Antifouling Surface Layers for Improved Signal-to-Noise of Particle-Based Immunoassays

• Published in: Langmuir 2009-12, Vol.25 (23), p.13510-13515
• Main Authors: Chen, Annie, Kozak, Darby, Battersby, Bronwyn J, Forrest, Robin M, Scholler, Nathalie, Urban, Nicole, Trau, Matt
• Format: Article
• Language: English
• Subjects: Animals; Biological Interfaces: Biocolloids, Biomolecular and Biomimetic Materials; Cattle; Chemistry; Colloidal state and disperse state; Exact sciences and technology; General and physical chemistry; GPI-Linked Proteins; Humans; Immunoassay - methods; Membrane Glycoproteins - analysis; Mice; Nanoparticles - chemistry; Polyethylene Glycols - chemistry; Sulfones - chemistry; Surface physical chemistry
• ISSN: 0743-7463; 1520-5827
• DOI: 10.1021/la903148n

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 × 10−2 molecules per nm2 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.