Scope and limitations of the TEMPO/EPR method for singlet oxygen detection: The misleading role of electron transfer

Article abstract of DOI:10.1016/j.freeradbiomed.2014.08.020

For many biological and biomedical studies, it is essential to detect the production of ¹O₂ and quantify its production yield. Among the available methods, detection of the characteristic 1270-nm phosphorescence of singlet oxygen by time-resolved near-infrared (TRNIR) emission constitutes the most direct and unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in combination with a singlet oxygen probe. This is based on the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6-tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has been widely employed, it can produce misleading data. This is demonstrated by the present study, in which the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the TEMPO/EPR method are compared for a set of well-known photosensitizers. The results reveal that the TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet excited state of the photosensitizer is efficiently quenched by TEMP, acting as electron donor. In such case, generation of the TEMP⁺ radical cation, followed by deprotonation and reaction with molecular oxygen, gives rise to an EPR-detectable TEMPO signal that is not associated with singlet oxygen production. This knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in chemical, biological, and medical studies.

Full text of DOI:10.1016/j.freeradbiomed.2014.08.020

Free Radical Biology and Medicine 77 (2014) 64–70
Contents lists available at ScienceDirect
Free Radical Biology and Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Original Contribution
Scope and limitations of the TEMPO/EPR method for singlet oxygen
detection: the misleading role of electron transfer
a
b
b
a
Giacomo Nardi , Ilse Manet , Sandra Monti , Miguel A. Miranda ,
a,
n
Virginie Lhiaubet-Vallet
Instituto de Tecnología Química UPV-CSIC, Universitat Politècnica de València, 46022 Valencia, Spain
Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, 40129 Bologna, Italy
a
b
a r t i c l e i n f o
a b s t r a c t
For many biological and biomedical studies, it is essential to detect the production of 1O2 and quantify its
production yield. Among the available methods, detection of the characteristic 1270-nm phosphorescence
of singlet oxygen by time-resolved near-infrared (TRNIR) emission constitutes the most direct and
unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in
combination with a singlet oxygen probe. This is based on the detection of the TEMPO free radical formed
after oxidation of TEMP (2,2,6,6-tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR
method has been widely employed, it can produce misleading data. This is demonstrated by the present
study, in which the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the
TEMPO/EPR method are compared for a set of well-known photosensitizers. The results reveal that the
TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet
Article history:
Received 25 June 2014
Received in revised form
19 August 2014
Accepted 20 August 2014
Available online 16 September 2014
Keywords:
EPR
Photosensitizer
Singlet oxygen
TEMPO
excited state of the photosensitizer is efficiently quenched by TEMP, acting as electron donor. In such case,
Time-resolved near-infrared emission
Free radicals
þd
generation of the TEMP
radical cation, followed by deprotonation and reaction with molecular oxygen,
gives rise to an EPR-detectable TEMPO signal that is not associated with singlet oxygen production. This
knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in
chemical, biological, and medical studies.
&
2014 Elsevier Inc. All rights reserved.
Singlet oxygen (molecular oxygen in the 1
one of the most important “reactive oxygen species.” Its reactions
include oxidation of lipids [1,2], proteins [3–5], and nucleic acids
6–8], which may trigger biological damage. This reaction cascade
can lead to undesired adverse effects, such as drug-induced
phototoxicity [9,10], but can also be exploited to produce bene-
Δ
state, or 1
O
) is
For many biological and biomedical studies, it is essential to
detect the production of O and quantify its production yield.
2
g
2
1
Among the available methods, detection of the characteristic
1270-nm phosphorescence of singlet oxygen by time-resolved
[
1
near-infrared (TRNIR) emission constitutes the most direct and
unambiguous proof [14,15]. However, the required equipment is
not always available in biochemical laboratories.
ficial effects as in photodynamic therapy [11,12].
1
Production of O
2
by a photosensitizer is a classical example of
An alternative indirect method that has been widely applied is
1
photoinduced energy transfer: after absorption of light, the photo-
sensitizer reaches its singlet excited state and subsequently
electron paramagnetic resonance (EPR) in combination with a O
2
1
2
probe. Upon reaction with O , the trapping molecule gives rise to
crosses to its triplet excited state. Then, the triplet ground state
a detectable spin-active species with a distinctive line pattern.
Thus, oxidation of TEMP (2,2,6,6-tetramethylpiperidine) by singlet
oxygen yields the TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl)
free radical easily detected by EPR (Fig. 1) [16]. Although the
TEMPO/EPR method has been widely employed [17–25], a sys-
tematic investigation of the scope and limitations of this technique
has never been performed. For instance, amines are widely known
for their ability to quench excited states, so a probable source of
artifacts may be the interaction between the excited photosensi-
tizer and TEMP [26–29]. The aim of the present study was to
compare the results obtained for the detection and quantification
of singlet oxygen by means of the direct method (TRNIR emission)
1
of molecular oxygen is promoted to the Δ
g
state through triplet–
triplet energy transfer [13].
Abbreviations: ACN, acetonitrile; BAHA, tris(4-bromophenyl)aminium hexachlor-
oantimonate; BP, benzophenone; CBZ, carbazole; EPR, electron paramagnetic
resonance; LFP, laser flash photolysis; NP, naphthalene; PET, photoinduced electron
transfer; PN, phenalenone; RB, rose Bengal; TEMP, 2,2,6,6-tetramethylpiperidine;
TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxyl; TRNIR, time-resolved near infrared
n
Corresponding author.
E-mail address: lvirgini@itq.upv.es (V. Lhiaubet-Vallet).
http://dx.doi.org/10.1016/j.freeradbiomed.2014.08.020
0891-5849/& 2014 Elsevier Inc. All rights reserved.

G. Nardi et al. / Free Radical Biology and Medicine 77 (2014) 64–70
65
1O2
Cl
Cl
N
H
N
O
Cl
Cl
O
TEMP
TEMPO
I
O
I
HO
O
OH
I
I
Rose Bengal(RB)
O
O
H
N
Phenalenone (PN) Benzophenone (BP) Naphthalene (NP)
Carbazole (CBZ)
Fig. 1. Structure of the molecules involved in the study and EPR signal of the TEMPO radical.
1
and the indirect O
2
trapping mode (TEMPO/EPR method), using a
Fluorescence lifetimes
set of well-known photosensitizers. The basis of the TEMPO
method and the chemical structure of the selected photosensiti-
zers are shown in Fig. 1. The results obtained reveal that the EPR
method leads to significant overestimation of singlet oxygen
production when the singlet or triplet excited state of the photo-
sensitizers is efficiently quenched by TEMP, acting as electron
donor.
Fluorescence decay was measured in air-equilibrated solutions
with a time-correlated single-photon counting apparatus (IBH
5000F) equipped with a TBX picosecond photon detection module.
A nano-LED pulsed excitation source at 331 and 278 nm was used
and the emission was collected at right angle at 341 or 320 nm
using a long-pass cutoff filter at 305 nm. Fluorescence decay
profiles were fitted using a monoexponential function of the decay
analysis software DAS6 provided by the manufacturer with
deconvolution of the instrumental response function.
Materials and methods
Chemicals
Laser flash photolysis measurements
TEMP, tris(4-bromophenyl)aminium hexachloroantimonate (BAHA),
phenalenone (PN), benzophenone (BP), naphthalene (NP), carbazole
The beam of a pulsed Nd:YAG laser, operating at 532 or 355 nm
20 ns FWHM, 2 Hz, 2.7 mJ/pulse), was suitably shaped to pass
(
(CBZ), rose Bengal (RB), and acetonitrile (ACN) were from Sigma–
through a 3-mm-high and 10-mm-wide rectangular window and
2
Aldrich. TEMP was freshly distilled at 152 1C before use.
provide a fairly uniform energy density of 9 mJ/cm incident onto
the sample cell. A front portion of 2-mm depth of the excited
solution was probed at right angle, the useful optical path for
analyzing light being 1 cm. All transient spectra were recorded
with 3 ml of sample solutions in 1 ꢂ 1-cm quartz cells; when
Absorption and fluorescence spectra
2
UV–Vis absorption spectra were recorded on a commercial
spectrophotometer (λ650; PerkinElmer). Fluorescence spectra
were measured using 1-nm steps and 0.5-s dwell time, at right-
angle detection (FLSP920; Edinburgh Instruments). Slits were kept
narrow to 1 nm for excitation and 1 or 2 nm for emission; where
necessary, a cutoff filter was used. All the measurements were
carried out at 295 K in quartz cuvettes with a path length of 1 cm.
The fluorescence spectra were obtained for air-equilibrated solu-
tions with A o 0.1 over the whole absorption range to avoid inner
filter effects and reabsorption of emission. Quenching of CBZ and
NP fluorescence intensity by TEMP upon excitation at 331 and
specified ACN solutions were bubbled for 10 min with Ar before
data acquisition. The absorbance of the samples was kept in the
range 0.30–0.40 at the laser wavelength. Stock solutions of the
quenchers were prepared, so that addition of microliter volumes
to the sample cell allowed us to obtain the appropriate quencher
concentration.
ꢁ
1
ꢁ1
The bimolecular rate constant k
q
(M
s
) for quenching of
the triplet states was calculated from the slope of linear plots of
ꢁ
1
the observed triplet decay rate constant kobs (s ) versus the
quencher concentration, applying Eq. (2),
2
78 nm, respectively, was performed by adding increasing
kobs ¼ k
0
þk
q
½Qꢀ;
ð2Þ
amounts of TEMP to the solution. For NP measurements, the
fluorescence intensities were corrected for the inner filter effect
due to absorption of TEMP at 278 nm. The following equation was
used to determine Ksv, the Stern–Volmer quenching constant:
where k
0
is the triplet decay rate constant in the absence of
quencher and [Q] is the quencher molar concentration (M).
Singlet oxygen TRNIR emission measurements
F
0
=F ¼ 1þKsv½Qꢀ:
ð1Þ
In Eq. (1), F
0
and F are the fluorescence intensities, respectively, in
The pulse of a Nd:YAG laser, operating at 355 or 266 nm (20-ns
FWHM), was used for excitation of the samples dissolved in air-
equilibrated acetonitrile. A preamplified (low impedance) Ge
photodiode (Applied Detector Corp., Model 403HS, time reso-
lution 300 ns), cooled at 77 K and equipped with a 5-mm-thick
the absence and presence of the quencher Q; [Q] is the quencher
concentration (M); and Ksv is the Stern–Volmer constant. The
bimolecular quenching rate constant k
dividing Ksv by the fluorescence lifetime.
ꢁ
1
ꢁ1
q
(M
s
) was obtained

66
G. Nardi et al. / Free Radical Biology and Medicine 77 (2014) 64–70
AR (anti-reflection)-coated silicon metal filter with wavelength
pass 41.1 μm and an interference filter at 1.27 μm, was used to
measure emission of singlet oxygen at 1270 nm in right-angle
geometry. The photodiode output current was fed into a digital
oscilloscope. All measurements were made at room temperature
in 1-cm-path length quartz cuvettes. The absorbance of the
samples was 0.30 at the laser wavelength. The singlet oxygen
indirect method (TEMPO/EPR), by comparison with phenalenone
(ϕ ¼ 0.95) [30] (Fig. 2). The values obtained are reported in
Δ
Table 1. The results of the TRNIR direct method are in accordance
with literature values [37–39]. However, the quantum yield of
benzophenone evaluated by the indirect method TEMPO/EPR was
significantly higher. This result is somewhat intriguing as, upon
addition of 50 mM TEMP to the benzophenone solution, the direct
TRNIR emission measurement led to nearly zero quantum yield
(Figs. 2B and 3A).
Δ
quantum yield (ϕ ) was determined using phenalenone in acet-
ref
onitrile (ϕ
Δ
¼ 0.95) as reference [30]. Singlet oxygen formation
quantum yield was calculated from the slope of linear plots
representing signal intensity at zero time versus laser pulse energy
Effect of added TEMP on singlet oxygen detection by TRNIR
ref
according to the equation ϕ
emission intensity for the sample at pulse end, Iref is the emission
intensity for the reference, and ϕ
Δ
¼ ϕ
Δ
(Isample/Iref), where Isample is the
1
To investigate the direct effect of TEMP on O
2
production, TRNIR
ref
Δ
is the quantum yield of singlet
emission measurements were performed with photosensitizers in
oxygen formation of the reference.
EPR trapping measurements
18
1
6
The EPR signal of the free radical TEMPO (g ¼ 2.0060, a
N
¼
1
7.3 G) generated by the reaction of singlet oxygen with TEMP was
14
PN
BP
recorded [31]. The measurements were performed in a Wildman
Suprasil/aqueous quartz-ware flat cell (volume 150 ml, length
1
2
10
6
0 mm) with a Bruker EMX 10/12 EPR spectrometer, using the
following parameters: microwave power, 20 mW; modulation
amplitude, 1.0 G; and modulation frequency, 100 kHz.
Aerated ACN solutions of 50 mM TEMP containing a photosensi-
tizer, with an absorbance of 0.4 at 280 nm, were irradiated using the
light produced by a Microbeam system (Model L-201), including a
8
6
4
2
0
NP
CBZ
Control
1
50-W xenon lamp coupled with a monochromator (Model 101);
EPR spectra were recorded at various irradiation times. The singlet
oxygen quantum yield (ϕ ) was determined using phenalenone in
¼ 0.95) as reference [30]. Singlet oxygen formation
was calculated from the slope of the plots of signal area versus
0
4
8
12
16
20
Δ
Irradiation time / min
ref
acetonitrile (ϕ
Δ
ref
Δ
(xsample/xref),
irradiation time according to the equation ϕ
where xsample is the coefficient of linear fit for the sample, xref is the
coefficient of linear fit for the reference, and ϕ
Δ
¼ ϕ
0
.12
PN
NP
ref
is the quantum yield
0.10
.08
0.06
Δ
of singlet oxygen formation of the reference.
0
In the case of BAHA oxidation, EPR spectra of aerated ACN
solutions of 50 mM TEMP were recorded before and after addition
of 0.1 BAHA equivalents.
0
0
0
.04
.02
.00
BP
Photoinduced electron transfer
CBZ
According to Rehm and Weller [32] the free energy change ΔG
BP+TEMP
for electron transfer is expressed by ΔG ¼ Eox ꢁ Ered ꢁ E
where Eox is the oxidation potential of the donor, Ered is the
reduction potential of the ground-state acceptor, E is the energy
n
þ C,
1.5
2.0 2.5 3.0 3.5 4.0 4.5
5.0
n
Energy / mJ
of the acceptor excited state, and C is a coulombic term accounting
for the electrostatic attraction of the produced ions. Neglecting the
C term, ΔG was calculated using the following values for the
potentials: Eox (TEMP) ¼ 1.0 V and Ered ¼ ꢁ1.2, ꢁ1.3, ꢁ2.6, and
Fig. 2. Quantification of 1
sensitizers. (A) Area of TEMPO signal in EPR as a function of irradiation time at
O formation for aerated acetonitrile solutions of photo-
2
280 nm, A2 ¼ 0.4 (control: ACN solution of 50 mM TEMP). (B) Initial intensity of
80
1
O TRNIR emission as a function of 266-nm laser energy, A266 ¼ 0.3; BP þ TEMP, a
2
solution of BP and 50 mM TEMP.
ꢁ
1.8 V vs SCE (Saturated Calomel Electrode) for PN [33], BP [20],
NP [34], and CBZ [35], respectively. In the cases of PN [36] and BP
20], the lowest triplet excited state energy was considered for E ,
[
n
Table 1
ꢁ
1
with values of 220 and 292 kJ mol , respectively, whereas this
parameter was associated with the singlet manifold for NP [34]
and CBZ [37], using values of 377 and 344 kJ mol , respectively.
Singlet oxygen quantum yield of each photosensitizer.
a
ꢁ
1
ϕΔ/TRNIR
ϕΔ/EPR
Phenalenone
Benzophenone
Naphthalene
Carbazole
0.95b
0.95b
0.50
0.50
0.21
c
0.35 (0.35)
Results and discussion
0.56 (0.5)d
0.15 (0.17)e
Determination of singlet oxygen quantum yield by TRNIR emission or
TEMPO/EPR
a
Literature values are in parentheses.
Reference, see Ref. [30].
From Ref. [39].
From Ref. [38].
From Ref. [37].
b
c
Singlet oxygen quantum yields (ϕ
Δ
) were determined for each
d
e
photosensitizer by both a direct method (TRNIR emission) and an

G. Nardi et al. / Free Radical Biology and Medicine 77 (2014) 64–70
67
1.0
0.8
0.6
0.4
0.2
0.0
0.03
0.02
0.01
1
0
0
0
0
.00
.75
.50
.25
.00
(A)
0
5
10
15
20
25
Time / ns
0.00
340 360 380 400 420 440 460 480 500
0
100
200
300
Wavelength / nm
Time /μs
1
.00
0
.04
.02
0
0.75
0
0
0
.50
.25
.00
0
3
00
320
340
360
380
400
0
50
100
150
200
250
Wavelength /nm
Time /μs
Fig. 4. (A) Steady-state fluorescence spectra of CBZ in aerated ACN solution with
increasing amounts of TEMP. [CBZ] 3.5 ꢂ 10ꢁ5 M, λex 331 nm. [TEMP] 0 (black),
Fig. 3. Luminescence decay of 1
B) PN with 0 (□), 5 (○), or 50 mM (Δ) TEMP. Incident 355-nm laser energy: 3.65
mJ/pulse, A355 ¼ 0.3.
2
O at 1270 nm in aerated ACN solution of (A) BP or
(
10 (red), 50 (blue), 100 mM (green). Inset: decay of CBZ at 341 nm in aerated ACN
solution with 0 (black) and 100 mM TEMP (blue). (B) Steady-state fluorescence
spectra of NP in aerated ACN solution with increasing amounts of TEMP. [NP]
ꢁ
6
2
.3 ꢂ 10 M, λex 278 nm. [TEMP] 0 (black), 10 (red), 50 mM (blue). Inset: decay of
the presence of increasing amounts of TEMP. As shown in Fig. 3, in
NP at 320 nm in aerated ACN solution with 0 (black) and 50 mM TEMP (blue).
1
the absence of TEMP the
2
O lifetime was 85 ms using either
phenalenone or benzophenone, in accordance with the literature
value [40]. Addition of TEMP to the solution had different conse-
more than 90% of triplet BP is quenched by TEMP, whereas more
than 90% of triplet PN is quenched by O under the conditions
2
quences depending on the photosensitizer: in the case of phenale-
employed. This fact explains the difference between BP and PN in
the singlet oxygen formation yields upon TEMP addition.
1
none TEMP acted as a poor quencher, shortening the
2
O lifetime
6
ꢁ1 ꢁ1
with k
q
¼ 1.3 ꢂ 10 M
s
[41], a result consistent with literature
As regards NP and CBZ, weak intensity of the LFP signals makes
their triplet states barely detectable in the presence of TEMP (for
example, Supplementary Fig. S4); this was attributed to a scarce
population of the triplet states caused by quenching of the precursor
singlet excited states. To check this hypothesis, the emission signals
of NP and CBZ were monitored in the presence of TEMP. The results
for CBZ are shown in Fig. 4A; they reveal a marked decrease in the
fluorescence intensity concomitant with the growth of a new
structured emission band with peaks at 400, 420, 450, and 470 nm.
This type of long-wavelength emission has been previously observed
in the presence of amines and attributed to charge transfer in the
excited state (excited CBZ carbanion and/or exciplex formation) [44].
data; in the case of BP, in addition to lifetime shortening, TEMP was
able to knock out the formation of 1
indicates that there is an interaction between TEMP and the
precursor of 1
, namely the excited triplet state of BP. In the case
of PN, addition of TEMP did not influence the initial TRNIR signal
2
O (Fig. 3A). This observation
O
2
1
1
intensity of O
Quenching of photosensitizer excited states by TEMP
In view of the effects produced by addition of TEMP on the O
2 2
, but affected the O lifetime (Fig. 3B).
1
2
TRNIR signal, the triplet–triplet transient absorption of the photo-
sensitizers was studied by laser flash photolysis in the presence of
TEMP (see Supplementary Figs. S2–S4). In the case of BP, triplet
q
The k determined by means of steady-state and time-resolved
8
8
ꢁ1 ꢁ1
emission measurements were 9.1 ꢂ 10 and 6.7 ꢂ 10 M
s
,
quenching by TEMP was observed with rate constant k
q
¼
respectively (Supplementary Fig. S5). The difference between the
two values suggests the contribution of static quenching, probably
due to formation of a ground-state complex.
9
ꢁ1 ꢁ1
1
.5 ꢂ 10 M
s
, in accordance with the literature [42,43]. In
the case of PN, although TEMP was also able to deactivate the
triplet excited state, the measured rate constant was 2 orders of
magnitude lower (k
constants for triplet quenching by oxygen are very similar for the
two ketones (ca. 2.5 ꢂ 10 M
Likewise, the singlet excited state of NP was quenched by TEMP
(Fig. 4B); the rate constant obtained from steady-state fluorescence
6
ꢁ1 ꢁ1
q
¼ 8.3 ꢂ 10 M
s
). In contrast, the rate
9
ꢁ1 ꢁ1
(k
q
¼ 1.9 ꢂ 10 M
s
, Supplementary Fig. S6) was similar to that
9
ꢁ1 ꢁ1
9
ꢁ1 ꢁ1
s
). Taking into account the
, as well as the k values and
resulting from time-resolved fluorescence (k
indicating that NP–TEMP ground-state complexation can be safely
ruled out. It should be noted that the efficient quenching of the
q
¼ 2.0 ꢂ 10 M
s
),
competition between TEMP and O
2
q
2
the TEMP and O concentrations (50 and 1.9 mM, respectively),

68
G. Nardi et al. / Free Radical Biology and Medicine 77 (2014) 64–70
Fig. 5. Proposed mechanism for the electron transfer oxidation of TEMP to TEMPO in the presence of molecular oxygen.
singlet excited photosensitizer by TEMP and the reduced 1O
2
production are not reflected in the EPR measurements of Table 1.
Overall, the results related to excited-state quenching by TEMP
suggest the possibility of an alternative electron transfer reaction
pathway for the oxidation of TEMP that produces a detectable
TEMPO signal not involving singlet oxygen.
1
0
0
0
0
.00
.75
.50
.25
.00
Electron transfer oxidation of TEMP to TEMPO
Photoinduced electron transfer (PET) is a feasible reaction between
ketone or aromatic hydrocarbon photosensitizers and amines [26–29].
Being a secondary amine, TEMP (Eox ¼ 1.0 V vs SCE) [45] could be
oxidized by PET if the process is thermodynamically allowed. Accord-
ing to Rehm and Weller [32], this requirement is fulfilled for all the
investigated photosensitizers. Indeed ΔG for electron transfer is in all
ꢁ
1
cases negative, being ca. ꢁ10, ꢁ70, ꢁ30, and ꢁ70 kJ mol for PN,
5
60
580
600
620
640
660
680
BP, NP, and CBZ, respectively.
Wavelength/nm
To actually prove that an electron transfer reaction can con-
tribute to the production of the TEMPO radical, TEMP was oxidized
by BAHA (also named magic blue), a known one-electron oxidizing
agent (Ered ¼ 1.17 V vs SCE) [46]. Addition of BAHA to a solution of
TEMP in acetonitrile led to the bleaching of the initial blue color,
which turned pale brown (Supplementary Fig. S7). The resulting
solution was analyzed by EPR, and an enhanced TEMPO signal was
observed under aerobic conditions (see Supplementary Fig. S8).
0
0
0
0
0
.08
.06
.04
.02
.00
The mechanistic scheme explaining these results is outlined in
þd
Fig. 5. Generation of the radical cation TEMP
is followed by
deprotonation and reaction of the resulting neutral radical with
molecular oxygen, finally leading to the TEMPO radical [47–49].
1
The case of rose Bengal, an archetypal O
2
photosensitizer
-0.02
1
2
The methodology was applied to RB, a O photosensitizer widely
0
10
20
30
40
used in cellular experiments. Steady-state fluorescence showed an
almost unaltered emission of RB singlet excited state in the presence
of 100 mM TEMP (Fig. 6A). This result is in accordance with time-
resolved measurements, which allowed determining a very low
Time / μs
7
ꢁ1 ꢁ1
18
16
quenching rate constant of ca. 10 M
s
(Fig. 6A, inset). Likewise,
the triplet excited state of RB was not affected by the presence of
the secondary amine (Fig. 6B). Thus, from these data one can
anticipate that the RB singlet oxygen quantum yield obtained by the
TEMPO/EPR method should correlate well with that reported in the
PN
1
1
4
2
literature (ϕ
Δ
¼ 0.54) [50]. This was confirmed by performing the
10
8
EPR experiment using PN as standard, which led to a very reasonable
value of 0.56 (Fig. 6C).
RB
6
4
Conclusion
2
0
Detection and quantification of singlet oxygen by means of the
TEMPO/EPR method is a useful and widely employed technique.
This method, however, may be misleading when the excited
0
4
8
12
16
20
Irradiation time / min
photosensitizer is capable of reacting with TEMP, acting as an
Fig. 6. (A) Emission of an aerated ACN solution of RB alone (black) or in the
þd
ꢁ6
electron donor. In this case, generation of the TEMP
radical
presence of 100 mM TEMP (blue). [RB] 4 ꢂ 10 M, λex 560 nm. Inset: Stern–Volmer
plot obtained from time-resolved measurements of RB in the presence of increasing
cation, followed by deprotonation and reaction with molecular
oxygen, gives rise to an EPR-detectable TEMPO signal that is not
associated with singlet oxygen production. The possibility of such
an electron transfer interference can be anticipated by means of
amounts of TEMP (λex
¼ 560 nm, λem¼573 nm). (B) Triplet–triplet absorption
decays monitored at 620 nm (λex 532 nm) of argon-bubbled ACN solution of RB
ꢁ5
(
3.3 ꢂ 10 M) alone (black) or in the presence of 100 mM TEMP (blue). (C) Area of
TEMPO signal in EPR as a function of irradiation time at 280 nm, A280 0.4.

G. Nardi et al. / Free Radical Biology and Medicine 77 (2014) 64–70
69
simple thermodynamic calculations based on redox potentials and
excited state energies. In addition, this source of artifacts can be
safely ruled out when no quenching of the photosensitizer singlet
and triplet excited states by TEMP is observed in fluorescence and
laser flash photolysis experiments, respectively. Thus the applica-
tion of such relatively simple techniques provides knowledge that
is essential for an appropriate and error-free application of the
TEMPO/EPR method of singlet oxygen detection in chemical,
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Acknowledgments
The Spanish government (CTQ2012-32621, RyC-2007-00476, PFIS
FI09/00312, Severo Ochoa Program SEV-2012-0267), the Carlos III
Institute of Health (Grant RIRAAF, RETICS Program RD12/0013/0009),
and the Generalitat Valenciana (Prometeo II/2013/005) are gratefully
acknowledged for financial support. Dr. A. Vidal-Moya is acknowl-
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