CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targetedflow-cytometric detection of acute myeloid leukaemia

Abstract

Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR)emitting nanosystems having a wide range of biomedical applications. Here, we reportthe development of a Au-NC based targeted fluorescent nano-bioprobe for theflow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ∼ 25–28 atoms showing bright red–NIR fluorescence (600–750 nm) and average size of ∼ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilizationin aqueous phase. The protein protected clusters were conjugated withmonoclonal antibody against CD33 myeloid antigen, which is overexpressed in ∼ 99.2% of the primitive population of AML cells, as confirmed by immunophenotypingusing flow cytometry. Au-NC-CD33 conjugates having average size of ∼ 12 nm retained bright fluorescence over an extended duration of ∼ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studiesrevealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effecton the cell viability and inflammatory response. Target specificity of the conjugates fordetecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ∼ 95.4% of leukaemia cells within 1–2 h compared to a non-specific uptake of ∼ 8.2% in human peripheral blood cells (PBMCs) which areCD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCsby leukaemia cells, thus confirming the flow cytometry results. This study demonstratesthat novel nano-bioprobes can be developed using protein protected fluorescentnanoclusters of Au for the molecular receptor targeted flow cytometry based detection andimaging of cancer cells.