Effects of Surface Epitope Coverage on the Sensitivity of Displacement Assays that Employ Modified Nanoparticles: Using Bisphenol A as a Model Analyte.

Yang Lu,John Gooding,Nanju Lee,Joshua Peterson,Erwann Luais

doi:10.3390/bios6030043

Abstract

With the ever-increasing use of nanoparticles in immunosensors, a fundamental study on the effect of epitope density is presented herein, with a small molecule epitope, on the performance of the displacement assay format in an enzyme-linked immunosorbent assay (ELISA). Thiolated bisphenol A (BPA) functionalized gold nanoparticles (cysBPAv-AuNPs) and specific anti-BPA antibodies are employed for this purpose. It is shown that the displacement of cysBPAv-AuNPs bound to the immobilized antibodies was influenced by both the avidity of bound cysBPAv-AuNPs and the concentration of free BPA to displace it. The importance of surface epitope density was that it changed the number of epitopes in close proximity to the antibody-binding site. This then influenced the avidity of cysBPAv-AuNPs bound to the immobilized antibody. Furthermore, the molar epitope concentration in an assay appears to affect the degree of antibody binding site saturation. Controlling surface epitope density of the functionalized nanoparticles and molar epitope concentration in an assay leads to a decrease of the concentration of free BPA required to displace the bound cysBPAv-AuNP, and hence better assay performance with regards to the D50 value and dynamic range in the displacement assay.

Highlights

The fabrication of nanoparticles and the exploration of their properties have been increasingly utilized in many branches of science, including chemistry, biology, physics, and engineering [1,2,3,4,5].A particular focus on these studies has been the unique size-dependent electrical and optical properties relative to the equivalent bulk materials [6,7,8]
We investigated the impact of the affinity between the surface-bound epitope and the antibody (IgM) on the sensitivity and specificity of a displacement electrochemical immunosensor [16]
Assay a microwell onplate) antibody–surface epitope binding in a displacement assay and subsequent assay sensitivity, allowed us to modify and optimize experimental conditions quickly and and the experiments were conductedassay with using multiple replicates for better statisticalThis analysis

Summary

Introduction

The fabrication of nanoparticles and the exploration of their properties have been increasingly utilized in many branches of science, including chemistry, biology, physics, and engineering [1,2,3,4,5].A particular focus on these studies has been the unique size-dependent electrical and optical properties relative to the equivalent bulk materials [6,7,8]. Biosensors 2016, 6, 43 is that they could replace proteins as the platform upon which small molecule epitopes are attached This is an attractive notion, as surface functionalization of nanoparticles confers far greater control over epitope density—and potentially assay performance—than protein analogues. We turn our attention to asking questions regarding the effects of the density of nanoparticle-bound epitopes on displacement assays, rather than competitive inhibition assays, as explored previously [12]. This is because displacement assays are much more compatible with the development of reagentless immunosensors that do not require washing and rinsing steps, as all of the reagents are surface bound [13,14,15]

Methods

Results

Conclusion