Antifouling surface layers for improved signal-to-noise of particle-based immunoassays.
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.
Chen
Annie
A
Biomarker Research and Development Centre, Level 5 East, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD 4072, Australia.
Kozak
Darby
D
Battersby
Bronwyn J
BJ
Forrest
Robin M
RM
Scholler
Nathalie
N
Urban
Nicole
N
Trau
Matt
M
D000818:Animals; D002417:Cattle; D058851:GPI-Linked Proteins; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008562:Membrane Glycoproteins / Q000032:analysis; D000090204:Mesothelin; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D013450:Sulfones / Q000737:chemistry
D058851:GPI-Linked Proteins; C000715527:MSLN protein, human; D008562:Membrane Glycoproteins; C000715528:Msln protein, mouse; D013450:Sulfones; C032605:2,2,2-trifluoroethanesulfonyl chloride; D011092:Polyethylene Glycols; D000090204:Mesothelin