Preparation of ferrocene-functionalized gold nanoparticles by primer extension reaction on the particle surface

Article abstract of DOI:10.1016/j.bmcl.2014.04.062

DNA molecules possessing multiple ferrocene (Fc) molecules as a redox active probe were prepared by the primer extension (PEX) reaction using a 2′-deoxyuridine-5′-triphosphate derivative in which Fc was connected to the C5-position of the uridine by a diethylene glycol linker. Gold nanoparticles (AuNP) covered with DNA possessing the Fc molecules were prepared by the PEX reaction on the surface. The AuNP-FcDNA conjugates exhibit a detectable electrochemical signal due to the Fc molecules. Possible application of the PEX reaction on AuNP is demonstrated for the detection of a single nucleotide mutation in the target DNA.

Full text of DOI:10.1016/j.bmcl.2014.04.062

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2661–2663
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preparation of ferrocene-functionalized gold nanoparticles
by primer extension reaction on the particle surface
⇑
⇑
Tadao Takada , Takaaki Tochi, Mitsunobu Nakamura, Kazushige Yamana
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
DNA molecules possessing multiple ferrocene (Fc) molecules as a redox active probe were prepared by
the primer extension (PEX) reaction using a 20-deoxyuridine-50-triphosphate derivative in which Fc
was connected to the C5-position of the uridine by a diethylene glycol linker. Gold nanoparticles (AuNP)
covered with DNA possessing the Fc molecules were prepared by the PEX reaction on the surface. The
AuNP–FcDNA conjugates exhibit a detectable electrochemical signal due to the Fc molecules. Possible
application of the PEX reaction on AuNP is demonstrated for the detection of a single nucleotide mutation
in the target DNA.
Received 14 March 2014
Revised 9 April 2014
Accepted 16 April 2014
Available online 24 April 2014
Keywords:
Polymerase extension reaction
DNA
Ó 2014 Elsevier Ltd. All rights reserved.
Ferrocene
Gold nanoparticles
Electrochemistry
Nucleic acid derivatives possessing functional molecules at
various positions have been designed and prepared for biochemical
and medicinal applications, such as the fluorescent and electro-
chemical detections of DNA.1 Many functional nucleic acid deriva-
tives have been synthesized by a phosphoramidite method using
an automated DNA synthesizer.1 Recently, an enzymatic method
based on the DNA polymerase reactions has proven to be effective,
facile, and applicable for the multiple incorporation of functional
molecules into DNA.2 The enzymatic method allows us to control
the number and arrangement of functional molecules, such as
fluorophore or redox active molecules on DNA.2 Fojta and Hocek
In this paper, we report the enzymatic incorporation of
ferrocene (Fc) molecules into DNA by the primer extension (PEX)
reaction using a deoxyuridine triphosphate (dUTP) labeled with
Fc as a redox reporter. We prepared Fc-modified deoxyuridine
triphosphate (Fc-dUTP) using an aqueous cross-coupling reaction
between 5-iodo-dUTP and Fc-derivatives having an ethynyl group.
The important finding is that the Fc-dUTP using a longer ethylene
glycol linker efficiently affords the full-length product (FcDNA) in
the PEX reaction. Moreover, the PEX reaction or the incorporation
of Fc molecules into DNA can proceed on gold nanoparticles
(AuNPs). We demonstrated that the PEX reaction on AuNPs is
applicable for the electrical detection of a single nucleotide
mutation in the target DNA.
have reported
a method for the synthesis of base-modified
deoxyribonucleoside triphosphates by an aqueous cross-coupling
reaction, and have successfully incorporated them into DNA.3
The multiple-labeling of DNA has some advantages in the detec-
tion of DNA and biomolecules because of the possible generation of
strong signals derived from the fluorescence and redox labels.4–9
Electrochemical sensors have become promising because they offer
a more convenient and cost-effective method for the detection of
DNA and biomolecule when compared to optical devices.10–12 In
fact, unique electrochemical sensors based on DNA probes and
aptamers targeting various kinds of substances from small
molecules, metal ions, and oligonucleotides to large proteins have
been developed.3,13
20-Deoxyuridine triphosphates possessing ferrocene as a redox
active probe at the C5-position of the pyrimidine ring were
prepared following the method developed by Hocek et al.3 Two
derivatives (FU1 and FU2) were prepared by the Sonogashira
cross-coupling reaction between 5-iododeoxyudirine triphosphate
and ferrocene derivatives bearing an ethynyl group (Fig. 1A). The
modification at the C5-position of the pyrimidine base is known
to be useful in the design of the triphosphate analogs with a high
tolerance to the functional groups in the polymerase reactions.3
In this study, the ferrocene ring was attached through the
propargyl group to the C5-position of uracil base (FU1). In FU2, a
diethyleneglycol linker was inserted as a spacer between the
ethynyl group and the ferrocene ring.
Two kinds of thermostable polymerases categorized to family B
(KOD Dash and vent (exo-)), showing a high tolerance to a wide
⇑
Corresponding authors. Tel./fax: +81 79 267 4661 (T.T.).
E-mail address: takada@eng.u-hyogo.ac.jp (T. Takada).
http://dx.doi.org/10.1016/j.bmcl.2014.04.062
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2662
T. Takada et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2661–2663
(A)
(A)
(B)
(C)
(B)
Figure 1. (A) Structures of 20-deoxyuridine triphosphate possessing ferrocene (Fc)
as a redox active probe at the C5-position of the pyrimidine ring (FU1 and FU2). (B)
Sequence of primer (P1) and template (T1) used for the primer extension (PEX)
reaction. P1 was labeled with
a
fluorescein at the 50-terminus. (indicated by
asterisk). (C) A 15% denaturing PAGE of the primer extension products obtained
using the Fc-labeled dUTP and polymerase KOD Dash (lanes 1–8) or Vent (exo-)
(lanes 9–16).
Figure 2. (A) Schematic illustration of enzymatic approach to prepare AuNP
modified with multiple Fc molecules on the surface by the PEX reaction. (B) Non-
denaturing 3% agarose gel electrophoresis of AuNP subjected to the PEX reaction
using FU2 as a substrate of the polymerase reaction. The PEX reaction was carried
out with a primer (P2) and template (T2) immobilized on AuNP (20 nm) using Vent
(exo-). The reaction was carried out at 37 °C for 30 min. Lanes 1–3: unmodified
AuNP, AuNP/D/P2, AuNP/D/P2/T2. Lanes 4 and 5: AuNP conjugates after the PEX
reaction using dUTP (lane 4) and FU2 (lane 5).
range of triphosphate analogs with modification at the
C5-position,7 were used to investigate the primer extension (PEX)
reaction using the FU1 or FU2. The single-nucleotide extension
and full-length extension reaction were investigated by denaturing
polyacrylamide gel electrophoresis (PAGE) (Fig. 1B and C). A
14-mer primer (P1) labeled with a fluorescein and 34-mer DNA
template (T1) were hybridized, and then subjected to the PEX
reaction using FU1 or FU2 as a substrate instead of dUTP for the
polymerase reaction. When the reaction mixture contained only
FU1 or FU2 (single nucleotide extension), electrophoretic bands
corresponding to the P1 disappeared after the reaction and a
slow-migrating band appeared (lanes 5 and 6 for KOD Dash, and
lanes 13 and 14 for Vent (exo-)), indicating the incorporation of
FU1 and FU2 by these polymerases. A full-length PEX product
was obtained for FU2 (lane 8 for KOD Dash, lane 16 for Vent
(exo-)), whereas FU1 did not afford the full-length product
efficiently. This is probably due to the steric hindrance of the bulky
ferrocene molecule that is close to the recognition site in the poly-
merase extension reactions. In contrast, the Fc of FU2 connected by
the ethylene glycol linker did not interfere with the polymerase
reaction, leading to an efficient enzymatic incorporation. A similar
positive effect of the ethylene glycol linker providing an efficient
incorporation has also been reported.14
oligonucleotides with a thiol group.16 Surface coverage of AuNP
with P2 was reduced by adding a short 10-mer dA (D) in order
to facilitate the hybridization between the primer and the tem-
plate, reduce the steric hindrance and enhance the polymerase
activity.15 A 36-mer template DNA (T2) was then hybridized with
P2 on the surface.
The PEX reaction on AuNP was analyzed by agarose gel electro-
phoresis (Fig. 2B). All electrophoretic bands were visualized by the
strong red color of the AuNP. The surface-modified AuNP with
D/P2 (lane 2) ran much slower than the unmodified AuNP (lane
1). After hybridization with T2, the AuNP conjugates showed
decreased mobility. The PEX reaction with Vent (exo-) polymerase
on the surface of AuNP was carried out by dUTP (lane 4) or FU2
(lane 5). After the PEX reaction, both AuNP conjugates showed
slower bands compared to the unreacted AuNP conjugates (lane
3). This difference in the electrophoretic mobility means that the
extension reaction on the surface occurred. The slightly slower
band of the AuNP conjugates by FU2 than that by dUTP may corre-
spond to the multiple incorporation of Fc molecules causing an
increased molecular size.
The preparation of AuNP/DNA conjugates possessing the Fc
molecules on the surface by the PEX reaction are schematically
depicted in Figure 2A. FU2 was used for this experiment because
FU2 had shown a better incorporation efficiency than FU1. The pri-
mer was immobilized on the surface of AuNP and hybridized with
the template DNA, and then the PEX reaction was carried out using
FU2 to incorporate the Fc molecules into DNA on the surface.15
AuNP modified with the primer DNA (P2) and diluter DNA (D)
(1:1) was prepared according to the standard protocol using
The electrochemical response of the AuNP/DNA conjugates
possessing the Fc molecules prepared by the PEX reaction was
investigated (Fig. 3A).17 The surface of 20 nm AuNP is covered with
the capture (C1), primer (P3), and diluter DNA (D) (1:1:1), and then
hybridized with the template DNA (T3). Template DNAs with A or T
next to the starting point of the extension reaction were used. After
hybridization, the PEX reaction was carried out using the FU2 as

T. Takada et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2661–2663
2663
template DNA sequences suggests that the present approach may
be applicable to identify the single nucleotide variation in DNA.
Electrochemical detection of nucleic acid sequences based on PEX
reactions has been reported.3,12,19 Our present approach contains
the combined use of a DNA polymerase reaction on nanoparticles
and immobilization of redox-modified DNA-nanoparticles on an
electrode surface. Therefore, the method presented herein is useful
for multiple incorporation redox molecules into DNA on an elec-
trode that should lead to a highly sensitive electrochemical detec-
tion of DNA.
(A)
We have described the enzymatic incorporation of ferrocene
(Fc) molecules into DNA using Fc-modified deoxyuridine triphos-
phates (Fc-dUTP) as a substrate for the DNA polymerase. The
Fc-dUTP using a longer ethylene glycol linker efficiently afforded
the full-length product (FcDNA) in the primer extension (PEX)
reaction. It was also shown that the PEX reaction or the incorpora-
tion of Fc molecules into DNA can proceed on gold nanoparticles
(AuNP). Electrochemical experiments showed that the AuNP–
FcDNA conjugates exhibit an electrochemical signal from the Fc
molecules. The electrochemical signal was reduced by a single base
mismatch in DNA, indicating the potential utility of the present
method for the detection of a single nucleotide mutation in DNA.
(B)
(C)
Acknowledgments
This work has been partly supported by a Grant-in-Aid for Sci-
entific Research from the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT) of Japanese Government.
Figure 3. (A) Immobilization of AuNP/DNA conjugates possessing the Fc molecules
by the PEX reaction on the electrode. Sequences of primer (P3), template (T3),
capture DNAs (C1) and (C2). The letter ‘N’ in T3 represents the A or T base. The gold
electrode was modified with C2, and the AuNP conjugates after the PEX reaction
were immobilized on the surface via hybridization between C1 and C2. (B) UV/vis
absorption spectrum of AuNP/DNA conjugates recovered from the electrode
through dehybridization in hot water. Inset in the figure shows pictures of the
electrode before and after immobilization of the AuNP conjugates. (C) Differential
pulse voltammetry (DPV) response of the electrode modified with AuNP conjugates
prepared using two template DNAs (T3-A, T3-T) or without template DNA.
Supplementary data
Supplementary data (synthesis of the triphosphate derivatives,
and experiment details) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.04.
062.
the substrate for Vent (exo-). We assumed that the Fc molecules
could be efficiently incorporated when N of the template DNA is
A (T3-A), whereas the PEX reaction is inhibited in T3-T due to
the mismatch base pair. After the PEX reaction, the AuNP conju-
gates were immobilized on the gold electrode through the hybrid-
ization of the capture C1 on AuNP with C2 on the electrode.
Immobilization of AuNP on the electrode was confirmed by the
color change of the electrode surface originated from the red color
of the AuNP particles. (Fig. 3B, inset). The AuNP conjugates can be
released from the surface by immersing the electrode in hot water.
The resulting solution was analyzed by UV/vis absorption mea-
surements. The plasmon absorption band with a maximum around
520 nm was observed (Fig. 3B). This is another indication for the
surface immobilization of the AuNP/DNA.
Electrochemical responses of the AuNP/DNA conjugates
immobilized on the electrode are shown in Figure 3C. As expected,
the electrochemical signals of the differential pulse voltammetry
(DPV) for the AuNP/DNA conjugates prepared using T3-A were
observed at ꢀ0.3 V versus Ag/AgCl, which contributed to the
oxidation peak of the Fc molecules on the surface.18 In contrast,
little electrochemical signal was observed for the AuNP/DNA
prepared without using the template DNA. These data indicate that
the observed electrochemical signal for the AuNP/FU2-DNA pre-
pared using T3-A is resulted from the oxidation of the ferrocene
(Fc) molecules. When T3-T was used as a template, the suppressed
electrochemical signal of Fc was observed. This signal reduction is
caused from the poor incorporation of the redox molecules by the
T–T mismatch base pair. The detectable difference in the electro-
chemical measurements between the matched and mismatched
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