期刊
LANGMUIR
卷 25, 期 23, 页码 13510-13515出版社
AMER CHEMICAL SOC
DOI: 10.1021/la903148n
关键词
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资金
- Australian Research Council [FF0455861]
- National and International Research Alliances Program [RM2007001266]
- NCI [NCI P50 CA083636]
- Australian Research Council [FF0455861] Funding Source: Australian Research Council
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 imrnunoassay. 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.
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