Bioconjugation and characterisation of gold colloid-labelled proteins
, Simon Attree, Robert Boyd, Neelam Kumarswami, James Noble
Mateusz Szymanski, Robert A. Porter
Biotechnology, Analytical Science, National Physical Laboratory, Teddington, Middlesex, TW11 0LW, UK
article info abstract
Received 16 November 2009
Received in revised form 12 February 2010
Accepted 17 February 2010
Available online 24 February 2010
Colloidal metal particles, in particular gold, have found many biological applications often as
probes in light and electron microscopy, and more recently since the 1980s in membrane-
based rapid immunoafﬁnity tests. The surface plasmon resonance absorbance properties in the
visible spectroscopy region of gold colloids make them useful tools in medical devices, as the
colloids are directly visible to the naked eye.
Despite the relative ease with which gold-protein conjugates can be prepared a major issue is
the manufacture of poor-quality and poorly characterised bioconjugates that can result in the
under performance of subsequent diagnostic tests. This paper describes the preparation of
good-quality conjugates for use in immunoassays by optimising the adsorption of antibodies
onto the surface of gold colloids, followed by their subsequent characterisation. The conjugates
were characterized for size, aggregation and quality using a range of techniques: UV–visible
(UV/Vis) absorption spectroscopy, transmission electron microscopy (TEM) and dynamic light
scattering (DLS). The biological activities of the conjugated products were also assessed using
an immunoassay format and electrochemical measurements.
By utilising a number of measurement techniques we aimed to gain a better understanding of
the extent of particle aggregation, and the resulting stability and activity of the biological
molecule on the surfaces of nanoparticles. The tools developed will enable researchers and
companies to ensure the sensitivity, quality and reproducibility of batches of nanoparticle bio-
Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
Colloidal metal particles, in particular gold, have charac-
teristically found biological applications as afﬁnity labels in
the diagnostic ﬁeld and also as probes in light and electron
microscopy (Horisberger, 1992; Sonvico et al., 2005; Jennings
and Strouse, 2007). Other prevalent areas of exploitation
include biosensors and drug delivery. The surface plasmon
resonance absorbance properties in the visible spectroscopy
region of metal colloids make them highly useful tools in
diagnostics, since the colloids are directly visible to the naked
eye, providing convenience for rapid testing. The wavelength
maxima at which these nanoparticles absorb and scatter light
depends on their size and shape, the surrounding medium
and their interaction with adjacent particles. Unlike ﬂuores-
cence or enzyme-detection systems, colloidal metal probes
are essentially inert and non-toxic with their optical
properties retained overtime, augmenting their use in
The performance of bio-nanoparticles in immunoassays
depends on both the chemical properties of the gold particles
as well as the bioactivity of the protein. Understandably the
Journal of Immunological Methods 356 (2010) 60–69
Abbreviations: ASV, Anodic stripping voltammetry; BSA, Bovine serum
albumin; cTnI, cardiac troponin I; DLS, Dynamic light scattering; EDTA,
Ethylenediaminetetraacetic acid; MUP, 4-Methylumbelliferyl phosphate
disodium salt; PdI, Polydispersity index; pI, Isoelectric point; RI, Refractive
index; Strep-AP, streptavidin–alkaline phosphatase conjugate; TEM, Trans-
mission Electron Microscopy; UV/VIS, Ultraviolet–visible.
⁎ Corresponding author. Tel.: +44 208 943 6927; fax: +44 20 8614 0573.
E-mail address: firstname.lastname@example.org (J. Noble).
Present Address: UCB-Celltech, NBE Molecular Evaluation ,216 Bath
Road, Slough, SL1 3EN.
0022-1759/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
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