Analytische Chemie
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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 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.