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 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.