One-step gold nanoparticle size-shift assay using synthetic binding proteins and dynamic light scattering

Gold nanoparticle (AuNPs) coupled dynamic light scattering (DLS) is a biosensing technique combining the light scattering properties of AuNPs with analyte recognition to produce an easy-to-use analytical tool. The principle of the assay is to immobilize bioreceptors specific to the target analyte onto AuNPs to produce nanobiosensors. When the analyte is added to the system, binding of target protein to the immobilized bioreceptors leads to size shift of the functionalised AuNPs. This specific shift in size can be quantified by DLS. It has proven effective in detecting multiple analytes such as metal ions, small chemicals and macromolecules. Previously, antibodies (Abs) have been used as bioreceptors in this technique. However, here we report the use of synthetic binding proteins, termed Affimer reagents, as bioreceptors. Anti-myoglobin Affimers were isolated by phage display and characterized by ELISA and surface plasmon resonance (SPR). Five Affimers showed strong binding to myoglobin and were immobilized onto AuNPs to produce the nanobiosensors for AuNPs coupled DLS assay. The nanobiosensors could selectively detect myoglobin with a limit of detection of 554 fM. AuNP size and concentration were tested and proved to be important factors in the technique.