Real matrix-matched standards for quantitative bioimaging of cytosolic proteins in individual cells using metal nanoclusters as immunoprobes-label: A case study using laser ablation ICP-MS detection.

The persistent lack of adequate matrix-matched reference materials still hinders the quantitative analysis of elements and biomolecules in biological samples by LA-ICP-MS. This fact is especially critical in cell cultures due to their complex matrix. In this work, we propose a novel matrix-matched calibration strategy, which fully mimics the matrix of cultured cells, by using the same cell line of the sample to create laboratory standards. As a model case, the quantitative imaging of two cytosolic proteins (MT2A and APOE) in individual HRPEsv cells was performed by LA-ICP-MS, both in cells subjected to inflammation with cytokine Interleukin-1α (IL-1α) and controls (CT). A single biomarker strategy using Au nanoclusters (AuNCs) as specific antibody labels was employed for the analysis of the selected proteins in individual cells by LA-ICP-MS. HRPEsv cells supplemented with suspensions containing nude AuNCs was employed to generate single-cell laboratory standards (HRPEsv cells@AuNCs). The preparation and characterization of the single-cell laboratory standards by both ICP-MS and LA-ICP-MS were optimized as well as the data treatment protocol required for obtaining the quantitative distribution of the proteins in individual cells. The mass of APOE and MT2A per cell in CT and IL1α-treated HRPEsv cells analysed by LA-ICP-MS using the proposed matrix-matched calibration were successfully corroborated with commercial ELISA kits. In addition, quantitative real time polymerase chain reaction (qPCR) analyses were performed to study the proteins gene expression.