Laser ablation inductively coupled plasma -mass spectrometry (LA-ICP-MS) is proposed for a better understanding of metals and proteins distribution in micrometre structures of human brain tissues. Simultaneous absolute quantitative imaging of Fe and ferroportin (FPN), in 5 m thick tissue sections of the stratum pyramidale of hippocampus CA1 region, was carried out for Alzheimer disease (AD) patients and healthy controls (HC). For the imaging of FPN by LA-ICP-MS, antibodies were labelled via carbodiimide crosslinking with fluorescent gold nanoclusters (AuNCs) of 2.2 nm diameter, enabling a high amplification (314 gold atoms per NC). Laboratory made gelatin standards containing Fe and Au were used for LA-ICP-MS calibration.Results showed that iron presents an increased concentration in AD donors compared with HC donors, whereas similar concentrations of FPN in AD donors with respect to HC donors were obtained. The average absolute FPN concentrations in selected areas obtained with the proposed AuNCs method were compared with the levels obtained by densitometric analysis with a traditional IHC approach, observing a similar trend in all cases.
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. Graphical abstract Gold nanoclusters (AuNCs) conjugated to a primary specific antibody serve as a label for amplified bioimaging of metallothioneins (MTs) by laser ablation coupled to inductively coupled plasma - mass spectrometry (ICP-MS) in human ocular tissue sections.
A sensitive methodology using antibody-conjugated gold nanoclusters (AuNCs) was developed for the quantitative bioimaging of specific proteins in biological tissues by laser ablation (LA) coupled to inductively coupled plasma-mass spectrometry (ICPMS). Determination of metallothioneins (MT1/2 protein isoforms) images in human retina tissue sections was carried out as a proof of concept. AuNCs used as label were conjugated to the selected antibody through carbodiimide coupling. A stoichiometry of AuNCs/available antibody of 1:1 was obtained. The high amplification provided by AuNC labels allowed for obtaining the distribution of MT1/2 in the neurosensory retina layers (5 μm thick sections) by LA-ICPMS. Elemental images of Au were quantified with gelatin matrix-matched standards and then converted to 2D quantitative images of MT1/2 concentration. For validation purposes, average concentrations of MT1/2 obtained in the human retinal layers by LA-ICPMS were successfully compared with those obtained with a commercial ELISA kit.
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.
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.
Antibodies conjugated with naturally abundant and isotopically enriched AgNCs are used for bioimaging of proteins in human retinal layers by LA-ICP-MS.
Flow injection analysis (FIA), developed for the automation of serial assays, has become a powerful tool most adequate for on‐line performing any sample preparation before final measurement (e.g. sample dissolution, dilutions, matrix removal, and analyte preconcentration). It is not surprising that the combination of FIA with atomic spectrometric techniques has enlarged the analytical potential of atomic methods and expanded their field of applications, allowing also for greener procedures. The variety of sample manipulation processes that can be covered today by FIA is amazing, and therefore, the general instrumentation required is reviewed in this contribution.
The description of the different flow strategies is carried out according to a hierarchy going from simple dilutions, reagent mixing, or standard additions, to more sophisticated flow manifolds such as those based on the use of two phases (e.g. gas–liquid, liquid–liquid, or solid–liquid) for separation/preconcentration purposes. Modern approaches allowing for on‐line decomposition/dissolution of solid samples (e.g. photo‐oxidation and microwave heating) are also described.
The coupling of the above‐mentioned flow methodologies to a variety of atomization/excitation/ionization sources (flames, quartz tubes, graphite furnaces, inductively coupled plasmas, microwave induced plasmas, glow discharges, etc.) is detailed, aiming to show the usefulness of this combination for atomic techniques based on either photon measurements (absorption, emission, and fluorescence) or ion measurements (mass spectrometry, MS).
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