Total protein concentration quantification using nanobeads with a new highly luminescent terbium( iii ) complex

Total protein concentration (TPC) is a key parameter in many biochemical experiments and its quantification is often necessary for isolation, separation, and analysis of proteins. A sensitive and rapid nanobead-based TPC quantification assay based on Förster Resonance Energy Transfer (FRET) has been developed. A new, highly luminescent Tb( iii ) complex has been synthesised and applied as donor in this FRET assay with an organic dye (Cy5) as acceptor. FRET-induced changes in luminescence have been investigated both at donor and acceptor emission wavelength using time-resolved luminescence spectroscopy with time-gated detection. In the assay, the Tb( iii ) complex and fine-tuned polyglycidyl methacrylate (PGMA) nanobeads ensure that an improvement in sensitivity and background reduction is achieved. Using 40 nm large PGMA nanobeads loaded with the Tb( iii ) complex, it is possible to determine TPC down to 50 ng mL −1 in just 10 minutes. Through specific assay components the sensitivity has been improved when compared to existing nanobead-based assays and to currently known commercial methods. Additionally, the assay is relatively insensitive to the presence of contaminants, such as non-ionic detergents commonly found in biological samples. Due to no need for any centrifugal steps, this mix-and-measure bioassay can easily be implemented into routine TPC quantification protocols in biochemical laboratories.