Excessive nonspecific binding often occurs when labeling cells with immuno-labeled gold nanoparticles (IgG-AuNPs). We have investigated the physical properties of IgG-AuNPs assembled with three different protocols in an attempt to understand and eliminate this non-specific binding. One of these protocols involves conjugating the secondary antibody AP124F via van der Waals (vdW) and/or electrostatic forces to the AuNPs, and the other two employ a PEG-linker, OPSS-PEG-NHS (OPN). In all three protocols we follow with PEG-SH to provide protection against aggregation in saline solution. OPN and PEG-SH chains of varying molecular weights were examined in different combinations to determine the optimally protective layer. The hydrodynamic radius and surface plasmon resonance (SPR) were monitored at each stage of assembly using a dynamic light scattering (DLS) instrument and spectrophotometer, respectively. SPR measurements indicate a different physical structure near the gold surface when the PEG-linker is bound to gold first and then bound to the antibody second (AP124F-[OPN-Au]) rather than vice versa ([AP124F-OPN]- Au). These observed structural differences may lead to differences in the amount of non-specific binding observed when immuno-labeling cells. SPR measurements also yielded a half-life of 27 minutes for the binding of the PEG-linker to the surface of the AuNPs and a half-life of 133 minutes for the hydrolysis of the NHS functional groups on the OPN molecule. These different reaction rates led us to add AP124F 40 minutes after the linker began binding to the AuNPs, so that the antibody can bind covalently to the correct end of the OPN linker.

Date Published: 4 March 2014
PDF: 6 pages
Proc. SPIE 8934, Optical Coherence Tomography and Coherence Domain Optical Methods in Biomedicine XVIII, 89342V (4 March 2014); doi: 10.1117/12.2041714

Published in SPIE Proceedings Vol. 8934:
Optical Coherence Tomography and Coherence Domain Optical Methods in Biomedicine XVIII
Joseph A. Izatt; James G. Fujimoto; Valery V. Tuchin, Editor(s)