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Issue 12, 2016
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A fluorescence-based method to directly quantify antibodies immobilized on gold nanoparticles

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Abstract

The ability to evaluate antibody immobilization onto gold nanoparticles is critical for assessing coupling chemistry and optimizing the sensitivity of nanoparticle-enabled biosensors. Herein, we developed a fluorescence-based method for directly quantifying antibodies bound onto gold nanoparticles. Antibody-modified gold nanoparticles were treated with KI/I2 etchant to dissolve the gold nanoparticles. A desalting spin column was used to recover the antibody released from the nanoparticles, and NanoOrange, a fluorescent dye, was used to quantify the antibody. We determined 309 ± 93 antibodies adsorb onto a 60 nm gold nanoparticles (2.6 × 1010 NP mL−1), which is consistent with a fully adsorbed monolayer based on the footprint of an IgG molecule. Moreover, the increase in hydrodynamic diameter of the conjugated nanoparticle (76 nm) compared to that of the unconjugated nanoparticle (62 nm) confirmed that multilayers did not form. A more conventional method of indirectly quantifying the adsorbed antibody by analysis of the supernatant overestimated the antibody surface coverage (660 ± 87 antibodies per nanoparticle); thus we propose the method described herein as a more accurate alternative to the conventional approach.

Graphical abstract: A fluorescence-based method to directly quantify antibodies immobilized on gold nanoparticles

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Supplementary files

Article information


Submitted
25 Jan 2016
Accepted
20 Apr 2016
First published
20 Apr 2016

Analyst, 2016,141, 3851-3857
Article type
Paper

A fluorescence-based method to directly quantify antibodies immobilized on gold nanoparticles

S. L. Filbrun and J. D. Driskell, Analyst, 2016, 141, 3851
DOI: 10.1039/C6AN00193A

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