pool by a collision and reaction cell instrument in cooperation with Micromass and used this instrument for speciation studies of peptides and proteins and demonstrated that by ICP-HEX-MS quantitative proteomics is feasible. Therefore, we more and more focused in the following years on metalloproteins and published a famous paper on “Metallobiomolecules: The basis of life, the challenge of atomic spectroscopy” (together with Luc Moens and Ryszard Lobinski). For detection of metalloproteins we applied typical workflows of biochemistry and proteomics, for which I had to extend my knowledge about biochemistry and proteomics. As a new analytical tool, we used a homemade laser ablation cell for sample introduction of metalloproteins after their separation by gel electrophoresis and extended this work by applying metal-tagging of antibodies for Western blot assays. For this purpose, proteins were separated in SDS-PAGE and electroblotted onto membranes. Specific detection of proteins even not containing any metal could be performed by laser ablation ICP-MS using the metal tagged antibodies for indirect detection. This research was interrupted in 2009 by a movement from ISAS (where atomic spectroscopy was declining) to BAM (the Federal Institute for Materials Research and Testing, Berlin) where this research direction was fostered. The experience we achieved at ISAS in the previously mentioned projects were now used here at BAM in the fourth decade for materials research and the development of a quantitative elemental microscope with cellular resolution. So, at the end of my career I am trying to apply all my knowledge and expertise to develop analytical methods and to apply multimodal spectroscopies to decipher the construction code of the cellular machinery, which is the most precise and complex machinery I have ever seen. If we were able to understand how this machinery works, we can better diagnose and treat a malfunction in case of the development of a disease.
Finally, I can conclude that lifelong learning starts before school but does not end at the end of this lecture. It looks like this heritage lecture will be focused on my career only, but this is not the case because some highlights of my career will be used to illustrate a few universal principles: how to have fun, how to find friends and how this all leads to an increase of joy and joy is the basis of new ideas (which must not always be related to your profession) and novel ideas are essential for a successful and satisfying career. So, this heritage lecture wants to answer the most important question of a life which was dedicated to plasma spectrochemistry:
1) Is it possible - at all - to have fun in this research direction?
2) Can we learn already today what we need tomorrow?
3) How can we still realize our scientific dreams of cutting edge research in times of cutting budgets? Which automatically leads to the next question:
4) Is necessity the mother of invention?
All questions will be answered! Controversial discussions (for angry or disappointed colleagues) will be stimulated and my visions of future research (for students and postdocs) and instrumental developments (for manufacturer) will be presented. Finally, conclusions will be drawn by the auditorium (everybody) and thanks will be given to Ramon Barnes (by me) already in advance!
Heritage Lecture
(2018)
After finishing my diploma thesis in plasma physics in 1981, I dreamt of a future in a research lab to develop novel fusion reactors for energy production or to study universal plasmas and their emission in the cosmos. This dream never became real, however I found my first job in a team to build up a new museum dedicated to “Energy”, and this first part of my career was already finished after a year, because the funding was not extended. So, I found immediately a new job as a young scientist in the institute for analytical sciences (originally ISAS: Institute for Plasmachemistry and Applied Spectroscopy) in 1982 to develop novel plasma ion sources for inorganic mass spectrometry. The first source of interest was based on a glow discharge for direct analysis of conducting solids (technically supported by Finnigan MAT, Bremen). Here I adopted the design of the Grimm type discharge for the first time, which was well known in optical emission spectroscopy, and coupled it to a quadrupole mass analyzer. The advantage of this design was that flat craters are produced by sputtering which made this source very powerful for in-depth analysis of technical layers. This then became the topic of my PhD, which was not originally planned, and I had to learn a lot about surface analysis. However, since the first project was too successful we established a small team (in cooperation with Jose Broekaert - an expert in ICP-OES) which started with the development of our own inductively coupled plasma ion source in 1986 coupled to a quadrupole and in 1989 to a sector field mass spectrometer (funded by the Minister of Science and Technology; again in cooperation with Finnigan MAT). The latter device was launched to the market in 1993 as the Element 1.
The second decade of my career was still related to instrumental development but mainly of glow discharge sources. In an EU funded project first an automated glow discharge sector field instrument was developed where the Grimm type geometry was combined with a fast flow concept (in cooperation with Volker Hoffmann at IFW in Dresden). This was done in cooperation with VG (which became later a part of Thermo Fisher Scientific together with Finnigan MAT), so that it is not surprising that this concept for the Element GD. This project was later continued in the third phase of my career, again funded by the EU and in cooperation with the group of Alfredo Sanz-Medel (Rosario Pereiro and Jorge Pisonero), to develop a fast flow, but now rf-powered GD ion source in combination with a time-of-flight mass spectrometer, which was later launched to the market commercially by Horiba Jobin Yvon (France) for in-depth profiling of thin layers even of non-conducting materials.
In the first decade of my career I started to study already “analytical chemistry” from the scratch because the instruments developed have been applied now for direct analysis of solid materials, technical layers and environmental samples. In case of environmental applications our ICP-MS (the quadrupole and the sector field instrument) was coupled with separation techniques, so that this period of instrumental development was dominated in the second decade by development of high efficiency sample introduction systems in combination with speciation studies of Pt group elements, arsenic, selenium and phosphorus (in DNA and phosphorylated proteins), Ni and Cr. Additionally, we continued with the analysis of solid ceramic materials (Al2O3, SiC, SiN) and ambient air-born particles. At the end of the second decade we complemented our instrumental pool by a collision and reaction cell instrument in cooperation with Micromass and used this instrument for speciation studies of peptides and proteins and demonstrated that by ICP-HEX-MS quantitative proteomics is feasible. Therefore, we more and more focused in the following years on metalloproteins and published a famous paper on “Metallobiomolecules: The basis of life, the challenge of atomic spectroscopy” (together with Luc Moens and Ryszard Lobinski). For detection of metalloproteins we applied typical workflows of biochemistry and proteomics, for which I had to extend my knowledge about biochemistry and proteomics. As a new analytical tool, we used a homemade laser ablation cell for sample introduction of metalloproteins after their separation by gel electrophoresis and extended this work by applying metal-tagging of antibodies for Western blot assays. For this purpose, proteins were separated in SDS-PAGE and electroblotted onto membranes. Specific detection of proteins even not containing any metal could be performed by laser ablation ICP-MS using the metal tagged antibodies for indirect detection. This research was interrupted in 2009 by a movement from ISAS (where atomic spectroscopy was declining) to BAM (the Federal Institute for Materials Research and Testing, Berlin) where this research direction was fostered. The experience we achieved at ISAS in the previously mentioned projects were now used here at BAM in the fourth decade for materials research and the development of a quantitative elemental microscope with cellular resolution. So, at the end of my career I am trying to apply all my knowledge and expertise to develop analytical methods and to apply multimodal spectroscopies to decipher the construction code of the cellular machinery, which is the most precise and complex machinery I have ever seen. If we were able to understand how this machinery works, we can better diagnose and treat a malfunction in case of the development of a disease.
Finally, I can conclude that lifelong learning starts before school but does not end at the end of this lecture. It looks like this heritage lecture will be focused on my career only, but this is not the case because some highlights of my career will be used to illustrate a few universal principles: how to have fun, how to find friends and how this all leads to an increase of joy and joy is the basis of new ideas (which must not always be related to your profession) and novel ideas are essential for a successful and satisfying career. So, this heritage lecture wants to answer the most important question of a life which was dedicated to plasma spectrochemistry:
1) Is it possible - at all - to have fun in this research direction?
2) Can we learn already today what we need tomorrow?
3) How can we still realize our scientific dreams of cutting edge research in times of cutting budgets? Which automatically leads to the next question:
4) Is necessity the mother of invention?
All questions will be answered! Controversial discussions (for angry or disappointed colleagues) will be stimulated and my visions of future research (for students and postdocs) and instrumental developments (for manufacturer) will be presented. Finally, conclusions will be drawn by the auditorium (everybody) and thanks will be given to Ramon Barnes (by me) already in advance!
Silver nanoparticles (AgNPs), have a high scientific and commercial impact due to their important antibacterial properties. However, there are serious concerns about their toxicological adverse effects as a consequence of their broad range of applications. Particularly, the impact of AgNPs on cells is not very well understood yet and there is a current demand to develop analytical methodologies providing information about the interaction and distribution of AgNPs at a single cell level. In this research, mass cytometry was used to introduce a new quantitative approach to study the uptake of AgNPs by individual THP-1 macrophages as a cell model system. Here, we show that this methodology provides not only multi-variate phenotypic information of individual cells but enables the quantitative analysis of AgNPs associated to cells in a single measurement by performing an external calibration using AgNPs suspension. Using differentiated THP-1 cells, we monitored and quantified the uptake of 50 nm AgNPs in a time and dose-dependent manner by mass cytometry. 7 to 120 AgNPs per cell (2 to 89 fg Ag/cell) were determined after exposure of differentiated THP-1 cells to low AgNPs concentrations of 0.1 and 1.0 mg L-1, at time points of 4 and 24 h. The results were validated by mass cytometric analysis of digested cells working as a conventional inductively coupled plasma mass spectrometry, ICP-MS. This study demonstrates the power of single cell analysis by mass cytometry even for low doses experiments as a new analytical tool for hitherto unaddressed questions in nanotoxicology.
Silver nanoparticles (AgNPs), have a high scientific and commercial impact due to their important antibacterial properties. However, there are serious concerns about their toxicological adverse effects as a consequence of their broad range of applications. Particularly, the impact of AgNPs on cells is not very well understood yet and there is a current demand to develop analytical methodologies providing information about the interaction and distribution of AgNPs at a single cell level. In this research, mass cytometry was used to introduce a new quantitative approach to study the uptake of AgNPs by individual THP-1 macrophages as a cell model system. Here, we show that this methodology provides not only multi-variate phenotypic information of individual cells but enables the quantitative analysis of AgNPs associated to cells in a single measurement by performing an external calibration using AgNPs suspension. Using differentiated THP-1 cells, we monitored and quantified the uptake of 50 nm AgNPs in a time and dose-dependent manner by mass cytometry. 7 to 120 AgNPs per cell (2 to 89 fg Ag/cell) were determined after exposure of differentiated THP-1 cells to low AgNPs concentrations of 0.1 and 1.0 mg L-1, at time points of 4 and 24 h. The results were validated by mass cytometric analysis of digested cells working as a conventional inductively coupled plasma mass spectrometry, ICP-MS. This study demonstrates the power of single cell analysis by mass cytometry even for low doses experiments as a new analytical tool for hitherto unaddressed questions in nanotoxicology.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is gaining importance for bioimaging cytometry to answer analytical, biological and biomedical questions. High sensitivity and spatial resolution make it an excellent tool for imaging of metal and heteroelement distribution in single cells. Comparable to CyTOF imaging mass cytometry, metal coded antibodies can be used for multiparametric analysis as well as quantification.
In this project, nanocrystals are investigated as new highly sensitive metal tags for identification and quantification of biomarkers, like Alzheimer’s or breast cancer, in clinical cell assays and tissue samples. Of high significance is the simultaneous analysis of several biomarkers at once, which is possible by special coding of lanthanide tags on the biomarker associated antibody. Nanocrystals show potential for sensitive measurement in MS due to high stability and signal amplification compared to tags with fewer metal atoms. For proof of principle, synthesis and characterization of lanthanide doped nanocrystals was performed by a nanoPET pharma GmbH with great reproducibility and homogenous size. In A549 cell cultures, the uptake and distribution of these nanocrystals within the monolayered cells was investigated by LA-ICP-MS measurements using subcellular resolution. The nanocrystals showed high sensitivity and the possibility of multiparametric analysis by doting different lanthanides. Additionally, stability of the bioconjugation of the nanocrystals and target antibodies was investigated using Dot Blot experiments and LA-ICP-MS.
Development and market introduction of new nanomaterials trigger the need for an adequate risk assessment of such products alongside suitable risk communication measures. Current application of classical and new nanomaterials is analyzed in context of regulatory requirements and standardization for chemicals, food and consumer products. The challenges of nanomaterial characterization as the main bottleneck of risk assessment and regulation are presented. In some areas, e.g., quantification of nanomaterials within complex matrices, the establishment and adaptation of analytical techniques such as laser ablation inductively coupled plasma mass spectrometry and others are potentially suited to meet the requirements. As an example, we here provide an approach for the reliable characterization of human exposure to nanomaterials resulting from food packaging. Furthermore, results of nanomaterial toxicity and ecotoxicity testing are discussed, with concluding key criteria such as solubility and fiber rigidity as important parameters to be considered in material development and regulation. Although an analysis of the public opinion has revealed a distinguished rating depending on the particular field of application, a rather positive perception of nanotechnology could be ascertained for the German public in general. An improvement of material characterization in both toxicological testing as well as end-product control was concluded as being the main obstacle to ensure not only safe use of materials, but also wide acceptance of this and any novel technology in the general public.
Single-cell analysis by ICP-MS/MS as a fast tool for cellular bioavailability studies of arsenite
(2018)
Single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) has become a powerful and fast tool to evaluate the elemental composition at a single-cell level. In this study, the cellular bioavailability of arsenite (incubation of 25 and 50 μM for 0-48 h) has been successfully assessed by SC-ICP-MS/MS for the first time directly after re-suspending the cells in water. This procedure avoids the normally arising cell membrane permeabilization caused by cell fixation methods (e.g. methanol fixation). The reliability and feasibility of this SC-ICP-MS/MS approach with a limit of detection of 0.35 fg per cell was validated by conventional bulk ICP-MS/MS analysis after cell digestion and parallel measurement of sulfur and phosphorus.
Online immunocapture ICP‑MS for the determination of the metalloprotein ceruloplasmin in human serum
(2018)
The human copper-protein ceruloplasmin (Cp) is the major copper-containing protein in the human body. The accurate determination of Cp is mandatory for the reliable diagnosis of several diseases. However, the analysis of Cp has proven to be difficult. The aim of our work was a proof of concept for the determination of a metalloprotein-based on online immunocapture ICP-MS. The immuno-affinity step is responsible for the enrichment and isolation of the analyte from serum, whereas the compound-independent quantitation with ICP-MS delivers the sensitivity,
precision, and large dynamic range. Off-line ELISA (enzyme-linked immunosorbent assay) was used in parallel to confirm the elution profile of the analyte with a structure-selective method. The total protein elution was observed with the 32S mass trace. The ICP-MS signals were normalized on a 59Co signal. The human copper-protein Cp could be selectively determined. This was shown with pure Cp and with a sample of human serum. The good correlation with off-line ELISA shows that Cp could be captured and eluted selectively from the anti-Cp affinity column and subsequently determined by the copper signal of ICP-MS.
An immunohistochemical method is described to visualize the distribution of metallothioneins 1/2 (MT 1/2) and metallothionein 3 (MT 3) in human ocular tissue. It is making use of (a) antibodies conjugated to gold nanoclusters (AuNCs) acting as labels, and (b) laser ablation (LA) coupled to inductively coupled plasma – mass spectrometry (ICP-MS). Water-soluble fluorescent AuNCs (with an average size of 2.7 nm) were synthesized and then conjugated to antibody by carbodiimide coupling. The surface of the modified AuNCs was then blocked with hydroxylamine to avoid nonspecific interactions with biological tissue. Immunoassays for MT 1/2 and MT 3 in ocular tissue sections (5 μm thick) from two post mortem human donors were performed. Imaging studies were then performed by fluorescence using confocal microscopy, and LA-ICP-MS was performed in the retina to measure the signal for gold. Signal amplification by the >500 gold atoms in each nanocluster allowed the antigens (MT 1/2 and MT 3) to be imaged by LA-ICP-MS using a laser spot size as small as 4 μm. The image patterns found in retina are in good agreement with those obtained by conventional fluorescence immunohistochemistry which was used as an established reference method.
In chemical elements with three or more stable isotopes, mass-dependent stable isotope fractionation is expressed by co-varying isotope ratios. In the three-isotope space ((δ’m2/δ’m1)/(( δ’m3/δ’m1)) these plot along a line with a slope (β), the so called ‘terrestrial fractionation line’. This partitioning of stable isotopes results from both kinetic and equilibrium reactions that are characterized by specific β-values.
For the natural range of isotope ratios of ‘novel’ stable isotope systems such as Si, Mg, Fe, Zn, Cu this information cannot be accessed because samples fall close to the delta-zero standard where the current measurement precision is too low to resolve small differences in β. We present a new approach to resolve deviations from a reference slope β by standard-sample bracketing against material offset from the natural range. We use this approach to explore the isotope fractionation mechanism in the mammalian food web. We have analyzed Mg stable isotope ratios in bone bioapatite of herbivore, omnivore and carnivore mammals. Positive shifts in δ26/24Mg along the trophic chain (from herbivore to carnivore) together with β= 0.513 suggest the presence of two isotope fractionation mechanisms operating during biomineralization. While positive shifts in δ26/24Mg are in favor of equilibrium isotope fractionation process, the proximity of β to the theoretically calculated β(kinetic) of typically 0.511 suggests the presence of a second component that fractionates stable isotopes kinetically. The herein presented approach is applicable to any element with 3+ stable isotopes analyzed by multi-collector inductively coupled plasma mass spectrometry.
The analytical potential of a nanosecond laser ablation inductively coupled plasma mass spectrometer (ns-LA-ICP-SFMS) system, equipped with an ultra-fast wash-out ablation chamber, is critically investigated for fast and highly spatially resolved (∼μm) qualitative elemental distribution within single cells. Initially, a low surface roughness (< 10 nm) thin In–SnO2 layer (total coating thickness ∼200 nm) deposited on glass is employed to investigate the size, morphology and overlapping of laser-induced craters obtained at different laser repetition rates, making use of Atomic Force Microscopy (AFM). Conical craters with a surface diameter of about 2 µm and depths of about 100 nm were measured after a single laser shot. Furthermore, the influence of the sampling distance (i.e. distance between the sample surface and the inner sniffer of the ablation chamber) on the LA-ICP-MS ion signal wash-out time is evaluated. A significant decrease of the transient 120Sn+ ion signal is noticed after slight variations (±200 μm) around the optimum sampling position. Ultra-fast wash-outs (< 10 ms) are achieved reducing the aerosol mixing from consecutive laser shots even when operating the laser at high repetition rates (25 – 100 Hz). Fast and highly spatially resolved images of elemental distribution within mouse embryonic fibroblast cells (NIH/3T3 fibroblast cells) and human cervical carcinoma cells (HeLa cells), incubated with gold nanoparticles (Au NPs) and Cd-based quantum dots (QDs), respectively, are determined at the optimized operating conditions. Elemental distribution of Au and Cd in single cells is achieved using a high scanning speed (50 µm/s) and high repetition rate (100 Hz). The results obtained for the distribution of fluorescent Cd-based QDs within the HeLa cells are in good agreement with those obtained by confocal microscopy. The size, morphology and overlapping of laser-induced craters in the fixed cells are also investigated using AFM, observing conical craters with a surface diameter of about 2.5 µm and depths of about 800 nm after a single laser shot.
Nanoparticles (NPs) have found a wide range of applications in research and industry. Thereby the interaction of NPs with biological systems like cells has become a major field of interest, ranging from medical applications to nanotoxicology. Size, shape and surface modification of the nanomaterials determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes.
In recent years, elemental imaging of biological samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is gaining more and more importance. Improvements concerning both spatial resolution (down to 1 µm) and signal-to-background ratio due to low-dispersion LA chambers make LA-ICP-MS particularly interesting for single cell analysis.
Here LA-ICP-MS was applied for the imaging of individual cells to study the uptake and intracellular processing of metal-containing nanostructures. The cells were incubated with different NPs under varying experimental conditions and afterwards fixed with para-formaldehyde and dried for LA analysis. High-spatial resolution LA-ICP-MS was achieved by careful optimisation of the laser ablation parameters.
Our findings show, that LA-ICP-MS is applicable to localize NP aggregates within cellular compartments. The uptake efficiency depends strongly on the physicochemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time.
The results demonstrate the potential of LA-ICP-MS providing insight into nanoparticle-cell interaction dependent on experimental parameters.
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.
Nanoparticles (NPs) have found a wide range of applications in research and industry. Thereby the interaction of NPs with biological systems like cells has become a major field of interest, ranging from medical applications to nanotoxicology. Size, shape and surface modification of the nanomaterials determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes.
Inductively coupled plasma mass spectrometry (ICP-MS) is a well-established analytical method offering high sensitivity and multi-element capability. By coupling a laser ablation (LA) system to an ICP-MS the analysis of different kinds of solid samples is possible. In recent years, it was shown that LA-ICP-MS can provide quantitative as well as distribution information of metal containing nanoparticles (NPs) in cell samples.
Here LA-ICP-MS was applied for the imaging of individual fibroblast cells to study the uptake and intracellular processing of NPs. Our results show that LA-ICP-MS can be used to localize nanoparticle aggregates within cellular compartments. The studied NPs accumulate in the perinuclear region in the course of intracellular processing, but do not enter the cell nucleus. The uptake efficiency depends strongly on the physico-chemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time.
ICP-MS was used to determine the composition of the nanomaterials as well as the number of NPs in cells after acid digestion of the samples.
Laser tissue soldering (LTS) based on indocyanine green (ICG)-mediated heat- denaturation of proteins might be a promising alternative technique for micro-suturing, but up to now the Problem of too weak shear strength of the solder welds in comparison to sutures is not solved. Earlier reports gave promising results showing that solder supported by carrier materials can enhance the cohesive strength of the liquid solder. In these studies, the solder was applied to the carriers by dip coating. Higher reliability of the connection between the solder and the carrier material is expected when the solder is bound covalently to the carrier material. In the present study a poly(ether imide) (PEI) membrane served as carrier material and ICG- supplemented albumin as solder substrate. The latter was covalently coupled to the carrier membrane under physiological conditions to prevent structural protein changes. As laser source a diode continuous-wave laser emitting at 808 nm with intensities between 250mW and 1500mW was utilized. The Albumin functionalized carrier membrane was placed onto the tunica media of explanted pig thoracic aortae forming an overlapping area of approximately 0.5×0.5 cm2. All tests were performed in a dry state to prevent laser light absorption by water. Infrared spectroscopy, spectro-photometrical determination of the secondary and Primary amine groups after acid orange II staining, contact angle measurements, and atomic force microscopy proved the successful functionalization of the PEI membrane with albumin. A laser power of 450mW LTS could generate a membrane-blood vessel connection which was characterized by a shear strength of 0.08±0.002MPa, corresponding to 15% of the tensile strength of the native blood vessel. Theoretically, an overlapping zone of 4.1mmaround the entire circumference of the blood vessel could have provided shear strength of the PEI membrane-blood vessel compound identical to the tensile strength of the native blood vessel. These in-vitro results confirmed the beneficial effects of solder reinforcement by carrier membranes, and suggest LTS with covalently bound solders on PEI substrates for further studies in animal models.
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.
Nanoparticles (NPs) have found a wide range of applications in research and industry. Thereby the interaction of NPs with biological systems like cells has become a major field of interest, ranging from medical applications to nanotoxicology. Size, shape and surface modification of the nanomaterials determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is an established quantitative multi-elemental analysis and mapping technique. In recent years, it was shown that LA-ICP-MS can provide quantitative as well as distribution information of metal containing nanoparticles (NPs) in cell samples.
Here LA-ICP-MS was applied for the imaging of individual fibroblast cells to study the uptake and intracellular processing of NPs. Our results show that LA-ICP-MS can be used to localize nanoparticle aggregates within cellular compartments. The studied NPs accumulate in the perinuclear region in the course of intracellular processing, but do not enter the cell nucleus. The uptake efficiency depends strongly on the physico-chemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time.
ICP-MS was used to determine the composition of the nanomaterials as well as the number of NPs in cells after acid digestion of the samples.
Imaging of elemental distributions in single cell assays as well as tissue sections can be performed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). This powerful technique offers precise spatially resolved measurements at trace and ultratrace levels and has been established as an excellent tool to answer analytical, biological and biomedical questions. To date, mass cytometry is already able to simultaneously detect up to 40 cellular targets due to conjugation of isotopically pure lanthanides to affinity binders, e.g. antibodies.
To further enhance the ability of multiparametric analysis to more than 100 analytes at once, we investigated lanthanide nanocrystals as new, highly sensitive metal tags for identification of targets in clinical cell assays and tissue samples. Multiparametric analysis will be possible by encoding the lanthanide composition of nanocrystals associated to the affinity binders. Nanocrystals showed remarkable potential for sensitive detection in MS due to high stability and signal amplification compared to e.g. polymer tags, carrying fewer metal atoms. Furthermore, the nanocrystals allow multimodal imaging due fluorescence of Eu3+ as well as contrast enhancing properties of Gd3+ in magnetic resonance imaging.
Synthesis of functionalized lanthanide nanocrystals for further bioconjugation was performed with high reproducibility and monodisperse size distribution. For proof of principle, the uptake and distribution of these nanocrystals within the monolayered cell line A549 were investigated by mapping the intensities at subcellular resolution using LA-ICP-MS. It could be shown, that the cells were efficiently labeled with the nanocrystals. Additionally, the bioconjugation of the nanocrystals to antibodies and particularly the preservation of the antibody specificity was investigated using Dot Blot experiments. All in all, the results imply high sensitivity and the possibility of multiparametric analysis by doting various lanthanides into the nanocrystals.
In der klinischen Diagnostik werden für zytometrische Messverfahren bereits eine Reihe von Reagenzien eingesetzt zur Markierung von Antikörper eingesetzt, um die Detektion von Biomarkern mittels Fluoreszenz- oder Flugzeitmassenspektrometrie zu ermöglichen.
Seit kurzem ist auch eine Imaging Mass Cytometry Kombination direkt erhältlich, wodurch der Nachweis von Biomarkern in Gewebeschnitten erreicht werden kann. Dazu wird eine Kopplung von Laser Ablation und induktiv gekoppeltem Plasma Massenspektrometrie eingesetzt, wobei ähnlich der Massenzytometrie, zuvor Antikörper mit Metallen markiert, und im Anschluss mit dem Gewebeschnitt inkubiert werden. Durch die hohe Ortsauflösung können die Biomarker lokalisiert, und zukünftig vielleicht auch quantifiziert werden.
Insbesondere Lanthanide eignen sich als Markierungsmetalle, da sie einen niedrigen Untergrund und chemisch ähnliches Verhalten zueinander aufweisen. Allein durch diese Elemente können bereits etwa 15 Parameter unterschieden werden, was durch isotopenreine Standards weiter gesteigert werden kann.
Vom Markierungsgrad abhängig werden unterschiedlich viele Metalle am Antikörper gebunden, und beeinflussen so die Sichtbarkeit im ICP-MS. Nanopartikel könnten daher eine deutliche Steigerung der Sensitivität bewirken. GdVO4 Nanokristalle scheinen bisher sehr vielversprechend und bieten neben multiparametrischen Anwendungen auch Multimodalität.
Die Synthese der Nanokristalle zeigte hohe Homogenität und Reproduzierbarkeit in Partikelgröße in der Zusammensetzung. Ein erstes Experiment mit einer Zellkultur konnte bereits die effiziente Markierung der Zellen unter Beweis stellen, wobei durch hohe Signalstärke auch subzelluläre Auflösung in der LA-ICP-MS erreicht werden konnte.
Imaging of elemental distributions in single cell assays as well as tissue sections can be performed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). This powerful technique offers precise spatially resolved measurements at trace and ultratrace levels and has been established as an excellent tool to answer analytical, biological and biomedical questions. To date, mass cytometry is already able to simultaneously detect up to 40 cellular targets due to conjugation of isotopically pure lanthanides to affinity binders, e.g. antibodies.
To further enhance the ability of multiparametric analysis to more than 100 analytes at once, we investigated lanthanide nanocrystals as new, highly sensitive metal tags for identification of targets in clinical cell assays and tissue samples. Multiparametric analysis will be possible by encoding the lanthanide composition of nanocrystals associated to the affinity binders. Nanocrystals showed remarkable potential for sensitive detection in MS due to high stability and signal amplification compared to e.g. polymer tags, carrying fewer metal atoms. Furthermore, the nanocrystals allow multimodal imaging due fluorescence of Eu3+ as well as contrast enhancing properties of Gd3+ in magnetic resonance imaging.
Synthesis of functionalized lanthanide nanocrystals for further bioconjugation was performed with high reproducibility and monodisperse size distribution. For proof of principle, the uptake and distribution of these nanocrystals within the monolayered cell line A549 were investigated by mapping the intensities at subcellular resolution using LA-ICP-MS. It could be shown, that the cells were efficiently labeled with the nanocrystals. Additionally, the bioconjugation of the nanocrystals to antibodies and particularly the preservation of the antibody specificity was investigated using Dot Blot experiments. All in all, the results imply high sensitivity and the possibility of multiparametric analysis by doting various lanthanides into the nanocrystals.
Invited for this month’s cover picture is the group of Dr. Knut Rurack at the Department of Analytical Chemistry; Reference Materials at the Bundesanstalt fuer Materialforschung und -pruefung (BAM) in Berlin (Germany). The cover picture shows how differences in color and fluorescence on a test strip can be easily read out with a mobile device. Two reference spots Frame the sensitive spot that indicates the presence of trace amounts of HgII below the threshold in a natural water sample. This dipstick contains a hybrid material that combines boron-dipyrromethene (BODIPY) probes sterically loaded into specifically tailored mesoporous silica particles, allowing for ultrasensitive HgII detection through enhanced fluorescence in a few seconds. The applicability in real water samples and fish extracts are also studied.
Gegenstand der vorzustellenden Arbeiten ist die Prüfung der Umweltbeständigkeit und -verträglichkeit von Materialien und Produkten hinsichtlich der Emission von potenziellen Schadstoffen in die Umwelt. Hierzu werden chemisch-physikalische Einflüsse (Bewitterung) und mikrobielle Beanspruchungen an Modellmaterialien evaluiert. So werden die Freisetzungsraten von Schadstoffen in Abhängigkeit der Beanspruchung beschrieben. Als Modellmaterialien kommen die Polymere Polystyrol (PS) und Polypropylen (PP) zum Einsatz, die jeweils mit polybromierten Flammschutzmitteln (PBFSM) versehen sind. Synergistische Effekte der Bewitterungsparameter und der mikrobiologischen Beanspruchung sollen dabei ebenso betrachtet werden, wie die gezielte Alterung. Auch findet eine Beschreibung des Verhaltens der ausgetragenen Schadstoffe in den Umweltkompartimenten Boden oder Wasser statt. Hier sind mit Hilfe der zu entwickelnden Screening- und non-Target-Analyseverfahren die Transformation und der Metabolismus durch Mikroorganismen zu beschreiben. Auch soll mit Hilfe der RFA und der LA-ICP-MS die Abreicherung der PBFSM in den Modellmaterialien beschrieben werden. Aus den Ergebnissen sollen Korrelationen zwischen den künstlichen Alterungsverfahren und realen Szenarien abgeleitet werden.
Analysis of single cells via LA-ICP-MS is a technique with great potential, however manual targeting of single cells is laborious and therefore microarraying of cells looks promising. In this work, we investigate the potential of a commercial non-contact piezo dispenser arraying system (S3, Scienion AG, Berlin), equipped with a novel technology for single-cell isolation called CellenONE™ (Cellenion, Lyon). Usually if one aims to create a microarray of single cells via spotting a suitably diluted cell suspension, one will observe a Poisson-distributed cell number per spot. CellenONE™ overcomes this problem by controlling the number of cells optically in the piezo dispense capillary (PDC) via image recognition to obtain true single cell arrays. The figures of merit of the customized and optimized setup will be presented. In a proof of concept experiment we investigated the trace elemental fingerprint of THP-1 cells by LA-ICP-TOF-MS (Analyte G2, Teledyne Cetac; icpTOF, TOFWERK) and quantified two metal cell dyes, mDOTA-Ho (CheMatech, Dijon), and Ir-DNA intercalator (Fluidigm, San Francisco). For that, matrix matched calibration standards after Wang et al. were successfully prepared using the same arraying system. We believe that this novel approach opens new ways for automated quantitative single cell LA-ICP-MS.
Many current nanomaterials can serve as contrast agents in cellular or tissue imaging, drug delivery vehicles or therapeutics, whereas others can cause toxic effects. In order to evaluate nano-bio interactions, the number of nanoparticles (NPs) inside cells as well as their localisation within cellular substructures is of particular interest. The cellular uptake depends on the primary characteristics of the NPs (e.g. size, shape, surface coating) and on the cell type.
Laser ablation inductively coupled plasma mass spectrometry (LA‑ICP‑MS) is more and more used to study the NP pathway from uptake, via intracellular processing up to cell division. High-spatial resolution laser ablation at single cell level is achieved using novel low-dispersion LA chambers and by careful optimisation of laser energy, ablation frequency and scan speed at small laser spot sizes down to 1 µm. Different examples from BAM, Division 1.1 and cooperation partners using LA-ICP-MS to localize and quantify metal-containing nanoparticles are shown. The results demonstrate the potential of LA-ICP-MS providing insight into NP uptake, intracellular distribution and cell-to-cell variation.
The synthesis, characterization, and application of mesoporous materials containing boron–dipyrromethene (BODIPY) moieties that allow the sensitive and selective detection of HgII in aqueous environments by fluorescence enhancement is reported. For this purpose, BODIPY dye I containing a thia‐aza crown ether receptor as the fluorescent probe for the detection of HgII in aqueous environments is encapsulated into mesoporous materials to avoid self‐quenching or aggregation in water. Determination of HgII is accomplished within a few seconds with high selectivity and sensitivity, reaching a limit of detection of 12 ppt. The determination of trace amounts of HgII in natural waters and in fish extracts is demonstrated by using our sensing material. The incorporation of the material into several μ‐PAD strips yields a portable, cheap, quick, and easy‐to‐handle tool for trace HgII analysis in water.
Hepcidin-25 was identified as the main iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry (MS/MS), high-resolution mass spectrometry HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.
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.
Roadmap for the purity determination of pure metallic elements – Basic rinciples and helpful advice
(2018)
High purity materials can serve as a realisation of the Système International d’Unitès (SI) unit amount of substance for the specific element. Solutions prepared from such high purity materials using gravimetric preparation and the concept of molar mass are used as calibration solutions in many fields of analytical chemistry. Calibration solutions prepared this way provide the traceability to the SI and are the metrological basis in elemental analysis.
The preparation and characterization of such primary pure substances, representing the realisation of the SI unit amount of substance, is undertaken only by a small number of National Metrology Institutes (NMI) and Designated Institutes (DI).
Many other NMIs and DIs, however, prepare elemental calibration solutions as calibrants for their measurement services, such as the certification of matrix Reference Materials or the provision of reference values for Proficiency Testing schemes. The elemental calibration solutions used for this purpose are not a direct service to customers, such as preparing secondary calibration solutions, but provide the source of traceability for the other services. Hence, it is necessary for the NMI or DI to obtain data on the purity of the pure metals or other materials used to prepare the solutions with measurement uncertainties meeting the needs of the above described services. This is commonly undertaken as a “fit for purpose” assessment, appropriate for the uncertainty requirement of the service provided to customers.
As a consequence, total purity measurements are a long-term strategy of CCQM-IAWG. Several studies were conducted (CCQM-P107, CCQM-K72 and CCQM-P149) on the measurement of the purity of zinc. From these studies, several conclusions can be drawn for the purity assessment of a pure (metallic) element. These conclusions will be put together in this document in order to assist all NMIs/DIs in performing a purity assessment, whenever needed.
Neurodegenerative diseases are one of the major problems for our ageing society. Alzheimer’s disease (AD) as the most common neurodegenerative disorder affects over 46.8 million people worldwide and the number will increase as the population ages. The diagnosis of AD is challenging and only half of the patients are identified yet and often only in late stages. One reason is that existing assays for identification and quantification of AD biomarkers lack accuracy and are poorly comparable.
This study is part of the EU project “ReMiND” aiming to develop accurate, reliable and traceable methods for the detection and quantification of known and suspected AD biomarkers. Our target is the tau protein, as brain load and distribution of tau is highly correlated with the clinical progression of AD. We intend to develop a measurement method for the accurate quantification of tau by means of inductively coupled plasma mass spectrometry (ICP-MS).
ICP-MS is a powerful method for the matrix independent quantitative analysis of target elements. Developed for the use in inorganic trace analysis, ICP-MS is nowadays emerging as a valuable tool for bioanalytical questions. Especially the use of ICP-MS for quantitative proteomics by measuring heteroatoms is highly promising, considering that established quantification methods like organic mass spectrometry depend on the existence of matched protein and peptide standards or labelling of the target protein. In this work, we applied isotope dilution analysis (IDA) using ICP-MS to quantify proteins of known stoichiometry via their sulphur content. Sulphur is present in two amino acids, cysteine and methionine, and hence is omnipresent in nearly all proteins. A NIST standard bovine serum albumin (BSA) was quantified using sulfur IDA to optimize sample preparation and method parameters. Our goal is to employ the developed method in a proof of concept study for the quantification of the AD biomarker tau extracted from brains of a mouse model for AD.
A set of 59 ancient magical artefacts, mainly made of lead, was selected from the collections of the Staatliche Museen zu Berlin in order to unravel their origins. All the selected artefacts have been studied for their Pb isotope compositions, which covered the whole range of the Mediterranean ore deposits. However, the majority (≈86%) were made of lead matching the small compositional range of the Laurion ore deposits. Only eight out of the 59 artefacts were made of recycled lead or lead from other ore deposits.
Additionally, all but two were approximately dated based on their inscriptions. The lead isotopic composition together with information obtained from the inscriptions, the resulting dating, the context of the find and the known history of each item allowed us to gain more detailed information about the origins of these magical artefacts. The Attic provenance of 36 curse tablets was confirmed, whereas for 11 curse tablets previously classified as non-Attic, the provenance was either confirmed and specified (six artefacts) or changed to Attic (five artefacts). Surprisingly, the majority (six out of eight) of the analysed curse tablets from the Egyptian collection showed a lead isotopic composition closely matching that of Laurion.
A Laurion-like lead isotopic composition was also observed for three of the four analysed oracular tablets from Dodona. Together with the dating information, this points to Laurion as the major and dominant lead source in the Aegean, at least during the fourth–third century B.C. The few curse tablets from earlier than the fourth–third century B.C. point to the use of multiple and thus isotopically more variable lead sources compared with the Roman times.
Absolute Isotope Ratios
(2018)
Measurement results and scientific models leading to important decisions in forensics, food fraud or climatology are based on isotope ratio data. Molar masses of multi-isotopic elements are as well based on isotope ratio data. Thus, in the case of Si, isotope ratios directly impact the redefinition of the SI base units kilogram and mole. Therefore, new strategies are required leading to new primary isotope reference materials, whose isotope ratios are traceable to the SI. This in turn will ensure the comparability of isotope ratio data and will render the traceability exception requested by the CCQM superfluous. Such new procedures will be developed for the key elements S, Si, Ca, Sr and Nd at relative uncertainty levels of ≤ 0.01 %.
In chemical elements with three or more stable isotopes, mass-dependent stable isotope fractionation yields correlated isotope ratios, m2/m1 and m3/m1. In three-isotope space, i.e. in a δ’m2/δ’m1 vs. δ’m3/δ’m1 plot, data align along a slope θ, the so called ‘triple isotope fractionation exponent’ that scales the two isotope ratios. Theoretical calculations predict small differences in θ for kinetic- and equilibrium isotope fractionation (Young et al. 2002) and thus the precise measurement of θ allows constraining the reaction mechanism. However, due to an apparent lack of precision of stable isotope analysis by MC-ICP-MS, θ is merely used as analytical quality control, i.e. for demonstration that samples and standards plot within their analytical precision in the range of theoretical θ-values originating in δ-zero.
We show how θ can be determined precisely enough by MC-ICP-MS to distinguish kinetic- and equilibrium isotope fractionation, even when isotopic differences between samples are low. For low magnitudes of isotope fractionation, we exploit new, isotopically fractionated isotope standard materials (Vogl et al. 2016). We determine quality norms regarding interferences and measurement conditions to warrant trueness and to maximize precision. We exemplary explore the measurement of the three-isotope composition of Mg stable isotopes, budget the uncertainty of θ-values, and report the first θ-values of carbonate-water pairs and bioapatite. Our measurement approach adds a new dimension to isotope data beyond the δ-scale that has a high potential to reveal different modes of (bio)mineral precipitation in the sedimentary and biological record and thus to contribute solving conundrums in the Earth and Life Sciences.
Bulk enstatite chondrites (EC) and their achondritic counterpart, the aubrites, have the lightest measured Si isotope composition of all rocks in the solar system. Some authors proposed that the enrichment of 28Si in EC metal by equilibrium or kinetic isotope fractionation processes in the solar nebula may have led to the negative δ30/28Si of bulk EC. Because EC are also depleted in refractory elements, loss of a heavy Si component with refractory elements might be an alternative explanation. To further constrain their genesis, we analysed for the first time in situ Si-isotope ratios simultaneously with major- and trace element abundances in silicate and metal phases of Sahara 97072 (EH3) and Indarch (EH4).
This presentation gives a short overview of isotope ratio measurements being carried out in the past few years at BAM in different fields such as plant metabolism, food web structures and archaeology. The corresponding isotope reference materials which have been certified at BAM in parallel are presented as well. Additionally an outlook is being provided on future iRM projects.
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.
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.
Mass spectrometric methods (MC-ICP-MS and TIMS) are without doubt the working horse of stable isotope analysis. However, drawbacks of these methods include the high costs for instruments and their operation, experienced operators and elaborate chromatographic sample preparation which are time consuming.
We propose a lower-cost and faster optical alternative for the analysis of isotope ratios of selected elements: high-resolution continuum source molecular absorption spectrometry (HR-CS-MAS). Stable isotope amount compositions of boron (B) and magnesium (Mg) were determined based on the absorption spectra of in-situ generated mono-hydrides and halide (MH and MX) using graphite furnace HR-CS-MAS. The use of a modular simultaneous echelle spectrograph (MOSES) helps to find the maximal isotope shift in the diatomic molecular spectra produced in a graphite furnace by using isotopic spike solutions. Isotopes of boron (10B and 11B) were studied via their hydrides for the electronic transition X 1Σ+ → A 1Π. The spectrum of a given sample is a linear combination of the 10BH molecule and its isotopologue 11BH. Therefore, the isotopic composition of samples can be calculated by a partial least square regression (PLS). For this, a spectral library was built by using samples and spikes with known isotope composition. Boron isotope ratios measured by HR-CS-MAS are identical with those measured by mass spectrometric methods at the 0.15 ‰ level. Similar results were obtained for a multiple isotope system like Mg (24Mg, 25Mg, and 26Mg), where isotope shifts of their isotopologues can be resolved in the MgF molecule for the electronic transition X 2Σ → A 2 Πi. The extension of this methodology to other elements like Li, Ca, Cu, and Sr is discussed.
For the first time, an international comparison was conducted on the determination of the purity of a high purity element. Participants were free to choose any analytical approach appropriate for their institute’s applications and services. The material tested was a high purity zinc, which had earlier been assessed for homogeneity and previously used in CCQM-K72 for the determination of six defined metallic impurities. Either a direct metal assay of the Zn mass fraction was undertaken by EDTA titrimetry, or an indirect approach was used wherein all impurities, or at least the major ones, were determined and their sum subtracted from ideal purity of 100 %, or 1 kg/kg. Impurity assessment techniques included glow discharge mass spectrometry, inductively coupled plasma mass spectrometry and carrier gas hot extraction/combustion analysis. Up to 91 elemental impurities covering metals, non-metals and semi-metals/metalloids were quantified. Due to the lack of internal experience or experimental capabilities, some participants contracted external laboratories for specific analytical tasks, mainly for the analysis of non-metals. The reported purity, expressed as zinc mass fraction in the high purity zinc material, showed excellent agreement for all participants, with a relative standard deviation of 0.011 %. The calculated reference value, w(Zn) = 0.999 873 kg/kg, was assigned an asymmetric combined uncertainty of + 0.000025 kg/kg and – 0.000028 kg/kg. Comparability amongst participating metrology institutes is thus demonstrated for the purity determination of high purity metals which have no particular difficulties with their decomposition / dissolution process when solution-based analytical methods are used, or which do not have specific difficulties when direct analysis approaches are used. Nevertheless, further development is required in terms of uncertainty assessment, quantification of non-metals and the determination of purity of less pure elements and/or for those elements suffering difficulties with the decomposition process.
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.
For the first time polyethylene (PE) frits were used to quantify sulphur in copper metal and its alloys by isotope dilution combined with LA-ICP-MS: an alternative approach for sample preparation. The properties of the PE frit meet the requirements for isotope dilution LA-ICPMS which are porous material, thermal and chemical resistance and high absorption efficiency. The breakthrough, however, as a support material, is the low sulphur blank, which is only two times of the gas flow blank (2.3-4.0 x 10⁴ cps). Additionally, the porosity of the frit was considered, as it directly affects the absorption efficiency for the sample solution, which is present in the cavities of the frit.
The absorption efficiency was studied by loading sulphur standards with varying sulphur amounts (0 - 80 µg S) onto the frits. The remaining sulphur which was not absorbed by the frit was rinsed off and was measured by ICP-MS. This indirect method shows that more than 99.5 % of the loaded sulphur was absorbed by the frit. The so prepared frits with increasing sulphur amount were measured by LA-ICP-MS showing a good linearity between 0 µg S and 40 µg S with a coefficient of determination, r2 of 0.9987 and a sensitivity of 3.4x10⁴ cps µgˉ¹ for 32S.
Three copper reference materials produced by BAM (BAM-M376a, BAM-228 and BAM-227) were selected to develop and validate the LA-ICP-IDMS procedure. The IDMS technique was applied to these samples as follows: the samples were spiked, dissolved, digested and then the digested solution was absorbed on the frits. The dried frit samples were then analyzed by LA-ICP-IDMS and it could be demonstrated that the sample solution dispersed on the frits did not influence the 32S/34S ratio significantly even though the sulphur intensities were fluctuating along the scan lines. Relative standard deviations of the isotope ratios were below 5 % in average between 3 lines (except for the pure spike solution and procedure blank). The measurement results were validated by comparing them with the results obtained by conventional ICP-IDMS. Plotting the mass fraction of sulphur in copper obtained by LA-ICP-IDMS versus those obtained by ICP-IDMS yields a linear curve with a correlation coefficient of 0.9999 showing a strong agreement between both techniques.
The metrological traceability to the SI for the mass fraction of sulphur in copper is established by an unbroken chain of comparisons, each accompanied by an uncertainty budget. Thus, the measurement results are considered reliable, acceptable and comparable within the stated measurement uncertainty. The metrological traceability chain from the kg down to the final mass fraction in the samples obtained by LA-ICP-IDMS is illustrated in this presentation.
This is the first time that PE frits were used to quantify sulfur in copper and its alloys by isotope dilution combined with LA-ICP-MS: an alternative approach for sample preparation. The following properties of the PE frit meet the requirements for isotope dilution LA-ICPMS:: porous material, thermo plastic (melting point >100oC), chemical resistance (nitric acid >70%) and high adsorption efficiency. The breakthrough, however, as a support material, especially when comparing the PE frit with other materials such gelatin or sodium silicate is the low blank, which is only two times of the gas flow blank (2.3-4.0 x 104 cps). Additionally, the porosity of the frit was considered, as it directly affects the adsorption efficiency for the sample solution, which is present in the cavities of the frit.
Adsorption efficiency was studied by depositing sulfur standards with varying sulfur amounts (0, 2, 5, 10, 20, 40 and 80 µg S) on the frits. The remaining sulfur which was not absorbed by the frit was rinsed off and was measured by ICP-MS. This indirect method shows that more than 99.5 % of the loaded sulfur was absorbed by the frit. Such high absorption efficiency is completely sufficient for a support material to be used in LA-ICP-IDMS. The so prepared frits whith increasing sulfur amount were measured by LA-ICP-MS showing a good linearity between 0 µg S and 40 µg S whit a correlation coefficient r2 of 0.9987 and sensitivy of 3.4x104 cpsµg-1 for 32S.
Three copper reference materials produced by BAM were selected to develop and validate the LA-ICP-IDMS procedure. The IDMS technique was applied to these samples as follows: the samples were spiked, dissolved, digested and then the digest was adsorbed on the frits.
This is the first time that PE frits were used to quantify sulfur in copper and its alloys by isotope dilution combined with LA-ICP-MS: an alternative approach for sample preparation. The following properties of the PE frit meet the requirements for isotope dilution LA-ICPMS: porous material, thermo plastic (melting point >100°C), chemical resistance (nitric acid >70%) and high adsorption efficiency. The breakthrough, however, as a support material, especially when comparing the PE frit with other materials such gelatin or sodium silicate is the low blank, which is only two times of the gas flow blank (2.3-4.0 x 10⁴ cps). Additionally, the porosity of the frit was considered, as it directly affects the adsorption efficiency for the sample solution, which is present in the cavities of the frit.
Adsorption efficiency was studied by depositing sulfur standards with varying sulfur amounts (0, 2, 5, 10, 20, 40 and 80 µg S) on the frits. The remaining sulfur which was not absorbed by the frit was rinsed off and was measured by ICP-MS. This indirect method shows that more than 99.5 % of the loaded sulfur was absorbed by the frit. Such high absorption efficiency is completely sufficient for a support material to be used in LA-ICP-IDMS. The so prepared frits with increasing sulfur amount were measured by LA-ICP-MS showing a good linearity between 0 µg S and 40 µg S with a correlation coefficient r2 of 0.9987 and sensitivity of 3.4x10⁴ cps µgˉ¹ for 32S.
Three copper reference materials produced by BAM were selected to develop and validate the LA-ICP-IDMS procedure. The IDMS technique was applied to these samples as follows: the samples were spiked, dissolved, digested and then the digest was adsorbed on the frits.
Imaging of Eu doped very small iron oxide nanoparticle in atherosclerotic plaques via LA-ICP-MS
(2018)
Atherosclerotic lesions can be visualized by magnetic resonance imaging (MRI) using very small iron oxide nanoparticles (VSOP). VSOP accumulate in atherosclerotic plaques and thus serve as an atherosclerosis probe. The aim of this project was to image the distribution of europium doped VSOP (Eu-VSOP) by laser ablation ICP-MS in histological thin cuts of the aortic root region of ApoE knockout mouse model that is rich in atherosclerotic plaques. In addition, it was investigated whether VSOP accumulation in the plaques correlates with other biomarkers of inflammation for example macrophages and altered endothelium to assess whether it correlates with instability or vulnerability of the plaque regions. For this purpose, antibodies were labeled with various lanthanide elements and correlated with the Eu-VSOP distribution using LA-ICP-MS in a multiplex measurement mode.