Antifouling surface layers for improved signal-to-noise of particle-based immunoassays

Abstract

A 10-fold improvement in the signal-to-noise (S/N) ratio of an optically encoded silica particle-based immunoassay was achieved through incorporating a protein resistant poly(ethylene glycol) (PEG) surface layer and optimizing antibody immobilization conditions. PEG was activated using 2,2,2-trifluoroethanesulfonyl chloride (tresyl) and required a minimum reaction time of 1.5 h. The activated PEG had a reactive half-life of approximately 5 h when stored in acidified dimethyl sulfoxide (DMSO). By increasing the protein incubation time and concentration, a maximum antibody loading on the particle surface of 1.6 x 10(-2) molecules per nm(2) was achieved. The assay S/N ratio was assessed using a multiplexed multicomponent optically encoded species-specific immunoassay. Encoded particles were covalently grafted or nonspecifically coated with either bovine or mouse IgG for the simultaneous detection of complementary anti-IgG "target" or uncomplementary anti-IgG "noise". The versatility and potential as a serum-based assay platform was demonstrated by immobilizing either a polyclonal antibody or an engineered single-chain variable fragment (scFv) capture probe on particles for the detection of the ovarian cancer biomarker, mesothelin (MSLN). The MLSN antigen was spiked into PBS buffer or 50% human serum. Both capture probe orientations, and media conditions showed similar low level detection limits of 5 ng/mL; however, a 40% decrease in maximum signal intensity was observed for assays run in 50% serum.

Figure 1. A, B, C & D. Optimization of assay loading via tresyl mediated immobilization of IgG

A) Tresyl-activation of PEG with time and subsequent degree of immobilization of IgG onto the particle surface. B) Longevity of the activated PEG particles in acidified DMSO C) Incubation time of IgG with tresyl-activated PEG particles (▲) and non-specific adsorption onto non-activated PEG particles (■). D) Bovine (□) and Mouse (▲) IgG immobilization with increasing incubation concentrations. All curves are the combination of two independent experimental setups and errors represent the calculated experimental standard deviation of each point.

Figure 2. Signal-to-Noise Ratio of Multiplexed Antibody-anti-Antibody Immunoassay

Species specific assay capturing a fluorescent target anti-bovine IgG and non-target anti-mouse IgG from solution on particles with non-specifically immobilized (adsorbed) Bovine IgG (● ○) and PEGylated particles with covalently attached Bovine IgG (▲, Δ) filled symbols represent target signal and open symbols non-target noise signal.

Figure 3. Mesothelin Immunoassay using

A) covalently immobilized pAb for antigen capture and fluorescently labelled scFv for display, and B) covalently immobilized scFv for capture and fluorescently labelled pAb as display of increasing concentrations of MSLN spiked in PBS buffer (■), and 50% human serum / 50% PBS buffer (▲). The assay dynamic ranges are determined by non-specific adsorption of display agents onto PEGylated particle platform surface (background noise, ◊), and unmodified particles (expected maximum signal, ♦)