A fluorescence-based assay for nanogram quantification of proteins using a protein binding ligand

Abstract

Fluorescence emission has been investigated in the context of estimation of proteins at nanogram levels. A Schiff base ligand with donor–acceptor substituents has been utilized as a fluorescent probe. The potency of this ligand is that it possesses the binding sites for both hydrophobic as well as hydrophilic groups in the proteins. The fluorescence emission of the probe was enhanced in the presence of nanogram levels of protein, which clearly signifies that even the least concentration of the protein is sufficient to perturb the environment around the probe. We demonstrate here that the fluorescence characteristic of the probe can be utilized to estimate even nanogram levels (66 ng–1 μg mL^–1) of protein. The major limitation of the currently available standard methods is the range of protein estimation, which terminates at microgram level and the interference due to the specificity of the amino acids, which vary from proteins to proteins. This fluorescence emission-based method is free from interference from any type of buffers, ionic strength of the medium and any specific amino acid residue and is a simple, rapid, single-step, sensitive method of estimation which can be applied to different classes of proteins.