Sensitive Luminometric Method for Protein Quantification in Bacterial Cell Lysate Based on Particle Adsorption and Dissociation of Chelated Europium

摘要

A sensitive and rapid assay for the quantification of proteins, based on sample protein adsorption to Eu~(3+)-chelate-labeled nanoparticles, was developed. The lanthanide ion of the surface-conjugated Eu~(3+) chelate is dissociated at a low pH, decreasing the luminescence signal. The increased concentration of the sample protein prevents dissociation of the chelate, leading to a high luminescence signal due to the nanoparticle-bound protein. The assay sensitivity for the quantification of proteins was 130 pg for bovine serum albumin (BSA), which is an improvement of nearly 100-fold from the most sensitive commercial methods. The average coefficient of variation for the assay of BSA was 8percent. The protein-to-protein variability was sufficiently low; the signal values varied within a 28percent coefficient of variation for nine different proteins. The developed method is relatively insensitive to the presence of contaminants, such as nonionic detergents commonly found in biological samples. The existing methods tested for the total protein quantification failed to measure protein concentration in the presence of bacterial cell lysate. The developed method quantified protein also in samples containing insoluble cell components reducing the need for additional centrifugal assay steps and making the concept highly attractive for routine laboratory work.