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Eingeladener Vortrag
- nein (69)
In this work, we report on the application of hyphenated gas source mass spectrometry to study and understand the mechanism of the reduction-distillation of ZnO using Al powder as reductant in its activated and inactivated form. The experiments revealed that the purity of the Zn metal produced were superior using activated Al with respect to inactivated Al, i.e., m5N8 (99.9998% metallic based) versus m5N3. The achieved purity levels of Zn and the absence of high volatile Cd, Mg, and Sb impurities in the gas phase and the material collected were explained with respect to the impurity elements free-energy values, vapor pressure data, and an observed scavenging effect of the Ta crucible, which was supported by the on-line observed mass spectrometric profiles of the residual gas.
Analytische Chemie 2008/2009
(2010)
An efficient solid phase extraction (SPE) method using bis-(2-ethylhexyl)-phosphate (HDEHP) coated reverse phase C18 support has been developed for the pre-concentration of Gadolinium (Gd) and Gd contrast agents widely used in magnetic resonance imaging (MRI). Enrichment of Gd in the ionic form has been compared with strong cation exchange material Chromabond SA (SCX), weak ion exchange material Chelex-100 and also with lanthanide specific HDEHP modified reverse phase C18. The determination of Gd and its complexes after enrichment were performed using inductively coupled plasma mass spectrometry (ICP-MS) and on the basis of 158Gd. Among the three SPE materials, HDEHP coated reverse phase C18 SPE has been found to be most efficient, yielding a hundredfold Gd enrichment with > 95% recovery for linear and cyclic contrast agents like Gd-DTPA (Magnevist), Gd-DOTA (Dotarem), Gd-BOPTA (Multihance), and Gd-BT-DO3A (Gadovist). The developed SPE method has been successfully applied to the surface water and waste water samples originated from different places in Berlin, Germany. The results were in good agreement with the results obtained with direct measurement with ICP-MS. The developed pre-concentration method can be efficiently used for the determination of trace levels of gadolinium in the environment even with less sensitive analytical techniques.
We report herein the utilization of a vacuum reduction distillation system (VRDS) coupled with mass spectrometry (MS) to characterize chemical transformations in thermal analysis. The system initially designed to monitor metal purification by distillation is applied for the first time to the catalytic reaction of in situ formed nickel oxide during the carbothermic reduction of zinc oxide.
Elementmassenspektrometrie und Metallomics als neue Herausforderung für die Lebenswissenschaften
(2009)
For the study of bladder cancer and the identification of respective tumor markers, blood and, in particular, urine constitute suitable sources of biological material, while both harboring their specific challenges for analytics concerning low-abundance biomarkers. Dissolved proteins and nucleic acids as well as cells and cell-bound molecules can be the analytes. In urine, exfoliated bladder tumor cells have to be identified and in blood, circulating tumor cells have to be detected among huge amounts of other cells. For the detection of both low-abundance cells and molecules, their specific enrichment prior to analysis is advantageous or even necessary. Adapted methods for the analysis of proteomes and subproteomes by 2D-gel electrophoresis, multidimensional chromatography and antibody arrays are discussed. Analysis of nucleic acid-based markers exploits the high amplification power of PCR and modified PCR combined with previous (subtransciptomes) or subsequent (microarray) enrichment to sensitively and specifically detect markers. DNA mutations, DNA-methylation status and apoptotic DNA fragments, as well as levels of ribonucleic acids including microRNAs, can be analyzed by means of these methods. Finally, the challenge of identifying circulating tumor cells and assigning them to their original tissue is critically discussed.
An immunoassay was developed for ochratoxin A (OTA) detection in wine and two completely different detection methods were compared: ICP-MS and photometry. For labelling, we applied secondary antibodies conjugated with gold nanoparticles for ICP-MS and with horseradish peroxidase for photometric detection, respectively. Detection limits of the assay were determined as 0.003 µg L-1 for both detection methods. OTA in wine was determined below the EU limit value of 2 µg kg-1. The measurement range was between 0.01 and 1 µg L-1 for ICP-MS and photometric detection. Assay precision detected by ICP-MS and photometry were similar showing that precision of the detection method has only a minor effect on total assay precision. By using BSA as a buffer additive to minimize nonspecific binding, we could also confirm that OTA strongly binds to BSA. In a comparison study of several additives, casein was found to be preferable.
A huge number of functions and reactions can be performed by proteins merely based on their amino acids and their spatial arrangement which results in a specific geometric and electronic environment. The diversity and efficiency of functions, however, can be increased by protein-bound cofactors which fulfil their task in close interaction with the surrounding amino acid side chains. Among these cofactors, metals play an important role. They can be bound to proteins directly via specific amino acids such as cysteine and histidine, for example in zinc finger proteins or in iron sulfur cluster proteins. Another possibility is their integration into the 3D-structure of a protein via a prosthetic group. Porphyrine or porphyrine-like molecules with copper or iron as central bound metal ions constitute important structures in this respect.
In this work the determination of Cd, Cu, Cr, Fe and Sb as organic and inorganic additives in in-house plastic materials (ABS, LDPE) using electrothermal vaporization combined with inductively coupled plasma optical emission spectroscopy is described. The influence of CCl2F2 as gaseous halogenation modifier was investigated. Especially for the carbide forming elements the sensitivity was improved and the memory effects were significantly reduced. Calibration was performed by external calibration and standard addition with aqueous standard solutions added directly into the sample boats. Absolute limits of detection (3s-criterion) range between 0.1 ng (Cd) and 9 ng (Fe) which corresponds to relative values of 0.1 mg kg-1 and 1.6 mg kg-1, respectively, taking 5 mg as typical sample mass. The detection limits are sufficient to monitor the element contents of plastic materials according to European directives such as European directive on the safety of toys. The developed ETV-ICP-OES method allows a fast analysis with a high sample throughput (3 minutes per analysis), low sample consumption and good trueness and precision for the analyzed elements. Sample preparation is reduced to ashing the samples in a muffle furnace. Furthermore, measurements are possible regardless of the chemical form in the additives. For verification the results obtained with the developed method were compared with measurement results of independent methods ICP-MS/OES after digestion. In addition Cd, Cr and Pb were determined in a solid plastic reference material BAM-H010 to confirm the applicability and accuracy of the method.
Speciation and imaging of Gd containing MRI contrast agents in environmental samples by ICP-MS
(2015)
Tributyltin is listed as one of the priority substances in the European Water Framework Directive (WFD). Despite its decreasing input in the environment, it is still present and has to be monitored. In the European Metrology Research Programme project ENV08, a sensitive and reliable analytical method according to the WFD was developed to quantify this environmental pollutant at a very low limit of quantification. With the development of such a primary reference method for tributyltin, the project helped to improve the quality and comparability of monitoring data. An overview of project aims and potential analytical tools is given.
A fast and mild method for iodine labelling of proteins is presented which is specifically designed and optimized for laser ablation (LA-)ICP-MS based proteomics. Single proteins (lysozyme, bovine serum albumin, cytochrome c and β-casein), whole proteomes (microsomal proteome of rats) and antibodies (anti-bovine casein, anti-bovine serum albumin) can be efficiently iodinated by means of potassium triiodide with minimal losses of antigen properties and antibody binding to iodinated proteins. A comparison with iodination by use of IODO-Beads is presented and it is shown that triiodide labelling is a fast, cheap and less laborious alternative without compromising the analytical figures of merit.
Hydrophilic interaction chromatography (HILIC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) were optimised for the direct determination of gadolinium-based contrast agents in tap water. In comparison to our previous work, a new developed zwitterionic HILIC column (ZIC-cHILIC) was used for speciation of Gd-containing contrast agents. The limit of quantification (LOQ) for the five contrast agents Gd-BOPTA, Gd-DPTA-BMA, Gd-BT-DO3A, Gd-DOTA and Gd-DTPA are in the range of 512 ng Gd per litre. Additionally, a new internal standard, Pr-DOTA, was investigated to correct intensity drifts, minor and major changes in the sample volumes and possible matrix effects. With the speciation method described, tap water samples from the area of Berlin were analysed and for the first time, three Gd species, Gd-BT-DO3A, Gd-DOTA and Gd-BOPTA, were found in tap water samples at concentrations of about 10–20 ng Gd per litre. These are the same Gd species which have been previously detected predominantly in surface waters of the Berlin area.
Technical background
(2015)
The major components of sector field instruments can be listed as follows and will be described in more detail in the following chapter. (Specific details for the different types of instruments will be given in the corresponding instrumental sections in Chapters 11-17.) The general principle of all mass spectrometers follows ionisation after sample introduction, Separation of the ions according to their mlz ratio and final detection at the detection unit. The transport and Separation of ions follow the principles of ion optics.
A micro-droplet generator (µDG) sample introduction system was coupled to a sector field ICP-MS instrument to investigate the analytical figures of merit with respect to single cell analysis. The sector field instrument was operated for the first time in a fast scanning mode (E-scan) with the shortest time resolution of 100 µs to measure the single droplet time resolved and using the original detector in a pulse counting mode without modification of the existing electronics. For reduction of the droplet diameter a triple pulse mode of the droplet generator was applied and a droplet diameter down to 23 µm has been achieved for this investigation with a 100% transport efficiency of droplets. Signal duration times of single droplets of less than 500 µs have been measured. Overall detection efficiencies in the range of 10-3 counts per atom have been achieved and absolute limits of detection range between 120 ag for Fe and 1.1 ag for Mg as a mean value from 1000 droplet events.
We have applied a micro droplet generator (µDG) for sample introduction of single selenized yeast cells into a sector field ICP-MS, which was operated in a fast scanning mode with sampling rates of up to 10 kHz, to measure single cells time resolved with 100 µs integration time. Selenized yeast cells have been used as a model system for preliminary investigation. The single cells to be measured have been embedded into droplets and it will be shown that the time duration of a single cell event always is about 400 to 500 µs, and thus comparable to the time duration of a droplet without a cell. A fixed droplet generation rate of 50 Hz produced equidistant signals in time of each droplet event and was advantageous to separate contribution from background and blank from the analytical signal. Open vessel digestion and a multielement analysis were performed with washed yeast cells and absolute amounts per single cell were determined for Na (0.91 fg), Mg (9.4 fg), Fe (5.9 fg), Cu (0.54 fg), Zn (1.2 fg) and Se (72 fg). Signal intensities from single cells have been measured for the elements Cu, Zn and Se, and histograms were calculated for about 1000 cell events. The mean elemental sensitivities measured here range from 0.7 counts per ag (Se) to 10 counts per ag (Zn) with RSD's from 49% (Zn) to 69% (Se) for about 1000 cell events.
In this work, sample losses of silver nanoparticles (Ag NPs) in asymmetrical flow field-flow fractionation (AF4) have been systematically investigated with the main focus on instrumental conditions like focusing and cross-flow parameters as well as sample concentration and buffer composition. Special attention was drawn to the AF4 membrane. For monitoring possible silver depositions on the membrane, imaging laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) was used. Our results show that the sample residue on the membrane was below 0.6% of the total injected amount and therefore could be almost completely avoided at low sample concentrations and optimized conditions. By investigation of the AF4 flows using inductively coupled plasma mass spectrometry (ICP-MS), we found the recovery rate in the detector flow under optimized conditions to be nearly 90%, while the cross-flow, slot-outlet flow and purge flow showed negligible amounts of under 0.5%. The analysis of an aqueous ionic Ag standard solution resulted in recovery rates of over 6% and the ionic Ag content in the sample was found to be nearly 8%. Therefore, we were able to indicate the ionic Ag content as the most important source of sample loss in this study.
ICP-MS is based on the formation of (preferentially monovalent positively)charged atomic ions in an inductively coupled Ar plasma at almost 10 000 K. The ions formed are transferred from the plasma source at ambient pressure into a mass Separator operated at high vacuum via a set of cones. The ions are separate«! according to their mass/charge ratio in the mass Separator (quadrupole, magnetic sector field or time-of-flight mass Separator). In most cases, the ions are detected using a secondaiy electron multiplier; in some set-ups (also) a Faraday cup can be used. Single-collector (scanning mass spectrometer usually used for quantitative elemental analysis) or multicollector (static Operation of mass spectrometer for precise isotope ratio analysis) configurations can be found.
A reversed phase high performance liquid chromatography coupled to an inductively coupled plasma mass spectrometer (HPLC-ICP-MS) approach in combination with isotope dilution analysis (IDA) for the separation and parallel quantification of nanostructured and ionic silver (Ag) is presented. The main focus of this work was the determination of the ionic Ag concentration. For a sufficient stabilization of the ions without dissolving the nanoparticles (NPs), the eluent had to be initially optimized. The determined Ag ion concentration was in a good agreement with results obtained using ultrafiltration. Further, the mechanism of the NP separation in the HPLC column was investigated. Typical size exclusion effects were found by comparing results from columns with different pore sizes. Since the recovery rates decreased with increasing Ag NP size and large Ag NPs did not elute from the column, additional interactions of the particles with the stationary phase were assumed. Our results reveal that the presented method is not only applicable to Ag NPs, but also to gold and polystyrene NPs. Finally, IDA-HPLC-ICP-MS experiments in single particle mode were performed to determine the particle cut-off size. The comparison with conventional spICP-MS experiments resulted in a similar diameter and particle size distribution.
Elemental imaging of biological samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) provides spatially resolved information on element distribution in thin sections. By rastering with a laser across the sample, a two-dimensional image of the elemental distribution can be reconstructed that shows the intensities of the respective elements. Using overlapping laser shots the area ablated from single cells or thin sections (thickness 5 to 10 µm) can be reduced significantly so that the pixel size of the intensity measurement is significantly reduced. Having in mind that a laser shot ablates thin biological samples completely, we can make use of a new concept for calibration in the laser ablation method: the concept of total consumption. This calibration strategy allows production of simple matrix matched standards and provides an internal standardisation by ink jet technology, where a metal containing inks is printed on as thin layer on top of a biological sample.
Different applications will be presented where our concepts have been applied. In the first example we used nanoparticle suspension of given particle numbers to quantify the uptake of metallic nanoparticles by biological cells. In the second example antibodies have been tagged by metals to measure protein expression in prostata cancer. In this approach application of house keeping proteins are investigated additionally to compensate variations in thickness and density of the biopsy samples. In the third application different nephrotoxic behaviour of Pt containing drugs have been investigated to study the local enrichement in kidney samples of mice treated with these three different compounds. Here the internal standard is required to allow intercomparisons between different individual mouse tissues.
At the end of the lecture future trends will be discussed for elemental microscopy.
Single particle ICP-MS (spICP-MS) has gained great influence in the analysis of engineered nanoparticles (NPs) due to its simplicity, speed and ability to obtain a particle number size distribution. Despite its many advantages, the method is hampered by matrix effects affecting the sensitivity of the instruments.
Consequently, over- or underestimated particle sizes might be obtained. To overcome these challenges, we present in this work the detection of both Ag isotopes with a quadrupole mass spectrometer for the application of isotopic dilution analysis (IDA) in combination with spICP-MS. Here, the isotopes are measured sequentially using the conventional spICP-MS integration time of 10 ms. Citrate stabilized Ag NPs of a spherical shape with the nominal diameters of 30, 40, 50 and 80 nm have been investigated.
The experimental concept of adding ¹⁰⁹Ag+ solutions to the NP suspensions resulted in the NP Spikes being only visible in the ¹⁰⁷Ag trace. Therefore, a maximum of 45% of the particles was detected compared to that by conventional spICP-MS. A modified mass flow equation was applied to determine the particle sizes, particle size distributions and particle number concentrations of various Ag NPs. The addition of different spike concentrations between 0.5 and 4 mg L⁻¹ ¹⁰⁹Ag resulted in similar particle diameters, suggesting that the calculated diameter might be independent of the spike concentration.
This would have the advantage that no size information would be needed before the analysis. By analyzing Ag NP suspensions in a simulated seawater matrix, we demonstrate its significant influence on the particle size determination using conventional spICP-MS. A lower transport efficiency of 6.1% was found in the matrix compared to 7.3% without the matrix. In our approach, the addition of the Matrix influenced the NP intensity stronger than the spike signal, resulting in slightly smaller diameters using IDA–spICP-MS with the matrix compared to the results without the matrix. On the other hand, the IDA–spICP-MS approach with the matrix can result in equivalent results for the particle sizes compared with
conventional spICP-MS using suspensions without the matrix. Due to the lower instrument sensitivity in the matrix, a diameter of 30 nm was found to be close to the detection limit of the instrument.
A reversed phase high performance liquid chromatography coupled to an inductively coupled Plasma mass spectrometer (HPLC–ICP-MS) approach in combination with isotope dilution analysis (IDA) for the separation and parallel quantification of nanostructured and ionic silver (Ag) is presented. The main Focus of this work was the determination of the ionic Ag concentration. For a sufficient stabilization of the Ions without dissolving the nanoparticles (NPs), the eluent had to be initially optimized. The determined Ag ion concentration was in a good agreement with results obtained using ultrafiltration. Further, the mechanism of the NP separation in the HPLC column was investigated. Typical size exclusion effects were found by comparing results from columns with different pore sizes. Since the recovery rates decreased with increasing Ag NP size and large Ag NPs did not elute from the column, additional interactions of the particles with the stationary phase were assumed. Our results reveal that the presented method is not only applicable to Ag NPs, but also to gold and polystyrene NPs. Finally, IDA-HPLC-ICP-MS experiments in single particle mode were performed to determine the particle cut-off size. The comparison with conventional spICP-MS experiments resulted in a similar diameter and particle size distribution.
Gold nanostructures that serve as probes for nanospectroscopic analysis of eukaryotic cell cultures can be obtained by the in situ reduction of tetrachloroauric acid (HAuCl4). To understand the formation process of such intracellularly grown particles depending on the incubation medium, the reaction was carried out with 3T3 fibroblast cells in three different incubation media, phosphate buffer, Dulbecco's Modified Eagle Medium (DMEM), and standard cell culture medium (DMEM with fetal calf serum). The size, the optical properties, the biomolecular corona, and the localization of the gold nanoparticles formed in situ vary for the different conditions. The combination of surface-enhanced Raman scattering (SERS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) microscopic mapping and transmission electron microscopy (TEM) provides complementary perspectives on plasmonic nanoparticles and non-plasmonic gold compounds inside the cells. While for the incubation with HAuCl4 in PBS, gold particles provide optical signals from the nucleus, the incubation in standard cell culture medium leads to scavenging of the toxic molecules and the formation of spots of high gold concentration in the cytoplasm without formation of SERS-active particles inside the cells. The biomolecular corona of nanoparticles formed in situ after incubation in buffer and DMEM differs, suggesting that different intracellular molecular species serve for reduction and stabilization. Comparison with data obtained from ready-made gold nanoparticles suggests complementary application of in situ and ex situ generated nanostructures for optical probing.
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.
Lecture 7: Speciation
(2017)
ICP-MS is a well-established analytical method which excels by high accuracy, high dynamic range and extremely low limits of detection for most metals. Furthermore ICP-MS offers a very high multi-element coverage so that many elements of the periodic table can be detected simultaneously. In this series of lectures, we want to focus on the historical developments, fundamentals, instrumentation and novel applications of ICP-MS in the life and material sciences.
Boron isotope amount ratios n(10B)/n(11B) have been determined by monitoring the absorption spectrum of boron monohydride (BH) in a graphite furnace using high-resolution continuum source molecular absorption spectrometry (HR-CS-MAS). Bands (0→0) and (1→1) for the electronic transition X1Σ+ → A1Π were evaluated around wavelengths 433.1 nm and 437.1 nm respectively. Clean and free of memory effect molecular spectra of BH were recorded. In order to eliminate the memory effect of boron, a combination of 2% (v/v) hydrogen gas in argon and 1% trifluoromethane in argon, an acid solution of calcium chloride and mannitol as chemical modifiers was used. Partial least square regression (PLS) for analysis of samples and reference materials were applied. For this, a spectral library with different isotopes ratios for PLS regression was built. Results obtained around the 433.1 nm and 437.1 nm spectral regions are metrologically compatible with those reported by mass spectrometric methods. Moreover, for the evaluated region of 437 nm, an accuracy of 0.15‰ is obtained as the average deviation from the isotope reference materials. Expanded uncertainties with a coverage factor of k = 2 range between 0.15 and 0.44‰. This accuracy and precision are compatible with those obtained by mass spectrometry for boron isotope ratio measurements.
The release of iron from iron nanoparticles (NPs) used as parenteral formulations appears to be influenced by the size and surface properties of the colloidal iron complex and the matrix. A clinically applied product Venofer® has been used as a model formulation to establish adequate analytical strategies to evaluate the fate of iron nanoparticles (NPs) in blood. First, the preparation was characterized by high resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS) and UV-vis absorption spectroscopy. This revealed the presence of monodisperse iron NPs with a hydrodynamic diameter of ∼15 nm and an iron core of ∼4 nm. Venofer® was then incubated with serum and whole blood in a quantitative study on the iron bioavailability from these NPs. Iron was speciated and quantified by using inductively coupled plasma mass spectrometry (ICP-MS). Iron solubilization levels of up to 42% were found in both fluids using isotope dilution of iron for quantification within the first hour of incubation even in the absence of the reticuloendothelial system. The monitoring of the iron-containing proteins present in serum was conducted by highperformance liquid chromatography with ICP-MS detection. It indicated that the dissolved iron ions are bound to transferrin. Quantitative speciation studies using isotope pattern deconvolution experiments concluded that the released iron saturated almost completely (up to 90%) the metal binding sites of transferrin. The remaining iron appeared also associated to albumin and, to a lesser extent, forming smaller sized particles. Thus, the methods presented here provide new insights into the fate of Venofer® nanoparticles and may be applied to other formulations.
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 metallic nanoparticles (Ag, Au) in single adherent cells (fibroblast cell line). Recently, we have developed staining techniques to image the protein (by a lanthanide containing chelate) and DNA (by an Ir-containing intercalator) distribution in single cells which can be combined with the nanoparticle studies. Additionally, we have developed an immunoassay by use of metal-tagged antibodies to identify the cell status of individual cells.
Using pneumatic nebulization or microdroplet generation, we have also studied the up-take of nanoparticles and toxic metals as well as essential elements in single suspension cells using different ICP-MS mass spectrometric concepts (sector field instrument, triple-quad instrument, time of flight (CyTOF) instrument). For this purpose, the ICP-MS was operated in the single particle mode. First results of single cell ICP-MS (scICP-MS) will be presented for up-take of metallic (Ag) nanoparticles by THP 1 and macrophage cells and the strength of the different instruments will be discussed.
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.
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.
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.
Previously applied methods for the quantification of sulphur in copper and other pure metals revealed a lack of SI-traceability and additionally showed inconsistent results, when different methods were compared.
Therefore, a reference procedure is required which allows SI-traceable values accompanied by a Sound uncertainty budget. In this study a procedure was developed for the quantification of total sulphur in copper at low concentration levels using inductively coupled plasma-isotope dilution mass spectrometry (ICP-IDMS). The major part of the copper matrix was separated by adding ammonia which forms a complex with the copper while releasing the sulphur followed by chromatographic separation using a weak cation resin. After that the sulphur fraction was further purified by chromatographic means using first an anion exchange method and second a chelating resin. The developed procedure shows high performance, especially concerning high efficiency in matrix removal (>99.999%) while keeping the recovery of sulphur above 80%. Procedure blanks are in the order of 3–53 ng resulting in LOD and LOQ values of 0.2 mg g1 and 0.54 mg g1, respectively. The procedure is sufficient to facilitate value assignment of the total sulphur mass fraction in reference materials. Additionally, relative measurement uncertainties were calculated to be below 1% and the measurement results were traceable to the SI. The procedure reported in this study is a new reference procedure for sulphur measurement in copper, being fit for two major purposes, certification of reference materials and assignment of reference values for inter-laboratory comparison.
An overview about different analytical approaches will be presented of how to detect metals in individual biological cells by use of ICP-MS. For this purpose, we are using different sample introduction systems for ICP-MS for detection, imaging and quantification of metals at cellular levels.
By use of laser ablation, we have studied the up-take by and distribution of nanoparticles in single cells. Recently we have developed staining techniques to measure protein and DNA content of cells and identifying the cell status by immunoassays using metal-tagging of antibodies. New research based on cell arrays will be shortly discussed.
Using pneumatic nebulization and microdroplet generation, we have also studied the up-take of nanoparticles and toxic metals as well as essential elements in single cells using different ICP-MS mass spectrometric concepts (sector field instrument, triple-quad instrument, time of flight (CyTOF) instrument).
The different ICP-MS based methods will be compared concerning their analytical figures of merit and their strengths and weaknesses will be evaluated.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been revealed as a convenient technique for trace elemental imaging in tissue sections, providing elemental 2D distribution at a quantitative level. For quantification purposes, in the last years several approaches have been proposed in the literature such as the use of CRMs or matrix matched standards. The use of Isotope Dilution (ID) for quantification by LA-ICP-MS has been also described, being mainly useful for bulk analysis but not feasible for spatial measurements so far. In this work, a quantification method based on ID analysis was developed by printing isotope-enriched inks onto kidney slices from rats treated with antitumoral Pt-based drugs using a commercial ink-jet device, in order to perform an elemental quantification in different areas from bio-images. For the ID experiments ¹⁹⁴Pt enriched platinum was used. The methodology was validated by deposition of natural Pt standard droplets with a known amount of Pt onto the surface of a control tissue, where could be quantified even 50 pg of Pt, with recoveries higher than 90%. The amount of Pt present in the whole kidney slices was quantified for cisplatin, carboplatin and oxaliplatin-treated rats. The results obtained were in accordance with those previously reported. The amount of Pt distributed between the medullar and cortical areas was also quantified, observing different behavior for the three drugs.
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.
We have developed lanthanide labeling strategies for antibodies to adapt conventional biochemical workflows like Western blot immunoassays for detection by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis with a special interest to apply the multi-element capabilities of ICP-MS for the design of multiplexed immunoassays. In this paper the lanthanide labeling of antibodies with MeCAT was investigated and the reaction conditions were optimized for application in a Western blot immunoassay analyzed by LA-ICP-MS. Furthermore, the MeCAT labeling strategy was compared with two other commercially available labeling reagents, MAXPAR and SCN-DOTA. As a proof-of-principle experiment chemically induced alterations of cytochrome P450 protein expression were investigated and the suitability of the differentially labeled antibodies for Western blot immunoassays of a complex liver microsomal protein fraction was tested. Limits of detection (LODs) in the lower fmol range were reached in the Western blot application using MeCAT and MAXPAR as element labeling reagents, whereas even sub-fmol LODs can be achieved in a dot blot experiment for the pure antibodies.
The β-secretase or β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the enzyme responsible for the formation of amyloid-β peptides, which have a major role in Alzheimer pathogenesis. BACE1 has a transmembrane sequence (TMS), which makes it unique among related proteases. We noticed that the BACE1 TMS contains an uncommon sulfur-rich motif. The sequence MxxxCxxxMxxxCxMxC spans the entire TMS, resembles metal ion binding motifs, and is highly conserved among homologues. We used a synthetic 31-mer model peptide comprising the TMS to study metal ion binding and oligomerization. Applying diverse biochemical and biophysical techniques, we detected dimer and trimer formation of the TMS peptide with copper ions. Replacement of the central Cys466 by Ala essentially abolished these effects. We show that the peptide undergoes a redox reaction with copper ions resulting in a disulfide bridge involving Cys466. Further, we find peptide trimerization that depends on the presence of monovalent copper ions and the sulfhydryl group of Cys466. We identified Cys466 as a key residue for metal ion chelation and to be the core of an oligomerization motif of the BACE1-TMS peptide. Our results demonstrate a novel metal ion controlled oligomerization of the BACE1 TMS, which could have an enormous therapeutic importance against Alzheimer disease.