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The interaction of nanoparticles (NPs) with cells has become a major field of interest, ranging from therapeutic applications to nanotoxicology. The cellular uptake depends on the primary characteristics of the NPs (e.g. size, shape, surface modification) and on the cells interacting with the particles. Thereby the quantification of NPs in cells is of particular importance to obtain information under different experimental conditions. Presently, the number of NPs internalized is often determined by inductively coupled plasma (ICP) optical emission spectrometry (OES) or ICP mass spectrometry (MS) after acid digestion of a cell suspension or a cell pellet. The result is an average value and no information about the distribution among cells or within a cell is available. Therefore we developed a method based on laser ablation (LA) in combination with ICP-MS to localise and quantify metallic NPs in single cells.
LA-ICP-MS is a powerful analytical method which offers excellent sensitivity at high spatial resolution and multielement capability without time-consuming sample preparation steps. Recently, LA-ICP-MS was established for elemental mapping of biological samples like tissues.
In our experiments, fibroblast cells were incubated with gold or silver containing nanoparticles and grown on sterile coverslips under standard conditions. For LA analysis the cells were fixed with formaldehyde and dried. Subcellular resolution is achived by careful optimisation of laser energy, ablation frequency and scan speed. The elemental distribution was determined by continuous ablation line by line of cells incubated with NPs. Our results show that LA-ICP-MS is able to detect NP aggregates within cellular substructures. After 24 h of incubation the NPs were found in the cytosol, preferencially in the perinuclear region, but do not enter the nucleus. Additionally, a quantification strategy at single-cell level was developed. For this purpose nitrocellulose membrane was spiked with Ag or Au nanoparticle suspension at different concentration levels and analysed by LA-ICP-MS. Based on this calibration the number of NPs taken up by individual cells was determined and variations within the cell population become visible. The cells show a strong dependence of NP uptake on concentration and incubation time.
Our results demonstrate the potential of LA-ICP-MS providing insight into NP uptake and intracellular distribution 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.
Inductively coupled plasma mass spectrometry (ICP-MS) is a well-established analytical method for multi-elemental analysis in particular for elements at trace and ultra-trace levels. It has found acceptance in various application areas during the last decade. ICP-MS is also more and more applied for detection in the life sciences. For these applications, ICP-MS excels by a high sensitivity, which is independent of the molecular structure of the analyte, a wide linear dynamic range and by excellent multi-element capabilities. Furthermore, methods based on ICP-MS offer simple quantification concepts, for which usually (liquid) standards are applied, low matrix effects compared to other conventional bioanalytical techniques, and relative limits of detection (LODs) in the low pg g−1 range and absolute LODs down to the attomol range.
In this chapter, we focus on new applications where the multi-element capability of ICP-MS is used for detection of lanthanoides or rare earth elements, which are applied as elemental stains or tags of biomolecules and in particular of antibodies.
Inductively coupled plasma mass spectrometry (ICP-MS) is a well-established analytical method for multi-elemental analysis in particular for elements at trace and ultra-trace levels. It has found acceptance in various application areas during the last decade. ICP-MS is also more and more applied for detection in the life sciences. For these applications, ICP-MS excels by a high sensitivity, which is independent of the molecular structure of the analyte, a wide linear dynamic range and by excellent multi-element capabilities. Furthermore, methods based on ICP-MS offer simple quantification concepts, for which usually (liquid) standards are applied, low matrix effects compared to other conventional bioanalytical techniques, and relative limits of detection (LODs) in the low pg g−1 range and absolute LODs down to the attomol range.
In this chapter, we focus on new applications where the multi-element capability of ICP-MS is used for detection of lanthanoides or rare earth elements, which are applied as elemental stains or tags of biomolecules and in particular of antibodies.