Aptamer-based colorimetric determination of early-stage apoptotic cells via the release of cytochrome c from mitochondria and by exploiting silver/platinum alloy nanoclusters as a peroxidase mimic.

An enzyme-free aptameric nanosensor is presented for apoptosis assay. The method exploits the peroxidase-mimicking property of silver/platinum alloy nanoclusters (Ag/Pt NCs) and uses a Cyt c binding ssDNA aptamer. An extra-strand polycytosine (C14) aptamer was designed as a template for synthesis of the Ag/Pt NCs. If cell lysate or purified Cyt c is placed in a polystyrene microplate, Cyt c will bind to the surface of the wells of a microtiterplate. On addition of Apt@Ag/PtNCs, it will associate with Cyt c and then catalytically oxidize colorless tetramethylbenzidine (TMB) in the presence of H2O2 to give a blue colored oxidation product (TMBox) due to the peroxidase-mimicking property of the Ag/Pt NCs. Under optimal conditions, the absorbance of TMB at 660 nm is linearly enhanced as the concentration of Cyt c increases from 50.0 fM to 500 nM, and the detection limit is ~10 pM. The assay is simple, sensitive and cost effective in that it is enzyme-free, antibody-free and label-free. Graphical abstractSchematic diagram of the apoptosis assay on the basis of microplate well-coated mitochondrial cytochrome c releasing by using Aptamer@Ag/Pt NCs.