Synthesis and application of a triazene?ferrocene modifier for immobilization and characterization of oligonucleotides at electrodes

Article abstract of DOI:10.1021/jo9024368

A new DNA modifier containing triazene, ferrocene, and activated ester functionalities was synthesized and applied for electrochemical grafting and characterization of DNA at glassy carbon (GC) and gold electrodes. The modifier was synthesized from ferrocenecarboxylic acid by attaching a phenyltriazene derivative to one of the ferrocene Cp rings, while the other Cp ring containing the carboxylic acid was converted to an activated ester. The modifier was conjugated to an amine-modified DNA sequence. For immobilization of the conjugate at Au or GC electrodes, the triazene was activated by dimethyl sulfate for release of the diazonium salt. The salt was reductively converted to the aryl radical which was readily immobilized at the surface. DNA grafted onto electrodes exhibited remarkable hybridization properties, as detected through a reversible shift in the redox potential of the Fc redox label upon repeated hybridization/denaturation procedures with a complementary target DNA sequence. By using a methylene blue (MB) labeled target DNA sequence the hybridization could also be followed through the MB redox potential. Electrochemical studies demonstrated that grafting through the triazene modifier can successfully compete with existing protocols for DNA immobilization through the commonly used alkanethiol linkers and diazonium salts. Furthermore, the triazene modifier provides a practical one-step immobilization procedure.

Full text of DOI:10.1021/jo9024368

pubs.acs.org/joc
Synthesis and Application of a Triazene-Ferrocene Modifier for
Immobilization and Characterization of Oligonucleotides at Electrodes
Majken N. Hansen, Elaheh Farjami, Martin Kristiansen, Lilia Clima,
Steen Uttrup Pedersen, Kim Daasbjerg, Elena E. Ferapontova,* and Kurt V. Gothelf*
Danish National Research Foundation: Centre for DNA Nanotechnology (CDNA) at Department of
Chemistry and iNANO, Aarhus University, Langelandsgade 140, DK-8000 Aarhus C, Denmark
e_ferapontova@chem.au.dk; kvg@chem.au.dk
Received November 13, 2009
A new DNA modifier containing triazene, ferrocene, and activated ester functionalities was
synthesized and applied for electrochemical grafting and characterization of DNA at glassy carbon
(GC) and gold electrodes. The modifier was synthesized from ferrocenecarboxylic acid by attaching a
phenyltriazene derivative to one of the ferrocene Cp rings, while the other Cp ring containing the
carboxylic acid was converted to an activated ester. The modifier was conjugated to an amine-
modified DNA sequence. For immobilization of the conjugate at Au or GC electrodes, the triazene
was activated by dimethyl sulfate for release of the diazonium salt. The salt was reductively converted
to the aryl radical which was readily immobilized at the surface. DNA grafted onto electrodes
exhibited remarkable hybridization properties, as detected through a reversible shift in the redox
potential of the Fc redox label upon repeated hybridization/denaturation procedures with a
complementary target DNA sequence. By using a methylene blue (MB) labeled target DNA sequence
the hybridization could also be followed through the MB redox potential. Electrochemical studies
demonstrated that grafting through the triazene modifier can successfully compete with existing
protocols for DNA immobilization through the commonly used alkanethiol linkers and diazonium
salts. Furthermore, the triazene modifier provides a practical one-step immobilization procedure.
Introduction
protocols include, e.g., anodized carbon using carbodiimide
chemistry, where the carboxylic groups in the electrode
surface are coupled to amine-modified DNA.6,7 Another
approach, most frequently used for gold surfaces, is DNA
chemisorption onto electrodes through an alkanethiol or
disulfide linker, producing functional DNA self-assembled
monolayers (SAMs).8,9 However, limited thermal10 and
Immobilization of DNA on surfaces is important for most
DNA-based sensor technologies.1,2 In recent years, DNA
immobilization on electrode surfaces for the development of
simple, cost-effective, andsensitiveelectrochemicalbiosensors
has attracted much attention.3-5 Covalent immobilization of
DNA on electrode surfaces is most often required for obtain-
ing stable DNA layers during hybridization and/or denatur-
ing analysis. Commonly used covalent DNA attachment
(6) Lucarelli, F.; Marrazza, G.; Turner, A. P. F.; Mascini, M. Biosens.
Bioelectron. 2004, 19, 515–530.
€
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Bertling, W. M.; Hassmann, J. Talanta 2002, 56, 875–885.
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4677.
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(2) Sassolas, A.;Leca-Bouvier, B. D.;Blum, L.Chem. Rev. 2008, 108, 109–139.
(3) Davis, F.; Higson, S. J. Biosens. Bioelectron. 2005, 21, 1–20.
(4) Drummond, T. G.; Hill, M. G.; Barton, J. K Nat. Biotechnol. 2003, 21,
1192–1199.
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M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321–335.
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2474 J. Org. Chem. 2010, 75, 2474–2481
Published on Web 03/23/2010
DOI: 10.1021/jo9024368
r
2010 American Chemical Society

Hansen et al.
JOCArticle
electrochemical stability11-13 of the Au-S bond restricts
application of the DNA-modified gold electrodes to mode-
rate temperatures and potentials.
SCHEME 1. General Scheme for Immobilization of Aryltria-
zenes on Surfaces
Recently, multistep procedures using diazonium salt deri-
vatives for the immobilization of DNA have been demon-
strated. The surfaces, e.g., carbon nanotube-,14 silicon-,15
or boron-doped, single-crystalline diamond films,16 are
functionalized with nitrophenyl groups, which are reduced
either chemically or electrochemically to the amine. The
DNA coupling is then performed by a glutaraldehyde
linker15 or by using maleimide chemistry.14,16 A more direct
approach utilizing a phenylmaleimide diazonium salt con-
jugated to Fc-labeled DNA was also reported,17 but hybri-
dization properties of the DNA-functionalized electrodes
were not shown, which was ascribed to reaction of the
diazonium species with the nucleic acid bases.18 Another
problem associated with conjugation of diazonium salts
derivatives to DNA is that they are only stable in aqueous
solutions at low pH of 2-3,19 at which depurination of DNA
may occur and cause degradation of the DNA.20
Here we introduce a new one-step electrochemical modi-
fication procedure of electrode surfaces by DNA through a
DNA-conjugated triazene modifier. Triazenes can be used as
masked diazonium salts, and they have been used for graft-
ing of molecules to surfaces.19,21 When masked as the
triazene, the diazonium salt is not liberated until it is
activated and grafting occurs, minimizing the chance of
undesired byproducts. A general scheme for the immobiliza-
tion of aryltriazenes via diazonium salts on surfaces is shown
in Scheme 1.
the triazene, triggering the spontaneous cleavage to the aryl
diazonium salt.25 A one-electron transfer from the electrode
reduces the diazonium group, whereby it decomposes to
nitrogen gas and the aryl radical, which immediately reacts
with the electrode surface and forms a covalent bond.
A common approach to provide unambiguous evidence
for successful immobilization of DNA onto the electrode
surfaces is to use a redox-labeled DNA species for graft-
ing.4,17,26 Successful DNA grafting can also be revealed
through specific hybridization of the immobilized DNA with
a redox-labeled complementary probe.1-5 This enables elec-
trochemical characterization of the DNA grafting efficiency
and properties of the produced DNA monolayers. The latter
approach is, however, highly dependent on the hybridization
efficiency of the immobilized DNA. To incorporate these
features in one molecular unit we have synthesized the
triazene ferrocene N-hydroxysucccinimide ester (TzFcNHS)
modifier 1 (Figure 1). This modifier integrates (i) an acti-
vated ester for conjugation to amine-modified DNA, (ii) the
triazene moiety for grafting to a surface, and (iii) a ferrocene
redox reporter group for monitoring grafting efficiency and
hybridization events. Application of this modifier provides a
unique quality control of DNA monolayers, since it enables
the direct and quantitative electrochemical characterization
of the grafted DNA at the electrode.
Tour et al. have demonstrated grafting of small molecules
via triazenes onto silicon22,23 and single-walled carbon
nanotubes.24 In their studies, conversion of the triazene to
the diazonium salt was done at pH 2, whereby the triazene is
protonated and cleaved. As an alternative we have used
Me2SO4, which was added directly to the DNA-triazene
conjugate solution already placed on the electrode. It is
assumed that Me2SO4 methylates the terminal nitrogen of
Results and Discussion
(11) Beulen, M. W.; Kastenberg, M. I.; van Veggel, C. J.; Reinhoudt,
D. N. Langmuir 1998, 14, 7463–7467.
For the synthesis of the TzFcNHS modifier 1, the com-
mercially available ferrocenecarboxylic acid was applied as
the starting material (Scheme 2). After conversion into the
methyl ester 2,27 a handle for the triazene moiety had to
be attached to the unsubstituted ferrocene Cp ring. By a
Friedel-Crafts acylation with succinic anhydride, 2 was
converted into 3 in moderate yield of 40%, followed by
removal of the excessive ketone in a Clemmensen reduction
to give 4.
(12) Finklea, H. O. In Electroanalytical Chemistry: A Series of Advances;
Bard, A. J., Rubinstein, I., Eds.; Marcel Dekker: New York, 1996; Vol. 19, pp
109-335.
(13) Ravenscroft, M. S.; Finklea, H. O. J. Phys. Chem. 1994, 98, 3843–
3850.
(14) Lee, C.-S.; Baker, S. E.; Marcus, M. S.; Yang, W.; Eriksson, M. A.;
Hamers, R. J. Nano Lett. 2004, 4, 1713–1716.
(15) Shabani, A.; Mak, A. W. H.; Gerges, I.; Cuccia, L. A.; Lawrence,
M. F. Talanta 2006, 70, 615–623.
(16) Shin, D.; Tokuda, N.; Rezek, B.; Nebel, C. E. Electrochem. Commun.
2006, 8, 844–850.
(17) Harper, J. C.; Polsky, R.; Wheeler, D. R.; Brozik, S. M. Langmuir
2008, 24, 2206–2211.
The triazene moiety was prepared from 4-(2-aminoethyl)-
aniline by first performing a selective Cbz protection of the
amine to yield 5. The aniline was then converted into a
diazonium salt by treatment with sodium nitrite under acidic
conditions and subsequently in situ trapped by dimethyla-
mine to give the triazene 6. The reaction had to be made in a
solvent mixture containing THF, MeCN, H2O, and HCl to
avoid precipitation of starting material or intermediates.
After deprotection of Cbz with H2 and 10% Pd/C, the amine
(18) Dolan, P. L.; Wu, Y.; Ista, L. K.; Metzenberg, R. L.; Nelson, M. A.;
Lopez, G. P. Nucleic Acids Res. 2001, 29, e107.
(19) Pinson, J.; Podvorica, F. Chem. Soc. Rev. 2005, 34, 429–439.
(20) Tamm, C.; Hodes, M. E.; Chargaff, E. J. Biol. Chem. 1952, 195,
49–63.
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(21) Delamar, M.; Hitmi, R.; Pinson, J.; Saveant, J.-M. J. Am. Chem. Soc.
1992, 114, 5883–5884.
(22) Chen, B.; Flatt, A. K.; Jian, H.; Hudson, J. L.; Tour, J. M. Chem.
Mater. 2005, 17, 4832–4836.
(23) Lu, M.; Chen, B.; He, T.; Li, Y.; Tour, J. M. Chem. Mater. 2007, 19,
4447–4453.
(24) Hudson, J. L.; Jian, H.; Leonard, A. D.; Stephenson, J. J.; Tour,
J. M. Chem. Mater. 2006, 18, 2766–2770.
(25) In a separate study we are investigating the reaction mechanism of
the acid and dimethylsulfate activation of triazenes for generation of
diazonium salts.
(26) Ferapontova, E. E.; Olsen, E. M.; Gothelf, K. V. J. Am. Chem. Soc.
2008, 130, 4256–4258.
ꢁ ꢀ
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J. Organomet. Chem. 2003, 682, 131–142.
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FIGURE 1. Formation, grafting, and characterization of the triazene-ferrocene-DNA conjugate. The surface coverage can then be
determined by the redox activity of ferrocene.
SCHEME 2. Synthesis of TzFcNHS Modifier 1 and Conjugation to 30-Amino-Modified ssDNA
in 7 was coupled to 4 using HBTU as the coupling reagent to
give the assembled triazene ferrocene ester 8 in a satisfying
yield of 89%. The use of HBTU or TBTU is preferred
compared to acid chloride formation or EDC or DCC
coupling, since it is a one-pot procedure which requires short
reaction times and can be performed in commercial grade
solvents. Furthermore, side products from the coupling
reagent can easily be removed by extraction.28
The methyl ester in 8 was hydrolyzed with LiOH to give
the unstable acid 9 of which pure 1H NMR spectrum could
not be obtained. The instability is caused by the acid lability
of the triazene moiety, and it is important to continue the
synthesis right after purification of the acid. In this last step,
an activated ester was formed in a reaction with NHS and
EDC giving the TzFcNHS modifier 1 in an overall yield
(28) van Staveren, D. R.; Metzler-Nolte, N. Chem. Rev. 2004, 104, 5931–
5985.
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FIGURE 2. (a) Schematic illustration of the hybridization and denaturing events. (b) Cyclic voltammogram of the Fc-labeled DNA-modified
GC electrode in PBS, pH 7, before (ss1) and after (ds1) hybridization, and after denaturation at 50 °C in water (ss2). Scan rate is 100 mV s-1, and
CVs recorded after the fifth scan are shown. (c) Normalized and background-subtracted anodic scans currents derived from the corresponding CVs.
of 26%. The NHS ester is relatively stable, and after storage
for 1 year at 4 °C it was still sufficiently preserved to
conjugate to amino-modified DNA.
for ss1. The observed quasi-reversibility of this electron
transfer (ET) process can have several origins such as the
relatively large distance between the surface and the ferro-
cene redox probe, a relatively poor electrolyte, permeability
of the electrografted layer and neighboring charge effects
caused by the formation of ferrocenium ions. The peak
current’s linear dependency of the potential scan rate is
characteristic for a surface-confined redox process,29 and
the amount of immobilized DNA, estimated by integration
of the Fc redox peaks, was 29 ( 4 pmol cm-2, which
approaches the best values shown for the compact DNA
monolayer surface coverage.30 The ET rate constant ks,
determined from the cathodic and anodic peak separation,31
was 0.28 ( 0.05 s-1. Similar data were obtained for the
DNA-modified gold electrodes via the TzFc-modifier
(Supporting Information, Figure S2a), where the represen-
tative CVs demonstrate a pair of the Fc redox peaks from
the Fc-labeled DNA grafted to the gold surface centered at
þ218 ( 4 mV. A close to compact DNA monolayer coverage
of 31 ( 9 pmol cm-2 was obtained from the peak area
integration, while the ET rate constant ks between DNA
ferrocenes and gold was close to 1 s-1, which, as expected, is
higher than what was observed with the glassy carbon
electrodes.32 The grafting was also attempted without the
The TzFcNHS modifier 1 was coupled to a 10-mer oligo-
nucleotide modified with a C6 amino linker at the 30-end
(Scheme 2). The reaction, which proceeded in 31% yield, was
performed in an aqueous 100 mM EPPS buffer containing 25
mM modifier and 50% DMF. After incubation at 45 °C for
16 h, the conjugate was HPLC-purified and redissolved in
phosphate buffer at pH 8. UV-vis of the conjugate showed
the expected absorbance at 260 nm from the DNA bases and
a smaller peak at 320 nm from the triazene moiety
(Supporting Information, Figure S1). As shown by HPLC,
the conjugate was stable after three months at -18 °C.
The produced DNA-triazene conjugate was in one step
activated by addition of Me2SO4 to release the diazonium
salt, which was then immediately in situ electrochemically
reduced to the aryl radical for immobilization on the elec-
trode surface at a fixed potential of -200 mV vs Ag/AgCl (3
M KCl) for 20 min. In a separate experiment, HPLC analysis
showed that the Me2SO4 treatment did not cause methyla-
tion of the DNA bases under the conditions applied for
grafting.
The representative cyclic voltammograms (CVs) of the
DNA-modified GC electrodes are shown in Figure 2. The
CVs display the redox peaks with a mean potential of 231 ( 5
mV, which correlates well with the Fc redox label conjugated
to the grafted DNA (Figure 2b,c). There is a small oxidative
peak at around -200 mV in the first scan (not shown), and
in the following scans the CV remains as shown in Figure 2b
(29) Bard, A. J.; Faulkner, L. R. Electrochemical Methods - Fundamen-
tals and Applications, 2nd ed.; Wiley: New York, 2000.
(30) Petrovykh, D. Y.; Kimura-Suda, H.; Whitman, L. J.; Tarlov, M. J.
J. Am. Chem. Soc. 2003, 125, 5219–5226.
(31) Laviron, E. J. Electroanal. Chem. 1979, 101, 19–28.
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(32) Liu, G.; Liu, J.; Bocking, T.; Eggers, P. K.; Gooding, J. J. Chem.
Phys. 2005, 319, 136–146.
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FIGURE 3. (a) Schematic illustration of the hybridization and dehybridization events of the 15-mer TzFc-modifier grafted DNA (red) with
MB-labeled 22-mer DNA (blue) and competitive removal of the surface hybrid by addition of a competitive 22-mer DNA strand (green) that
forms a duplex with the full MB-labeled 22-mer DNA. (b) Differential pulse voltammogram (DPV) of DNA-modified GC electrode (ss1) after
grafting with 15-mer TzFc-DNA conjugate; (ds1) after hybridization with a complementary 22-mer MB-labeled DNA, (ss2) after
dehybridization using the competitive hybridization with excess amount of 22-mer ss-DNA complementary to the MB-labeled DNA; (ds2)
and (ss3) repetition of hybridization/dehybridization process, potential step 2 mV, modulation amplitude 50 mV and apparent scan rate
10 mV s-1. (c) Structure of the modifier (MB-NHS ester) used to label DNA with the MB probe.
presence of Me2SO4 and without applying a reductive po-
tential to the electrode. In either case, no grafting occurred as
deduced from the absence of redox signals from Fc in the
CVs performed after washing the electrode.
ss1 signal. Thus, the stability of the produced C-C bond
at the surface (346 kJ mol-1 33
was sufficient to enable
)
denaturation of the DNA duplex at elevated temperatures.
However, the weaker bond between gold and carbon resulted
in higher susceptibility of this bond to breakage: in the course
of repeated cycles hybridization and thermal denaturation,
the signal from the Fc redox label constantly decreased
consistent with a lower amount of gold surface-tethered
DNA.
Hybridization properties of the electrode-grafted DNA
were probed with a label-free cDNA (cDNA) sequence.
After hybridization the voltammetric responses from the
DNA-modified electrodes changed. For dsDNA-modified
GC electrodes mean potential of Fc shifted -15 mV
(Figure 2b,c), and the ks increased slightly to 0.34 ( 0.04 s-1
.
To further investigate the hybridization/denaturation at
the GC electrode surfaces we employed a cDNA sequence
conjugated to methylene blue (MB) (Figure 3a). Application
of MB as an additional redox probe enables us to simulta-
neously monitor both the grafted TzFc-DNA and the
MB-cDNA electrochemically. The signals from the two
redox probes are well resolved since MB has a redox poten-
tial 0.6 V more cathodic than the Fc redox probe.34 A 15-mer
DNA sequence was grafted onto the GC electrode via the
TzFcNHS modifier as described above. A complementary
30-amino-modified 22-mer DNA sequence was conjugated to
MB via an activated ester (Figure 3c). In solution, the
melting temperature (Tm) for the 15-mer hybrid between
the two strands was measured to be 60.7 °C.
In addition, the amount of electrochemically active Fc
groups, and thereby the amount of the covalently bound
DNA, remained the same. After hybridization to the DNA-
grafted gold electrodes, the redox potential for the Fc label
shifted -14 mV, consistent with the data obtained for DNA
grafted onto the GC electrodes (Supporting Information,
Figure S2b), while ks remained essentially the same. The peak
width increased after hybridization, and we assume that this
is caused by a contribution to the overall electrochemical
signal from a minor population of single-stranded TzFc-
grafted DNA at the electrode.
For both electrodes, denaturation was performed to re-
generate the original ssDNA-modified electrode state. After
the electrode was heated to 50 °C in pure water, the signal
from the Fc label returned to the initial signal observed in
CVs before hybridization with the cDNA (Figure 2 and
Supporting Information, Figure S2b). For the GC electrode
the magnitude of the electrochemical signal from the Fc
redox label also appeared to be identical to the original
First the Fc signal from the TzFc-DNA grafted electrode
was detected by CV (Supporting Information, Figure S3)
(33) Rayner-Canham, G. Descriptive Inorganic Chemistry, 2nd ed.; W. H.
Freeman: New York, 2000.
(34) Ferapontova, E. E.; Gothelf, K. V. Electroanalysis 2009, 21, 1261–
1266.
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and differential pulse voltammetry (DPV) verifying a
successful grafting of the DNA (Figure 3b, ss1). Then
MB-cDNA in a PBS buffer containing 5 mM MgCl2 was
added to the modified electrode and allowed to hybridize for
1 h at room temperature. After hybridization, two redox
peaks, correlating both with the grafted Fc-conjugated DNA
and with the hybridized MB-labeled cDNA, were observed
(Figure 3b, ds1). A significant difference in the magnitude of
the two signals is observed. In connection to this it should be
kept in mind that, in contrast to cyclic voltammetry, the
amount of surface-confined redox active molecules in DPV
cannot be directly estimated by integration of the peak areas.
In first approximation35 this amount is directly proportional
to the peak currents and, inversely, to the square of the
number of electrons n involved in the ET reaction. For the
MB label n is two and for Fc it is one, and therefore, more
pronounced peaks from the MB-labeled cDNA are consis-
tent with smaller peaks from the Fc-labeled DNA and
correspond to the same amount of DNA when divided by n2.
For regeneration of the ssDNA electrode, we applied
another strategy than the water denaturation described
above. The MB-cDNA sequence is designed with a single-
stranded toehold extending from the duplex in ds1, and this
toehold hybridizes instantly with a 220mer DNA sequence
which was added to the solution. This sequence is fully
complementary to the MB-cDNA conjugate (Tm 71.9 °C).
The 15-mer hybrid of TzFc-DNA and cDNA was therefore
outcompeted, and the 22-mer duplex formed in solution was
washed away from the electrode. After this procedure, only
the Fc signal from the DNA grafted onto the electrode was
observed in DPVs recorded with the DNA-modified electro-
des (Figure 3b, ss2). This hybridization and subsequent
competitive removal of MB-cDNA was repeated on the
same electrode, giving the expected reappearance and dis-
appearance of the MB signal (Figure 2b, ds2 and ss3). As
observed for hybridization of the immobilized DNA to a
nonlabeled DNA sequence (Figure 2), the hybridization to
the MB-labeled cDNA also induced cathodic shift in the Fc-
redox potential. In addition, a decrease in the magnitude of
the Fc signal is also observed, and we assume that this is
caused by shielding of the Fc moiety by the MB group.
presence of a cDNA sequences. The formed C-C bond
linking DNA to the GC surface was more stable than the
commonly utilized chemisorption approach using thiol-
modified DNA on gold electrodes. Denaturation at 50 °C
water did not diminish the Fc signal and thereby the elec-
trode surface coverage. Further proof for the stability of and
accessibility to the grafted DNA surfaces was made by
hybridizing with a MB-labeled complementary probe where-
upon signals from both redox probes could be observed.
During two cycles of hybridization and dehybridization, the
initial Fc signal was restored.
By the application of this modifier we have demonstrated
that the aryltriazene is a useful functionality for introduction
of an aryldiazonium precursor in DNA. By tuning the
conditions for release of the free diazonium salt with the
electrochemical grafting a dense monolayer of DNA, which
is fully capable for hybridization, could be immobilized on
GC and gold electrodes. The increased stability of the layers
immobilized on GC renders this an attractive alternative to
the thiol-gold immobilization protocol. The triazene modi-
fier may also be used for grafting DNA and also other
biomolecules to other surfaces and materials, such as gra-
phite, carbon nanotubes, and graphene.
Experimental Section
General Procedures, Instrumentation, and Materials. All che-
micals used for organic synthesis, buffer solutions, and Me2SO4
were purchased from commercial suppliers. Flash chromatogra-
phywasperformedusingsilicagel60(230-400 mesh). All solvents
were HPLC grade; THF was distilled from Na and Et3N from
CaH2. NMR spectra were recorded at 400 MHz (1H NMR) or
100 MHz (13C NMR) and calibrated to the residual solvent peak.
RP-HPLC was performed on a system with a fraction collector
and UV detection at 260 nm, fitted with a column (3 υm, 50 ꢀ
4.6 mm). Solvents were MeCN in 0.1 M triethylammonium
acetate (TEAA). Concentration determinations of oligonucleo-
tides were performed by UV-vis spectroscopy. MALDI spectra
were recorded on a MALDI-TOF spectrometer.
Cyclic voltammetry (CV) and differential pulse voltammetry
(DPV) experiments were done using a three-electrode potentio-
stat equipped with GPES 4.9.006 software. Analysis of the
voltammetric data and polynomial background correction was
performed using GPES software facilities. An Ag/AgCl, 3 M
KCl electrode was used as reference and a Pt wire as the auxiliary
electrode.
Conclusion
Gold disk working electrodes (diameter 2 mm) were mechani-
cally polished to a mirror luster stepwise in 1 μm diamond and
in 0.1 μm alumina slurries on microcloth, ultrasonicated in
ethanol/water bath for 10 min, electrochemically polished by
cycling in 1 M H2SO4, as described elsewhere,35,36 washed with
water, and further kept in absolute ethanol for 30 min. The disk
glassy carbon (GC) working electrodes (diameter 0.3 mm) were
pretreated similarly to gold electrodes: mechanically polished,
ultrasonicated, washed with water, and further kept in absolute
ethanol for 15 min.
The gold electrode surface area was determined from the
surface oxide reduction peak in 1 M H2SO4.37,38 Surface rough-
ness was found to be around 2.5. Before modification, a Teflon
cap was placed on the top part of the electrode, thus forming a
5-μL volume well-like microcell, with the bottom representing
We have synthesized a new triazene-ferrocene-NHS
ester 1 which is conjugated to amino-modified DNA and
applied for one-step immobilization of DNA on electrodes.
Furthermore, the ferrocene redox label is used to determine
the density and stability of the immobilized layer by the total
charge of the redox peaks and to detect hybridization to
cDNA by a shift in the redox potential. Remarkably close to
compact DNA monolayer surface coverages of 29 ( 4 and 31
( 9 pmol cm-2 were observed for immobilization on GC and
gold electrodes, respectively. The ks determined from the
cathodic and anodic peak separation was close to 1 s-1 for
gold and 0.28 ( 0.05 s-1 for the electrochemically slower
carbon electrodes. The grafted DNA retained its ability for
hybridization, which could be detected by a cathodic shift in
the Fc label redox potential for both electrodes in the
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the electrode surface with a diameter of 2 mm and a well wall
stirred overnight at rt and the organic solvent evaporated in
vacuo. The aqueous phase was extracted with EtOAc (3 ꢀ 15 mL)
and washed with brine and the product purified by flash
chromatography (CH2Cl2/EtOAc 2:1) as white crystals (1.44
g, 5.66 mmol, 77%): 1H NMR (CH3OD) δ (ppm) 7.31 (m, 5H),
6.94 (d, J=8.4 Hz, 2H), 6.66 (d, J=8.4 Hz, 2H), 5.04 (s, 2H), 3.25
(t, J=7.4 Hz, 2H), 2.64 (t, J=7.4 Hz, 2H); 13C NMR (CH3OD) δ
(ppm) 158.7, 146.7, 138.4, 130.4, 130.1, 129.4, 128.9, 128.7,
116.9, 67.2, 43.8, 36.3; ESI-TOF high-acc (m/z) calcd for
C16H18N2O2 ([M þ Na]þ) 293.1266, found 293.1264; mp
81.1-81.5 °C.
height of 1.6 mm.35
The electrolyte was an aqueous 20 mM phosphate buffer, pH
7.0, containing 0.15 M NaCl (PBS). The reproducibility of the
measurements was verified with at least three equivalently
prepared electrodes. All nucleotides were synthesized by stan-
dard automated oligonucleotide synthesis by DNA Technology
A/S (Risskov, Denmark). Millipore water (18.2 MΩ) was used
throughout the work.
DNA Sequences. Grafting of DNA to gold and GC electrodes
and hybridization: 50-TGG TAC GTTA-(CH2)6-NH2-30 (TzFc
modified) and 50-TAA CGT ACC A-30 (cDNA).
Benzyl 4-(3,3-Dimethyltriaz-1-enyl)phenethylcarbamate (6).
Benzyl 4-aminophenethylcarbamate (2.06 g, 8.09 mmol) was
dissolved in THF (17 mL), H2O (12 mL), MeCN (8.5 mL), and
concd HCl (4.1 mL) and cooled to 0 °C. A solution of sodium
nitrite (0.74 g, 10.76 mmol) in THF (2.5 mL) and H2O (2.5 mL)
was added slowly. The mixture was then added dropwise to a
40% aqueous solution of dimethylamine (22 mL) under vigor-
ous stirring and immediately extracted with CH2Cl2 (3 ꢀ 15 mL).
The combined organic phases were washed with H2O and dried
over Na2SO4, and the product was purified by flash chromato-
graphy (EtOAc/CH2Cl2 1:24) as light yellow crystals (2.22 g,
6.82 mmol, 84%): 1H NMR (CDCl3) δ (ppm) 7.35 (m, 7H), 7.14
(d, J = 8.4 Hz, 2H), 5.10 (s, 2H), 4.81 (bs, 1H), 3.45 (t,
J = 6.8 Hz, 2H), 3.33 (bs, 6H), 2.80 (t, J = 6.8 Hz, 2H); 13C
NMR (CDCl3) δ (ppm) 156.4, 149.7, 136.7, 135.8, 129.6, 128.6,
128.1, 120.8, 66.7, 42.3, 35.7; ESI-TOF high-acc (m/z) calcd
for C18H22N4O2 ([M þ Na]þ) 349.1640, found 349.1639; mp
56.8-57.4 °C.
Grafting of DNA to GC, hybridization with MB-labeled
DNA and strand exchange: 50-H2N-(CH2)6-TAC GTG AAC
CTA CTG-30 (TzFc modified), 50-TCA GCA TCA GTA GGT
TCA CGT A-(CH2)6-NH2-30 (MB modified), and 50-TAC GTG
AAC CTA CTG ATG CTG A-30 (cDNA).
Methyl Ferrocenecarboxylate (2). To ferrocenecarboxylic
acid (4.00 g, 17.4 mmol) in MeOH (120 mL) was added
BF3 Et2O (12.3 mL) and the mixture refluxed overnight. A
3
5% NaHCO3 solution was added to adjust to pH 8, and the
reaction mixture was extracted with CH2Cl2 (3 ꢀ 50 mL). The
organic phases were washed with brine and dried over MgSO4.
The solvent was evaporated in vacuo to yield dark orange
crystals (4.12 g, 16.96 mol, 98%). Analytical data were in
accordance with literature data:27 13C NMR (CDCl3) δ (ppm)
188.9, 172.3, 71.4, 71.1, 70.2, 69.8, 51.7; ESI-TOF high-acc
(m/z) calcd for C12H12FeO2 MS ([M þ Na]þ) 267.0084, found
267.0084.
Methoxycarbonyl-1-ferrocenesuccinic Acid (3). Methyl ferro-
cenecarboxylate 2 (4.12 g, 16.96 mmol) and succinic anhydride
(3.70 g, 36.93 mmol) dissolved in CH2Cl2 (28 mL) were added
dropwise to a solution of AlCl3 (10.61 g, 79.58 mmol) in CH2Cl2
(28 mL). The reaction was stirred for 2 h at rt and poured onto
ice (150 mL), and the organic phase was removed. The aqueous
layer was acidified with concd HCl, and orange crystals were
collected by filtration (2.28 g, 6.64 mmol, 40%). Analytical data
were in accordance with literature data:27 1H NMR (CH3OD) δ
(ppm) 4.91 (s, 4H), 4.87 (t, J=1.8 Hz, 4H), 4.60 (t, J=1.8 Hz,
2H), 4.57 (t, J=1.8 Hz, 2H), 3.81 (s, 3H), 3.09 (t, J=6.8 Hz, 2H),
2.65 (t, J=6.8 Hz, 2H); 13C NMR (CH3OD) δ (ppm) 204.3,
176.5, 172.7, 80.9, 74.9, 74.3, 74.0, 72.8, 71.8, 52.3, 35.5, 28.5;
ESI-TOF high-acc (m/z) calcd for C16H16FeO5 ([M þ Na]þ)
367.0245, found 367.0255.
10-Methoxycarbonyl-1-ferrocenebutyric Acid (4). To a stirred
mixture of zinc dust (3 g, 45.9 mmol) and HgCl2 (0.25 g, 0.92
mmol) was added H2O followed by concd HCl (0.2 mL) which
was added dropwise. The solution was stirred for 10 min at rt
before it was filtered and the precipitate (Zn-Hg) washed
carefully with MeOH.
[4-(2-Aminoethyl)phenyl](3,3-dimethyl)triazene (7). Com-
pound 6 (807 mg, 2.47 mmol) was dissolved in MeOH (10 mL)
and 10% Pd/C (81 mg) was added. The suspension was stirred 1 h
under hydrogen (1 atm) at rt and filtered through Celite and the
solvent evaporated in vacuo to give the product as an orange oil
(452 mg, 2.35 mmol, 95%): 1H NMR (CDCl3) δ (ppm) 7.35 (d,
J=8.4 Hz, 2H), 7.15 (d, J=8.4 Hz, 2H), 3.31 (bs, 6H) 2.94 (t,
J=6.8 Hz, 2H), 2.64 (t, J=6.8 Hz, 2H); 13C NMR (CDCl3) δ
(ppm) 150.7, 138.0, 130.2, 121.5, 44.2, 39.7; ESI-TOF high-acc
(m/z) calcd for C10H16N4 ([M þ H]þ) 193.1453, found 193.1457.
Methyl 10-[N-(4-(3,3-Dimethyltriaz-1-enyl)phenethyl)butyl-
amide]ferrocene-1-carboxylate (8). 10-Methoxycarbonyl-1-fer-
rocenesuccinate (236 mg, 0.71 mmol) and 7 (137 mg, 0.71 mmol)
were suspended in dry DMF (7.5 mL) and Et3N (0.8 mL).
HBTU (271 mg, 0.71 mmol) was added and the mixture stirred
for 100 min at rt. After addition of H2O, the solution was
extracted with EtOAc (3 ꢀ 20 mL). The product was purified
by flash chromatography (EtOAc/CH2Cl2 1:2) as an orange oil
(318 mg, 0.63 mmol, 89%): 1H NMR (CDCl3) δ (ppm) 7.37 (d,
J=8.4 Hz, 2H) 7.15 (d, J=8.4 Hz, 2H), 5.49 (bs, 1H), 4.70 (s, 2H),
4.33 (s, 2H), 4.07 (s, 2H), 4.04 (s, 2H), 3.78 (s, 3H), 3.51 (q,
J=6.6 Hz. 2H), 3.33 (bs, 6H), 2.80 (t, J=6.6 Hz, 2H), 2.25 (t, J=
The Zn-Hg (2.4 g) was added in small portions to a stirred
solution of 3 (0.5 g, 1.45 mmol) in AcOH (6.5 mL) and concd
HCl (9.9 mL) over a period of 20 min. The slurry was stirred for
1 h at rt, H2O (100 mL) was added, and the remaining Zn-Hg
was removed by filtration. The solution was extracted with
CH2Cl2 (3 ꢀ 50 mL) and washed, and the combined organic
phases were washed with brine and evaporated in vacuo to yield
an orange oil (466 mg, 1.41 mmol, 97%). Known compound:27
1H NMR (CH3OD) δ (ppm) 4.72 (t, J=2.0 Hz, 2H), 4.43 (t, J=
2.0 Hz, 2H), 4.12 (s, 4H), 3.79 (s, 3H), 2.31 (t, J=7.6 Hz, 2H),
2.29 (t, J = 7.6 Hz, 2H) 1.76 (k, J = 7.6 Hz, 2H); 13C NMR
(CH3OD) δ (ppm) 177.2, 174.1, 91.2, 73.2, 72.4, 71.7, 70.9, 70.2,
52.1, 34.4, 28.8, 27.3; ESI-TOF high-acc (m/z) calcd for
C16H18FeO4 ([M þ Na]þ) 353.0452, found 353.0463.
7.6 Hz, 2H), 2.10 (t, J=7.6 Hz, 2H), 1.77 (k, J=7.6 Hz, 2H); 13
C
NMR (CDCl3) δ (ppm) 172.7, 172.1, 149.7, 135.9, 129.3, 120.8,
89.9, 71.9, 70.7, 69.8, 69.1, 51.6, 40.6, 36.3, 35.2, 28.0, 26.8;
ESI-TOF high-acc (m/z) calcd for C26H32FeN4O3 ([M þ Na]þ)
527.1722, found 527.1722.
10-[N-(4-(3,3-Dimethyltriaz-1-enyl)phenethyl)butylamide]-
ferrocene-1-carboxylic Acid (9). LiOH (90 mg, 3.76 mmol) was
added to a solution of 8 (77 mg, 0.15 mmol) in THF (6 mL) and
water (0.5 mL). The mixture was refluxed for 96 h before the
THF was evaporated in vacuo. AcOH (20%) was added drop-
wise until the product precipitated (pH 6-7), and the aqueous
solution was extracted with EtOAc (3 ꢀ 10 mL) and washed with
brine. The product was immediately purified by flash chroma-
tography (EtOAc f EtOAc/MeOH 1:1) as an unstable orange
oil (61 mg, 0.12 mmol, 82%). It was not possible to obtain pure
spectroscopic data, and the compound should be used immedi-
ately in the proceeding reaction step due to degradation.
Benzyl 4-Aminophenethylcarbamate (5). To a solution of 4-(2-
aminoethyl)aniline (1 g, 7.34 mmol) in THF (30 mL) and NaOH
(1 M, 7.3 mL) was added benzyl chloroformate (1.03 mL, 7.35
mmol) in THF (10 mL) dropwise at 0 °C. The solution was
2480 J. Org. Chem. Vol. 75, No. 8, 2010

Hansen et al.
JOCArticle
Succinimidyl 10-[N-(4-(3,3-Dimethyltriaz-1-enyl)phenethyl)-
butylamide]ferrocene-1-carboxylate (1, TzTcNHS). EDC (110
mg, 0.57 mmol) was added to a solution of 9 (134 mg, 0.27
mmol) and NHS (65 mg, 0.56 mmol) in CH2Cl2 (8 mL), and the
reaction was stirred for 1.5 h at rt. The solution was then washed
with water and brine. The product was purified by flash chroma-
tography (EtOAc/CH2Cl2 1:1) as a red brown oil (93 mg, 0.16
mmol, 58%): 1H NMR (CDCl3) δ (ppm) 7.36 (d, J=7.6 Hz, 2H),
7.15 (d, J=7.6 Hz, 2H), 5.54 (bs, 1H), 4.82 (s, 2H), 4.51 (s, 2H),
4.26 (s, 2H), 4.24 (s, 2H), 3.50 (q, J=6.2 Hz, 2H), 3.33 (bs, 6H),
2.81 (m, 6H), 2.33 (t, J=7.2 Hz, 2H), 2.11 (t, J=7.2 Hz, 2H), 1.79
(k, J=7.2 Hz, 2H); 13C NMR (CDCl3) δ (ppm) 172.8, 169.7,
167.5, 149.7, 136.0, 129.3, 120.8, 91.0, 73.6, 71.5, 71.0, 70.2, 64.6,
40.7, 36.2, 35.3, 28.1, 26.8, 25.8;ESI-TOF high-acc(m/z) calcd for
C29H33FeN5O5 ([M þ Na]þ) 610.1729, found 610.1724.
1.2 V over 5 min followed by electrode cycling in a blank solu-
tion (1 M HClO4, 100 mVs-1) 10 times between 0 and 1.4 V.
Modification of the gold electrodes with the TzFc-DNA
conjugate was performed by grafting the conjugate under
potentiostatic control for 20 min at -200 mV vs Ag/AgCl
(3 M KCl) from a 20 μL drop of 10 μM TzFc-DNA conjugate
solution in PBS (20 mM pH 7.0, 0.15 M NaCl), placed on the top
of the gold electrode. Modification of the GC electrodes with the
TzFc-DNA conjugate was performed by grafting for 20 min at
-400 mV vs Ag/AgCl (3 M KCl) from a 20 μL drop of 15 μM
DNA conjugate solution in PBS (20 mM pH 7.0, 0.15 M NaCl).
The triazene functional group was activated by the addition of
1 μL of a 100 mM solution of Me2SO4 in 100 mM PBS directly to
the DNA drop (5 mM Me2SO4). After electrodeposition, the
electrodes were rinsed with water to remove any nonspecifically
adsorbed conjugate species.
On-Surface Hybridization/Denaturation Assay. Twelve
microliters of a 42 μM solution of cDNA in PBS containing
4.2 mM MgCl2 was placed on top of the DNA-modified elec-
trode. Hybridization was performed for 1 h at rt, after which the
electrodes were washed with PBS. Denaturation was performed
by placing the electrodes in 50 °C water for 5 min.
For measuring the combined redox signal from immobilized
TzFc-DNA conjugate on the GC electrode surface and methy-
lene blue, the hybridization process was performed by placing
12 μL of a 15.8 μM solution of MB-labeled DNA in PBS
containing 5 mM MgCl2 on the electrode. After 1 h of hybridi-
zation at room temperature, the electrode was carefully washed
with PBS buffer, and DPV was measured. For dehybridization
excess amounts (200 pmol) of the 220mer complementary to the
MB-labeled DNA in PBS containing 5 mM MgCl2 was added
and allowed to hybridize for 2 h before DPV was performed.
Triazene-Ferrocene-DNA Conjugate (10-mer). 30-Amino-
modified DNA (3 nmol) was evaporated to dryness and the
pellet dissolved in 133 mM EPPS buffer pH 8.8 (7.5 μL)
containing 33% DMF. A solution of 100 mM 9 in DMF
(2.5 μL) was added and the reaction incubated overnight at
45 °C. The product was purified by RP HPLC (TEAA pH 8.5):
(0-40%; 20 min) rt, 17.1 min; yield 995 pmol, 31%; ESI MS
(m/z) calcd for ([M þ Na ]þ) 3708.6, found 3709.1.
Triazene-Ferrocene-DNA Conjugate (15-mer). 50-Amino-
modified DNA (6 nmol) was evaporated to dryness and the
pellet dissolved in 133 mM EPPS buffer pH 8.8 (7.5 μL)
containing 33% DMF. A solution of 100 mM 9 in DMF
(2.5 μL) was added and the reaction incubated overnight at
45 °C. The product was purified by RP HPLC (TEAA pH 8.5):
(0-40%; 20 min) rt, 16.8 min; yield 2578 pmol, 43%; ESI MS
(m/z) calcd for ([M þ Na ]þ) 5203.4, found 5203.4.
Methylene Blue-DNA Conjugate. A solution containing 30-
amino-modified DNA (105 nmol) and methylene blue NHS
ester (1.8 mg, 2.8 μmol) in 0.75 M sodium tetraborate buffer pH
8.5 with 14% DMSO was incubated at rt for 20 h.
Acknowledgment. The Danish National Research Foun-
dation funded this work. DNA Technology A/S, Risskov, is
acknowledged for performing ESI-MS of the formed con-
jugates. E.E.F. thanksthe Carlsberg Foundation forfinancial
support, and E.F. thanks the Ministry of Science, Research
and Technology (MSRT) of Iran for financial support.
After lyophilization the pellet was dissolved in H2O and
purified by RP HPLC (TEAA pH 7.0): (0-40%, 20 min) rt,
14.17 min; yield 49 nmol, 47%; MALDI MS TOF ([M þ Na ]þ)
calcd 7333, found 7332.
Electrode Modification with the Triazene-Ferrocene-DNA
Conjugate. In order to measure the combined redox signal from
ferrocene and methylene blue, a two-step activation process for
the GC surface was performed before the grafting step, accord-
ing to methodology described elsewhere.38 In brief, it was an
electrochemical activation in 1 M KOH by a potential step to
Supporting Information Available: UV spectrum of the
TzFc-DNA conjugate, cyclic voltammograms of functionalized
goldelectrodes, and1H, 13C NMR, andMSspectra. Thismaterial
is available free of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 8, 2010 2481