Sensitive method for determination of protein and cell concentrations based on competitive adsorption to nanoparticles and time-resolved luminescence resonance energy transfer between labeled proteins

A sensitive mix-and-measure method for the determination of protein and cell concentrations was developed. It is based on the competitive adsorption between the analyte and donor- and acceptor-labeled proteins to carboxylate-modified polystyrene nanoparticles. A high time-resolved luminescence resonance energy transfer (TR-LRET) signal is detected in the absence of the analyte due to the close proximity of the nanoparticle-adsorbed labeled proteins. The increased concentration of the analyte decreases the adsorption of the labeled proteins, leading to the loss of proximity and thus a decrease in the TR-LRET. The detection limit of the assay was 2.6 ng of proteins, which is higher than that of the most sensitive commercial methods. The method was also applied to cell counting, and 200 eukaryotic cells were measured in a microtiter well under the optimized conditions. The average coefficient of variation for both developed assays was approximately 10%, and the protein-to-protein variability for 11 different proteins was no more than 20%. The developed method requires no labeled particles, making the concept optimally applicable to varying targets as the material of the particle may be selected according to the application.