Chemische Charakterisierung und Spurenanalytik
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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.
Inductively coupled plasma-mass spectrometry (ICP-MS) is a well-established analytical method offering high sensitivity and multi-element analysis. ICP-MS has found acceptance in various application areas ranging from material analysis to applications in the life sciences. Within the last 15 years new strategies for the sensitive detection and accurate quantification of biomolecules in complex biomedical samples have been developed. Recent instrumental improvements have contributed to this progress.
As most of the biomolecules do not contain endogenous metals etectable with ICP-MS, bioconjugation with artificial metal-containing tags based on metal-loaded chelate complexes or nanoparticles is increasingly applied to determine biomolecules indirectly. Especially, the combination of immunohistochemical workflows using lanthanoid-tagged antibodies and ICP-MS detection provides new insights in the complexity and interdependency of cellular processes. Single-cell ICP-MS, also termed as mass cytometry, allows high-dimensional analysis of biomarkers in cell populations at single-cell resolution. For that purpose, lanthanoid isotope labelled antibodies are used to detect their corresponding target molecules. The visualisation of the elemental distribution is possible with laser ablation ICP-MS (LA-ICPMS) at high spatial resolution. Especially, the combination of LA with ICP time-of-flight mass spectrometry, also referred to as imaging mass cytometry (IMC), opens new possibilities for multiparametric tissue imaging at the single-cell level and even below. The lanthanoid localisation and concentration can be linked to their conjugated antibody target providing valuable information about surface markers, intracellular signalling molecules to measure biological function, and the network state of an individual cell in a tissue.
This book chapter focuses on new applications, where the multi-element capabilities of ICP-MS are used for the detection of lanthanoids applied as artificial elemental stains or tags for biomolecules and in particular antibodies.