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.