Simultaneous determination of proteins with micrometric resolution is a significant challenge. In this study, laser ablation (LA) inductively coupled plasma -mass spectrometry (ICP-MS) was employed to quantify the distribution of proteins associated to the eye disease age-related macular degeneration (AMD) using antibodies labelled with three different metal nanoclusters (MNCs). PtNCs, AuNCs and AgNCs contain hundreds of metal atoms and were used to detect metallothionein 1/2 (MT1/2), complement factor H (CFH) and amyloid precursor protein (APP) in retina, ciliary body, retinal pigment epithelium (RPE), choroid and sclera from human cadaveric eye sections. First, the labelling of MNCs bioconjugated primary antibodies (Ab) was optimised following an immunolabelling protocol to avoid the non-specific interaction of MNCs with the tissue.Then, the LA and ICP-MS conditions were studied to obtain high-resolution images for the simultaneous detection of the three labels at the same tissue section. A significant signal amplification was found when using AuNCs, AgNCs and PtNCs labelled Ab of 310, 723 and 1194 respectively. After the characterisation of MNCs labelled immunoprobes, the antibody labelling was used for quantitative determination of MT1/2, CFH and APP in the RPE-choroid-sclera, where accumulation of extracellular deposits related to AMD was observed, suggesting that this method is fully suitable for the simultaneous detection and quantification of at least three different proteins.

Laser ablation (LA) coupled to ICP-MS is regarded as a versatile tool for direct trace elemental and isotopic analysis of solids. The development of new strategies for quantitative elemental mapping of biological tissues is one of the growing research areas in LA-ICP-MS. On the other hand, latest advances are related to obtaining not only elemental distribution of heteroatoms but also molecular information. In this vein, mapping of specific proteins in biological tissues can be carried out with LA-ICP-MS by using metal-labelled immunoprobes. However, although LA-ICP-MS is in principle a quantitative technique, critical requirements should be met for absolute quantification of protein distribution. In this review, progress based on the use of metal-labelled antibodies for LA-ICP-MS mapping of specific proteins are tackled. Critical requirements to obtain absolute quantitative mapping of specific proteins by LA-ICP-MS are highlighted. Additionally, illustrative examples with the advances carried out so far using LA-ICP-MS are collected.

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The authors describe the use of platinum nanoclusters (PtNCs) as bimodal labels in a competitive immunoassay for immunoglobulin E (IgE). Both fluorometry and inductively coupled plasmamass spectrometry (ICP-MS) are used. Optimization of the PtNCs synthesis process using lipoic acid as ligand was carried out. The time for synthesis and the effect of NaOH added to the PtNCs precursor mixture was optimized with the aim to obtain PtNCs with strong fluorescence and low size dispersity. Maximal fluorescence was obtained at excitation/emission wavelengths of 455/620 nm. The average diameter (1.5 nm) and crystal structure (face-centered cubic structure) of the PtNCs were determined by HR-TEM. It was calculated that each PtNC contains 116 Pt atoms at average. Labelling of the antibody (Ab) against IgE with PtNCs was optimized in terms of recognition capabilities and fluorescence signal. A molar ratio (Ab:PtNCs) of 1:11 is found to be best. A competitive immunoassay for IgE was developed and detection was carried out by using both ICP-MS (by measuring 195 Pt) and fluorometry. The limit of detection (LOD) of the fluoroimmunoassay is 0.6 ng mL-1 of IgE. The LOD of the ICP-MS method is as low as 0.08 ng mL-1. The method was evaluated by analyzing four (spiked) serum samples by ICP-MS. No sample pretreatment excepting dilution is needed. Results compared favorably with those obtained by a commercial ELISA kit.

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