Electrogenerated chemiluminescence of triazole-modified deoxycytidine analogues in N,N-dimethylformamide

Article abstract of DOI:10.1039/c1cp22116g

Triazole-modified deoxycytidines have been prepared for incorporation into single-stranded deoxyribonucleic acid (ssDNA). Electrochemical responses and electrogenerated chemiluminescence (ECL) of these deoxycytidine (dC) analogues, 1-4, were investigated as the monomers. Cyclic voltammetry and differential pulse voltammetry techniques were used to determine the oxidation and reduction potentials of 1-4, along with the reversibility of their electrochemical reactions. The dC analogues, in N,N-dimethylformamide containing 0.1 M tetra-n-butylammonium perchlorate as electrolyte, exhibited weak relative ECL efficiencies following the annihilation mechanism, while these efficiencies were enhanced with the use of benzoyl peroxide following the coreactant mechanism. It was shown that these nucleosides could generate excited monomers, and excimers as seen by the red-shifted ECL maxima relative to their corresponding photoluminescence peak wavelengths.

Full text of DOI:10.1039/c1cp22116g

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
PCCP
Cite this: Phys. Chem. Chem. Phys., 2011, 13, 17405–17412
www.rsc.org/pccp
PAPER
Electrogenerated chemiluminescence of triazole-modified deoxycytidine
analogues in N,N-dimethylformamidew
Kalen N. Swanick, David W. Dodd, Jacquelyn T. Price, Allison L. Brazeau,
Nathan D. Jones, Robert H. E. Hudson and Zhifeng Ding*
Received 29th June 2011, Accepted 11th August 2011
DOI: 10.1039/c1cp22116g
Triazole-modified deoxycytidines have been prepared for incorporation into single-stranded
deoxyribonucleic acid (ssDNA). Electrochemical responses and electrogenerated chemiluminescence
(ECL) of these deoxycytidine (dC) analogues, 1–4, were investigated as the monomers. Cyclic
voltammetry and differential pulse voltammetry techniques were used to determine the oxidation and
reduction potentials of 1–4, along with the reversibility of their electrochemical reactions. The dC
analogues, in N,N-dimethylformamide containing 0.1 M tetra-n-butylammonium perchlorate as
electrolyte, exhibited weak relative ECL efficiencies following the annihilation mechanism, while these
efficiencies were enhanced with the use of benzoyl peroxide following the coreactant mechanism. It was
shown that these nucleosides could generate excited monomers, and excimers as seen by the red-shifted
ECL maxima relative to their corresponding photoluminescence peak wavelengths.
ECL applications in the detection and diagnostics of deoxyribo-
1. Introduction
nucleic acid (DNA) involve electrochemistry and spectroscopy,
Electrogenerated chemiluminescence or electrochemiluminescence
and have many distinct advantages over other spectroscopy-based
detection systems11 such as a lack of scattered light interference
(ECL) is the process in which radicals are electrochemically
and the use of electrochemistry-based sensors17 which offer high
generated in solution and react through electron transfer to form
excited states that emit light.1 In the 1960s, Hercules, Bard et al.,
sensitivity. In fact, a common practice to detect DNA via ECL is
and Visco et al. reported on the first ECL studies.2–4 Since then,
ECL has become a powerful analytical technique5–14 in immuno-
to immobilize luminophore-labelled DNA double or single strands
at an electrode,18,19 which can be measured with the emitted light
assays, food and water testing, trace metal determination, and
upon redox reactions of the luminophore along with a coreactant
biomolecule detection. Two main ECL systems are used:
in the solution. The immobilized single strands can be employed to
annihilation and coreactant ECL.1 Annihilation ECL is observed
recognize complementary strands followed by ECL detection.19,20
when a luminophore species in solution is scanned to its first
Modified nucleosides could find potential applications as lumines-
oxidation and reduction potentials at an electrode. The excited
cent probes incorporated into single-stranded deoxyribonucleic
acid (ssDNA) for sequence interrogation using ECL methods.20,21
species are formed from the generation of a radical cation and a
radical anion of the species in the vicinity of the electrode. The
Single nucleotide polymorphisms (SNPs) are single-base variations
emission of light results from the excited state species. An
in the genetic code that occur about once every 1000 bases
along the 3-billion base pair human genome.22 The ability to
alternative to annihilation ECL is a coreactant system, which is
performed with one directional potential scanning at an electrode
detect SNPs is of prime importance as mutations can be directly
in a solution containing the luminophore species and an added
responsible for, or make one more susceptible to diseases such
coreactant reagent such as benzoyl peroxide, BPO.15,16 Radicals
as asthma, diabetes, atherosclerosis, schizophrenia, and various
cancers.22 Fluorescent modified nucleosides, when in the context
are generated from the luminophore, and intermediates from
BPO that will decompose to produce powerful oxidizing species
of an oligomer, are potential candidates for the detection of
nucleic acids with single nucleobase variations.23–28
and react with the reduced luminophore. This generates an
excited species, which upon decay emits light.
Modifications of nucleosides in nucleic acid chemistry are well
established.29–31 However, there have been a few reports on
modified deoxycytidine (dC) nucleosides,32–35 although the ECL
behaviour of tris-2,20-bipyridylruthenium(II) (Ru(bpy)32+) has
Department of Chemistry, The University of Western Ontario,
1151 Richmond Street, London, Ontario N6A 5B7, Canada.
E-mail: zfding@uwo.ca; Tel: +1 519 661 2111 x86161
w Electronic supplementary information (ESI) available. CCDC
reference numbers [CCDC NUMBER(S)]. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c1cp22116g
been widely studied since the 1970s.36 In the above context,
2+
Ru(bpy)3
can be attached to a target ssDNA as an ECL
label, then hybridize with its complementary strand of ssDNA
immobilized on the surface of an electrode to measure the ECL
c
This journal is the Owner Societies 2011
Phys. Chem. Chem. Phys., 2011, 13, 17405–17412 17405

View Article Online
performing a click reaction, using the Huisgen 1,3-dipolar
cycloaddition reaction which is the premier example of a
click reaction between alkynes and azides.41,42 The selective
1,4-disubstituted products of the click reaction were obtained
by using a copper(I) catalyst.43,44 Compounds 1–4 were
synthesized by reacting 5-ethynyldeoxycytidine with one of
the four corresponding aryl azides in the presence of a Cu(I)
catalyst, generated from CuSO4 and sodium ascorbate, in a
1 : 1 THF–H2O solution. The crude products of 1–4 were
purified using column chromatography and characterized
1
by H and 13C{1H} NMR spectroscopies and HRMS.38 The
purified triazole-containing compounds were then used for
CV, DPV and ECL analysis.
2.3 Electrochemical preparation
CV, DPV and ECL experiments were conducted using a 2 mm
diameter Pt disc inlaid in a glass sheath as the working
electrode (WE), a coiled Pt wire as the counter electrode
(CE), and a coiled Ag wire as the quasi reference electrode
(RE). Prior to an experiment, the electrochemical cell was
rinsed with acetone and deionized water, then immersed in 5%
KOH in isopropanol for 4 h. The cell was rinsed with copious
amounts of deionized water, immersed in 1% HCl for 4 h, and
finally thoroughly rinsed with ultrapure water. The cell was
dried at 120 1C for 12 h, and then cooled to room temperature.
The CE and RE were rinsed with acetone and ultrapure
water, then sonicated in DMF for 15 min, in ethanol for
5 min, and finally in ultrapure water for 5 min before being
thoroughly rinsed again with ultrapure water. The electrodes
were then dried at 120 1C for 5 min, then left to cool to room
temperature.
Scheme 1 Molecular structures of compounds 1–4.
response of the double-stranded DNA (dsDNA). Our objective
was to create a metal-free DNA sensor based on dC in
combination with ECL for the detection of SNPs.
Our group previously synthesized triazole-modified deoxy-
cytidine nucleosides, 1–4 (Scheme 1).37,38 These compounds
are easy to prepare, and are potential candidates for use as
metal-free ECL labels that can be incorporated into ssDNA
for the detection of SNPs. The modified nucleosides contain
four different aromatic groups, 1–4, that have been appended
to dC as desirable targets for potential uses in nucleic acid
chemistry. Compound
2 has been reported previously
although characterization data were lacking.32
Here we report the electrochemical behaviour of these
triazole-containing dC nucleosides, 1–4, employing cyclic
voltammetry (CV) and differential pulse voltammetry (DPV).
ECL of these four compounds were also investigated via
annihilation by scanning between the first oxidation and
reduction potentials, and coreactant mechanisms by adding
BPO and scanning the potential in the cathodic region. The
majority of DNA-based biosensors have used uracil for
studies.39,40 At the time of writing, there were no reports of
ECL of any modified dC nucleosides in the literature.
The WE was polished with a felt polishing pad using a
1.0 mm alumina suspension in ultrapure water (Milli-Q,
Millipore) for 5 min followed by a 0.05 mm alumina suspension
in ultrapure water to obtain a mirror surface and finally
washed with copious amounts of ultrapure water (Buehler
Ltd., Lake Bluff, IL). Then the WE electrode was electro-
chemically polished by cycling in 0.1 M aqueous H2SO4
solution for 400 segments between the potentials of 1.400
and ꢀ0.600 V at 0.5 V sꢀ1 to obtain a clean and more
reproducible Pt surface.45 The oxidation and reduction of
H2SO4 produce an ordered structure of polycrystalline Pt
surfaces.46 The electrodes were then washed repeatedly with
ultrapure water, then dried with a stream of Ar gas over the Pt
disc area and left to dry for 12 h at room temperature.
All solutions for electrochemical and ECL experiments
were prepared in the electrochemical cell placed inside an
N2-filled drybox that possessed little oxygen and moisture.
The solutions of 1–4 were in the concentration range between
2.0 ꢁ 10ꢀ3 and 2.7 ꢁ 10ꢀ3 M in anhydrous DMF (Sure/Sealt
bottle from Aldrich) containing 0.1 M TBAP as supporting
electrolyte. For coreactant systems, 5.0 ꢁ 10ꢀ3 M BPO was
added to each solution of 1–4. The electrodes were immersed
in the solution and connected by a copper wire inserted
through the air-tight Teflon cap. The assembly was moved
out of the drybox to perform electrochemistry and ECL
experiments. After completion of each experiment, the electro-
chemical potential window was calibrated using Fc as the
2. Experimental
2.1 Chemicals
Commercial products and chemical reagents were used as
received. 9,10-Diphenylanthracene (DPA, 97%), benzoyl
peroxide (BPO, reagent grade, Z 98%) and ferrocene
(Fc, 98%) were purchased from Aldrich (Mississauga, ON).
The supporting electrolyte, tetra-n-butylammonium perchlorate
(TBAP, electrochemical grade), was purchased from Fluka.
All solutions were prepared using anhydrous N,N-dimethyl-
formamide (DMF, 99.8%) in a Sure/Sealt bottle, bought from
Aldrich, that was immediately transferred into an N2-filled
drybox prior to use.
2.2 Synthesis of modified triazolyldeoxycytidine nucleosides
The synthesis of compounds 1–4 (Scheme 1) was published
elsewhere.38 In brief, these compounds were obtained by
c
17406 Phys. Chem. Chem. Phys., 2011, 13, 17405–17412
This journal is the Owner Societies 2011

View Article Online
internal standard. The redox potential of Fc/Fc+ was taken as
The ECL spectra were obtained by replacing the PMT
with a spectrometer (Cornerstone 260, Newport, Canada)
attached to a CCD camera (Model DV420-BV, Andor Tech-
nology, Belfast, UK). The camera was cooled to ꢀ55 1C prior
to use, and controlled by a computer for operation and data
acquisition. The intensities versus wavelengths (spectra) were
recorded by Andor Technology program. Similar to the
pulsing experiments, the samples were pulsed at 10 Hz within
each compound’s potential window. The exposure time of the
spectra was set to 60 s for both the annihilation and coreactant
systems. Vertical lines/spikes observed in the spectra were
from cosmic rays from the CCD camera.
0.470 V vs. NHE.47
2.4 Electrochemical instrumentation
The CVs were conducted on a CHI 610A electrochemical
analyzer (CH Instruments, Austin, TX). The experimental
parameters for CVs are listed here: 0.000 V initial potential
in the experimental scale, positive or negative initial scan
polarity, 0.1 V sꢀ1 scan rate, 4 sweep segments, 0.001 V sample
interval, 2 s quiet time, 1–5 ꢁ 10ꢀ5 A Vꢀ1 sensitivity. The
potential range depended on the particular compound.
Four DPVs were taken for each compound on the CHI
610A, two for anodic scans (forward and reverse scans in the
experimental potential scale between 0.000 V and the upper
limit potential value of the compound obtained from CV
experiments) and two for cathodic scans (forward and reverse
scans in the experimental potential scale between 0.000 V and
the low limit potential value of the compound obtained from
CV experiments). The experimental parameters for DPVs are
as following: 0.004 V increments, 0.05 V amplitude, 0.5 s pulse
width, 0.0167 s sampling width, 0.2 s pulse period, 2 s quiet
time, 1–5 ꢁ 10ꢀ5 A Vꢀ1 sensitivity.45
2.6 ECL efficiency calculations
ECL quantum efficiencies (QE) were calculated relative to
DPA (the reported relative ECL efficiency, FECL, of DPA
was taken 100% or 1.0 in DMF)48,49 by integrating both the
ECL intensity and the current value versus time for each
compound, as described in eqn (1),10,50,51
,
0
R
1
0
R
1
b
b
R
R
ECL dt
ECL dt
B
B
B
@
C
C
C
A
B
B
B
@
C
C
C
A
a
a
F ¼ 100 ꢁ
ð1Þ
x
b
b
Current dt
Current dt
a
a
st
x
2.5 ECL instrumentation
where x stands for the compound (1–4) and st represents DPA.
The ECL cell was specifically designed to have a flat Pyrex
window at the bottom for detection of generated light from the
WE and was sealed with a Teflon cap with a rubber O-ring for
CV, DPV, and ECL measurements. The ECL data along with
CV data were obtained using the CHI 610A coupled with a
photomultiplier tube (PMT, R928, Hamamatsu, Japan) held
at ꢀ750 V with a high voltage power supply. The ECL
collected by the PMT under the flat Pyrex window at the
bottom of the cell was measured as a photocurrent, and
transformed into a voltage signal, using a picoammeter/
voltage source (Keithley 6487, Cleveland, OH). The potential,
current signals from the electrochemical workstation, and the
photocurrent signal from the picoammeter were sent simulta-
neously through a DAQ board (DAQ 6052E, National
Instruments, Austin, TX) in a computer. The data acquisition
system was controlled using a custom-made LabVIEW program
(ECL_PMT610a.vi, National Instruments, Austin, TX). The
photosensitivity on the picoammeter was set manually in order
to avoid the saturation.
3. Results and discussion
3.1 Electrochemistry and its correlation to electronic
structures
The electrochemical behaviours of compounds 1–4 were
studied in order to determine the oxidation and reduction
potentials. Fig. 1 shows the CVs of 1 in DMF solution
containing 0.1 M TBAP as supporting electrolyte and the
blank DMF solution containing 0.1 M TBAP at a scan rate of
0.1 V sꢀ1 within potential ranges between 0.000 and 2.169, and
between 0.000 and ꢀ1.889 V, respectively. When the potential
was scanned initially from 0.000 to 2.169 V, compound 1
underwent the first oxidation at a peak potential of 1.858 V
followed by a continuous rise in current in the potential scan
until 2.169 V. There was no cathodic peak when the applied
potential was scanned back, indicating the irreversibility of the
electrochemical oxidation reaction. The radical cations might
ECL pulsing experiments were conducted by using a
potentiostat (Model AFCBPI, Pine Instrument Co., Grove
City, PA), an EG&G PAR 175 Universal Programmer
(Princeton Applied Research, Trenton, NJ), and the PMT
with the picoammeter in a similar manner. The assembly was
able to perform the pulsing experiments without a delay in a
relative fast time pace. The data acquisition for the current,
potential and ECL signals was carried out using another
homemade LabVIEW program (ECL_PAR610a.vi). For
coreactant systems, the applied potential was pulsed at the
WE in the cathodic region (in the experimental potential scale
between 0 and the low limit potential value for the compound
reduction as obtained from CV experiments) with a pulse
width of 0.1 s or 10 Hz.
Fig. 1 Cyclic voltammograms of 1 (solid lines) in DMF containing
0.1 M TBAP as supporting electrolyte, and blank solution (dotted lines),
with the initial scan from 0.000 to 2.169 V (anodic scan), and the initial
scan from 0.000 to ꢀ1.889 V (cathodic scan), with a scan rate of 0.1 V sꢀ1
.
c
This journal is the Owner Societies 2011
Phys. Chem. Chem. Phys., 2011, 13, 17405–17412 17407

View Article Online
Fig. 3 Differential pulse voltammograms of 1 (solid lines) in DMF
containing 0.1 M TBAP as supporting electrolyte, and blank solution
(dotted lines), with the initial scan from 0.000 to 2.118 V (top, anodic
scan), and the initial scan from 0.000 to ꢀ1.929 V (bottom, cathodic
scan) and their reverse scans.
0
The formal potentials (E0 ) of 1 can be determined from DPVs
by eqn (2):52,53
0
E0 = Emax + DE/2
(2)
where Emax represents the peak potential in the DPV and DE
represents the pulse height (50 mV).
The oxidation peak at 1.826 V in the anodic scan and the
reduction peak at ꢀ1.785 V in the cathodic scan were more
visible (Fig. 3), than in CV (Fig. 1) since DPV suppresses the
background signal and enhances sensitivity.45
Fig. 2 Ball-and-stick representation of 1. Except for OH and NH2
protons, H-atoms are omitted for clarity. Please see Fig. S2, in the
ESIw for labeled atoms. The final solution was submitted to the
IUCR CIF checking program and had some Alert level A’s or B’s
associated with the lack of complete data, however the general
structure can be observed for 1, similar to our previously reported
solid state structure of 2.38
The electrochemical behaviours of compounds 2–4 were
also characterized using CV and DPV, from which it can be
concluded that compounds 2–4 undergo irreversible oxidation
and reduction reactions, and their radical cations and anions
are not stable.
All the electrochemical data have been summarized in
Table 1. The electrochemical gaps, which are the potential
difference between the formal potentials of the first reduction
and oxidation, match well with their corresponding HOMO–
LUMO energy gaps. The excited state gap taken from the PL
emission wavelength from Table 2 shows similar energy values
as the experimental energy values in Table 1.
undergo further chemical reactions (EC mechanism). From
Fig. S4 (ESIw) of our previous publication, the HOMO orbital
revealed by the density function theory calculation is the linear
combination of pz atomic orbitals of S, C, N and O atoms in
the thiophene, triazole and deoxycytidine rings.38 The electron
withdrawn from compound 1 upon oxidation was delocalized
in the molecule because of the co-planar structure determined
by X-ray crystallography (Fig. 2). Note that the crystal
structure is our best estimation since it did not pass cif file
checking (see details in the ESIw). Similarly, compound 1
demonstrated an irreversible reduction peak at ꢀ1.835 V when
the applied potential was scanned from 0.000 V to the cathodic
region. Based on the DFT calculation, the LUMO orbital was
mostly contributed from pz atomic orbitals of S, C and N
atoms in the thiophene and triazole rings. Dimerization would
probably happen on the thiophene ring. It should be noted
that some small multiple oxidation and reduction peaks
were observed in consecutive cycling of the applied potential,
due to the electrochemical reactivity of the intermediates from
the EC reaction mechanisms (see more details in the ECL
section).
From Table 1, there was a general trend of decreasing
oxidation potential with increasing conjugation. The addition
of a second thiophene ring in 3 decreases the oxidation
potential from 1.851 V in 1, to 1.488 V in 3. This trend,
however, was not apparent in the reduction potentials of 1–4.
Noticeably, compound 4 underwent up to 4 reduction
reactions as summarized in Table 1. Nevertheless, the trend
of smaller electrochemical gaps, as seen in CV and DPV, with
increasing conjugation was observed from our experimental
Typical DPVs of compound 1 conducted in a similar
manner as in CVs shown in Fig. 1 are illustrated in Fig. 3,
where the first irreversible oxidation and reduction peaks of 1
are well displayed. Again, isolation of the anodic and cathodic
DPVs avoids any additional redox peaks as seen in Fig. 4.
This agrees well with the observation from CVs because
of the electrochemical reactivity of the intermediates.
Fig. 4 Cyclic voltammogram and ECL–voltage curve of 1 scanned at
0.1 V sꢀ1 with the initial potential at 0.000 to 2.069 V then scanned to
ꢀ1.889 V and back to 0.000 V.
c
17408 Phys. Chem. Chem. Phys., 2011, 13, 17405–17412
This journal is the Owner Societies 2011

View Article Online
Table 1 Electrochemical (from DPV) and quantum chemistry calculation data of compounds 1–4
E0
E0
Electrochemical
Theoretical
Theoretical
Theoretical
0
0
Ep,a
Ep,c
oxidationa/V reductiona/V oxidationa/V reductiona/V gap/eV
HOMOc/eV LUMOc/eV HOMO–LUMO gap/eV
1
2
1.826
1.268
ꢀ1.785
ꢀ2.081/
ꢀ2.449
ꢀ1.948
ꢀ1.310/
ꢀ1.550/
ꢀ1.806/
ꢀ2.480
1.851
1.293
ꢀ1.810
ꢀ2.106/
ꢀ2.474
ꢀ1.973
ꢀ1.335/
ꢀ1.575/
ꢀ1.831/
ꢀ2.505
3.66
3.40
ꢀ5.90
ꢀ5.88
ꢀ1.74
ꢀ1.70
4.15b
4.18c
3
4
1.463
0.765/1.365
1.488
0.790/1.390
3.46
2.13
ꢀ5.90
ꢀ5.93
ꢀ1.96
ꢀ3.02
3.94c
2.92c
a
b
In V vs. NHE at 0.1 V sꢀ1 scan rate. Energy value obtained from previously reported data, DFT/B3LYP/6-31G* calculations.38 c Energies
obtained from DFT/B3LYP/6-31G* calculations.
Table 2 ECL data of compounds 1–4 in annihilation and coreactant systems
Annihilation scanning QEa/% Scanning QEa/% Coreactant pulsing QEa/%
l
max/nm PL lmax/nm Shiftsb lmax(ECL)ꢀlmax(PL)/nm
1
2
3
4
0.40–0.52
0.39–0.41
0.71–1.08
0.10–0.24
0.33–1.48
0.73–0.86
0.24–0.39
0.54–2.33
0.71
0.20
0.25
1.87
515
395/577 380
375
140
15/197
208
171
588
556
380
385
a
ECL quantum efficiencies (QE) measured in DMF relative to DPA (FECL = 100% or 1.0 in DMF).48,49 b The shifts represent the difference
between the ECL (in DMF) and PL (in EtOH)38 emission wavelengths. Only small shifts (o5 nm) in PL emission maxima were observed when
varying the solvent composition from EtOH to DMF.
data and from previous DFT quantum chemistry calculations
that our group has reported.38 Here, the HOMO–LUMO gap
decreases in 3, 3.94 eV, relative to 1, 4.15 eV, having greater
delocalization of the p electrons due to increased conjugation
in the aromatic system, Table 1. For compounds 1 and 2,
the gaps, of 4.15 eV and 4.18 eV, were larger due to smaller
conjugated systems. Synthesizing compounds with an
additional thiophene ring, 3, and extended aromatic system,
4, the HOMO–LUMO gaps for these conjugated systems
decreased to 3.94 eV for 3 and 2.92 eV for 4. These values
followed a similar trend as the experimental HOMO–LUMO
gaps from the oxidation and reduction potentials of 1–4 as
seen in Table 1. The gap observed in 2 from experimental data
was smaller, 3.40 eV, than 1, 3.66 eV, this may be due to the
slightly out-of-plane aromatic system of 1 compared to the
in-plane system of 2. However, the HOMO–LUMO gap
calculations were calculated from a solid-state representation
of each compound, whereas, the redox, ECL and spectral
data were obtained from solutions of these compounds.
We must take into consideration the free rotation of the
compounds in solution. Here, we would expect variations in
the data as seen in the differences between the theoretical and
experimental data.
oxidation and nor an anodic peak for reduction in CVs
(Fig. 1). This is evidence that both radicals were not stable.
Cations underwent chemical reactions to generate reduction-
active species while radical anions generated redox-active
species. However, we cannot determine which are the species.
At both positive and negative potential limits, weak ECL
was observed. A slightly stronger emission of light was
observed in the positive potential region. The radical cations
appear to be more stable than the radical anions for 1, as
stronger ECL was detected in the region of positive potential,
Fig. 4. The weak ECL was expected since both radical cations
and anions were not stable as observed from the irreversibility
of its electrochemical behaviours. The ECL given off from 1,
written as ThdC, was proposed via annihilation of radical
+
cations (ThdCꢂ ) with radical anions (ThdCꢂꢀ) generated
electrochemically, eqn (3) and (4).
ThdC - ThdCꢂ + eꢀ
+
(3)
(4)
ThdC + eꢀ - ThdCꢂ
ꢀ
An excited species, ThdC*, and a ground-state species, ThdC,
of 1, are the products of the annihilation as seen in eqn (5).
The excited species then relaxes to its ground state, ThdC, as
seen in eqn (6) and emits light.
3.2 ECL via annihilation mechanisms
+
ꢀ
ThdCꢂ + ThdCꢂ - ThdC* + ThdC
(5)
(6)
Fig. 4 shows the CV overlaid with the ECL–voltage curve of 1
that was run simultaneously in the potential range between its
first oxidation and reduction peaks.
ThdC* - ThdC + hn1
During these continuous scans, we observed additional
bumps/peaks in the middle of the potential window. These
peaks were not from the neutral compound as demonstrated in
Fig. 1: when the applied potential was scanned separately for
oxidation and reduction peaks, these additional peaks were
not present. In addition, there was neither a cathodic peak for
Fig. 4 also shows that ECL can only be detected after one
complete cycle of the potential sweep. In practice, two cycles
or four segments of the potential sweep were conducted for
each compound.
Compound 2 displayed similar ECL emission to that from
compound 1, and demonstrated stronger ECL intensity in the
c
This journal is the Owner Societies 2011
Phys. Chem. Chem. Phys., 2011, 13, 17405–17412 17409

View Article Online
anodic region than in the cathodic region. For 3, ECL
emission was observed in the region of negative potential
and 4 did not show a significant increase in positive or negative
potential regions. Nevertheless, weak ECL was generated
through annihilation mechanisms from compounds 3–4,
because of the irreversible redox reactions, resulting in unstable
radical ions. The ECL efficiencies were determined as the
photons emitted per redox event relative to the standard,
DPA, as expressed in eqn (1).50 These ECL efficiencies
(Table 2) were low relative to the standard, which were in a
range between 0.40 to 0.52% for 1, 0.39 to 0.41% for 2, and
0.71 to 1.08% for 3. Compound 4 had the smallest ECL
efficiency, ranging between 0.10 to 0.24%.
An increase in photocurrent as shown in Fig. 5 was
observed when scanning to the negative potential. The use of
BPO contributes to the enhanced ECL intensities of 1–4
relative to the intensities observed in annihilation studies.
It is important to note that there was a relatively weak
ECL peak in Fig. 5 that was observed right after the BPO
reduction potential, ꢀ0.780 V, but even before the reduction
+
of compound 1. In fact, the ThdCꢂ generated through
reactions (7)–(9) can react with BPOꢂꢀ, eqn (10), to produce
the excited species, ThdC*:
+
ꢀ
ThdCꢂ + BPOꢂ - ThdC* + BPO
(10)
Compounds 2–4, in the same coreactant system, displayed
higher intensities of ECL than in annihilation systems relative
to DPA as seen in Table 2. ECL was observed in all com-
pounds after the reduction of BPO, following the same
mechanism, eqn (10).
3.3 ECL via coreactant mechanisms
Fig. 5 shows the CV overlaid with the simultaneous ECL–
voltage curve of 1 containing BPO. The applied potential was
only needed to scan in the cathodic region. In this way the
short-life radical anions, ThdCꢂꢀ, generated can immediately
react with their counterparts.1,54 Sometimes the radical cations
can be generated directly from the coreactant.
It was discovered that ECL was enhanced when 5.0 ꢁ 10ꢀ3 M
BPO was added to the solutions of 1–4. The proposed ECL
coreactant mechanism for 1 begins when a negative potential is
applied to the system, initially at 0.000 V, as seen in Fig. 5 for
compound 1. Upon scanning to more negative potential, BPO is
first reduced to its radical anion, BPOꢂꢀ, at ꢀ0.780 V, eqn (7).
The radical anion, BPOꢂꢀ, immediately decomposes to form a
The intensity of ECL varied from 1–4. Compounds 1 and 4
displayed the highest ECL intensities with photocurrents
around 100 nA for 1 and 180 nA for 4. The efficiencies for
these compounds were in a range between 0.33 to 1.48% for 1
and 0.54 to 2.33% for 4. Compounds 2 and 3 had lower ECL
intensities, relative to 1 and 4, with photocurrents around
85 nA for 2 and 30 nA for 3. The efficiencies were in a range
between 0.73 to 0.86% for 2 and 0.24 to 0.39% for 3.
ECL was enhanced when the applied potential was pulsed in
the cathodic region between 0.000 V and the low limit
potential value for the compound reduction as obtained from
CV experiments with a pulse width of 0.1 s. Instead of slowly
scanning the potential from 0.000 V to ꢀ2.278 V shown in
Fig. 5, the working electrode was pulsed quickly between these
potential values and an increase in intensity of ECL was
observed. The efficiencies for these compounds were 0.71%
for 1, 0.20% for 2, 0.25% for 3, and 1.87% for 4. Pulsing
generates radical cations and anions in a faster alternative
pace and reduces the decay of the unstable radicals, leading to
greater occasions for the cations and anions to meet, react,
and emit light.
ꢂ
ꢂ
strong oxidizing intermediate radical, C6H5CO2 , and
ꢀ
C6H5CO2 , eqn (8). This intermediate radical, C6H5CO2 , reacts
with 1 and generates the radical cation of 1, ThdCꢂ+, eqn (9).
ꢀ
BPO + eꢀ - BPOꢂ
(7)
(8)
(9)
ꢀ
ꢀ
BPOꢂ - C6H5CO2 + C6H5CO2
ꢂ
+
ꢀ
C6H5CO2 + ThdC - ThdCꢂ + C6H5CO2
ꢂ
With the applied potential scanned to more negative potential,
the radical anion of 1, ThdCꢂꢀ, was generated at ꢀ1.835 V,
eqn (4). The radical cation, ThdCꢂ+, combines with the
radical anion, ThdCꢂꢀ, and generates an excited species,
ThdC*, of 1, as stated previously with the annihilation
mechanism in eqn (5) and (6), which relaxes down to its
ground state, ThdC, and light is emitted.
3.4 ECL spectroscopy
Fig. 6 shows the coreactant ECL spectra of 1–4 with BPO
during the pulsing of the applied potential in the cathodic
regions (till the reduction of the compounds). The spectrum
for 1 in the coreactant system was fitted to one peak centered
at 515 nm, Fig. 6(a) (see spectra with curve-fitting, Fig. S1,
in the ESIw). The peak at 515 nm was red-shifted relative to
that in PL by 140 nm. The long wavelength ECL can be
assigned to emission from excimers.38 Excimers are excited
states of dimers that can be observed in ECL of organic
compounds.15,16 The formation of excimers might result from
dimerization of the radical anion and cation or stacking of the
monomer in the excited state with a monomer in the ground
state due to the p conjugation of the modified nucleobase.50
A possible mechanism for excimer growth in ECL for 1 is
shown in eqn (11)–(13).55 ThdC* can react with the ground
state species ThdC to form an excimer as seen in eqn (11).
Another possible route for excimer formation involves the
radical cation and anion species to form the excimer,
Fig. 5 Cyclic voltammogram and ECL–voltage curve of 1 with
5.0 ꢁ 10ꢀ3 M BPO scanned at 0.1 V sꢀ1, with the initial scan from
0.000 to ꢀ2.278 V and back to 0.000 V.
c
17410 Phys. Chem. Chem. Phys., 2011, 13, 17405–17412
This journal is the Owner Societies 2011

View Article Online
peak to red-shift relative to the photoluminescence (PL) peaks
as previously reported.38
4. Conclusions
We have determined the redox potentials and calculated the
ECL efficiencies of a series of modified triazolyldeoxycytidine
nucleosides, 1–4. With increasing conjugation in the lumino-
phore, we observed a decrease in the separation between
first oxidation and reduction peaks of the compounds. The
annihilation ECL efficiencies were weak relative to DPA,
ranging from 0.40 to 0.52% for 1, 0.39 to 0.41% for 2, 0.71
to 1.08% for 3, and 0.10 to 0.24% for 4. The efficiencies
increased in the coreactant systems with ECL efficiencies
ranging from 0.33 to 1.48% for 1, 0.73 to 0.86% for 2, 0.24
to 0.39% for 3, and 0.54 to 2.33% for 4. The ECL spectra were
red-shifted relative to the corresponding PL spectra previously
reported with wavelengths ranging from 515 nm for 1, 395 and
577 nm for 2, 588 nm for 3, and 556 nm for 4. The generation
of excimers in 1–4, with the monomer still present in 2, was
observed in the ECL systems studied. The radical anions and
cations generated in solution follow two pathways, via electron
transfer, and combination of electron transfer and dimeriza-
tion, forming excited species and excimers. Incorporating
these modified nucleosides into ssDNA, and hybridizing the
modified ssDNA with a complementary ssDNA or a single-
base mis-matched ssDNA may allow for an ECL readout
which could give information on the nature of the mismatch,
thereby leading to an effective means of SNP typing. ECL is a
fast, cost effective technique that requires low quantity and
is highly selective, sensitive, and tunable with our electro-
chemical instrumentation and detection systems. Using ECL
detection with these modified nucleosides is in progress
towards SNP typing of genes pertinent to various human
genetic disorders.
Fig. 6 ECL spectra of 1–4 in DMF containing 5.0 ꢁ 10ꢀ3 M BPO
and 0.1 M TBAP as supporting electrolyte and pulsing between
potential ranges from (a) 0.000 to ꢀ2.278 V, t = 60 s for 1,
(b) 0.000 to ꢀ2.452 V, t = 60 s for 2, (c) 0.000 to ꢀ2.517 V, t = 60 s
for 3, and (d) 0.000 to ꢀ2.126 V, t = 60 s for 4. ECL intensities were
normalized by their respective peak heights.
(ThdC)2*, eqn (12). The excimer can then relax down to its
ground state and emit light, eqn (13). For compound 1, the
two routes are possible:
ThdC + ThdC* - (ThdC)2*
(11)
(12)
(13)
+
ꢀ
ThdCꢂ + ThdCꢂ - (ThdC)2*
(ThdC)2* - (ThdC)2 + hn2
The energy of the resulting excited species, (ThdC)2*, was
lower than that of the reactants and relaxes down to the
ground state, ThdC, and a longer wavelength than the mono-
mer will be observed, similar to the spectrum of 2, Fig. 6(b).
For 2, the spectrum showed two peaks, one at 395 nm and the
second broad band at 577 nm. The peaks were red-shifted by
15 and 197 nm with respect to the PL peak seen at 380 nm.
From eqn (5), (6), and (9)–(13), we observed excited monomer
and excimer formation from the two given pathways. It
appears that as conjugation increases in the modified nucleo-
sides, ECL emission becomes more red-shifted. Compound 3,
Fig. 6(c), showed one maximum at 588 nm and displayed the
most significant red-shift of 208 nm relative to the PL peak at
380 nm. Furthermore, the maximum wavelength of compound
4, Fig. 6(d), was at 556 nm, red-shifted to PL by 171 nm
respectively. The growth in excimer formation was more easily
observed with 2, Fig. 6(b), and the most red-shifted com-
pounds were 2, 3 and 4. We can conclude that compounds 1–4
can easily form excimers, however there was a combination of
monomers and excimers present in ECL system 2 by pulsing
the electrode potential.
Acknowledgements
We thank the Natural Sciences and Engineering Research
Council (NSERC, Canada) for generous financial support
through the Discover Grant and Research Tools and Instruments
Grant programs. DWD acknowledges an NSERC Canada
Graduate Scholarship (CGS-D). Additionally, we would like to
acknowledge Dr P.J. Ragogna, and Dr D. W. Shoesmith, for use
of their instruments, John Vanstone and Jon Aukema (Electro-
nics Shop), Yves Rambour (Glassblowing Shop) for their
technical service, and the Department of Chemistry at The
University of Western Ontario for supporting graduate research.
References
1 A. J. Bard, in Electrogenerated Chemiluminescence, ed. A. J. Bard,
Marcel Dekker, Inc., New York, 2004.
2 D. M. Hercules, Science, 1964, 145, 808–809.
3 R. E. Visco and E. A. Chandross, J. Am. Chem. Soc., 1964, 86,
5350–5351.
4 K. S. V. Santhanam and A. J. Bard, J. Am. Chem. Soc., 1965, 87,
139–140.
5 R. Pyati and M. M. Richter, Annu. Rep. Prog. Chem., Sect. C,
2007, 103, 12–78.
6 M. M. Richter, Chem. Rev., 2004, 104, 3003–3036.
The crystal structures of 1 (this work) and 238 illustrate the
coplanar shape of the conjugated aromatic ring systems for
these four compounds, allowing the p electrons to delocalize
over the aromatic ring systems and therefore lowering the
band gap between the HOMO and LUMO. This enhances the
effective conjugation length and consequently causes emission
c
This journal is the Owner Societies 2011
Phys. Chem. Chem. Phys., 2011, 13, 17405–17412 17411

View Article Online
7 S. Kulmala and J. Suomi, Anal. Chim. Acta, 2003, 500, 21–69.
8 K. A. Fahnrich, M. Pravda and G. G. Guilbault, Talanta, 2001, 54,
34 D. Peters, A.-B. Hornfeldt, S. Gronowitz and N. G. Johansson,
¨
Nucleosides Nucleotides, 1992, 11, 1151–1173.
35 M. Mizuta, J.-I. Banba, T. Kanamori, R. Tawarada, A. Ohkubo,
M. Sekine and K. Seio, J. Am. Chem. Soc., 2008, 130, 9622–9623.
36 N. E. Tokel-Takvoryan, R. E. Hemingway and A. J. Bard, J. Am.
Chem. Soc., 1973, 95, 6582–6589.
37 K. N. Swanick, D. W. Dodd, R. H. E. Hudson, Z. Ding and
N. D. Jones, 19th QOMSBOC, Toronto, Canada, 2008.
38 D. W. Dodd, K. N. Swanick, J. T. Price, A. L. Brazeau, M. J.
Ferguson, N. D. Jones and R. H. E. Hudson, Org. Biomol. Chem.,
2010, 8, 663–666.
39 P. Kocalka, N. K. Andersen, F. Jensen and P. Nielsen, ChemBioChem,
2007, 8, 2106–2116.
40 V. Munshi, M. Lu, P. Felock, R. J. O. Barnard, D. J. Hazuda,
M. D. Millers and M.-T. Lai, Anal. Biochem., 2008, 374, 121–132.
41 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004–2021.
42 H. C. Kolb and K. B. Sharpless, Drug Discovery Today, 2003, 8,
1128–1137.
43 P. Wu and V. V. Folkin, Aldrichimica Acta, 2007, 40, 7–17.
44 P. Wu, A. K. Feldman, A. K. Nugent, C. J. Hawker, A. Scheel,
´
B. Voit, J. Pyun, J. M. J. Frechet, K. B. Sharpless and V. V. Folkin,
¨
531–559.
9 A. W. Knight, TrAC, Trends Anal. Chem. (Pers. Ed.), 1999, 18,
47–62.
10 D. Laser and A. J. Bard, J. Electrochem. Soc., 1975, 122, 632–640.
11 W. Miao, Chem. Rev., 2008, 108, 2506–2553.
12 L. Hu and G. Xu, Chem. Soc. Rev., 2010, 39, 3275–3304.
13 S. Krishnan, E. G. Hvastkovs, B. Bajrami, D. Choudhary, J. B.
Schenkman and J. F. Rusling, Anal. Chem., 2008, 80, 5279–5285.
14 J. F. Rusling, E. G. Hvastkovs, D. O. Hull and J. B. Schenkman,
Chem. Commun., 2008, 141–154.
15 R. Y. Lai, J. J. Fleming, B. L. Merner, R. J. Vermeij, G. J. Bodwell
and A. J. Bard, J. Phys. Chem. A, 2004, 108, 376–383.
16 J.-P. Choi, K.-T. Wong, Y.-M. Chen, J.-K. Yu, P.-T. Chou and
A. J. Bard, J. Phys. Chem. B, 2003, 107, 14407–14413.
17 T. G. Drummond, M. G. Hill and J. K. Barton, Nat. Biotechnol.,
2003, 21, 1192–1199.
18 X. Xu and A. Bard, J. Am. Chem. Soc., 1995, 117, 2627–2631.
19 C. A. Marquette and L. J. Blum, Anal. Bioanal. Chem., 2008, 390,
155–168.
20 W. Miao and A. J. Bard, Anal. Chem., 2003, 75, 5825–5834.
21 K. Furukawa, H. Abe, J. Wang, M. Uda, H. Koshino, S. Tsuneda
and Y. Ito, Org. Biomol. Chem., 2009, 7, 671–677.
22 E. Pinnisi, Science, 1998, 281, 1787–1789.
Angew. Chem., Int. Ed., 2004, 43, 3928–3932.
45 A. J. Bard, Z. Ding and N. Myung, in Structure & Bonding,
Springer Berlin/Heidelberg, 2005, vol. 118, pp. 1–57.
46 R. M. Cervino, W. E. Triaca and A. J. Arvia, J. Electroanal. Chem.,
1985, 182, 51–60.
23 S. Tyagi, D. P. Bratu and F. R. Kramer, Nat. Biotechnol., 1998, 16,
49–53.
24 A. P. Silverman and E. T. Kool, Chem. Rev., 2006, 106,
3775–3789.
47 S. K. Haram, B. M. Quinn and A. J. Bard, J. Am. Chem. Soc.,
2001, 123, 8860–8861.
25 X. Li and D. R. Liu, Angew. Chem., Int. Ed., 2004, 43, 4848–4870.
26 S. Tyagi and F. R. Kramer, Nat. Biotechnol., 1996, 14, 303–308.
27 A. Okamoto, K. Tanaka, T. Fukuta and I. Saito, J. Am. Chem.
Soc., 2003, 125, 9296–9297.
48 K. M. Omer, S.-Y. Ku, K.-T. Wong and A. J. Bard, Angew. Chem.,
Int. Ed., 2009, 48, 9300–9303.
49 R. Bezman and L. R. Faulkner, J. Am. Chem. Soc., 1972, 94,
6317–6323.
28 A. Okamoto, K. Tanaka, T. Fukuta and I. Saito, ChemBioChem,
2004, 5, 958–963.
29 J. Zhang, X. J. Sun, K. M. Smith, F. Visser, P. Carpenter, G. Barron,
Y. S. Peng, M. J. Robins, S. A. Baldwin, J. D. Young and C. E. Cass,
Biochemistry, 2006, 45, 1087–1098.
50 C. Booker, X. Wang, S. Haroun, J. Zhou, M. Jennings, B. L.
Pagenkopf and Z. Ding, Angew. Chem., Int. Ed., 2008, 47,
7731–7735.
51 W. L. Wallace and A. J. Bard, J. Phys. Chem., 1979, 83,
1350–1357.
30 L. F. Liu, Y. F. Li, D. Liotta and S. Lutz, Nucleic Acids Res., 2009,
37, 4472–4481.
31 J. A. Secrist, J. R. Barrio and N. J. Leonard, Science, 1972, 175,
646–647.
52 A. J. Bard and L. R. Faulkner, Electrochemical methods, funda-
mentals and applications, John Wiley & Sons Inc., New York,
2nd edn, 2001.
53 H. H. Girault, Electrochimie Physique et Analytique, Presses Poly-
technique et Universitaires Romandes, Lausanne, Switzerland,
2001.
54 Z. Ding, B. M. Quinn, S. K. Haram, L. E. Pell, B. A. Korgel and
A. J. Bard, Science, 2002, 296, 1293–1297.
55 I. Prieto, J. Teetsov, M. A. Fox, D. A. Vanden Bout and
A. J. Bard, J. Phys. Chem. A, 2001, 105, 520–523.
´
32 N. K. Andersen, L. Spa
´
cilova
´
, M. D. Jennings, P. Kocalka, F. Jensen
and P. Nielsen, Joint Symposium of the 18th International Round-
table on Nucleosides, Nucleotides and Nucleic Acids and the
35th International Symposium on Nucleic Acids Chemistry, Kyoto,
Japan, 2008.
33 M. E. Hassan, Nucleosides Nucleotides, 1991, 10, 1277–1283.
c
17412 Phys. Chem. Chem. Phys., 2011, 13, 17405–17412
This journal is the Owner Societies 2011