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

Gold nanoparticles (AuNPs) have attracted significant interest for biosensing applications because of their distinctive optical properties including light scattering. Dynamic light scattering (DLS) is an analytical tool used routinely for measuring the hydrodynamic size of colloids and nanoparticles in liquid environment. By combining the light scattering properties of AuNPs with DLS, a label-free, facile and sensitive assay has been developed. There have been several reports showing that NPcoupled DLS size shift assays are capable of quantitative analysis for target analytes ranging from metal ions to proteins as well as being a tool for biomolecular interaction studies. The principle of the assay developed is to immobilise bioreceptors (antibodies, oligonucleotides or synthetic binding proteins) specific to the target analyte onto AuNPs to produce nanobiosensors. When the analyte is added to the system, binding of the target protein to the immobilised bioreceptors leads to a size increase of the functionalised AuNPs. The hydrodynamic diameter (DH) can then be measured by DLS for complete quantitation. However, the ability to use synthetic binding proteins (Affimers) in optical sensing has not been investigated. Here, antimyoglobin (Mb) Affimers were selected by biopanning of a phage display library and subcloned into a bacterial plasmid for expression in a prokaryotic system. These Affimers were then expressed and characterised before being used as bioreceptors in the NP-coupled DLS size shift assay. The Affimer functionalised AuNPs were compared to those using polyclonal antibodies (IgG) as bioreceptors. The Affimer nanobiosensors could selectively detect Mb with a limit of detection of 554 fM when multiple Affimer clones were immobilized onto the AuNPs, which was comparable to IgG based nanobiosensors (LOD = 148 fM). These findings suggest that in general a polyclonal reagent is optimum for the assay. In addition, other factors, such as AuNP size and concentration, related to the assay were investigated. The detection range of the size shift assay could be tailored to each analyte by selecting the appropriate AuNP size and concentration. This fundamental data will serve as a base for future studies of using Affimers in DLS based sensing applications.