Gold nanoclusters as elemental label for the sequential quantification of apolipoprotein E and metallothionein 2A in individual human cells of the retinal pigment epithelium using single cell-ICP-MS

Gold nanoclusters (AuNCs) with a diameter of 1.99 nm on average were synthesized and applied as labels in immunoprobes for the determination of cytosolic proteins in individual human retinal pigment epithelium (HRPEsv) cells by single cell – inductively coupled plasma – mass spectrometry (sc-ICP-MS). For quantitative purposes, the number of gold atoms per immunoprobe (i.e., the amplification factor) was determined; 466 gold atoms on average were obtained. Human metallothioneins (MT), including the 2A isoform (MT2A), and apolipoprotein E (APOE) play an important role under inflammation and oxidation processes in the RPE. The new single biomarker strategy introduced was applied to the sequential determination of MT2A and APOE in HRPEsv cells under pro-inflammatory and control conditions through the development of immunoassays with the corresponding AuNCs immunoprobes and the measurement of the 197Au+ signal by sc-ICP-MS. In addition, 56Fe+ signal was measured as constituent element of HRPEsv cells in order to check the integrity of the cells after the immunoassay and to confirm the number of cell events detected when monitoring the protein label (197Au+). Optimisation of parameters related with the sample preparation for the analysis of cytosolic proteins in intact HRPEsv cells was carried out. The method was successfully applied to the determination of both proteins in control cells and cells treated with the recombinant human interleukin-1α. Quantitative results obtained per cell for the average protein amounts of APOE and MT2A using the sc-ICP-MS procedure were corroborated with commercial ELISA kits.