Studies on the attachment of DNA to silica-coated nanoparticles through a Diels-Alder reaction

Article abstract of DOI:10.1081/NCN-200059170

A new method has been investigated for the functionalization of gold nanoparticles with DNA. Silica-coated nanoparticles functionalized with a maleimide have been prepared. These particles are designed to react with modified DNA containing a diene functionality at one end of the molecule. The result would be the formation of a more stable attachment of the DNA to the particle through a Diels-Alder reaction. This covalent attachment would not be susceptible to ligand exchanges, which are known to occur in the conventional DNA functionalization of gold nanoparticles. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200059170

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STUDIES ON THE ATTACHMENT OF DNA TO SILICA-
COATED NANOPARTICLES THROUGH A DIELS-ALDER
REACTION
M. Proupin-Perez a , R. Cosstick a , L. M. Liz-Marzan b , V. Salgueiriño-Maceira b & M. Brust a
a Centre for Nanoscale Science, The University of Liverpool, Department of Chemistry ,
Liverpool, UK
b Department of Physical Chemistry , The University of Vigo , Vigo, Spain
Published online: 15 Nov 2011.
To cite this article: M. Proupin-Perez , R. Cosstick , L. M. Liz-Marzan , V. Salgueiriño-Maceira & M. Brust (2005) STUDIES ON
THE ATTACHMENT OF DNA TO SILICA-COATED NANOPARTICLES THROUGH A DIELS-ALDER REACTION, Nucleosides, Nucleotides
and Nucleic Acids, 24:5-7, 1075-1079, DOI: 10.1081/NCN-200059170
To link to this article: http://dx.doi.org/10.1081/NCN-200059170
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):1075–1079, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200059170
STUDIES ON THE ATTACHMENT OF DNA TO SILICA-COATED
NANOPARTICLES THROUGH A DIELS-ALDER REACTION
5
M. Proupin-Perez and R. Cosstick
Centre for Nanoscale Science, Department of
Chemistry, The University of Liverpool, Liverpool, UK
5
L. M. Liz-Marzan and V. Salgueiri˜no-Maceira
Department of Physical Chemistry,
The University of Vigo, Vigo, Spain
5
M. Brust
Centre for Nanoscale Science, Department of Chemistry, The University of
Liverpool, Liverpool, UK
5
A new method has been investigated for the functionalization of gold nanoparticles with DNA.
Silica-coated nanoparticles functionalized with a maleimide have been prepared. These particles are
designed to react with modified DNA containing a diene functionality at one end of the molecule. The
result would be the formation of a more stable attachment of the DNA to the particle through a Diels-
Alder reaction. This covalent attachment would not be susceptible to ligand exchanges, which are known
to occur in the conventional DNA functionalization of gold nanoparticles.
INTRODUCTION
Nucleic acid–coated gold nanoparticles are finding an increasing number of
diverse applications. In particular, the ability of the DNA to direct nanoparticle
assembly through hybridization can be used as the basis of a simple sensor system
in biomedical diagnostics[1] and as a means to form supramolecular structures.[2,3]
The conventional method for preparing DNA-functionalized gold nanoparticles
uses DNA chemically derived with either a 3’- or 5’- terminal thiol group and results
in the DNA bound to the surface of the particle through an S-Au bond.[4] Particle
stabilility increases with increasing DNA coverage, but hybridisation efficiency
decreases with increasing DNA coverage. This method requires the DNA to be
purified and isolated by conventional techniques prior to attachment to the gold. In
addition the thiol bond to the gold surface is not sufficient to accomplish a
permanent linkage, and in the presence of other thiols (e.g., DTT) equilibrium is
Address correspondence to M. Proupin-Perez, Centre for Nanoscale Science, Department of Chemistry, The
University of Liverpool, Crown St., Liverpool L69 7ZD, UK; E-mail: mproupin@liv.ac.uk or mproupin@yahoo.es
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M. Proupin-Perez et al.
established with dynamic ligand exchange. We have started to examine a more
direct method for the preparation of DNA functionalized gold particles in which the
DNA is bound to the surface through a permanent carbon–carbon bond. The
strategy involves attaching silica-coated gold particles to the 5’-end of DNA as the
final step in the automated synthesis using a Diels-Alder reaction. This procedure
should result in very stable gold nanoparticles that have a permanent attachment to
the DNA and can be isolated simply by centrifugation of the particles following
deprotection of the DNA.
RESULTS AND DISCUSSION
Gold nanoparticles are obtained by mixing an aqueous solution of a gold salt
with a solution of a stabilizing agent, citrate, which leads to reduction of the metal
and at the same time adsorbs to the gold surface, stabilizing the colloid through
electrostatic stabilizing forces. More stable nanoparticles are obtained by covering
the surface of gold with a silica shell.[5] The prevention of flocculation is more
effective than in the common stabilization with citrate as the electrostatic stabilizing
interactions are replaced by covalent bonds. The resulting silica-coated nano-
particles react easily with alkoxysilane derivatives through condensation of terminal
OH groups. In our case, a ligand containing a free maleimide has been attached to
the silica-coated nanoparticles. The gold nanoparticles have been covered in a first
step with 3-aminopropyltrimethoxysilane (APS) and a buffered solution of sodium
silicate. The molecules of APS are linked to the silica nanoparticles through the
amine group and the silane groups directed away from the particle surface. There
are variants of this method, for example, based in the use of sulphur instead
nitrogen as binding atom.[6] This generates a shell about 3 nm thick. In a second
step, condensation of the silica groups was performed through base-catalysed
hydrolysis and condensation of tetraethylorthosilicate (TEOS) in ethanol. With this
procedure a net around the particle is generated that avoids any ligand exchange.
The thicker the shell, the more homogenous in size and shape the nanoparticles
are. In this approach, nanoparticles with 60 nm of total diameter have been
prepared. After eliminating the excess ammonia and TEOS by centrifugation, N-
(propyltrimethoxysilane)maleimide was added in ethanol. The ligand was added in
a ratio of 1:20 (nanoparticle: ligand). After refluxing for 45 minutes, the excess of
the maleimide was eliminated by centrifugation. The presence of the maleimide
can be detected by the appearance of two absorption bands at 1,730 cmÀ1 in the
IR-spectrum due to the carbonyl groups (Figure 1).
The synthesis of two different maleimides has been studied: N-(propyltrime-
thoxysilane)maleimide, and N-(propyltriethoxysilane)maleimide. The synthesis of
these maleimide derivatives presented several difficulties due to the presence of
the silane groups. One of the most conventional strategies to synthesise maleimides
is the condensation of an anhydride and an amine at high temperature.[7] Thus,
the dienophile was synthesised in two steps (see Figure 2). In the first step,

M. Proupin-Perez et al.
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FIGURE 1 Synthesis of silica-coated gold nanoparticles.
N-propyltriethoxysilane is added to maleic anhydride in dichloromethane to obtain
N-(propyltriethoxisilane)maleamic acid as an intermediate. The reaction took place
readily (1 h) and the product was isolated as white crystals simply by removing
the solvent.
The cyclization of the maleamic acids to the maleimides has been investigated
by two different methods. In the first one, the N-(propyltriethoxisilane)maleamic
acid was treated with triethylamine and trimethylsilylchloride (TMSCl) in DCM
over night. Poor results were obtained with this method and very low yields of the
maleimide were obtained (10–17% yield after distillation). In a second method, the
maleamic acid was treated with zinc chloride and hexamethaldisalazane at 80°C.[8]
Under these conditions the N-(propyltriethoxisilane)maleimide was obtained in 40%
1
yield and its structure confirmed by H NMR.
N-(propyltrimethoxysilane)maleamic acid was synthesised from N-propyltrime-
thoxysilane and maleic anhydride. The same features explained for the ethyl
analogue were found. Both maleamic acids were obtained as white crystalline solids
by simply removing the solvents. However, in some experiments N-(propyltrime-
thoxysilane)maleamic acid was obtained contaminated with the product of the

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M. Proupin-Perez et al.
FIGURE 2 Synthesis of N-(propyltriethoxysilane)maleimide and N-(propyltrimethoxysilane)maleimide.
FIGURE 3 Synthesis of diene-derivatized phosphoramidites. A) Derived from 1,3-hexacyclohexene. B) Derived
from 8,10-dodecadien-1-ol.

M. Proupin-Perez et al.
1079
polymerization of the silane groups and the prevention of the polymerization of the
methoxysilane groups has shown to be difficult. The cyclization of N-(propyltrime-
thoxysilane)maleamic acid has led to a mixture of polymers following the two
methods described for the ethyl analogue and the isolation of the N-(propyltrime-
thoxysilane)maleimide was unsuccessful (Figure 3).
As a reactive partner for the maleimide in the Diels-Alder, an oligonucleotide
derivatized with a diene is required. In order to attach a diene to the 5’- end we
have prepared a DNA strand of two different phosphoramidites with 8,10-
dodecadien-1-ol (commercially available) and 1,3-hexacyclohexene.[9] The Diels-
Alder reaction between the diene-derivatized oligonuclotide and the maleimide-
functionalized nanoparticle is still under investigation. DNA strands that had not
reacted with the silica-coated gold nanoparticles will stay in the supernatant, while
the DNA-gold aggregates will precipitate.
CONCLUSIONS
In this approach, we have functionalized with a maleimide more stable silica-
coated gold nanoparticles that can be used in presence of another ligands in
solution without ligand exchanges. Modified DNA containing a diene at the 5’- end
of the molecule has been prepared for reaction with the particles through a Diels-
Alder reaction leading to a permanent C–C bonding. This could be done before
deprotection and cleavage from the solid support of the DNA and would allow the
separation of the DNA functionalized particles from the free DNA strands just by
simple centrifugation.
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