Single Cell-ICP-ToF-MS for the Multiplexed Determination of Proteins: Evaluation of the Cellular Stress Response.

An automated and straightforward detection and data treatment strategy for the determination of the protein relative concentration in individual human cells by single cell-inductively coupled plasma-time-of-flight mass spectrometry (sc-ICP-ToF-MS) is proposed. Metal nanocluster (NC)-labeled specific antibodies for the target proteins were employed, and ruthenium red (RR) staining, which binds to the cells surface, was used to determine the number of cell events as well as to evaluate the relative volume of the cells. As a proof of concept, the expression of hepcidin, metallothionein-2, and ferroportin employing specific antibodies labeled with IrNCs, PtNCs, and AuNCs, respectively, was investigated by sc-ICP-ToF-MS in human ARPE-19 cells. Taking into account that ARPE-19 cells are spherical in suspension and RR binds to the surface of the cells, the Ru intensity was related to the cell volume (i.e., the cell volume is directly proportional to (Ru intensity)3/2), making it possible to determine not only the mass of the target proteins in each individual cell but also the relative concentration. The proposed approach is of particular interest in comparing cell cultures subjected to different supplementations. ARPE-19 cell cultures under two stress conditions were compared: a hyperglycemic model and an oxidative stress model. The comparison of the control with treated cells shows not only the mass of analyzed species but also the relative changes in the cell volume and concentration of target proteins, clearly allowing the identification of subpopulations under the respective treatment.

Determination and localization of specific proteins in individual ARPE-19 cells by single cell and laser ablation ICP-MS using iridium nanoclusters as label.

Single cell-inductively coupled plasma-mass spectrometry (sc-ICP-MS) and laser ablation (LA)-ICP-MS have been complementary employed to develop a comprehensive study of APOE and claudin-1 expression in ARPE-19 cells submitted to a glucose treatment (100 mM, 48 h) that induces oxidative stress conditions. Results were compared with control cells. The determination of the two proteins by ICP-MS was sequentially carried out using specific immunoprobes labelled with IrNCs that offer a huge amplification (1760 ± 90 atoms of Ir on average). A novel sample introduction system, the microFAST Single Cell set-up, was employed for sc-ICP-MS analysis. This introduction system resulted in a cellular transport efficiency of 85 ± 9% for ARPE-19 cells (91 ± 5% using a PtNPs standard). After the proper immunocytochemistry protocol with the specific IrNCs immunoprobes in cell suspensions (sc-ICP-MS), the mass of APOE and claudin-1 in individual ARPE-19 cells was obtained. Average detection limits per cell by sc-ICP-MS were 0.02 fg of APOE and 3 ag of claudin-1. The results of sample analyses obtained by sc-ICP-MS were validated with commercial ELISA kits. The distribution of both target proteins in individual cells (fixated in the chamber wall) was unveiled by LA-ICP-MS. The high amplification provided by the IrNCs immunoprobes allowed the identification of APOE and claudin-1 within individual ARPE-19 cells. High resolution images were obtained using a laser spot of 2 × 2 µm.

Iridium nanoclusters as high sensitive-tunable elemental labels for immunoassays: Determination of IgE and APOE in aqueous humor by inductively coupled plasma-mass spectrometry.

Iridium nanoclusters (IrNCs) stabilized with citrate were synthesized and then the ligand was exchanged by lipoic acid (LA). The IrNCs@LA were bioconjugated through carbodiimide chemistry with specific antibodies to prepare IrNCs-labelled immunoprobes. The IrNCs-immunoprobes were employed in competitive immunoassays for immunoglobulin E (IgE) and apolipoprotein E (APOE) determination by detecting the iridium through inductively coupled plasma - mass spectrometry (ICP-MS). The IrNCs@LA have a 1.89 nm diameter at average and each NC contains 250 Ir atoms. Labelling of specific antibodies with IrNCs was optimized in terms of recognition capabilities and signal amplification by ICP-MS. Amplification and detection limits can be tuned by selecting the IrNCs:Ab molar ratio. An immunoprobe prepared by mixing a 10:1 IrNC:Ab molar ratio was selected for the determination of IgE and APOE in aqueous humor, achieving a signal amplification of 1760 iridium atoms per molecule of the sought protein and limits of detection in the tens of pg mL-1 of protein. The IrNCs-immunoprobes were evaluated for IgE determination in serum samples as well as for IgE and APOE in aqueous humor (from controls subjects and patients affected by primary open angle glaucoma) by ICP-MS, being required just sample dilution as pre-treatment. Results were corroborated with commercial ELISA kits.

Nanoparticles as labels of specific-recognition reactions for the determination of biomolecules by inductively coupled plasma-mass spectrometry.

Inductively coupled plasma - mass spectrometry (ICP-MS) is an essential tool for quantitative ultratrace elemental and isotopic determinations in the biological and biomedical fields. In addition, sensitive and multiplexed quantification of target biomolecules in biological fluids and single-cells by ICP-MS can be carried out using metal elements or their isotopes as labels of immunoreaction and hybridization specific-recognition reactions. Following similar strategies, the bioimaging of biomolecules in tissues and single-cells by laser ablation (LA)-ICP-MS can be also achieved. Sensitivity can be further increased by resorting to amplification strategies based on the use of labels containing several atoms of a given elemental (or isotopic) reporter, such as inorganic nanoparticles (NPs). In this review, we intend to highlight the progress achieved in this active research area. Following the introduction, a short report of the characteristics of the most relevant NPs used as labels of specific-recognition reactions for ICP-MS detection, as well as the most common labelling routes are given. Then, the applications of NPs-labelled detection probes used in combination with ICP-MS (either with liquid nebulization or LA sampling) for identification and determination of proteins and oligonucleotides in biological samples are thoroughly reviewed. In the conclusions section, the challenges faced and the expected advances in this topic are underlined.