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Organisationseinheit der BAM
Eingeladener Vortrag
- nein (68)
The analysis of biomolecules requires highly sensitive and selective detection methods capable of tolerating a complex, biological matrix. First applications of biomolecule detection by ICP-MS relied on the use of heteroelements as a label for quantification. However, the combination of immunoassays and ICP-MS facilitates multiparametric analyses through elemental tagging, and provides a powerful alternative to common bioanalytical methods. This approach extends the detection of biomarkers in clinical diagnosis, and has the potential to provide a deeper understanding of the investigated biological system. The results might lead to the detection of diseases at an early stage, or guide treatment plans. Immunoassays are well accepted and established for diagnostic purposes, albeit ICP-MS is scarcely applied for the detection of immune-based assays. However, the screening of biomarkers demands high throughput and multiplex/multiparametric techniques, considering the variety of analytes to be queried. Finally, quantitative information on the expression level of biomarkers is highly desirable to identify abnormalities in a given organism. Thus, it is the aim of this review to introduce the fundamentals, and to discuss the enormous strength of ICP-MS for the detection of different immunoassays on the basis of selected applications, with a special focus on LA-ICP-MS.
Elemental imaging of biological samples (bio-imaging) using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) provides spatially resolved information on element distribution (qualitative and quantitative) in thin sections of biological samples. By rastering with a laser across the sample, a two-dimensional image of the elemental distribution can be reconstructed that shows the relative intensities of the respective elements. However the method is hampered by a lack of internal standards and quantification concepts, which will be discussed in this lecture in more detail.
In liquid analysis the internal standard is used for drift correction and calibration and thus it is required that the standard should have similar physical and chemical properties similar to the analyte element during the pneumatic nebulization process, the transport, ionization and transmission into the ICP-MS. In laser ablation it should correct additionally for differences in the ablation process by laser instabilities or changes of sample properties to compensate variations or drift effects during the LA process.
Actual research demonstrates that LA-ICP-MS is capable of being used as an imaging tool with cellular resolution. The aim of this investigation was the method development for LA-ICP-MS to extend the versatility to quantitative and multiplexing imaging of single eukaryotic cells. For visualization of individual cells selected, lanthanide-labeled antibodies were optimized for immuno-imaging of single cells with LA-ICP-MS. The molar content of the artificial introduced labels per cell was quantified using self-made nitrocellulose-coated slides for matrix-matched calibration and calculated amounts were in the range of 3.1 to 17.8 atmol per cell. Furthermore, the quantification strategy allows a conversion of 2D intensity profiles based on counts per second (cps) to quantitative 2D profiles representing the molar amount of the artificial introduced elemental probes per pixel for each individual cell.
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.
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.
We are using laser ablation (LA)-ICP-MS to image the local distribution of elements (metals and hetero-elements) directly or (metallo-)proteins by metal-tagged antibodies in cells and tissue indirectly. Different applications will be discussed to demonstrate the state of the art and to visualize the elemental distribution pattern in soft bio-materials (tissue, single cells). In the first application Pt-containing drugs for cancer treatment are investigated and elemental distribution pattern are shown for tissue samples from animal experiments. Different standardization and quantification schemes including isotope dilution analysis will be discussed. In the second application, which is dedicated to toxicological research, the up-take of nano-particles by single cells are discussed and metal containing stains are used to visualize the distribution of nano-particles, proteins and DNA in a single cell simultaneously. This information is correlated with the distribution of the nanoparticles to identify the cell compartments where nano-particles are enriched. Quantification schemes have been developed to transform the measured intensities into number of particles up taken by the cells. In the third and last application LA-ICP-MS is applied to visualize the local distribution of proteins, which are used as bio-markers for prostate cancer. For this purpose, biopsy samples from patients have been simultaneously stained by eight differently metal-tagged antibodies in a multiplex approach. Detection of house-keeping proteins serves as internal standards to overcome differences in protein expression. Additionally ink-jet printing of metal doped inks onto the surface of these tissue samples has been applied for internal standardization and drift corrections.
Finally future trends to develop an “elemental microscope” will be discussed.