Iron Dysregulation in Alzheimer's Disease: LA-ICP-MS Bioimaging of the Distribution of Iron and Ferroportin in the CA1 Region of the Human Hippocampus.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by cognitive decline and neuropathological hallmarks, including ß-amyloid (Aß) plaques, Tau tangles, synaptic dysfunction and neurodegeneration. Emerging evidence suggests that abnormal iron (Fe) metabolism plays a role in AD pathogenesis, but the precise spatial distribution of the Fe and its transporters, such as ferroportin (FPN), within affected brain regions remains poorly understood. This study investigates the distribution of Fe and FPN in the CA1 region of the human hippocampus in AD patients with a micrometer lateral resolution using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). For this purpose, we visualized and quantified Fe and FPN in three separated CA1 layers: stratum molecular-radial (SMR), stratum pyramidal (SP) and stratum oriens (SO). Additionally, chromogenic immunohistochemistry was used to examine the distribution and colocalization with Tau and Aß proteins. The results show that Fe accumulation was significantly higher in AD brains, particularly in SMR and SO. However, FPN did not present significantly changes in AD, although it showed a non-uniform distribution across CA1 layers, with elevated levels in SP and SO. Interestingly, minimal overlap was observed between Fe and FPN signals, and none between Fe and areas rich in neurofibrillary tangles (NFTs) or neuritic plaques (NP). In conclusion, the lack of correlation between Fe and FPN signals suggests complex regulatory mechanisms in AD Fe metabolism and deposition. These findings highlight the complexity of Fe dysregulation in AD and its potential role in disease progression.

Bimodal determination of immunoglobulin E by fluorometry and ICP-MS by using platinum nanoclusters as a label in an immunoassay.

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 plasma - mass spectrometry (ICP-MS) are used. Optimization of the PtNCs synthesis 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 red 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 intensity. 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 195Pt) 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. Graphical abstract Schematic representation of the bimodal quantification (fluorescence and ICP-MS) of immunoglobulin E (Ig E) in human serum using antibody against human Ig E, labelled with several platinum nanoclusters (NCs) as immunoprobe. Elemental mass spectrometry (MS) allows high amplification of the signal because of the high number of platinum atoms per nanocluster (~116 Pt/NC).

Fluorescent silver nanoclusters as antibody label in a competitive immunoassay for the complement factor H.

Silver nanoclusters (AgNCs) were investigated as labels for the development of a fluoroimmunoassay for the complement factor H (CFH). The reductive one-pot synthesis of AgNCs using lipoic acid as a ligand was optimized by varying the concentration of NaBH4, the temperature and the reaction time. The average diameter and crystal structure of the AgNCs (which display red fluorescence) were determined by HR-TEM. The silver concentration was quantified by ICP-MS. Labelling of the antibody against CFH with AgNCs was optimized. The antibody was labeled with the AgNCs without compromising the recognition capabilities of the antibody. A competitive fluoroimmunoassay was then developed. Fluorescence is measured at excitation/emission maxima of 430/660 nm. The assay has a 0.4 ng mL-1 detection limit and a linear range that extends from 1.2 to 23 ng mL-1. The results compare favorably with those obtained by a commercial ELISA kit. The method was applied to the determination of CFH in spiked human serum. Graphical abstract Schematic presentation of the method for the determination of complement factor H (CFH) protein in human blood by using a CFH-antibody labelled with fluorescent silver nanoclusters.

Laser ablation ICP-MS for simultaneous quantitative imaging of iron and ferroportin in hippocampus of human brain tissues with Alzheimer's disease.

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 µm 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.

Quantitative mapping of specific proteins in biological tissues by laser ablation-ICP-MS using exogenous labels: aspects to be considered.

Laser ablation (LA) coupled with inductively coupled plasma mass spectrometry (ICP-MS) is 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, the latest advances are related to obtaining not only the elemental distribution of heteroatoms but also molecular information. In this vein, mapping of specific proteins in biological tissues can be done with LA-ICP-MS by use of 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 is reported. Critical requirements to obtain absolute quantitative mapping of specific proteins by LA-ICP-MS are highlighted. Additionally, illustrative examples of the advances made so far with LA-ICP-MS are provided. Graphical abstract In the proposed critical review, last advances based on the use of metal-labelled antibodies and critical requirements for LA-ICP-MS quantitative mapping of specific proteins are tackled.

Quantitative Imaging of Specific Proteins in the Human Retina by Laser Ablation ICPMS using Bioconjugated Metal Nanoclusters as Labels.

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 197Au+ 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.

Bioimaging of metallothioneins in ocular tissue sections by laser ablation-ICP-MS using bioconjugated gold nanoclusters as specific tags.

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.