Abstract
The biomolecule interface is a key element in immunosensor fabrication, which can greatly influence the sensor performance. This paper explores the effects of surface epitope coverage of small molecule functionalized nanoparticle on the apparent affinity (avidity) of antibody in a competitive inhibition assay using bisphenol-A (BPA) as a model target. An unconventional two-antibody competitive inhibition ELISA (ci-ELISA) using thiolated BPA modified gold nanoparticles (cysBPAv-AuNP) as a competing reagent was devised for this study. It was shown that the antibody complexation with cysBPAv-AuNPs required a minimum number of surface epitopes on the nanoparticle to form a sufficiently strong interaction and reliable detection. The binding of cysBPAv-AuNP to anti-BPA antibodies, for limited antibody binding sites, was enhanced by a greater number of epitope-modified nanoparticles (cysBPAv-AuNP) as well as with higher epitope coverage. Increasing the molar concentration of epitope present in an assay enhanced the binding between anti-BPA antibodies and cysBPAv-AuNP. This implies that, to increase the limit of detection of a competitive inhibition assay, a reduced molar concentration of epitope should be applied. This could be achieved by either lowering the epitope coverage on each cysBPAv-AuNP or the assay molar concentration of cysBPAv-AuNP or both of these factors.
Highlights
Due to the size-dependent optical and electronic properties of gold nanoparticles (AuNPs), the volume of literature on their application in sensing is immense [1,2,3]
The purpose of this paper is to demonstrate the application of AuNPs modified with small molecule epitopes for binding to antibodies in a competitive inhibition format
We explored the interaction between an antibody and multivalent surface epitopes of a small molecule in a competitive inhibition assay
Summary
Due to the size-dependent optical and electronic properties of gold nanoparticles (AuNPs), the volume of literature on their application in sensing is immense [1,2,3]. AuNPs have been used to enhance refractive index changes [4, 5], increase the surface-volume ratio, provide high conductivity, and accelerate electron transfer to a redox active species [6,7,8]. The organic molecules could be a ligand or an epitope to which an antibody could bind. Amplification of analytical signals that are made possible by gold nanoparticles is even more important for small molecule detection than macromolecule detection. The antibody binding of small molecules to a biosensing interface is notoriously hard to detect otherwise. The vast majority of studies employing organic molecule functionalized AuNPs are motivated towards detecting protein [3, 8, 10] rather than small molecules such as pesticides, endocrine disruptors, and pharmaceutical or veterinary drugs
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