Bioimaging of specific proteins by single cell laser ablation ICP-MS employing iridium nanoclusters as elemental labels

Resumen del trabajo presentado en el 15th European Workshop on Laser Ablation (EWLA 2022), celebrado en Berna (Suiza), del 12 al 15 de julio de 2022Cell populations in all biological systems are well-known for their heterogeneous nature. In fact, individual cells of the same line, under same physiological conditions and external stimuli, may show different biomolecule expression. Therefore, differences among cell populations can be difficult to elucidate unless biological systems are studied on a cell-by-cell basis. At present, there is a need for innovative analytical techniques that allow for the determination of elements and biomolecules in individual cells. In this work, LA-ICP-MS and single cell (sc) ICP-MS are presented as complementary strategies for the determination of specific proteins in cell cultures. On the one hand, LA-ICP-MS has demonstrated a huge potential for determining the spatial distribution of metals in individual cells. Although the bioimaging of endogenous cellular proteins still remains a challenge, an immunoassay in fixated cells using immunoprobes conjugated with an elemental label can be employed for proteins detection. On the other hand, sc-ICP-MS has been proposed for high-throughput cell-by-cell quantitative determination of target proteins within cell cultures [1]. Therefore, combining these two techniques it is possible to determine the concentration of sought proteins in single cells as well as to obtain their spatial distribution along the cell structures. In the proposed methodology, well characterized small iridium nanoclusters (IrNCs) providing a high signal amplification were employed as labels for the bioimaging of membrane and cytosolic proteins in individual retinal pigment epithelia cells (ARPE-19 cell line) by LA-ICP-MS (NWR193 laser equipped with a TwoVol2 ablation cell and DCI interface coupled to a 7900 ICP-MS). Protein quantification in individual cells was studied by sc-ICP-MS (microFAST Single Cell system coupled to a 7900 ICP-MS) and the proteins distribution was evaluated by LA-ICP-MS. The novel approach was based on immunoassays (cells fixed on chamber slides for LA-ICP-MS and cell suspensions for sc-ICP-MS) using IrNCs conjugated to specific antibodies for determination of the sought proteins under control and stress conditions (glucose treatment, 100 mM).This work was financially supported through project PID2019-107838RB-I00 Agencia Estatal de Investigación (AEI)/10.13039/501100011033) and project AYUD/2021/51289 - FICYT. P. Menero- Valdés acknowledges the FPU Grant (Ref. FPU19/00556; Ministry of Education)

Metal nanoclusters as elemental labels for the determination of specific proteins related to eye neurodegenerative diseases in individual cells by single cell and laser ablation ICP-MS

Resumen del trabajo presentado en el Colloquium Spectroscopicum Internationale XLII (CSI XLII), celebrado en Gijón (España), del 30 de mayo al 3 de junio de 202

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.This work was financially supported through projects PID2019- 107838RB-I00/Agencia Estatal de Investigacion ´ (AEI)/10.13039/ 501100011033) in Spain and AYUD/2021/51289 - FICYT. P. MeneroVald´es acknowledges the FPU Grant with Ref. FPU19/00556 (Ministry of Education, Spain). Authors would like to acknowledge David Clases (University of Graz, Austria) for the support with SPCaL software and the technical support provided by Servicios Científico-T´ecnicos of the University of Ovied

Towards Automated and High-Throughput Quantitative Sizing and Isotopic Analysis of Nanoparticles via Single Particle-ICP-TOF-MS

The work described herein assesses the ability to characterize gold nanoparticles (Au NPs) of 50 and 100 nm, as well as 60 nm silver shelled gold core nanospheres (Au/Ag NPs), for their mass, respective size, and isotopic composition in an automated and unattended fashion. Here, an innovative autosampler was employed to mix and transport the blanks, standards, and samples into a high-efficiency single particle (SP) introduction system for subsequent analysis by inductively coupled plasma–time of flight–mass spectrometry (ICP-TOF-MS). Optimized NP transport efficiency into the ICP-TOF-MS was determined to be >80%. This combination, SP-ICP-TOF-MS, allowed for high-throughput sample analysis. Specifically, 50 total samples (including blanks/standards) were analyzed over 8 h, to provide an accurate characterization of the NPs. This methodology was implemented over the course of 5 days to assess its long-term reproducibility. Impressively, the in-run and day-to-day variation of sample transport is assessed to be 3.54 and 9.52% relative standard deviation (%RSD), respectively. The determination of Au NP size and concentration was of 107Ag/109Ag particles (n = 132,630) over the course of the measurements was determined to be 1.0788 ± 0.0030 with high accuracy (0.23% relative difference) when compared to the multi-collector–ICP-MS determination