Fluorometric determination of the CCAAT/enhancer binding protein alpha by using gold nanoparticles and a labeled protein-binding DNA.

A method is described for the determination of the CCAAT/enhancer binding protein alpha (C/EBPα) which is a regulator in adipocyte differentiation. The method is based on quenching of the red fluorescence (with excitation/emission maxima at 548/562 nm) of Cy3-labeled DNA if it becomes adsorbed on positively charged gold nanoparticles (AuNPs). Fluorescently labeled dsDNA that can bind C/EBPα is introduced as a fluorescent probes. The dsDNA is electrostatically adsorbed on the positively charged AuNPs to quench their fluorescence. In the presence of C/EBPα, it will bind dsDNA which then diffuses away. The fluorescence of the AuNPs becomes restored. The fluorescent signal increases linearly in the 0.05 to 600 ng·mL μM C/EBPα concentration range, and the detection limit is 29 pg·mL. The method is specific and was applied to analyze cell lysates and in-situ. Graphical abstractSchematic representation of a fluorometric method for determination of the CCAAT/enhancer binding protein alpha (C/EBPα). Fluorescently labeled dsDNA that can bind C/EBPα is introduced as a fluorescent probes. The dsDNA is electrostatically adsorbed on the positively charged AuNPs to quench their fluorescence. In the presence of C/EBPα, it will bind dsDNA which then diffuses away. The fluorescence of the AuNPs becomes restored.